THE NEURAL BASIS OF ESTRADIOL

THE NEURAL BASIS OF ESTRADIOL CONDITIONED TASTE AVERSION LEARNING AND ESTRADIOL ANOREXIA

by

Houri Hintiryan

A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PSYCHOLOGY)

May 2009

Copyright 2009

Houri Hintiryan

UMI Number: 3355410 Copyright 2009 by Hintiryan, Houri All rights reserved

INFORMATION TO USERS

The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.

______________________________________________________________ UMI Microform 3355410 Copyright 2009 by ProQuest LLC All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

_______________________________________________________________ ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106-1346

Acknowledgments I would like to express my deepest gratitude to my faculty advisor, Dr. Kathleen Chambers, for all of her guidance and support throughout my graduate career. I am forever grateful to my mentor and my friend. I also would like to thank my teammate and dear friend Nicholas Foster, who unselfishly devoted his time and efforts to helping me throughout the years. Last, but not least, I would like to thank my husband and best friend Raffie, who patiently and lovingly supported and encouraged me throughout this challenging journey.

ii

Table of Contents Acknowledgments

ii

List of Tables

viii

List of Figures

ix

Abstract

xi

Chapter 1 Neural Regulation of Eating Behavior 1.1 Introduction 1.2 The Dual Center Theory of Ingestive Behavior 1.3 Key Neural Substrates of Eating Behavior 1.3.1 Hypothalamus Organization of the Hypothalamus Connections of the Hypothalamus Connections within Hindbrain Structures Hypothalamic Nuclei and Neuropeptides Regulating Eating 1.3.1.1 Arcuate Nucleus (ARC) Anabolic Peptides within the Arcuate Neuropeptide Y (NPY) Agouti Related Protein (AgRP) Galanin Catabolic Peptides within the Arcuate Proopiomelanocortin (POMC) and CocaineAmphetamine Related Transcript (CART) Interactions between NPY/AgRP and POMC/CART Neurons 1.3.1.2 Lateral Hypothalamus (LH) Anabolic Peptides within the Lateral Hypothalamus Melanin-Concentrating Hormone (MCH) Orexin 1.3.1.3 Paraventricular Nucleus (PVN) 1.3.1.4 Dorsomedial Hypothalamus (DMH) 1.3.1.5 Ventromedial Hypothalamus (VMH) 1.3.1.6 Tuberomammillary Nucleus (TM) 1.3.2 Hindbrain Structures 1.3.2.1 Area Postrema (AP) 1.3.2.2 Nucleus of the Solitary Tract (NST) 1.3.2.3 Parabrachial Nucleus (PBN) The Lateral Parabrachial Nucleus The Medial Parabrachial Nucleus 1.3.3 Forebrain Structures 1.3.3.1 Amygdala (Amg)

1 1 2 3 4 4 4 6 8 9 10 10 12 14 16 16 17 18 19 19 20 23 24 26 28 29 29 31 33 33 37 37 37

iii

1.3.3.2 Bed Nucleus of Stria Terminalis (BNST) 1.4 Short-term Regulation of Eating 1.4.1 Glucose and the Glucostat Hypothesis 1.4.2 Peripheral Feeding Signals Ghrelin 1.4.3 Peripheral Satiety Signals Cholecystokinin (CCK) Peptide YY (PYY) Glucagon-like Peptides-1and 2 (GLP-1/GLP-2) 1.5 Long-term Regulation of Eating 1.5.1 Set Point Theory of Body Weight 1.5.2 Peripheral Adiposity Signals Leptin Insulin Amylin 1.6 Conclusion

40 42 43 46 46 49 49 52 54 56 57 58 58 64 67 69

Chapter 2 Estradiol and Eating Behavior 2.1 Estradiol: Overview 2.2 Estradiol and Eating Behavior Tonic and Phasic Effects of Estradiol Estradiol and Anorexia Nervosa 2.3 Neural Substrates of Estradiol Hypophagia C-fos Like Immunoreactivity (c-FLI) Studies Central Implant Studies Lesion Studies 2.4 Estradiol and the Lateral Parabrachial Nucleus (PBN) 2.5 Estradiol, Neuropeptides and Eating Cholecystokinin (CCK) and Estradiol Neuropeptide Y (NPY) and Estradiol Melanin-Concentrating Hormone (MCH) and Estradiol

71 71 73 76 76 86 86 87 89 90 91 92 94 95

Chapter 3 Conditioned Taste Aversion Learning 3.1 Conditioned Taste Aversion: Overview Attributes of Conditioned Taste Aversion Learning Evolutionary Significance of Conditioned Taste Aversion 3.2 Gustatory (CS) and Malaise (US) Information Neural Gustatory Pathway (CS Pathway) Neural Visceral Pathway (US Pathway) 3.3 Neural Substrates of Conditioned Taste Aversion Learning 3.3.1 The Vagus and Nucleus of the Solitary Tract (NST) 3.3.2 The Area Postrema (AP) 3.3.3 The Parabrachial Nucleus (PBN) Lateral Parabrachial Nucleus (lateral (PBN) Medial Parabrachial Nucleus (medial PBN)

97 97 97 99 100 100 101 104 106 107 112 113 117

iv

3.3.4 The Amygdala (Amg) 3.3.5 The Insular Cortex (IC) 3.3.6 Configuring the Conditioned Taste Aversion Neural Pathway 3.4 Neurotransmitters and Conditioned Taste Aversion Learning Methodological Issues 3.4.1 Acetylcholine (ACh) Synthesis of Acetylcholine Cholinergic Receptors and Receptor Sites Acetylcholine and Learning Acetylcholine and Conditioned Taste Aversions 3.4.2 Histamine (HA) Synthesis of Histamine Histamine Receptors and Receptor Sites Histamine and Learning Histamine and Conditioned Taste Aversions 3.5 Estradiol and Conditioned Taste Aversion Learning Conditioned Taste Aversions Revisited Neural Substrates of Estradiol Conditioned Taste Avoidance Neurochemical Mediation of Estradiol Conditioned Taste Avoidance

121 123 125 130 130 134 134 134 136 136 142 142 143 144 145 146 147 148 149

Chapter 4 Conducted Experiments 4.1 Outline of Experiments 4.2 Primary Aims 4.2.1 Primary Aim 1 Specific Aim 1.1 Specific Aim 1.2 Specific Aim 1.3 4.2.2 Primary Aim 2 Specific Aim 2.1 Specific Aim 2.2 4.3 Secondary Aims 4.3.1 Secondary Aim 1 4.3.2 Secondary Aim 2 4.4 General Methodology 4.4.1 Subjects and Husbandry 4.4.2 Chemical Agents Conditioning Agents Lesioning Agent 4.4.3 Surgical Procedures Anesthetics and Surgical Techniques Analgesics, Antibiotics, and Post-operative Care

153 153 154 154 155 159 161 162 165 165 166 166 169 170 170 171 171 172 172 172 176

v

4.4.4 Behavior Testing Conditioned Taste Avoidance/Aversion (CTA) Procedure Measurements of Fluid Intake Measurements of Food Intake 4.4.5 Histological Staining Neuron-Specific Nuclear Protein 4.4.6 Statistical Analyses Description of Analyses Assessment of CTA Formation Assessment of Extinction Assessment of Fluid Intake Assessment of Food Decrement Additional Analyses 4.5 Presentation of Experiments 4.5.1 Experiment 1 Methods Results Discussion 4.5.2 Experiment 2 Methods Results Discussion 4.5.3 Experiment 3 Methods Results Discussion 4.5.4 Experiment 4 Methods Results Discussion 4.5.5 Experiment 5A Methods Results Discussion 4.5.6 Experiment 5B Methods Results Discussion

177 177 179 179 180 180 182 182 183 184 184 185 186 187 187 187 188 194 196 197 197 200 201 203 203 204 206 207 213 219 222 223 234 251 252 252 256 263

vi

4.5.7 Experiment 5 Discussion CTA1: Estradiol: 6 Hour CS-US Interval CTA1: Estradiol: 0 Hour CS-US Interval CTA2: Lithium Chloride: 0 Hour CS-US Interval Estradiol (CTA1) and Lithium Chloride (CTA2) Unilateral Lesions and CTAs Experiment 5b and CTAs: Some Important Issues Summary of CTA Findings Estradiol Hypophagia Summary of Experiment 5 Findings 4.6 General Discussion Dissociating Estradiol CTA from Estradiol Hypophagia What is Estradiol CTA? How is the Lateral PBN Involved in Estradiol CTA? What is Meant by “Attenuated CTA”? 4.7 Future Direction Excitotoxic Lesions CTA Behavioral Procedure Measurements of Food and Fluid Intake Additional Considerations

264 264 266 271 272 275 278 281 281 284 285 285 288 291 294 296 296 298 299 299

References

300

vii

List of Tables Table 1. Effects of Peripherally and Centrally Administered Cholinergic Antagonists on Conditioned Taste Aversions

139

Table 2. Histamine and Estradiol Conditioned Taste Avoidance

151

Table 3. Criteria for Acquisition of a CTA: Changes in Sucrose Consumption Across Acquisition and Either Post-Acquisition Test 1 or Post-Acquisition Test 2

213

Table 4. Number of Animals that Acquired or Did Not Acquire an Estradiol Conditioned Taste Avoidance Based on Criteria for Conditioned Taste Avoidance Qualification

218

viii

List of Figures Figure 1. Neural Connectivity Among Hypothalamic Nuclei, Hindbrain Structures and Forebrain Structures

8

Figure 2. Neural Circuitry of Conditioned Aversions

130

Figure 3. Methodology for Determining Chemical Mediation of Estradiol Conditioned Taste Avoidance

164

Figure 4. Effect of Two Different Time Intervals between Acquisition and Post-acquisition Testing on Estradiol Conditioned Taste Avoidance and Calorie Intake

193

Figure 5. Effect of Two Different Doses of Estradiol on Conditioned Taste Avoidance and Calorie Intake

199

Figure 6. Effect of a 24 hour CS-US Interval on Estradiol Conditioned Taste Avoidance

204

Figure 7. Effect of Various CS-US Intervals on Estradiol Conditioned Taste Avoidance

215

Figure 8. Replication of the Effect of a 6 hour CS-US Interval on an Estradiol Conditioned Taste Avoidance

217

Figure 9. Number of Animals in each Estradiol and Oil Group that Acquired or Did Not Acquire Estradiol Conditioned Taste Avoidance for Each CS-US Interval

218

Figure 10. Methodology for Determining Chemical Mediation of Estradiol Conditioned Taste Avoidance

221

Figure 11. Neuron-Specific Nuclear Protein (NeuN) Stain of an Ibotenic Acid Lesion of Lateral Amygdala

226

Figure 12. Neuron-Specific Nuclear Protein (NeuN) Stain of Ibotenic Acid Lesions of Lateral Parabrachial Nucleus

230

Figure 13. Sucrose and Water Intake for Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus Across 7 Acquisition Trials of an Estradiol Conditioned Taste Avoidance

236

ix

Figure 14. Sucrose and Water Intake for Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus Across Extinction Phases of an Estradiol Conditioned Taste Avoidance

242

Figure 15. Chocolate Milk Intake in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus During Acquisition and Extinction of a Lithium Chloride Conditioned Taste Aversion

244

Figure 16. Size of Lateral Parabrachial Nucleus Lesion in Bilateral E and Bilateral Oil Groups on Right and Left Sides of the Brain

246

Figure 17. Amount of Food Consumed During Twenty Four Hour Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus

247

Figure 18. Amount of Food Consumed During Twelve Hour Dark Phase Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus

249

Figure 19. Amount of Food Consumed During Twelve Hour Light Phase Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus

251

Figure 20. Neuron Specific Nuclear Protein (NeuN) Stain of Electrolytic Lesions of the Lateral Parabrachial Nucleus

257

Figure 21. Sucrose Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Acquisition and the First PostAcquisition Test of a of a Lithium Chloride Conditioned Taste Aversion

259

Figure 22. Sucrose Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Extinction of a Lithium Chloride Conditioned Taste Aversion

260

Figure 23. Chocolate Milk Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Acquisition and PostAcquisition Tests of an Estradiol Conditioned Taste Avoidance

261

Figure 24. Chocolate Milk Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Extinction of an Estradiol Conditioned Taste Avoidance

262

Figure 25. Amount of Food Consumed During Twenty-Four Hour Periods that Followed Estradiol Injections in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus

263

x

Abstract When consumption of a novel tasting substance is followed by administration of a chemical agent that produces physiological changes indicative of malaise, animals will reduce their consumption of the substance during subsequent encounters. This learned response is traditionally referred to as a conditioned taste aversion (CTA). Studies have shown that the hormone estradiol is capable of producing this learned gustatory aversion. In addition, estradiol produces reductions in food intake and body weight, a phenomenon that is referred to as its anorectic effects. As a consequence of this anorectic effect, we question whether estradiol truly can induce CTA learning. Therefore, one of the purposes of the experiments presented in this dissertation was to test the dissociability of estradiol CTA and estradiol anorexia. The second purpose of this thesis was to examine the neural basis of estradiol CTA and estradiol anorexia. Four approaches were adopted to test the ability of estradiol to condition independent of its ability to produce reductions in eating. First, we show that estradiol can produce a CTA in the absence of its anorectic effects. Second, we demonstrate that a low dose of estradiol that produces reductions in eating does not produce CTA. Next, we show that contingent pairing is necessary for a CTA since non-contingent pairing does not result in the gustatory aversion. Finally, we dissociate the conditioning effect of estradiol from its anorectic effect by showing that both excitotoxic and electrolytic lesions of the lateral parabrachial nucleus (PBN) either attenuated or blocked an estradiol CTA, while leaving estradiol anorexia unaffected. Together, all of the data suggest that estradiol can condition a gustatory xi

aversion. The data also suggest that estradiol elicits a CTA based on its aversion inducing properties since lesions of the lateral PBN, an area that processes visceral information, blocked the CTA. Third, the data show that the lateral PBN is necessary for estradiol CTA. Finally, the studies show that although the lateral PBN is involved in different types of anorexia, it is not involved in estradiol anorexia.

xii

CHAPTER 1 Neural Regulation of Eating Behavior 1.1 Introduction Ingestion is a complex behavior that encompasses four different phases that are distinguished from one another using behavioral and neurological criteria (Watts, 2000). Briefly, the initiation phase consists of a change in the internal drive state, which shifts the organism’s attention to food. The procurement phase follows and involves the various aspects of foraging, while the consummatory phase entails the stereotyped action patterns of eating. During this latter phase, associations regarding the nutritional load and hedonic valence of the food also are formed. Satiation or a competing motivated behavior, leads to the last stage, namely the termination of a meal. This chapter will discuss the main neural areas and neuropeptides involved in the initiation and termination of a meal, bypassing the mechanisms involved in foraging and in the motor regulation of consummatory behavior. The latter half of the chapter will be designated to the neural regulation of body weight. Thus, the focus of the chapter will remain on the maintenance of energy homeostasis, the process by which energy intake is matched to energy expenditure. Although environmental and social stimuli, such as time of day, financial circumstances, and the presence of food and people, should not be discarded as important factors in motivating eating behavior, they too are beyond the scope of this chapter.

1

1.2 The Dual Center Theory of Ingestive Behavior For an extended period of time, the predominant conception regarding the neural control of eating revolved around the “dual center” theory. This hypothesis gained solidity following the publication of the classic paper The Physiology of Motivation, in which Stellar (1954) purported that most motivated behaviors, including eating, were controlled by two specific brain structures: one that activated the behavior and another that played an inhibitory role. Following this proposal, the prevalent conception from the 1950’s to the 1970’s was that eating was under the control of two functional centers, namely a feeding center and a satiety center. Stellar reported evidence that was available at the time on the neural control of eating that substantiated this binary theory. To that point, it had been demonstrated that following lesions of the ventromedial nucleus of the hypothalamus (VMH), animals would exhibit hyperphagia, which eventually would lead to severe obesity (Hetherington & Ranson, 1940; Anand & Brobeck, 1951; Brobeck, Tepperman & Long, 1943). Electrical stimulation of this area had the opposite effect, resulting in decreased eating (Anand & Dua, 1955). On the contrary, bilateral lesions of the lateral hypothalamus (LH) lead to hypophagia (Anand & Brobeck, 1951a), while stimulation studies showed that activation of this area increased eating (Anand & Brobeck, 1951b; Delgado & Anand, 1953). Predicated on these observations, the VMH appropriately got labeled the satiety center, while the LH earned the title of the feeding center.

2

Due to recent advancements in the field of food intake and body weight regulation, it is clear that not only does ingestive behavior extend beyond the VMH and the LH within the diencephalic structure, it also involves regions of the hindbrain and the forebrain. As such, the idea of functional centers has become obsolete. Presently, the effects of the neuropeptides that are localized within a neural structure are used in combination with lesion studies to assess the role of a particular area in eating behavior. In addition, there are separate mechanisms that work in conjunction to control the size of individual meals and to regulate body weight. Although the neural machinery is highly conserved for these separate processes (control of meal frequency, meal size, and body weight), the molecular signals that initiate each process appear to be disparate. 1.3 Key Neural Substrates of Eating Behavior Since the hypothalamus is the central player in eating behavior, the specific nuclei of the diencephalic structure and their residing neuropeptides will be covered first. Subsequently, discussion of hindbrain areas, like the area postrema (AP), nucleus of the solitary tract (NST), and the parabrachial nucleus (PBN), and of forebrain structures, like the amygdala (Amg) and the bed nucleus of the stria terminalis (BNST), that work in concert with the hypothalamus to regulate eating and body weight will follow.

3

1.3.1 Hypothalamus Organization of the Hypothalamus The hypothalamus contains numerous nuclei, some of which are divided further into subnuclei (Simerly, 1995). Generally, along the medial-lateral axis, the hypothalamus is organized into three zones. Starting from the most lateral, they are the (1) lateral, (2) medial, and (3) periventricular zones. Each of these contains nuclei that have been shown to play an integral role in eating. The lateral zone includes the lateral hypothalamus (LH), while the medial zone contains the dorsomedial (DMH) and ventromedial (VMH) nuclei. The paraventricular nucleus (PVN) resides in the periventricular zone, while the arcuate (ARC) extends across the medial and periventricular zones. The mammillary nucleus (MM), also an important regulator of eating behavior, lies in between the medial and periventricular zones. Along the anterior-posterior axis, the hypothalamus is divided into several regions as well. Starting with the most anterior, the hypothalamic nuclei discussed in this chapter are organized into the following regions: (1) anterior, (2) tuberal, and (3) mammillary regions. The LH extends across all of these regions, while the PVN is restricted to the anterior region. The DMH, VMH, and ARC nuclei are found in the tuberal region. The most posterior of the nuclei are the mammillary bodies, which reside in the mammillary region. Connections of the Hypothalamus The nuclei of the hypothalamus are heavily interconnected, but they also share neural connections with the AP, NST, and PBN in the hindbrain and with the 4

Amg and BNST of the forebrain. The connectivity among these eating-related structures gives insight into the potential circuitries underlying eating behavior. Of course, fibers of the hypothalamus innervate many more regions of the brain; however, since these are the eating-related structures covered in this chapter, discussion regarding the connections of the hypothalamus will be limited to these neural areas (see Figure 1 for neural connections discussed below). Within the hypothalamus the ARC is a key regulator of eating. It receives its densest afferent projections from other hypothalamic structures including the PVN, while weaker inputs come from the LH (Simerly, 1995). It receives projections from extra-hypothalamic structures implicated in eating like the lateral subdivision of the PBN, NST, central nucleus of the Amg and the BNST (Ricardo & Koh, 1978; Li, Chen & Smith, 1999). Tracing studies have shown that there are reciprocal connections between the LH and the ARC, PVN, DMH, and VMH (Simerly, 1995). The LH also shares reciprocal connections with the PBN and NST where it receives viscero-sensory information pertaining to food (Luiten, ter Horst & Steffens, 1987; Larsen, Moller & Mikkelson, 1991; Simpson & Raubenheimer, 2000). Finally, the LH is the target of afferent projections from the Amg (Simerly, 1995). Like the LH, the PVN receives afferents from forebrain regions such as the BNST and central Amg (Sawchenko & Swanson, 1983) and hindbrain structures like the PBN (Thompson & Swanson, 1998). In turn, the PVN sends information

5

to the NST (Hosoya, Sugiura, Okado, Loewy & Kohno, 1991) and to the PBN (Moga, Saper & Gray, 1989; Moga, Herbert, Hurley, Yasui, Gray et al., 1990). Most of the inputs received by the DMH are provided by other hypothalamic nuclei such as the LH, ARC, VMH, and PVN (Simerly, 1995; Thompson & Swanson, 1998). Much like the PVN and LH, although to a lesser degree, it is the target of the PBN (Thompson & Swanson, 1998). The efferent projections of the DMH also are mainly limited to the hypothalamus, with the strongest recipient being the PVN (Simerly 1995). Although the VMH sends weak inputs to eating-related areas (Canteras, Simerly & Swanson, 1994), it receives strong inputs from the Amg, the LH, the PBN, and the NST (Fulwiler & Saper, 1985). Connections within Hindbrain Structures Since the neural connectivity of the AP, NST, and PBN are going to be important in the circuitry of peptides that regulate eating, the connections amongst the structures are briefly introduced here. Neuroanatomical studies have established that the AP receives information from the viscera via the afferent vagus nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985). In addition, it sends and receives information from the NST and the PBN, although predominantly from the lateral subdivision of the PBN (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985). The PBN and NST also share connections with one another (Lowey & Burton, 1979; Saper & Lowey, 1980). Finally, the PBN has bi-directional communication with the LH, VMH, and PVN (Moga et al., 1990) and the central 6

Amg and BNST (Bernard, Peschanski & Besson, 1989; Moga et al., 1989; Moga et al., 1990; Bernard, Carroue & Besson, 1991). Anatomical studies have shown that the NST and central Amg share reciprocal connections (Norgren, 1978; Ricardo & Koh, 1978; Gray & Magnuson, 1987). This connection has been confirmed by studies using electrical stimulation of the central Amg and measurement of c-fos (the product of the immediate early gene c-Fos and an indicator of neuronal activity). The results showed that stimulation of the amygdaloid nucleus produced activation of c-fos in the NST (Petrov, Jhamandas & Krukoff, 1996).

7

Figure 1. Neural Connectivity Among Hypothalamic Nuclei, Hindbrain Structures and Forebrain Structures. DMH VMH ARC

LH

BNST PVN Amg Medial PBN

Lateral PBN

NST

Area Postrema Vagus Nerve Figure 1. Neural connectivity among hypothalamic nuclei, hindbrain structures and forebrain structures. Not all connections are depicted. Double arrows ( ) indicate reciprocal connections. AP: Area Postrema; NST: Nucleus of the Solitary Tract; PBN: Parabrachial Nucleus; PVN: Paraventricular Nucleus; ARC: Arcuate; VMH: Ventromedial Hypothalamus; DMH: Dorsomedial Hypothalamus; LH: Lateral Hypothalamus; Amg: Amygdala; and BNST: Bed Nucleus of the Solitary Tract

Hypothalamic Nuclei and Neuropeptides Regulating Eating The role of each hypothalamic nucleus is largely defined by the neuropeptides that are localized within each structure. These structures and their regulatory peptides are discussed below. 8

1.3.1.1 Arcuate Nucleus (ARC) The different populations of neuropeptides that are localized within the ARC largely define its role in eating behavior. This periventricular nucleus contains peptides that are involved in both catabolic and anabolic effector systems (Kaiyala et al., 1995; Schwartz, Woods, Porte, Seeley & Baskin, 2000). Briefly, catabolic systems work to decrease food intake and increase energy expenditure and are activated by positive energy states. Anabolic systems do the opposite by increasing eating and decreasing energy expenditure and are activated by negative energy states. Numerous studies using various types of techniques ranging from immunohistochemistry to in situ hybridization have identified two distinct populations of neurons in the ARC. One of these populations contains the orexigenic protein Neuropeptide Y (NPY), which is coexpressed with a second feeding related agent, Agouti Related Protein (AgRP; Broberger, de Lecea, Sutcliffe & Hokfelt, 1998; Hahn, Breininger, Baskin & Schwartz, 1998). Galanin is a third orexigenic that is synthesized in the ARC, but is not colocalized with any other peptide. A second population of neurons in the ARC co-expresses peptides that have the opposite effect on eating. These anorexigenics are proopiomelanocortin (POMC) and cocaine-amphetamine related transcript (CART; Kristensen, Judge, Thim, Ribel, Christjansen et al., 1998). These different neuropeptides with their various effects on eating define the diverse role the ARC plays in eating behavior. 9

Anabolic Peptides within the Arcuate Nucleus Neuropeptide Y (NPY) Neuropeptide Y is an orexigenic peptide that potently stimulates eating. This is an effect that consistently has been demonstrated across various species of animals (Clark, Karla, Crowley & Karla, 1984; Kuldosky, Glazner, Moore, Low & Woods, 1988; Morley, Hernandez & Flood, 1987; Pau, Pau & Spies, 1985) and across the life span of rats, ranging from the time of peri-natal development (Capuano, Barr & Leibowitz, 1986) to late adulthood (Pinch, Messori, Zoli, Ferraguti, Marrama et al., 1992). Infusions of the peptide produce hyperphagia when infused into the lateral ventricles or directly into the PVN (Stanley & Leibowitz, 1985; Stanley, Kyrkouli, Lampert & Leibowitz, 1986), the LH, and the VMH (Stanley, Chin & Leibowitz, 1985). Evidence implicates the perifornical area (PFA) as the most sensitive hypothalamic site for NPY infusions (Stanley, Willet, Donias, Ha & Spears, 1993). The PFA is immediately adjacent to the LH and stimulation of this area has been shown to produce increases in eating (Stanley et al., 1993). Infusions of NPY into the PFA produce the strongest effects on eating probably due to the substantial NPYergic connections it receives from the ARC (Booth & Thibault, 2000). Extrahypothalamic infusions of NPY have no effect on eating (Morley et al., 1987). Interestingly, NPY increases the motivation to eat since following NPY infusions, animals will work extremely hard at a lever press in order to get a food reward, they will eat food adulterated with an unpalatable agent such as quinine, and they will drink milk that is paired with an 10

electric shock (Flood & Morley, 1991; Jewett, Cleary, Levine, Schaal & Thompson, 1992). Further, intracerebroventricular infusions of NPY reduce energy expenditure (Billington, Briggs, Grace & Levine, 1991; Billington, Briggs, Harker, Grace & Levine, 1994), stimulate lipogenesis in the liver and in white adipose tissue (Billington et al., 1991; Zarjevski, Cusin, Vettor, Rohner-Jeanrenaud & Jeanrenaud, 1993), and increase circulating levels of insulin (Zarjevski et al., 1993). These effects were independent of the peptide’s effects on eating since since restricting the animals’ consumption following the infusions resulted in elevated lipogenesis and circulating insulin. It is not surprising then that chronic administration of the peptide readily leads to obesity in both mice and rats (Stanley et al., 1986; Zarjevski et al., 1993). Evidence implicates the involvement of NPY in a prominent anabolic effector system that includes the ARC and PVN. Although NPYergic neurons in the ARC heavily innervate other hypothalamic nuclei, they most densely project to the PVN (Bai, Yamano, Shiotani, Emson, Smith, et al., 1985; Elias, Saper, Maratos-Flier, Tritos, Lee et al., 1998b), another diencephalic structure shown to be involved in the suppression of eating (Leibowitz, Hammer & Chang, 1981; Aravich & Scalfani, 1983). This ARC-PVN connection has been implicated in the NPY correctional mechanism during states of depleted energy. For instance, NPY synthesis in the ARC and its subsequent release in the PVN are both elevated in response to fasting (Sahu, Karla & Karla, 1988), lactation (Li, Chen & Smith, 1998; Smith, 1993), physical exercise (Lewis, Shellard, Koeslag, Boer, McCarthy 11

et al., 1993), and in the period of insulin deficiency during which cells are glucose deprived (Sahu, Sninsky, Phelps, Dube, Karla, et al., 1992). These findings suggest that NPY is released to increase eating in order to correct for the ensuing negative energy state. Further, they demonstrate the important interaction between the ARC and PVN in regulating energy homeostasis. Despite the transparent importance of this neuropeptide in energy homeostasis, NPY knock out mice continue to display normal patterns of eating (Erickson, Clegg & Palmer, 1996; Hollopeter, Erickson, Seeley, Marsh & Palmiter, 1998). This finding may suggest that compensatory mechanisms exist which take over the responsibilities of NPY during its absence. Agouti Related Protein (AgRP), discussed in the next section, is a possible candidate that assumes this role. Evidence for this hypothesis is provided by the finding that AgRP mRNA is upregulated in NPY knock out mice (Marsh, Miura, Yagaloff, Schwartz, Barsh et al., 1999). Nevertheless, NPY appears to be a critical regulator of energy intake. Many molecules that regulate energy intake interact with NPY to induce their effects on eating whether it is for the cessation of an individual meal or the control of body weight through glucose, leptin, or insulin. These interactions will be discussed in later sections. Agouti Related Protein (AgRP) Agouti Related Protein is synthesized exclusively in the ARC and is coexpressed with NPY. Messenger RNA for AgRP is found in all NPYergic 12

neurons of the ARC and thus far has not been localized in any other region of the brain (Broberger et al., 1998). Similar to NPY, AgRP is an orexigenic compound that increases eating when infused centrally into the lateral ventricles (Ollman, Wilson, Yang, Kerns, Chen et al., 1997; Hagan, Rushing, Pritchard, Schwartz, Strack et al., 2000). Its overexpression in transgenic mice produces increased eating and culminates in obesity (Graham, Shutter, Sarmiento, Sarosi & Stark, 1997). Although intracerebroventricular infusions of NPY produce more potent hyperphagia compared to infusions of AgRP (Stanley & Leibowitz, 1984; Morley et al, 1987), the hyperphagic actions of AgRP are longer lived compared to that of NPY, lasting for several days as opposed to several hours (Hagan et al., 2000). There is evidence suggesting that AgRP induces its hyperphagic effects through melanocortin receptors 3 and 4 (MC3/MC4) of α-melanocyte stimulating hormone (α -MSH), an anorexigenic compound. Alpha-MSH is thought to restrict eating and constrain weight gain by acting as an agonist for MC3/MC4 receptors. This implies an inhibitory role on eating by these melanocortin receptors. It is demonstrated that AgRP acts as an antagonist for MC3/MC4 receptors (Ollmann et al., 1997; Quillan, Sades, Wei, Jimenez, Ji et al., 1998) suggesting that the peptide induces its effects on eating by inhibiting the satiogenic effects of melanocortin receptors. Since the long-term actions of AgRP are still present following the blockade of MC3 and MC4 receptors (Hagan et al., 2000), mechanisms other than the α-MSH circuitry must aid in the orexigenic action of the peptide.

13

Galanin In the ARC, NPY/AgRP and POMC/CART neurons (discussed in the following section) have received considerable attention and have constituted the focus of most research; however, galanin, another neuronal peptide that drives feeding, also is localized in this hypothalamic structure (Bartfai, Fisone & Langel, 1992; Merchenthaler, Lopez & Negro-Vilar, 1993; Crawley, 1995). Three galanin receptors have been identified and all three have been shown to densely populate the hypothalamus (Bartfai et al., 1992; Smith, Walker, Artymyshyn, Bard, Borowsky et al., 1998). Infusions of low amounts of the peptide into the lateral (Schick, Samsami, Zimmermann, Eberl, Endres et al., 1993) and third (Dube, Horvarth, Leranth, Karla, Karla, 1994) ventricles produce short-lived (~30 minutes) increases in eating. Although galanin also is produced in the periphery, in particular in the pancreas, urogenital tract, and in ganglia innervating the heart and the gut (Melander, Hokfelt, Rokaeus, Fahrenkrug, Tatemoto et al., 1985; McDonald, Brooks, Rokaeus, Tinner & Staines, 1992), this effect of galanin on eating appears solely to be mediated centrally because intraperitoneal injections of the agent have no effect on consumption (de Pedro et al., 1995). As such, galanin is discussed in this section as opposed to the section on peripheral signals that control eating. Sitespecific neural injections of galanin have shown that the peptide elicits its most potent effects in the PVN (Kyrkouli, Stanley & Leibowitz, 1986; Corwin, Robinson & Crawley, 1993) although the peptide also acts in the DMH (Kyrkouli et al., 14

1986), VMH (Schick, Yaksh & Go, 1993), Amg (Kyrkouli et al., 1986), and the AP/NST (Corwin et al., 1993). Although the peptide is not colocalized with other neuropeptides in the ARC, it colocalizes with corticotrophin-releasing (CRH) hormone and thyrotropin-releasing hormone (TRH) in the anterior PVN (reviewed in Leibowitz, 1998), both of which produce reductions in eating (Arase, York, Shimazu, Shargill & Bray, 1998 for CRH; Lin, Chu & Leu, 1983 for TRH). It may be that galanin inhibits CRH and TRH neurons to preclude their anorexigenic effects. Galanin interacts with other chemicals in the CNS to exert its hyperphagic effects. One of these agents is the neurotransmitter norepinephrine. Administration of adrenergic receptor (α2) antagonist blocks the hyperphagic effects of galanin (Kyrkouli, Stanley, Hutchinson, Seirafi & Leibowitz, 1990), while leaving NPY-elicited feeding unaltered. Importantly, the authors showed that infusions of the antagonists alone did not affect baseline measurements of eating. Together, these suggest that galanin may produce its effects through the release of norepinephrine. This finding is substantiated by an in vivo microdialysis study, which showed that intra-paraventricular injections of galanin increased hypothalamic norepinephrine release within the PVN (Kyrkouli, Stanley & Leibowitz, 1992).

15

Catabolic Peptides within the Arcuate Nucleus Proopiomelanocotin (POMC) and Cocaine-Amphetamine Related Transcript (CART) The second population of neurons in the ARC important in the regulation of energy homeostasis involves the coexpressed anorexigenics POMC and CART (Kristenson et al., 1998). It has been demonstrated that POMC also is produced in the PVN and the DMH (Koylu, Couceyro, Lambert, Ling, DeSouza et al., 1997). Proopiomelanocotin is the precursor for α-MSH and is classified as part of the melancortin family (Cochet, Chang & Cohen, 1982). The inhibitory effect that POMC exerts on eating appears to be mediated by α-MSH via MC4 receptors (Poggioli, Vergoni & Bertolini, 1986; Fan, Boston, Kesterson, Hruby & Cone, 1997). Deletions targeted against POMC (Yaswen, Diehl, Brennan & Hochgeschwender, 1999) and MC4 (Huszar, Lynch, Fairchild-Huntress, Dunmore Fang et al., 1997) receptors both lead to increases in eating and obesity in mice, suggesting the importance of this peptide in the inhibition of eating. Interestingly, in humans, mutations in either the POMC (Krude, Biebermann, Luck, Horn, Brabant et al., 1998) or the MC4 receptor gene (Vaisse, Clement, Durand, Hercberg, Guy-Grand et al., 2000) are associated with obesity. Most studies available on CART and eating suggest an anorexic role for the peptide. Infusions of CART into the lateral ventricles consistently have been shown to decrease food consumption in sated animals (Kristensen et al., 1998; Edwards, Abbot, Sunter, Kim, Dakin et al., 2000; Abbott et al., 2001). On the 16

contrary, infusions of the peptide antiserum significantly increase eating in satiated rats (Kristensen et al., 1998; Lambert, Couceyro, McGirr, Dall Vechia, Smith et al., 1998). A comprehensive study by Abbott et al. (2001) has shown the more dynamic effects of CART on eating behavior. The authors demonstrated that intracerebroventricular infusions of the peptide produce reductions in eating in both sated and fasted animals; however, the reductions were more dramatic in sated animals. Furthermore, infusions of the peptide produced significant increases in eating when injected into particular nuclei such as the LH, PVN, VMH, DMH, and ARC. This increase was followed by a significant decrease in eating 4-24 hours following the injection into the ARC and DMH. The significant decrease may be either a compensatory response to the increase in eating or due to a delayed anorectic effect of the peptide. This reduction of eating following an initial increase in eating by CART is observed in both sated and fasted animals, suggesting that the effect is not dependent on the energy state of the animal. Taken together, these findings insinuate an anorectic and possibly a site-specific orexigenic mechanism of CART. Interaction between NPY/AgRP and POMC/CART Neurons The two populations of neurons, NPY/AgRP and POMC/CART, form connections with other hypothalamic nuclei. Within the ARC itself, it appears that the two populations share reciprocal connections (Cowley, Smart, Rubinstein, Cerdan, Diano et al., 2001), suggesting an interaction between these peptides. Evidence for such an interaction has been demonstrated. For instance, it is reported 17

that one of the mechanisms by which NPY stimulates eating is by inhibiting anorexigenic POMC/CART neurons (Roseberry, Liu, Jackson, Cai & Friedman, 2004). Additionally, both populations of neurons in the ARC send projections to the PVN and to the LH (Bagnol, Lu, Kaelin, Day, Ollmann et al., 1999; Elias, Aschkenasi, Lee, Kelly, Ahima et al., 1999). Specifically, they form synapses with lateral hypothalamic neurons that contain the orexigenic agents melanin concentrating hormone (MCH) and orexin or “hypocretin” (Elias et al., 1998b; discussed in the section that follows). It could be purported then that NPY/AgRP work in concert with these compounds to increase eating, while POMC/CART inhibit the actions of these agents in an attempt to suppress eating. Indirect evidence for this possibility is provided by the finding that CART is coexpressed with MCH in the LH (Elmquist, Elias & Saper, 1999; Broberger, 1999; Yaswen et al., 1999), suggesting a potential interaction by which both CART and MCH influence energy intake. 1.3.1.2 Lateral Hypothalamus (LH) The LH is a large nucleus comprised of several subnuclei, is highly interconnected within the hypothalamus and with the rest of the brain, and is traversed by the medial forebrain bundle (MFB) and the fornix (fx). The LH is considered to play a major role in feeding behavior in part due to the orexigenic neuropeptides that are synthesized within this particular nucleus. Both lesion (Anand & Brobeck, 1951) and stimulation (Delgado & Anand, 1953) studies have corroborated this hypothesis although the effects of these studies may have been 18

due to damage and/or stimulation of fibers of passage such as the MFB. Many independently conducted studies have confirmed that the LH contains neurons that express the orexigenic compounds melanin concentrating hormone (MCH; Bittencourt & Elias, 1998) and orexin (Saper, 2000), otherwise known as hypocretin (de Lecea, Kilduff, Peyron, Gao, Foye et al., 1998), which may better define the role of the LH in eating behavior. Unlike NPY and AgRP in the ARC, MCH and orexin are not coexpressed within the same neurons (Broberger et al., 1998); however, the neurons synthesizing the two peptides are intermingled and have similar wide-ranging projection sites (Bittencourt, Presse, Arias, Peto, Vaughn et al., 1992; Peyron, Tighe, van de Poll, de Lecea, Hellar et al., 1998). Additionally, unlike the ARC, the LH appears to contain only orexigenic agents and no anorexic agents have been identified in the nucleus. Anabolic Peptides within the Lateral Hypothalamus Melanin Concentrating Hormone (MCH) Studies spanning from intracerebroventricular infusions to transgenic MCH null mice have repeatedly shown the stimulatory effect of MCH on eating. Infusions of MCH into the ventricles increase food intake in both rats (Rossi et al., 1997; Ludwig et al., 1998) and mice (Della-Zuana et al., 2002), which occur throughout the light and dark phases of the day (Rossi & Bloom, 1997). Deletions targeted against the MCH precursor protein prepro-MCH gene result in lean, hypophagic animals (Shimada et al., 1998), while transgenic mice that overexpress MCH are obese and hyperphagic (Ludwig et al., 2001). It must be taken into 19

account that because the prepro-MCH gene codes for other agents, this phenotype cannot be attributed solely to the absence of MCH. Another piece of evidence linking MCH with feeding behavior is the finding that both short-term (Fellmann et al., 1993) and long-term (Presse et al., 1996; Herve & Fellmann, 1997) food deprivation increase MCH immunoreactivity in the rat suggesting its role as an anabolic signal to restore enery homeostasis. Orexin A second set of neuropeptides that are expressed in the LH and in the adjacent PFA are orexins A and B (Sakurai et al., 1998), otherwise known as hypocretins 1 and 2 (de Lecea, Kilduff, Peyron, Gao, Foye, et al., 1998), respectively. Both orexin A and B appear to be involved in feeding behavior, although the former has a more potent effect (Tritos, Vicent, Gillette, Ludwig, Flier et al., 1998). It should be noted that genetically eliminated orexin leads to hypophagia in addition to narcolepsy (Chemelli, Willie, Sinton, Elmquist, Scammell, et al., 1999). This suggests a potential interaction between eating and wakefulness. As such, it may be that orexin affects eating partly by affecting the overall arousal state of the animals. Messenger RNA levels of the orexin precursor protein, prepro-orexin, are upregulated during fasting states (Sakurai, Amemiya, Ishii, Matsuzaki, Chemelli et al, 1998), a finding that is similar to the orexigenic peptides discussed thus far. Injections of both orexin A and B into the lateral ventricles produce a feeding response (Sakurai et al., 1998), although the hyperphagic effect of orexin A outlasts that of orexin B. On the contrary, blocking 20

orexin A receptors decreases eating (Haynes, Jackson, Chapman, Tadayyon, Johns et al, 2000). Evidence shows that although intracerebroventricular infusions of orexin A produce increases in eating, they do not affect body weight (Yamanaka, Sakurai, Katsumoto, Yanagisawa & Goto, 1999). This finding is not too surprising given that orexin A has been shown also to increase energy expenditure (Wang, Osaka & Inoue, 2001). In situ hybridization studies aiming to identify location of the receptors for orexin report that orexin A receptor is most abundantly expressed in the VMH, whereas orexin B receptor is predominantly found in the PVN (Trivedi, Yu, MacNeil, van der Ploeg, Guan et al., 1998; Marcus, Aschkenasi, Lee, Chemelli & Saper, 2001) although both types of receptors are localized outside of the hypothalamus as well. Although most of the aforementioned studies with orexin have been performed in murine animals, the orexin circuitry within the hypothalamus is involved in homeostatic mechanisms in non-human primates as well. Orexinsynthesizing neurons are found exclusively in the LH-PFA area and in the DMH. In fasted animals, there is increased c-fos activation in these hypothalamic orexincontaining neurons (Diano, Horvarth, Urbanski Sotonyi & Horvarth, 1993). This suggests that the orexin circuit in the hypothalamus of non-human primates also is involved in signal transduction associated with metabolic alterations. Orexin immunoreactive fibers from the LH and DMH form synapses on ARC NPY/AgRP neurons (Horvath, Diano, Anthoney & van der Pol, 1999) and on POMC cell bodies (Guan, Saotome, Wnag, Funahashi, Hori, et al., 2001), 21

suggesting a potential circuitry for orexin-induced feeding. Further, triple immunofluorescence has revealed that NPY and POMC neurons contain orexin receptors implying that these neurons are influenced by the peptide (Funahashi, Yamada, Kageyama, Takenoya, Guan, et al., 2003). Lending support for an orexinNPY interaction is the finding that intracerebroventricular infusions of orexin produce c-fos protein activation in ARC NPYergic neurons (Yamanaka, Kunii, Nambu, Tsujino, Sakai, et al., 2000). In a review article, Funahashi, Takenoya, Guan, Kageyama, Yada et al. (2003) report findings of unpublished physiological studies that provide further evidence for the interaction of NPY and POMC with orexin. They report that orexin increases [Ca]2+ in isolated NPY neurons and decreases [Ca]2+ in POMC neurons in the ARC suggesting an excitatory effect of orexin on NPY and an inhibitory effect on POMC. The LH also has reciprocal connections with the NST where it receives viscero-sensory information pertaining to food (Simpson & Raubenheimer, 2000). In fact, the NST has been shown to receive dense orexin innervation from the LH (Date, Ueta, Yamashita, Yamaguchi, Matsukura, et al., 1999; Peyron et al., 1998) and orexin A depolarizes 90% of NST neurons tested (Yang & Ferguson, 2003). Since the NST innervates the dorsal motor nucleus of the vagus, which in turn, sends fibers that innervate the entire GI system (Grabauskas & Moises, 2003; Hayakawa, Takanaga, Tanaka, Maeda & Seki, 2003; Wu, Gao, Yan, Owyang & Li, 2004), it is possible that the NST serves as the mediator between hypothalamic orexin information and the periphery. Finally, in the rat LH, there are reciprocal 22

connections between the orexinergic and MCHergic neurons (Guan, Uehara, Lu, Wang, Funahashi, et al., 2002) indicating that these two peptides interact with one another in controlling food intake similar to the NPY/AgRP and POMC/CART neuronal populations in the ARC. 1.3.1.3 Paraventricular Nucleus (PVN) The role of the PVN in eating was discovered following the finding that parasagittal knife cuts of what was thought to be at the level of the VMH produced increases in eating and weight gain. It was discovered that the optimum site for these knife cuts was at the PVN and not the VMH (Gold, Jones, Sawchenko & Kapatos, 1977). Studies followed which showed that lesions of the PVN in the absence of any damage to the VMH produced obese rats (Aravich & Scalfani, 1983; Leibowitz et al., 1981), a finding that was initially demonstrated in dogs (Heinbecker, White & Rolf, 1944). This was the introduction of the PVN as a key player in the eating system. Supplementing the lesion studies are immunohistological staining experiments that further implicate the PVN as an important structure in regulating eating. Similar to the ARC and the LH, the role of the PVN in eating also is characterized by the peptides that are synthesized within the hypothalamic nucleus. Cocaine and amphetamine-regulated transcript (Koylu et al., 1997), POMC (Koylu et al., 1997), corticotropin releasing hormone (CRH; Sawchenko, Swanson & Vale, 1984), thyrotropin-releasing hormone (TRH; Segersen, Kauer, Wolfe, Mobtaker, Jackson & Lechan, 1987; Kow & Pffaf, 1991), and cholecystokinin (CCK; Mezey, 23

Reisine, Skirboll, Beinfeld & Kiss, 1986) are all synthesized in neurons residing in the PVN and all produce reductions in eating (Arase, York, Shimazu, Shargill & Bray, 1998 for CRH; Lin, Chu & Leu, 1983 for TRH). Taken together, these two types of studies confer an inhibitory role of PVN in eating. On the other hand, the ARC-PVN connection implicates the latter structure in the initiation of eating as well. Arcuate orexigenic NPYergic neurons densely project to the PVN (Bagnol, Lu, Kaelin, Day, Ollmann, Gantz, et al., 1999; Elias et al., 1998b), a connection that has been implicated in the NPY correctional mechanism during states of energy depletion (Sahu et al., 1988; Li et al., 1998; Smith, 1993; Lewis, Shellard, Koeslag, Boer & McCarthy, et al., 1993). The stimulatory effect of noradrenaline on eating (Leibowitz, 1978) and the inhibitory effect of serotonin (Smith, York & Bray, 1999) when infused into the PVN further evinces the dual role this nucleus plays in the eating system. The role of the PVN in serotonin hypophagia (Leibowitz, 1990) is not surprising given that the structure is innervated by the raphe nucleus (Larsen, Hay-Schmidt, Vrang & Mikkelsen, 1996), which is a rich source of serotonin. 1.3.1.4 Dorsomedial Hypothalamus (DMH) The DMH is another hypothalamic nucleus that is an important player in food intake. Like most other neural structures discussed thus far, the dual role of the DMH in eating has been confirmed through various methods. Although most orexin/hypocretin cells are localized within the LH and in the adjacent PFA, some also are localized in the DMH (de Lecea et al., 1998), suggesting the role of the 24

nucleus in feeding responses. The appetite suppressing neuropeptides POMC/CART and the hypophagia-inducing neurotransmitter serotonin also is produced in the DMH (Koylu et al., 1997 for POMC/CART; Frankfurt & Azmitia, 1983 for serotonin) demonstrating its role in satiation. Ablation studies also implicate the DMH in energy regulation. Electrolytic (Bellinger, Bernardis & Brooks, 1979; Bernardis, 1975), kainic acid (Bellinger & Williams, 1983) or ibotenic acid (Belinger, 1987; Chou, Scammell, Gooley, Gaus, Saper, et al., 2003) lesions of the DMH produce hypophagic animals that maintain lower body weights. Dorsomedial hypothalamic lesioned (via ibotenic acid) animals are able to respond to a 24-hour fasting period, which results in a further decrease in body weight. They do so by increasing food intake until their postoperative body weight is restored (Chou et al., 2003). This suggests that DMH lesioned animals are still able to regulate intake and body weight, but just at a reduced level. Another interesting finding regarding the DMH and eating involves the role of the nucleus in circadian rhythms. Chou et al. (2003) showed that ibotenic acid lesions of the DMH disrupt the circadian rhythmicity of many behaviors including eating. Animals in the experimental lesion group showed an overall decrease in eating compared to controls; however, the pattern of eating throughout the light/dark cycles also was disrupted. For instance, the animals exhibited increases in eating during the light cycle and decreases during the dark phase compared to controls. The same experiment showed that while lesions of the LH resulted in 25

overall reductions in eating, the ablations did not cause any circadian alterations. That is, the animals continued to show the greatest eating during the dark phase. The involvement of the DMH in circadian rhythms is significant considering that the suprachiasmatic nucleus (SCN) of the hypothalamus is often thought to be the sole regulator of circadian rhythmicity (Schwartz & Gainer, 1977; Silver, LeSauter, Tresco & Lehman, 1996). 1.3.1.5 Ventromedial Hypothalamus (VMH) Following the observations that lesions of the VMH lead to immediate, voracious feeding, the VMH, at the time, was justifiably classified as the neural satiety center. Since this area, once thought to be crucial for the induction of satiety has been the focus of many debates, its history warrants some attention. A group of researchers examining the effects of VMH lesions on eating reported that the animals would initiate eating for several hours immediately following surgery (Balagura & Devenport, 1970; Harrell & Remley, 1973; Becker & Kissileff, 1974). One group reported that some of the lesioned rats started eating prior to completely recovering from anesthesia and died as a result of choking (Brooks, Lockwood & Wiggins, 1946). Soon after, evidence against this hypothesis rapidly piled. Researchers claimed that VMH lesions lead to obesity, not due to hyperphagia, but due to vagally mediated metabolic abnormalities such as fat metabolism (Han, 1968; Cox & Powley, 1981), hyperinsulinemia (Hales & Kennedy, 1964), and decreased gastric secretions (Ridley & Brooks, 1965; Weingarten & Powley, 1980) that accompanied the lesions. This hypothesis gained 26

support from studies suggesting that the weight gain was not entirely due to elevated levels of eating. In these studies, VMH-lesioned animals accumulated more fat compared to pair-fed control animals (Han, 168; Cox & Powley, 1981). The finding that restricted lesions of the PVN, which spare the entire VMH, result in hyperphagia and obesity (Aravich & Scalfani, 1983; Leibowitz et al., 1981) put an end to the ‘VMH as the satiety center’ claim. Additionally, Gold (1973) illustrated that extremely circumscribed electrolytic lesions of the VMH did not produce alterations in eating in male rats; however, when the lesions overflowed to surrounding areas, the magnitude of the elevated eating and obesity was proportional to the extent of overflow. Despite the evidence against the VMH as a structure important in eating behavior, the role of the nucleus in energy homeostasis has been resurrected. The discovery of sexual dimorphism in VMH lesion weight gain provided explanation for some of the disparate findings. It was shown that female rats and mice gained substantially more weight following lesions compared to males (Cox, Kakolewski & Valenstein, 1969; Kakolewski, Cox & Valenstein, 1968; King & Frohman, 1986; Sanders et al., 1973), which explained why some research groups did not find changes in eating following lesions since most animals used in lesion studies were male. Although the role of the VMH as the satiety center of the brain is entirely outdated, currently, the role of the VMH in eating should not be discarded.

27

1.3.1.6 Tuberomammillary Nucleus (TM) This hypothalamic nucleus is seldom discussed as an important nucleus involved in eating behavior; however, the TM is the major production site of the neurotransmitter histamine (Garbarg, Barbin, Geger & Schwartz, 1974; Panula, Yang & Costa, 1984; Steinbusch & Mulder, 1985; Wouterlood & Steinbusch, 1991), which has been shown to be a potent inhibitor of eating. Findings of research manipulating both central and peripheral levels of histamine implicate this neurotransmitter as a powerful appetite suppressor in cats, rats, goats, and chicks (Machidori, Sakata, Yoshimatsu, Ookuma, Fujimoto, et al., 1992; Clineschmidt & Lotti, 1973; Kawakami, Bungo, Ohgushi, Ando, Shimojo, et al, 2000). Ookuma and colleagues (1993) have shown that infusions of -fluromethylhistamine (FMH), a “suicide” inhibitor of histidine decarboxylase (HDC), into the third cerebroventricle induces feeding behavior. Studies investigating the significance of histamine in ingestive behaviors were initiated following the observation that some antidepressant and antipsychotic drugs stimulated appetite and led to weight gain in humans (Kalucy, 1980). Examinations attempting to link these drugs inducing weight gain revealed that they possess anti-histaminergic properties. In particular, binding assays have shown them specifically to be potent histamine 1-receptor (H1R) blockers (Hill & Young, 1978; Taylor & Richelson, 1980). The involvement of H1R in histamine’s hypophagic abilities is substantiated by several studies. Intraperitoneal injections of metorpine, which elevates brain histamine levels by inhibiting the conversion of 28

histamine to the inactive metabolite methylhistamine, suppresses food intake (Leklin et al., 1994, Lelklin et al., 1995). Leklin and Tuomisto (1997) demonstrated that this hypophagia is abolished with pretreatment of the H1R antagonist mepyramine, suggesting that histamine exerts its depressive effects through H1R. It is reported that H1R antagonists elicit feeding when they are infused into the third cerebroventricle (Fukagawa et al., 1989), into the PVN (Ookuma et al., 1989) or into the VMH (Sakata et al., 1988). Interestingly, reduction of neuronal histamine appears to have a hyperphagic effect on eating. 1.3.2 Hindbrain Structures 1.3.2.1 Area Postrema (AP) Neuroanatomical studies have established that the AP receives information from the gut via the afferent vagus nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985). This cranial nerve serves as a major afferent input to the brainstem from the gastrointestinal system (Martin, Rogers, Novin & VanderWeele, 1977). As such, it is an important structure in detecting gastric distention, malaise, gastric emptying, among other gastrointestinal mediated responses that are pertinent for eating behavior. The AP also receives input from hypothalamic structures such as the PVN and the DMH (Shapiro & Miselis, 1985), which implies that this circumventricular structure also receives information regarding eating from other brain regions. In addition, it sends and receives information from the NST and the PBN, predominantly the lateral subdivision, (van der Kooy & Koda, 1983; Shapiro

29

& Miselis, 1985), both structures that are integrally important in the energy regulation (discussed below). Direct evidence for the role of the AP in eating from lesion studies is difficult to gather since ablations of the structure produce illness in animals. Therefore, findings of such studies must be interpreted with caution. One study showed that animals with lesions of the AP, which are allowed 6 weeks to recover, displayed hypophagia and decreased weight (Lutz, Mollet, Rushing, Riediger & Scharrer, 2001), which was interepreted as a role of the nucleus in feeding. Unfortunately, since inactivation of this area potentially produced chronically sick animals, this conclusion should be revisited. It may have been that the lesioned animals were expressing illness-inudced food decrements and weight loss. A second approach to determining the role of the AP in eating has been to inspect the effects of anorexigenics in AP-lesioned animals. Studies have shown that the appetite suppressors amylin (Lutz et al., 2001), calcitonin gene-related peptide (Lutz, Senn, Althaus, del Prete, Ehrensperger, et al., 1998a), CCK (Edwards, Ladenheim & Ritter, 1986), and estradiol (Bernstein, Courtney & Braget, 1986) are rendered ineffective in animals with lesions of the AP and in some of the cases the surrounding NST, implying that neurons in this area are necessary for the expression of hypophagia produced by these substances. Once again, although animals in the studies mentioned above were allowed a substantial amount of time to recover following lesion surgeries, they all continued to express hypophagia and lowered body weight, which taints the results of these studies. For 30

instance, since the animals were already at the lower end of their weight range, the effects of the anorexigenics may have been masked by the decrease in eating and body weight already produced by the lesions themselves (Roy & Wade, 1977). One way to potentially circumvent this problem is through the use of temporary AP lesions (approximately 1 hour inactivation), which may preclude the illness of animals. One disadvantage to this approach is that the role of the AP in short-term satiety agents can be best studied since the brief length of the inactivation should overlap with the time the satiogenics produce their effects. A possible approach for examining the role of the AP in long-term satiety agents using temporary AP lesions exists. First, the length of time the agent produces its reductions in eating needs to be determined. If the AP is going to be inactivated for an hour and the agent produces reductions in eating up to 5 hours following its administration, then the agent can be administered 4 hours before the inactivation begins. This way, the final hour that the agent is active will overlap with the 1 hour inactivation of the AP. 1.3.2.2 Nucleus of the Solitary Tract (NST) The NST and the PBN (discussed in the section that follows) are major relays for taste and malaise information, both of which play a critical role in eating behavior. The taste and malaise pathways will be discussed in further detail in the following chapter and therefore only a summary will be provided here. From the cranial nerves, gustatory information is transmitted to the rostral portion of the NST (Torvik, 1956). The information is then projected to the ipsilateral medial or 31

“gustatory” PBN (Norgren, 1978, 1984), which in turn sends this information to forebrain structures through the thalamus. A second, less defined route by which taste information reaches the forebrain involves mono-synaptic projections from the medial PBN to the central nucleus of the Amg, the substantia innominata, the LH, the BNST, and the insular cortex (Norgren, 1974, 1976; Saper & Loewy, 1980; Shipley & Sanders, 1982; Fulwiler & Saper, 1984; Moga, Herbert, Hurley, Yasui, Gray, et al., 1990; Bernard, 1993). The pathway for malaise information follows very closely to the taste pathway. This information is carried from the viscera by the vagus nerve to the AP (Baldino & Wolfson, 1985), which transmits the information to the caudal NST (Cechetto, 1987). The NST also receives direct connections from the vagus nerve that carries gustatory (Hamilton & Norgren, 1984) and visceral information (Kalia & Sullivan, 1982). Next, the caudal NST sends extensive projections to the ipsilateral lateral PBN (Lowey & Burton, 1979; Saper & Lowey, 1980), which is the relay station for ascending visceral information from the NST to the thalamus, hypothalamus, and the Amg (Cechetto, 1987). The caudal NST is thought to partially be involved in tumor anorexia. As the name suggests, tumor anorexia is the subsequent decreases in eating and body weight that are observed following the growth of tumors (Mordes & Rossini, 1981). A role for the peptide tumor necrosis factor (TNF) in tumor anorexia has been proposed given that the agent causes reductions in eating and body weight (Beutler & Cerami, 1987; Socher, Friedman & Martinez, 1988). Combined lesions 32

of the AP and the caudal NST are shown to attenuate anorexia produced both by Leydig tumors, which cause elevations in estradiol, and by TNF (Bernstein, 1996); however, since these two regions are sites of vagal afferent projections, any attenuation could have been due to an interference with vagal function (Bernstein, 1996). The illness caused by lesions of the AP further complicates the interpretation of these findings. More compelling evidence for the role of the NST in eating is provided by studies showing that the nucleus is a site of production for eating-related agents. For instance, although the ARC is the main site for POMC synthesis, a small population of neurons in the NST also manufactures the catabolic peptide (Sawchenko, Swanson & Joseph, 1982). Similarly, the satiety-eliciting agents glucagon-like peptides 1 and 2 (GLP-1/GLP-2) are produced in the NST (Jin, Han, Simmons, Towle, Lauder, et al., 1988; Larsen, Tang-Christensen, Holst & Ørsky, 1997). As will be discussed shortly, the NST also is a key target for peripheral signals that regulate meal initiation and termination and body weight, further insinuating involvement of the nucleus in eating behavior. 1.3.2.3 Parabrachial Nucleus (PBN) The Lateral Parabrachial Nucleus The parabrachial nucleus, particularly the lateral subdivision, is implicated in eating behaviors. However, the results of studies examining the effects of lateral PBN lesions on eating and body weight have been contradictory. One of the earliest studies on the lateral PBN and eating showed that electrolytic lesions of the 33

structure produced increases in eating and body weight in female Sprague-Dawley rats 3 weeks following the surgery and obesity 12 weeks following surgery (Nagai, Ino, Yamamoto, Nakagawa, Yamano, et al., 1987). This would suggest that the lateral PBN plays a role in satiety; however, since the researchers used electrolytic lesions, the possibility that the destruction of fibers of passage produced the hyperphagia could not be discarded. More importantly however, histological examination of the lesions revealed extensive damage which included the lateral PBN, the superior cerebellar peduncle, the external and central nucleus of the inferior colliculus, the sagulum nucleus, the dorsal nucleus of the lateral lemniscus, and the ventral spino-cerebellar tract. In another study, neither lesions of the lateral nor medial PBN affected eating during a 60-minute test following 24-hour food deprivation. Surprisingly, in a companion paper, non-deprived medial PBN and lateral PBN-lesioned rats showed reductions in baseline eating (Trifunovic & Reilly, 2002), a finding that was inexplicable to the authors since in prior studies, lesions had no effect on baseline levels of consumption. Finally, a study in which lesions were restricted to the external portion of the lateral PBN found that approximately a week following ibotenic acid lesions, animals did not exhibit an increase in weight (Li, Spector & Rowland, 1994), but rather their intake and weights were similar to those of the sham operated controls. The role of the lateral PBN in eating behavior has been revealed by those studies that have investigated the involvement of this structure in the effects of the satiety agents CCK and serotonin. Excitotoxic lesions of the entire lateral PBN, 34

which do not affect baseline levels of consumption, abolish CCK-induced anorexia, while destruction of the medial PBN leaves CCK hypophagia intact (Trifunovic & Reilly, 2001). In addition, injections of CCK produce c-Fos-like immunoreactivity (c-FLI) activation in the lateral PBN (Inagaki, Shiotani, Yamano, Shiosaka, Takagi, et al., 1984) and lesions that include the lateral PBN abolish c-FLI normally expressed in the VMH by an injection of the anorexigenic CCK (Nagai et al., 1987). Taken together, these findings suggest that the decrease in consumption produced by CCK involve the lateral PBN and possibly its projections to the VMH. Studies implicate the lateral PBN in serotonin-induced hypophagia as well. Unilateral infusions of the serotonergic agonist D-fenfluramine (serotonin releaser and reuptake inhibitor) into the lateral PBN of male rats reduce food intake, while infusions of the agent in surrounding brain regions have no effect (Simansky & Nicklous, 2002). Administering the selective serotonin1B receptor agonist into the lateral PBN also produces decrements in eating (Lee et al., 1998; Simansky & Nicklous, 2002), which is blocked by a selective serotonin1B receptor antagonist (Simansky & Nicklous, 2002), but not by serotonin2C antagonists (Kennett & Curzon, 1988). These finding implicate the lateral PBN in serotonin-induced anorexia. Unfortunately, the results of lesion studies have not been as unequivocal. A study examining the effects of ibotenic acid lesions of the lateral PBN on serotonin-induced anorexia found that the lesions blocked the hypophagia normally observed following injection of a serotonin agonist dexfenfluramine (DFEN; Li et al, 1994). This finding was contradicted in a study by Trifunovic and Reilly 35

(2001), which showed that excitotoxic lesions of the entire lateral PBN did not affect DFEN hypophagia, while destruction of the medial PBN abolished the serotonin agonist induced anorexia. On the other hand, immunohistochemical studies specifically implicate the external lateral PBN in DFEN-induced anorexia. The serotonin agonist normally produces c-fos activation in the lateral PBN, central Amg, and the laterodorsal BNST in intact animals, which is eliminated in lateral PBN-lesioned animals (Li et al., 1994). This suggests that DFEN does not directly act on serotonin receptors in the central Amg and the BNST and that its activation of these forebrain structures is a secondary response to activation of neurons in the external lateral PBN. Hence, the projections from the lateral PBN to the central Amg and the BNST may be critical in serotonin-induced anorexia. The lateral PBN also contains receptors shown to modulate feeding responses. Stimulation of any of the opioid receptors (μ, κ, δ subtypes) produces increases in eating in mammals, including rats (Polidori, de Caro & Massi, 2000; Silva, Hadjimarkou, Rossi, Pasternak & Bodnar, 2001), mice (Asakawa Inui, Momose, Ueno & Fujino, 1998), and rabbits (Gosnell & Lipton, 1986) and the lateral PBN contains all three receptor subtypes (Mansour, 1995). A study by Wilson, Nicklous, Aloyo & Simansky (2003) showed that infusions of a μ opioid receptor agonist DAMGO into the lateral PBN increased food intake in male rats, while infusions into the surrounding areas were ineffective. Additionally, the authors showed that infusions of a non-selective opioid antagonist completely 36

blocked the hyperphagic effect of DAMGO without affecting baseline levels of food consumption. The Medial Parabrachial Nucleus The medial PBN has been shown to be involved in the negative contrast effect observed in consummatory behaviors. When animals are shifted from a low to a high reward situation, they exhibit a suppressed level of the low reward compared to animals that receive only the low reward (Flaherty & Checke, 1982; Flaherty & Rowan, 1985; Capaldi & Sheffer, 1992). This negative contrast effect is often discussed as an emotional response to shifted rewards. Similar results are obtained when shifts are made from high concentration to low concentration of sucrose (Becker & Flaherty, 1982; Becker et al., 1984). For instance, animals given a 4% sucrose solution after being exposed to a 34% sucrose solution, consume less of the 4% solution than control animals given 4% sucrose solution alone. This negative contrast effect is eliminated in animals with ibotenic acid lesions of the medial PBN (Grigson, Spector & Norgren, 1994) suggesting that an intact medial PBN is essential for this consummatory effect and is involved in the emotional responses to shifted rewards pertaining to food. 1.3.3 Forebrain Structures 1.3.3.1 Amygdala (Amg) The role of the Amg in eating behavior is varied and complex. Electrolytic lesions of various nuclei within the limbic structure have revealed disparate results. Studies have shown that lesions of the basolateral Amg produce aphagia (Fonberg, 37

1971), while lesions of the basomedial nucleus result in hyperphagia (Fonberg, 1966). Predicated on these findings, Fonberg purported a dual control of eating by the limbic structure (Fonberg, 1974). Since these studies used electrolytic lesioning techniques, the possibility that the results are due to damage to fibers of passage should be considered. Research using excitotoxic kainate acid lesions of the central nucleus of the Amg have shown that destruction of this area results in decreases in eating (Hajnal, Sandor, Jando, Vida, Czurko, et al., 1992). Some researchers ascribe the effects on eating imposed by amygdaloid lesions to encroaching damage to the striatum. To test this possibility, various groups of researchers have made either discrete lesions of the central, lateral, and medial amygdala or combined lesions of these nuclei with lesions of the striatum (Schoenfeld & Hamilton, 1981). The results indicated that lesions restricted to the amygdaloid nuclei had no effect on food intake or body weight, while lesions that encroached into the striatum did. This finding is supported by a second study which illustrated that discrete damage to the central nucleus did not affect eating or body weight, while damage to the striatum decreased both (Dacey & Grossman, 1977). As such, these lesion studies alone do not implicate the amygdala in eating behavior. Instead, other methods of determining its role in eating are necessary. Similar to the effects produced by medial PBN ablations, electrolytic lesions of the medial, but not the lateral Amg, have been shown to disrupt the negative contrast effect of consummatory behavior. Neurally intact animals initially exposed to a 34% sucrose solution drink less of a 4% sucrose solution 38

compared to animals given only the 4% solution. This effect is abolished in animals with medial amygdaloid lesions (Becker et al., 1984) suggesting a role for the nucleus in food-related rewards. Although the complications due to lesion studies cast some doubt on the role of the Amg on eating, studies utilizing different techniques solidify the structure’s role in eating. For instance, the central Amg has been shown to be involved in the circuitry for serotonin-induced anorexia. Administration of the serotonin agonist DFEN produces the induction of c-fos in the central Amg and the laterodorsal BNST (Li et al., 1994). Further, activation in these areas is eliminated following lesions of the external lateral PBN, which also eliminate the serotoninproduced hypophagia. This finding shows that projections from the lateral PBN to the central Amg and BNST are involved in the serotonin anorexia. Since lesion studies have not confirmed the exact role of the Amg in eating, it is difficult to decipher whether the activation of the central Amg following DFEN infusion in the lateral PBN are indicative of activation or inhibition. If the central Amg is involved in feeding behavior, it could be that the central Amg is inhibited following lateral PBN administration of DFEN. If the central Amg is involved in satiation, then the central Amg is probably being activated by the lateral PBN-infused DFEN. Nevertheless, the loss of activation in the central Amg following lesions of the lateral PBN and DFEN application implies that the nucleus is associated with the decreases in eating produced by the serotonergic agonist.

39

There is evidence of an opioid signaling pathway between the NST and the central Amg that solidifies the role of the central Amg in eating behavior. First, bilateral infusions of an μ receptor agonist, DAMGO, in both the NST (Kotz, Billington & Levine, 1997) and the central Amg (Gosnell, 1988) result in increases in feeding behavior. Second, infusions of an opioid antagonist naloxone into the central Amg blocks the feeding induced by DAMGO (Gosnell, 1988). Although, this effect has not been demonstrated in the NST, it has been shown that naloxone in the central Amg blocks NPY-induced feeding (Kotz, Billington & Levine, 1995). Predicated on these findings, a study was conducted which showed that hyperphagia induced by infusing DAMGO into the NST was inhibited by infusions of an opioid antagonist injected into the central Amg (Giraudo, Kotz, Billington & Levine, 1998). The reverse was true as well. These results suggest a potential opioid-opioid signaling pathway between these two structures. 1.3.3.2 Bed Nucleus of the Stria Terminalis (BNST) The involvement of the BNST in serotonin-induced anorexia has been discussed in the previous section (Li, Spector & Rowland, 1994); however, this structure has been shown to play an important role in the suppression of eating produced by CRF as well (Ciccocioppo, Fedeli, Economidou, Policani, Weiss, et al., 2003). Since the BNST contains receptors for both CRF, particularly the CRF2 subtype (Chalmers, Lovenberg & de Souza, 1995) and receptors for the CRF antagonist, N/OFQ, researchers purported the structure to be important in CRF induced anorexia and for its reversal by N/OFQ (Ciccocioppo, et al., 2003). A 40

series of studies showed that microinjections of CRF into the BNST produced marked hypophagia in food-deprived rats, while injections into the central Amg had no effect. Further, injections of N/OFQ into the BNST blocked the anorexia produced by infusions of CRF made into both the BNST and into the ventricles. Injections of the antagonist into the central Amg, VMH or PVN had no effect on CRF produced decrements in eating. Equally important is the finding that injections of N/OFQ into the BNST did not affect basal levels of eating (Ciccocioppo, et al., 2003; Ciccocioppo, Cippeitelli, Economidou, Fedeli & Massi, 2004). The BNST also may be involved in reductions in eating produced by estradiol. The hormone produced c-fos activation in the laterodorsal region of the BNST 24 hours following its injection, which is precisely when reductions in eating produced by estradiol are observed (Chambers, Hintiryan & So, unpublished manuscript). The serotonin agonist DFEN also produces activation in this same region, which is eliminated with lesions of the lateral PBN along with serotonininduced anorexia. This suggests that the conncections between the lateral PBN and the BNST in serotonin anorexia. Whether lateral PBN lesions have the same effect on estradiol-induced BNST activation and estradiol anorexia remains to be determined. Although not directly tested, evidence suggests a role for the BNST in the orexigenic effects of both orexin and MCH. Immunohistochemical studies have verified that orexin and MCH fibers originating from the PFA and LH innervate the 41

BNST (Peyron et al., 1998; Bittencourt et al., 1992). Given that this forebrain structure receives projections from hypothalamic structures and neuropeptides that are heavily involved in eating, a role for the BNST in eating behavior could be a possibility. 1.4 Short-term Regulation of Eating There are separate mechanisms that work in conjunction to control the size of individual meals and to regulate body weight. Although the neural machinery is highly conserved for these separate processes (control of meal size/frequency and body weight), the molecular signals that initiate each process are disparate. The phrase short-term control of eating refers to the initiation or termination of an individual meal as opposed to long-term control, which refers to body weight regulation over an extended period of time. There is a plethora of identified peripheral satiety signals that are implicated in the cessation of a meal, while those involved in the feeding response, termed here as peripheral feeding signals, are more limited. Prior to discussing these signals individually, it should be clarified that the term satiation refers to the physiological process that begins during a meal and causes the termination of eating (Blundell, 1979; Smith, 1998). In the literature, this is differentiated from postprandial satiety, which is the interval between meals during which an animal does not engage in eating. The central nervous system can gain access to information regarding the initiation or termination of a meal carried by peripheral signals by at least two mechanisms. The first involves the vagus nerve that terminates on the AP (Shapiro 42

& Miselis, 1985) and the NST (Cechetto, 1987) and the second involves the blood, by diffusion through the blood-brain barrier or through areas where the blood-brain barrier is poorly developed (i.e. the AP or ARC). From the NST, direct and indirect projections through the PBN reach the PVN, DMH, ARC, LH, central Amg and the BNST (Saper, 2000). 1.4.1 Glucose and the “Glucostat” Hypothesis One of the first peripheral compounds speculated to be involved in the regulation of eating was glucose. The popular “glucostat” hypothesis purported that circulating levels of glucose serve as the negative feedback signal to the central nervous system, such that decreases in the fuel initiate a meal, while the elevated glucose levels following ingestion subsequently cause the termination of a meal (Mayer, 1955; Anand, Chhina, Sharma, Dua & Singh, 1964). There is evidence to support the involvement of glucose levels in the initiation and termination of a meal. Infusions of 2-deoxyglucose (2DG), a competitor of glucose that causes hypoglycemia, into the hepatic portal vein stimulate immediate eating (Novin, VanderWeele & Zidek, 1973). Since the hepatic portal vein transports blood from the small intestines into the liver, this finding suggests that the liver contains receptors that are glucose sensitive, meaning they respond to decreases in the level of glucose. Glucose responsive cells on the other hand are glucose receptors that respond to increases in the level of glucose. The liver has been shown to contain glucose responsive receptors as well since infusions of glucose into the hepatic

43

portal vein during a meal reduce the size and duration of the first meal of the dark cycle (Langhans, Grossman & Geary, 2001). Vagotomies have been shown to eliminate the feeding response elicited by the infusion of 2DG into the hepatic portal vein (Novin et al., 1973), suggesting that the information regarding initiation of a meal signaled by decreased glucose levels is carried from the liver to the central nervous system via the Xth cranial nerve, perhaps by the hepatic branch of nerve. Since glucose is the sole source of fuel for the brain, it is intuitive then that it too contains receptors that are directly sensitive to the availability of the metabolic fuel. Glucose can pass through the blood-brain barrier (Nicholls & Kuffler, 1964) thereby accessing the brain through the blood. Hindbrain areas such as the AP (Bernstein et al., 1986) and the NST (Edwards et al., 1986) contain glucose receptors (Bird, Cardone & Contreras, 1983 for the AP; Yettefti, Orsini & Perrin, 1997 for the NST). As such, these areas can gain access to circulating levels of glucose through their connection with the vagus nerve or directly via the blood. Originally, the VMH was hypothesized to be the locus of glucoreceptors or the “glucostat” that initiated satiety. Immunohistochemical studies have revealed that several other hypothalamic nuclei that are involved in energy homeostasis like the LH, the ARC, the PVN, and the DMH all stain for glucose receptors (DunnMeynell, Govek & Levine, 1997; Fukuda, Ono, Nishino & Sasaki, 1984). In vitro electrophysiological studies have shown that about 40% of the neurons in the LH 44

are glucose sensitive, while glucose responsive neurons are sparse (Ashford, Boden & Treherne, 1990). The situation is the opposite in the VMH, where most of the neurons are glucose responsive, while glucose sensitive neurons are rare. These findings would suggest that the glucose receptors in the LH are involved in producing the feeding response, while those in the VMH are involved in the inhibition process. In the section devoted exclusively to the role of the VMH in eating behavior, it was mentioned that contradictory findings regarding the effects of VMH lesions on eating had left researchers wondering if the structure even played a role in the behavior. However, this study establishes the hypothalamic structure as a glucose responsive site and provides evidence for its significance in eating behavior. Following the hypothesis of glucose regulation of eating, several other peripheral compounds involved in eating were discovered. Among these, ghrelin is involved in the initiating of eating, while cholecystokinin (CCK), peptide YY (PPY), glucagon-like peptide-1 (GLP-1), and gastrin-releasing peptide (GRP) are involved with the termination of a meal. Each of these agents, as will be discussed in more detail shortly, is released from the gastrointestinal tract, but also is produced in the brain. Thus, it is difficult to decipher whether the effects these peptides exert on eating are primarily of a peripheral or central source.

45

1.4.2 Peripheral Feeding Signal Ghrelin Ghrelin, like the peptides discussed below, is a peripheral signal that modulates food intake and recently has received considerable attention. Unlike most of the gastrointestinal peptides that will be discussed in this section, ghrelin drives the feeding response rather than suppressing it. Ghrelin is a growth hormone releasing secretagogue (GHS) that is predominantly manufactured and released from the stomach (Kojima, Hosoda, Date, Nakazato, Matsuo, et al., 1999; Date, Kojima, Hosoda, Sawaguchi, Mondal, et al., 2000) and was initially discovered as a ligand for the GHS receptor (Kojima et al., 1999). Lower amounts of the peptide are produced by the kidneys (Mori et al., 2000), bowel (Date et al., 2000), and the hypothalamus (Kagotani, Sakata, Yamazaki, Nakamura, Hayashi, et al., 2001), specifically in the ARC (Cowley, Smith, Diano, Tschöpp, Pronchuck, et al., 2003). The signals that stimulate ghrelin release are not known; however, since ghrelin levels increase during fasting in rats (Kojima et al., 1999), peak preprandially in humans (Cummings, Purnell, Frayo, Schmidova, Wisse, et al., 2001) and decrease following a meal (Cox, 1998; Tschöpp, Smiley & Heiman, 2000), a hunger-inducing role of the hormone is suggested. In humans, ghrelin levels rise in response to weight loss due to dieting (Cummings, Weighle, Frayo, Breen, Ma, et al., 2002) or due to pathological states such as cancer anorexia or during eating disorders (Otto, Cuntz, Fruehauf, Wawarta, Folwaczny, et al., 2001). The finding that provides direct evidence for its involvement in food intake is that 46

intracerebroventricular infusions of the peptide dose-dependently increase food intake and subsequently lead to obesity in rats (Wren, Seal, Cohen, Brynes, Frost, et al., 2001). More specifically, infusions of ghrelin into the PVN (Olszewski, Grace, Billington & Levine, 2003) and into the ARC (Bagnasco et al, 2003), produce increases in eating, thereby suggesting a role for the hormone in these nuclei. The hormone also is effective in producing hyperphagia in humans (Wren et al., 2001). The endocrine hunger effect of ghrelin could be mediated via two mechanisms. The first involves the vagus nerve since ghrelin receptors can be found on vagal afferent neurons. Additionally, vagotomies abolish the initiation of eating that is observed following intravenous injections of ghrelin (Date, Murakami, Toshinai, Matsukura, Nijima, et al., 2002). The second route, which has been overlooked in the literature, is via the blood stream and into the ARC. This is a possibility considering the ARC is a site for ghrelin hyperphagia and has a penetrable blood brain barrier (Broadwell & Brightman, 1978). Studies attempting to elucidate the neural mechanisms underlying ghrelinmediated meal initiation have focused on the ARC and NPY for several reasons. First, ghrelin may be produced in the ARC (Kojima et al., 1999). Second, specifically NPYergic neurons in the ARC contain GHS receptors (Willensen, Kristensen & Romer, 1999). Third, direct intra-arcuate infusions of ghrelin produce increases in eating (Wren et al., 2001). Electron microscopy has revealed that immunoreactive ghrelin dendrites receive afferent connections from unknown 47

terminals in the ARC (Lu, Guan, Wang, Uehara, Yamada, et al., 2002). Intracerebroventricular administration of ghrelin increases NPY mRNA (Shintani et al., 2001) and antagonism of NPY and AgRP abolish the feeding response elicited by ghrelin (Nakazato, Murakami, Date, Kojima, Matsuo, et al., 2001). Together, these findings suggest a possible relationship between ghrelin and NPY and possibly AgRP. Another hypothalamic pathway involved in ghrelin feeding involves the orexin-synthesizing neurons of the LH. One study showed that ghrelin containing axon terminals synapse on orexinergic neurons in the LH (Toshinai, Date, Murakami, Shimada, Mondal, et al., 2003). Further, ghrelin is rendered ineffective in the absence of orexin as illustrated by orexin knock-out mice (Toshinai, Date, Murakami, Shimada, Mondal, et al., 2003). There is evidence of a second orexigenic compound, namely NPY, which may be involved in ghrelin feeding. For instance, blocking synthesis of orexin A and orexin B (via anti IgG) weakens the effects of ghrelin on eating. Blocking NPY receptors in addition to blocking orexin exaggerates this attenuation suggesting the involvement of NPY in the ghrelin-orexin pathway. This combined attenuation is greater than the sum of the independent reductions produced by each of the peptides alone, suggesting an interaction between orexin and NPY rather than an additive effect. However, ghrelin infused into the lateral ventricles produces activation in orexinergic neurons, which is not reversed following administration of NPY anti IgG, suggesting that NPY is not necessary for orexin-mediated ghrelin action on eating. 48

The role of MCH in ghrelin elicited feeding also has been examined. The evidence to date suggests that ghrelin’s actions are independent of MCH. First, ghrelin does not produce activation in MCH neurons like it does in orexincontaining neurons (Toshinai et al., 2003). Second, pretreatment with anti-MCH IgG does not affect ghrelin’s hyperphagic effects. Although most studies examining the effects of ghrelin on eating have focused on the ARC, hindbrain areas such as the AP and NST also may be important in the hyperphagic effects of this peripheral peptide. Growth hormone secretagogue receptors are also expressed in these structures (Guan, Yu, Palyha, McKee, Geighner, et al., 1997) and infusions directly into the third and fourth ventricles, which activate these hindbrain structures, produce increases in eating comparable to those reported after infusions into the ARC (Faulconbridge, Cummings, Kaplan & Grill, 2003). 1.4.3 Peripheral Satiety Signals Cholecystokinin (CCK) Cholecystokinin is a peptide that is released by the duodenum in response to a meal (Moran & Schwartz, 1994) and serves as a negative feedback signal that controls the size of a meal (Gibbs, Young & Smith, 1973). Cholecystokinin also is produced neurally in the hypothalamus and in the neocortex (Rehfeld, 1978), in quantities that exceed those produced in the small intestines. Specifically, CCK is produced in PVN neurons that also express CRF (Mezey, Reisine, Skirboll, Beinfeld & Kiss, 1986). The satiating effects of CCK have been thoroughly and 49

consistently demonstrated across various species and varying routes of administration (Gibbs et al., 1973; Gibbs, Falasco & McHugh, 1976; Gibbs & Smith, 1977). In the LH, CCK terminals release the peptide in response to both vagal stimulation (Schick, Schusdziarra, Yaksh & Go, 1994) and after intragastric meal load (Schick, Yaksh & Go, 1986). Studies exploring the neural circuitry of CCK have led to some interesting findings. For instance, when connections between the hindbrain and forebrain are severed by supracollicular transections, peripherally administered CCK becomes effective in decreasing intake of a sucrose solution in 24-hour fasted rats (Grill & Smith, 1988). This result suggests that hindbrain structures are involved in the satietogenic effects of the peptide. It also suggests that the termination of a meal can occur in the absence of hypothalamic influences (Grill & Smith, 1988). This hypothesis is corroborated by a separate study demonstrating that decerebrate animals continue to decrease their intake of sucrose following intragastric infusions of milk (Grill, 1980). Evidence to the contrary also exists. Bilateral PVN lesions block the satiating effects of CCK (Crawley & Kiss, 1985). Additionally, central infusions of CCK into discrete hypothalamic nuclei (i.e. DMH, PVN, VMH, and LH) produce decreases in eating (Blevins et al., 2000) implicating a role for CCK satiation in the diencephalic structure. Given the complexity of eating behavior, it is not improbable that redundancy exists within the different levels of the eating system. It may be that when the feedback signal CCK is released by the duodenum due to food intake, the hindbrain structures are involved in terminating an 50

individual meal; however, hypothalamic structures come into play with the peptide’s involvement in the circuitry of long-term satiety. For instance, leptin, a compound involved in the regulation of body weight (discussed in later sections), increases the satietogenic potency of CCK, which is accompanied by increased cfos activation in the AP and the NST (Emond, Schwartz, Ladenheim & Moran, 1999) than that produced by either CCK or leptin alone. Although c-fos expression in the PVN does not differ from control levels in response to CCK administration, the combination of leptin and CCK produces greater neuronal activation in the PVN compared to that produced by leptin alone (Barrachina, Martinez, Wang, Wei & Yvette, 1997; Wang et al., 1998; Emond et al., 1999). Together, these findings suggest an interaction between the PVN and NST in the augmented satietogenic response to CCK during weight regulation. This is not surprising considering that reciprocal connections between the NST and PVN exist (ter Horst et al., 1989). Evidence for the involvement of the NST in CCK is supplemented by lesion studies. Inactivation of the commissural and medial NST, which is the target of afferents from the gastric branch of the vagus nerve (Leslie, Gwyn & Hopkins, 1982; Shapiro & Miselis, 1985), eliminates the satietogenic effects of CCK (Edwards et al., 1986). This peripheral information regarding energy state can reach the hypothalamus via the NST, which has both direct and indirect connections to the hypothalamus and other neural areas implicated in eating (Saper, 2002). For instance the NST sends projections directly or indirectly through the

51

PBN to the PVN, DMH, ARC and LH, the central amygdala, and bed nucleus of the stria terminalis. Peptide YY (PYY) Peptide YY is a hormone released in the periphery that mediates food intake. Peptide YY is synthesized in the endocrine L-cells of the ileum and is released from the small intestines post-prandially in proportion to the caloric content of a meal (Adrian, Savage, Sagor, Allen, Bacarese-Hamilton, et al., 1985; Pedersen-Bjergaard, Host, Kelbaek, Schifter, Rehfeld, et al., 1996). One of the major forms of PYY that is most commonly studied is PYY3-36. This form of PYY is found in intestinal endocrine cells and in the circulation (Eberlein, Eysselein, Schaeffer, Layer, Grandt, et al., 1989; Grandt, Schimiczek, Beglinger, Layer, Goebell, et al., 1994). Exogenous administration of the hormone produces hypophagia in both rats and humans (Batterham, Cowley, Small, Herzog, Cohen, et al., 2002). Evidence thus far suggests that PYY produces hypophagia by acting on NPY and POMC in the ARC (Batterham et al., 2002), which it can directly access through the blood due to the poorly developed blood-brain barrier at the level of the ARC (Broadwell & Brightman, 1978). Studies show that there is a significant increase in c-fos expression in the lateral ARC and specifically in the POMC neurons following a single injection of PYY3-36, which led researchers to further investigate the role of the ARC in PYY3-36 hypophagia. It was found that central infusions of PYY3-36 directly into the ARC produce reductions in eating (Batterham et al., 2002; Batterham & Bloom, 2003) further corroborating this hypothesis. 52

Finally, Battterham et al. (2002) showed that infusions of PYY3-36 depolarized 19 out of 22 ARC POMC neurons tested further solidifying the role of ARC POMC neurons in the hormone’s satietogenic properties. Peptide YY also has been shown to act on ARC NPYergic neurons. Studies investigating the receptor subtype involved in PYY3-36 satiation have speculated the Y2 receptor to be involved (Batterham et al., 2002). The Y2 receptor is an inhibitory receptor expressed on NPY neurons in the ARC (Broberger, Landry, Wong, Walsh & Hökfelt, 1997). Additionally, PYY3-36 is shown to be an agonist for NPY Y2 receptors (Grandt et al., 1994). Batterham et al. (2002) demonstrated that infusions of aY2 agonist into the ARC decreased eating, while infusions into the PVN were ineffective, providing evidence for the possibility of ARC Y2 receptor mediation in PYY3-36 hypophagia. The finding that infusions of PYY3-36 are ineffective in Y2 knockout mice also substantiates this claim (Batterham et al., 2002). However, although there is elevated neuronal activity in POMC neurons in response to PYY3-36 administration, the POMC neurons do not express Y2 receptors (Broberger et al., 1997). It could be that PYY initially inhibits NPY and this inhibition in turn disinhibits POMC neurons. This NPY and POMC connection is a strong possibility considering that these two populations of neurons send and receive reciprocal collaterals (Cowley, Smart, Rubinstein, Cerdan, Diano, et al., 2001). The brainstem has been shown to express Y2 receptors (Lynch, Walker & Snyder, 1989; Dumont, Fournier, St-Pierre, Schwartz & Ouirion, 1990) raising the 53

possibility for PYY brainstem access in vivo. Patch-clamp studies have demonstrated that the inhibitory effect of PYY in the brainstem is in part due to the inhibition of glutamatergic transmission between the NST and DMH via the activation of Y2 receptors (Browning & Travagli, 1997). Glucagon-Like Peptide-1 and 2 (GLP-1/GLP-2) Like PYY, GLP-1 and GLP-2 are produced in the endocrine L-cells of the ileum and are released post-prandially (Bell, Santerre & Mullenbach, 1983; Drucker, 1998). This peptide has been shown to induce satiety in both rodents (Turton, O’Shea, Gunn, Beak, Edwards, et al., 1996; Tang-Christensen, Larsen, Goke, Fink-Jensen, Jessop, et al., 1996) and in humans (Flint, Raben, Astrup & Holst, 1998; Näslund, Gutniak, Skogar, Rossner & Hellström, 1998), while antagonism of the GLP-1 receptor has been shown to increase eating (Turton et al., 1996). Aside from being produced in the gastrointestinal system, GLP-1 also is synthesized in the brain, specifically in the caudal NST and the medullary reticular nucleus (Jin et al., 1988; Larsen et al., 1997), both of which receive vagal inputs from the gastrointestinal system. It has been shown that the hypophagic actions of GLP-1 are at least partially mediated via the vagus nerve (Balks, Holst, von zur Mulen & Brabant, 1997). Glucagon-like peptide-1 immunoreactive terminals can be found in the PVN, VMH, DMH, LH (Grossman, 1975; Jin et al., 1988), and in the Amg (Jin et al., 1988). It has been demonstrated that all of these areas along with structures in the brainstem (Shimizu, Hirota, Ohboshi & Shima, 1987) contain 54

GLP-1 receptors, suggesting a role for this hormone in each of these neural structures. Intra-hypothalamic infusions of GLP-1 directly into discrete nuclei have shown that the DMH, VMH, and the LH are all involved in the satietogenic effect of GLP-1 (Schick, Zimmermann, vorm Walde & Schusdziarra, 2003). Infusions of this hormone into the medial Amg however have no effect on eating (Schick et al., 2003). Interestingly, infusions of GLP-1 into the third ventricle produces c-fos activation in the PVN, ARC, central Amg, AP, NST, and lateral PBN (van Dijk et al., 1996). Since infusions of this peptide into the third ventricle could not sufficiently diffuse to the brainstem, this finding suggests that the centrally infused GLP-1 acts directly on one of the diencephalic nuclei and that the hindbrain structures like the AP, NST, and the lateral PBN indirectly are involved in the circuitry of the peptide. It should be mentioned that the inhibitory effect of GLP-1 on eating may be due to this hormone’s antagonistic effect on gastric emptying, which is the movement of food from the stomach into the small intestines (Näslund et al., 1998; Schirra, Leicht, Hildebrand, Beglinger, Arnold, et al., 1998). In humans, administration of GLP-1 slows gastric emptying of both liquid (Wettergren, Schjøldager, Mortensen, Myhre, Christiansen, et al., 1993) and solid (Näslund et al., 1999) foods. Additionally, this peripheral peptide affects metabolism by causing the release of insulin and inhibiting the breakdown of glucagon (Ørskov,

55

1992) each of which could produce a positive energy state and reduce the amount of intake. 1.5 Long-term Regulation of Eating The neural mechanisms that are involved in body weight regulation will be discussed in this section. Not surprisingly, the neural structures involved in this process are identical to those involved in short-term eating although the molecular signals that activate the circuits of weight control are different. Despite the fact that the amount and type of food ingested by individuals varies from one meal to the next and fluctuates across days, most adults across a wide range of mammalian species, including humans, have the ability to maintain energy homeostasis (Edholm, 1977; Stallone & Stunkard, 1991). That is, adults are capable of matching precisely their energy intake with their energy expenditure over long intervals of time. Following a calorie-restricted diet, rats will show a marked increase in eating when given ad libitum access to chow. This hyperphagia ensues until body weight returns to baseline levels. Once baseline is achieved, the rats revert to their normal levels of consumption (Harris, Kasser & Martin, 1986). This maintenance of body weight set point has been demonstrated with force-fed rats as well. When animals are force-fed to gain weight, they consequently reduce their intake when allowed to consume at will until their body weights return to baseline levels (Wilson, Meyer, Cleveland & Weigle, 1990). Additionally, when animals are fed a diet that is either high or low in calorie, they will appropriately adjust the amount of food that they eat in order to maintain their body weight 56

(Cabanac & Lafrance, 1991). Interestingly, peripheral satiety signals lose their efficacy in calorically-restricted animals forced to lose weight (Cabanac & Lafrance, 1991), indicating that signals regulating body weight work in conjunction with gastric factors to exert their effects. Body weight regulation also is apparent in humans. Evidence for this is provided by the Bioshpere 2 study. In this experiment, eight volunteers (4 females and 4 males) maintained themselves within a self-sustainable microcosm for 2 years. Due to the scarcity of harvested food, the participants were subjected to severe caloric restriction for 2 years. The data showed that within the first 6-9 months, all of the subjects lost weight, which returned to baseline levels approximately 6 months following the end of the project (Walford, Mock, MacCallum & Laseter, 1999). 1.5.1 The Set Point Theory of Body Weight One largely debated theory about the control of body weight that is highly influential is the set point theory. The set point theory of body weight purports that each individual has a programmed weight that the body will battle to maintain in the face of any changes. This theory asserts that body weight is controlled by an “adipostat”, which receives a signal from adipose tissue, informing the central nervous system about body mass. If this feedback signal from the body fat has deviated from the programmed set point, regulatory mechanisms are activated in order to correct for the error. Despite its vast influence, the validity of this set point theory is controversial and is the subject of much debate (Berthoud, 2002).

57

This ability of adult mammals to maintain neutral energy balance compellingly indicates a robust biological mechanism involved in the regulation of body weight set point. But how is the feeding system regulated and what cues are involved in maintaining body weight over a long-term basis? There are three main molecules that act as adiposity signals and are thought to regulate body weight. It is agreed that in order for an agent to be considered an adiposity signal, it must fulfill three requirements. It should (1) be secreted in proportion to body fat mass, (2) affect both eating and body weight, and (3) act on neural substrates implicated in eating (Schwartz & Seeley, 1997; Woods, Seeley, Porte & Schwartz, 1998). Leptin, insulin, and amylin are three hormones that meet these criteria and are considered to be the main signaling molecules regulating body weight. 1.5.2 Peripheral Adiposity Signals Leptin Leptin is a hormone that is synthesized by adipocytes and is released into the bloodstream (Zhang, Proenca, Maffei, Barone, Leopold, et. al, 1994). This hormone becomes active after being cleaved of 21 amino acids, a process that evidence suggests is activated by insulin secretion following increased glucose levels after a meal (Zhang, Proenca, Maffei, Barone, Leopold, et al., 1994; Saladin, de Vos, Guerre-Millo, Leturque, Girard, et al., 1995). Leptin is considered to be an important adiposity signal that activates the central nervous system’s negative feedback loop in the control of body weight. As an adiposity signal, leptin affects 58

both eating and body weight. Centrally administered leptin into the lateral ventricles reduces food intake in both obese and lean mice (Campfield, Smith, Guisez, Devos & Burn, 1995). Further, leptin administered Leptin deficient ob/ob (Zhang et al., 1994), leptin resistant db/db (Campfield et al., 1995), and leptin receptor deficient fa/fa Zucker (Chua, Chung, Wu-Peng, Zhang, Liu, et al., 1996) mice exhibit profound hyperphagia and obesity, suggesting the hormone’s involvement in body weight regulation. In addition, levels of this hormone circulate in proportion to body fat content in humans (Havel, Kasim-Karakas, Mueller, Johnson, Gingerich, et al., 1996) and in animals (Ahren, Mansson, Gingerich & Havel, 1997; Landt, Gingerich, Havel, Mueller, Schoner, et. al., 1998). For example, when body fat is high, leptin levels are low, and eating is sustained at a lower level, while the opposite is true when body fat is reduced. Although leptin levels generally circulate in proportion to body fat, acute changes in the hormone level independent of adiposity also occur. For instance, short-term fasting decreases leptin levels (Weigle, Duell, Connor, Steiner, Soules, et al., 1997; Dubac, Phinney, Stern & Havel, 1998), while re-feeding results in increases in the hormone (Weigle et al., 1997). Although these short-term eating restrictions are not sufficient to affect adiposity, they still produce reductions in leptin levels. Since even very low doses of centrally administered leptin produce reductions in eating and body weight (Campfield et al., 1995), it is speculated that the brain is the main target for the actions of the hormone. This is supported by the 59

fact that leptin receptors have been localized in areas integrally involved in eating such as on the NPY/AGRP neurons and POMC/CART neurons of the ARC (Håkansson, Brown, Ghilardi, Skoda & Meister, 1998 and Shioda, Funahashi, Nakjo, Yada, Maruta, et al., 1998 for NPY/ARGP; Joseph, Pilcher & Knigge, 1985; Khachaturian, Lewis, Haber, Akil & Watson, 1984 for POMC; Broberger et al., 1998; Kristensen et al., 1998; Vrang, Larsen, Clausen & Kristensen, 1999 for CART). Leptin receptors also are localized on the orexin/hypocretin and MCH neurons in the LH (Horvath et al., 1999 for orexin/hypocretin; Bittencourt et al., 1992 for MCH), and on the CRH neurons in the PVN, VMH, DMH, AP, NST, and PBN (Schwartz, Baskin, Bukowski, Kuijper, Foster et al.,1996 for PVN; Mercer, Hoggard, Williams, Lawrence, Hannah, et al., 1996; Elmquist, Ahima, Elias, Flier & Saper, 1998 for VMH and DMH; Elmqist, Ahima, Maratos-Flier, Flier & Saper, 1997 for AP; Mercer, Moar, Findlay, Hoggard & Adam, 1998 for the NST;Grill, Schwartz, Kaplan, Foxhall, Breininger, et al., 2002 for PBN). The mechanism by which leptin accesses the central nervous system remains unclear. It is possible that the hormone gains access to the brain from circumventricular organs lacking a blood-brain barrier such as the AP or median eminence (Broadwell & Brightman, 1976). Alternatively, there may be a saturable transport system for leptin through the blood-brain barrier (Banks, Kastin, Huang, Jaspan & Maness, 1996) although potential neural access through the vagus also should be considered.

60

Although all of the aforementioned brain areas contain leptin receptors, the ARC is thought to be the central site for leptin action. Specifically, studies have concentrated on leptin responsive NPY/AgRP and POMC/CART neurons in this hypothalamic nucleus. Direct evidence for this hypothesis is provided by the finding that infusions of leptin into the ARC produce marked hypophagia in rats (Satoh, Ogawa, Katsuura, Hayase, Tsuji, et al., 1997) and that intracerebroventricularly infused leptin is rendered ineffective by lesioning the ARC (Tang-Christensen, Holst, Hartmann & Vrang, 1999; Dawson, Pelleymounter, Millard, Liu & Eppler, 1997). Specifically, it is speculated that leptin induces reductions in eating by inhibiting the NPY/AgRP neurons in the ARC. First, NPY mRNA and leptin receptors are coexpressed within the ARC nucleus (Mercer et al., 1996). Second, there is a decrease in NPY mRNA in the ARC following leptin administration (Wang, Bing, Al-Barazanji, Mossakowaska, Wang, et al., 1997; Stephens, Basinski, Bristow, Bue-Valleskey, Burgett, et al., 1995). On the contrary, there is an increase in the expression of NPY mRNA when leptin levels are low, for instance during fasting, or in ob/ob leptin deficient mice (Bray & York, 1979; Stanley et al., 1985; Chen, Charlat, Tartaglia, Woolf, Weng, et al., 1996; Iida, Murakami, Ishida, Mizuno, Kuwajima, et al., 1996). Further, this ARC NPY overexpression observed during leptin deficiency is eliminated upon leptin administration (Ahima, Prabakaran, Mantzoros, Ou, Lowell, et al., 1996; Stephens et al., 1995). Finally, electrophysiological studies have shown that leptin hyperpolarizes and as such inhibits ARC NPY/AgRP neurons (Spanswick, Smith, 61

Groppi, Gogan & Ashford, 1997; Roseberry, Liu, Jackson, Cai & Friedman, 2004). All of the evidence together implicates a role for NPY/AgRP in leptin regulation of energy homeostasis. The ARC POMC neurons are a second major target for leptin. Evidence for this is provided by the finding that POMC neurons and leptin receptors are coexpressed within the ARC (Cheung, Clifton, & Steiner, 1997). In leptin deficient ob/ob mice, there is a decrease in POMC expression (Ahima et al., 1996) and electrophysiological studies confirm that leptin depolarizes, and therefore activates, POMC neurons in normal animals (Cowley et al., 2001). Further, deletions of leptin receptors located only on POMC neurons in the ARC leads to animals that are overweight (Balthasar, Coppari, McMinn, Lu, Lee, et al., 2004). However, the extent of the weight gain in these mutated animals is less than the increase observed in leptin resistant db/db mice, suggesting that leptin receptive POMC neurons are not solely responsible for leptin mediated weight control. A relationship between leptin and CART also has been identified. Decreased levels of CART mRNA are found in situations of leptin deficiency such as in the ob/ob mouse or during starvation (Kristensen et al., 1998). This reduction in CART mRNA is reversed upon leptin administration indicating that the ARC neuropeptide is regulated by the adiposity signal. The DMH, VMH, and PVN also play an important role in the leptin hypothalamic circuitry. Although intravenous administration of leptin produces cfos activation in the PVN, studies show that leptin receptors are sparse within this 62

hypothalamic nucleus, while they are most abundant in the ARC, VMH, and DMH (Mercer et al., 1996; Fei, Okano, Li, Lee, Zhao, et al., 1997). It has been hypothesized that injections of leptin activate the PVN through these other hypothalamic leptin-responsive neurons. A double-labeling study has shown that PVN afferents from leptin responsive neurons in the DMH and VMH activate the PVN, which implicates a leptin circuit involving these three structures (Elmquist et al., 1998b). Given that brainstem areas such as the AP, NST, and the PBN contain leptin receptors (Grill et al., 2002), these structures also are potential targets of leptin. Intracerebroventricular infusions of leptin into the fourth ventricle, which probably diffuse to the AP and NST, produce suppression of eating that is comparable to those produced by infusions into the lateral ventricles, which diffuse to more forebrain structures (Grill et al., 2002). In addition, a double labeling study has shown that following the infusions of the retrograde tracer, cholera toxin-b, into the PVN and intravenous infusions of leptin, double labeled cells are found in the lateral PBN, while none are found in the NST (Elmquist et al., 1998a). This suggests that direct or indirect activation of the PBN by leptin causes activation in the PVN, indicating a potential PBN-PVN circuit in the central pathway of leptin. It was mentioned in the beginning of this chapter that there are separate neural mechanisms that work in conjunction to control the size of individual meals and to regulate body weight. One example of this interaction involves leptin and CCK. It has been shown that leptin increases the satietogenic potency of CCK, 63

which is accompanied by increased c-fos activation in the NST (Emond et al., 1999). Although c-fos expression in the PVN does not differ from control levels in response to CCK administration, the combination of leptin and CCK produces greater neuronal activation in the PVN compared to that produced by leptin alone (Barrachina, et al., 1997; Wang et al., 1998; Emond et al., 1999). Given that (1) the NST contains both leptin receptors (Grill et al., 2002) and CCK synthesizing neurons (Mantyh & Hunt, 1984; Herbert & Saper, 1990) and that (2) the NST projects to the PVN (ter Horst et al., 1989), it is possible that leptin activation of NST CCKergic neurons produces the augmented response, which in turn is projected to the PVN. Together, these findings implicate a NST-PVN pathway in the leptin-augmented CCK satiation. Insulin Although leptin is the most studied adiposity signal, insulin was the first hormone discovered as a weight regulating compound. Insulin is a hormone released primarily by the pancreas and much like leptin, circulates in proportion to adiposity levels (Bagdade, Bierman & Porte, 1967). Insulin also is known to affect eating and body weight and acts on the hypothalamus to do so. For instance, infusions of the hormone into the ARC or into the third ventricle decrease food intake (Woods, Lotter, McKay & Porte, 1979; McGowan, Andrews, Kelly & Grossman, 1990), suggesting that insulin acts on the hypothalamus and also on brainstem structures to produce its hypophagic response. There are dissimilarities between leptin and insulin. Unlike leptin, insulin has a known transport system 64

into the brain (Schwartz, Sipols, Kahn, Lattermann, Taborsky, et al., 1990; Schwartz, Bergman, Kahn, Taborsky, Fisher, et al., 1991). In addition, unlike leptin, insulin deficiency does not lead to obesity. In both rats (Havel, Uriu-Hare, Liu, Stanhope, Stern, et al., 1998) and humans (Hathout, Sharkey, Racine, Ahn, Mace, et al., 1999), insulin deficient diabetes leads to hyperphagia (Leedom & Meehan, 1989), but adiposity and circulating leptin levels remain low. This is probably due to the fact that insulin is necessary for the lipid synthesis and storage (Zhang et al., 1994; Saladin et al., 1995), which in turn produces leptin (Zhang et al., 1994). Bringing leptin levels back to non-diabetic levels in this model eliminates the hyperphagia, while infusions of insulin have no effect (Sindelar, Havel, Seeley, Wilkinson, Woods, et al., 1999). This suggests that it is the deficiency of leptin that results in hyperphagia and not deficiency in insulin in the diabetes mellitus model. Evidence for the contrary also exists. One study showed that insulin deficiency-induced hyperphagia was reversed upon central and peripheral infusions of insulin (Sipols, Baskin & Schwartz). If this is true, then the hyperphagia in a state of insulin deficiency could be a result of insulin deficit rather than from a paucity of leptin. Insulin, like leptin, potentially exerts its hypophagic effect through its interactions with ARC NPYergic neurons. The ARC densely expresses insulin receptors (Baskin, Wilcox, Figlewicz & Dorsa, 1988), suggesting a role for insulin in the hypothalamic nucleus. Second, the hyperphagia resulting from insulin deficient diabetes is accompanied by increases in NPY expression in the 65

hypothalamus (Williams, Grill, Lee, Cardosa, Okpere & Bloom 1989), which is reversed upon both central and peripheral infusions of insulin (Sipols, Baskin & Schwartz, 1995). This leads to the conclusion that insulin inhibits NPY neurons in the ARC and thereby reduces eating. Arcuate AGRP also is a target for insulin. Intracerebroventricular infusions of insulin eliminate ARC AGRP immunostaining normally observed within the ARC in control animals (Dunbar, Lapanowski, Barnes & Rafols, 2005) suggesting inhibition of this feeding-related neuropeptide by insulin. Moreover, perifusion of hypothalamic tissue, including the anterior hypothalamus and the PVN, with insulin blocks the release of AGRP (Breen, Conwell & Wardlaw, 2005) further implicating an interaction between insulin and AGRP. There is evidence to suggest that insulin also exerts its effects on eating via the short-term satiety agent CCK. Sub-threshold doses of intracerebroventricularly administered insulin enhance the effectiveness of peripherally delivered subthreshold doses of CCK, such that there is a 50% reduction in intake (Figlewicz, Stein, West, Porte & Woods, 1986). These findings suggest that insulin in the CNS augments sensitivity to peripheral CCK. A follow up study has demonstrated that centrally infused insulin also enhances sensitivity to central CCK (Figlewicz, Sipols, Seeley, Chavez, Woods, et al., 1995), showing that the interaction between these two agents occurs both centrally and perhaps peripherally. This provides another example of how short-term satiety signals interact with adiposity signals to regulate food intake and body weight. 66

Amylin Amylin is a hormone produced by the β cells of the pancreas (Westermark, Wernstedt, Wilander & Sletten, 1986; Cooper, Willis, Clark, Turner, Sim, et al, 1987) in response to a meal and is the third adiposity signal discussed in this chapter. It is released in proportion to body fat mass (Butler, Chou, Carter, Wang, Bu, et al., 1990) and is transported through the blood-brain barrier (Banks, Kastin, Maness, Huang & Jaspan, 1995). Denervation of the vagus (Lutz, del Prete & Scharrer, 1994) or the splanchnic (Edwards, Power & Young, 1998) does not disrupt amylin-induced anorexia. Injections of amylin reduce food intake by about 55% (Bhavsar, Watkins & Young, 1998), while blocking this hormone’s receptors with an antagonist elevates eating (Rushing, Hagan, Seeley, Lutz, D’Alessio, et al., 2001) and body fat mass (Rushing et al., 2001). Additionally, both male (GebreMedhin, Mulder, Pekny, Zhang, Tornell, et al., 1997) and female (Devine & Young, 1998) mice lacking the gene encoding for amylin become overweight, suggesting an endogenous role of amylin in body weight regulation. The effects of amylin appear to be predominantly centrally rather than peripherally mediated. Intracerebroventricularly infused amylin more potently produces reductions in eating compared to intraperitoneally administered amylin (Bhavsar, Watkins & Young, 1997; Lutz, Rossi, Althaus, del Prete & Scharrer, 1998). Studies focused on localizing the central cite of amylin’s hypophagic effect have reported that peripheral administration of amylin produces c-fos activation in the AP, NST, PBN, the BNST, and central Amg (Riediger, Zuend, Becskei & Lutz, 67

2004; Rowland, Crews & Gentry, 1997). Interestingly, c-fos immunoreactivity is blocked in all of the aforementioned areas following AP lesions (Riediger et al., 2004). This suggests that the circumventricular organ is the initial site of action for the hormone and that the other brain regions are activated by amylin via their direct or indirect connections with the AP. Further, inactivation of the AP blocks the anorectic effect of peripherally delivered amylin (Edwards et al., 1998; Rowland & Richmond, 1999), while dexfenfluramine anorexia remains unaltered. This is an important finding because it suggests that the lesions of the AP do not destroy all satietoogenic circuits. Further, the lesions of the AP do not affect amylin-produced c-fos activation in rostral areas of the brain (Rowland & Richmond, 1999) and lateral ventricle infusions of the hormone in these lesioned animals are effective in producing marked reductions in eating (Lutz et al., 1998). These results suggest that amylin also may directly act on hypothalamic sites independent of its effects on the AP. In support of this hypothesis, direct intra-hypothalamic infusions of amylin have been shown to reduce food intake (Chance, Balasubramaniam, Zhang, Wimalawansa & Fischer, 1991), although the precise regions were not specified in the report. Investigations into the specific mechanisms underlying amylin anorexia have found an interaction between the hormone and CCK once again suggesting that these adiposity signals interact with meal-related signals to regulate meal size and ultimately body weight. Doses of CCK and amylin that are individually ineffective, result in very potent inhibition of intake when they are co-administered 68

(Bhavsar et al., 1998) and selectively blocking amylin receptors decreases the satietogenic effectiveness of CCK (Lutz Tschudy, Rushing & Scharrer, 2000). 1.6 Conclusion The biological basis of eating and body weight regulation is varied and complex. Mechanisms that control short-term and long-term satiety work in conjunction to maintain energy homeostasis. Research on eating behavior has advanced tremendously from the time of the ‘dual center theory’ of ingestive behavior, when the lateral hypothalamus was thought of as the feeding center, while the ventromedial hypothalamus was considered the satiety center. Presently, we know that the intricate connections between neural structures of the forebrain telencephalon, forebrain diencephalon, and the hindbrain all work in concert to control eating and to regulate body weight. Forebrain telencephalic structures involved in eating include the amygdala and the bed nucleus of the stria terminalis. Besides the lateral hypothalamus and the ventromedial hypothalamus, forebrain diencephalic structures include other hypothalamic nuclei as well. These include the arcuate, the paraventricular nucleus, the dorsomedial nucleus, and tuberomammillary nucleus. Hindbrain structures like the area postrema, nucleus of the solitary tract, and the parabrachial nucleus are all implicated in ingestive behavior. These neural areas control eating behavior with the help of several anabolic and catabolic peptides that are localized within the neural structures themselves. The anabolic neural peptides that stimulate eating are plentiful and include neuropeptide Y, agouti related protein, galanin, melanin-stimulating 69

hormone, and orexin. Catabolic peptides that inhibit eating include proopiomelanocortin, cocaine-amphetamine regulated transcript, corticotrophin releasing hormone, and thyrotropin releasing hormone. These neural structures and peptides are activated or inhibited via peripheral satiety and feeding signals in the control of short-term satiety, which is satiety related to individual meals. These satiety signals such as cholecystokin, peptide YY, and glucagon-like peptides 1 and 2 and feeding signals such as ghrelin are released mainly from the gastrointestinal tract in response to the presence or the absence of a meal. In addition, there are separate molecules that serve to control long-term satiety, which is the satiety that regulates body weight. Although the neural machinery is highly conserved for short-term and long-term satiety, the molecular signals that initiate each process are disparate. The signals involved in long-term body weight regulation include the adiposity signals leptin, insulin, and amylin, which act in conjunction with shortterm satiety signals to regulate body weight. Given that energy homeostasis is essential for survival, the intricate and complex systems evolved to insure its accurate functioning is expected.

70

CHAPTER 2 Estradiol and Eating Behavior Because the reduction in eating produced by estradiol is a subsidiary focus of the experiments reported in this dissertation, a separate chapter is dedicated to the discussion of the decreases in eating and body weight produced by this hormone. 2.1 Estradiol: Overview Estradiol is part of a group of steroid sex hormones called estrogens and is present in both males and females. The primary source of estradiol in the circulatory system is the granulosa cells in the ovaries of female rodents and the aromatization of testosterone in the testes of male rodents (de Jong, Hey & Molen, 1973; Shoham & Schachter, 1996). As such, it is thought of primarily as a gonadal hormone. However, evidence demonstrates that in humans and in rats estrogens are synthesized outside of the gonads as well. For instance, in post-menopausal women and in men, estradiol is produced as a product of androgen aromatization, which occurs locally at various sites like the skin and adipose tissue (for review see Nelson & Bulun, 2001; Grodin, Siiteri & MacDonald, 1973). In rats, the presence of aromatase in bone suggests that estradiol is produced at this site as well (Eyre, Bland, Bujalska, Sheppard, Stewart, et al., 1998). There also is evidence for the neural synthesis of estradiol in rodents. There is a baseline concentration of estradiol in the lateral parabrachial that is significantly increased by stimulation of the cervical branch of the vagus nerve (Saleh, Saleh, Deacon & Chisholm, 2002). 71

The vagal stimulation alone does not affect circulating levels of the hormone, suggesting that the increase in estradiol observed in the lateral PBN is synthesized neurally. In addition, aromatase activity is found in both the hypothalamic preoptic area and in the amygdala (Balthazart, Foidart & Hendrick, 1990; Chambers, Thornton & Roselli, 1991), suggesting that the hormone may be produced at these sites. Recent research shows that estradiol may be produced in astrocytes in response to brain injury (Peterson, Saldanha & Schlinger, 2001), suggesting that estradiol may be synthesized generally throughout the brain and not simply in discrete structures. Interestingly, aromatase activity also has been illustrated in fetal and neonatal brains of vertebrates (MacLusky & Naftolin, 1981) demonstrating the possible ubiquitous nature of estradiol. In cycling female rats, physiological levels of estradiol vary across the various phases of the reproductive cycle. Low physiological levels range from 630 pg/ml and occur during the diestrous, metestrous, and the estrous phases, while high levels range from 22-88 pg/ml during proestrous (Butcher, Collins & Fugo, 1974; Cecchini, Chattoraj, Fanous, Panda, Brennan, et al., 1983; Dupon & Kim, 1973; Hawkins, Freedman, Marshall & Killen, 1975; Henderson, Baker & Fink; 1977; Lu, LaPolt, Nass, Matt & Judd; 1985; for review, see Overpeck, Colson, Hohmann, Applestine & Reilly, 1978). In adult male rats, physiological levels range from 1.2 to 3.1 pg/ml (Hawkins et al., 1975; Södersten, DeJong, Vreeburg & Baum, 1974). In humans, the range of circulating estradiol is between 31-164 pg/ml for females (Kim, Hosseinian & Dupon, 1974; Skinner, England, Cottrell & 72

Selwood, 1974) and from 21-46 pg/ml for males (Angsusingha, Kenny, Nankin & Taylor, 1974; Haug, Aakvaag, Sand & Torjesen, 1974). 2.2 Estradiol and Eating Behavior There is a substantial amount of evidence illustrating the role of estradiol in ingestive behaviors. Physiological levels of estradiol have been associated with systemic variations in the amount of food intake across the reproductive cycle of animals. Specifically, in rats (Wade, 1972), mice (Blaustein, Gentry, Roy & Wade, 1976), and hamsters (Morin & Fleming, 1978) decreases in consumption are observed during proestrous, when estradiol levels are elevated. Cyclic changes in food intake have been observed in female mammals as well where decreases in consumption are observed during the follicular phase, while increases are associated with the luteal phase in guinea pigs (Czaja & Goy, 1975), rhesus monkeys (Czaja, 1975; Kemnitz, Eisele, Lindsay, Engle, Perelman, et al., 1984), baboons (Gilbert & Gillman, 1956), and humans (Cohen, Sherwin & Fleming, 1987). These variations in eating observed across the reproductive cycles are shown to be inversely associated with circulating levels of estradiol that accompany the separate phases such that during the follicular phase, when endogenous estradiol levels are highest, eating is at its lowest, while the opposite is true of the luteal phase. The role of estradiol as an anorectic hormone is further supported by studies showing that ovariectomized rats demonstrate hyperphagia and increase in meal size which are accompanied by increases in body weight (Blaustein & Wade, 1976; 73

Asarian & Geary 2002). Similarly, ovariectomies in rhesus monkeys results in increased eating which persists for 3 weeks and also results in weight gain (Sullivan, Daniels & Cameron, 2003). Further, estradiol treatment, which mimics physiological levels of the hormone, is sufficient to normalize both eating and body weight in ovariectomized rats (Geary & Asarian, 1999b) and rhesus monkeys (Czaja & Goy, 1975). Administering estradiol exogenously in animals has validated that it is the steroid hormone responsible for the decreases in eating. The ability of estradiol to produce unconditioned decrements in food intake has been demonstrated across different species, implementing various routes of administration, and different forms of the estrogenic steroid (Wade, 1975; Bernstein, Courtney & Braget, 1986; Sandberg, David & Stewart, 1982; Miceli & Fleming, 1983; Tritos, SegalLieberman, Vezeridis & Maratos-Flier, 2004). The consistent results unequivocally establish estradiol as a satiety-inducing agent. Further, in rats, the decrease in eating produced by the hormone is expressed as a decrease in meal size, which is absent from compensatory increases in meal frequency (Asarian & Geary, 2002), suggesting that the hormone regulates eating by advancing satiety. Evidence strongly suggests that estradiol acts as a long-term satiety agent, that is, it reduces eating as a means of regulating body weight (Tarttelin & Gorski, 1973; Wade 1972). After ovariectomy, there is a transient increase in food intake and body weight; however, after the increase in eating subsides and returns to control intact levels, body weight stabilizes and remains 20-25% higher than the 74

weight of sham operated animals (Tarttelin & Gorski, 1973; Gentry & Wade, 1976). In addition, ovariectomies of rats and mice produce 10-30% increase in body adiposity (Mystkowski & Schwartz, 2000; Wade & Gray, 1979). Finally, estradiol fails to induce decreases in food intake in females that do not gain weight after ovariectomy further suggesting that estradiol affects eating as a means of regulating body weight (Wade, 1972). In addition, evidence shows that estradiol affects body adiposity. Leshner and Collier (1973) showed that ovariectomy double body fat content in female rats. Others have shown that treatments with estradiol reverse the increase in adiposity produced by the ovariectomies (Roy & Wade, 1977). Studies with aromatase knockout mice (ArKO), which cannot synthesize endogenous estradiol, have shown that both male and female ArKO mice progressively accumulate more adipose tissue compared to their wild type littermates (Jones, Thorburn, Britt, Hewitt, Wreford, Proietto et al., 2000). This finding corroborates the results of previous studies implicating estradiol in lipid homeostasis. Research conducted in mutant null mice implicates the importance of the αreceptor subtype in estradiol-induced reductions in eating. While wild type (WT) animals displayed hypophagia and reduced body weight following estradiol injection, WT animals treated with oil and α-receptor knock out mice (αERKO) did not show any alterations in their daily intake or in their body weight (Geary, Asarian, Chan, Korach, Pfaff et al., 1999).

75

Tonic and Phasic Effects of Estradiol Based on the data discussed above, the inhibitory effect of estradiol on eating and body weight can be divided into two categories: phasic and tonic (Drewett, 1973, 1974). The phasic or “acute” effects refer to the ability of cyclic estradiol to produce temporary decreases in eating, while the tonic or “chronic” effects apply to the long-term suppression of body weight that is maintained by estradiol and is eliminated upon ovariectomy. It is suggested that these two separate effects operate via different physiological and neural mechanisms (Geary, 2001). Estradiol and Anorexia Nervosa According to the Diagnostic and Statistical Manual of Mental Disorders IV (DSM IV; American Psychiatric Association, 1994), anorexia nervosa is categorized as an Axis I disorder, which is thought to be a transient psychopathological state that is superimposed on otherwise healthy individuals. Anorexia nervosa is characterized by a failure to maintain 85% of ideal body weight, an intense fear of gaining weight, unrealistic focus on weight, distortions of body image, and physical manifestations that include, but are not limited to amenorrhea. Eating disorders in general predominantly afflict females varying from 12 to 22 years of age and anorexia nervosa is no exception. Not surprisingly then adolescence has been identified as a major risk factor for the development of this infirmity (Halmi, Casper, Eckert, Goldberg & Davis, 1979).

76

Estradiol has now been implicated as a key player in anorexia nervosa (Gustavson & Gustavson, Noller, Melton, O’Brien, et al, 1989; Young, 1991). There are two facts that logically associate the hormone to the eating disorder. First, the onset of anorexia nervosa parallels the drastic increase in estradiol that occurs in females during the onset of puberty. At least 40% of identified cases of anorexia occur in females 15-19 years of age according to the National Eating Disorder Association. Second, the effects of this hormone mimic two of the primary symptoms of the eating disorder; decreased eating and increased physical activity. In addition to producing decreases in eating and body weight, injections of the steroid cause increases in physical activity. Female rats given access to running wheels generally run 10 km in 10 days (Ramsden, Berchtold, Kesslak, Cotman & Pike, 2003). This level of activity varies across the estrus cycle, with the highest levels of activity observed during proestrus when plasma estradiol is elevated (Wollnik & Turek, 1988) despite the decreased caloric intake observed during this phase. Further, ovariectomies decrease running wheel activity in female rats and implantation of ovarian tissue returns activity levels to control levels or to the levels of intact females (Slonaker, 1924). These findings indirectly implicate estradiol in elevated activity levels. More directly, estradiol implants in ovariectomized Swiss-Webster mice increase running-wheel activity (Garey, Morgan, Frohlich, McEwen & Pfaff, 2001) solidifying the role of the hormone in the regulation of locomotor activity.

77

Although scarce, evidence directly tying estradiol to anorexia nervosa also exists. The first of this evidence came from studies conducted by Gustavson and Gustavson, using conditioned taste aversion (CTA) as their paradigm and rats as the animal model. Traditionally, a CTA is a form of associative learning where an “aversion” is acquired toward a novel taste stimulus that has been paired with an agent that produces aversive internal states such as nausea or gastrointestinal malaise (Garcia, Kimmeldorf & Koelling, 1955; Barker, Domjan & Best, 1977). Estradiol has been found to be capable of inducing CTAs in both female and male rats. However, the dose required to induce CTA is greater for females than males. Gustavson et al. (1989b) showed that subcutaneous injections of 0.4 mg/kg of estradiol cypionate were sufficient to produce a CTA in males, but was ineffective in females. This sexually dimorphic behavior involving estradiol and CTA led these researchers to suggest estradiol as an originating cause of anorexia nervosa. Since males appeared to be more sensitive to the hormone, they proposed that during fetal and early development, the CNS of some females become partially masculinized, which results in an increased sensitivity to the nauseating and thus the conditioning effects of estradiol. A previous study by these investigators provided evidence for this hypothesis, namely, that prenatal masculinization leads to increased sensitivity to estradiol-induced food aversions. Female rats prenatally masculinized due to exposure to testosterone showed a lower dose threshold for developing estradiol-induced food aversions as adults (Gustavson, Gustavson, Noller, Melton, O’Brien & Pumariega, 1989a), which led to persistent changes in 78

food intake and body weight maintenance. They extended these results to humans by suggesting that pollutants, maternal stress, or pharmacological drugs that elevate androgen levels might cause fetal masculinization of female fetuses and consequently increased sensitivity to the nauseating effects estradiol (Schou, 1968). The researchers hypothesized that these masculinized females frequently get nauseated when estradiol production rises during puberty and hence develop food aversions, which limits the number of acceptable foods these girls consume. Subsequently, this produces a general decrease in consumption and hence body weight. Although this weight loss results in a reduction of estradiol production and consequently an increase in ingestion, the resulting weight gain restores the production of the hormone, which once again leads to decreases in eating. It was speculated that this vicious cycle potentially manifests itself into a starvation system, leading to the development of anorexia nervosa. Studies examining the role of food aversions in various anorexia symptoms provide support for the food aversion driven anorexia hypothesis. For example, it has been demonstrated that continuous exposure to a target diet during a chronic illness state leads to a CTA to that diet, which in turn leads to significant weight loss (reviewed in Bernstein & Borson, 1986). The hypothesis for the origin of anorexia proposed by Gustavson et al. becomes clearer when the behavioral effects of hormones during fetal development and during adulthood are considered. The presence or absence of prenatal hormones dictates the neural development of fetuses. For instance, male fetuses 79

exposed to testosterone develop a “male” sexually dimorphic nucleus in the brain, such that during adulthood, when these males are re-exposed to the hormone, the hormones activate the “male” brain, which results in the exhibition of “male” sexual behaviors. A disruption in the hormone balance during fetal development disrupts behavior expression during adulthood. For instance, if male fetuses are not exposed to testosterone during fetal development, they develop more of a “female” brain or a smaller sexually dimorphic nucleus. When these males are exposed to estrogen and progesterone during puberty or adulthood, the “female” brain is activated and they display “female” sexual behaviors and prefer male partners. Similar disruptions can occur in female fetuses. In utero estrogenic exposure has major organizational effects on the brain development of female fetuses such that when these females are re-exposed to the hormones in adulthood, their behavior is affected. Exposure to estrogens in utero leads to masculinization of the female brain or a larger sexually dimorphic nucleus. As such, in adulthood, when these females are exposed to estrogens and progesterone, they exhibit male sexual behaviors such as mounting (masculinization) and decreased lordosis and impaired ovarian function (defeminization: Hines, Alsum, Roy, Gorski & Goy, 1987; Hines & Goy, 1985). Gustavsons’ hypothesis of anorexia is clearer once it is superimposed on this hormonal model of development and adulthood exposure. They propose that prenatal exposure to hormones that masculinize the female brain during the organizational period leads to masculinized behavior in adulthood when these females are exposed to estrogen during puberty. In their hypothesis, this 80

masculinized behavior is a sensitized reaction estrogen, which serves as a potent unconditioned stimulus for the acquisition of food aversions and hence reduced food intake. Although the examples used to demonstrate the effects of prenatal hormones and adulthood behavior have focused on sexual behavior, one study has showed the effects prenatal hormonal alteration in food preference. For instance, female fetuses exposed to TCDD, a dioxin that produces hormonal changes, showed reduced preference for saccharin as adults (Amin, Moore, Peterson & Schantz, 2000). Their preference for saccharin was reduced to levels of saccharin preference exhibited by males. This study shows that alterations in prenatal hormones affect eating behaviors in addition to reproductive behaviors and supplies credence to Gustavsons’ hypothesis of the origins of anorexia. Synthetic estrogens also are shown to produce masculinization effects during fetal development. For instance, diethylstilbestrol (DES), a synthetic nonsteroidal estradiol, is known to produce masculinization syndrome in female rats. One study demonstrated that the sexually dimorphic nucleus in the preoptic area of female rats was masculinized by treatment with either testosterone or DES. The size of this nucleus in the females was similar to size of the sexually dimorphic nucleus of normal male rats (Dohler, Coquelin, Davis, Hines, Shryne & Gorski, 1984). Although the researchers did not test for sexual behaviors expressed as adults, these females probably would display male sexual behaviors when exposed to estrogen and progesterone in adulthood. Female guinea pigs exposed to synthetic estrogens in utero display masculine typical sexual behaviors as adults, 81

instead of female sexual behaviors, when they are exposed to estradiol and progesterone (Hines, Alsum, Roy, Gorski & Goy, 1987; Hines & Goy, 1985). The Gustavson et al. study (1989) discussed above, which produced masculinized brains in female rats by prenatally treating them with testosterone also had produced masculine females by prenatally exposing them to DES. These female rats exhibited behavior identical to those prenatally treated with testosterone; namely, they developed estradiol-induced food aversions at lower doses compared to non-treated controls. It should be mentioned that these findings raise the possibility that the aromatization of testosterone to estradiol may be producing the “masculinization” effects since no differences are detected between the brains of female rats treated prenatally with testosterone or DES. Evidence for this hypothesis exists. One study showed that female guinea pig fetuses exposed to testosterone in utero displayed male sexual behaviors in adulthood. However, exposing these females to both testosterone and ATD, an aromatase inhibitor, prevented the display of masculine sexual behaviors by the female guinea pigs (Roy, 1992; Choate & Resko, 1994). This suggests that the aromatization of testosterone into estradiol is primarily responsible for the masculine behaviors displayed by these females. This possibility is further bolstered by the fact that prenatal aromatase activity in vertebrates has been demonstrated (MacLusky & Naftolin, 1981). Along these same lines, it would be interesting to see whether in utero exposure to dihydrotestosterone (DHT), which does not aromatize into estradiol, would preclude females from displaying masculine behaviors in 82

adulthood. If masculine behaviors are not displayed in these females, this too would suggest that prenatal exposure to estradiol is responsible for masculinized behaviors exhibited during adulthood. Evidence from human research supports the hypothesis regarding the origins of anorexia laid out by Gustavson et al. (1991). This evidence can be pulled from the daughters of women who were treated with DES during pregnancy. If exposure to DES masculinizes the brains of female fetuses such that during adulthood, these women display an increased sensitivity to the aversive affects of estrogen in adulthood, then the daughters of women exposed to DES during pregnancy should be at an increased risk for developing anorexia. Physicians began to prescribe DES to pregnant women from about 1947-1971 in hopes of avoiding miscarriages and circumventing other complications associated with pregnancy (Hammes & Laitman, 2003). The FDA encouraged the discontinuation of its use in 1971 when the synthetic estrogen became associated with vaginal and cervical cancer. Children of mothers who were administered DES also were affected, suffering from reproductive tract abnormalities, increased infertility, and eating disorders (Bibbo, Al-Naqeeb, Baccarini, Gill, Newton, et al., 1975; Gustavson et al., 1991). Interestingly, as Chambers and Bernstein (2003, 2005) point out, the time of DES usage corresponds to the increase in the reported incidence of anorexia nervosa in 14-20 year old females in the United States beginning in 1965 and continuing through 1976 (Jones, Fox, Babigian & Hutton, 1980). More direct evidence for the role of DES in eating disorders is provided by 83

the findings that women prenatally exposed to DES were 5 times more likely to show inexplicable weight loss and to be diagnosed with an eating disorder such as anorexia nervosa or bulimia nervosa, compared to women who are not exposed to the drug (Gustavson, Gustavson, Noller, O’Brien, Melton et al., 1991). Evidence that anorexics show an exaggerated anorexic effect of estradiol provides strong support for the role of this hormone in the eating disorder. It has been reported that in anorexic patients, estrogen therapy increases the vomiting related to estrogenic vaginal smears (Moulton, 1942). How this increased sensitivity to estradiol is produced is still in question. As stated above, Gustavson et al. (1989a,b) proposed in utero “masculinization” produced by various agents like testosterone and DES as potential culprits. However, there is evidence that prenatal exposure to testosterone may serve a protective role. For instance, young women with twin brothers have more healthy eating behavior than women with twin sisters (Culbert, Breedlove, Burt & Klump, 2008). Moreover, disordered eating is lower in both females and males with a male twin, suggesting that exposure to embryonic testosterone may play a preventative role. These findings also suggest that prenatal exposure to estradiol may provoke disordered eating since both males and females with a female twin exhibited disordered eating. However, these findings seem contradictory since evidence of prenatal aromatase activity exists (MacLusky & Naftolin, 1981). If in utero testosterone is capable of aromatizing into estradiol and estradiol is playing a facilitatory role, then how would in utero testosterone be serving a protective role against the eating disorder? 84

One possibility for the contradictory results may lie in the prenatal aromatizability of testosterone. The MacLusky and Naftolin (1981) study showed aromatase activity in the brains of fetuses and neonates. Although testosterone may aromatize within the fetus, it may not be able to convert to estradiol inside of the embryonic fluid, if the embryonic fluid is the only way twin fetuses are exposed to each other’s hormones. As such, female twins exposed to testosterone due to their male siblings may be protected. Even still, the logic of this hypothesis that testosterone protects females by making them more masculine contradicts the hypothesis set forth by Gustavson et al. (1989a,b), who claim that making the females more masculine is precisely the culprit in predisposing them to disordered eating. Taken together, all of these data demonstrate a role for estradiol in anorexia. It is clear that females with anorexia do exhibit increased sensitivity to the effects of estradiol; however, the originating cause of this sensitization to estradiol remains in question although prenatal exposure to estradiol continues to be a strong possibility. Studies showing the masculinization of female brains treated prenatally with testosterone or DES provide evidence for the masculinization hypothesis although further investigation is warranted. The effects of prenatal exposure to pollutants and maternal stress remain unexplored. Progress towards testing this hypothesis demands identification of neural areas that mediate the anorectic effects of estradiol. In the next section, existing research that has investigated possible neural sites for this effect of estradiol will be discussed.

85

2.3 Neural Substrates of Estradiol Hypophagia To date, the neural site(s) of estradiol action regarding its ability to inhibit feeding have not been equivocally identified; however, there have been many studies implicating several brain regions as potential candidates. These structures include the area postrema (AP), the nucleus of the solitary tract (NST), the parabrachial nucleus (PBN), the paraventricular nucleus of the hypothalamus (PVN), and the amygdala (Amg). C-fos Like Immunoreactivity (c-FLI) Studies Immunohistological studies conducted in our lab suggest a role for the AP, lateral PBN and the PVN in estradiol-elicited reductions in consumption. Ovariectomized female rats were injected with sesame oil or 50μg/kg dose of estradiol benzoate at the end of the light phase of the light/dark cycle and their brain were processed for c-FLI 24 hours later. Estradiol activated c-FLI expression in the AP, the lateral PBN, the PVN, and in the amygdala (central, lateral, basolateral, and medial subnuclei) 24 hours following its administration, which is precisely the time when reductions in eating produced by exogenous administration of this hormone are initially expressed (Chambers, Hintiryan & So, unpublished manuscript; Tartellin & Gorski, 1973). Decreases in eating also are observed 24 hours following the increase in plasma estradiol levels in cycling rats (Asarian & Geary, 2002; Griffin & Ojeda, 1996). Although c-fos activation provides only an indirect association between estradiol and these neural structures, it provides a good starting point in identifying neural substrates of estradiol hypophagia. 86

Immunohistological studies conducted in other labs have shown that in food-deprived ovariectomized rats, eating increases the expression of c-FLI in the NST, PVN, and the central nucleus of the Amg (ceAmg), all areas that are involved in consumption (discussed in Chapter 1, “Neural Regulation of Eating”). Activation of c-FLI is found within these same neural areas when ovariectomized females are pretreated with a dose of estradiol that produces blood levels of estradiol observed during proestrous (Eckel & Geary, 2001). Since c-FLI activation may be indicative of activation or inhibition, estradiol may be inhibiting feeding-induced neuronal activation within these brain regions. Unfortunately, immunohistochemical staining of the lateral PBN was excluded from this study. Central Implant Studies Initial studies delving into the neural areas involved in estradiol hypophagia included the medial Amg. Bilateral implants of estradiol benzoate into the medial Amg produced decreases in food intake and body weight over a period of 72 hours (Donohoe & Stevens, 1981) implicating this limbic structure in estradiol hypophagia. Seminal studies on the neural localization of estradiol hypophagia also focused on the ventromedial nucleus of the hypothalamus (VMH) since at the time this area was branded the ‘satiety center’ (Stellar, 1954) of the brain. Although there is evidence contrary to the now archaic notions of ‘satiety’ and ‘hunger’ centers, nevertheless, the VMH has been shown to be involved in satiety processes (Cox, Kakolewski & Valenstein, 1969; King & Frohman, 1986). These studies showed that microinjections of pure estradiol into the VMH (Palmer & 87

Gray, 1986) and the VMH/arcuate (ARC; Butera & Czaja, 1984) decreased eating in ovariectomized rats. Based on these findings, it was concluded that the VMH was a neural area involved in mediating decreases in consumption produced by estradiol. Further, it was demonstrated that amygdaloid implants continue to cause suppression in eating and body weight in ovariectomized rats sustaining lesions of the VMH/ARC region (Donohoe & Stevens, 1981). Together, these results imply that the steroid may act independently on both structures to produce its hypophagic effects. Subsequent studies however, showed that infusing a more diluted form of estradiol (3:1 or 10:1 mixture of estradiol to cholesterol), which restricts steroid diffusion within the brain, into the VMH had no effect on eating and body weight. On the other hand, implants of this diluted estradiol into the PVN decreased both intake and body weight (Butera & Beikirch, 1989). Consistent with this failure to find an involvement of the VMH, are the results of studies showing that neither VMH electrolytic lesions in female rats (Kemnitz et al., 1977) nor gold thioglucose lesions of the VMH in mice (Blaustein, Gentry, Roy & Wade, 1976) block estradiol-induced reductions in food intake. Retroactively, it was speculated that the neural administration of pure estradiol produced a vast number of changes that could have independently affected eating. This may have also been true for the medial Amg implant studies since the researchers used the undiluted form of the steroid.

88

Lesion Studies Lesion studies also implicate the AP in hypophagia that is induced by estradiol. Male rats were given a target diet the night before they were placed under chronic vehicle treatment or estradiol treatment that produced supraphysiological blood levels of estradiol (116.2 or 169.4 pg/ml). The estradiol-treated males showed a decrease in consumption of their target diet across 5 days while the control animals did not. A subsequent study showed that these estradiol-treated males did not express hypophagia to the target diet following thermal lesions of the AP (Bernstein et al., 1986). The results of inactivation studies examining the role of the PVN in estradiol-induced hypophagia have been mixed. Some evidence has been provided by the finding that PVN lesions render estradiol ineffective in producing food decrements (Butera, Willard & Raymond, 1992). On the other hand, these findings have been inconsistent with the findings of Dagnault and Richard (1994) and Hrupka and Geary (unpublished data as cited in Geary, 2000), which show that lesions of this hypothalamic nucleus do not block the effect of the steroidal hormone on eating. The reason for these inconsistencies remains unexplored; however, despite these incongruous findings, the central implant studies discussed above and the studies examining the neuropeptides involved in estradiol hypophagia discussed below, strongly suggest a role for the PVN in estradiol hypophagia.

89

Because the primary focus of this dissertation is the role of the PBN in estradiol hypophagia, a separate section is dedicated to the interaction of estradiol and this neural structure. 2.4 Estradiol and the Lateral Parabrachial Nucleus (PBN) As discussed in chapter 1, “Neural Regulation of Eating”, the PBN has been implicated in eating. In particular, evidence suggests that the lateral PBN is involved in satiation (Nagai et al., 1987). Although there is no direct evidence that the lateral PBN plays a role in the hypophagic effects of estradiol, several pieces of evidence are suggestive. First, both α and β estrogen receptors have been identified in the lateral PBN (Shughrue, Lane & Merchenthaler, 1997), suggesting a role for the hormone in this neural area. Second, although research on the neural production of estradiol is scarce, one study suggests that this hormone either is produced in the lateral PBN or that it accumulates in this structure following vagal stimulation (Saleh, Saleh, Deacon & Chisholm, 2002). The authors showed that there is a baseline concentration of estradiol in the lateral PBN that can be changed by stimulation of the cervical branch of the vagus nerve. Estrogen significantly increases in response to vagal stimulation in ovariectomized female and intact male rats, reaching a maximum 90 minutes after stimulation is initiated, while it remains constant in non-stimulated controls. The authors also showed that vagal stimulation alone does not affect circulating levels of the hormone, suggesting that the increase in estradiol observed in the lateral PBN is not due to peripheral increases in the hormone. The third piece of indirect evidence links the PBN 90

specifically to estradiol hypophagia. This evidence involves the neural connectivity of the structure. The PVN, which remains the most likely candidate for involvement in the decrements in eating produced by estradiol, is predominantly innervated by the lateral PBN (Krukoff, Harris & Jhamandas, 1993), suggesting a potential interaction between these two areas in mediating estradiol produced changes in eating. In fact, this PVN-lateral PBN connection has been shown to be involved in serotonin-induced anorexia (Li, Spector & Rowland, 1994). Fourth, it is demonstrated that c-FLI is activated in the central, crescent, and external lateral subnuclei of the PBN 24 hours after injection of 50μg/kg of estradiol benzoate, which is precisely the time when reductions in eating produced by endogenous and exogenous estradiol are expressed (Chambers, Hintiryan & So, unpublished manuscript; Griffin & Ojeda, 1996). Although the lateral PBN appears to be a good candidate for the site of estradiol hypophagia, no studies examining this possibility have been conducted. 2.5 Estradiol, Neuropeptides and Eating Although most research involved in determining the neural regulation of estradiol hypophagia has focused on establishing the site of the hormone’s action, some attention has been centered on the neurochemical mediation of estradiolinduced reductions in eating. This research, which also gives insight into the neural structures important for the hormone’s actions, shows that there is an interaction between estradiol and CCK, NPY, and MCH.

91

Cholecystokinin (CCK) and Estradiol A vast amount of research has established CCK as a satiety-inducing agent (Moran & Schwartz, 1994). Further, it is suggested estradiol produces its hypophagic effects by increasing the potency of CCK (for review, see Geary, 2001). For instance, chronic estradiol injections augment the satiating potency of CCK in ovariectomized animals (Lindén, Uvnäs-Moberg, Forsberg, Bednar & Södersten, 1990; Butera, Campbell & Cataldo, 1993; Geary, Trace, McEwen & Smith, 1994). This augmented decrease in eating produced by CCK in estradiol treated animals could be the additive hypophagic effects of the two anorectic compounds; however, the decrease in eating produced by the CCK and estradiol combination was greater than the decrease produced by adding the reduction in eating produced by each of these agents alone. Therefore, the augmented decrease was most likely not due to the additive satiogenic effects of CCK and estradiol. Studies using estradiol treatment that mimics the reproductive cycles of rats have revealed corroborating evidence for the augmented decrease in eating when CCK is added to the estradiol treatment regimen (Asarian & Geary, 1999a). Further support for an estradiol-CCK interaction is provided by the results of three additional studies. Estradiol treatment increases the satiating effects of intralipid, an agent whose satiating action is mediated partly by CCK (Yox, Brenner & Ritter, 1992). On the other hand, estradiol treatment is ineffective in altering the satiating effect of l-phenylalanine, an agent whose effects on eating are independent of CCK action (Greenberg, 1998). These results suggest that estradiol potentiates the satiating 92

properties of agents that release CCK in female rats. Another study which confirms the interaction of estradiol and CCK in satiety showed that the administration of a CCKA antagonist during a day that mimics estrus estradiol levels, prevented the expected decrease in the size of meals, while administration of the intestinal peptide on a day that mimics diestrus estradiol levels, had no effect (Asarian & Geary, 1999b). These data indicate that the satiogenic CCK signaling is increased during a period when estradiol levels are elevated. Studies investigating c-FLI protein activation also substantiate the interaction between estradiol and CCK and suggest potential neural substrates for the site for this action. Eckel, Houpt and Geary (1998) showed that injections of CCK octapeptide induced c-FLI activation in the NST, PVN and in the central Amg, which also were increased in animals treated with estradiol. Research conducted in mutant null mice has revealed the nature of the interaction between CCK and estradiol. Estradiol or oil treatments were given to either wild type (WT) or α-estradiol receptor knock out (αERKO) mice. WT animals treated with estradiol displayed hypophagia and reduced body weight, while eating remained unaltered in WT animals treated with oil and in αERKO mice (Geary, Asarian, Chan, Korach, Pfaff et al., 1999). In addition, a CCKA antagonist prevented the decrease in food intake in the WT mice injected with estradiol, but it did not have an effect in oil treated control animals. The CCKA antagonist also did not affect eating in αERKO mice treated with estradiol, which is what one would expect since estradiol did not affect eating in these animals. These 93

data suggest a role for CCK in estradiol hypophagia. Moreover, the caudal NST is implicated as one of the sites of this estradiol-CCK interaction. Central implants of estradiol benzoate above the caudal NST decreased eating and increased CCKinduced c-FLI in the estradiol alpha receptors of the caudal NST (Thammacharoen, Lutz, Geary & Asarian, 2008). Neuropeptide Y (NPY) and Estradiol It has been consistently and thoroughly demonstrated that NPY is a potent appetite stimulator (Clark, Karla, Crowley & Karla, 1984; Morley, Hernandez & Flood, 1987; Pau, Pau & Spies, 1985). Hypothalamic sites such as the ARC, PVN and VMH that contain NPY synthesizing neurons have been shown to concentrate estradiol following administration of the radioactively labeled hormone (Sar & Stumpf, 1981; Morell, Krieger & Pfaff, 1986; Sar, Sahu, Crowley & Karla, 1990). This morphological inter-relationship suggests a potential link between the two peptides. A direct examination of this possibility was conducted by Bonavera, Dube, Karla and Karla (1994) in a two-part experiment. The first study demonstrated a decrease in the expression of NPY in the PVN and in the surrounding PFA in ovariectomized female rats treated with estradiol for 18 days via a Silastic capsule and behaviorally showing decreases in eating and in body weight compared to the control oil animals. Hypothalamic levels of β-endorphin, another orexigenic agent (Morley, Levine, Yim & Lowy, 1983), were unaffected by estradiol treatment, although these animals also showed reductions in eating and body weight. These results suggest that (1) the effects of estradiol were specific to 94

NPY and (2) the reductions in the neuronal peptide were not a result of a negative energy state produced by the estradiol-produced decreases in eating. If the changes in NPY expression were in response to general decreases in eating in body weight, one would expect that there would be an increase in this orexigenic peptide since NPY levels increase in response to energy depletion (Sahu, Sninsky, Phelps, Dube, Karla, et al., 1992). The second study in the two-part experiment examined the effects of estradiol treatment on the release of NPY from the PVN and VMH. Increases in eating observed during the onset of the dark phase are accompanied by increases in NPY release in the PVN in the rat (Karla, Dube, Sahu, Phelps & Karla, 1991; Sahu & Karla, 1993). As such, the animals treated with estradiol for 18 days via a Silastic capsule were sacrificed at the onset of the dark phase. Results showed that both basal and KCl-induced release of NPY in the PVN was significantly decreased in rats treated with estradiol, while levels of the neuropeptide release in the VMH remained unchanged. Together, the findings of this two-part experiment suggest that estradiol produces its hypophagic effects by decreasing the release of NPY in the PVN. Whether estradiol acts directly on the PVN to reduce NPY release or whether the hormone acts indirectly through another neural structure such as the ARC is not known and warrants further investigation. Melanin-Concentrating Hormone (MCH) and Estradiol Research also suggests an interaction between estradiol and the orexigenic peptide MCH (Rossi, Choi, O’Shea, Miyoshi, Ghatel et al., 1997; Ludwig, Tritos, 95

Mastaitis, Kulkami, Kokkotou et al., 2001). Murray, Baker, Levy and Wilson (2000) showed that subcutaneous administration of estradiol benzoate significantly decreased the expression of MCH precursor protein in the zona incerta and in the LH 24 and 48 hours following the injection, which is the time when decreases in eating are elicited by estradiol following its administration (Chambers, Hintiryan & So, unpublished manuscript). This finding was corroborated by a separate study which investigated the effects of chronic estradiol treatment on the expression of MCH (Mystkowski, Seeley, Hahn, Baskin, Havel et al., 2000). The results showed that estradiol treatment for 18 days resulted in decreased eating and body weight, which were accompanied by decreases in hypothalamic MCH expression. This decreased hypothalamic MCH expression was not observed in pair-fed controls, which were given the same decreased amount of food as the estradiol group, but did not receive estradiol benzoate. In fact, there was a significant increase in MCH expression in pair-fed animals. This is in line with results showing an increase in MCH in response to energy depletion (Fellmann, Risold, Bahjaoui, Compagnone, Bresson, et al., 1993; Herve & Fellmann, 1997).

96

CHAPTER 3 Conditioned Taste Aversion Learning 3.1 Conditioned Taste Aversion (CTA): Overview Attributes of Conditioned Taste Aversion Learning When consumption of a novel tasting substance is followed by administration of a chemical agent that produces physiological changes indicative of malaise, animals will reduce their consumption of the substance during subsequent encounters. This learned response is traditionally referred to as a conditioned taste aversion (CTA). A CTA is acquired through a learning process that is similar to the traditional classical conditioning paradigm. For instance, pairing a distinctively flavored taste stimulus (the conditioned stimulus or CS) with an agent that normally elicits nauseating effects (the unconditioned stimulus or US), will lead to the subsequent avoidance of the taste (the conditioned response or CR). Although CTAs follow the general scheme of classical conditioning, there are two important differences that distinguish them from most other types of the paradigmatic learning and render them a very powerful form of learning. First, aversions to novel taste stimuli can be learned easily in a single trial, while most classically conditioned responses develop following several to numerous pairings of the CS with the US (Mackintosh, 1974). In the classic illustration of Pavlov’s experiment, a CR of salivation was elicited by the CS tone only after several pairings of the meat powder (US) and the tone (Pavlov, 1927). Early studies of eyeblink conditioning where the CS tone is paired with a US air puff to the eye show that learning in rabbits occurs after approximately 140 trials (Thompson & Krupa, 1994) 97

and after 60 pairings in humans (Reynolds, 1945). The one-trial attribute of CTAs has been demonstrated extensively in studies using lithium chloride (LiCl). As a powerful poison, LiCl is considered to be the putative illness-inducing agent and is the most commonly used US in studies investigating the underlying mechanisms of CTA learning. Lithium chloride is a salt of lithium and has properties similar to sodium, although the precise mechanism by which this agent acts is not quite understood. The second characteristic of CTAs that separates them from other forms of Pavlovian conditioning involves the temporal contiguity between the CS and US presentations. In CTAs, associations between the CS tastants and US toxins still occur if the interval between the CS and US is as long as 4-12 hours, while in traditional classical conditioning, delays of over a few minutes or even seconds can greatly disrupt learning. The earliest studies exploring this phenomenon showed that an apomorphine CTA towards a saccharin solution could be formed with an inter-stimulus interval as long as 75 minutes (Garcia, Ervin & Koelling, 1966). Laboratories have reported significant aversions to a 4% sucrose solution when exposure to x-radiation was administered 6 hours following the CS presentation and gustatory aversions were readily established toward a 0.1% saccharin solution when the CS and the x-radiation US were separated by as much as 12 hours (Smith & Roll, 1967). The mechanisms underlying such a lengthy interval remain undetermined; however, studies show that it is neither due to regurgitation nor due to lingering aftertaste. The regurgitation hypothesis cannot be upheld in the case of rapidly metabolized substances such as sucrose solutions, which are no longer present in the system throughout the 1-12 hour 98

delay (Revusky, 1968). Additionally, this hypothesis cannot be applied to rats since the emetic response is blocked by the cardiac sphincter in this species. The aftertaste hypothesis has not received support either, since CTA to a novel solution is not disrupted when either the animal’s pallet is rinsed or when other more familiar flavors are ingested during the CS-US interval (Kalat & Rozin, 1971). Such a lengthy CS-US interval is extraordinary considering that traditional conditioning experiments deal in seconds and fractions thereof. For instance, in a study that paired the sound of a buzzer to an electric shock, researchers found that the most efficient conditioning occurred when the inter-stimulus interval was 667 milliseconds (Kappauf & Schlosberg, 1937). Optimal CS-US interval delay in eye-blink conditioning is approximately 400-450 milliseconds in humans (Kimble, 1947; Reynolds, 1945) and 250 milliseconds in rabbits (Coleman & Gormezano, 1971). Evolutionary Significance of Conditioned Taste Aversion The aforementioned qualities of CTAs designate them as a potent form of learning, which is common to most animals including humans (Bernstein, 1978, 1985; Gustavson, Gustavson, Young, Pumariega & Nicolaus, 1989). Evolutionarily, this type of learning is imperative for the survival of a species. Omnivorous animals that survive after ingestion of a novel toxic substance and learn quickly and unequivocally to avoid that distinctive taste will increase their chances of survival. Possessing the capability to bridge very long CS-US intervals and to disregard interpolated familiar CSs as culprits for the illness further confers selective advantage.

99

The classic study by Garcia and Keolling (1966) illuminates the biological predisposition of animals to associate particular tastes with illness. In this study, male rats were presented with both audiovisual and gustatory cues. The audiovisual cues consisted of “bright-noisy” water, which was provided by connecting an incandescent lamp (5 watt) and a clicking relay into the drinkometer circuit such that each time the animal licked, a bright flash was accompanied by the emission of a noise. The gustatory stimulus was “tasty” water, which was water mixed with flavors. Both audiovisual and gustatory stimuli were paired with radiation. Aversions developed only to the gustatory cues (tasty water) and not to the audiovisual stimulus (brightnoisy water). On the other hand, when both cues were paired with a foot shock, aversions were formed only to the audiovisual stimuli. These findings suggest that biologically, animals are predisposed to associate taste with internal malaise. Auditory and visual cues, on the other hand, are not so readily associated with gastrointestinal discomfort, but are readily associated with peripheral pain. 3.2 Gustatory (CS) and Malaise (US) Information Since the formation of a CTA requires the association between taste (CS) and malaise (US) information, the neural areas involved in individually processing both of these stimuli become potential candidates as mediators of the learned behavior. Neural Gustatory Pathway (CS Pathway) From taste receptors on the tongue, gustatory information is transmitted to the trigeminal (V), to the lingual branch of the glossopharyngeal (IXth) and to the chorda tympani and superficial petrosal branches of the facial (VIIth) cranial nerves (Torvik, 100

1956; Hamilton & Norgren, 1984). From these nerves, gustatory information is transmitted to the rostral portion of the nucleus of the solitary tract (NST; Torvik, 1956) located in the brainstem. The information is then projected to the ipsilateral medial or “gustatory” parabrachial nucleus (PBN; Norgren, 1978, 1984), which transmits the information to the contralateral ventroposterior medial thalamus (VPMpc; Saper & Loewy, 1980). The VPMpc is the final relay of taste information before its final destination in the gustatory cortex of the dysgranular region of the insular cortex (IC; Cechetto & Saper, 1987). A second, yet less defined, route by which taste information reaches the forebrain involves mono-synaptic projections from the medial PBN to the central nucleus of the amygdala (Amg), the substantia innominata, the lateral hypothalamus (LH), the bed nucleus of stria terminalis (BNST), and the IC (Norgren, 1974, 1976; Saper & Loewy, 1980; Shipley & Sanders, 1982; Fulwiler & Saper, 1984; Moga, Herbert, Hurley, Yasui & Gray, 1990; Bernard, 1993). Neural Visceral Pathway (US Pathway) There are three routes by which malaise information produced specifically by LiCl reaches the central nervous system (CNS). First, malaise information is transmitted via the vagus nerve (parasympathetic) second, through the splanchnic nerve (sympathetic) and third, through a humoral route via the area postrema (AP). Visceral information from the gut, liver and cardiorespiratory system is carried by the vagus nerve (Baldino & Wolfson, 1985) to the caudal NST (Cechetto, 1987). Next, the caudal NST sends extensive projections to the ipsilateral lateral PBN (Lowey 101

& Burton, 1979; Saper & Lowey, 1980), which is the relay station for ascending visceral information from the NST to the thalamus, hypothalamus, and Amg (Cechetto, 1987). Specifically, the lateral PBN has a contralateral projection to the thalamus, in particular, to the ventroposterior lateral parvocellular nucleus of the thalamus (VPLpc; Cechetto & Saber, 1987). The VPLpc is the final relay station of visceral information being carried to the granular IC (Cechetto & Saper, 1987). The brain-gut axis involving the vagus and the AP also has been established as a potential route through which LiCl-induced malaise information is carried into the CNS. Neuroanatomical studies have well established that the AP receives visceral input via the afferent vagus nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985), a major afferent input to the brainstem from the gastrointestinal system. In addition, it sends and receives information from the NST and the PBN, predominantly the lateral subdivision, (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985), thereby transmitting information regarding malaise. A second route through which malaise information is carried to the brain is via the splanchnic nerves. Application of LiCl into the intestines or the duodenum elicits discharges in the afferent splanchnic nerve (Niijima & Yamamoto, 1994). Additionally, the magnitude of the LiCl-elicited neural discharges is greater for the splanchnic than for the vagus nerve. The authors ascribed this effect to a more important role of the splanchnic compared to the vagus in carrying information regarding LiCl produced malaise information. The greater activation produced by LiCl in the splanchnic however could also be due to the transmission of pain information 102

produced by the injection and not necessarily due to the transmission of malaise information. One study examining the effects of sympathectomy on emetic responses showed that bilateral sympathectomy alone had no effect on copper sulfate-induced emesis (Wang & Borison, 1951); however, the combined vagotomy and sympathecotomy increased the threshold for the drug produced emesis above that observed by vagotomies alone. This suggests that communication between the vagus and splanchnic or the concomitant transmission of information of the two play a critical role in emetic responses. To our knowledge, there is no study showing the effect of splanchnic denervation specifically on the acquisition of a CTA. Although the projections of the splanchnic still need to be elucidated, research examining various behaviors has shown a potential connection between the splanchnic and the NST. Microinfusions of GABA into the commissural NST markedly reduce sympathetic splanchnic nerve activity (Sato, Colombari & Morrison, 2001). Studies examining the connection between peripheral osmoreceptors and the CNS have also focused on the targets of the afferent splanchnic nerve. The researchers showed that gastric infusions of hypertonic saline produce c-fos activation in the AP and the NST, which was attenuated following bilateral splanchnic denervation (Carlson & Osborn, 1998), suggesting possible direct or indirect connections between the nerve and the brainstem areas. Finally, information regarding the US can reach the CNS through the blood. The blood-brain-barrier is poorly developed at the level of circumventricular organs such as the AP (Lang & Marvig, 1989) and arcuate nucleus of the hypothalamus 103

(Broadwell & Brightman, 1978). As such, the structure serves as a potential passage for circulating toxins from the blood into the CNS (Grant & Borison, 1988; Lang & Marvig, 1989). Evidence for the involvement of this structure in CTAs is provided in the following section. 3.3 Neural Substrates of Conditioned Taste Aversion Learning Lesioning techniques provide valuable information regarding the role of a particular neural structure in CTAs; however, prior to delving into evidence that have utilized lesioning techniques, it is important to mention a few important general limitations of the procedure. When lesioning a brain area results in the subsequent abolition of a behavior, this may mean one of several things. First, the lesion may have caused a deficit in motivation such that the animal no longer cares to learn the task. Second, there could be an attention deficit so that the subject is no longer able to focus on the stimuli. These two possibilities could be eliminated if the lesioned subjects are able to show a different type of learning suggesting that the animals are motivated to learn and are capable of attending to stimuli. Finally, it may be that there is an initial loss of function, but with time, the behavior may return due to subsiding stress or swelling caused by the mechanics of the lesion. To avoid this, subjects must be given sufficient time to recover following lesions prior to proceeding with behavior testing. Behavior testing also could be repeated several months following the lesioning to ensure that the result of the ablation is a permanent chronic effect, rather than a temporary acute one.

104

When interpreting the results of lesion studies involving CTAs, there are further considerations that need to be addressed, some of which differ from the aforementioned explanations. The abolition of a CTA following a lesion may mean one of several things. All of these possibilities must be given serious consideration when interpreting the results of lesion effects on the acquisition of CTAs. (1) The ability to perceive the CS tastant is destroyed. (2) The ability to process the gustatory stimulus is destroyed. Reilly, Grigson and Norgren (1993a) explain this as the ability to use the CS to predict important events in the internal or external environment. For instance, the animals should be able to perceive a taste as novel or familiar in order to predict the future consequences of ingesting that particular food. (3) The ability to perceive the US may be damaged. (4) The ability to process the US stimulus is destroyed. This refers to the ability of the US to predict important events in the internal and external environment. For example, the ability to perceive the specific negative sensory properties of a malaiseinducing agent as familiar allows the animal to predict that a novel taste consumed after experiencing those negative sensations is unlikely to be the cause of them even though they return after consuming the novel taste. (5) The ability to associate the CS with the US is eliminated. (6) The ability to bridge the temporal interval between the offset of the CS and the onset of the US may be affected. (7) The ability of the animal to express the learned behavior is destroyed. 105

Two brain access routes have been identified as important for conveying US information during the acquisition of a CTA, the AP and the vagus. Although the splanchnic is also a possible route, as indicated above, information regarding its role in CTAs is non-existent. Three neural areas have been implicated in transmitting both CS and US information. These areas are (1) the parabrachial nucleus (PBN), (2) the amygdala (Amg), and (3) the insular cortex (IC). Since the focus of this proposal is the PBN, more attention will be given to this nucleus compared to any of the others. 3.3.1 The Vagus Nerve and the Nucleus of the Solitary Tract (NST) As mentioned previously, the vagus nerve carries information regarding LiCl from the gastrointestinal system into the CNS. Intraperitoneally and intraduodenally administered LiCl causes gradual discharges in the afferent vagus nerve (Nijima & Yamamoto, 1994). This information regarding malaise is then carried from the vagus to the NST. Evidence corroborating this comes from a study which showed that the expression of the protein c-fos found in the NST after LiCl administration is eliminated following bilateral vagotomies (Yamamoto, Shimura, Sako, Azuma & Bai, 1992). Additionally, aside from LiCl, various other agents used as USs in CTAs, such as hypertonic saline and copper sulfate, also produce c-fos in the caudal NST (Sakai & Yamamoto, 1997). Moreover, the expression of the protein is correlated with the strength of the CTA produced following the administration of these agents. That is, stronger CTAs elicited by higher doses of LiCl result in higher expression of the protein in the caudal NST. Given that this region is essential for respiration, lesions of

106

the caudal NST would result in death. As such, ablation studies examining the role of the caudal NST in CTAs have not been conducted. Both intraoral and intragastric infusions of sucrose produce activation of c-fos in the rostral NST (Yamamoto & Sawa, 2000). This is not unexpected considering that the rostral region of the NST is important for processing gustatory information; however, this region does not appear to be involved in the acquisition of CTAs. Rats with electrolytic lesions of the rostral NST express impaired gustatory processing, but they are still capable of developing a CTA (Shimura, Grigson & Norgren, 1997; Grigson, Shimura & Norgren, 1997a,b). As such, it remains unclear how gustatory information important for CTAs gets from the taste receptors to the PBN. 3.3.2 The Area Postrema (AP) The AP is located in the caudal region of the brainstem, more specifically at the ventral midline of the IV ventricle near the obex. This area has many features that would implicate its involvement in CTAs. First, the blood-brain barrier is poorly developed at the level of the AP. As such, the structure serves as a passage for circulating toxins from the blood into the CNS (Grant & Borison, 1988; Lang & Marvig, 1989). Second, the neural connections of this circumventricular structure also suggest a function of this structure in CTAs. As indicated above, neuroanatomical studies have well established that the AP receives visceral input via the afferent vagus nerve (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985), which is a major afferent input to the brainstem from the gastrointestinal system. The AP also sends and receives information from the NST and the lateral PBN (van der Kooy & Koda, 1983; Shapiro 107

& Miselis, 1985), two structures that are important in the formation of a CTA (Chang & Scott, 1984; Swank & Bernstein, 1994; Grigson, Shimura & Norgren, 1997a,b for the NST; DiLorenzo, 1988; Ivanova & Bures, 1990b; Flynn, Grill, Schulkin & Norgren, 1991 for the PBN). Additionally, the AP receives input from structures integrally involved in eating such as the paraventricular nucleus (PVN) and the dorsomedial (DMH) hypothalamus (Shapiro & Miselis, 1985) suggesting that the structure may receive information regarding ingested foods. Third, in humans, the AP is regarded as a chemoreceptor trigger zone for emetic responses (Borison & Wang, 1953). This is supported by findings showing that thermal lesions of the AP in humans relieve intractable vomiting (Lindstrom & Brizzee, 1962). Finally, electrical stimulation of the AP alone can serve as the US in a CTA paradigm (Gallo, Arnedo, Agüero & Puerto, 1988), thus implicating the circumventricular structure in conditioned gustatory aversions. Given these characteristics, the AP is speculated to be a US agent detector in a CTA model. C-fos immunoreactivity is evident in the AP following the intraperitoneal administration of LiCl (Yamamoto et al., 1992; Swank, Schafe & Bernstein 1995; Thiele, Roitman & Bernstein 1996; Sakai & Yamamoto, 1997; Yamamoto & Sawa, 2000) indicating that the chemical produces some increased activity in this region of the brain. As mentioned earlier, the AP could potentially detect US agents either through the blood (Coil, Rogers, Garcia & Novin 1978; Grant & Borison, 1988; Lang & Marvig, 1989) or from the vagus (Coil et al., 1978). Examination of this possibility through electrophysiological studies shows that the AP contains neurons that respond 108

to LiCl that is administered directly into the blood (Adachi, Kobashi, Miyoshi & Tsukamoto, 1991) or into the IV ventricle (Adachi et al., 1991) suggesting a more humoral route for the illness-inducing agent. Additionally, vagotomies do not disrupt acquisition of LiCl CTAs and based on this result it has been suggested that for this learned behavior, LiCl acts directly on the AP, via the bloodstream, rather than indirectly through the vagus nerve (Martin, Cheng & Novin 1978); however, one cannot eliminate the possibility that the AP mediates LiCl CTAs when activated by a vagal as well as a humoral stimulus. If LiCl CTAs can be induced via two pathways, vagus to AP and humoral to AP, then blocking the AP would eliminate the ability to acquire CTA but blocking the vagus would not because the humoral to AP pathway would still be intact. Since the humoral pathway was not blocked in the study mentioned above, all one can say is that CTAs are not supported by vagal input alone. Lesion studies substantiate the importance of the AP in the acquisition of CTAs induced by various agents. Permanent lesions of the AP eliminate CTAs caused by LiCl (Ossenkopp, 1983; Rabin, Hunt & Lee, 1983a; Wang, Lavond & Chambers, 1997a), gamma radiation (Ossenkopp, 1983; Rabin et al., 1983a), histamine (Rabin et al., 1983a), and estradiol (Bernstein, Courtney & Braget, 1986). Temporary cooling lesions of this structure also block LiCl CTAs (Wang et al., 1997a). The AP however, does not affect ethanol- (Rabin et al., 1983a), amphetamine- (Berger, Wise & Stein, 1973; Rabin et al., 1983a), nicotine- (Ossenkopp & Giugno, 1990) or apomorphine (Wang, Lavond & Chambers, 1997b) CTAs. These latter findings are important, since they suggest that the inability of an animal to develop a CTA when LiCl, radiation, 109

histamine and estradiol are used as USs is not due to a deficit in motivation or attention since lesioned animals are able to learn CTA when certain USs are used. Several of the aforementioned possible explanations for the disrupted CTA following the lesions of the AP can be eliminated due to some important studies. It has been proposed that the abolition of a CTA following AP lesions may be due to the inability of the rats to detect the CS or to recognize it as novel, thereby blunting the acquisition of the learned response (Kosten & Contreras, 1989). A study using the reversible cooling lesion technique has shown evidence against both of these hypotheses by inactivating the AP only during the duration of US (Wang et al., 1997a,b). Since the AP is functional when the animals first encounter the novel CS before acquisition and after acquisition on extinction day 1, blocking of a LiCl CTA cannot be due to the inability of an animal to detect the CS nor to an inability to process it as novel. The lesion of the AP also is probably not due to the ability of an animal to express the learned behavior since the structure is intact during extinction test 1. Additionally, permanent lesions of the AP made following acquisition of a LiCl or radiation CTA, but before extinction day 1, have no effect (Rabin et al, 1983b), on the acquisition of a CTA, implying that the animals are still able to express the learned behavior with a dysfunctional AP. Since the AP is an important structure implicated in human emetic responses, destruction of the AP probably interferes with the detection or the processing of illness responses. The temporary lesion of the AP made exclusively throughout the duration of the US (Wang & Chambers, 1997) suggests that the disruption of the LiCl CTA is 110

due to either an interference with malaise information processing or even the associative processing, since the formation of a LiCl CTA is thought to be quite rapid (within 15 minutes; Houpt & Berlin, 1999). Evidence supports the former hypothesis. Lesions of the AP not only block LiCl CTAs, but they also eliminate some behavioral and physiological effects normally produced by the agent. For instance, following the administration of a high dose of LiCl, AP-lesioned animals do not display ‘lying on the belly’ or delayed gastric emptying compared to sham operated controls (Bernstein, Chavez, Allen & Taylor, 1992). Since emesis in rats is blocked by the cardiac sphincter, behavioral responses like ‘lying on the belly’ and physiological responses such as delayed stomach emptying, are commonly used as indices of malaise. Although this finding suggests that the lesions disrupt the ability of animals to detect or process malaise information rather than interfering with the association process, it is conceivable that the lesions of the AP could have disrupted both. Emphasis has been placed on the former hypothesis, namely that lesions of the AP disrupt malaise information processing, but it is worth noting that the AP also receives some gustatory information via the lingual-tonsilar branch of the IXth cranial nerve (Hamilton & Norgren, 1984) implicating it as a possible site of association. In addition, lesions of the AP do not affect the development of a CTA when the CS (saccharin) and the US (hypertonic NaCl) are administered concurrently (Arnedo, Gallo, Agüero & Puerto, 1990), which could mean that the AP is necessary for bridging the time interval between the CS and US when the CS and US are administered sequentially.

111

3.3.3 The Parabrachial Nucleus (PBN) A second neural structure important for CTA acquisition is the PBN, which is also located in the brainstem. This pontine nucleus is separated into two main subdivisions by the superior cerebellar peduncle or brachium conjuctivum: the medial “gustatory” PBN, which receives taste information (Norgren & Leonard, 1971, 1973; Perrotto & Scott, 1976; Cechetto & Saper, 1987) and the lateral “visceral” PBN, which receives malaise information in addition to information regarding cardiovascular, respiratory, and nociceptive processes (Chamberlin & Saper, 1992; Bernard, Huang & Besson, 1994; Bester, Menendez, Besson & Bernard, 1995). It is primarily the lateral subdivision of the PBN that sends information to and receives inputs from the AP (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985), making it the second neural structure potentially involved in US detection in the CTA circuit. Administration of LiCl is known to evoke c-fos immunoreactivity in the central, crescent dorsal, and external lateral subnuclei of the PBN and the external medial PBN (Yamamoto et al., 1992; Chambers & Wang, 2004). The external lateral nucleus is thought to be the putative lateral PBN target zone for ascending LiClinduced viscerosensory information (Sakai & Yamamoto, 1997; Yamamoto et al., 1992). More importantly, there is strong correlation between the strength of a CTA developed to a sweet solution and c-fos activation in the central, crescent, and external lateral PBN and the external medial PBN following radiation, rotation, and LiCl (Sakai & Yamamoto, 1997; Chambers & Wang, 2004). On the other hand, substances that do not induce a CTA such as electric foot shock, strychnine, and physiological saline, 112

elicit only very modest induction of the protein in the PBN (Sakai & Yamamoto, 1997) suggesting activation in the region is indicative of US processing. Initial studies investigating the role of the PBN in CTAs performed lesions of the entire nucleus and examined the effects of the ablations on the learned behavior. It has been found that bilateral temporary tetrodotoxin (TTX) lesions (Ivanova & Bures, 1990b) and permanent electrolytic lesions (Hill & Almli, 1983; Di Lorenzo, 1988; Yamamoto & Fujimoto, 1991; Yamamoto, Shimura, Sako, Yasoshima & Sakai, 1994) of the entire PBN consistently abolish the formation of a LiCl CTA, which implicates involvement of the whole neural structure in the acquisition process. This finding sparked interest in the role of the individual subdivisions of the pontine nucleus in the acquisition of CTAs. The Lateral Parabrachial Nucleus Inactivation of the lateral subdivision of the PBN using various lesioning techniques has yielded congruous results. Confined electrolytic (Sakai &Yamamoto, 1998) or ibotenic acid (Reilly & Trifunovic, 2000a,b, 2001; Trifunovic & Reilly, 2002) lesions of the nucleus block the acquisition of a LiCl CTA. Reversible TTX lesions (Ivanova & Bures, 1990a,b; Bielavska & Bures, 1994) or cooling lesions (Wang & Chambers, 2002) made after the CS such that it overlaps with the US duration also disrupt the associative learning. The lateral PBN is also involved in CTAs induced by different USs other than LiCl. Cytotoxic lesions of the pontine nucleus block the acquisition of a morphine-induced CTA (Nader, Bechara & van der Kooy, 1996), while cooling lesions block apomorphine CTAs (Chambers & Wang, 2004). 113

The lesions of this subnucleus have been shown to either attenuate (Reilly & Trifunovic, 2001) or have no effect (Sakai, Tanimizu, Sako, Shimura & Yamamoto, 1994) on neophobic responses to the novel CS. In both cases however, the lateral PBN lesion still blocks the LiCl CTA. In the first study, the authors claimed that the disruption in CTA was not due to the effect of the lesion on neophobia. They argued that lesions of the lateral PBN do not affect neophobic responses to a capsaicin solution, (Reilly & Trifunovic, 2001) yet animals with these lesions are unable to develop a LiCl CTA to this solution (Reilly & Trifunovic, 2001). As such, the authors claimed that the acquisition of a CTA is not predicated on neophobic responses to the taste, rather, it is independent of these responses because the lesions disrupt the learning despite the effects on neophobia. A direct argument against the neophobia hypothesis is provided by reversible lesions of the lateral PBN (Wang & Chambers, 2002). Cooling lesions made between the CS and the US such that the lesion overlaps only with the US duration, also disrupt the formation of a CTA. If the disruption in CTAs with lateral PBN lesions was due to abolished neophobia, then inactivation of the nucleus made only during the US should not disrupt the learning. Additional support for this hypothesis comes from a study finding that animals with lesions of the lateral PBN continue to develop strong aversions when intragastric hypertonic sodium chloride serves as the US (Cubero, Lopez, Navarro & Puerto, 2001). This finding is important not only because it substantiates that lateral PBN lesions leave gustatory capabilities intact but also because it demonstrates that these lesions leave the attentional state and motivational 114

state of the animal undisturbed. The temporary cooling lesion study also provides evidence against the hypothesis that the lesions of this subdivision may be due to disruptions in the ability of the animal to express the learned behavior since the nucleus is functional during the first extinction trial. Permanent lesions of this nucleus made after the CS-US pairing do not affect the expression of the learned behavior (Sakai & Yamamoto, 1998) further negating this hypothesis. Although the evidence above allows for the dismissal of most alternative explanations, it leaves open the possibility that the blockade of the CTA is either due to a disruption in the transmission of malaise information or a disruption in associative processes. The evidence available thus far suggests that lesions of the lateral PBN abolish CTAs by causing a disruption in ascending malaise information rather than affecting the associative process. First, the lateral PBN (especially the external subnucleus) receives the greatest part of the afferents projecting from the AP (Lanca & van der Kooy, 1985; Shapiro & Miselis, 1985; Milner, Joh & Pickel, 1986; Miceli, Post & van der Kooy, 1987). In neurally intact animals, electrical stimulation of the AP can serve as a US in a CTA paradigm; however, this is disrupted in lateral PBN lesioned animals (Agüero, Arnedo, Gallo & Puerto, 1993). Given that the AP is critical for malaise information and emetic responses (Borison & Brizzee, 1951), this finding would suggest that the lateral PBN is important in processing the malaise information it receives from the AP. Second, animals with lesions of the lateral PBN are also unable to learn other behaviors that involve LiCl-toxicosis. For instance, animals with lateral PBN lesions are also unable to develop a CTA towards a mild capsaicin solution 115

(Reilly & Trifunovic, 2000a), which the authors claim is non-gustatory trigeminal stimulus (Silver & Finger, 1991). As such, since the lateral PBN does not receive information regarding the trigeminal stimulus, disruption of the CTA cannot be due to a disruption in association. It should be noted that although capsaicin is considered by researchers to be solely a trigeminal stimulant (Reilly & Trifunovic, 2000a; Shimura et al., 1997) there is some evidence that the agent may also activate taste receptors (Silver & Finger, 1991; Travers, Urbanek & Grill, 1999). To our knowledge, no studies investigating the effect of capsaicin on medial or lateral PBN activation have been conducted. Therefore, the possibility that some neurons in the lateral PBN mediate both trigeminal and US information cannot be completely dismissed. A third piece of evidence that suggests the abolition of CTAs following lateral PBN lesions are due to disruption in the transmission of malaise information is provided by a study that used morphine as a US. Ibotenic acid lesions of the lateral PBN precluded the formation of a CTA (Nader et al., 1996). These same animals also were not able to develop a morphine-elicited conditioned place aversion, and on the basis of these results it was suggested that the lesions had interrupted the transmission of malaise information rather than disabling the associative process. This conclusion assumes that the lateral PBN does not process information regarding place. If the lateral PBN does process place information, then the inability of lesioned animals to acquire a conditioned place aversion may be due to the inability of the animals to process information regarding place and not the US properties of morphine. This hypothesis however has not been investigated. 116

Fourth, lesions of the lateral PBN impair CTAs produced by intraperitoneal injections of an AP-dependent muscarinic receptor antagonist methyl-scopolamine (Gallo, Arnedo, Agüero & Puerto, 1990; Berger, Wise & Stein, 1993), which crosses the blood-brain barrier; however, the lesions are ineffective against CTAs produced by intraventricularly infused methyl-scopolamine (Cubero & Puerto, 2000). If the lateral PBN was involved in association, then the lesions should disrupt CTAs produced by the US delivered by both routes of administration. On the contrary, it only disrupts the CTA produced by the route that involves the malaise afferent pathway and not the intracerebroventricular one that bypasses it. Once again, based on one study, one cannot completely discard the association possibility. It could be that the lateral PBN, in addition to other areas like the amygdala and the insular cortex, serves as a place of association. Therefore, in the absence of the PBN, these forebrain structures could substitute as the association areas. As a result, a CTA could still be formed when a US is infused intraventricularly despite a dysfunctional PBN. The Medial Parabrachial Nucleus Lesions of the medial subdivisions of the PBN also disrupt CTAs. Both electrolytic (DiLorenzo, 1988; Flynn et al., 1991; Reilly et al, 1993a) and excitotoxic (Spector, Norgren & Grill, 1992; Sakai et al., 1994; Sakai & Yamamoto, 1998; Trifunovic & Reilly, 2002) lesions of the medial PBN block CTAs even when very high doses of LiCl are used (DiLorenzo, 1988; Grigson, Reilly, Shimura & Norgren, 1998; Flynn et al., 1991; Spector et al., 1992; Reilly et al, 1993a; Sakai et al., 1994; Sakai & Yamamoto, 1998; Trifunovic & Reilly, 2002). Additionally, medial PBN 117

lesioned animals fail to learn a second CTA 8 months following the first, indicating the absence of recovery following permanent lesions of this area (Scalera, Spector & Norgren, 1995). Lesions of the medial PBN are not involved in morphine-induced CTAs (Bechara, Martin, Pridgar & van der Kooy 1993). This suggests that lesions of the area cannot be due to the absence of attentional capacities or motivational factors of the subjects. It has been demonstrated that the blockade of CTA following medial PBN lesions is not due to the inability of animals to bridge the temporal interval between the offset of the CS and the onset of the US. This possibility was directly tested and rejected by using a methodology that minimizes the demand on memory. Neurologically intact animals injected with LiCl immediately before intraoral infusions of a CS that is delivered every 5 minutes over 30 minutes shows a reversal of taste reactivity from ingestive to aversive. On the contrary, medial PBN lesioned animals do not show these changes in taste reactivity responses (Spector et al., 1992). Additionally, in a concurrent CTA design, i.e. when a LiCl solution is used both as the CS and US thereby minimizing the inter-stimulus interval, animals with medial PBN lesions still do not acquire a CTA (DiLorenzo, 1988). The abolition of CTAs following medial PBN lesions also is not due to a disruption in motivation nor in the ability of animals to express the learned behavior. Permanently lesioning this area after the acquisition of a CTA does not affect the learned behavior (Grigson, Shimura & Norgren, 1997b). This finding also argues against the hypothesis the medial PBN lesions disrupt the ability of the animals to 118

detect the CS since post-acquisition lesioned animals continue to show the gustatory aversion learning. Another possible explanation for the blockade of CTAs in medial PBN lesioned animals is that the ablations interfere with gustatory processing. Taste reactivity testing of orofacial responses in non-deprived animals with medial PBN lesions shows that these animals display blunted responses to alanine, a sweet tasting amino acid (Flynn et al., 1991). These animals also display increased intake of highly concentrated sucrose solution and quinine, which neurally intact animals normally avoid (Flynn et al., 1991). Sakai and Yamamoto (1998) demonstrate that animals with medial PBN lesions also show equal preferences for quinine, HCl, and sucrose solutions, while animals with lateral PBN lesions do not exhibit this impaired sensibility for sapid stimuli. Animals with medial PBN lesions also show attenuation of neophobia to sucrose, which could potentially explain the abolished CTA following its inactivation (Scalera, Grigson, Shimura, Reilly & Norgren, 1992; Reilly et al., 1993a; Sakai et al., 1994). These findings are not surprising given that the medial PBN is a major relay for ascending taste information. Cleverly designed experiments suggest that the blockade of a CTA by lesions of the medial PBN is not due to disruption in either gustatory or visceral information processing, but due to a selective disruption in the associative mechanism responsible for taste aversion learning. In a series of experiments, Grigson et al. (1998) show that rats with electrophysiologically guided ibotenic acid lesions of the medial PBN fail to acquire a CTA to an alanine solution that is paired with LiCl. These same animals 119

however are able to develop a conditioned taste preference for a flavor that is enhanced with a non-caloric sweetner. A conditioned flavor preference (CFP) procedure pairs two flavors: one distinctive flavor, the CS+, which is mixed with a nutrient, and a second distinctive flavor, the CS-, which is not mixed with a nutrient. Following several acquisition trials during which animals get single-bottle presentations of either the CS+ or CS-, the animals are given a two-bottle test of preference for the two flavors but the nutrient is excluded from the CS+. The animals choose the flavor that is paired with the nutrient (CS+). The ability of lesioned animals to develop a CFP indicates that the animals are able to detect and process gustatory information. Further, these same animals are able to learn an aversion to a dilute (0.01mM) capsaicin solution paired with LiCl, indicating that these animals also are able to process malaise information. These findings are consistent with other experiments. Animals with excitotoxic lesions of the medial PBN, which fail to develop a LiCl CTA, are able to develop a conditioned place aversion with the identical US (type and dose), which is ineffective in producing a CTA (Reilly et al., 1993a). These findings provide strong evidence that medial PBN lesions preclude the integration of the CS with US stimulus. Supporting evidence for this hypothesis can be found in studies that do not involve lesions. Electrophysiological studies show that there are neurons in the medial PBN that respond to both gustatory and visceral information. Hermann and Rogers (1985) implicate the interstitial zone of neurons in the caudal medial PBN and the subadjacent parvocellular reticular formation in containing such neurons. In a separate study, they also show that these two specific areas receive projections from both the 120

gustatory and visceral NST further validating the existence of these co-responsive neural cells (Hermann, Kohlermann & Rogers, 1983). It is mainly for this reason that in the realm of CTA learning, the PBN is thought to be the first area where integration of CS and US information. The evidence presented thus far suggests that the PBN is critically involved in the acquisition process of a CTA. Interestingly, although an intact PBN is necessary, it is not sufficient to produce a CTA. Decerebrated rats with an intact PBN do not acquire CTAs (Grill & Norgren, 1978). Additionally, when the bilateral decerebration is made following the CS presentation either 15 or 5 minutes before the US presentation, animals remain capable of learning the behavior (Buresova & Bures, 1973). This suggests that CS gustatory processing at the level of the brain stem (i.e. in the NST and the medial PBN) is insufficient for CTA and that gustatory processing in the forebrain is essential for the development of a gustatory aversion. 3.3.4 The Amygdala (Amg) A third structure important for CTA acquisition is the amygdala (Amg). Studies investigating the involvement of the Amg in CTAs have mainly focused on two amygdalar nuclei: the central and basolateral. Intraperitoneal injection of LiCl induces c-fos activation in the central (Yamamoto et al., 1992; Gu, Gonzalez, Chen & Deutsch, 1993; Lamprecht & Dudai, 1995; Yamamoto, Sako, Sakai & Iwafune, 1997; Spencer & Houpt, 2001) and basolateral (Koh & Bernstein, 2005) nuclei although the expression is higher in the former. C-fos induction is also present in the central Amg following sucrose consumption, while the expression of the proto-oncogene is 121

relatively low in the basolateral nucleus relative to the central nucleus (Yamamoto et al., 1997). Additionally, expression of c-fos is also found in both of these amygdaloid nuclei following the pairing of a novel CS and US (Koh & Bernstein, 2005) further suggesting their potential role in CTAs. Despite the initial contradictory findings across studies due to differences in lesioning techniques and behavioral testing, some conclusions have been drawn regarding the roles of the central and basolateral nuclei in CTAs. Bilateral ibotenic acid lesions of the central Amg do not affect the acquisition of a CTA (Yamamoto & Fujimoto, 1991; Sakai & Yamamoto, 1999; Morris, Frey, Kasambira & Petrides, 1999), while lesions of the basolateral Amg, which spare the bi-directional PBNinsular fibers that pass through the central nucleus (Frey, Morris & Petrides, 1997), abolish CTAs (Yamamoto, Fujimoto, Shimura & Sakai, 1995; Morris et al., 1999; Sakai & Yamamoto, 1999). A study looking at the role of the Amg in different stages of CTAs shows that bilateral temporary lesions of the Amg made by TTX infusions before the CS so that the inactivation overlaps only during the CS duration do not disrupt the development of a CTA, while temporary lesions of the area made after the CS presentation, during the US activation, block the CTA (Gallo, Roldan & Bures, 1992; Roldan & Bures, 1994). The findings of this experiment suggest several things. It means that the blockade of CTA following amygdalar lesions is not due to the ability of animals to (1) detect or process gustatory information, (2) express the learned behavior, (3) pay attention to CS or US stimuli, (4) be motivated to perform the learned behavior, or (5) bridge the 122

temporal gap between the CS and the US presentations. In this fashion, temporary lesions provide extremely valuable information that permanent lesions are unable to provide. On the other hand, the findings do suggest that the Amg is involved in either the processing of malaise information or in the CS-US association. Investigations that disassociate the latter two possibilities unfortunately have not been conducted. 3.3.5 The Insular Cortex (IC) A fourth structure implicated in CTA acquisition is the IC. The IC is generally divided into three layers that are hierarchically organized. The granular is the most dorsal, the dysgranular is the middle layer, and the agranular, most ventral. Moreover, the three layers of the IC are stratified into rostral and caudal areas (for review, see Sewards, 2004). Studies (Hanamori, Kunitake, Kato & Kannan, 1998a,b) show that the rostral area is exclusively gustatory, while the caudal portion is predominantly visceral. In addition, in between these two regions, there is an area that receives convergent gustatory, chemoreceptive, baroreceptive, and nociceptive information. Given the complexity of the IC organization, interpreting IC lesion studies has been difficult when the precise location of the lesions has not been specified. There has been one study that attempted to show the differential effect of the rostral, central, and caudal IC lesions on CTA learning. The results showed that ibotenic acid lesions of the central IC, but not the rostral or caudal, disrupted formation of the CTA (Nerad, Ramirez-Amaya, Ormsby & Bermudez-Rattoni, 1996). For the remainder of the IC lesion studies discussed in this section, the area that is lesioned will be considered rostral, central or caudal depending on the stereotaxic coordinates 123

set forth for each of these areas by Nerad et al. (1996). Some researchers report that bilateral lesions of the central IC made prior to the CS presentation impair, but do not abolish, the acquisition of a LiCl CTA (Braun, Lasitar & Kiefer, 1982; Dunn & Everitt, 1988; Bermudez-Rattoni & McGaugh, 1991; Sakai & Yamamoto, 1999). This means that the lesions accelerated the extinction rate of the CTA, but did not completely block the learning. On the other hand, different laboratories show that permanent (Braun, Slick & Lorden, 1972) or temporary TTX (Gallo et al., 1992) lesions of the central IC made before the CS, but not after (Gallo et al., 1992; Roldan & Bures, 1994), completely abolish the CTA. The temporary lesion study provides valuable information regarding the role of the IC lesions in gustatory aversions. For instance, it shows that the blockade of a CTA following the IC lesions is not due to the animal’s inability to (1) detect or process visceral information, (2) express the learned behavior, (3) pay attention to the CS or US stimuli, (4) be motivated to perform the learned behavior, or (5) bridge the temporal gap between the CS and the US presentations. On the other hand, the findings do suggest that the IC is involved in either the detection or processing of gustatory information or in the association process. Follow up studies show that animals with permanent IC ablations are able to detect the saccharin solution since they showed a preference for the CS solution over water, similar to the neurally intact control animals (Braun et al., 1972; Dunn & Everitt, 1988). Taken together, these studies suggest that the lesions of the IC do not interfere with the detection of the CS, but rather with the processing of the gustatory information, i.e. the lesions destroy the ability to process the CS as novel. In fact, there is some evidence for this 124

attenuated neophobia hypothesis. The study by Dunn & Everitt (1988) shows that both electrolytic and ibotenic acid lesions of the IC reduce the neophobic response of animals to the CS. It should be mentioned that since the authors used a different rat atlas than Nerad et al. (1996), it is difficult to compare the area they lesioned to those mentioned above; however, given the description of the histology, it is likely that the caudal, central, and rostral areas of the IC as described by Nerad et al (1996) sustained damage. In summary, the IC appears to be involved in the acquisition of CTAs, most probably by affecting the processing of gustatory information. This idea is further supported by the fact that bilateral cortical spreading depression or anesthesia induced before the CS presentation abolishes a CTA, without interfering with the ability of animals to show unconditioned signs of illness (Buresova & Bures, 1973; Bures & Buresova, 1989b). In contrast, bilateral cortical spreading depression or anesthesia applied after the CS, such that the depression overlaps with the US, does not affect the associative learning process (Buresova & Bures, 1973; Bures & Buresova, 1989). These results suggest that the acquisition of a CTA requires cortical involvement for the processing of gustatory information, while subcortical areas are sufficient for illness processing. 3.3.6 Configuring the Conditioned Taste Aversion Neural Pathway Although the individual aforementioned neural areas play an important role in CTAs, acquisition of this associative learning involves the interaction of all of these brain structures. Lesioning of individual neural structures is very informative in terms 125

of determining whether or not the structure alone is involved; however, individually, they do not give a complete picture of the CTA neural circuitry. Thus far, it is known that the AP, PBN, Amg, and the IC are all involved in the acquisition process of LiCl CTAs, but how are these areas interconnected? Figure 2 depicts the possible neural circuitry that underlies conditioned taste aversion learning when LiCl is used as the US. It is well established that the AP and the PBN, primarily the lateral subdivision, have reciprocal connections (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985; Miceli et al., 1987), establishing the initial circuitry of CTA learning. It should be mentioned that lesions of the lateral PBN disrupt CTAs produced by AP-dependent (i.e. methyl-scopolamine) and AP-independent (i.e. ethanol) USs (Cubero et al., 2001) suggesting that CTA-producing agents may act directly on the lateral PBN or indirectly via a different structure. Studies have demonstrated the importance of the connections between the PBN, IC, and the Amg in CTAs by performing combined lesions of these areas. It appears to be the case that the intact ipsilateral connections between the PBN and the IC, between the PBN and Amg, and between the Amg and the IC are important in the acquisition of CTAs. The first study in a series conducted by Gallo and Bures (1991) shows that neither unilateral de-cortication of the IC made before the CS presentation nor unilateral TTX lesions of the entire PBN made during the inter-stimulus interval overlapping the US disrupt a CTA, an effect that has been substantiated by other studies (Buresova & Bures, 1973). More interestingly, combined ipsilateral lesions of 126

the PBN and the IC do not block the formation of a CTA, while contralateral lesions of these structures inhibit the learning. Together, these findings suggest that ipsilateral connections between the IC and the PBN are critical in the acquisition of a CTA. A series of studies conducted by Bielavska and Roldan (1996) shows the significance of the connections between the PBN and the Amg. The initial part of the study demonstrates that separate unilateral TTX lesions of the individual structures do not affect CTA acquisition. The following study demonstrates that ipsilateral concomitant destruction of both structures during the CS-US interval do not impair CTA development, while contralateral lesions of the two structures abolish CTAs. This implicates the importance of ipsilateral connections between the PBN and Amg in CTA acquisition. If contralateral connections between the PBN and Amg were critical, the animals with lesions of the PBN and the Amg on the same side of the brain should not have acquired a CTA since this pattern of lesion would have effectively disrupted the contralateral communication between the two structures. Identical results are obtained when the connections between the Amg and IC are examined. Namely, the results show that neither separate unilateral TTX lesions of the IC prior to CS presentation nor unilateral TTX lesions of the Amg made between the CS-US interval have an effect on the acquisition of a LiCl CTA. Similarly, unilateral lesions of the Amg and the IC made on the same side of the brain do not disrupt a CTA; however, when the lesions of each of the structures are made on opposite hemispheres, CTA formation is precluded, once again suggesting that

127

ipsilateral connections between the Amg and the IC are necessary for the formation of the gustatory aversion. One more piece of evidence that stresses the importance of the connections between the basolateral Amg and the IC in CTA acquisition is provided by a study that involves long-term potentiation (LTP). Long-term potentiation is an increase in the excitability of a neuron to a certain input due to repeated exposure to that particular input (Bliss & Lømo, 1973). Long-term potentiation has been proposed as a model for synaptic plasticity underlying learning and memory (Bliss & Collingridge, 1993; Escobar & Bermudez-Rattoni, 2000). Tetanic stimulation of the basolateral Amg induces LTP in the IC of rats in vivo (Escobar, Chao & Bermudez-Rattoni, 1998). More interestingly, inducing LTP in the IC in this manner prior to CTA training enhances retention of the learned task (Escobar & Bermudez-Rattoni, 2000). Studies investigating the connections between the lateral PBN and the basolateral Amg have assessed their importance in CTA acquisition. Tracing studies have established at least 4 pathways through which the lateral PBN projects to the Amg. The first is a direct connection to the central Amg (Norgren, 1984; Sakai & Yamamoto, 1999). The second involves a cortical route via the IC (Saper & Loewy, 1980; Sakai & Yamamoto, 1999) to the basolateral Amg (Saper & Loewy, 1980; Saper, 1982; Yamamoto, Azuma & Kawamura, 1984; Turner & Herkenham, 1991). The third route involves projections from the lateral PBN to the zona incerta (ZI; Krukoff et al., 1993; Bianchi, Corsetti, rodella, Tredici & Gioia, 1998; Sakai & Yamamoto), which projects to the midline and intralaminar thalamic nuclei (MITC; 128

Sakai & Yamamoto, 1999), which in turn projects to the basolateral Amg (Groenewegen & Berendse, 1994). Although not replicated in the research of Yamamoto, one study showed that the dorsal PBN cells accumulate Evans Blue following the infusion of the retrogradely transported fluorescent label into the basolateral Amg (Woolf & Butcher, 1982). This suggests that there potentially is a direct connection from the lateral PBN to the basolateral Amg. In order to assess the importance of these connections from the lateral PBN to the Amg in CTA acquisition, single and combined lesions of these areas have been conducted. Sakai &Yamamoto (1999) showed that bilateral ibotenic acid lesions of the ZI, IC, central Amg, and MITC alone did not abolish LiCl CTAs, while individual lesions of the lateral PBN and the basolateral Amg completely blocked the learning. Additionally, combined lesions of the ZI with either the central Amg or the IC blocked the CTA, while lesions of both the IC and central Amg did not. On the basis of these results, it was concluded that the route from the lateral PBN through the ZI and MITC to the basolateral Amg is of significant importance for CTA acquisition followed by the cortical route. Figure 2 depicts the possible neural circuitry that underlies conditioned taste aversion learning when LiCl is used as the US.

129

Figure 2. Neural Circuitry of Conditioned Aversions.

Forebrain

Insular Cortex C

Midbrain

Central Amg

Zona Incerta

Hindbrain

Medial PBN

Basolateral Amg

Midline & Intralaminar Thalamic Nuclei

Lateral PBN

Area Postrema

Figure 2. Depicts the possible neural circuitry that underlies conditioned taste aversion learning when LiCl is used as the US. The figure illustrates ipsilateral connections among the structures, which are thought to be necessary in the learned gustatory aversion. Not all connections are depicted. Double arrows indicate reciprocal connectivity ( ).

3.4 Neurotransmitters and Conditioned Taste Aversion Learning Methodological Issues Methodologically, studying the neurochemical basis of CTAs is extremely difficult. Two issues have hampered progress in this area of study and must be taken into account when designing CTA neurochemical studies: (1) US pre-exposure effect and (2) state-dependent learning.

130

Generally, the way in which the role of a neurochemical has been examined in CTAs has involved blocking the receptors of interest prior to the CS-US pairing by peripherally administering the appropriate antagonist. For instance, it has been hypothesized that histamine mediates estradiol-induced CTAs via its H1 receptors (Rice, Lopez & Garcia, 1987; Rice, 1989; Hintiryan et al., 2005). As such, an H1 antagonist would be peripherally administered prior to the CS-estradiol pairing to determine whether blocking the histamine receptors affect the subsequent formation of a CTA; however, pre-exposure effects may confound the interpretation of these results. In CTA experimental designs, exposing animals to an agent (exposure agent) capable of producing a CTA before either the same (intra) or a different (inter) agent (conditioning agent) is paired with a taste solution on acquisition day attenuates or blocks the subsequent formation of the CTA. The experience of pretreatment is referred to as the US pre-exposure effect. This effect can be found when pretreatment is given less than 24 hours before conditioning (proximal) or greater than 24 hours before (distal). It is generally believed that only agents that produce CTAs themselves can serve as pre-exposing agents. Preliminary studies suggest that for agents capable of inducing CTAs, doses that are too low to promote acquisition can still produce a pre-exposure effect (Merwin & Doty, 1994). Pre-exposure effects have been illustrated with a wide range of agents. Proximal pre-exposure to LiCl 30 minutes before the pairing of saccharin with a second injection of LiCl significantly disrupts the formation of a LiCl CTA (Domjan & Best, 1977). Distally pre-exposing animals to LiCl blocks a LiCl CTA when identical doses of LiCl are used (Domjan & Best, 1977). 131

Correspondingly, distal pre-exposure to radiation blocks a radiation-induced CTA (Rabin, Hunt & Lee 1989), distal pre-exposure to ethanol blocks ethanol-induced CTA (Berman & Cannon, 1974), and distal pre-exposure to estradiol blocks CTAs produced by estradiol (Merwin & Doty, 1994). Inter-agent pre-exposure studies have shown that previous experience with LiCl is also effective in disrupting CTAs produced by radiation (Rabin et al., 1989), ethanol (Cannon, Baker & Berman, 1977; Rabin et al., 1989), and estradiol (De Beun, Peeters & Broekkamp, 1993), and that pre-exposure to estradiol accelerates the extinction of LiCl CTA (Chambers & Hayes, 2002; Yuan & Chambers, 1999). One way to show whether an antagonist is acting as a pre-exposing agent or an antagonist is to have two groups that receive the antagonist prior to the CS-US pairing: one group is given the CS-US pairing while the antagonist is actively blocking the receptors and a second group receives the pairing while the antagonist is no longer active. If the CTA is blocked in both cases, then it would suggest that the antagonist is acting as a pre-exposure agent rather than a true antagonist. A second way to possibly avoid pre-exposure effects is to administer the antagonists centrally rather than peripherally; however, studies on pre-exposure and state-dependent effects (discussed in the following paragraph) of neurally infused drugs are non-existent. As such, preexposure effects should be kept in mind when examining the effects of centrally infused drugs and CTAs. The histamine and estradiol study mentioned above is complicated by one other factor: state-dependent learning. The state dependent learning hypothesis states that 132

the failure to provide identical states during the training and testing days may interfere with the retrieval of a previously learned avoidance response on conditioning day (Overton, 1974). For instance, in the study where an H1 antagonist is peripherally administered prior to the CS, the animals are conditioned to the novel tastants in a drug state (the H1 antagonist), but are tested for saccharin preference in a non-drug state. Therefore, the failure to acquire a CTA may be due to a state-dependent learning process. One way to avoid state-dependent learning is to administer the antagonist AFTER the CS presentation. This way, the animals will be trained and tested under identical, non-drug states. Although this may eliminate state-dependency and preexposure effects, it does not eliminate possible conditioning effects if the antagonist has conditioning abilities. When two separate 1.5meq/kg doses of LiCl are given after the CS, separated in time by 90-120 min, the subsequent CTA is stronger than when only one 3.0meq/kg dose injection of LiCl is given. Likewise, it has been shown that administering an H1 antagonist after the CS, but before the US estradiol augments the CTA (Hintiryan et al., 2005). As such, in an attempt to circumvent state-dependency and pre-exposure effects the neurotransmitter antagonist may be delivered after the CS; however, if the agent has conditioning capabilities, then the data will be confounded with an augmented CTA. Due to such methodological issues, studies investigating the involvement of neurotransmitters in CTAs have been sparse. Of those studies performed, most of the attention has been focused on the muscarinic cholinergic receptors, while histamine has been the focus of fewer studies. 133

3.4.1 Acetylcholine (ACh) Synthesis of Acetylcholine Acetylcholine is synthesized by the combination of choline and acetate, an ion which is provided by the cofactor acetyl-CoA with the aid of the enzyme choline acetyltransferase (ChAT). Post-synaptic potentials induced by ACh are short-lived and are enzymatically deactivated by acetylcholinesterase (AChE). Research examining the effects of ACh on various behaviors has used these enzymes to manipulate levels of the neurotransmitter. Acetylcholine is synthesized in many regions of the brain including those involved in CTA learning such as the AP (Tago et al., 1989), PBN (Tago et al., 1989; Tafti et al., 1997), Amg (Ichikawa et al., 1997) and possibly the IC (Lopez-Garcia, Bermudez-Rattoni & Tapia, 1990; Berman, Hazvi, Neduva & Dudai, 2000). Cholinergic Receptors and Receptor Sites Cholinergic neurons project extensively throughout the brain. Due to the limited scope of this paper, the discussion regarding these cholinergic projections will be restricted to the previously mentioned brain areas that are involved in CTA learning, namely the AP, PBN, Amg, and IC. Two types of ACh receptors exist: nicotinic receptors, which are ionotropic in nature and muscarinic, which are metabotropic. Most research on ACh and CTAs has focused on the role of muscarinic receptors. There is scarce information regarding the role of nicotinic receptors in memory formation as it relates to CTAs. As such, the discussion in this section will focus only on the metabotropic receptors. The 134

muscarinic ACh receptors are classified as M1-M5 according to their biochemical properties (Bonner, Buckley, Young & Brann, 1987; Bonner, Young, Brann & Buckley, 1988; Buckley, Bonner & Brann, 1988; Buckley, Bonner, Buckley & Brann, 1989). Scopolamine, a commonly used antagonist in CTAs, which also will be used in the experiments proposed in this proposal, is a non-selective muscarinic receptor antagonist that blocks all subtypes. A high density of muscarinic receptors, identified by radiolabeled 3H-QNB (quinuclidinyl benzilate) binding, can be found in the AP (Pedigo & Brizzee, 1985) and the dorsal nucleus of the lateral PBN (Wamsley, Lewis, Young & Kuhar, 1981). It is not surprising to find these receptors in the AP given that it is considered to be a trigger zone for emetic responses (Borison & Brizzee, 1951) and that the muscarinic receptor antagonist scopolamine is agreed to be the most effective treatment against motion sickness-induced emesis (Brand & Perry, 1966; Wood, 1979; Waldrop, 1982). With respect to the PBN, the M1, M2, and M3 receptors have been found in both the medial (Mallios, Lydic & Baghdoyan, 1995) and the entire lateral PBN (Christie & North, 1988; Mallios et al., 1995). A high density of M1 receptors can be found in the basolateral Amg and the IC (Spencer, Horvarth & Traver, 1986; Wall, Yasuda, Hory, Flagg, Martin et al., 1991; Flynn & Mash, 1993) as well. The IC also contains a large number of M2 receptors (Spencer et al., 1986; Wall et al., 1991; Flynn & Mash, 1993). Given that the M3 receptor distribution frequently coincides with that of M1 sites, this subtype also is probably found in the basolateral Amg and the IC (Flynn & Mash, 1993). 135

Acetylcholine and Learning In general, cholinergic mechanisms are implicated in learning, memory, and attention (Decker & McGaugh, 1991; Fibiger, 1991; Hasselmo & Bower, 1993; Muir, Page, Sirinathsinghji, Robbins & Everitt, 1993). Specifically, there is evidence to suggest the involvement of ACh in habituation (Pearson, 1973), and acquisition of classical eye-blink conditioning (Downs, Cardozo, Schneiderman, Yehle, Vandercar et al., 1972), passive avoidance (Buresova, Bures, Bohdanecky & Weiss, 1964), active avoidance (Meyers, Roberts, Riciputi & Domino, 1964), and maze learning (Whitehouse, 1967). It is not surprising then that cholinergic mechanisms are also reported to be involved in CTAs (Deutch, 1978; Bermudez-Rattoni, Coburn, Fernandez, Chavez & Garcia, 1987; Lopez-Garcia et al., 1990). Acetylcholine and Conditioned Taste Aversions One piece of evidence that would suggest that ACh is a mediator of CTAs, is if the neurotransmitter administered alone produces a CTA. Acetylcholine has been shown to be capable of producing the learned gustatory response. Intraperitoneal injections of cholinergic agonists that cross the blood-brain barrier are capable of producing CTAs (Preston & Schuster, 1981; Romano & King, 1987). Intracerebral infusion of these agonists into the medial septum and lateral ventricles also result in CTAs, whereas infusions into the ventral hippocampus do not (Myers & de Castro, 1977). This site-specific effect suggests that ACh is not producing some general effect that is acting as the US.

136

Several studies using ACh receptor antagonists claim to provide support for the involvement of cholinergic mechanisms in CTAs. The muscarinic receptor antagonist atropine delivered peripherally before the intraoral infusions of saccharin solution, blocks the CTA normally produced by a high dose of LiCl (Deutsch, 1978). The CTA however, is not blocked when atropine is given after the CS and during exposure to LiCl. Although anti-cholinergic activity appears to interfere with fear (Plotnik, Molenauer & Snyder, 1974) and nociception (Pert, 1975), this blocking effect of atropine is not due to an antidote effect. That is, atropine sulfate does not attenuate the effects of LiCl since atropine sulfate co-administered with LiCl produces an aversion that is equal in strength to a CTA produced by LiCl alone (Deutch, 1978). Although the authors claim that these studies show the involvement of ACh in LiCl CTAs, preexposure and state-dependency may be confounding the findings. In fact, atropine sulfate administered peripherally has been shown to produce a CTA (Preston & Schuster, 1981; Romano & King, 1987), which suggests that the agent may serve as a pre-exposure agent. Interestingly, ACh antagonists also interfere with radiation CTAs when backwards conditioning is employed, that is, when the US is administered prior to the CS. Atropine sulfate, but not atropine methylnitrate, given subcutaneously 30 minutes prior to radiation, blocks the formation of a radiation CTA (Gould & Yatvin, 1972; Gould & Yatvin, 1973). Since atropine sulfate crosses the blood-brain barrier, while atropine methylnitrite does not, this suggests central rather than peripheral effects of the antagonist. A different study has shown that atropine administered after the 137

radiation also blocks the CTA (Gould & Yatvin, 1973). This suggests that the abolition of the CTA is not due to pre-exposure effects. Equally important, the researchers showed that the half-life of peripherally injected atropine is 3 days. This would mean that the animals were both trained and tested for a CTA under the influence of the drug, thus also eliminating the state-dependency hypothesis as a possible explanation for the ability of atropine to block CTA. Central effects of atropine on CTAs have also been investigated. Atropine sulfate delivered into the lateral ventricles prior to the CS presentation blocks a LiCl CTA (Deutch, 1978), an effect that seems to be centrally, rather than peripherally mediated, since intraperitoneal injections of atropine methylnitrite does not interfere with the LiCl CTA (Deutch, 1978). The same study showed that the intraperitoneal injection of a LiCl and atropine sulfate mixture produced a CTA comparable in strength to LiCl injection alone (Deutch, 1978). This implies that in this study, atropine did not produce a CTA and thus the effects may not be due to pre-exposure effects; however a direct test of this possibility was not conducted using a method similar to distal pre-exposure. If distal pre-exposure to atropine blocked a LiCl CTA, then this would suggest that pre-exposure effects may have played a role since the blockade of the CTA would have occurred even after the ACh receptors were no longer actively blocked. Table 1 summarizes the effects of peripherally and centrally administered atropine on CTAs.

138

Table 1. Effects of Peripherally and Centrally Administered Cholinergic Antagonists on Conditioned Taste Aversions. ACh Antagonist Atropine

Scopolamine

Infusion Site

Methodology

Outcome

CS ---> ACh antagonist ---> LiCl

Peripheral

ACh antangonist ---> CS--> LiCl

Peripheral

ACh antagonist ---> Radiation ---> CS

No CTA Gould & Yatvin, 1973

Peripheral Radiation ---> ACh antagonist ---> CS Lateral Ventricles ACh antagonist ---> CS ---> LiCl

No CTA Gould & Yatvin, 1973 No CTA Deutsch, 1978

Peripheral

ACh antagonist ---> CS ---> LiCl

Peripheral

CS --> ACh antagonist ---> LiCl

Amygdala

ACh antagonist ---> CS ---> LiCl

Amygdala

CS ---> ACh antagonist ---> LiCl

Insular Cortex Insular Cortex

ACh antagonist ---> CS ---> LiCl CS ---> ACh antagonist ---> LiCl

CTA

Reference

Peripheral

Deutsch, 1978

No CTA Deutsch, 1978

No CTA Evenden et al., 1992 CTA

Kral, 1971

No CTA Castellanos et al., 2000 CTA

Castellanos et al., 2000

No CTA Ferreiro et al., 2002 CTA Ferreiro et al., 2002

Similar results have been obtained using the muscarinic receptor antagonist scopolamine. Intraperitoneal injection of scopolamine (0.1 and 0.3mg/kg) given 30 minutes prior to (Evenden, Lavis & Iversen, 1992), but not after (Kral, 1971) exposure to sucrose solution, blocks a LiCl CTA. Microinjection of the muscarinic ACh antagonist scopolamine into the gustatory IC before, but not during LiCl exposure impairs the CTA (Castellanos, Salas, Gonzalez, Roldan & Garcia, 2000; Ferreira, Gutierrez, de la Cruz & Bermudez-Rattoni, 2002) suggesting the importance of ACh in the acquisition of taste memory and further implicating the role of cortical ACh in gustatory information processing. Similarly, scopolamine administered into the Amg 10 minutes before the CS presentation also blocks a LiCl CTA indicating the role of the cholinergic system in the limbic structure as well (Castellanos et al., 2000). On the contrary, infusing the antagonist during the inter-stimulus interval so that it is present during LiCl exposure does not affect the learned behavior. Microinjection of scopolamine into the IC 20 minutes prior to CS presentation on extinction day 1 only, 139

does not affect the CTA, suggesting that the muscarinic antagonist does not affect the sensation of taste, the hedonic valence of the taste or other faculties required to perform the task (Naor and Dudai, 1996). As discussed previously, this blockade potentially could be due to either state dependency or due to drug pre-exposure effects. Table 1 summarizes the effects of peripherally and centrally administered scopolamine on CTAs. Despite possible pre-exposure effects and state-dependent learning that may account for the findings in the previous studies, there is sufficient evidence to suggest the involvement of cholinergic mechanisms in the processing of gustatory stimuli. Microdialysis studies show that there is a release of ACh in the IC in response to a novel saccharin or quinine taste stimulus in comparison to rats that drink familiar water (Shimura, Zuzuki & Yamamoto, 1995; Miranda, Ramirez-Lugo & Bermudez-Rattoni, 2000). Further, this increment is attenuated as the novel stimuli become familiar (Miranda et al., 2000) indicating an inverse relationship between the levels of ACh and the familiarity of taste. This suggests that ACh in the IC is involved in assessing the novelty of a particular taste. Corroboration for this hypothesis is provided by a study that shows the involvement of ACh in neophobic responses. Blocking muscarinic ACh receptors with scopolamine in the IC before the CS presentation prevents a novel solution from becoming familiar (Gutierrez, Tellez & Bermudez-Rattoni, 2003). That is, the animals continue to show neophobia toward the sweet solution when cholinergic receptors are blocked. This effect is not due to state-dependency since scopolamine administered both before training and test days revealed the same finding. 140

Additionally, scopolamine administered after the CS presentation also prevents the solution from becoming familiar. Finally, this effect also is not due to the aversive qualities of scopolamine, since infusions of the cholinergic antagonist into the IC do not produce a CTA. Interestingly, ACh may modulate the induction of LTP that is observed in the IC following the tetanic stimulation of the basolateral Amg (Escobar et al., 1998). Microinfusions of atropine (Jones, French, Bliss & Rosenblum, 1999) or scopolamine (Ramirez-Lugo, Miranda, Escobar, Espinosa & Bermudez-Rattoni, 2003) into the IC reduces the induction of LTP in the basolateral-IC projection and simultaneously blocks the increased retention of LiCl CTA that is normally observed with the induction of LTP. Although studies have shown that infusions of muscarinic antagonists into the IC and Amg following the CS do not affect a LiCl CTA, no studies have examined the effect of these antagonists in the PBN. The evidence presented thus far implicates cholinergic mechanisms in gustatory information processing; however, the neurotransmitter may be involved in the processing of malaise information as well. Stimulation of the vagus nerve normally produces excitatory potentials in the “visceral” region of the IC (Barnabi & Cechetto, 2001). Administration of atropine, a muscarinic receptor antagonist, attenuates these vagally evoked cortical responses, suggesting a cholinergic mechanism in the transmission of visceral information. On the other hand, GABAA and α-adrenergic receptor antagonists have no effect on cortical excitatory outputs induced by vagal stimulations. 141

Further, ACh has been implicated in emetic responses. The muscarinic antagonist scopolamine is one of the most effective treatments for motion or space sickness (Wood, 1979; Waldrop, 1982). Ex juvantibus reasoning suggests the involvement of ACh in human emetic responses. As mentioned previously, one of the brainstem areas that mediates emetic responses is the AP (Borison & Brizzee, 1951), which has connections with both the medial and lateral PBN (Leslie & Gwyn, 1984; Shapiro & Miselis, 1985). It would not be surprising then to find that cholinergic mechanisms are involved in the processing of US stimuli in the brainstem, particularly in the PBN. 3.4.2 Histamine (HA) Synthesis of Histamine Histamine is synthesized by the combination of the amino acid histidine through pyridoxal phosphate and histidine decarboxylase. It is broken down into its major inactive catabolite 3-(tele)-methylhistamine through the combined actions of hitamine-N-methyltransferase, aldehyde dehydrogenase, and S-adenosyl-methionine (SAM). Histamine may also be catabolized into imidazole acetic acid by diamine oxidase. The 3-(tele)-methylhisamine can be further metabolized to N-methylimidazoleacetic acid by monoamine oxidase as well. Several investigators have implemented immunohistological methods in attempts to localize and map the distribution of HA in the CNS using neural tissue of rats and guinea pigs (Garbarg, Barbin, Feger & Schwartz, 1974; Steinbusch & Mulder, 1985; Wouterlood & Steinbusch, 1991). The data are summarized only briefly here. 142

Histamine cell bodies are generally confined to a region in the hypothalamus, specifically in the tuberomammillary nucleus. Additionally, scattered HA cell bodies are found throughout the posterior hypothalamus that extend to the middle portion of the third ventricle. Some histaminergic cell bodies also are localized in the medial forebrain bundle. A similar organization is described in humans, with the exception that the neurons are greater in number (Airaksinen, Paetau, Paliarui, Reinikainen, Riekkinen, et al., 1991). Histaminergic Receptors and Receptor Sites Four types of HA receptors have been identified up to date: H1-4, the H4 being the most recent. All four are G-protein coupled receptor subtypes (Gantz, Munzert, Tashiro, Schäffer, Wang, et al., 1991; Lovenberg, Rowland, Wilson, Jiang, Pyati, et al., 1999; Hough, 2001). Localization of the H4 receptor in the central nervous system remains to be established; however, studies show that both the medial and lateral PBN contain H1 (Palacios et al., 1981; Lintunen, Sallmen, Karlstedt, Fukui, Eriksson, et al., 1998 et al., 1998) and H2 (Ruat, Traiffort, Buthenet, Schwartz, Hirschfeld, et al., 1990) receptor subtypes. The H3 receptor is an autoreceptor that inhibits the release of HA (Arrang, Garbarg & Schwartz, 1983, 1987). H1 (Palacios et al., 1981; Lintunen et al., 1998; Lozeva, Tuomisto, Sola, Plumed, Hippeläinen, et al., 2001), H2 (Martinez-Mir, Pollard, Moreau, Traiffort, Ruat, et al., 1993) and H3 (Pillot, Heron, Cochois, TardivelLacombe, Ligneau, et al., 2002) are also found in the AP, Amg, and the IC.

143

Histamine and Learning The role of HA in learning has been contradictory. Initial studies have shown that depletion of neuronal HA retards the learning in an active avoidance response (Kamei, Okumura & Tasaka, 1993), and this deficit is reversed upon infusions of HA into the lateral ventricles. Histamine 1 receptor blockers have been shown to cause decrements in the same learning paradigm, suggesting that HA facilitates learning via H1 receptors (Kamei & Tasaka, 1991). Similar facilatory effects are observed with H3 antagonists, which increase the production of neuronal HA. This elevated performance is observed with different learning paradigms including passive avoidance (Meguro, Yanai, Sakai, Sakurai, Maeyama, et al., 1995), object recognition (Giovannini, Bartolini, Bacciottini, Greco & Blandina, 1999), elevated plus maze (Onodera et al., 1998), and object recognition (Giovannini et al., 1999). In contrast, other researchers have shown that intraperitoneal injections of the HA precursor (L-Histidine) impairs acquisition in the active avoidance paradigm, while an inhibitor of HA synthesis has the opposite effect (Rubio, Begega, Santin, Miranda, & Arias, 2001). Similar results have been obtained when the neurotransmitter is infused into the basolateral Amg and ventral hippocampus (Alvarez & Ruarte, 2002). A study using the elevated plus-maze illustrates that an infusion of HA into the ventral hippocampus inhibits learning, which is blocked by an H1 antagonist (Alvarez, Ruarte & Banzan, 2001). Interestingly, it is speculated that HA may facilitate learning through its interactions with acetylcholine. Histamine 3 agonists, which decrease neuronal histamine cause the inhibition of cortical ACh release (Clapham & Kilpatrick 1992; 144

Arrang, Drutel & Schwartz, 1995), implicating a relationship between the two agents. This hypothesis is supported by the finding that administration of HA antagonizes the learning deficits caused by the ACh antagonist scopolamine (Miyazaki, Imaizumi & Onodera, 1995). Histamine and Conditioned Taste Aversions Much less attention has been focused on the involvement of the neurotransmitter HA in CTAs. Studies investigating the cellular basis of CTAs have targeted peritoneal mast cell degranulation as a potential mediator of the gustatory learning (Persinger & Fiss, 1978). Mast cells, which contain biological agents such as serotonin and dopamine, also are known to be large depositories for HA (Persinger, 1977). Evidence supporting the mast cell hypothesis is provided by three different findings. First, ionizing radiation, which has been used in many CTA studies as an unconditioned stimulus (Garcia et al., 1955; Garcia, Ervin & Koelling, 1967; Levy, Carrol, Smith & Hofer, 1974; Rabin, Hunt & Lee, 1982) activates degranulation of peritoneal mast cells (Seyle, 1965). Second, this radiation-induced degranualtion is abolished by the intraperitoneal administration of pyrilamine, an H1 antagonist (Smith, 1958). Third, administration of another H1 antagonist, chlorpheniramine maleate, before pairing a saccharin solution with gamma radiation, prevents the formation of a CTA normally produced by that dose of radiation (Levy, Carrol, Smith & Hofer, 1974). Additional evidence for the involvement of HA in mediating CTAs comes from the fact that the neurotransmitter itself is capable of inducing the learned response. Both intraperitoneal injections (Rabin et al., 1983a) and intraventricular infusions into 145

the IV ventricle (Rabin et al., 1982) of HA produce CTAs. This is not surprising considering that like ACh, HA, specifically through its H1 receptor subtype, is also implicated in emetic responses. Histamine 1 receptor antagonists that cross the bloodbrain-barrier are effective in preventing motion sickness in humans (Brand & Perry, 1966; Wood & Graybiel, 1970; Graybiel, Wood, Knepton, Hoche & Perkins, 1975), while those that do not, remain ineffective (Kohl, Homick, Cintron & Calkins, 1987), suggesting a central role of the histaminergic system in malaise. Administration of chlorpheniramine maleate (CM), before pairing a saccharin solution with gamma radiation, prevents the formation of a CTA normally produced by that dose of radiation (Levy et al., 1974); however, this effect is probably due to statedependent learning since the CTA is no longer abolished when the anti-HA is given after the sucrose presentation on acquisition day (Levy, Carrol & Smith, 1975). The same result is found in a separate study that also used radiation as the US. Administration of CM before the CS on acquisition day completely blocked the radiation CTA; however, when CM was given before the CS on both acquisition and extinction day 1, the CTA was no longer abolished (Rabin et al., 1982). 3.5 Estradiol and Conditioned Taste Aversion Learning There are a considerable number of chemical agents that are capable of inducing CTAs. One agent that has been studied extensively as an unconditioned stimulus in CTA experiments is the hormone estradiol. The ability of estradiol to produce a conditioned decrement in food intake has been demonstrated across different species, routes of administration, and conjugated forms for both males and females 146

(Mordes, Longscope, Flatt, MacLean & Rossini, 1984; Bernstein et al., 1986; Rice et al., 1987; Miele, Rosellini & Svare, 1988; Ganesan & Simpkins, 1990; Ganesan & Simpkins, 1991; De Beun, Jansen, Smeets, Niesing, Slangen et al., 1991; Peeters et al., 1992; De Beun et al., 1993; Merwin & Doty, 1994; Ossenkopp, Rabi & Eckel, 1996; Yuan & Chambers, 1999). The successful treatments have included subcutaneous injection of estradiol, estradiol benzoate, and estradiol cypionate, subcutaneous implantation of a melted estradiol pellet, an estradiol-filled Silastic capsule, and an osmotic mini-pump filled with estradiol benzoate, oral administration of 17α-ethinyl estradiol, and intravascular administration of a brain-enhanced estradiol chemical delivery system. The species have included Sprague-Dawley, Wistar, and Long-Evans rats, Crl: CD-1(ICR)BR and Rockland-Swiss mice, and humans. Although it unequivocally has been found that high supraphysiological doses of estradiol produce strong CTAs, low physiological doses appear to be ineffective in both females and males. In one study, a supraphysiological dose of 17β-estradiol produced strong CTAs to a saccharin solution, while physiological doses, such as 0.4 and 2μg/kg, did not (De Beun et al., 1991). Conditioned Taste Aversions Revisited There is an assumption that those agents capable of inducing CTAs produce some type of illness and thus all reductions in consumption that occur after pairing an agent with a novel taste are based on aversive conditioning; however, this assumption has been challenged. It has been suggested that conditioned reductions in intake can result from states that differ from illness (Booth, 1977). For instance, pairing a food 147

with a satiety-producing agent will elicit a learned reduction in the consumption of that particular food (Booth, 1985). More recently, it has been suggested that CTAs induced by some agents are qualitatively different than those produced by the classic illness agent LiCl (Hunt & Amit, 1987; Parker, 1988; Zalaquett & Parker, 1989). For instance, animals conditioned with LiCl avoid sucrose on subsequent exposures. These animals also increase their aversive orofacial responses to the CS when the solution is forcibly infused into their oral cavities via a cheek fistula. On the other hand, although morphine (Parker, 1988) and amphetamine (Zalaquett & Parker, 1989) produce reductions in the consumption of the taste CS, the animals do not show an increase in aversive orofacial responses when exposed to the CS. Because of these concerns and the fact that estradiol is a long-term satiety hormone, our lab has decided to use the terminology conditioned taste avoidance for estradiol CTAs since it is behaviorally descriptive and is not laden with inferences as to its illness producing properties. The learned gustatory aversions produced by LiCl will be referred to as aversions. Neural Substrates of Estradiol Conditioned Taste Avoidance Neural mechanisms of CTAs have primarily focused on CTAs produced by LiCl. Limited attention has been paid to CTAs produced by estradiol. One study demonstrated that estradiol-induced CTAs may be mediated by the AP. Male rats placed under chronic estradiol treatment decrease their food consumption. Additionally, when offered a novel diet while under estradiol treatment, animals show a preference for the novel diet, while the control animals prefer their familiar diet, suggesting that the estradiol treated animals develop a CTA to the diet they were 148

exposed to under estradiol treatment. Following thermal lesions of the AP, these animals no longer express estradiol-induced hypophagia or CTA (Bernstein et al., 1986). A preliminary study in our lab shows that injections of 10- and 50μg of estradiol induces the expression of c-FLI in the crescent, external, and dorsal nuclei of the lateral PBN, and the external medial PBN 2- and 24 hours following injection (Chambers, unpublished data). Expression is found in the central lateral PBN for 50μg/kg when measured 2 and 24 hours after injection, but for 10μg/kg it is found only when measured 2 hours following injection. We speculate that the 2-hour measurement is associated with acquisition of a CTA, while the 24-hour measurement is probably associated with the unconditioned effects of estradiol since decrements in eating are observed 24 hours following subcutaneous injection. Neurochemical Mediation of Estradiol Conditioned Taste Avoidance Studies examining the neurochemical basis of estradiol CTAs are extremely limited. A preliminary study conducted in our laboratory shows that pre-treating animals with peripherally administered scopolamine methyl nitrate, which does not cross the blood-brain barrier, 20 minutes prior to implantation of a 30mm estradiol capsule, attenuates a CTA normally produced by estradiol (Ikramullah & Chambers, unpublished data). This attenuation could in fact be due to the blockade of muscarinic ACh receptors and not due to pre-exposure effects or state-dependent learning since the antagonist was administered after the CS. Further, the study was not contaminated by conditioning effects of the antagonist. If scopolamine methyl nitrate had conditioning 149

properties, the CTA would have been augmented, but instead, it was attenuated. Together, these suggest that ACh may be mediating the conditioning effects of estradiol. An association between estradiol and HA also has been demonstrated. Application of 17-β estradiol increases the secretion of HA that has been triggered by compound 48/80, a mast cell degranulator (Vliagoftis, Dimitriadou, Boucher, Rozniecki, Correia et al., 1992). This elevated release of HA is blocked by the application of the estrogen receptor antagonist, tamoxifen (Vliagoftis et al., 1992). Predicated on these findings, it is purported that CTAs produced by estradiol are mediated by HA. An experiment was conducted in our lab to directly test this hypothesis. Peripheral administration of the H1 receptor antagonist chlorpheniramine maleate (CM) 24 hours or 20 minutes before pairing sucrose with estradiol blocked the acquisition of a subsequent CTA (Hintiryan et al., 2005). Three explanations were suggested to account for this result: (1) the anti-histaminergic properties of CM antagonize the action of HA, which mediates CTAs elicited by estradiol (the histaminergic hypothesis), (2) CM given before acquisition but not before each extinction test creates a state-dependency that precludes retrieval of the CTA during post-acquisition testing if CM is not present (the state-dependency hypothesis), or (3) CM given before acquisition acts as a pre-exposure agent and consequently blocks the CTA (Hintiryan et al., 2005). Two additional studies were conducted in order to clarify which of these hypotheses best explained the findings. To examine the possibility of state-dependent 150

learning, CM was administered before the CS on both acquisition and extinction days. The estradiol CTA was blocked, providing evidence against the state-dependent hypothesis. Since CM has a depressive effect on drinking, its administration prior to the CS in each of these experiments resulted in decreased consumption of the CS on acquisition day. In order to circumvent this problem and to provide additional evidence against the state-dependent hypothesis, CM was injected after sucrose consumption on acquisition day. As such, the animals were not under a drug state during their exposure to sucrose and the drug did not compromise their drinking. Further, if HA mediated estradiol CTAs, CM should block the CTA whether it was given before or after the CS presentation on acquisition day. Administering CM after sucrose consumption actually augmented the CTA rather than blocking or attenuating it. This provided evidence against the histaminergic hypothesis as well as the statedependent hypothesis. Taken together, the results suggest that some other explanation than a histaminergic or state-dependency hypothesis must account for the blocking of the estradiol CTA observed when CM is administered before sucrose consumption during acquisition. A summary of the HA and estradiol studies is provided in Table 2. Table 2. Histamine and Estradiol Conditioned Taste Avoidance. Acquisition Extinction Experiment Distal Pre-exposure Proximal Pre-exposure Taste Stimulus Post-exposure Pre-exposure Taste Stimulus Outcome Sucrose Estradiol Sucrose 1 CM No CTA CM Sucrose Estradiol Sucrose No CTA Sucrose Estradiol CM 2 CM Sucrose No CTA Sucrose CM ---> Estradiol 3 Sucrose Stronger CTA

151

Closer examination of the literature suggests that the effects of CM follow a pattern that supports its role as a pre-exposure and conditioning agent. The pattern of results for the first two experiments is similar to the pattern found with proximal preexposure to LiCl in CTAs induced by LiCl (Domjan & Best, 1977) and in CTAs induced by estradiol (De Beun et al., 1993; Chambers & Hintiryan, unpublished data). When CM was given 20 minutes before sucrose consumption on acquisition day, it blocked the estradiol-induced CTA. In the third experiment, CM was injected immediately after sucrose consumption and estradiol was implanted 20 minutes later. This post-exposure to CM augmented the estradiol-induced CTA. This finding is similar to what has been observed with LiCl. A stronger CTA is produced when two separate doses of 1.5meq/kg LiCl are injected immediately and 35 or 70 minutes after sucrose consumption than when a single dose of 3.0meq/kg LiCl is injected immediately after consumption (Domjan, Foster & Gillan, 1979). All of these results taken collectively suggest that peripheral administration of CM has aversive properties and that its effect on estradiol CTAs is due to these properties. It serves as a pre-exposure agent to the conditioning agent estradiol, thus blocking the subsequent formation of the CTA, and it does not act as an antihistaminergic agent. Recent evidence in our lab showing that CM can induce a CTA is consistent with this hypothesis. As such, it is not possible to examine the neurochemical basis of estradiol CTAs using peripheral administration of antagonists. On the other hand, there are agents that selectively induce CTA when infused centrally, that is, they induce CTA when infused into some neural sites but not others. This 152

suggests that central infusions of neurotransmitter antagonists may be a potential solution to the problem of studying the chemical mediation of conditioned gustatory aversions.

153

CHAPTER 4 Conducted Experiments 4.1 Outline of Experiments The experiments conducted and discussed in the present chapter addressed the following two Primary Aims: (I) to determine the ability of estradiol to produce a conditioned taste avoidance (CTA) by testing - the unconditioned hypothesis - the negative contrast hypothesis - the aversive hypothesis (II) to conduct preliminary experiments that would allow the examination of the neurochemical basis of estradiol elicited CTA. A total of five experiments, comprising of nine studies, were conducted in order to address each of these aims. In addition to the two Primary Aims, the experiments addressed the following two Secondary Aims: (i) to compare CTAs produced by lithium chloride and estradiol and (ii) to investigate the neural basis of estradiol anorexia 4.2 Primary Aims 4.2.1 Primary Aim 1 Primary Aim 1 is to test the capability of estradiol to induce a conditioned taste avoidance (CTA). 154

In this dissertation, the ability of estradiol to produce a CTA is challenged. We introduce two alternative hypotheses that could account for reductions in sucrose consumption observed after pairing it with estradiol. The first is termed the unconditioned hypothesis and it purports that reductions in CS consumption are a result of the unconditioned effects of estradiol on eating. The second is the negative contrast hypothesis, which proposes estradiol conditioning based on a relative preference model rather than an avoidance model. Under Primary Aim 1, experiments were designed to determine whether either of these hypotheses could account for the reduction in CS consumption after the CS has been paired with estradiol. Specific Aim 1.1: To test the unconditioned hypothesis by determining whether the unconditioned effects of estradiol on food consumption contribute to the apparent reduction in CS consumption observed after pairing the hormone with the novel tastant. When consumption of a novel tasting substance is followed by administration of a chemical agent that produces physiological changes indicative of malaise, animals will reduce their consumption of the substance during subsequent encounters. This learned response is traditionally referred to as a conditioned taste aversion (CTA). The ability of estradiol to produce a CTA when administered exogenously has been demonstrated across different species, routes of administration, and forms of the hormone in both males and females (Mordes, Longscope, Flatt, MacLean & Rossini, 1984; Bernstein et al., 1986; Rice et al., 1987; Miele, Rosellini 155

& Svare, 1988; Ganesan & Simpkins, 1990; Ganesan & Simpkins, 1991; De Beun, Jansen, Smeets, Niesing, Slangen et al., 1991; Peeters, Smets & Broekkamp, 1992; De Beun et al., 1993; Merwin & Doty, 1994; Ossenkopp, Rabi & Eckel, 1996; Yuan & Chambers, 1999). There also is substantial evidence illustrating the role of estradiol in the unconditioned regulation of ingestive behaviors. Physiological levels of estradiol have been associated with systemic variations in the amount of food intake across the reproductive cycle of certain mammals (Wade, 1972; Blaustein, Gentry, Roy & Wade, 1976; Morin & Fleming, 1978; Czaja & Goy, 1975; Czaja, 1975; Kemnitz, Eisele, Lindsay, Engle, Perelman, et al., 1984; Gilbert & Gillman, 1956; Cohen, Sherwin & Fleming, 1987). These variations in eating are shown to be inversely associated with circulating levels of estradiol that accompany the separate phases. During the follicular phase, when endogenous estradiol levels are highest, eating is at its lowest, while the opposite is true of the luteal phase. Administering estradiol exogenously in ovariectomized female rats has validated that it is estradiol that is responsible for the decreases in eating, a finding likewise demonstrated across various species, methods of administration, and forms of the estrogenic steroid (Wade, 1975; Bernstein, Braget & Courtney, 1986; Sandberg, David & Stewart, 1982; Miceli & Fleming, 1983; Tritos, Segal-Lieberman, Vezeridis & Maratos-Flier, 2004). The consistent results unequivocally establish estradiol as an anorexigenic agent.

156

Due to the timing of the unconditioned effects exerted on eating behavior by the steroid, the role of estradiol as a true unconditioned stimulus in a CTA paradigm is called into question. The traditional CTA procedure consists of an acquisition (training) phase during which animals consume a novel substance such as a sweet solution (CS), before exposure to the unconditioned stimulus (US) and a postacquisition (testing) phase during which the animals are again exposed to the CS but not the US. The formation of a CTA is assessed by comparing the amount of sweet solution drunk during the acquisition trial and the first post-acquisition test. A significant reduction in intake across tests indicates that conditioning has occurred. In studies using estradiol as the US, reductions in consumption of the CS have been observed when the first post-acquisition test is given 24 (Miele, Rosellini & Svare, 1988; Merwin & Doty, 1994), 48 (Hintiryan, Hayes & Chambers, 2005; Rice et al., 1987) or 72 (DeBeun et al., 1991; 1993; Ossenkopp, Rabi & Eckel, 1996) hours following the CS-US pairing. However, the unconditioned decreases in food consumption produced by estradiol also have been shown to occur 24-72 hours following the administration of the hormone. For instance, decreases in eating have been observed 24 hours following the increase in plasma estradiol levels in cycling rats (Asarian & Geary, 2002; Griffin & Ojeda, 1996) and 20 (Riviera & Eckel, 2005), 24 (Tartellin & Gorski, 1973), and 48 hours (Asarian & Geary, 2002; Santollo, Wiley & Eckel, 2007) following the exogenous administration of estradiol in ovariectomized rats. In addition, a study conducted in our laboratory showed that a 50μg/kg dose of estradiol benzoate produced food decrements 24 hours following its 157

subcutaneous administration, which was sustained at least 72 hours following the injection (Chambers, Hintiryan & So, unpublished manuscript). Consequently, for estradiol-induced CTAs any decrease observed in CS consumption during the postacquisition test potentially may be due to the unconditioned reduction in eating produced by estradiol and not due to the US stimulus properties of the hormone. Two approaches were used to determine whether the expression of CTA after pairing estradiol with a novel taste solution is due to the unconditioned reduction in eating induced by this hormone. In the first approach, experiments were designed to test the hypothesis that there is a correlation between expression of estradiol hypophagia and estradiol CTA such that CTA is not expressed unless hypophagia is activated. Specifically, if the unconditioned hypothesis is correct, then one would expect each of the following to be evident: (1) For a dose of estradiol that is capable of inducing CTA, the expression of the CTA should be present only during the time of unconditioned suppression of food intake (Experiment 1). (2) A dose of estradiol that does not induce CTA also should not trigger unconditioned suppression of food intake at the time of the post-acquisition test, while a dose of estradiol that does induce CTA should also trigger unconditioned suppression (Experiment 2). (3) Contingent pairing of the taste solution with estradiol should not be a requirement for expression of the apparent CTA (Experiment 3).

158

In the second approach, experiments were designed to test the hypothesis that the same neural areas mediate estradiol hypophagia and estradiol CTA. Specifically, if the unconditioned hypothesis is correct, then one would expect both the unconditioned and conditioned reductions in food consumption to be mediated by the same neural areas. Experiments 5a and 5b were designed to begin testing this hypothesis by determining whether lesions of the lateral parabrachial nucleus block expression of both estradiol CTA and hypophagia. Specific Aim 1.2: To test the negative contrast hypothesis by determining whether an estradiol CTA could be formed if an inter-stimulus interval greater than 30 minutes is employed. The ability of estradiol to produce a CTA has been challenged by a second hypothesis. Recently, it has been suggested that the CTAs produced by reinforcing agents are qualitatively different than those induced by the illness agent LiCl (Hunt & Amit, 1987; Parker, 1988, 1995). Reinforcing agents are a class of agents that are effective positive reinforcers in a drug self-administration paradigm and that produce conditioned place preferences (Reicher & Holman, 1977; Spyraki, Fibiger & Phillips, 1982; van der Kooy, Swerdlow & Koob, 1983; Wise, Yokel & DeWitt, 1976). These agents can induce CTAs at the same doses that are rewarding (van der Kooy et al., 1983; Wise et al., 1976), but they do not produce conditioned aversive taste reactions (Parker, 1995; Parker & Brosseau, 1990). One hypothesis that has been suggested to account for this apparent contradiction is based on the results of reward-comparison studies, which have revealed an anticipatory negative contrast effect (Grigson, 1997). 159

In these studies, animals are given access to a less preferred solution (e.g., 15% sucrose) followed by access to a more preferred solution (e.g., 32% sucrose; Capaldi & Sheffer, 1992; Flaherty & Checke, 1982; Flaherty & Grigson, 1988; Lucas, Timberlake, Gawley & Drew, 1990). Following several pairings, they learn that the presence of the less preferred solution predicts the future availability of the more preferred solution, and consequently reduce their consumption of the less preferred solution. Accordingly, it has been suggested that when consumption of the CS is followed by an injection of a reinforcing agent, the CS becomes devalued so that on subsequent exposures, animals reduce their consumption in anticipation of receiving the more rewarding stimulus, the reinforcing drug (Grigson, 1997). Given the recent finding that intermediate doses of intracranially administered estradiol might be reinforcing in male Syrian hamsters (DiMeo & Wood, 2006), one might argue that reductions in the CS following injections of estradiol are due to negative contrast effects (Grigson, 1997). Although it is difficult to make comparisons between doses administered intracranially and those administered peripherally in CTA studies, the possibility that the doses of estradiol used to induce CTA potentially may be reinforcing precludes the abandonment of the negative contrast hypothesis. As discussed in an article by Reilly, Bornovalova, and Trifunovic (2004), in order for the anticipatory negative contrast effect to be expressed, the time interval between the presentation of the less preferred solution and the more preferred solution should be of reasonable length. This would allow the animals to effectively make comparisons between the solutions. This is supported by the findings that 160

anticipatory contrast effects are most evident when the solutions are presented sequentially with no delay, while the effect is eliminated when the interval between the two presentations approaches 30 minutes (Flaherty & Checke, 1982; Flaherty, Grigson, Checke & Hnat, 1991). If the CTAs formed by estradiol are due to negative contrast effects, then CSUS intervals greater than 30 minutes should not result in the gustatory avoidance. Therefore, one of the purposes behind Experiment 4 was to test the effect of CS-US intervals that were longer than 30 minutes on the formation of an estradiol CTA. This experiment also was designed to determine the longest CS-US interval that would support an estradiol CTA (see Primary Aim 2). As such, several CS-US intervals were tested in six separate studies, all of which included intervals much longer than 30 minutes. Specific Aim 1.3: To test the aversive hypothesis by determining whether lesions of the lateral parabrachial nucleus block estradiol CTA. If estradiol truly conditions (Specific Aim 1.1) and it is not do so based on its reinforcing properties (Specific Aim 1.2), then we will test to see if the hormone produces a CTA based on its aversion producing properties (Specific Aim 1.3). This hypothesis may be examined by determining the role of the lateral PBN in estradiol CTA. The lateral PBN is a relay station for visceral information that is carried from the vagus to the area postrema. The area postrema is a neural substrate that processes information regarding malaise and emesis (reference) and it sends strong projections to the lateral PBN (van der Kooy & Koda, 1983; Shapiro & Miselis, 1985). This 161

suggests that information regarding aversive states is projected and processed in the lateral PBN. In addition, lesions of the lateral PBN block CTAs produced by the putative illness inducing agent LiCl (Sakai &Yamamoto, 1998; Reilly & Trifunovic, 2000a,b). More importantly, it is determined that lesions of the pontine nucleus block the CTA learning not because of a disruption in the associative process, but because of a disruption in the US processing (reference). Taken together, if lesions of the lateral PBN block estradiol CTA, then this would suggest that estradiol too conditions avoidance to a sucrose solution based on its aversion-producing properties. This possibility is tested in Experiment 5. 4.2.2 Primary Aim 2 Primary aim 2 is to conduct preliminary experiments that would allow for the examination of the neurochemical basis of estradiol CTA. In order to study the neurochemical basis of estradiol CTAs, it is critical to determine the length of time the neurotransmitter receptors of interest would have to remain inactivated following the estradiol injection. With LiCl CTAs, the receptors are kept inactive for 2 hours, which is the estimated time that LiCl remains in the system before it is cleared below functional levels. However, a 50μg/kg injection of estradiol benzoate produces estradiol levels that remain in circulation for an extended period of time, thus requiring neurotransmitter receptors to be blocked for greater lengths of time (Wooley & McEwen, 1993). In addition, the relationship between plasma levels of estradiol and the time a CTA is developed has not been established, thereby making it difficult to determine the amount of time necessary to keep 162

neurotransmitter receptors inactivated. These same issues have precluded the use of temporary lesions in the investigation of the neural circuitry of estradiol CTAs. A possible solution to this problem may be to implement chronic versus acute inactivation of the receptors to ensure the antagonism of the receptors throughout the duration of estradiol activation. However, this chronic blockage of receptors could directly or indirectly affect the behaviors of interest and thus confound the data. Such chronic inactivation could also result in aversive effects in the animals. As such, this option was considered, but discarded. The deterioration of the CS trace potentially could be used to circumvent this problem. In male rats, CTA learning can support up to a 12-hour delay between the taste and toxin when radiation is used as the US (Garcia et al., 1966; Smith & Roll, 1967). Presumably, a CTA cannot be formed with intervals greater than 12 hours because the CS trace deteriorates and consequently cannot be associated with the US. The longest inter-stimulus interval allowed for the formation of an estradiol CTA has not been established. Once this information is obtained, then it could be used to determine when the neurotransmitter antagonists need to be infused in order to assess the agents’ influence on estradiol CTAs. For instance, suppose we wanted to examine the effect of the muscarinic receptor antagonist scopolamine on estradiol CTAs and we know that scopolamine infused into the brain inactivates receptors for at least 1 hour. If the maximum time interval allowed for an estradiol CTA is 6 hours, then scopolamine and estradiol would be administered 6 hours following the CS presentation (see Figure 3). In this fashion, the cholinergic receptors would be 163

blocked during the time that is critical for the formation of the estradiol CTA. The high blood levels of estradiol would not matter since a CTA cannot be formed due to the extended interval between the CS and the US. This same methodology also could be employed to study the effects of temporary lesions of the lateral PBN on estradiol CTA. In the above example, the injection of estradiol would be made 5 hours after the CS exposure period and the lateral PBN would be blocked for 1 hour. Figure 3. Methodology for Determining Chemical Mediation of Estradiol Conditioned Taste Avoidance.

CTA

No CTA Time receptors will be inactivated

CS trace

2

3

4

5

6 7 Estradiol Time between CS-US interval (hrs)

Figure 3. Depicts the methodology that will be used to test the role of acetylcholine and histamine in estradiol induced CTAs. If, hypothetically, 6 hours is the longest CS-US interval that supports an estradiol CTA, then acetylcholine and histamine antagonists that are active for at least 1 hour, will be infused 6 hours following the CS. The estradiol injections will be made immediately following the infusions of the antagonists.

164

Specific Aim 2.1: To determine the longest CS-US interval that supports an estradiol CTA. No studies to date have been conducted on the effects of CS-US length on estradiol CTA. As such, several interval lengths were tested to hone in on our target inter-stimulus interval. In Experiment 4, four studies with six different interval lengths and one replication study were conducted in order to find an interval that produced a strong estradiol CTA. It is important that the interval chosen produces a CTA in the majority of the animals tested. If we block an estradiol CTA with the antagonists, we want to be certain that it was because the infusions blocked the conditioning and not because the interval did not produce a CTA in some of the control animals. In order to find a site of infusion for the antagonists, Experiments 5a and 5b were conducted in order to see if lesions of the lateral PBN affected estradiol CTA with the CS-US interval determined in Experiment 4. For this lesion study, it also is important that the majority of the control animals acquire a CTA with the predetermined inter-stimulus interval. If lesions abolish the estradiol CTA, we want to be able to say with certainty that it was due to the lesion and not due to the possibility that those animals did not acquire a CTA in the first place due to the lengthy CS-US interval. Specific Aim 2.2: Do lesions of the lateral parabrachial nucleus block estradiol CTA when an extended inter-stimulus interval is used? As discussed earlier, one of the purposes behind Experiment 4 was to determine the maximum CS-US time interval that supported an estradiol CTA. The 165

next step after determining this critical interval was to find the neural site of infusion. In other words, we needed to determine a neural site that is involved in estradiol CTA where the neurotransmitter antagonists could be infused. Since the lateral PBN extensively has been shown to be involved in the acquisition of LiCl CTA, it is hypothesized that lesions of the lateral PBN would also prevent acquisition of estradiol CTA. 4.3 Secondary Aims Examination of the role of the lateral PBN in estradiol CTA and anorexia warrants investigation for two additional reasons. These constitute the two secondary aims below. 4.3.1 Secondary Aim 1 Secondary aim 1 is to determine whether estradiol CTAs are similar to gustatory aversions produced by the putative illness inducing agent lithium chloride. Lesioning the lateral PBN will show whether the neural circuitry of CTAs produced by estradiol and LiCl are similar. By doing so, we may be able to shed light into the underlying mechanism of estradiol CTAs. For instance, it would be valuable to know if estradiol, like LiCl, is able to condition an avoidance to sucrose based on its ability to produce an aversion. Lithium chloride (LiCl) is considered the putative illness inducing agent and all other USs used in CTA studies are compared to this chemical agent. As an illness producing agent, conditioning to the CS when LiCl is used is thought to be due to an aversive state. Estradiol, like LiCl, has been shown to be aversive. For instance, it has been demonstrated that at high doses the 166

hormone produces aversive orofacial responses in male rats (Ossenkopp et al., 1996) and it produces nausea in humans (Gustavson et al., 1989). However, CTAs formed when the hormone is used as a US may be due to states other than illness, such as satiety (Booth, 1977), and therefore different from LiCl. Investigating the similarities and differences in the neural substrates between the two agents will shed some light on the mechanism by which estradiol produces CTA. Specifically, comparing the neural substrates involved in estradiol and LiCl CTAs would test the aversive hypothesis of estradiol CTA. Conditioned taste avoidance induced by estradiol shares some similarities with those produced by LiCl. First, the area postrema (AP) is necessary for CTAs produced by both the steroid hormone (Bernstein et al., 1995) and by LiCl (Ossenkopp, 1983; Rabin et al., 1983; Wang et al., 1997a,b). Second, both produce c-fos activation in similar brain regions i.e. the AP, the NST, crescent lateral PBN, and medial PBN (Yamamoto et al., 1992; Chambers & Wang, 2004). Third, they each can serve as pre-exposure inter-agents for one another. That is, distal and proximal pre-treatment with LiCl attenuates an estradiol CTA (Hintiryan & Chambers, 2003), while pre-exposure to estradiol attenuates a LiCl CTA (Chambers & Hayes, 2002). Researchers use this cross-familiarization of drugs to suggest common underlying neural mechanisms for the chemical agents. Fourth, the doseresponse curve for LiCl is linear and there is some evidence that this is true for estradiol as well. After pairing different doses of estradiol with a saccharin solution, consumption is reduced in fluid deprived rats given high doses of estradiol (50- and 167

250μg/kg). Consumption is not reduced in those given a low dose of 0.4μg/kg and for animals given intermediate doses of 2- and 10μg/kg, the consumption amount falls in between the high and low doses, although the consumption amounts for these doses were not statistically different than either the high or the low dose consumption levels (De Beun et al., 1991). Fifth, a parametric study conducted in our laboratory has revealed that the strength of the CTA produced by LiCl and estradiol varies as a function of CS-US interval length such that the shorter CS-US intervals produce stronger CTAs compared to the longer CS-US intervals (Chambers, Hintiryan & Foster, 2006 unpublished data). On the other hand, the two agents produce CTAs that appear to be qualitatively different. Only high supraphysiological doses of estradiol (100μg/kg) have been shown to produce aversive orofacial responses to saccharin following conditioning (Ossenkopp et al., 1996), while even very low doses of LiCl (2 ml/kg) have been shown to produce aversive orofacial responses (Parker, 1995) during taste reactivity testing. This is despite the fact that a 100μg/kg dose of estradiol probably conditions more strongly than a 2ml/kg dose of LiCl. Unpublished studies in our laboratory indicate that LiCl doses between 2- and 4ml/kg produce CTAs similar in strength to a 50μg/kg dose of estradiol. Although low supraphysiological doses like those comparable to 50μg/kg dose, produce CTAs, they do not produce negative orofacial responses in female rats (Flanagan-Cato, Grigson & King, 2001). It has been demonstrated consistently that both excitotoxic (Reilly & Trifunovic, 2000a,b, 2001; Trifunovic & Reilly, 2002) and electrolytic (Sakai 168

&Yamamoto, 1998) lesions of the lateral PBN eliminate CTAs produced by LiCl. Reversible TTX lesions (Ivanova & Bures, 1990a,b; Bielavska & Bures, 1994) or cooling lesions (Wang & Chambers, 2002) made after the CS such that it overlaps with LiCl duration also disrupt the associative learning. The lateral PBN also is involved in CTAs induced by different USs other than LiCl. Cytotoxic lesions of the pontine nucleus block the acquisition of morphine-induced CTA (Nader, Bechara & van der Kooy, 1996), while cooling lesions block apomorphine CTAs (Chambers & Wang, 2004). Given that the lateral PBN is involved in CTAs induced by LiCl (as well as other various US agents), determining whether this area is involved in estradiol CTAs will provide evidence for or against the hypothesis that estradiol CTAs are similar to those produced by LiCl. 4.3.2 Secondary Aim 2 Secondary Aim 2 is to determine whether the lateral parabrachial nucleus is critical for estradiol elicited anorexia. Since the role of estradiol in anorexia nervosa is reported in detail in the “Estradiol and Anorexia Nervosa” section of Chapter 2, only a summary of the data will be presented here. Physiological levels of estradiol produce both marked reductions in food consumption (Geary & Asarian, 1999) and increases in physical activity (Wollnik & Turek, 1988). Because the symptoms of the eating disorder anorexia nervosa can be mimicked by high doses of estradiol, this hormone has been implicated in the eating disorder. Briefly, it has been suggested that environmental estrogen-like agents contribute to the onset of anorexia nervosa in pubertal girls by 169

sensitizing areas of the brain that control estradiol-regulated eating responses (Gustavson et al., 1991). Associations have been found between exposure to such agents and an increased incidence of the eating disorder (Bibbo, Al-Naqeeb, Baccarini, Gill, Newton, et al., 1975; Gustavson et al., 1991). However, progress towards testing this hypothesis demands identification of neural areas that mediate the anorectic effects of estradiol. Attempts have been made to identify these neural areas in the forebrain, but its localization remains unestablished. It is well documented that the lateral PBN of the brainstem contains estrogen receptors (Shughrue, Lane & Merchenthaler, 1997) and is involved in ingestive behaviors (Nagai, Ino, Yamamoto, Nakagawa, Yamano, et al., 1987) thereby making the lateral PBN a potential mediator of estradiol induced anorexia. Preliminary studies in our laboratory show that estradiol produces protein activation in the lateral PBN 24 and 48 hours following its administration, which is precisely the time when decreases in food intake are observed after injections of estradiol. Conducting lesions of the lateral PBN and testing for estradiol produced suppression of eating possibly will help identify a neural area for estradiol anorexia. 4.4 General Methodology 4.4.1 Subjects and Husbandry Female Sprague-Dawley rats obtained from Harlan Laboratories (San Diego, CA) were used for each of the experiments. The subjects were 60 days old and weighed approximately 190 grams at the start of each experiment. They were housed individually in a room that was temperature (21-22oC), humidity (51%), and light 170

controlled (12 hr light:12 hr dark cycle). The animals in Experiment 4 series were housed in pairs and a divider was used to separate the animals during behavior testing. Each cage (North Kent Plastics) measured 58 x 38 cm and had a solid bottom that was covered with wood chips. The rats were allowed at least 1 week to adapt to their living conditions before surgery. Rats had ad libitum access to rat chow (Rodent Blox; protein 24%, fat 4%, fiber 4.5%; Harlan Teklad 8604) and tap water throughout the duration of the experiments. The experiments were conducted according to the standards set by the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHEW Publication 80-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD) and the institutional guidelines of the University of Southern California. 4.4.2

Chemical Agents

Conditioning Agents Crystalline estradiol and estradiol benzoate were purchased from Steraloids (Wilton, NH). Each dose of estradiol was dissolved in 0.25ml of sesame oil and was injected subcutaneously in the amounts of 1- (Experiment 2), 10- (Experiment 1), and 50µg/kg of body weight (Experiments 1-5). Sesame oil in the same amount was used as vehicle. In Experiment 4a, each of the animals received an intraperitoneal injection of 0.9% NaCl in addition to their assigned drug. The reason for this is discussed in the methods section for Experiment 4a. Lithium chloride dissolved in 0.9% NaCl was administered in the dose of 10ml/kg as a conditioning agent in Experiment 5. 171

Lesioning Agent Ibotenic acid (Ascent Scientific, Princeton, New Jersey) was dissolved in PBS to achieve a concentration of 20μg/μl. Due to the low solubility of ibotenic acid in PBS, the ibotenic acid was titrated with 1M NaOH until the solution was completely dissolved. Approximately 5μl of NaOH was needed to thoroughly dissolve a 250μl solution of ibotenic acid. The final pH of the solution was approximately 7.5 and the bottle was kept refrigerated throughout the experiment. The same bottle of ibotenic acid was utilized across 10 days of surgery since the solution is stable for two weeks under proper conditions of storage (Filer, Lacy & Peng, 2005; Nielson Schousboe, Hansen, Krogsgaard-Larsen, 1985) and does not get decarboxylated to muscimol. Pilot studies in our laboratory have shown that a bottle of ibotenic acid is effective 11 days after it is dissolved. Surgeries for Experiment 5a were conducted over a 10 day period, suggesting that the batch of dissolved ibotenic acid used throughout the surgeries was still effective. 4.4.3 Surgical Procedures Anesthetics and Surgical Techniques (1) Ovariectomies (a) Anesthesia. Animals in each of the experiments were ovariectomized. Animals in Experiments 1-4, and 5b were ovariectomized using intraperitoneal injections of a ketamine-xylazine solution, which was a mixture of 5 parts of Ketaject (equivalent to 100mg of ketamine: Phoenix Pharmaceutical, Inc) to 1 part xylazine (Lloyd Laboratories) in the dose of 0.12ml/100g of body weight. For Experiment 5a, 172

the ovariectomies were performed under halothane anesthesia (Halocarbon Laboratories, River Edge, NJ). The halothane was vaporized (Fluotec 3) and mixed with oxygen by a Fraser Harlake VMC small animal inhalation anesthesia machine (Datex-Ohmeda, Madison, WI). This gas mixture was delivered to each rat through a nose cone. The percent of halothane in the gas mixture was maintained between 1.5 and 2.0 and the oxygen flow rate was maintained between 3.5 and 4.0 L/min, while the animals were ovariectomized. (b) Surgical Technique. Ovariectomies included bilateral incision of the skin and muscles (1-2 cm from the midline and anterior to the hip), removal of the ovaries, and tying of the fallopian tubes. For Experiment 5a, the ovariectomies were performed the same day as the excitotoxic lesions. For Experiment 5b, the ovariectomies were performed several weeks after the electrolytic lesions. Since recovery from electrolytic lesions is more difficult and longer in length than excitotoxic lesions, we decided not to overwhelm the animals with an additional surgery. As such, the ovariectomies were performed after the animals had fully recovered from their electrolytic lesions. (2) Neural Lesions (a) Anesthesia. Excitotoxic (Experiment 5a) and electrolytic (Experiment 5b) lesions were performed under ketamine-xylazine anesthesia. In Experiment 5a, once the ovariectomy was completed, the animals were removed from the halothane. Once the animals started to recover from the halothane, they were injected intraperitoneally with 5 to 1 ketamine-xylazine mixture in the dose of 0.06ml/100g of 173

body weight to prepare for neural surgeries. In order to minimize possibility of overdose, the injection anesthesia was administered after the animals started to recover from the halothane and in half of the dose normally required to anesthetize an animal. The animals were given supplemental injections of ketamine-xylazine in amounts ranging from 0.02-0.04ml to maintain deep anesthetic state throughout the remainder of the neural surgeries in both experiments. (b) Surgical Technique (i) Coordinates. For Experiment 4, the lateral parabrachial (PBN) nucleus was approached at a 30° angle in an attempt to avoid the transverse sinus. The coordinates according to Paxinos and Watson (1998) 12.84 (AP), 2.0 (ML), and 7.275 (DV) were used for the lateral PBN. For Experiment 5b, these same coordinates were used in 3 of the animals and 13.12 (AP), 2.0 (ML), and 7.85 (DV) were used in 3 other animals. To increase the accuracy of placement, the ML for bregma was measured by calculating the midpoint of the coronal suture. This was accomplished by placing the micropipette (Experiment 5a) or electrode (Experiment 5b) in the groove on left side of skull above the end of the coronal suture and recording the coordinate. The pipette or electrode was then moved into the groove of the right side above the coronal suture and the coordinate recorded. The two coordinates were subtracted. The midpoint was shifted 0.125ml to the right. The length of the skull from groove to groove calculated from each arm never differed more than 60µm. In addition, for Experiment 5a, the surgery was performed using a microscope to increase accuracy of the placement. 174

(ii) Excitotoxic Lesions (Experiment 5a). Excitotoxic lesions of the entire lateral PBN were made. The excitotoxic lesioning technique with ibotenic acid was chosen in order to avoid damaging fibers of passage such as the superior cerebellar peduncle and the ventral spinocerebellar tract or any other fibers passing through the pontine nucleus. When infused into the brain, ibotenic acid causes selective degeneration of cell bodies while sparing not only fibers of passage, but also nerve terminals from extrinsic origin and glial elements (Schwarcz, Kohler, Fuxe, Hokfelt & Goldstein, 1979; Markowaska, Bakke, Walther & Ursin, 1985; Edwards & Johnson, 1991). Ibotenic acid was delivered via non-filamented micropipettes (outside tip diameters 40-60μm) in the amount of 0.2μl/10minutes (Reilly & Trifunovic, 2000a,b; 2001). To minimize the spread of the neurotoxin along the track, the micropipette remained in situ for an additional 10 minutes following the 10-minute infusion. Control sham lesioned animals were treated the same way as lesioned animals except infusions of phosphate buffered saline (PBS) in the place of ibotenic acid were made. Two new micropipettes were used for each rat. The micropipettes were attached and glued to PE100 tubing, which was attached and glued to PE20, which then was attached to a 10μl Hamilton syringe situated in a micro-injector pump (CMA/100, Carnegie Medicin, Stockholm, Sweden). The PE tubes, micropipettes, and Hamilton syringe were filled with mineral oil to ensure the creation of an airtight seal. The tips of the pipettes were painted with red nail polish for better visibility and thus improved accuracy during surgery. This procedure was tested in pilot animals 175

and did not have any effect neurally or behaviorally. A microscope was used during the surgery to increase visibility of the micropipette tips. The ibotenic acid and PBS were backfilled manually into the pipettes via a micro-injector. (iii) Electrolytic Lesions (Experiment 5b). An insect pin coated with epoxylite was used as the electrode for the electrolytic lesions. A current of 0.1mAmps was delivered for 10 seconds by a lesion maker (Grass Instruments, West Warwick, RI). The tip of the electrode was exposed approximately 0.3mm. Control sham lesioned animals were treated the same way as lesioned animals except no electric current was passed through the electrode. Analgesics, Antibiotics, and Post-Operative Care For Experiment 5, the analgesic buprenex (buprenorphine hydrochloride: Reckitt & Colman Products: Hull, England) and the antibiotic dualcillin (penicillin benzathine: Western Medical, Arcadia, CA) were administered subcutaneously in the amounts of 0.02ml (0.017mg/kg) and 0.05ml (43,000 units/kg), respectively, to each rat the day of surgery. The same dose of buprenex also was given the day following surgery in order to alleviate any persisting discomfort. No buprenex was administered in Experiments 1-4 after ovariectomies. Generally, it has been our observation that animals remove their stitches after ovariectomies if their pain is alleviated with analgesics. Consequently, when only ovariectomies are performed, buprenex is not administered to the animals; however, it was our concern that the double surgeries (the neural lesion and ovariectomy) would cause too much pain and discomfort to the animals not to administer analgesics. As such, each animal in 176

Experiment 5 received an injection of buprenex. Additional post-operative care for Experiment 5 consisted of weighing and general inspection of the animals. Each animal also was given Saltine crackers following surgery. Since the rats find the novel crackers palatable, they begin eating shortly after recovering from the anesthesia. This is not the case with their Rodent Blox. We also have observed that the Saltine crackers increase the animals’ water intake immediately after surgery. Since eating and drinking are signs of recovery, the crackers were given after surgery to aid in the animals’ return to physiological baseline. For Experiment 5b, whenever necessary, intraperitoneal injections of Ringer solution, which contained calcium, potassium, and magnesium, were made to assist the recovery process. Those animals that were otherwise immobile and lethargic, started to display increased activity following the injections of the Ringer solution. 4.4.4 Behavior Testing Behavioral testing commenced following a 1-week recovery period after ovariectomies for Experiments 1-4, while a 2-3 week recovery period was allowed for Experiments 5a and 5b. Conditioned Taste Avoidance/Aversion (CTA) Procedure For Experiments 1-4, animals were subjected to one CTA procedure, which was divided into the following four periods: (1) preconditioning, (2) acquisition, (3) post-acquisition recovery, and (4) post-acquisition test. For Experiment 5, two of the groups in study a and all of the animals in study b were subjected to a second CTA

177

procedure. Each of the CTA procedures consisted of (1) preconditioning, (2) acquisition, (3) post-acquisition recovery, and (4) extinction. All drinking solutions used during each period were stored under refrigeration and were given to the rats at the beginning of the dark portion of the light/dark cycle. During the preconditioning phase, a 100ml cylinder containing refrigerated tap water was added to the cages of the animals for 1 hour. It has been our observation that preconditioning rats with cold water immediately after the lights switch off increases the likelihood that non-deprived rats will drink the novel sucrose solution presented during acquisition. On acquisition day, a 100ml cylinder containing a novel sucrose solution (10% w/v) or chocolate milk (Experiment 5) was added to the cages. One hour later, the amount of fluid consumed was recorded, the cylinder was removed, and each rat was administered its assigned conditioning agent. The amount of time between the exposure to the CS and the US injection was 0 minutes in Experiments 1, 2, and 5 while it ranged from 3-12 hours in Experiment 4 and was 24 hours in Experiment 3. Depending on the experiment, the post-acquisition test was conducted 35 to 215 hours after acquisition. Animals were left undisturbed during the interval between acquisition and post-acquisition testing or extinction. For Experiment 5, daily extinction tests were initiated two days after acquisition. The extinction tests were conducted in the same manner as the acquisition tests except no conditioning agent was administered. Extinction tests were given daily until group differences, if present, were no longer statistically significant.

178

Measurements of Fluid Intake For Experiment 5a, water consumption was measured at the same time as sucrose intake for the first CTA (CTA1, described in methods section for Experiment 5a). If any significant changes in sucrose consumption were detected across the groups, the water intake data would help determine whether the changes were due to factors related to the CTA, changes in neophobic responses (Reilly & Trifunovic, 2001), or due to alterations in general drinking behavior (i.e. adipsia or polydipsia; Edwards & Johnson, 1991). Measurements of Food Intake In addition to CTA testing, food intake measurements were conducted in the same animals for Experiments 1, 2, and 5. In Experiments 1and 2 food intake measurements were made every 24 hours just prior to each period of the CTA procedure while in Experiment 5, baseline measurements of food intake were initiated at least two weeks following the final estradiol injection of the CTA procedure. For Experiment 5a, food measurements were made every 12 hours, once immediately before the lights turned on and once immediately before the lights turned off. For Experiment 5b, food measurements were made every 24 hours, immediately before the lights turned off. For both Experiments 5a and 5b, two injections of estradiol were administered 24 hours apart. Each injection was made immediately before the lights turned off. Given the natural variability in eating data and the fact that we occasionally have observed decreases in eating in control animals following oil injections, we adopted this two injection methodology to increase the 179

reliability of obtaining differences between the oil and estrogen groups following injections. This two injection method was not used in Experiments 1 and 2 since CTA and eating were tested at the same time; only a single estradiol injection is required for CTA testing and two injections would interfere with the CTA procedure. 4.4.5 Histological Staining Cresyl violet staining is typically used to assess the accuracy and extent of excitotoxic lesions. However, due to difficulties we encountered during the infusion of ibotenic acid (discussed in Experiment 5a section), the lesions were going to be extremely difficult to read with cresyl violet. Further, gliosis from damaged cells typically serves as the marker for reading lesions with cresyl violet staining and gliosis fades over the course of time. One study showed that gliosis had faded dramatically 7 weeks following excitotoxic lesions of the lateral PBN (Calingasan & Ritter, 1993). Since the behavior testing for Experiment 5a lasted up to 5 months following the lesioning, cresyl violet staining was not a viable option, assuming that gliosis would not be present at the conclusion of the experiment. As such, we decided to stain for neuron-specific nuclear protein (NeuN), a protein found exclusively in neurons. The absence of neurons due to the lesion would be indicative of a lesion, thus simplifying localization and extent of the lesion. Neuron Specific Nuclear Protein (NeuN) Staining At the conclusion of the behavior testing of Experiment 5, each rat was anesthetized with an overdose injection of ketamine:xylazine (5:1) and transcardially perfused with 300ml of room temperature 0.9% NaCl followed by 300ml of a 180

refrigerated 4% paraformaldehyde solution. The brains were extracted and post-fixed in 4% paraformaldehyde overnight at 4°C. For cryoprotection, they were transferred to a jar filled with a solution of 20% sucrose PBS for 24-48 hours. Coronal brain sections corresponding to the lateral PBN (according to the coordinates of Paxinos and Watson, 1998) were sliced at 36-40µm thickness with a microtome (American Optical Company, Buffalo, New York). The NeuN staining protocol adopted by our laboratory was a modified version of a protocol provided by Millipore (Temecula, CA). The free-floating method was used and each step was performed on a rotator. The sections were transferred to a 3% hydroxide absolute methanol for 20 minutes to inactivate the endogenous peroxidases. Following a no soak wash, they were rinsed in PBS 3 times (2 minutes each) and treated with 3% horse serum (Vector Laboratories, Inc. Burlingame, CA) and 0.2% Triton (Sigma, St Louis, MO) PBS for 30 minutes. Without rinsing, the sections were transferred to a 1% horse serum and 0.4% Triton PBS with 1:1000 primary anti-NeuN antibody (biotinylated, Mouse IgG, Millipore, Temecula, CA) for 48-72 hours at 4C. After 48-72 hours of incubation and a no soak wash, the slices were rinsed 3 times with PBS (2 minutes each) and were transferred to an avidin-biotin complex reagent solution (ABC Standard kits, Vector Laboratories, Inc. Burlingame, CA) for 1 hour at room temperature. Next, the sections were soaked and rinsed 3 times with PBS (2 minutes each). A peroxidase substrate kit was used to visualize the antigen-antibody reaction sites in the brain. The sections were placed in a solution made by 3,3'-diaminobenidine and nickel 181

enhancer (DAB kit; Vector Laboratories, Burlingame, CA) for 5-7 minutes depending upon the color of the sections and were rinsed 10 times with PBS (2 minutes each) to stop the reaction. The sections were individually mounted onto gelatin-coated glass slides coated, air- dried, and cover slipped with Cytoseal (Stephens Scientific, Kalamazoo, MI). 4.4.6 Statistical Analyses Description of Analyses Our data were analyzed by taking advantage of modern statistical procedures that avoid the problems of traditional statistics. Conventional statistical procedures such as the ANOVA are fraught with deleterious problems when assumptions of normality and equal variances are violated. Violations of these assumptions lead to poor probability coverage, low power, and inability to control for Type I errors and bias, which means that the probability of rejecting can drop as the population means become more unequal (Wilcox, 2001, 2003). Traditional approaches to achieve normality and equality of variances (i.e. transformations) also have been shown to be inadequate (Wilcox & Keselman, 2003). Reliable modern statistical techniques have been developed to replace such conventional methods of analyses. Due to the nonnormality and heteroscedasticity of our data, more modern robust statistical methodologies were implemented in order to attain reliable results. A percentile bootstrap with 20% trimmed means, which is not predicated on the assumptions of normality or equal variances, was performed to analyze these data. In general, this method randomly samples with replacement from the actual data to produce a data 182

set called a bootstrap sample. Next, the trimmed mean is computed. In the twosample case, the p-value is the probability that a bootstrap trimmed mean from the first group is less than the bootstrap trimmed mean of the second. The value of p is computed by repeatedly generating bootstrap samples. This procedure can be generalized to a repeated measures design (Wilcox, 2003b). Additionally, this method controls the family- wise error rate using adjusted p-values. It has been found to perform well among a range of methods when working with real data, including when working with small sample sizes (Wilcox, 2003b; Wu, 2002). For each comparison, a critical significance level is reported along with a significance level. A statistically significant result is obtained when the significance level is lower than the critical significance value. The p-value was set at 0.05 for all comparisons. The percentile bootstrap equivalent of dependent-t and ANOVA with repeated measures was performed on the data. This type of robust analysis previously has been used to analyze CTA data (Hintiryan, Hayes & Chambers, 2005, 2006). Assessment of CTA Formation For Experiments 1-4, within-group analyses of sucrose consumption across acquisition and the post-acquisition test was used to determine whether a CTA had been acquired. For Experiment 5, within-group analyses of sucrose consumption across acquisition and the first extinction test was used to assess acquisition. The post-acquisition test in Experiments 1-4 is equivalent to the extinction test 1 for Experiment 5. We have used different designations to prevent possible confusion 183

since, as indicated above, extinction of the CTA was assessed in Experiment 5 but not in the other experiments. Between-group comparisons of sucrose consumption were made for both the acquisition trial and the post-acquisition test (Experiments 1-4) or extinction test 1 (Experiment 5) to assess differences in acquisition strength. Assessment of Extinction Only between-group analyses were performed on the extinction data of Experiment 5. Due to the nature of the bootstrap analyses, the extinction trials for Experiment 5a were divided into phases. The estradiol CTA (discussed in Experiment 5a methods) consisted of 4 phases and each of the phases included 5 extinction days. The sum of the extinction trials in each phase was calculated and group-wise comparisons were performed across all of the phases. For Experiment 5a, the extinction data for the second CTA where LiCl was used as the US was not divided into phases. Analyses were performed on individual days since the animals extinguished from the CTA in 4 days. Assessment of Fluid Intake For Experiment 5a, water intake was analyzed in the same manner as the sucrose data for CTA1. Water intake was divided into 4 phases and each phase consisted of 5 days. These 5 days for each phase were the same for the sucrose data from CTA1. The sum of the water intake in each phase was calculated and groupwise comparisons were performed across all of the phases.

184

Assessment of Food Decrement Experiments 1 and 2 During acquisition and post-acquisition testing, the animals had access to both food and a sucrose solution. As such, a pattern of food intake developed such that following the injection, there was a decrease in food consumption in each of the groups, including the oil groups. Investigation into this pattern revealed that the control oil animals were voluntarily decreasing their food intake in order to compensate for the caloric intake of the sucrose solution. Therefore, the food and sucrose intake data was converted into calories and the total calorie intake of the individual days was used in the statistical analysis. Sucrose has 3.75 Cal/g, which translates to 0.375 Cal/ml and the chow pellets have 3.3 Cal/g. Baseline day for each of these experiments was the average of the caloric intake 24 and 48 hour periods prior to the estradiol injection. Within- and between-group analyses were performed on the total calories consumed during baseline days compared to the total calories consumed in the 24 hour period starting on the post-acquisition trial to assess any changes in caloric intake within and between groups. Experiment 5 Predicated on our experience in Experiments 1 and 2, food intake measurements were made independently of the CTA behavior testing. Therefore, food intake was not converted into calories and analyses were performed on raw data measurements (grams). Baseline day for Experiments 5a and 5b was the average of 185

the 4-day intake prior to the first estradiol injection. Within-group analyses were performed to assess differences between baseline and post injection days. Betweengroup comparisons were performed to detect differences in the amount of food consumed on each of the days. Additional Analyses Two additional types of analyses were performed that were specific to Experiments 4 and 5. They are introduced here, but are discussed in further detail in the methods sections for the respective experiments. Comparison of Number of Animals that Acquired CTA across Groups In Experiment 4 series, it was necessary to statistically compare the number of animals that acquired a CTA across the different groups. A percentile bootstrap equivalent to binomial analysis was performed and Rom’s method was used to control the family-wise error rate. Details regarding the criteria for CTA quantification are provided in the methods section for Experiment 4. Comparisons of Lesion Size between Lesioned Groups In Experiment 5a, comparisons of lesion size were made between the lesioned groups that received oil and estradiol. The lesion size of each animal was determined by the number of cells each animal had below the lower limit set for its corresponding AP level (discussed in detail in the methods for Experiment 5a). Comparisons between the two groups were made for the right and left sides. Between-group analyses were performed to assess differences in lesion size between the two groups. 186

4.5 Presentation of Experiments 4.5.1 Experiment 1 PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis Correlation between expression of hypophagic and CTA based on time of testing (Strategy 1). In Experiment 1, we employed the first of the three strategies outlined in the introduction to test the hypothesis that the unconditioned effects of estradiol on food consumption contribute to the apparent CTA. This strategy involved correlating the period of time during which a particular dose of estradiol triggers unconditioned reductions in consumption with its ability to induce CTA. To do this, we used two different time intervals between acquisition and post-acquisition testing, a 2-day interval, when unconditioned reductions in consumption produced by estradiol presumably are evident and a 9-day interval, when these reductions in intake presumably are no longer evident. If the unconditioned effects of estradiol are the reason for CTA expression, than both CTA and unconditioned reduction of food consumption should be expressed at the 2-day time period but neither should be expressed at the 9-day time period. Methods Sixty-five animals were randomly divided into one of the following six groups: (1) Oil-D2, (2) EB10-D2, (3) EB50-D2, (4) Oil-D9, (5) EB10-D9, and (6) EB50-D9, with n=11 for all groups except the Oil-D9 group which had n=10. Both of the Oil, EB-10, and EB-50 groups received an injection of sesame oil, 10μg/kg of 187

estradiol benzoate (EB), or 50μg/kg of EB, respectively, immediately following the 1 hour drinking of sucrose on acquisition day. The post-acquisition test was held 2 days (47 hours) after acquisition for the three D2 groups and 9 days (215 hours) following acquisition for the three D9 groups. Body weight and food measurements were made throughout the duration of the experiment. Results Assessment of CTA Formation Groups Oil-D2, EB10-D2, and EB50-D2. Both of the 2-day estradiol groups developed a CTA, while the oil group did not (see Figure 4). Within-group analysis showed that groups EB10-D2 and EB50-D2 drank significantly less sucrose during the post-acquisition test than the acquisition trial (critical significance=0.05, significance=0.00 in each case). Between-group comparisons revealed that the estradiol groups did not differ from the oil group in the amount of sucrose consumed during the acquisition trial, but during the post-acquisition test, the EB10-D2 and EB50-D2 groups drank significantly less sucrose than the Oil-D2 group (critical significance=0.05, significance=0.00 in each case). In addition, comparisons between each of the estradiol groups and the oil group revealed significant interaction effects across acquisition and the post-acquisition test (critical significance=0.05 and significance=0.00 in each case). Moreover, the EB50-D2 group formed a stronger CTA than the EB10-D2 group. Although these two groups consumed similar amounts of sucrose during 188

acquisition, the EB50-D2 group consumed less sucrose during the post-acquisition test than the EB10-D2 group (critical significance=0.05, significance=0.013). There also was a significant interaction effect across acquisition and the post-acquisition test in sucrose consumption when the EB10-D2 and EB50-D2 groups were compared (critical significance=0.05, significance=0.014). Groups Oil-D9, EB10-D9, and EB50-D9. As was true for the 2-day groups, both of the 9-day estradiol groups developed a CTA, while the oil group did not. Withingroup analyses showed that the EB10-D9 and EB50-D9 groups drank significantly less sucrose during the post-acquisition trial than the acquisition trial (critical significance=0.05, significance=0.00; see Figure 4). Although both the EB10-D9 group and the EB50-D9 group did not differ statistically from the Oil-D9 group in the amount of sucrose they drank during the acquisition trial they drank significantly less sucrose than the control group during the post-acquisition trial (critical significance=0.05, significance=0.00 in each case). Comparisons between each of the estradiol groups and the control oil group also showed a significant interaction effect across acquisition and the post-acquisition test (critical significance=0.05, significance=0.00 in each case). Similar to the 2-day estradiol groups, the EB50-D9 group acquired a stronger CTA than the EB10-D9 group. Although the EB50-D9 group consumed significantly more sucrose during the acquisition trial than the EB10-D9 group, this group also showed a greater reduction in sucrose consumption during the post-acquisition trial than the EB10-D9 group (critical significance=0.05, significance=0.002). 189

Furthermore, there was a significant interaction effect across acquisition and postacquisition test sucrose consumption between these two estradiol groups (critical significance=0.00, significance=0.05). Therefore, the difference found between the two estradiol groups in consumption during the acquisition trial does not warrant consideration. D2 Groups versus D9 Groups. Comparisons between the Oil-D2 and Oil-D9 groups revealed that these two oil groups did not differ in the amount of sucrose consumed during the acquisition or post-acquisition trials and they showed no interaction effect across these two tests. Similarly, comparisons made between the two EB10 groups (EB10-D2 versus EB10-D9) and the two EB50 groups (EB50-D2 versus EB50-D9), revealed no differences in sucrose consumption during or across both of these trials. As such, whether the post-acquisition test was held 2 days or 9 days following acquisition and whether a 10- or 50μg/kg dose was used, CTAs of comparable strength (relative to dose of estradiol) were acquired. Assessment of Caloric Decrement Groups Oil-D2, EB10-D2, and EB50-D2. Both doses of estradiol produced decreases in caloric intake across baseline and the post-acquisition test day while oil did not (see Figure 4). Within-group analyses showed that both the EB10-D2 and EB50-D2 groups consumed fewer calories on the day of the post-acquisition test compared to the baseline time period (critical significance=0.254-0.038, significance=0.00 in each case; see Figure 4). On the other hand, the Oil-D2 group did not reduce its caloric intake from the baseline time to the post-acquisition day. 190

In addition, between-group analyses revealed that both the EB10-D2 and EB50-D2 groups ingested significantly fewer calories during the post-acquisition test day than the Oil-D2 group (critical significance=0.0254, significance=0.00 in each case). Furthermore, there was a significant interaction effect across baseline and post-acquisition levels of consumption between the Oil-D2 and EB10-D2 groups and the Oil-D2 and EB50-D2 groups (critical significance=0.017, significance=0.00 in each case). The evidence for differences between the two estradiol groups in the extent of the decrease across baseline and the post-acquisition test day was not clearly discernable. These two groups did not differ in baseline levels of consumption, but showed differences in consumption during the post-acquisition test period (critical significance=0.025, significance=0.02). However, there was no significant interaction effect detected across the two time periods between the two estradiol groups. Groups Oil-D9, EB10-D9, and EB50-D9. Unlike the 2-day groups, neither dose of estradiol produced decreases in calorie intake. Within-group analyses showed that caloric intake was not reduced during the post-acquisition test day compared to the baseline time period for any of the 9-day groups. The three groups also did not differ in the amount of calories ingested during the baseline time period or during the postacquisition test day and there were no significant interactions across these two time periods.

191

D2 Groups versus D9 Groups. Comparisons made between the two oil groups (OilD2 and Oil-D9) showed no differences in calorie intake during either the baseline time period or during the post-acquisition trial day. Although the two EB10 groups consumed comparable calories during the baseline time period, the EB10-D2 group ingested significantly fewer calories during the post-acquisition test day than the EB10-D9 group (critical significance=0.05, significance=0.00). There also was a significant interaction effect across the two time periods for these two EB10 groups (critical significance=0.05, significance=0.01). Similarly, groups the two EB50 groups did not differ in calorie intake during the baseline time period, but group EB50-D2 ingested significantly fewer calories during the post-acquisition test day than group EB50-D9 (critical significance=0.05, significance=0.00). Finally, comparisons of intake across the baseline and post-acquisition time periods between these two EB50 groups revealed a significant interaction effect (critical significance=0.05, significance=0.00).

192

Trimmed Mean Sucrose Intake (ml)

Figure 4. Effect of Two Different Time Intervals between Acquisition and Postacquisition Testing on Estradiol Conditioned Taste Avoidance and Calorie Intake. 20

Oil-D2

EB10-D2

EB50-D2

Oil-D9

EB10-D9

EB50-D9

16 12

d

8 4

ab

ab

abc

abc

0 Acq

Post-Acq

Acq

Post-Acq

Acquisition Trial & Post-Acquisition Test

Trimmed Mean Total Calorie Intake

90

Oil-D2

EB10-D2

EB50-D2

Oil-D9

EB10-D9

EB50-D9

80

70 ab

60

abc

50 Base

Post-Acq

Base

Post-Acq

Baseline & Post-Acquisition Test

Figure 4 Caption. Upper Graphs. Trimmed mean sucrose consumption of ovariectomized female rats during the acquisition (Acq) trial and the post-acquisition (Post-Acq) test. The acquisition trial consisted of pairing a 10% sucrose solution with a subcutaneous injection of either sesame oil vehicle (Oil) or a 10or 50μg/kg dose of estradiol benzoate (EB10 or EB50, respectively). The post-acquisition test was given either 2 days (D2) or 9 days (D9) following acquisition for each of the three groups. aSignificant reduction in sucrose consumption across Acq and Post-Acq. bSignificantly lower than the Oil group during Post-Acq and significant interaction across Acq and Post-Acq when compared to the Oil group. c Significantly lower than the EB10 group during Post-Acq and significant interaction across Acq and Post-Acq when compared to the EB10 group. dSignificantly lower than the EB50 group during Acq. Lower Graphs. Trimmed mean total calorie intake of ovariectomized female rats during the baseline time period (Base) and the post-acquisition (Post-Acq) test day. For the Base time period, caloric intake was measured during the 24 and 48 hour periods prior to the injection of either sesame oil (OilD2) or estradiol benzoate in the doses of 10- (EB10-D2) or 50μg/kg (EB50-D2) and a mean for these two days was computed. For the Post-Acq test day, caloric intake was measured during the 24 hour period that occurred 2 days following injection and that started at the onset of the post-acquisition trial. Caloric intake during the Post-Acq test day included consumption of lab chow and sucrose while caloric intake during the Base time period included only lab chow. aSignificant reduction in total calorie intake across Base and Post-Acq days. bSignificantly lower than Oil-D2 group during the PostAcq day and significant interaction across Base and Post-Acq days when compared to the Oil group. c Significantly lower than EB10-D2 group during the Post-Acq day.

193

Discussion The results of Experiment 1 revealed that estradiol benzoate, at both 10- and 50µg doses, is able to condition an avoidance of sucrose whether the unconditioned effects of the hormone on eating are present or absent. Groups tested for CTA 2 days after receiving estradiol benzoate exhibited both CTA to sucrose and a general diminution of caloric intake. Although groups assessed for CTA 9 days after estradiol benzoate injection also displayed CTAs, they no longer displayed reduced caloric intake. Unlike the 2-day groups, the 9-day groups had returned to their baseline level of caloric consumption during their post-acquisition test day. These results are inconsistent with the hypothesis that the hypophagic effects of estradiol contribute to the expressed CTA and they are strongly supportive of the notion that estradiol is a true conditioning agent. Furthermore, the fact that the CTAs developed by the 2-day and 9-day groups were of comparable strengths (within each dose) suggests that the conditioned and unconditioned effects of estradiol are independent, because if they were not, one would expect the unconditioned effects to contribute to the CTA strength in the 2-day groups. Examination of differences between the two estradiol doses provides additional support for dissociation between the CTA observed after pairing this hormone with a sucrose solution and the unconditioned effects of estradiol on calorie intake. The high dose of estradiol clearly induced a stronger CTA than the low dose in the 2-day groups, as evidenced by the higher dose group showing a greater extent of decrease in sucrose consumption across acquisition and the post-acquisition test. 194

However, there was no difference between the two estradiol groups in the extent of decrease in calorie intake across acquisition and post-acquisition days. Despite the strength of these results, there is one other possibility that should be addressed. One cannot discount the slim possibility that the unconditioned effect of estradiol specifically inhibit sucrose consumption for a much longer period of time than it affects lab chow intake. However, given the high palatability valence of sucrose and the fact that rats will forgo lab chow for sucrose (observed in the Oil groups), we consider this possibility to be unlikely. In addition, the results of Experiment 3 increase the unlikelihood of this possibility (see the discussion of that experiment). Before delving into the details of the next experiment, it is worth noting that there are inconsistencies in the ability of a 10µg/kg dose of estradiol to induce a CTA. DeBeun et al. (1991) showed in a previous study that this dose of estradiol was ineffective in producing a CTA when the post-acquisition test was given 3 days after acquisition. We failed to replicate this in Experiment 1, demonstrating that the 10µg/kg dose produced a CTA whether the test was held 2 days or 9 days following acquisition. There are three major dissimilarities in the methodologies between the two studies. DeBeun et al. used fluid deprived Wistar rats and estradiol while the present study used Sprague-Dawley rats that had ad libitum access to water throughout the duration of the experiment and were injected with estradiol benzoate. Because fluid deprivation reduces sensitivity to the US, this factor could account for why the 10µg/kg dose did not induce CTA in fluid deprived rats but produced CTA 195

in nondeprived rats (Chambers, Sengstake, Yoder & Thornton, 1981; Grote and Brown, 1973; Hamdani & White, 2007; Sengstake, Chambers & Thrower, 1978). Moreover, Sprague-Dawley rats have been found to be less sensitive to LiCl than Fischer-344, as evidenced by the finding that Sprague-Dawley rats require a substantially higher dose of LiCl to achieve the same slow rate of extinction as Fischer-344 (Brownson, Sengstake & Chambers, 1994), which raises the possibility that a similar difference in sensitivity exists between Sprague-Dawley and Wistar rats when estradiol is used as the US. Finally, the conjugated form of estradiol, estradiol benzoate, has been shown to have varying kinetic and dynamic properties compared to estradiol (McEwen, 1981), which could alter the effects the hormone has on behavior. However, additional evidence does not support any of these differences as a reason for the disparate findings regarding CTAs and a 10µg/kg dose of estradiol. In another study conducted in our lab, we failed to induce CTA in nondeprived Sprague-Dawley female rats with a 10µg/kg dose of estradiol benzoate even after 7 sucrose-estradiol pairings (Chambers, Hintiryan & So, unpublished manuscript). At the present time, analysis of all of the evidence suggests that the best explanation for the differences in the ability to acquire a CTA with a 10µg/kg dose of estradiol is that this dose may be a threshold dose for inducing CTA. 4.5.2 Experiment 2 PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis Correlation between expression of hypophagic and CTA based on varying doses of estradiol (Strategy 2). 196

Although the results of Experiment 1 make a strong argument against the hypothesis that the unconditioned effects of estradiol on food consumption contribute to the apparent CTA, reproducing these findings using a different methodology would further solidify our finding. This leads us to the second strategy, which involved correlating the ability of doses of estradiol to induce CTA with their ability to trigger unconditioned reductions in consumption at the time of CTA expression. To do this, we chose to try a 1µg/kg dose, basing our decision on previous findings that doses as low as 0.5µg/kg reduce food intake (Zucker, 111969), but a 2µg/kg does not induce CTA (DeBeun et al, 1991). Additionally, we chose to use unconjugated estradiol instead of estradiol benzoate to verify that despite the form of estradiol used, the conditioned and unconditioned effects of the hormone are dissociable. Methods Thirty animals were randomly assigned to one of the three following groups (n=10 per group): Group (1) Oil (sesame oil), (2) E21 (1μg/kg dose of estradiol), and (3) E250, (50μg/kg dose of estradiol). Oil and estradiol injections were given immediately following the 1 hour sucrose exposure on acquisition day. The postacquisition trial was given 2 days (47 hours) following acquisition. Results Assessment of CTA Formation The high dose of estradiol induced a CTA while the low dose of estradiol and oil vehicle did not. Within-group comparisons showed that the E250 group drank significantly less sucrose during the post-acquisition test compared to the acquisition 197

trial (critical significance=0.05, significance=0.00; see Figure 5), while groups Oil and E21 drank comparable amounts across both trials. Between-group analysis revealed that while the E250 animals did not differ from the Oil and E21 animals in the amount of sucrose they consumed during the acquisition trial, they did drink significantly less sucrose than the Oil and E21 animals during the post-acquisition trial (critical significance=0.05 in each case, significance=0.00-0.02). In addition, there was significant interaction effect across acquisition and post-acquisition trials between the E250 group and both the Oil and E21 groups (critical significance=0.05 in each case, significance=0.00-0.019). The Oil and E21 groups did not differ in the amount sucrose consumed during either the acquisition trial or the post-acquisition test and there was no significant interaction effect across acquisition and the postacquisition test for these groups. Assessment of Caloric Decrement Both estradiol groups showed decreases in caloric intake while the Oil group did not. Within-group analysis revealed that compared to baseline, group Oil did not show reductions in calorie on the post-acquisition day, while groups E21 and E250 ingested significantly fewer calories (critical significance=0.05, significance=0.00 in each case; see Figure 5). Between-group analysis revealed that the caloric intake of the Oil group during the baseline time period was comparable to that of both the E21 and E250 groups. However, both of the estradiol groups differed significantly from the Oil group in the amount of calorie intake on the post-acquisition test day (critical significance=0.033-0.05, significance=0.004-0.018). In addition, there was a 198

significant interaction effect across the baseline and post-acquisition time periods between groups Oil and E21 (critical significance=0.02, significance=0.04) and Oil and E250 groups (critical significance=0.02, significance=0.012). The caloric intake of the two estradiol groups did not differ during the baseline or the post-acquisition time periods. In addition, no significant interaction was detected between the two groups across the two days.

90

20 16

Oil

E21

E250

12 8 4 0

ab

Acq

Post-Acq

Acquisition Trial & Post-Acquisition Test

Trimmed Mean Total Calorie Intake

Trimmed Mean Sucrose Intake (ml)

Figure 5. Effect of Two Different Doses of Estradiol on Conditioned Taste Avoidance and Calorie Intake.

E21

Oil

E250

80 ab

70

ab

60 Base

Post-Acq

Baseline & Post-Acquisition Test

Figure 5 Caption. Left Graph. Trimmed mean sucrose consumption during the acquisition (Acq) trial and the postacquisition (Post-Acq) test for animals that received either sesame oil (Oil) or estradiol in the dose of either 1- (E21) or 50μg/kg (E250) on Acq day. aSignificant reduction in sucrose consumption across Acq and Post-Acq. b Significantly different sucrose consumption than Oil and E21 groups during PostAcq and significant interaction across Acq and Post-Acq when compared to the Oil and E21 groups. Right Graph. Trimmed mean total calorie intake of ovariectomized female rats during the baseline time period (Base) and the post-acquisition (Post-Acq) test day. For the Base time period, caloric intake was measured during the 24 and 48 hour periods prior to the injection of either sesame oil (Oil) or estradiol in the doses of 1-(E21) or 50μg/kg (E250) and a mean for these two days was computed. For the Post-Acq test day, caloric intake was measured during the 24 hour period that occurred 2 days following injection and that started at the onset of the post-acquisition test. Caloric intake during the Post-Acq test day included consumption of lab chow and sucrose while caloric intake during the Base time period included only lab chow. aSignificant reduction in total calorie intake across Base and Post-Acq . bSignificantly lower than Oil group during the Post-Acq trial day and significant interaction across Acq and Post-Acq when compared to the Oil group.

199

Discussion The results of Experiment 2 provide additional evidence against our hypothesis that the unconditioned effects of estradiol contribute to the conditioning effects of the hormone. Specifically, the findings indicate that although a low dose of estradiol produces significant reductions in eating, it does not produce a CTA. It is the case that the 1µg group did show a small, albeit non-significant, drop in sucrose consumption. However, all of the statistical outcomes strongly indicate that this slight drop is unlikely to be meaningful. First, the amount of sucrose consumed by the Oil and E21 groups during the acquisition and post-acquisition trials did not differ, neither the Oil nor the E21 group decreased consumption across acquisition and postacquisition testing, and there was no significant interaction across these two trials between these two groups. Second, the sucrose consumption of both of the Oil and E21 groups differed from the E250 group during the post-acquisition test but not the acquisition trial and there was a significant interaction across these two trials between the E250 group and both the Oil group and the E21 group. Taken together, these results clearly indicate that the E21 group did not develop a CTA. In contrast, the 1µg dose of estradiol produced significant reductions in calorie intake across the baseline and post-acquisition time periods. Furthermore, in contrast to the CTA findings, the caloric intake of both the E21 and E250 groups differed from the Oil group during the post-acquisition time period but not the baseline time period, both the E21and E250 group decreased intake across the two time periods but the Oil group did not, and there was a significant interaction across 200

these two time periods between each of the estradiol groups and the Oil group. Therefore, the results of Experiment 2 show that a 1μg dose of estradiol produces significant reductions in eating without producing a CTA, while a 50µg/kg dose of estradiol produces significant decreases in caloric intake and produces a CTA. Similar to the findings in Experiment 1, these data suggest that the unconditioned effects of estradiol do not account for the conditioning effects of the hormone. As a final note, utilization of both estradiol and estradiol benzoate revealed that they produced similar effects. At 50µg/kg, both types of estradiol were shown to diminish calorie intake and to condition an avoidance of sucrose. This consistency across hormone variants increases confidence in the veracity of the effects we have demonstrated. 4.5.3 Experiment 3 PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis Correlation between expression of hypophagic and CTA based on non-contingent pairing of the CS and US (Strategy 3). As a final means of determining whether the unconditioned effects of estradiol on eating contribute to the CTA observed after pairing this hormone with a novel sucrose solution, we employed the third strategy outlined in the introduction. This strategy involved manipulating the length of the interval between consumption of sucrose and administration of estradiol. Contingent pairing of the taste solution with estradiol should not be a requirement for expression of the apparent CTA if unconditioned effects of estradiol play a role in this expression. In CTAs, 201

associations between the CS tastants and US toxins still occur if the interval between the CS and US is as long as 4-12 hours. The earliest studies exploring this phenomenon showed that an apomorphine CTA towards a saccharin solution could be formed with an inter-stimulus interval of 1.4 hours (Garcia, Ervin & Koelling, 1966). Other laboratories reported significant aversion to a 4% sucrose solution when exposure to x-radiation was administered 6 hours following the CS presentation and gustatory avoidance was readily established toward a 0.1% saccharin solution when the CS and the x-radiation US were separated by as much as 12 hours (Smith & Roll, 1967). However, administration of a US after a certain interval fails to result in CTA. For instance, CS-US intervals greater than 12 hours do not support radiation CTAs (Smith & Roll, 1967) and a 24 hour inter-stimulus interval prevents acquisition of a lithium chloride CTA (Houpt & Berlin, 1999). Predicated on this feature of CTAs, we proposed to find an interval that does not support an estradiol CTA. A 50μg/kg dose of estradiol was used as the US since this dose was demonstrated to produce strong unconditioned effects. Based upon pilot data, the CS-US interval 24 hours was chosen and the post-acquisition test was held approximately 2 days following the US administration. If the strength of the unconditioned effect is the only factor determining whether a CTA is detected 47 hours later, then regardless of the CS-US interval, an injection of a 50μg/kg dose of estradiol should produce significant reductions in sucrose consumption. On the other hand, if this interval fails to result in an estradiol CTA, just as they do with radiation and LiCl CTAs, then this would support a conditioning hypothesis instead. 202

Methods Twenty two animals were randomly assigned to one of the following two groups (n=11 per group): (1) E250-24, which received an injection of estradiol 24 hours following the 1 hour exposure to the CS on acquisition day or (2) Oil-24, which received an injection of sesame oil 24 hours following the CS on acquisition day. The post-acquisition test for this experiment was held 47 hours following the injections administered on acquisition day. Results A 24 hour CS-US interval did not support an estradiol CTA (see Figure 6). Analysis of the acquisition and post-acquisition test data revealed that neither the Oil24 nor the E250-24 showed a difference in the amount of sucrose drunk across the two days. In addition, the E250-24 group did not differ statistically from the Oil-24 group in the sucrose consumption levels during the acquisition or post-acquisition tests and no interaction effects were detected between the two groups across the two days.

203

Trimmed Mean Sucrose Intake (ml)

Figure 6. Effect of a 24 hour CS-US Interval on Estradiol Conditioned Taste Avoidance. 20

Oil-24 16

E250-24

12 8 4 0 0

Acq

Post-Acq

Acquisition Trial & Post-Acquisition Test Figure 6 Caption. Trimmed mean sucrose consumption on acquisition (Acq) and post-acquisition (Post-Acq) days for animals that received either a 50μg/kg dose of estradiol (E250-24) or sesame oil (Oil-24) 24 hours following the 1 hour exposure to sucrose on Acq day. No significant differences were found.

Discussion Consistent with Experiments 1 and 2, the results of Experiment 3 also failed to support the hypothesis that the hypophagic effects of estradiol on eating contribute to CTAs induced by this hormone. Instead, they strongly suggest that CTA expression solely is due to the conditioning capability of estradiol. If reductions in sucrose consumption observed during post-acquisition testing were due entirely to the unconditioned effects of the hormone, then uncoupling sucrose and estradiol should still result in reductions of sucrose consumption when tested during the time that estradiol produces its unconditioned hypophagic effects, especially when a potent 50µg/kg dose of estradiol is used. The results of Experiment 3 demonstrate that this is not the case. A 24 hour CS-US interval prevented the expression of a CTA. The post-acquisition test for this interval was held 47 hours following the 204

injection of estradiol, when the unconditioned effects of estradiol are still evident. The results of Experiments 1 and 2 demonstrated that a 50μg/kg dose of estradiol produces significant reductions in caloric intake 47 hours after injection and other labs have shown hypophagia 24 to 72 hours following its administration (Tartellin & Gorski, 1973; Rivera & Eckel, 2005; Asarian & Geary, 2002; Santollo, Wiley & Eckel, 2007), suggesting that the unconditioned effects of the hormone were present at the time of post-acquisition testing. The lack of a CTA during a time when unconditioned effects of estradiol were present strongly suggests a dissociable mechanism for estradiol conditioning and estradiol hypophagia. The results of Experiment 1 showed that if the post-acquisition test is conducted at a time when the unconditioned effects of estradiol on calorie intake are no longer expressed (9 days after estradiol injection) animals show significant reductions in sucrose consumption, which indicates the acquisition of a CTA. As mentioned in the discussion of Experiment 1, these results could alternatively be accounted for by a slower decay in hypophagia expressed specifically towards sucrose than the hypophagia expressed towards lab chow. This hypothesis is refuted by the findings of Experiment 3. The animals with a 24 hour CS-US interval did not show a reduction in sucrose consumption during their post-acquisition test, which was given at a time when estradiol hypophagia is expressed. With estradiol not having an effect upon sucrose consumption in the near-term when the unconditioned effects are probably evident (47 hours after estradiol injection), it is unlikely that it has an effect in the long-term either (9 days after estradiol injection). 205

The results of Experiment 3 also address Secondary Aim 1 of this dissertation, which is to compare CTAs produced by estradiol to those produced by the putative illness inducing agent LiCl. It has been demonstrated that a 24 hour inter-stimulus interval precludes the acquisition of a LiCl (Houpt & Berlin, 1999). In Experiment 3, we demonstrated that this interval also was ineffective in producing a CTA, providing an additional piece of evidence as to how the CTAs produced by the two USs are similar. 4.5.4 Experiment 4 PRIMARY AIM 1 – SPECIFIC AIM 1.2: Testing the Negative Contrast Hypothesis and PRIMARY AIM 2 – SPECIFIC AIM 2.1: Determining the Longest CS-US Interval Supporting Estradiol CTA. Experiment 4 was conducted for two reasons. The first reason was to test the negative contrast hypothesis of estradiol conditioning (PRIMARY AIM 1-SPECIFIC AIM 1.2). Anticipatory negative contrast effects are not established when the interval between the presentation of the two stimuli exceed 30 minutes (Flaherty & Checke, 1982; Flaherty, Grigson, Checke & Hnat, 1991). If the reductions in sucrose observed after pairing it with estradiol are due to negative contrast, then CS-US intervals greater than 30 minutes should not result in the gustatory avoidance. In this experiment, intervals much greater than 30 minutes were used. The second rationale behind Experiment 4 was to determine the longest CSUS interval that supports an estradiol CTA (PRIMARY AIM 2-SPECIFIC AIM 2.1), 206

which would allow for the examination of the neurochemical basis of estradiol CTA. Since no data to date exists on estradiol CTA and extended CS-US intervals, several interval lengths needed to be tested to hone in on the interval of interest. As such, inter-stimulus lengths of 3, 6, 7, 8, 9, and 12 hours were tested in Experiment 4. Methods Groups One hundred seventy two animals were used in 4 separate studies to assess a 3, 6, 7, 8, 9, and 12, CS-US interval on the formation of an estradiol CTA. For each interval, two groups of animals were tested; one group was injected with sesame oil and another group was injected with 50μg/kg of estradiol after 1 hour access to a sucrose solution on acquisition day. Procedures Normally in experiments with no CS-US interval, the CS is given for the first hour of the dark cycle and immediately following this, each animal is injected with the US. The post-acquisition test is held 47 hours after the pairing. However, in order to keep the conditions during acquisition and post-acquisition trials as similar as possible for these studies with varying inter-stimulus intervals, the time between the US injection and post-acquisition testing had to be shortened for each group in proportion to the length of its CS-US interval. Experiment 4a. 3 and 6 hours Forty eight animals were randomly assigned to one of the following four groups (n=12 per group): (1) E250-3 or (2) E250-6, which received estradiol 3 or 6 207

hours, respectively, following CS exposure or (3) Oil-3 or (4) Oil-6, which received oil 3 or 6 hours, respectively, following CS exposure. The post-acquisition test for the 3-hour and 6-hour CS-US interval was held 44 and 41 hours, respectively, following the injection administered on acquisition day as opposed to our standard 47 hours. The four groups in this study were a part of a larger study that included animals that received injections of LiCl. In order to minimize the number of animals used in the study, one control group was used for both the estradiol and LiCl experimental groups. As such, the Oil-3 and Oil-6 control groups received injections of both sesame oil and saline. In order to equate the control and experimental groups, the E250-3 and E250-6 groups also were given an additional injection of saline. The additional injection of saline did not affect the sucrose consumption of the Oil-3 or Oil-6 since these groups behaved similar to the other control oil groups. Sucrose consumption across acquisition and post-acquisition trials either stayed the same or increased for all of the Oil groups. For instance, the Oil-3, Oil-6, Oil-7, Oil8, and Oil-12 groups of Experiment 4 showed no changes in their sucrose consumption across the 2 days, while the Oil-9 group showed a significant increase in sucrose consumption across the 2 days (see Figure 7). Experiment 4b. 7 and 8 hours Fifty two animals were randomly assigned to one of the following four groups (n=13 each): (1) E250-7 or (2) E250-8, which received estradiol 7 or 8 hours, respectively, following CS exposure or (3) Oil-7 or (4) Oil-8, which received oil 7 or 208

8 hours, respectively, following CS exposure. Post-acquisition testing was held 40 and 39 hours, respectively, following the injection administered on acquisition day. Experiment 4c. 9 and 12 hours Forty nine animals were randomly assigned to one of the following four groups: (1) E250-9 (n=13) or (2) E250-12 (n=12), which received estradiol 9 or 12, respectively, following CS exposure or (3) Oil-9 (n=12) or (4) Oil-12 (n=12), which received sesame oil 9 or 12 hours, respectively, following CS exposure. Postacquisition testing was held 38 and 35 hours following the injections administered on acquisition day. Experiment 4d. 6 hours (Replication) Statistical analyses (discussed below) revealed that the 6 hour interval was the longest interval that supported a strong estradiol CTA in the majority of the animals tested. Since all of our neurochemical studies were predicated on this interval, it was imperative to the reliability of this finding. Therefore, another study was conducted to test the reproducibility of the results. Twenty three animals were randomly assigned to one of the following two groups: (1) E250-6R (n=12), which received estradiol 6 hours following CS exposure or (2) Oil-6R (n=11), which received oil 6 hours after CS exposure. The post-acquisition test was held 41 hours following the injections administered on acquisition day. Additional Statistical Analyses As discussed in the beginning of the chapter, the longest CS-US interval was going to be used to ascertain whether neurotransmitter antagonists would block an 209

estradiol CTA. If we block an estradiol CTA with the antagonists, we want to be certain that it was because the infusions blocked the conditioning and not because the interval did not produce a CTA in some of the animals. Also, in order to find a site of infusion for the antagonists, we conducted Experiment 5, where we tested to see if lesions of the lateral PBN affected estradiol CTA with the CS-US interval determined in Experiment 4. At this point, it too is important that the majority of the animals acquire a CTA with the predetermined inter-stimulus interval. If lesioned animals do not express an estradiol CTA, we want to say with certainty that it was due to the lesion and not due to the possibility that those animals had not acquired a CTA. For instance, in the lateral PBN lesion study, we could have a situation in the intact estradiol control group where only 6 out of 12 animals develop a strong estradiol CTA, which would lead to a significant group decrease across acquisition and postacquisition day. This would indicate that the estradiol control group developed a CTA; however, this situation could lead to complications in the interpretation of our results. If we showed that the lateral PBN lesioned experimental group did not develop an estradiol CTA, we would not be able to conclusively state that the lesions obliterated the CTA since it is possible that some of those animals would not have developed the CTA to begin with due to the interval. In order to circumvent such a situation, we needed to choose an interval that would allow the majority, if not all, of the animals in the control estradiol CTA group to develop a CTA. As such, solely examining group effects in this experiment did not suffice and the number of animals that developed a CTA within each group needed to be considered. 210

Thus, the following secondary analyses were performed on the data. A strict set of criteria, outlined below, were set forth for the qualification of a CTA. A percentile bootstrap equivalent to a binomial analysis was used to compare the number of animals that developed a CTA in each experimental group to both its respective oil group and to other experimental groups. It should be mentioned that using this analysis has an important limitation that needs to be addressed. It allows only two groups to be compared at a time and does not control for family-wise error rate when multiple comparisons are made. This limitation is not catastrophic since methods, such as Rom’s method, have been developed for adjusting p-values in such situations (Wilcox, 2003). Since multiple comparisons were made, Rom’s method was used to adjust p-values for this part of the analysis. Thus, for this analysis, a significance level and a critical significance level based on Rom’s method is reported. A significant result is obtained when the significance level is lower than the critical significance level. Criteria for CTA Qualification The amount of sucrose drunk from acquisition to post-acquisition test and, if necessary, extinction test 2 was used to determine whether or not an animal acquired a CTA. These criteria were based mainly on data collected on estradiol CTAs and from control oil or saline groups in both the present studies and past studies conducted in our laboratory. CTA. A CTA was acquired if the animal displayed a decrease greater than 2ml across acquisition and post-acquisition test 1. If the animal had decreased by 2ml or 211

less on post-acquisition test 1, then on post-acquisition test 2, the animal had to display a decrease greater than or equal to 1ml. No CTA. An animal did not acquire a CTA if it displayed an increase, no change, or a decrease by 1ml in sucrose consumption across acquisition and the postacquisition test 1. There was also no CTA if an animal displayed a decrease by 2ml across acquisition and post-acquisition test and displayed no change or an increase on post-acquisition test 2. These specifications are summarized in Table 3 and work well given the present data. As will be discussed shortly, based on these criteria, the majority of the animals in the control groups did not develop a CTA. This does not appear to be due to the fact that the criteria for CTA were too stringent since for the shortest intervals, most of the experimental animals acquired a CTA. Also, it would be expected that as the CS-US interval increases, the strength of the CTA decreases since after a certain interval length, a CTA is no longer acquired (i.e. 12 hours for radiation (Smith & Roll, 1967). Based on our criteria, the data showed that as the inter-stimulus interval increased, the number o f animals acquiring a CTA decreased (for summary see Table 4).

212

Table 3. Criteria for Acquisition of a CTA: Changes in Sucrose Consumption Across Acquisition & Either Post-Acquisition Test 1 or Post-Acquisition Test 2.

Acquired a CTA Did Not Acquire a CTA

Acquisition to Post-Acquisition Test 1

Acquisition to Post-Acquisition Test 2

Decrease > 2 ml

------------------------------

Decrease = 2ml

Decrease > 1

No change

------------------------------

Any increase

------------------------------

Decrease = 1 ml

------------------------------

Decrease = 2 ml

No change or increase

Results Assessment of CTA Formation Experiments 4a-4d Analysis of the acquisition and post-acquisition test 1 data for each group revealed that none of the control groups developed a CTA (see Figure 7). The Oil-3, Oil-6, Oil-7, Oil-8, and Oil-12 groups showed no difference in the amount of sucrose drunk across the two days. The Oil-9 group showed an increase in sucrose consumption across acquisition and post-acquisition test 1 (critical significance=0.05, significance=0.018). Analysis of the experimental groups revealed that groups E250-3, E250-6, and E250-7 developed a CTA (critical significance=0.05, significance=0.00 for E250-3 and E250-6; critical significance=0.05, significance=0.04, for E250-7), while groups E250-8, E250-9, and E250-12 did not (see Figure 7). Comparisons of the experimental groups with their respective oil groups across acquisition and post-acquisition test 1 revealed that although the E250-3 and E250-6 groups did not differ from their respective control groups (Oil-3 and Oil-6, 213

respectively) in their acquisition trial sucrose consumption, they significantly differed in their levels of consumption during post-acquisition test 1 (critical significance=0.05, significance=0.00 in each case). Although according to betweengroup analysis the E250-7 group acquired a CTA, it did not significantly differ from its respective Oil group (Oil-7) during acquisition or post-acquisition test 1 consumption. Similarly, the E250-8, E250-9, and E250-12 groups did not differ statistically from their respective control groups in their sucrose consumption levels during acquisition or post-acquisition test 1. Since within-group analysis revealed that a 7 hour inter-stimulus interval produced a CTA, but between-group analyses showed that this group did not differ from its control group in sucrose consumption, it was questionable whether this interval should be used as the longest interval to produce a CTA. As mentioned in the methods section to this experiment series, it was imperative that the majority of the animals in the estradiol control groups of future lesion and neurochemical studies acquire a CTA. Therefore, a secondary set of analyses was warranted.

214

Trimmed Mean Sucrose Intake (ml)

Figure 7. Effect of Various CS-US Intervals on Estradiol Conditioned Taste Avoidance. 20

Oil-3 16

Oil-6

E250-3

Oil-7

E250-6

E250-7

12

a

8

ab

ab 4 0 0

Acq

Post-Acq

0

Acq

Post-Acq

0

Acq

Post-Acq

Trimmed Mean Sucrose Intake (ml)

Acquisition Trial & Post-Acquisition Test 20

Oil-8 16

E250-8

Oil-9

E250-9

Oil-12

E250-12

12 8 4 0 0

Acq

Post-Acq

0

Acq

Post-Acq

0

Acq

Post-Acq

Acquisition Trial & Post-Acquisition Test

Figure 7 Caption. Trimmed mean sucrose consumption during acquisition (Acq) and post-acquisition (Post-Acq) test 1 for animals that received a 50μg/kg dose of estradiol either 3 (E250-3), 6 (E250-6), 7 (E250-7), 8 (E250-8), 9 (E250-9), or 12 (E250-12) hours following the 1 hour exposure to sucrose on Acq day with their respective control oil groups. aSignificant reduction in sucrose consumption across Acq and Post-Acq tests for E250-3, E250-6, and E250-7 groups. bSignificant difference in sucrose consumption during Post-Acq test from Oil group for E250-3 and E250-6.

Results of Binomial Analyses Binomial analysis of the data revealed that the number of animals acquiring a CTA in the E250-3 and E250-6 groups significantly differed from the number of animals that developed a CTA in the Oil-3 and Oil-6 groups, (critical significance=0.0051, significance=0.003 and 0.0002, respectively). There were no significant differences between the number of animals that developed a CTA in the 7, 8, 9, and 12 hour interval groups and their respective controls.

215

Additional analyses with the 6 hour interval group revealed that the number of animals that developed a CTA in this group significantly differed from the number of animals that developed a CTA in the E250-8, E250-9, and E250-12 groups, each of which did not show a CTA using within-group analysis (critical significance=0.0051 for each, significance=0.00009-0.00132; see Table 4 and Figure 9). The number of animals that acquired a CTA in the E250-6 did not differ from the number of animals that developed a CTA in the E250-3 group, which did acquire a CTA according to within-group analysis. Comparison of the E250-7 group with each of the other experimental groups revealed that the number of animals that developed a CTA in this group did not differ from the number of animals that developed a CTA in groups E250-8, E250-9, and E250-12 each of which did not acquire a CTA according to within-group analyses (see Table 4 and Figure 9). Finally, there was a significant difference in the number of animals that acquired a CTA in the E250-6 and E250-7 groups (critical significance=0.05, significance=0.03). More animals acquired a CTA with the 6 hour interval than the 7 hour interval (see Table 4 and Figure 9). 6 hour CS-US Replication Study The findings for the E250-6 group were replicated in the E250-6R group. The E250-6R group showed a significant decrease in sucrose consumption across acquisition and post-acquisition test 1 (critical significance=0.05, significance=0.00), while Oil-6R did not (critical significance=0.05, significance= 0.432; see Figure 8). 216

Between-group analysis revealed that although the groups did not differ in the amount of sucrose they drank during the acquisition test, the differed significantly on the amount they consumed during post-acquisition test 1 (critical significance=0.05, significance=0.024). In addition, there was a significant interaction effect between the Oil-6R and E250-6R groups across these two tests (critical significance=0.05, significance=0.038). Binomial analyses showed that the number of animals that developed a CTA in the E250-6R group significantly differed from the number of animals that developed a CTA in the Oil-6R group (critical significance=0.05, significance=0.00, see Table 4 and Figure 9). Finally, for both the E250-6 and E2506R groups, 11/12 animals acquired a CTA.

Trimmed Mean Sucrose Intake (ml)

Figure 8. Replication of the Effect of a 6 hour CS-US Interval on an Estradiol Conditioned Taste Avoidance. 20

Oil-6R 16

E250-6R

12 ab

8 4 0 0

Acq

Post-Acq

Acquisition Trial & Post-acquisition Test Figure 8 Caption. Trimmed mean sucrose consumption during acquisition (Acq) and post-acquisition (Post-Acq) test 1 for animals that received a 50μg/kg dose of estradiol 6 (E250-6R) hours following the 1 hour exposure to sucrose on Acq day with its respective control oil group (Oil-6R). aSignificant reduction in sucrose consumption across Acq and Post-Acq tests. bSignificant difference in sucrose consumption during Post-Acq test from Oil group.

217

Table 4. Number of Animals that Acquired or Did Not Acquire an Estradiol Conditioned Taste Avoidance Based on Criteria for Conditioned Taste Avoidance Qualification. Group Oil-3 E250-3

CTA 2 10

No CTA 10 2

Oil-6 E250-6

2 11

10 1

Oil-7 E250-7

2 7

11 6

Oil-8 E250-8

3 3

10 10

Oil-9 E250-9

0 2

12 11

Oil-12 E250-12

0 3 Replication Study Oil-6R 3 E250-6R 11

12 9 8 1

Figure 9. Number of Animals in each Estradiol and Oil Group that Acquired or Did Not Acquire Estradiol Conditioned Taste Avoidance for Each CS-US Interval.

Number of Animals

15

15

CTA

No CTA

CTA

10

10

5

5

0

E2-3

E2-6

E2-6R

E2-7

E2 -8

E2-9

E2 -12

0

O-3

O-6

O-6R

No CTA

O-7

O-8

O-9

O-12

Groups Figure 9 Caption. Left Graph. Number of animals in each of the estradiol groups in Experiments 4a-d that acquired or did not acquire a CTA when the CS-US interval was 3 (E2-3), 6 (E2-6, E2-6R), 7 (E2-7), 8 (E2-8), 9 (E2-9) or 12 (E2-12) hours. All animals received the same dose of estradiol (50µg/kg). As the interval increases, the number of animals that acquired a CTA decreased, while the number of animals that did not acquire a CTA increased.

218

Right Graph. The number of animals in each of the oil groups in Experiments 4a-d that acquired or did not acquire a CTA when the CS-US interval was 3 (O-3), 6 (O-6, O-6R), 7 (O-7), 8 (O-8), 9 (O-9) or 12 (O-12) hours. Regardless of interval length, the number of animals that acquired or did not acquire a CTA stayed relatively the same.

Discussion One of the goals of Experiment 4 was to test whether intervals greater than 30 minutes would still result in an estradiol CTA. It was determined that even an interstimulus interval of 3 and 6 hours resulted in a strong CTA. This finding refutes the hypothesis that reductions in sucrose consumption following its pairing with a CS are due to negative contrast effects. This finding further solidifies estradiol as an effective US in a CTA learning paradigm. The second goal of the present experiment was to determine the longest CSUS interval that would support an estradiol CTA. Although the E250-7 group showed significant reductions in sucrose consumption across acquisition and postacquisition test 1, it was decided that 6 hours is the longest CS-US interval to support an estradiol CTA given that (a) between-group analyses did not find a difference in sucrose consumption between E250-7 and Oil-7 on acquisition and post-acquisition test days, (b) a binomial analysis revealed that the number of animals that acquired a CTA in the E250-7 group did not differ from the number of animals that acquired a CTA in the Oil-7 group, and (c) the number of animals that acquired a CTA in E250-7 did not differ from the number of animals that acquired a CTA in E250-8, E250-9, and E250-12,

219

all of the groups that did not acquire a CTA according to within-group analyses conducted on the data. Therefore, the decision was made to use the 6 hour interval as the longest interval that supported a strong estradiol CTA. This interval was tested in two different batches of animals and in both experiments this interval produced a strong CTA. In fact for both studies, 11 out of 12 animals acquired a CTA. In conducting the study to ascertain the effects of antagonists on the estradiol CTA, the receptors of interest probably should be administered 5.75 hours after the CS exposure (see Figure 10). The antagonists will be delivered 5.75 hours after the CS presentation instead of 6 hours since ideally one should allow some time for the antagonist to take effect in case the effect is not immediate. This inactivation should last for 2 hours or 7.5 hours after CS exposure. Since there is some evidence of CTA with a 7 hour CS-US interval, extending the receptor blockage time to cover the 7 hour time period would be wise. The estradiol injection should be made 6 hours following the CS and 15 minutes following infusion of the antagonist. As such, we can be certain that at any time when estradiol is capable of producing a CTA, the receptors of interest will be inactivated. Although not a specific goal of the experiment, the results of Experiment 4 also failed to support the hypothesis that the hypophagic effects of estradiol on eating contribute to CTAs induced by this hormone. Like Experiment 3, they strongly suggest that CTA expression solely is due to the conditioning capability of estradiol. The results of the present study demonstrated that an 8, 9, and 12 hour CS-US 220

interval prevented the expression of a CTA. The post-acquisition tests for these intervals were held 35-39 hours following the injection of estradiol, which is during the time when the unconditioned effects of the hormone still are evident (results of Experiment 1, Tartellin & Gorski, 1973; Rivera & Eckel, 2005; Asarian & Geary, 2002; Santollo, Wiley & Eckel, 2007). This suggests that a CTA was not acquired despite the unconditioned effects of the hormone providing evidence for the dissociation between estradiol conditioning and estradiol hypophagia. Figure 10. Methodology for Determining Chemical Mediation of Estradiol Conditioned Taste Avoidance. Weak CTA  No CTA Time receptors will be kept inactivated

CTA

CS trace

2

3

4

5

6 7 Estradiol Time between CS-US interval (hrs)

8

Figure 10. Depicts the methodology that will be used to test the role of acetylcholine and histamine in estradiol induced CTAs. Six hours is the longest CS-US interval that supports an estradiol CTA. As such, acetylcholine and histamine antagonists that are active for at least 2 hours, will be infused 5.75 hours following the CS. The estradiol injections will be made approximately 15 minutes following the infusions of the antagonists.

221

4.5.5 Experiment 5A PRIMARY AIM 1- SPECIFIC AIM 1.1: Testing the Unconditioned Hypothesis Determining Whether Lateral Parabrachial Lesions Block Estradiol CTAs and Hypophagia and PRIMARY AIM 1: Testing the Aversive Hypothesis of Estradiol CTA and PRIMARY AIM 2 – SPECIFIC AIM 2.2: Determining the Ability of Lateral Parabrachial Lesions to Block Estradiol CTAs with Extended CS-US Intervals. In Experiment 5, animals with excitotoxic (Experiment 5a) and electrolytic (Experiment 5b) lesions of the lateral PBN were tested for estradiol CTA, LiCl CTA, and estradiol anorexia. Conducting this study was important for 4 reasons. First, it would determine whether lesions of the lateral PBN could eliminate estradiol CTA and/or estradiol anorexia thereby lending further support to the conclusion that the conditioned and unconditioned effects of the hormone are dissociated (PRIMARY AIM 1-SPECIFIC AIM 1.1). Second, it would help ascertain whether lesions of the lateral PBN would block an estradiol CTA when a 6 hour CS-US interval was used (PRIMARY AIM 2-SPECIFIC AIM 2.2). If so, then this would provide an infusion site for the neurotransmitter antagonists in the experiment examining the chemical mediation of estradiol CTA. In addition, if the lateral PBN is involved in estradiol CTA, then this provides one more piece of evidence that likens estradiol CTA to LiCl CTA (SECONDARY AIM 1) and will give insight into the underlying mechanism of 222

estradiol CTA (i.e. if estradiol conditions on the basis of its aversion-inducing properties; PRIMARY AIM 1-SPECIFIC AIM 1.3). Finally, Experiment 5 could potentially reveal a neural substrate for estradiol anorexia (SECONDARY AIM 2). Methods Groups Prior to histology, sixty-five rats were randomly assigned to one of the following four groups: (1) Intact Oil (n=12), which were neurally intact animals that received oil (2) Intact E (n=13), which were neurally intact animals that received estradiol (3) Lesion Oil (n=20), which were lesioned animals that received oil (4) Lesion E (n=20), which were lesioned animals that received estradiol Following histology, fifty-three rats were revealed to be in one of the following six groups: (1) Intact Oil (n=11), which were neurally intact and received oil (2) Intact E (n=13), which were neurally intact and received estradiol (3) Unilateral Oil (n=7), which were unilaterally lesioned and received oil (4) Unilateral E (n=6), which were unilaterally lesioned and received estradiol (5) Bilateral Oil (n=7), which were bilaterally lesioned and received oil (6) Bilateral E (n=9), which were bilaterally lesioned and received estradiol One Intact Oil, 6 Lesion Oil and 5 Lesion E animals were excluded from the statistical analyses due to either misplaced lesions, suboptimal lateral PBN sectioning, or poor staining. 223

Technical Difficulties with the Lesioning Technique Prior to discussing the method used to assess lesion accuracy, the technical difficulties encountered during the lesioning process will be addressed. In addition, we faced several other technical challenges that also are addressed in this section. 1. The Infusion Process 1a. The Problem. Laboratories that have performed ibotenic acid lesions of the lateral PBN have used micropipettes with a 40-60µm tip and a 10 minute infusion rate. However, for our experiment, blood dried at the tip of our micropipettes, thus clogging it. The clogging was facilitated by the slow infusion rate. As a consequence, insufficient amounts of ibotenic acid were infused into the lateral PBN. The Solution. In subsequent excitotoxic pilot studies, our solution to this problem was to abandon the micropipettes for 30G cannulae with beveled tips. The wider tip allowed more ibotenic acid to be infused, while the beveled tips circumvented the problem of clogging the tip with blood or brain tissue. In addition, a faster infusion rate (i.e. 0.2µl/1min) was used which averted the problem of clogging due to dried blood without producing physical lesions at the infusion site. The faster infusion rate did not allow sufficient time for blood to clog at the tip and if it did, the faster rate probably helped to unclog the debris. 1b. The Problem. A second reason for our incomplete infusions was that our pump was not exerting enough pressure to push out the ibotenic acid from the clogged pipettes. This probably was because we had smaller PE tubing attached to the Hamilton syringe in the pump and that was attached to larger PE tubing attached to 224

the micropipette. Going from the smaller PE to larger PE was potentially drowning the pressure that was being applied into the micropipette. The Solution. We attached the Hamilton syringe in the pump to the cannula via a single PE size tubing. As such, the pressure was not diluted. 2. Cresyl Violet Staining and Patchy NeuN Staining 2a. The Problem. Cresyl violet staining is typically used to assess the accuracy and extent of excitotoxic lesions. However, lesions are often difficult to read using this type of staining. In addition, due the complications faced during the infusion process, we anticipated small lesions, which would make the reading of the lesions even more difficult with cresyl violet. Finally, due to our lengthy behavior testing, gliosis, which is used as a marker for lesions with cresyl violet staining, would have faded and exacerbated the problem. The Solution. We needed to adopt a new staining method and needed to perfect the technique for optimal staining. We decided to stain for a compound that is found exclusively in neurons. As such, we chose to stain for neuron-specific nuclear protein (NeuN). 2b. The Problem. The lateral PBN is naturally heavily myelinated, which makes even NeuN staining of the pontine nucleus difficult. Reading lateral amygdala lesions with NeuN staining works flawlessly (see Figure 11). However, this was not the case with the lateral PBN. The Solution. Our protocol for NeuN staining had to be tailored specifically for the lateral PBN. The sections were sliced at 36-40μm instead of the usual 50μm 225

thickness. The incubation period for the primary antibody was extended (from 1 hour to 48-72 hours), the concentration was lowered (from 1:600 to 1:1000), and the temperature at the time of incubation was decreased (from room temperature to 4C). Figure 11. Neuron-Specific Nuclear Protein (NeuN) Stain of an Ibotenic Acid Lesion of Lateral Amygdala.

Figure 11 Caption. Magnified (4x) photomicrograph of a NeuN stained lateral amygdala from a rat with a unilateral ibotenic acid lesion. The extent of the lesion is outlined with dashed lines.

3. Insufficient Neuronal Death 3a. The Problem. In follow up pilot studies in which 30G cannulae and a 1 minute infusion rate were used to infuse ibotenic acid, the concentration of ibotenic acid was insufficient to kill all of the neurons in the lateral PBN. NeuN staining revealed surviving cells in the region, which made it difficult to read lesions. The Solution. We tested 3 potential solutions to this problem: (a) we increased the concentration of the ibotenic acid. Increasing the concentration helped kill more cells although there still was not a complete annihilation of the neurons at the infusion site, (b) we dissolved the ibotenic acid in a ringer solution that contained calcium. We hoped to facilitate neuronal death by increasing the influx of calcium 226

into the neuron. This did not alter the number of cells affected by the ibotenic acid, (c) we changed the neurotoxin from ibotenic acid to NMDA (0.02µl, 10µg/µl; dissolved in PBS). Unlike the ibotenic acid, the NMDA dissolved rapidly in PBS. No NaOH was necessary. The animals rapidly recovered from the anesthesia, i.e. 20 minutes following the surgery compared to the 2 hour recovery when ibotenic acid was used, and the NMDA killed all of the neurons at its point of infusion. Predicated on these pilot studies, for our next lateral PBN excitotoxic lesion study we will use 0.2µl/1minute NMDA (10µg/µl; solvent PBS) infused via 30G cannulae attached to a Hamilton syringe via PE1 tubing (Scientific Commodities Inc., Lake Havasu, AZ). Assessing Lesion Accuracy Due to the malfunction of the micropipettes, clear lesions of the lateral PBN were not obtained. Excitotoxic lesions should be easy to read with NeuN staining and the area intended for a lesion should not contain any neurons (see Figure 11). Although a paucity of cells could be seen in our lateral PBN sections, complete annihilation of the neural area was not obtained (see Figure 12). As a result, cell counts were performed for the lateral PBN to determine whether an animal was considered lesioned. Following NeuN staining, the lateral PBN sections of each animal were examined under a light microscope. For each animal that received infusions of ibotenic acid, the section that contained the maximum reduction in neurons in the lateral PBN was chosen for analysis. The option of analyzing more sections to assess 227

the extent of the lesions was considered, but eventually discarded since the lesions were not sufficiently extensive in the majority of the ibotenic acid animals. In 3 randomly selected ibotenic acid animals, a reduction in the number of cells (compared to a control) was observed only on the section that contained the maximum lesion. Differences in the number of neurons were not obtained in the sections anterior or posterior to the section with the maximum lesion. As such, only the section with the maximum reduction of neurons was analyzed. After choosing the section with maximum lesion for both the left and right sides, the AP level of each section was recorded (AP range -9.18 to -9.80) according to the Paxinos and Watson (1998) rat atlas. Next, sections corresponding to the same AP levels (AP range -9.18 to -9.8) were chosen from the PBS-infused animals. A minimum of ten sections from the control animals for each of the AP levels for each side was chosen. For instance, for animals that had their maximum lesion at AP -9.8 on the left side, 10 sections corresponding to AP level -9.8 on the left side of PBSinfused animals were chosen for cell count analysis. The same was done for the right side. Once the appropriate sections were chosen, magnified (10x) photomicrographs were taken and printed. The lateral PBN of each animal was divided into 5 subnuclei: dorsal, ventral, central, external, and crescent. Because the intermediate nucleus was difficult to identify in most sections, it was included in the central subsection of the lateral PBN. The neurons in each of the subnuclei were manually counted although only the total number of cells in the lateral PBN was used 228

for the analysis. During the cell counts, the experimenter was blind to the condition of the animal. Once the cells were counted, the photomicrographs were identified as either the ibotenic acid or PBS control and the data was recorded. Given the complications faced during the infusion process and given the subjective nature of performing cell counts, we decided to choose a conservative method of identifying lesions. For each AP level (both left and right sides) of the PBS-infused animals, the range, mean, 20% trimmed mean, median, and the 25% inter-quartile range was computed in order to assess the most conservative method of deciding whether an animal was considered lesioned. Based on these calculations, the lower end of the computed range was the most conservative number. Hence, an ibotenic acid animal was considered bilaterally lesioned if the cell count of that animal was lower than the lower end of the range of the PBS animals at that AP level for both the left and right sides.

229

Figure 12. Neuron-Specific Nuclear Protein (NeuN) Stain of Ibotenic Acid Lesions of Lateral Parabrachial Nucleus.

A

B

C

Figure 12 Caption. Magnified (4x) photomicrograph of a NeuN stained parabrachial nucleus of a (A) sham lesioned PBS-infused animal , (B) unilaterally lateral PBN lesioned animal, and (C) bilaterally lateral PBN lesioned animal.

230

Procedures Followed During CTA Testing Two separate CTA procedures were conducted in these animals (CTA1 and CTA2). CTA1: Estradiol CTA1 consisted of 7 acquisition trials: 4 trials with a CS-US interval of 6 hours and 3 trials with 0 minute CS-US interval. 6 hour CS-US interval. 4 acquisition trials Initially, this study was set up to assess the involvement of the lateral PBN in estradiol CTA where the inter-stimulus interval was 6 hours. This interval was chosen based on Experiment 4 where it was discovered that 6 hours was the maximum CS-US interval that supported a strong estradiol CTA. Although in our previous experiment 6 hours was sufficient to produce a CTA and this was replicated in a second independent study, the Intact E control animals, which received an injection of estradiol 6 hours following the 1 hour access to sucrose, did not develop a CTA in the present experiment. Repeated acquisition trials with the 6 hour CS-US interval were given since it is demonstrated that multiple acquisition trials strengthen CTA (Garcia, Kimeldorf & Hunt, 1956). On each acquisition day, animals were given access to a 10% sucrose solution and 6 hours later were injected with either sesame oil or estradiol depending on their group assignment. Acquisition trials were held every 48 hours.

231

0 hour CS-US interval. 3 acquisition trials Because the Intact E animals did not acquire a CTA after 4 acquisition trials with the 6 hour inter-stimulus interval, it was decided that a 0 minute CS-US interval would be employed. Three additional acquisition trials were given where each animal received a 10% sucrose solution for 1 hour. Following the 1 hour CS exposure, each animal was immediately injected with either sesame oil or estradiol. Two days after the final acquisition trial, animals were given one extinction trial each day for 21 days. Measurements of Fluid Intake Throughout the CTA1 procedure, measurements of water intake also were recorded. Water intake from a 100ml cylinder bottle was measured every 24 hours immediately before the lights turned off. Some researchers have shown that lesions of the lateral PBN produce increases in general fluid intake (Edwards & Johnson, 1991), while others have reported reductions in neophobic responses to novel solutions like sucrose (Reilly & Trifunovic, 2001). As such, if lesioned animals drank increased amounts of sucrose on acquisition day compared to non-lesioned animals, the fluid intake data would help to decipher between these two possibilities. Additionally, if the lesioned animals drank more sucrose across extinction tests, the water intake data would help to determine whether this increased drinking was due to a disruption in general drinking behavior (i.e. polydipsia) or whether it was due to an attenuated CTA.

232

CTA2: Lithium Chloride. 1 acquisition trial After animals were extinguished from their first CTA, the eating portion of the experiment was conducted (discussed below). Following food consumption testing, each animal was given a second CTA. Two studies on second CTAs conducted in our laboratory have revealed that when animals extinguish from their first CTA, they can acquire a second CTA of equal strength (Ycaza, Lavond, and Chambers, unpublished data). All of the animals in the Lesion E group acquired an estradiol CTA although they drank significantly more sucrose than the Intact E group during extinction, a finding that was apparent before histology was conducted. At the time, this lack of acquisition effect was considered to be due to two possibilities: (a) our lesions were incomplete since we discovered many problems with our glass pipettes after the surgeries were conducted or (b) we made sufficient lesions, but unlike LiCl, an estradiol CTA does not work through the lateral PBN, which was part of our rationale for conducting the study. Since it consistently has been demonstrated that LiCl CTAs are eliminated following lesions of the lateral PBN, we decided to inject the Lesion E group with LiCl to see whether or not they could acquire a LiCl CTA. The following two groups were used: (a) Intact animals that had received estradiol during CTA1 and were given LiCl during CTA2 (Intact E-Li) and (b) Lesioned animals that had received estradiol during CTA1 and were given LiCl during CTA2 (Lesion ELi).

233

The second CTA with LiCl was conducted in the same manner as estradiol CTA1 with a few differences. First, only one acquisition trial was given because all of the animals acquired a CTA. Second, during acquisition, the animals were given access to NesQuik Reduced Fat Chocolate milk for 1 hour. Pilot data from our laboratory has shown that despite previously having developed a CTA to sucrose, animals will readily drink the chocolate milk. Additionally, we have shown that it can be used as an effective CS in a CTA paradigm when LiCl or estradiol is used as the US. Third, immediately following access to the CS, each animal received a 10ml/kg injection of 0.15M LiCl. Daily extinction tests were initiated 47 hours later and commenced until the groups had extinguished. Measurements of Food Intake After completion of CTA testing, the animals had to be moved to a holding vivarium and then to a new permanent vivarium in a new building. Therefore, in order to allow sufficient time for them to adapt to their new environment, food intake measurements were not initiated until approximately one month after the last extinction test for CTA1. The measurements were made every 12 hours, once immediately before the lights turned off and once immediately before the lights turned on, for 5 consecutive days.

234

Results CTA1: Estradiol: Assessment of CTA Formation 6 hour CS-US interval. 4 acquisition trials None of the estradiol groups acquired a CTA. However, while none of the estradiol groups exhibited a change in sucrose consumption across the first 4 acquisition trials, all of the oil groups showed an increase in consumption (see Figure 13). Within-group analyses revealed an increase in sucrose consumption from acquisition 1 (A1) to post-acquisition test 4 for each of the oil groups (PA4; critical significance=0.005 in each case, significance=0.00). For all of the estradiol groups, sucrose consumption during A1 was not significantly different from their consumption during PA4. Between-groups analyses of consumption during PA4 confirmed the difference in sucrose consumption between the estradiol and oil groups. For all but one comparison, sucrose consumption was higher in the oil groups than the estradiol groups during PA4 (critical significance=0.05, significance=0.28 for Bilateral E vs. Intact Oil; critical significance=0.02-0.033, significance=0.00-0.032 for remaining 8 estradiol and oil comparisons).

235

Trimmed Mean Sucrose Intake (ml)

Figure 13. Sucrose and Water Intake for Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus Across 7 Acquisition Trials of an Estradiol Conditioned Taste Avoidance. 24 22 20 18 16 14 12 10 8 6 4 2 0

Intact Oil Intact E

0

A1

Bi Oil Bi E

Uni Oil Uni E

PA1/A2

PA2/A3

PA3/A4

PA4/A5

a

a

b

PA5/A6

PA6/A7

PA7

Acquisition & Post-Acquisition Tests

Trimmed Mean Water Intake (ml)

50

Intact Oil Intact E

40

Uni Oil Uni E

Bi Oil Bi E

30 c

20 10 0 0

A1

PA1/A2

PA2/A3

PA3/A4

PA4/A5

PA5/A6

PA6/A7

PA7

Acquisition & Post-Acquisition Tests

Figure 13 Caption. The amount of sucrose (upper graph) or water (lower graph) drunk by animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus across the 7 acquisition trials of CTA1 that received either estradiol (E) or sesame oil (Oil). The first 4 acquisition trials (A1 to A4) consisted of a 6 hour CS-US interval, while the final 3 acquisition trials (A5 to A7) had a 0 hour CS-US interval. The post-acquisition trials for each acquisition training day (PA1 to PA7) were held 48 hours following the acquisition trial. aSignficant decrease in sucrose following the 0 hour pairing (PA4/A5-PA5/A6) for Intact E and Uni E group and across PA5/A6PA6/A7 for the Bi E group. bSignficantly less water than each of the Oil groups on PA7.

0 Hour CS-US interval. 3 acquisition trials None of the Oil groups showed a decrease in sucrose consumption across the final 3 acquisition trials (see Figure 13). According to within-group analyses, no

236

statistically significant changes in sucrose consumption were detected across the 3 acquisition trials for any of the Oil groups. Between-group analyses revealed that the three Oil groups did not differ from one another in the amount of sucrose consumed during any of the acquisition trials. Each of the estradiol groups developed a CTA but they differed in the number of trials required to do so. The Intact E and Unilateral E groups immediately developed a CTA following the first acquisition trial. Both groups showed decreases in sucrose consumption across acquisition 5 (PA4/A5) and post-acquisition test 5 (PA5/A6; critical significance=0.017, significance=0.001 for Intact E; critical significance=0.014, significance=0.00 for Unilateral E). On the contrary, the Bilateral E group required two acquisition trials before developing a CTA. This group did not show changes in sucrose consumption across A5 and PA5/A6 but showed a significant decrease in sucrose consumption across acquisition 6 (A6) and extinction 6 (PA6/A7; critical significance=0.014, significance=0.00) and across acquisition 7 (A7) and post-acquisition test 7 (PA7; critical significance=0.017, significance=0.015). Between-group analyses corroborated within-group analysis, namely that unlike the Intact E and Unilateral E groups, the Bilateral E group did not acquire a CTA after the first paired acquisition trial, but did develop a CTA following the second sucrose-estradiol pairing. None of the estradiol groups differed in the amount of sucrose they drank during acquisition of the first immediate pairing (PA4/A5). However, the Bilateral E group drank significantly more sucrose during post237

acquisition test 5 (PA5/A6) compared to the Intact E and Unilateral E groups (critical significance=0.01 in each case, significance=0.01-0.011). The estradiol groups however did not differ in their sucrose consumption levels during tests PA6/A7 or PA7. No significant interaction effects were detected between the estradiol groups across PA4/A5 through PA7. In addition, the Bilateral Oil group did not differ from the Intact Oil group in the amount of sucrose consumed across any of the acquisition trials. This suggests that the greater sucrose consumption of the Bilateral E group was not due to the effects of the lesions on sucrose consumption per se. Although the Bilateral E group did not acquire a CTA on the first 0 hour CSUS interval acquisition trial, similar to the Intact E and Unilateral E groups, it did significantly differ from each of the oil groups in the amount of sucrose it drank on days PA4/A5 and PA5/A6. The Intact E, Unilateral E, and Bilateral E groups drank significantly less sucrose on these days compared to the each of the Oil groups (critical significance=0.02-0.025, significance= 0.00-0.012 for PA4/A5; critical significance= 0.01-0.038, significance=0.00-0.021 for PA5/A6). Relationship between Sucrose and Water Intake For most comparisons, changes in sucrose consumption were not accompanied by concomitant changes in water intake. However, whenever a relationship between sucrose and water consumption was found, it was an inverse relationship.

238

6 hour CS-US interval None of the significant changes in sucrose consumption observed in the oil groups across A1 and PA4/A5 were accompanied by changes in water intake (see Figure 13). Similarly, on PA4 when each of the oil groups drank more sucrose than each of the estradiol groups (except for the Bilateral E vs. Intact Oil comparison), they each showed comparable amounts of water intake compared to the estradiol groups. 0 Hour CS-US interval The significant decreases in sucrose consumption across the first paired acquisition trial (PA4/A5 and PA5/A6) for the Intact E and Unilateral E groups were not accompanied by significant changes in water intake. The Bilateral E group did not show changes in sucrose consumption across PA4/A5 and PA5/A6, but did show significant reductions in sucrose across PA5/A6 and PA6/A7 and PA6/A7 and PA7. Water intake for this group did not differ across PA4/A5 and PA5/A6 and PA5/A6 and PA6/A7, but the Bilateral E group showed significant increase in water intake only across PA6/A7 and PA7 (critical significance=0.014, significance=0.00). In addition to not acquiring a CTA following the first 0 hour CS-US interval acquisition trial, the Bilateral E group drank more sucrose than the Intact E and Unilateral E groups during PA5/A6. However, the Bilateral E group drank similar amounts of water as the two other estradiol groups on PA5/A6. Similarly, no relationship between sucrose and water intake were found when the Bilateral E group was compared to each of the oil groups. The Bilateral E group drank less sucrose 239

than each of the oil groups on PA4/A5 and PA5/A6, but showed no difference in water intake compared to the control groups. During the test that followed the final acquisition trial (PA7), an inverse relationship was found in the amount of sucrose and water consumed by the estradiol and oil groups. The estradiol groups drank less sucrose but more water than the oil groups (see Figure 13). Between-group analyses showed that on PA7, each of the estradiol groups drank significantly less sucrose than each of the Oil groups (critical significance=0.012, significance=0.00 in each case), but drank significantly more water than the Oil groups (critical significance=0.012 in each case, significance=0.00-0.01). No statistical differences in sucrose or water consumption were detected among the Oil groups or among any of the estradiol groups on PA7. CTA1: Assessment of CTA Extinction Lesions of the lateral PBN alone had no effect on sucrose consumption. Between-group analyses revealed that the Bilateral Oil group did not differ from the Intact Oil in the amount of sucrose consumed during each of the 4 extinction phases (see Figure 14). In addition, no difference between these two groups was detected in the amount of sucrose consumed across the 4 extinction phases, that is, there was no significant interaction. Each of the oil groups drank significantly more sucrose than each of the estradiol groups during each of the 4 extinction phases (critical significance=0.0250.05 for Phases 1-4, significance=0.0 for Phase 1; significance=0.00-0.004 for

240

Phase 2; significance=0.0-0.008 for Phase 3; significance=0.0-0.03 for Phase 4, see Figure 14). Although the estradiol groups did not differ from each other in the amount of sucrose they drank during Phases 1 and 2, the Bilateral E group drank significantly more sucrose than the Intact E and Unilateral E groups during extinction Phases 3 and 4, (critical significance=0.025-0.05, significance=0.0-0.02). However, no significant differences in extent of the increase across phases 3 and 4 were detected among the estradiol groups. In addition, no significant interactions were detected across extinction phases 1 and 2 either for any of the estradiol groups. The higher levels of sucrose consumption displayed by the Bilateral E group during Phases 3 and 4 were not due to a general increase in fluid consumption. No statistical differences were detected in the amount of water consumed between the Bilateral E group and the Intact E or Unilateral E groups during Phases 3 and 4 (see Figure 14).

241

Average Trimmed Mean Sucrose Intake (ml)

Figure 14. Sucrose and Water Intake for Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus Across Extinction Phases of an Estradiol Conditioned Taste Avoidance. 40

Uni Oil Uni E

Intact Oil Intact E

35 30

Bi Oil Bi E

25 20 a

15

a

10 5 0 0

Phase1

Phase2

Phase3

Phase4

Average Trimmed Mean Water Intake (ml)

Extinction Phases 45

Intact Oil Intact E

40 35

Uni Oil Uni E

Bi Oil Bi E

30 25 20 15 10 5 0 0

Phase1

Phase2

Phase3

Phase4

Extinction Phases Figure 14 Caption. Amount of sucrose (upper graph) or water (lower graph) drunk by animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus across extinction phases of CTA1 that received either estradiol (E) or sesame oil (Oil). Each extinction phase consisted of 5 extinction days. Data on graph represents the trimmed mean of the sum for each phase. a Significantly more sucrose consumed compared to the two other estradiol groups on extinction phases 3 and 4.

Relationship between Sucrose and Water Intake As was true for the acquisition data, there was an inverse relationship between the amount of water and sucrose consumed by the oil and estradiol groups during

242

extinction. During Phases 1 and 2, when each of the Oil groups drank significantly more sucrose than each of the estradiol groups, they also drank significantly less water than each of the estradiol groups (critical significance=0.025-0.05 for Phases 1 and 2, significance=0.00-0.16 for Phase 1, significance=0.0-0.026 for Phase 2; see Figure 14). Similarly, although the Bilateral E group showed increased sucrose consumption during Phases 3 and 4 compared to the Intact E and Unilateral E groups, no statistical differences were detected in the amount of water consumed between these groups during these phases of extinction. CTA2: Lithium Chloride (LiCl): Assessment of CTA Formation Each of the groups developed a CTA following a single pairing of the CS with LiCl (see Figure 15). Within-group analyses showed that the Intact E-Li, Unilateral E-Li, and the Bilateral E-Li groups each showed a significant drop in chocolate milk consumption across acquisition and extinction day 1 (E1; significance=0.0, critical significance=0.05 in each case). Each of the groups reduced their drinking of the CS to the same level on E1 (see Figure 15). Between-group analyses showed that the Unilateral E-Li and Bilateral E-Li groups drank significantly more chocolate milk on acquisition day compared to the Intact E-Li group (critical significance=0.05 in each case, significance=0.26-0.049). However, despite this difference in acquisition day consumption, the three groups did not differ in the amount of CS consumed on E1.

243

In addition, no significant interaction was detected among the groups across acquisition day and E1. CTA2: Lithium Chloride: Assessment of Extinction The Unilateral E-Li and Bilateral E-Li groups drank significantly more chocolate milk than the Intact E-Li group on extinction days 2-4 (E2-E4; critical significance=0.033-0.05 for E2, significance=0.03-0.49; critical significance=0.0330.05 for E3, significance=0.03-0.04; critical significance=0.025 in each case for E4, significance=0.02-0.025; see Figure 15). No significant differences in the rate of increase in consumption were detected.

Trimmed Mean Chocolate Milk Intake (ml)

Figure 15. Chocolate Milk Intake in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus During Acquisition and Extinction of a Lithium Chloride Conditioned Taste Aversion. 20 18 16 14 12 10 8 6 4 2 0

Intact E-Li

Uni E-Li

Bi E-Li b b b b

a a a

0

Acq

E1

b b

E2

E3

E4

Acquisition Trial & Extinction Tests Figure 15 Caption. Amount of chocolate milk consumed by animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus across acquisition (Acq) and each extinction test (E1-E4) of CTA2 that received lithium chloride (LiCl). aSignificantly less sucrose from Acq. b Significantly more sucrose than the Intact E-Li group.

244

Comparison of Lesion Size between Lesioned Groups (Bilateral E versus Bilateral Oil) Comparisons of lesion size were made between the Bilateral E and Bilateral Oil-E groups. The lesion size of each animal was determined by the number of cells each animal had below the lower limit set for its corresponding AP level. Comparisons between the two groups were made for the right and left sides. Analysis revealed that the two groups did not differ in amount of damage the animals sustained in the left or side of the brain (see Figure 16).

Number of Cells Below Lower Range

Figure 16. Size of Lateral Parabrachial Nucleus Lesion in Bilateral E and Bilateral Oil Groups on Right and Left Sides of the Brain.

Right Bilateral E Bilateral Oil-E Left

-100

-80

-60

-40

-20

0

Figure 16 Caption. Represents the average number of cells below the lower limit set. No significant differences were detected in lesion size between the two groups.

Assessment of Food Decrement: 24 hour Within-group analysis of the 24 hour food measurement revealed that compared to baseline (Base) each of the groups, including the Oil groups, decreased their food consumption 24 (24E21) and 48 (48E21) hours following the first injection 245

(48E21 was also 24 hours after the second injection, 24E22; critical significance=0.017-0.02 for 24 hours, significance=0-0.004; critical significance=0.014-0.02 for 48 hours, significance=0.00 in each case; see Figure 17). However, 72 hours following the first estradiol injection (72E21/48E22), the Oil groups returned to baseline levels of eating, while food consumption for each of the estradiol groups remained significantly lower than baseline (critical significance=0.012-0.017 for 72 hours, significance=0.00 in each case). Between-group analysis showed that none of the groups differed from one another in the amount of food consumed during baseline. However, although 48 hours following the first estradiol injection (48E21/24E21), each of the oil groups showed reductions in eating compared to baseline, they still consumed more food compared to each of the estradiol groups during that time period (critical significance=0.02-0.025; significance=0.00-0.01). That is, the estradiol groups showed greater reductions in eating at this time period compared to each of the Oil groups. None of the Oil groups differed from one another in their food consumption levels during this time period. Similarly, the estradiol groups did not differ in their food consumption level during this 48E21/24E21 time period. Additional between-group analysis showed that each of the Oil groups consumed more food than each of the estradiol groups 72 hours following the first estradiol injection (72E21/48E22; critical significance=0.017-0.02, significance=0.0 in each case). Each of the estradiol groups sustained their reduced consumption until

246

96 hours after the first injection (96E21/72E22; critical significance=0.011-0.03, significance=0.00-0.013).

Trimmed Mean 24 hr Food Consumption (g)

Figure 17. Amount of Food Consumed During Twenty Four Hour Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus. 25

Intact Oil Intact E

Bi Oil Bi E

Uni Oil Uni E

20

15

10 Base/E2 1

24E2 1 E2 2

a a a ab ab ab

a a a

48E2 1 24E2 2

72 E2 1 48E2 2

a a a

96E2 1 72E2 2

120E2 1 96E2 2

DAYS

Figure 17 Caption. Twenty-four hour eating data for animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus that received either a 50µg/kg dose of estradiol (E2) or sesame oil (Oil). Baseline (Base) consisted of the average of the two 24 hour periods before the first estradiol injection (E21). A second estradiol injection (E22) in the same dose was administered 24 hours after E21. Comparisons in 24 hour food consumption were made between Base and 24 (24 E21), 48 (48 E21/24 E22), 72 (72 E21/48 E22), and 96 (96 E21/72 E22) hour post E21. a Signficantly different compared to baseline. bSignificantly different from control Oil groups.

Assessment of Food Decrement: 12 hour Dark Phase Within-group analysis of the 12 hour eating period for the dark phase revealed that when compared to baseline, each of the groups, including the Oil groups, decreased their food intake during the dark phase of the 48E21/24E22 hour period (critical significance=0.011-0.014, significance=0.00 in each case; see Figure 18). However, during the dark phase of the 72E21/48E22 hour period, the Oil groups either returned to baseline or surpassed their baseline levels of consumption, while each of the estradiol groups maintained a reduced consumption level. Statistical 247

analyses revealed that the Unilateral-Oil group had returned to baseline, while the Intact Oil and Bilateral Oil groups showed increased consumption levels (critical significance=0.013-0.017, significance=0.00-0.012) while each of the estradiol groups sustained their suppressed consumption (critical significance=0.011-0.14, significance=0.00-0.004). Between-group analysis showed that none of the groups differed in the amount of food they consumed during the baseline period. At the 48E21/24E21 time period, only the Intact E and Unilateral E groups showed lower food intake than at least one of the Oil groups (significant difference between Intact E and Intact Oil, critical significance=0.02, significance=0.00 and significant difference between Unilateral Oil and each of the Oil groups, critical significance=0.017-0.02, significance=0.00-0.016). Additional between-group analysis showed that each of the Oil groups consumed more food than each of the estradiol groups 72E21/48E22 (critical significance=0.017-0.02, significance=0.0-0.014). The estradiol groups maintained the reduced intake until 96E21/72E22 (critical significance=0.011-0.014, significance=0.00-0.004).

248

Trimmed Mean 12hr Food Consumption (g) Dark Phase

Figure 18. Amount of Food Consumed During Twelve Hour Dark Phase Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus. 20

Intact Oil Intact E

15

Uni Oil Uni E

a a a a ab ab

10

0

Base/ E2 1

24E2 1 E2 2

48E2 1 24E2 2

Bi Oil Bi E

ab ab ab

72E2 1 48E2 2

96E2 1 72E2 2

120E2 1 96E2 2

Days Figure 18 Caption. Twelve hour dark phase eating data for animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus that received either a 50µg/kg dose of estradiol (E2) or sesame oil (Oil). Baseline (Base) consisted of the average of the two 24 hour periods before the first estradiol injection (E21). A second estradiol injection (E22) in the same dose was administered 24 hours after E21. Comparisons in 12 hour food consumption during the dark phase were made between Base and 24 (24 E21/ E22), 48 (48 E21/24 E22), 72 (72 E21/48 E22), and 96 (96 E21/72 E22) hour post E21. aSignficantly different compared to baseline. bSignificantly different from control Oil groups.

Assessment of Food Decrement: 12 hour Light Phase Analysis of the 12 hour eating period for the light phase revealed that compared to baseline each of the groups, including the Oil groups, decreased their food intake during the light phase of the 24 hour period following the first injection (critical significance=0.014-0.03, significance=0.00-0.012; see Figure 19). For the Bilateral Oil group, this decrease lasted until the 48E21/24E22 hour period (critical significance=0.02, significance=0.00). However, the Intact Oil and Unilateral Oil groups had returned to their baseline levels of consumption by the 48E21/24E22 hour period, while the Bilateral Oil group returned to baseline by the 72E21/48E22 hour 249

period. On the other hand, the Intact E group maintained a reduced consumption level until the 72E21/48E22 hour period while the Unilateral E and Bilateral E groups showed reduced consumption until the 96E21/72E22 hour period (critical significance=0.012-0.017, significance=0.00 in each case). Between-group analysis showed that none of the groups differed in the amount of food they consumed during the baseline period. During the 48E21/24E21 period, each of the estradiol groups consumed less food than the Intact Oil and the Unilateral Oil groups (critical significance=0.02-0.025, significance=0.00-0.008). No significant differences were detected between the estradiol groups and the Bilateral Oil group during the 48E21/24E21 period. Additional between-group analysis showed that each of the Oil groups consumed more food than each of the estradiol groups in the light phase of the 72 hour period following the first injection (critical significance=0.017-0.025, significance=0.00-0.02).

250

Trimmed Mean 12hr Food Consumption (g) Light Phase

Figure 19. Amount of Food Consumed During Twelve Hour Light Phase Periods that Followed Estradiol Injections in Animals with Excitotoxic Lesions of the Lateral Parabrachial Nucleus.

Intact Oil Intact E

4

Uni Oil Uni E

2 ab ab ab ab

0

0

Base/ E2 1

24E2 1 E2 2

48 E2 1 24E2 2

Bil Oil Bi E

ab ab ab

72E2 1 48E2 2

96 E2 1 72E2 2

120 E2 1 96E2 2

Days

Figure 19 Caption. Twelve hour light phase eating data for animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus that received either 50µg/kg dose of estradiol (E2) or sesame oil (Oil). Baseline (Base) consisted of the average of the two 24 hour periods before the first estradiol injection (E21). A second estradiol injection (E22) in the same dose was administered 24 hours after E21. Comparisons in 12 hour food consumption during the light phase were made between Base and 24 (24 E21/ E22), 48 (48 E21/24 E22), 72 (72 E21/48 E22), and 96 (96 E21/72 E22) hour post E21. aSignficantly different compared to baseline. bSignificantly different from control Oil groups.

Discussion Due to the complications encountered during the experimentation process for Experiment 5a, solid conclusions could not be made based solely on the findings of this study. A more complete and clear picture develops when the results of Experiment 5a are combined with the results of Experiment 5b. As such, the findings of these two experiments are discussed in combination following the results of Experiment 5b.

251

4.5.6 Experiment 5B Since the lesioning technique in Experiment 5a was fraught with problems, subsequent pilot studies were conducted in order to correct for some of the complications. One of these pilot studies was conducted in order to determine whether electrolytic lesions would affect LiCl CTA, estradiol CTA, and estradiol anorexia. With the results of Experiment 5a, it was difficult to conclusively state whether the very confined bilateral lesions of the lateral PBN affected acquisition of the CTAs although they did accelerate extinction of both an estradiol and LiCl CTAs. Therefore, one of the purposes behind Experiment 5b was to determine whether more extensive lesions of the lateral PBN would preclude CTA acquisition when estradiol and LiCl were used as the USs. Further, in Experiment 5a, weak lesions did not appear to affect estradiol anorexia. As such, the secondary purpose of Experiment 5b was to determine whether the inability of lesions to affect estradiol hypophagia was due to insufficient lesions. If so, then the more extensive lesions should inhibit estradiol induced decrements in eating. Methods Groups Eight ovariectomized rats were assigned to one of the two following groups: (1) Bilateral E (n=6), which received bilateral electrolytic lesions or (2) Intact E (n=2), which received sham lesions

252

Following histology, the animals were divided into three of the following groups: (1) Bilateral E (n=3), which had accurate, extensive bilateral lesions of the lateral PBN (2) Unilateral E (n=3), which had accurate and extensive unilateral lesions of the lateral PBN (3) Intact E (n=2), which received sham lesions Assessing Lesion Accuracy Following NeuN staining, each of the sections was mounted on gelatin coated slides and cover slipped. Each section was then examined under a light microscope for physical damage in the lateral PBN (see Figure 20). Sections represented contain the maximum damage that was observed on any of the sections. Conditioned Taste Aversive Procedure Two separate CTA behavior tests were conducted in these animals. The second CTA was conducted after each animal had extinguished from the first CTA (if acquired). CTA1: Lithium Chloride. 1 acquisition trial Since it consistently has been demonstrated that lesions of the lateral PBN block LiCl CTA and since one of the purposes behind conducting Experiment 5a was to insure accuracy of placement, the decision was made to administer LiCl first. The first CTA was a LiCl CTA that consisted of a single acquisition trial. On acquisition day, each of the animals was given 1 hour access to a 10% sucrose solution.

253

Immediately following the CS exposure, each animal was administered an intraperitoneal injection of LiCl (10ml/kg of body weight). CTA2: Estradiol. 3 acquisition trials The second CTA was an estradiol CTA and consisted of three acquisition trials in an effort to keep things as consistent as possible between Experiment 5a and 5b. After animals were extinguished from their first CTA, preconditioning for the second CTA began. One month separated the final extinction trial of CTA1 and the first acquisition trial of CTA2. Conditioned taste avoidance 2 was conducted in the same manner as CTA1 with some minor differences: (a) three acquisition trials were given as opposed to one and (b) during acquisition the animals were given access to NesQuik Reduced Fat Chocolate milk in place of 10% sucrose for 1 hour. Immediately following access to the CS, each animal received a 50µg/kg injection of estradiol. Acquisition trials were held every 48 hours. Extinction tests were initiated 48 hours following the final acquisition trial and continued until the groups had extinguished, if a CTA was acquired. Measurements of Food Intake Two weeks following the final extinction test of CTA2, baseline measurements of food intake were initiated. Measurements were made every 24 hours immediately before the lights turned off. Five days of baseline intake were taken before the first estradiol injection. Two estradiol injections separated by 24 hours were made. Measurements were taken until animals reached baseline levels of 254

intake. The intake of the 5 baseline days was averaged and used as the baseline level of food consumption. Statistical Analyses Due to the low number of animals in each group (n=3 for bilaterally and unilaterally lesioned and n=2 for sham lesioned), formal statistical analyses could not be performed on the data. Despite the lack of analyses, the graphs and the percentages of the animals in each group that acquired a CTA reveal the clear deficits produced by electrolytic lesions. In addition, the criteria set for CTA in Experiment 3 were used to assess whether the animals acquired a CTA. Explanation of Procedures Prior to discussing the results, there is an important issue that should be addressed regarding Experiment 5b. First, we tested an estradiol and LiCl CTA with a 0 hour CS-US interval. We made the decision not test the effect of a 6 hour CS-US interval on estradiol CTA in these animals. Experiment 5b was conducted as a pilot study to insure that making more extensive lesions of the lateral PBN with the same coordinates as those used in Experiment 5a would block LiCl CTA. This would show that the coordinates we used in Experiment 5a were accurate. When the LiCl CTA was blocked, the decision was made to test for an estradiol CTA. Since the 6 hour CS-US interval did not produce a CTA in Experiment 5a, the decision was made to test for an estradiol CTA with no interval. If we had used the 6 hour CS-US interval and the animals did not acquire a CTA, it would have been unclear whether it was due to the lesions or whether that particular batch of animals was not going to 255

acquire a CTA with this longer CS-US interval. The reason for this is because the control group only included 2 animals, thus making it difficult to decipher between these two possibilities. Although we could have used a 6 hour CS-US interval in a third CTA, we have no information on the ability of animals to acquire a third CTA, so interpretation of the results would have been problematic. Results About the Electrolytic Lesions In addition to sustaining complete bilateral lesions of the lateral PBN, each of the lesioned animals sustained some collateral damage. These included unilateral destruction of the superior cerebellar peduncle (see Figure 20, C, D, & E), unilateral and potentially bilateral damage to the ventral portion of the medial PBN (see Figure 20, E), and potentially unilateral damage of the medial external PBN (see Figure 20, D). The lesions did not extend anteriorily past the lateral PBN.

256

Figure 20. Neuron Specific Nuclear Protein (NeuN) Stain of Electrolytic Lesions of the Lateral Parabrachial Nucleus. A. Lateral PBN

Superior Cerebellar Peduncle

Medial PBN

Medial Ext PBN

B.

C.

D.

257

Figure 20 Continued. Neuron Specific Nuclear Protein (NeuN) Stain of Electrolytic Lesions of the Lateral Parabrachial Nucleus. E.

Figure 20 Caption. Magnified (4x) photomicrographs of NeuN stained brains of animals with a sham lesion (A), an electrolytic unilateral lesion (B), or electrolytic bilateral lesions (C, D, & E) of the lateral parabrachial nucleus. Dashed lines were used to outline the lesioned structure and solid lines were used to outline the intact structure. Bilaterally lesioned animals also sustained damage to the superior cerebellar peduncle on one (C & D) or both (E) sides, potential unilateral lesion to the medial external PBN (D), and damage to the ventral portion of the medial PBN (E). Sections represented contain the maximum lesion that was observed on any of the sections.

CTA1: Lithium Chloride Assessment of CTA Formation As depicted in the graph, animals that sustained extensive electrolytic lesions of the lateral PBN did not show reductions in sucrose consumption across acquisition (Acq) and post-acquisition test (PA) and did not acquire a CTA (see Figure 21). On the contrary, the Intact Li and Unilateral Li groups both show reductions in sucrose consumption across the two days. According to our criteria set for CTA in Experiment 3, none (0/3) of the animals in the Bilateral Li group acquired a CTA. On the other hand, 100% of the animals in the Unilateral Li group (3/3) and the Intact Li group (2/2) developed a CTA. It should be mentioned that there was overlap between the Bilateral Li group

258

and the other two groups in the amount of sucrose consumed during the acquisition trial but no overlap during the post-acquisition test. Figure 21. Sucrose Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Acquisition and the First Post-Acquisition Test of a of a Lithium Chloride Conditioned Taste Aversion. Mean Sucrose Intake (ml)

30

Intact Li

25

Uni Li

Bi Li

20 15 10 5 0

0

Acq

Post-Acq

Acquisition Trial & Post-Acquisition Test Figure 21 Caption. Mean sucrose consumption across acquisition (Acq) and post-acquisition (PostAcq) for animals with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic lesion of the lateral parabrachial nucleus. These animals received a 10ml/kg dose of lithium chloride (Li) as the US.

CTA1: Lithium Chloride Assessment of Extinction All of the Bilateral Li animals continued to drink more sucrose than the Intact Li and Unilateral Li animals during each of the extinction trials (see Figure 22). In addition, the data suggest that the Unilateral Li group drank more sucrose in the later days of extinction than the Intact Li group. In the Unilateral Li group, all (3/3) of the animals had reached acquisition level consumption by extinction day 6 (E6). On the contrary, neither (0%) of the Intact Li animals had reached acquisition consumption

259

levels by E6. One of the 2 in this group extinguished on extinction test 9 (E9), while the other continued to show decreased sucrose consumption on E9. Figure 22. Sucrose Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Extinction of a Lithium Chloride Conditioned Taste Aversion.

Mean Sucrose Intake (ml)

40 35

Intact Li

Bi Li

Uni Li

30 25 20 15 10 5 0 0

E2

E3

E4

E5

E6

E7

E8

E9

Extinction Tests Figure 22 Caption. Mean sucrose consumption across extinction tests for animals with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic lesion of the lateral parabrachial nucleus. These animals received 10ml/kg dose of lithium chloride (Li) as the US.

CTA2: Estradiol Assessment of CTA Formation Animals that did not acquire a LiCl CTA in CTA1 also did not develop an estradiol CTA even after 3 pairings (see Figure 23). In the Bilateral E group, none (0/3) of the animals developed a CTA after the 3 pairings. Interestingly, 66% (2/3) of the animals in the Unilateral E group acquired a CTA after the first pairing, and 100% (3/3) of the animals acquired a CTA after the second pairing (similar to the

260

findings of the Bilateral E group of Experiment 5a). In the Intact E group, all (2/2) of the animals acquired a CTA after first pairing. Unlike the LiCl CTA1, all of the animals in the Bilateral E group drank more sucrose during both the acquisition trial and the post-acquisition test than all of the animals in the Intact E and Unilateral E groups. Figure 23. Chocolate Milk Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Acquisition and Post-Acquisition Tests of an Estradiol Conditioned Taste Avoidance.

Mean Chocolate Milk Intake (ml)

45

Intact E

40

Uni E

BiE

35 30 25 20 15 10 5 0

0

A1

PA1/A2

PA2/A3

PA3

Acquisition & Post-Acquisition Tests Figure 23 Caption. Mean chocolate milk consumption across acquisition (Acq) and post-acquisition (Post-Acq) tests for animals with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic lesion of the lateral parabrachial nucleus. These animals received 50µg/kg dose of estradiol (E) as the US.

CTA2: Estradiol Assessment of Extinction All of the animals in the Bilateral E group continued to drink more sucrose than all of the animals in the Intact E and Unilateral E groups throughout extinction testing (see Figure 24). Similar to the LiCl CTA extinction, the Unilateral E group drank more sucrose in the later stages of extinction compared to the Intact E group. 261

Each of the animals in the Unilateral E group drank more sucrose on extinction days 11 and 12 compared to each of the Intact E animals.

Mean Chocolate Milk Intake (ml)

Figure 24. Chocolate Milk Intake in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus During Extinction of an Estradiol Conditioned Taste Avoidance. 45

Intact E

40 35

Uni E

Bi E

30 25 20 15 10 5 0 0

E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12

Extinction Tests Figure 24 Caption. Mean chocolate milk consumption across extinction days (E2-E12) for animals with either a sham (Intact) lesion or a unilateral (Uni) or bilateral (Bi) electrolytic lesion of the lateral parabrachial nucleus. These animals received 50µg/kg dose of estradiol (E) as the US.

Assessment of Estradiol Hypophagia All of the groups reduced their food consumption after estradiol injection (see Figure 25). For the Intact E, Unilateral E, and Bilateral E groups, 100% of the animals had reduced their food intake 48 (48E1/24E1) and 72 (72E1/48E2) hours following the first estradiol injection.

262

Figure 25. Amount of Food Consumed During Twenty-Four Hour Periods that Followed Estradiol Injections in Animals with Electrolytic Lesions of the Lateral Parabrachial Nucleus.

Mean Food Consumption (g)

25.0

Intact E

22.5

Uni E

Bi E

20.0 17.5 15.0 12.5

0

Base/ E2 1

24E2 1 E2 2

48E2 1 24E2 2

72E2 1 48E2 2

96E 21 72E 22

120 E2 1 96E2 2

Days

Figure 25 Caption. Twenty four hour eating data for animals with no (Intact), unilateral (Uni), or bilateral (Bi) lesions of the lateral parabrachial nucleus that received a 50µg/kg dose of estradiol (E2). Baseline (Base) consisted of the average of the five 24 hour periods before the first estradiol injection (E21). A second estradiol injection (E22) in the same dose was administered 24 hours after E21.

Discussion The results of the behavioral tests for Experiment 5b are discussed in conjunction with those of Experiment 5a in the following section. However, the electrolytic lesions themselves need to be addressed prior to discussing the behavioral results. As aforementioned, in addition to sustaining complete bilateral lesions of the lateral PBN, each of the lesioned animals sustained some collateral damage. These included unilateral destruction of the superior cerebellar peduncle, unilateral damage to the ventral portion of the medial PBN, and potentially unilateral damage of the medial external PBN. The effect of unilateral lesions of structures on our results is not problematic. A previous study has demonstrated that unilateral lesions of even the entire PBN (the lateral and medial subdivisions combined, along

263

with damage to the superior cerebellar peduncle) did not affect acquisition of CTA (Gallo & Bures, 1991). As such, these extraneous unilateral damages probably were not responsible for the blocked acquisition of CTA (discussed below in results section) we obtained. The collateral damage that may be more worrisome is the potential bilateral damage caused to the superior cerebellar peduncle. It is difficult to speculate on the effect of such lesions on CTA acquisition. However, all 3 bilaterally lesioned animals behaved identically. That is, none of them acquired a CTA after a single pairing of LiCl with sucrose or after three pairings of estradiol with chocolate milk. If the bilateral lesions of the superior cerebellar peduncle were responsible for the abolition of CTA in one of the animals, then the two other animals that sustained only unilateral damage to this structure should have exhibited CTA. Therefore, the potential bilateral lesion of this structure does not change the conclusions of the study, namely that complete bilateral lesions of the lateral PBN block CTA whether LiCl or estradiol is used as the US. 4.5.7 Experiment 5 Discussion CTA1: Estradiol: 6 Hour CS-US Interval One of the reasons for conducting Experiment 5a was to determine whether inactivation of the lateral PBN would block an estradiol CTA if an extended CS-US interval was utilized. In the series of studies conducted in Experiment 4, it was determined that 6 hours was the longest inter-stimulus interval that supported a strong and reliable estradiol CTA. As such, Experiment 5a was conducted with the 264

intention of determining whether the lateral PBN was critical for an estradiol CTA when a CS-US interval of 6 hours was used. However, in Experiment 5a the first injection of estradiol did not produce a CTA in the Intact E control animals when it was administered 6 hours after the 1 hour exposure to sucrose. Given that multiple injections increase the strength of CTA (Garcia, Kimeldorf & Hunt, 1956), 3 additional injections were given in hopes of acquiring a CTA. Analysis of the data revealed that even after 4 injections, none of the estradiol groups developed a gustatory avoidance toward the CS. Although none of the estradiol groups developed a CTA, they also did not exhibit a change in sucrose consumption across the first 4 acquisition trials. On the contrary, each of the oil groups showed an increase in consumption across this same time period, a behavior that is often observed in control animals most likely due to attenuated neophobia, the palatability of the sucrose, and learned preference due to calories provided by the CS. As such, although the estradiol group did not show reductions in CS consumption, they also failed to show increases in sucrose consumption like the oil groups. This finding is indicative of a negative effect of the hormone injections that was not experienced by the control animals. This negative impact was sufficient to prevent increases, but insufficient in producing decreases. We can only provide speculations as to why the Intact E animals did not acquire a CTA with a 6 hour inter-stimulus interval. This interval was tested in two separate studies in Experiment 4 and in both instances produced a CTA in 11 out of 12 animals. One possible explanation is batch effects. Perhaps the batch of animals 265

used for Experiment 5a was less sensitive to estradiol and as a consequence, the inter-stimulus interval was too long to sustain a CTA. As mentioned above, the estradiol appeared to have some negative effect although it was not sufficient to produce decreases in sucrose. This result is consistent with the suggestion that the animals may have been less sensitive to the US injections. A second possibility is that the PBS infused into the lateral PBN produced some damage or alteration in the functioning ability of the neurons such that the CTA with the 6 hour CS-US interval was obliterated. It has been the observation of some researchers that iontophoretically infused PBS produces damage to neurons. A control group that received no infusion into the brain would have answered this question; however, no such group was included. An alternative way of conducting this study is discussed in the Future Directions section below and will include this control group. CTA1: Estradiol: 0 Hour CS-US Interval Given that none of the groups developed a CTA after 4 acquisition trials with a 6 hour CS-US interval, the decision was made to administer estradiol immediately following exposure to sucrose. Three such paired acquisition trials were given. The analysis revealed that unlike the Intact E group, the Bilateral E group did not develop a CTA after the first pairing. In fact, the sucrose consumption level of the Bilateral E group following the first paired acquisition trial lay between the Intact E and Intact Oil control groups. So even though the Bilateral E group differed from the Intact E group, it also did not behave like the Intact Oil control group. This may have been due to the fact that each of the oil animals had increased their consumption across the 266

first 4 acquisition trials, while the estradiol injections had precluded such alterations across the same time period. Nevertheless, animals with bilateral lesions did not acquire a CTA after one pairing. The second pairing was sufficient to produce a CTA in the Bilateral E group and the amount of sucrose the Bilateral E and Intact E groups drank on the second pairing did not differ from one another. Prior to discussing the potential reasons for the inability of the Bilateral E group to acquire a CTA following the first pairing, it is important to point out three factors that do not explain this finding. First, it is highly unlikely that this finding was due to the effects of lesions per se on sucrose consumption. The Bilateral Oil and Intact Oil groups did not differ in the amount of sucrose they drank across the acquisition trials. This means that the lesions alone did not increase sucrose consumption such that the Bilateral E group drank more sucrose in general and as a consequence did not express CTA. One also cannot argue that the Bilateral E group sustained more neural damage than the Bilateral Oil and as a consequence the lesions may have affected drinking since analyses performed on lesion size between these two groups revealed no significant difference. Finally, the lack of CTA in the Bilateral E group was not due to a general effect in drinking produced by the lesions. The animals in the Bilateral E group did not show changes in the amount of water they drank across the same time period. There are four possible hypotheses that could explain the inability of the Bilateral E group to acquire a CTA after the first pairing. The first two hypotheses involve the role of the lateral PBN in pre-exposure effects. First, this finding simply 267

may be due to either CS and/or US pre-exposure effects. Because the 6 hour CS-US interval was too long to support acquisition of a CTA in this batch of animals, the Bilateral E group, in essence, had received 4 non-contingent exposures to sucrose and 4 non-contingent injections of estradiol. Both types of pre-exposure regimens (CS and US) have been shown to attenuate CTAs. For example, non-contingent preexposure to the CS, attenuates subsequent formation of CTA in neurally intact animals (Benedict & Ayres, 1971; Rescorla, 2000). Similarly, distal pre-exposure (greater than 24 hours) to estradiol blocks a CTA produced by estradiol in neurally intact animals (Merwin & Doty, 1994). However, the data do not support this hypothesis. If pre-exposure effects precluded the Bilateral E group from acquiring a CTA after the first pairing, then this should have been the case for the Intact E group as well. The Intact E group also received 4 non-contingent CS and US exposures, but this group immediately acquired a CTA, suggesting that pre-exposure effects could not explain the inability of the Bilateral E group to develop a CTA after the first pairing. The second hypothesis also involves pre-exposure effects. This hypothesis purports that bilateral lesions of the lateral PBN either allow for the expression of pre-exposure effects or they enhance pre-exposure effects. As such, the Bilateral E group did not acquire a CTA following the first pairing while the Intact E group did. This hypothesis assumes that the lateral PBN exerts some inhibitory effect on preexposure effects such that when it is intact and activated, pre-exposure effects are not expressed and a normal CTA is acquired. On the other hand, its inactivation removes 268

the inhibition and allows the expression of pre-treatment effects. This assumption faces some opposition from the fact that neurally intact animals pre-exposed to estradiol show attenuated estradiol CTA (Merwin & Doty, 1994). If an intact lateral PBN prevented the expression of pre-exposure effects, then these neurally intact animals should not exhibit attenuated CTA when pre-treated with estradiol. A third possible explanation for the inability of the Bilateral E group to acquire a CTA after the first pairing is that this finding was a chance occurrence and that the finding is not reproducible. This is presented as a possibility due to the acquisition findings of LiCl CTA2, which are discussed below. The fourth hypothesis to account for the inability of the Bilateral E group to acquire a CTA after the first pairing is that the lateral PBN is involved in estradiol CTA. It is easier to discuss this possibility when the results of Experiment 5b also are taken into consideration. In Experiment 5b, complete electrolytic lesions of the lateral PBN were achieved and the extensive lesions prevented the acquisition of an estradiol CTA even after 3 acquisition trials were administered. Taken together, the results of Experiment 5a and 5b suggest that incomplete ablations of the lateral PBN weaken estradiol CTA such that more pairings are required to acquire the learned avoidance, while complete inactivation of the nucleus entirely obliterates the CTA. Although determining the particular role the pontine nucleus plays in estradiol CTA was outside the scope of Experiment 5, conjectures could be made based on extensive research that has been conducted on the role of the lateral PBN in LiCl CTA. These are discussed in the General Discussion section. 269

A solid conclusion as to which of these last two hypotheses best explains the acquisition finding should not be drawn in the absence of the extinction data. Examination of the extinction data revealed that the Bilateral E group drank significantly more sucrose than the Intact E group during the final 2 phases of extinction. As was true for the acquisition data, the increased consumption in sucrose was not an effect of the lesions per se since there were no differences detected in CS consumption between the Intact Oil and Bilateral Oil groups during these phases of extinction. Once again, the argument could be made that the Bilateral E group may have sustained more neural damage than the Bilateral Oil and as a consequence the lesions may have affected drinking; however, the two groups did not differ in lesion size. Furthermore, this increase in sucrose consumption during extinction was not due to a disruption in the general drinking mechanism. The Bilateral E and the Intact E groups did not differ in the amount of water consumption during these final 2 phases of extinction. Given that we have eliminated these possibilities as potential causes for increases in CS consumption during extinction and given that the Bilateral E group required more acquisition trials to develop a CTA, the conclusion that increases in sucrose during extinction are a reflection of an attenuated learned gustatory avoidance is viable. Taking the acquisition and extinction findings in combination, that more pairings are required to acquire the learned avoidance and that animals show increased CS consumption during extinction, the data lend support to the hypothesis that partial lesions of the lateral

270

PBN weaken estradiol CTA. This hypothesis is further supported by the finding that complete lesions inhibit CTA learning all together. CTA2: Lithium Chloride: 0 Hour CS-US Interval The intact and bilaterally lesioned animals that had received an estradiol CTA (Intact E and Bilateral E) also received a LiCl CTA as their second gustatory aversion (Intact E-Li and Bilateral E-Li). Analysis revealed that both of the groups acquired a LiCl CTA after one pairing of the chocolate milk with LiCl. Furthermore on acquisition day, the Bilateral E-Li groups drank more chocolate milk than the Intact E-Li but consumed similar amounts during the first post-acquisition test. In addition, there was no difference in the extent of decrease following the pairing, which suggests that the strength of acquisition for both groups was similar. The results of a previous study support this conclusion (Chambers and Wang, 2004). In that study, CTAs were induced with three different doses of LiCl (2, 10, and 20 ml/kg). The animals showed dose-dependent differences in the rate of increase in sucrose consumption during extinction such that the rate increased significantly with each decrease in dose. Comparisons between the two lowest doses revealed that there were no differences in the amount of sucrose consumption during the first four extinction tests but those animals given the lowest dose consumed more sucrose during the later stages of extinction. Thus, increased consumption of the CS during the later phases of extinction can reflect a weaker avoidance. Although water intake was not measured during the CTA2 procedure, the LiCl CTA2 animals already had been tested for changes in their drinking patterns 271

during estradiol CTA1. The data showed that partial bilateral lesions did not lead to increases in water intake. As such, the increases in CS consumption observed across the 4 extinction days in these animals cannot be due to alterations in the general drinking system. Once again, conclusions as to what these data mean are more easily drawn taking the results of Experiment 5b into consideration. Experiment 5b showed that complete electrolytic lesions of the lateral PBN eliminated LiCl CTAs. Taken together, the data suggest that partial lesions of the lateral PBN attenuate LiCl CTAs, which was expressed as increased CS consumption during extinction, while complete lesions eliminate the aversion produced by the putative illness-inducing agent all together. Estradiol (CTA1) and Lithium Chloride (CTA2) Direct statistical comparisons between the estradiol CTA1 and the LiCl CTA2 were not made even though they were tested in the same animals. There were three reasons for this decision. First, it was not known whether comparable doses of estradiol and LiCl were used. That is, we did not know whether a 50µg/kg dose of estradiol and a 10ml/kg dose of LiCl produced CTAs that were comparable in strength. The 10ml/kg dose of LiCl was chosen not on the basis of a comparison with the estradiol dose used, but on the basis of previous studies which have shown that lesions of the lateral PBN block LiCl CTAs when this dose is used. Second, three pairings of the CS with estradiol were made, while only a single pairing was made between the CS and LiCl. Third, the animals had received 4 non-contingent 272

sucrose and estradiol exposures immediately prior to receiving contingent pairings of sucrose and estradiol. Regardless of the lack of direct statistical comparisons, two differences between the Bilateral E and the Bilateral E-Li groups were clear. The first of these differences was that animals with identical neural lesions showed different behaviors for estradiol and LiCl CTAs. Previous research indicates that the doses of estradiol and LiCl used in Experiment 5 readily induce CTA after only one pairing. However, for the estradiol CTA in Experiment 5a, lesioned animals did not acquire a CTA after the first pairing, while one pairing was sufficient to acquire a CTA for LiCl. This suggests that the LiCl CTA was more easily acquired than the estradiol CTA. There are 4 possible explanations for this finding. One potential explanation for this was discussed in “CTA1: 0 Hour CS-US Interval” section above, namely that the finding that the Bilateral E group did not acquire a CTA after the first pairing was by chance and irreproducible. However, as discussed above, given all of the data, this possibility is not very likely. A second reason is that the area that was lesioned is involved in different aspects of estradiol and LiCl CTA. For instance, the area lesioned affects acquisition for estradiol CTA, but does not for LiCl CTA. This would suggest that the subnuclei of the lateral PBN that control estradiol and LiCl CTA acquisition differ. A third reason for the difference between the estradiol and LiCl acquisition behavior could be accounted for by the fact that the estradiol CTA was tested first, followed by the LiCl CTA. That is, the fact that the LiCl CTA was a second CTA might have affected the behavior of the animals. Research in our lab indicates that when the time interval between two 273

CTAs is short (1 week), the second CTA is weaker than the first CTA but when the time interval is long (9 weeks), the second CTA is stronger than the first CTA (Ycaza, Lavond, and Chambers, unpublished manuscript). The CS and US were the same for both CTAs and for both intervals. Given that the time interval between the two CTAs in Experiment 5a was 8.5 weeks, these second CTA data suggest that the greater facility with which the LiCl CTA was acquired was a function of the long interval. The fourth and most likely explanation for the difference in the number of acquisition trials required for estradiol and LiCl CTAs is dose effects. Studies in our lab have shown that a 50µg/kg dose of estradiol produces a CTA that is comparable in strength to a CTA produced by a 2-4ml/kg dose of LiCl. The dose used for LiCl CTA2 was 10ml/kg, which should produce a stronger CTA compared to the 50µg/kg dose of estradiol. Given that lesions of the lateral PBN probably attenuated the CTAs by reducing the effectiveness of both USs, the effectiveness of the 50µg/kg of estradiol potentially was reduced such that it necessitated a second acquisition, while the CTA produced by LiCl was strong enough and not sufficiently reduced to affect acquisition. Given this rationale, if a lower dose of LiCl had been used, we would have increased our likelihood of blocking the acquisition in CTA2. A second clear difference between the Bilateral E and Bilateral E-Li animals was that the animals recovered from the LiCl CTA much sooner than from the estradiol CTA. On average, the animals recovered from the LiCl CTA in four days, while it took about 15 days for them to recover from the estradiol CTA. This delayed extinction in the estradiol animals probably could be explained by the multiple 274

pairings the Bilateral E group received versus the single pairing of the Bilateral E-Li, although differences in dose strength could have played a role as well. Unilateral Lesions and CTAs In Experiment 5a, animals with unilateral lesions did not acquire an estradiol CTA after four 6 hour CS-US interval acquisition trials. However, this group acquired a CTA immediately following the first 0 hour CS-US acquisition. This finding is not surprising given that research on unilateral lesions of the PBN has shown that these lesions alone are ineffective in abolishing acquisition of CTA (Bielavska & Roldan, 1996). These unilateral lesions also did not affect extinction of the estradiol CTA. Not surprisingly, unilateral lesions did not affect acquisition of LiCl CTA either. Both the Unilateral E-Li and Intact E-Li groups acquired a CTA after a single pairing of the chocolate milk with LiCl and they did not differ in the extent to which they each decreased their consumption from acquisition to post-acquisition day. However, animals with these lesions showed increases in CS consumption during extinction when LiCl was used as the US. The Unilateral E-Li animals, similar to the Bilateral E-Li group, drank significantly more chocolate milk compared to the Intact E-Li group on each of the 4 extinction days. To our knowledge, the effect of unilateral lesions on LiCl has not been studied in previous investigations. This finding suggests that partial unilateral lesions attenuate LiCl CTA. The results of Experiment 5b corroborate this conclusion. The results showed that each of the animals in the Unilateral Li group had reached acquisition consumption levels by 275

extinction day 6, while each of the animals in the Intact Li group continued to show reductions in sucrose consumption up until at least extinction day 9. Although a strong possibility, the effects of unilateral lesions on LiCl CTA should be tested in a more extensive study which includes a larger number of animals (discussed in the Future Directions section below). Complete unilateral lesions of the lateral PBN also may attenuate estradiol CTA. This could be observed by the separation in the extinction data between the Intact E and Unilateral E groups in Experiment 5b, where the unilaterally lesioned group showed increased CS consumption in the later days of extinction. Each of the animals in the Unilateral E group drank more chocolate milk on extinction days 11 and 12 compared to each of the Intact E animals. This was observed despite the fact that the animals had received 3 pairings of estradiol with the CS. In addition, the effect of the unilateral lesion on LiCl CTA probably was more evident due to the fact that the animals had received only a single pairing. However, once again, the effect of unilateral lesions on estradiol CTAs should be tested in a more complete study with more animals. It is curious that in Experiment 5a animals with identical unilateral lesions of the lateral PBN showed different behaviors for estradiol and LiCl CTAs.

For

instance, partial unilateral lesions of the lateral PBN accelerated extinction of LiCl CTA but not estradiol CTA. There are two possible explanations for this. First, the Unilateral E animals received 3 acquisition trials compared to the one pairing the Unilateral E-Li animals received. The multiple acquisitions may have produced a 276

stronger CTA such that it would take a longer time to extinguish. Perhaps we would have observed an attenuated estradiol CTA in the Unilateral E group if we had extended extinction testing. The second explanation for this difference in the estradiol and LiCl extinction in unilaterally lesioned animals is that the area lesioned in the Unilateral E group affects extinction of LiCl CTAs, while it has no effect on estradiol CTA extinction. Given the findings of Experiment 5b, namely that complete lesions of the lateral PBN possibly attenuate both LiCl and estradiol CTA, this would suggest that the parabrachial subnuclei involved in the extinction of CTA are not different for the two USs. One finding from Experiment 5a that is difficult to explain is the fact that both partial bilateral and unilateral lesions of the lateral PBN affected LiCl CTA similarly. Both the Unilateral E-Li and Bilateral E-Li groups showed elevated CS consumption on acquisition day and increased sucrose consumption across the 4 extinction days compared to the Intact E-Li groups. However, this was not the case with the Unilateral Li and the Bilateral Li groups from Experiment 5b. The LiCl CTA was completely blocked in bilaterally lesioned animals. On the other hand, the unilaterally lesioned animals acquired the LiCl CTA, although it appeared to be weaker than the CTA exhibited by the intact controls as evidenced by higher levels of sucrose consumption in the later days of extinction. As such, one can argue that the reason the bilateral and unilateral lesions of Experiment 5a yielded similar results was due to some aspect of the lesions. It could be that the unilateral lesions were extensive enough on one side to produce attenuated CTAs comparable to those 277

produced by the less extensive bilateral lesions. However, examination of the extent of the lesions showed that the unilateral lesions were not more extensive than the lesions on either the left or the right side of the bilateral lesions. Another possibility involves our conservative method of determining lesions. It is possible that critical cells were lesioned on the “non-lesioned” side of the unilateral animals and as such the bilateral and unilateral lesions showed the same effect on LiCl CTA. Once again the specific location of the lesions in unilaterally lesioned animals may have played a role. Finally, the extinction rate for the LiCl was generally fast. The animals extinguished from the CTA in 4 days. As such, it may be that the extinction rate was too fast to detect a just-noticeable difference. Experiment 5b and CTAs: Some Important Issues One issue with Experiment 5b that should be addressed is that only a single pairing was made for the LiCl CTA. It has been argued that if lesions of certain structures block CTA learning when only one pairing is given, it is possible that the lesions only reduced the sensitivity of the animals to the US. One way researchers have circumvented this problem is to perform multiple acquisition trials. However, although these animals received one LiCl pairing, they did receive 3 estradiol acquisition trials. Despite the multiple pairings, the animals did not acquire an estradiol CTA even, suggesting that the lateral PBN lesions did not simply reduce sensitivity to the US. Nevertheless, replication of the study should include multiple pairings of the CS with LiCl (discussed below).

278

A second issue with Experiment 5b is that the bilaterally lesioned group drank more sucrose (CTA1) and chocolate milk (CTA2) during their first exposure to these CSs on acquisition day compared to the intact and unilaterally lesioned animals for both the LiCl and estradiol CTAs, respectively. Since water intake was not measured in these animals, it is difficult to tell whether these bilaterally lesioned animals drank more during the acquisition test due to attenuated neophobia or due to an alteration in their general drinking mechanism. Similarly, our study did not include a lesioned group that received an injection of oil or saline. As such, it is difficult to say whether the lesions per se affected sucrose consumption such that these animals did not express CTA. Based on the available data, it is not possible to ascertain why the bilaterally lesioned animals drank more sucrose on acquisition day. There is contradictory evidence in the literature regarding lesions of the lateral PBN and neophobic responses to novel CSs. Some studies have shown that lesions eliminate neophobic responses (Reilly & Trifunovic, 2001), while others have shown no effect (Sakai, Tanimizu, Sako, Shimura & Yamamoto, 1994). However, even if the bilateral lesions blocked neophobic responses to the CS, evidence in the literature suggests that the absence of a CTA in lateral PBN lesioned rats is not predicated upon the absence of a neophobic response (Reilly & Trifunovic, 2001). One study showed that lesions of the lateral PBN eliminated neophobia to alanine, but had no effect on neophobic reactions to capsaicin. However, the lesions blocked the CTA to both capsaicin and alanine. This finding suggests that the neophobia deficit in lesioned animals is independent of, rather than responsible for, the absence of 279

conditioned aversions in lateral PBN lesioned rats. If the absence of neophobia was responsible for the absence of CTA acquisition, then only the animals in which the neophobic response was eliminated (the alanine) should have shown CTA deficits. Therefore, in Experiment 5b, even if the increased CS consumption during the first acquisition test was due to elimination of the neophobic response, the bilaterally lesioned animals still should have been able to express a CTA. The fact that they did not express a CTA is consistent with the hypothesis that they were unable to acquire a CTA. Although it is difficult to ascertain whether complete lesions of the lateral PBN produced increases in sucrose consumption, evidence suggests that this too should not preclude the acquisition of a CTA. In general, there is no relationship between the amount of sucrose consumed during the first acquisition test and the ability to acquire a CTA. For instance, the Bilateral E-Li and Unilateral E-Li groups in CTA2 of Experiment 5a drank significantly more sucrose than the Intact E-Li group during the first acquisition test. However, the three groups consumed comparable amounts during extinction test 1 and no significant interaction was detected among the groups across acquisition and extinction test 1. Similarly, in Experiment 1, the EB50-D9 group drank more sucrose on acquisition day compared to the two other groups; however, this group still acquired a strong CTA. This suggests that elevated acquisition test consumption levels do not preclude the acquisition of a CTA if the learning has taken place nor do they predict the strength of a CTA that is acquired. Therefore, even if the increased amount of sucrose and 280

chocolate milk that were consumed by the bilaterally lesioned rats during the acquisition test of Experiment 5b were the result of increases in general levels of CS drinking, this should not have prevented them from acquiring the CTA. Summary of CTA Findings There are a few conclusions that can be drawn based on the combined CTA data of Experiments 5a and 5b. First, the data suggests that partial bilateral lesions of the lateral PBN attenuate both estradiol and LiCl CTA, while extensive lesions of the nucleus block acquisition of a CTA induced by both agents. Second, our data suggests that unilateral lesions of the lateral PBN also attenuate LiCl and possibly estradiol CTAs, although this claim warrants further investigation. Our data on CTA and the lateral PBN thus far replicate, but also extend on previous research. Research consistently has shown that both excitotoxic (Trifunovic & Reilly, 2002) and electrolytic (Sakai &Yamamoto, 1998) lesions block acquisition of a LiCl CTA. However, no study in the literature has examined the effect of lateral PBN lesions on estradiol CTA. To our knowledge, no study to this point has examined the effects of unilateral lesions of the lateral PBN on extinction of CTAs produced by either agent. Estradiol Hypophagia Relationship between CTA and Hypophagia. Each animal from Experiment 5a also was tested for the hypophagic effects of estradiol. The data showed that the lesions of the lateral PBN alone did not affect baseline levels of consumption since none of the animals differed in the amount of food they consumed during baseline. This probably was not due to the fact that the lesions in Experiment 5a were incomplete 281

since complete lesions of the nucleus did not affect baseline consumption levels either. Furthermore, the data revealed that despite exhibiting an attenuated estradiol CTA, the animals that sustained partial bilateral ibotenic lesions did not express attenuated hypophagic effects following the hormone injections. The Intact E, Unilateral E, and Bilateral E groups each significantly reduced their food consumption following estradiol injections and each recovered simultaneously from the suppressed levels of eating. This decrease in consumption was observed in the 12 hour dark, 12 hour light, and in the 24 hour food intake. This provides some evidence for the dissociative nature of the conditioned and unconditioned effects of estradiol. The lack of lesion effect on estradiol hypophagia may be due to two factors. First, once again it could have been due to the fact that our excitotoxic lesions of the pontine nucleus were not extensive enough to affect eating. Nevertheless, the limited area of the lateral PBN that was lesioned was sufficient to attenuate the estradiol CTA, but not the estradiol hypophagia, suggesting separate neural substrates for the two behaviors. The results of Experiment 5b provide evidence for this possibility. Even complete lesions of the lateral PBN left estradiol hypophagia undisturbed suggesting that the partial lesions of Experiment 5a could not account for why those animals continued to exhibit estradiol hypophagia. To our knowledge, no other study has examined the effects of the lateral PBN in estradiol anorexia. Second, food intake measurements for Experiment 5a were made one month following the end of the CTA1 procedure. This means that measurements of food intake were made approximately 73 days after the lesions were produced. 282

Given this lengthy period time between the lesions and the testing for food consumption, the possibility of recovery of function cannot be dismissed. The results of Experiment 5b do not support this possibility. Food intake measurements for Experiment 5b were initiated approximately 37 days following the electrolytic lesions. Although the interval between the lesions and the time of testing was much shorted in Experiment 5b compared to Experiment 5a, the effects on estradiol hypophagia were the same, namely that lateral PBN lesions left estradiol hypophagia undisturbed. As such, finding an effect of lateral PBN lesions on CTA but not hypophagia substantiates the conclusions of Experiments 1-3 that the conditioned and unconditioned effects of estradiol are dissociable. If the unconditioned effects of estradiol were responsible for the hormone’s ability to condition, then lesions of the lateral PBN should have eliminated both CTA and hypophagia induced by estradiol. However, the lesions eliminated CTA without affecting hypophagia. Oil Groups and Reductions in Food Intake. As reported in the results section of Experiment 5a, for the 24-hour eating measurement, each of the estradiol and oil groups showed significant reductions in food intake in the 24 and 48 hour periods following the first estradiol injection compared to baseline. However, in the 48 hour period following the first injection, each of the estradiol groups showed significantly greater reduction in eating compared to each of the oil groups. In addition, each of the oil groups recovered by the 72 hour time period following the first injection, while the estradiol groups continued to show reductions in eating during this time period. So, clearly there was a difference between the oil and estradiol groups. In 283

pilot studies, we have observed fluctuations in day-to-day consumption of ovariectomized female rats and statistical analysis of these increases and decreases sometimes yielded significance. It is likely that the reduction in consumption exhibited by the oil animals was simply part of the typical pattern of fluctuations. Experiment 5b and Estradiol Hypophagia: 24 hour Measurements. An important issue regarding Experiment 5b and estradiol hypophagia that should be addressed is that only 24 hour food measurements were made in place of 12 hour measurements as was done in Experiment 5a. The results of Experiment 5a indicated that the separate 12 hour dark and 12 hour light measurements did not contribute any noteworthy information that was not revealed in the 24 hour measurements. The lateral PBN lesioned animals in Experiment 5b showed significant reductions in food intake following estradiol injections and as such the lack of having dark and light phase eating data was not critical. Summary of Experiment 5 Findings In summary, Experiment 5 answered three out of the four questions it set out to answer. First, the findings determined that complete lesions of the lateral PBN block an estradiol CTA without affecting estradiol anorexia and hence the two behaviors are dissociable (PRIMARY AIM 1-SPECIFIC AIM 1.1). Second, experiment 5 provides one more piece of evidence that likens estradiol CTA to LiCl CTA (SECONDARY AIM 1) namely that partial lesions of the lateral PBN attenuate, while complete lesions eliminate CTAs. Third, it provided information regarding the neural substrates of estradiol anorexia (SECONDARY AIM 2), namely 284

that the lateral PBN is either not involved or it is one of several areas that can mediate estradiol anorexia and as such is not essential. Unfortunately, with Experiment 5, we were unable to ascertain whether lateral PBN lesions affect estradiol CTA when a 6 hour CS-US interval is used (PRIMARY AIM 2-SPECIFIC AIM 2.2). As such, we have not determined an infusion site for the neurotransmitter antagonists in the experiment in which we will examine the chemical mediation of estradiol CTA. 4.6 General Discussion Dissociating Estradiol CTA from Estradiol Hypophagia The results of all five experiments support the conclusion that the effects of estradiol on conditioning are separable from the effects of estradiol on food consumption. Experiment 1 showed that an estradiol CTA is formed at a time when unconditioned effects of the hormone have subsided. Experiment 2 demonstrated that even though a low dose of estradiol produced unconditioned reductions in eating, it did not produce a CTA. Experiments 3 and 4 showed that non-contingent pairing of sucrose with estradiol did not result in a CTA even though the post-acquisition test was held at a time when unconditioned effects of the hormone are still evident. Finally, Experiment 5 showed that lesions of the lateral PBN attenuate (Experiment 5a) or block (Experiment 5b) estradiol CTA without affecting estradiol hypophagia. Each of these findings provides evidence against the hypothesis that the unconditioned effects of estradiol contribute to the conditioning capability of estradiol. 285

It should be mentioned that research conducted with different species of animals also has attempted to demonstrate that estradiol’s hypophagic effect is separate from its conditioning abilities. Predicated on the finding that progesterone attenuates the unconditioned hypophagic effects of estradiol (Wade, 1975), Ganesan (1994) tested to see whether progesterone antagonized the conditioning ability of estradiol benzoate in Rockland-Swiss albino mice. If the unconditioned and conditioned effects of the hormone were not dissociable, then progesterone should antagonize a CTA produced by estradiol benzoate. The results of the study showed that progesterone did not alter an estradiol CTA, suggesting independent underlying mechanisms for the unconditioned and conditioned effects of estrogen. Although a clever approach, this study suffered from one major flaw. The study demonstrating the antagonistic effects of progesterone on estradiol hypophagia used female rats as subjects, while Ganesan used female mice. It could be that progesterone does not have the same attenuating effects on estradiol hypophagia in mice as it does in rats. However, this possibility was not tested in control animals in the Ganesan study. Ganesan (1994) also used another approach to establish the true conditioning ability of estradiol. As aforementioned, estradiol has been shown to produce reductions in eating across many species of animals Wade, 1972; Blaustein, Gentry, Roy & Wade, 1976; Morin & Fleming, 1978; Czaja & Goy, 1975;Czaja, 1975; Kemnitz, Eisele, Lindsay, Engle, Perelman, et al., 1984; Gilbert & Gillman, 1956; Cohen, Sherwin & Fleming, 1987). Interestingly however, this hormone has the opposite effect on eating in the Mongolian gerbil (Ganesan, 1994; Maass & Wade, 286

1977; Raible & Gorzalka, 1985). In a three part study, Ganesan (1994) showed that despite the hyperphagic effect of estradiol benzoate in these gerbils, the hormone produces a CTA in this species indicating that reductions in sucrose consumption cannot be due the unconditioned effects. However, one problem with this conclusion is that even if these results do represent a conditioning effect of estradiol, one is left wondering whether gerbils are an exception in showing dissociation of the unconditioned and conditioned effects of estradiol on eating as they are in exhibiting hyperphagia as the unconditioned response to estradiol. Another problem is that the dose of estradiol benzoate used to induce a CTA in one of the three experiments was an extremely high dose (50mg), while the doses used to test for the hormone’s effects on eating in a separate experiment were much lower (10mg). In the study examining the conditioning abilities of estradiol, the group used to control for unconditioned effects of the hormone (water paired with estradiol) drank significantly less sucrose compared to the control oil group (sucrose paired with oil) on test day, suggesting that the high dose of estradiol benzoate may have suppressed the consumption of sucrose. It should be mentioned that the lower sucrose consumption of the waterestradiol group on test day compared to the sucrose-oil group also could be a mere reflection of a neophobic response. Test day constituted the second sucrose exposure for the sucrose-oil group whereas it was the first exposure for the water-estradiol group. However, comparing sucrose consumption on first exposure for both of these groups suggests that the water-estradiol group showed a greater neophobic response to the CS than the sucrose-oil group, indicating a possible contribution of estradiol to 287

the reduced consumption. In any case, it is difficult to draw unambiguous conclusions from this study. Our research extends these previous conclusions by clearly demonstrating the conditioning ability of estradiol using doses that were shown to produce both conditioned and unconditioned effects in rats. What is Estradiol CTA? In this dissertation, we have demonstrated that the unconditioned effects of estradiol on eating do not account for the ability of the hormone to produce a gustatory avoidance. We also have discussed the unlikelihood that estradiol produces an avoidance to sucrose based on its reinforcing properties. So what property of estradiol allows it to induce avoidance? There are two alternative hypotheses that could account for the conditioning ability of estradiol. One is based on satiety while the other is based on the ability of estradiol, like LiCl, to produce an aversion. The first hypothesis of estradiol CTA is one based on conditioned satiety. It has been suggested that conditioned reductions in intake can result from states that differ from illness (Booth, 1977). For instance, pairing a food with a satietyproducing agent will elicit a learned reduction in the consumption of that particular food (Booth, 1985). Some of these agents include cholecystokinin, cocaineamphetamine regulated transcript, and glucagon-like peptide-1 (Deutsch & Hardy, 1977; Aja, Robinson, Mills, Ladenheim & Moran, 2002; Thiele et al., 1997). Given that estradiol is considered a long-term satiety agent, it is possible that it conditions on the basis of its satiety-inducing properties. That is, following its administration, it produces feelings of satiety rather than illness and as such produces conditioned 288

reductions in sucrose consumption. It has been demonstrated that CTAs are formed rapidly following the pairing of the CS with the US (Houpt & Berlin, 1999). A CTA is expressed 15 minutes following the pairing of saccharin with lithium chloride. If this holds true for estradiol as well, then in order for estradiol to condition on the basis of satiety, the animals must be in a state of satiation quickly following estradiol injection. However, a study conducted in our laboratory has shown that a 250µg/kg dose of estradiol benzoate does not produce reductions in eating during the interval beginning immediately following its administration until 1.5 hours or 11.5 hours after it was administered (Chambers and Hintiryan, 2009). Because females show hypophagia when measurements are made 24 hours after estradiol administration, satiety must be triggered sometime between 11.5 hours and 24 hours after estradiol administration. This would suggest that animals are not in a state of satiation immediately following the injection of estradiol. Based on the information at hand, one would assume that CTAs acquired following the pairing of a CS with estradiol probably are not due to conditioned satiety although further experiments are required to make that determination. For instance, the speed at which an estradiol CTA is formed would need to be determined. It may be that unlike LiCl, an estradiol CTA takes a much longer time to be established, which could bring the conditioned satiety hypothesis back into contention. However, the results of Experiment 5 provide evidence against the conditioned satiety hypothesis. We showed that despite estradiol anorexia expression, the hormone was ineffective in conditioning an avoidance to sucrose. If estradiol CTA was conditioned satiety, then an estradiol 289

CTA should have been expressed in lateral PBN lesioned animals since satiety by estradiol was still expressed in these animals. The second more probable hypothesis is that estradiol conditions on the basis of its aversion-producing properties. This aversive hypothesis purports that estradiol produces aversive “internal” feelings like nausea and malaise such that when a CS is paired with the hormone, the animals feel ill. As a consequence, the animals will acquire a conditioned taste aversion. In male rats, high doses of estradiol (100µg/kg; Ossenkopp, Rabi & Eckel, 1996) have been shown to produce aversive orofacial reactions such as gaping, a gag-like response which evidence suggests is a marker for internal aversive states (Parker, 1995). Therefore, the aversive hypothesis remains a possibility when high doses of estradiol are used as a US in a CTA paradigm. In addition, estradiol has been shown to produce nausea and vomiting in humans (Schou, 1968), which corroborates the aversive hypothesis. It is not known whether lower doses of estradiol (i.e. 50µg/kg) produce aversive orofacial responses. In addition, the taste reactivity study conducted with the high dose of estradiol used male rats, which have been shown to be more sensitive to estradiol compared to females (Gustavson, Gustavson, Young, Pumariega & Nicolaus, 1989). Therefore, this dose may not produce the same negative orofacial responses in females as it did in males. The results of Experiment 5 provide evidence for the conditioned aversive hypothesis for estradiol CTA. First, we showed that the lateral PBN was necessary for expression of estradiol CTA. The lateral PBN receives information regarding the 290

viscera from the area postrema, which processes information regarding nausea and malaise. As such, the lateral PBN is one of the relay stations in the ascending visceral pathway. Second, the lateral PBN also is essential for the expression of CTA produced by the putative illness inducing agent LiCl. This blockade of LiCl CTA following lateral PBN lesions is not due to a disruption in the associative process, but due to a disruption in the US information processing (see discussion below). Taken together, the data suggest that CTAs produced by estradiol are based on the hormone’s aversion producing properties. This is not surprising considering that the hormone produced nausea and emesis in humans (Schou, 1968). How is the Lateral PBN Involved in Estradiol CTA? The results of Experiment 5 demonstrate that an intact lateral PBN is essential for the expression of an estradiol CTA. Although the role this pontine nucleus plays in estradiol CTA was beyond the scope of the experiment, comprehensive research conducted on the lateral PBN and different agents suggest a disruption in US processing rather than an associative role. The most obvious of the evidence is the fact that the pontine nucleus is the second relay station for afferent visceral information (Lowey & Burton, 1979; Saper & Lowey, 1980). The lateral PBN (especially the external subnucleus) receives the greatest part of the afferents projecting from the AP (Lanca & van der Kooy, 1985; Shapiro & Miselis, 1985; Milner, Joh & Pickel, 1986; Miceli, Post & van der Kooy, 1987), which is critical for malaise information and emetic responses (Borison & Brizzee, 1951). Based on this

291

information, one could make a strong argument that the lateral PBN plays an essential role in US information processing for estradiol CTA as well. A second piece of evidence for this hypothesis is provided by research that has investigated the role of the nucleus in LiCl CTA. In addition to not acquiring a CTA, animals with lesions of the lateral PBN also are unable to learn other behaviors that involve LiCl-toxicosis. For instance, they are unable to develop a CTA towards a mild capsaicin solution (Reilly & Trifunovic, 2000), which the authors of the study claim is a non-gustatory trigeminal stimulus (Silver & Finger, 1991). As such, since the lateral PBN does not receive information regarding the trigeminal stimulus, disruption of the CTA cannot be due to a disruption in association. It should be noted that although capsaicin is considered by researchers to be solely a trigeminal stimulant (Reilly & Trifunovic, 2000; Shimura et al., 1997) there is some evidence that the agent may also activate taste receptors (Silver & Finger, 1991; Travers, Urbanek & Grill, 1999). To our knowledge, no studies investigating the effect of capsaicin on medial or lateral PBN activation have been conducted. Therefore, the possibility that some neurons in the lateral PBN mediate both trigeminal and US information cannot be dismissed. Another piece of evidence that suggests the abolition of CTAs following lateral PBN lesions is due to disruption in the transmission of malaise information is provided by a study that used morphine as a US. Ibotenic acid lesions of the lateral PBN precluded the formation of a CTA (Nader et al., 1996). These same animals also were unable to develop a morphine-elicited conditioned place aversion, and on 292

the basis of these results it was suggested that the lesions had interrupted the transmission of malaise information rather than disabling the associative process. This conclusion assumes that the lateral PBN does not process information regarding place, a possibility that has not been investigated. If the lateral PBN does process place information, then the inability of lesioned animals to acquire a conditioned place aversion may be due to the inability of the animals to process information regarding place and not the US properties of morphine. A final piece of evidence is that lesions of the lateral PBN impair CTAs produced by intraperitoneal injections of an AP-dependent muscarinic receptor antagonist methyl-scopolamine (Gallo, Arnedo, Agüero & Puerto, 1990; Berger, Wise & Stein, 1993), which crosses the blood-brain barrier; however, the lesions are ineffective against CTAs produced by intraventricularly (lateral ventricles) infused methyl-scopolamine (Cubero & Puerto, 2000). If the lateral PBN was involved in association, then the lesions should disrupt CTAs produced by the US delivered by both routes of administration. On the contrary, it only disrupted the CTA produced by the route that involves the malaise afferent pathway and not the intracerebroventricular route that bypasses it. Once again, based on one study, one cannot completely discard the association possibility. It could be that the lateral PBN, in addition to other areas like the amygdala and the insular cortex, serves as a site of association. If this is the case, then in the absence of the lateral PBN, these forebrain structures could substitute as the association areas. As a result, a gustatory

293

aversion could still be formed when a US is infused intraventricularly despite a dysfunctional PBN. What is Meant by “Attenuated CTA”? In Experiment 5a, it was reported that animals with weak bilateral lesions of the lateral PBN exhibited an attenuated estradiol CTA because they showed increased sucrose consumption across the final two phases of extinction. But what exactly is meant by the phrase “attenuated CTA” when there is increased consumption during extinction? There was a time when extinction was viewed as a function of the acquisition process. That is, the strength of acquisition was reflected in the extinction process such that stronger CTAs took longer to extinguish compared to weaker CTAs. Taking this view, it could be argued that the Bilateral E group from estradiol CTA1 exhibited increased sucrose consumption during the final 2 phases of extinction because the animals showed weakened acquisition, which also was evidenced by the fact that it took 2 pairings for this group to acquire an estradiol CTA. As indicated above, dose response studies with LiCl have shown that for some dose comparisons, the only difference detected is in the later phases of extinction and this most certainly can be interpreted as a difference in strength of acquisition (Chambers and Wang, 2004). Currently, however, a more complex view of extinction has been adopted, which must be taken into consideration. Extinction is no longer viewed as a function of just acquisition, but is a reflection of (1) the tastemalaise association, (2) the ability to retain the association in memory, (3) the facility with which the memory of the association is retrieved, and (4) the ability to relearn 294

that consumption of the taste substance is followed by positive consequences. This final point about extinction representing the ability of an animal to relearn replaces the older view that extinction is simply forgetting or unlearning the previous association. Evidence for this assertion comes from the observation that rate of reacquisition is faster after extinction, spontaneous recovery of the conditioned behavior after extinction training has been completed, and reinstatement of the extinguished behavior with unpaired presentation of the CS after the US is given alone (Bouton, 1993; Delamater, 1996; Rescorla, 2001). Taking all of this into consideration then, the increased CS consumption during phases 3 and 4 of extinction in the Bilateral E group might be a reflection of the strength of the avoidance acquired, but the possibility of it being a reflection of the extinction process alone should not be dismissed. Although animals in LiCl CTA2 (Bilateral E-Li) acquired CTAs comparable in strength to the control animals, they also showed increased CS consumption across their 4 days of extinction. This finding could mean that extinction was independent of acquisition strength since despite similar acquisitions, the animals showed different extinction behavior. On the other hand, it is the case that even though statistical analyses did not reveal significant differences between the bilaterally lesioned and intact animals in the strength of CTA acquisition, floor effects could have obscured any possible differences that otherwise would have been observed.

295

4.7 Future Direction Due to the complications we experienced in Experiment 5a and the low numbers of animals in Experiment 5b, a follow up experiment (Experiment 6), which will comprise of two studies, will be conducted. There are two reasons for completing such a study. First, it is important to verify our conclusion that the lateral PBN is essential for acquisition of estradiol CTAs when the CS-US interval is 0 hours but it is not essential for estradiol anorexia (Experiment 6a). Second, given that lesions of this structure abolish estradiol CTAs, we also need to determine whether this structure mediates estradiol CTAs when the CS-US interval is longer than 0 hours (Experiment 6b). If it does, this will give us a neural site for infusion of antagonists and will allow us to proceed with determining the chemical mediation of estradiol CTA. As such, the 6 hour CS-US interval, along with two other intervals, will be tested. Excitotoxic Lesions Excitotoxic lesions of the lateral PBN will be performed. However, in this experiment, NMDA (10µg/µl; solvent PBS) infusions in the amount and rate of 0.2µl/1minute will be made via a 30G cannulae. The cannulae will be attached to a Hamilton syringe via PE1 tubing (Scientific Commodities Inc., Lake Havasu, AZ), which will be situated in a micro-pump. Ibotenic acid will be replaced with NMDA since this chemical has been less problematic for us in the lab. For instance, it easily dissolves in PBS, it is stable and does not breakdown into a different compound like ibotenic acid, and NMDA 296

produces complete annihilations of the neurons at the infusion site in the lateral PBN. We recognize that other studies that have examined lesions of the lateral PBN on CTA have used ibotenic acid as the neurotoxin. However, if complete lesions of the lateral PBN are made (as indicated with NeuN staining) then replacing the toxin should not make a difference as long as there are no surviving neurons. Prior to conducting this major study, a pilot study will be conducted to test all parameters and coordinates for the lateral PBN and to ensure that the volume of NMDA is sufficient to lesion the entire lateral PBN and that NMDA is destroying all of the neurons at the site of infusion. Groups for Experiment 6a: 0 Hour CS-US Interval Sixty-four ovariectomized rats will randomly be assigned to one of the following 3 groups: (1) Intact Oil (n=12), which will be PBS infused animals that receive oil (2) Intact E (n=12), which will be PBS infused animals that receive estradiol (3) Lesion Oil (n=20), which will be NMDA lesioned animals that receive oil (4) Lesion E (n=20), which will be lesioned animals that receive estradiol Groups for Experiment 6b: 6 Hour CS-US Interval Seventy-four ovariectomized rats will randomly be assigned to one of the following 5 groups: (1) Intact Oil (n=12), which will be PBS infused animals that will receive oil (2) Intact E (n=12), which will be PBS infused animals that receive estradiol (3) Lesion Oil (n=20), which will be NMDA lesioned animals that receive oil 297

(4) Lesion E (n=20), which will be lesioned animals that receive estradiol (5) Intact Interval Test Group1 (n=10), which will be randomly selected from the larger batch. (6) Intact Interval Test Group 2 (n=10), which will be randomly selected from the larger batch. Before neural surgery is performed, we will test the 6 hour CS-US interval in these animals first to determine whether the interval works in that particular batch of animals. If this interval fails to produce an estradiol CTA, a 5 hour CS-US interval will be tested. If this fails, a 4 hour interval will be tested in the Intact Interval Test Group 2. Also, there was concern that PBS infusions may be producing some alterations in the brain that affect CTAs when an extended CS-US interval is employed. The Intact Interval Test Group also would control for this possibility. The animals will not receive neural infusions of any sort. The behavior of this group could then be compared to the behavior of the Intact E group, which will receive infusions of PBS into the lateral PBN, to determine whether the PBS infusions affect behavior. CTA Behavioral Procedure Experiment 6a: 0 Hour CS-US Interval Each animal will be given 2 CTAs. An estradiol CTA with a 0 hour CS-US interval will be given as CTA1. The second CTA will be a LiCl CTA to enable

298

comparisons between estradiol and LiCl CTAs. Three acquisition trials will be given for both estradiol and LiCl. Experiment 6b: 6 Hour CS-US Interval The interval that produces a CTA in the Intact Interval Test Groups will be tested in the lesioned and sham-lesioned animals. Each of these animals will be given 2 CTAs, an estradiol CTA first and be a LiCl CTA second. Three acquisition trials will be given for both estradiol and LiCl. Measurements of Food and Fluid Intake Measurements of water intake will be made throughout the CTA testing. After the completion of CTA testing, food intake measurements will be made in the same animals. Additional Considerations It should be mentioned that potentially we could have a situation where the longest CS-US interval for the animals in Experiment 6b is 6 hours and that lesions of the lateral PBN block the estradiol CTA with this interval. However, due to batch effects, in the infusion study, it may be that 5 hours is the longest CS-US interval supporting an estradiol CTA, an interval that has not been tested lateral PBN lesioned animals. Although this type of situation may present itself, it is not necessarily a problem since it is the longest interval producing a CTA that is critical and not the specific amount of time.

299

References Abbott, C.R., Rossi, M., Wren, A.M., Murphy, K.G., Kennedy, A.R., Stanley, S.A., et al. (2001). Evidence of an orexigenic role for cocaine- and amphetamineregulated transcript after administration into discrete hypothalamic nuclei. Endocrinology, 142, 3457-3463. Adachi, A., Kobashi, M., Miyoshi, N., & Tsukamoto, G. (1991). Chemosensitive neurons in the area postrema of the rat and their possible functions. Brain Research Bulletin, 26, 137-140. Adrian, T.E., Savage, A.P., Sagor, G.R., Allen, J.M., Bacarese-Hamilton, A.J., Tatemoto, et al. (1985). Effect of peptide YY on gastric, pancreatic, and biliary function in humans. Gastroenterology, 89, 494-499. Agüero, A., Arnedo, M., Gallo, M., & Puerto, A. (1993). Lesions of the lateral parabrachial nuclei disrupts aversion learning induced by electrical stimulation of the area postrema. Brain Research Bulletin, 30, 585-592. Ahima, R.S., Prabakaran, D, Mantzoros C., Qu, D., Lowell, B., Maratos-Flier, E., et al. (1996). Role of leptin in neuroendocrine response to fasting. Nature, 18, 250252. Ahren, B., Mansson, S., Gingerich, R.L., & Havel, P.J. (1997). Regulation of plasma leptin in mice: influence of age, high-fat diet, and fasting. American Journal of Physiology, 273, R113-R120. Airaksinen, M.S., Paetau, A., Paliarui, L., Reinikainen, K., Riekkinen, P., Suomalainen, R., et al. (1991). Histamine neurons in human hypothalamus: anatomy in normal and Alzheimer’s diseased brains. Neuroscience, 44, 465-481. Alvarez, E.O., Ruarte, M.B., & Banzan, A.M. (2001). Histaminergic systems of the limbic system on learning and motivation. Behavioural Brain Research, 124, 195-202. Amin, S., Moore, R.W., Peterson, R.E., & Schantz, S.L. (2000). Gestational and lactational exposure to TCDD or coplanar PCBs alters adult expression of saccharin preference behavior in female rats. Neurotoxicology & Teratology, 22, 675-682. Anand, B.K., & Brobeck, J.R. (1951a). Hypothalamic controls of food intake in rats and cats. Yale Journal of Biology & Medicine, 24, 123-129.

300

Anand, B.K., & Brobeck, J.R. (1951b). Localization of a “feeding center” in the hypothalamus of the rat. Proceedings of the Society of Experimental Biology & Medicine, 77, 323-324. Anand, B.K., Chhina, G.S., Sharma, K.N., Dua, S., & Singh, B. (1964). Activity of single neurons in the hypothalamus feeding centers: effect of glucose. American Journal of Physiology, 207, 1146-1154. Anand, B.K., & Dua, S. (1955). Feeding responses induced by electrical stimulation of the hypothalamus in cat. Indian Journal of Medical Research, 43, 113-122. Angsusingha, K., Kenny, F.M., Nankin, H.R., & Taylor, F.H. (1974). Unconjugated estrone, estradiol, FSH, and LH in prepubertal and pubertal males and females. Journal of Clinical Endocrinology & Metabolism, 39, 63-68. Arase, K., York, D.A., Shimazu, H., Shargill, M., & Bray, G.A. (1998). Effects of corticotrophin releasing factor on food intake and brown adipose tissue thermogenesis. American Journal of Physiology, 55, E225-E259. Aravich, P.F., & Scalfani, A. (1983). Paraventricular hypothalamic lesions and medial hypothalamic knife cuts produce similar hyperphagic syndromes. Behavioral Neuroscience, 97, 970-983. Arnedo, M., Gallo, M., Agüero, A., & Puerto, A. (1990). Effects of medullary afferent vagal axonomy and area postrema lesions on short-term and long-term NaCl-induced taste aversion learning. Physiology & Behavior, 47, 1067-1074. Arrang, J.M., Drutel, G., & Schwartz, J.C. (1995). Characterization of histamine H3 receptors regulating acetylcholine release in rat entorhinal cortex. British Journal of Pharmacology, 114, 1518-1522. Arrang, J.M., Garbarg, M., & Schwartz, J.C. (1983). Autoinhibition of histamine release mediated by a novel class (H3) of histamine receptor. Nature, 302, 832837. Arrang, J.M., Garbarg, M., & Schwartz, J.C. (1987). Autoinhibition of histamine synthesis mediated by presynaptic H3-receptors. Neuroscience, 23, 149-157. Asakawa, A., Inui, A., Momose, K., Ueno, N., Fufino, M.A., & Kasuga, M. (1998). Endomorphins have orexigenic and anxiolytic activities in mice. NeuroReport, 9, 2265-2267.

301

Asarian, L., & Geary, N. (1999a). Estradiol (E) increases the satiating potency of intraduodenal infusions of intralipid (IL) but not of L-phenylalanine (L-PHE) in ovariectomized rats. Society for Neuroscience Abstracts, 25, 1556. Asarian, L., & Geary, N. (1999b). Cyclic estradiol treatment phasically potentiates endogenous cholecytokinin’s satiating action in ovariectomized rats. Peptides, 20, 445-450. Asarian, L., & Geary, N. (2002). Cyclic estradiol treatment normalizes body weight and restores physiological patterns of spontaneous feeding and sexual receptivity in ovariectomized rats. Hormones & Behavior, 42, 461-471. Ashford, M.L.J., Boden, P.R., & Treherne, J.M. (1990). Glucose-induced excitation of hypothalamic neurons is mediated by ATP-sensitive K+ channels. Pflügers Archives, 415, 479-483. Babicky, A., Ostadalova, J., Kolar, J., & Bibr, B. (1970). Use of radioisotope techniques for determining the weaning period in experimental animals. Physiologia Bohemoslovaca, 19(6), 457-467. Bagdade, J.D., Bierman, E.L., , & Porte, D. Jr. (1967). The significance of basal insulin levels in the evaluation of the insulin response to glucose in diabetic and nondiabetic subjects. Journal of Clinical Investigations, 46, 1549-1557. Bagnasco, M., Tulipano, G., Melis, M.R., Argiolas, A., Cocchi, D., & Muller, E.E. (2003). Endogenous ghrelin is an orexigenic peptide acting in the arcuate nucleus in response to fasting. Regulatory Peptides, 111, 161-167. Bagnol, D., Lu, X.Y., Kaelin, C.B., Day, H.E., Ollmann, M., Gantz, I., et al. (1999). Anatomy of an endogenous antagonist: relationship between Agout-related protein and proopiomelanocortin in brain. Journal of Neuroscience, 19, RC26. Bahjaoui-Bouhaddi, M., Fellmann, D., Griffond, B., & Bugnon, C. (1994). Insulin treatment stimulates the rat melanin-concentrating hormone-producing neurons. Neuropeptides, 27, 251-258. Bai, F.L., Yamano, M., Shiotani, Y., Emson, P.C., Smith, A.D., Powell, J.F., & Tohyama, M. (1985). An arcuato-paraventricular and –dorsomedial hypothalamic neuropeptide Y-containing system which lacks noradrenaline in the rat. Brain Research, 331, 172-175.

302

Balagura, S., & Devenport, L.D. (1970). Feeding patterns of normal and ventromedial hypothalamic lesioned male and female rats. Journal of Comparative & Physiological Psychology, 71, 357-364. Baldino, F., & Wolfson, B. (1985). Postsynaptic actions of neurotensin on preoptic-anterior hypothalamic neurons in vitro. Brain Research, 325, 161-170. Balks, H.J., Holst, J.J., von zur Muhlen, A., & Brabant, G. (1997). Rapid oscillations in plasma glucagon-like peptide-1 (GLP-1) in humans: cholinergic control of GLP-1 secretion via muscarinic receptors. Journal of Clinical Endocrinology & Metabolism, 82, 786-790. Balthasar, N., Coppari, R., McMinn, J., Lu, S.M., Lee, C.E., Tang, V., et al. (2004). Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron, 42, 983-991. Balthazart, J., Foidart, A., & Hendrick, J.C. (1990). The induction by testosterone of aromatase activity in the preoptic area and activation of copulatory behavior. Physiology & Behavior, 47, 83-94. Banks, W.A., Kastin, A.J., Maness, L.M., Huang, W., & Jaspan, J.B. (1995). Permeability of the blood-brain barrier to amylin. Life Sciences, 57, 1993. Banks, W.A., Kastin, A.J., Huang, W.E.A., Jaspan, J.B., & Maness, L.M. (1996). Leptin enters the brain by a saturable system independent of insulin. Peptides, 17, 305-311. Barker, L.M., Best, M.R., & Domjan, M. (1977). Learning mechanisms and food selection. Wako, TX: Baylor University Press. Baskin, D.G., Wilcox, B.J., Figlewicz, D.P., & Dorsa, D.M. (1988). Insulin and insulin-like growth factors in the CNS. Trends in Neuroscience, 11, 107-111. Barrachina, M.D., Martinez, V., Wang, L., Wei, J.Y., & Yvette T. (1997). Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proceedings of the National Academy of Sciences, 94, 10455-10460. Bartfai, T., Fisone, G., & Langel, U. (1992). Galanin and galanin antagonists: molecular and biochemical properties. Trends in Pharmacological Sciences, 13, 312-317.

303

Barnabi, F., & Cechetto, D.F. (2001). Neurotransmitters in the thalamus relaying visceral input into the insular cortex in the rat. American Journal of Physiology, 281, R1665-1674. Batterham, R.L., & Bloom, S.R. (2003). The gut hormone peptide YY regulates appetite. Annals of the New York Academy of Sciences, 994, 162-168. Batterham, R.L., Cowley, M.A., Small, C.J., Herzog, H., Cohen, M.A., Dakin, C.L., et al. (2002). Gut hormone PYY(3-36) physiologically inhibits food intake. Nature, 418, 650-654. Bechara, A., Martin, G.M., Pridgar, A., & van der Kooy, D. (1993). The parabrachial nucleus: a brain stem substrate critical for mediating the aversive motivational effects of morphine. Behavioral Neuroscience, 107, 147-160. Becker, E.E., & Kissileff, H.R. (1974). Inhibitory controls of feeding by the ventromedial hypothalamus. American Journal of Physiology, 226, 383-396. Becker, H.C., & Flaherty, C.F. (1982). Influence of ethanol on contrast in consummatory behavior. Psychopharmacology, 77, 253-258. Becker, H.C., Jarvis, M.F., Wagner, G.C., & Flaherty, C.F. (1984). Medial and lateral amygdalectomy differentially influences consummatory negative contrast. Physiology & Behavior, 33, 707-712. Bell, G.I., Santerre, R.F., & Mullenbach, G.T. (1983). Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature, 302, 716-718. Bellinger, L.L. (1987). Ingestive behavior of rats with ibotenic acid lesions of the dorsomedial hypothalamus. American Journal of Physiology, 252, R938-946. Bellinger, L.L., Bernardis, L.L., & Brooks, S. (1979). The effect of dorsomedial hypothalamic nuclei lesions on body weight regulation. Neuroscience, 4, 659-665. Bellinger, L.L., & Williams, F.E. (1983). Aphagia and adipsia after kainic acid lesioning of the dorsomedial hypothalamic area. American Journal of Physiology, 244, R389-399. Belvins, J.E., Stanley, B.G., Reidelberger, R.D. (2000). Brain Research, 860, 1-10. Berger, B.D., Wise, C.D., & Stein, L. (1973). Area postrema damage and bait shyness. Journal of Comparative & Physiological Psychology, 82, 475-479.

304

Berman, R.F., & Cannon, D.S. (1974). The effect of prior ethanol experience on ethanol-induced saccharin aversions. Physiology & Behavior, 12, 1041-1044. Berman, D.E., Hazvi, S., Neduva, V., & Dudai, Y. (2000). The role of identified neurotransmitter systems in the response of insular cortex to unfamiliar taste. Journal of Neuroscience, 20, 7017-23. Bermudez-Rattoni, F., Coburn, K.L., Fernandez, J., Chavez, A.F., & Garcia, J. (1987). Potentiation of odor by taste and odor aversions in rats are regulated by cholinergic activity of dorsal hippocampus. Pharmacology, Biochemistry & Behavior, 26, 553-559. Bernard, J.F., Alden, M., & Besson, J.M. (1993). The organization of the efferent projections from the pontine parabrachial area to the Amygdaloid complex: A Phaseolus vulgaris leucoagglutinin (PHA-L) study in the rat. Journal of Comparative Neurology, 329, 201-229. Bernard, J.F., Carroue, J., & Besson, J.M. (1991). Efferent projections from the external parabrachial area to the forebrain: a Phaseolus vulgaris leucoagglutinin study in the rat. Neuroscience Letters, 122, 257-260. Bernard, J.F., Huang, G.F., & Besson, J.M. (1994). The parabrachial area: Electrophysiological evidence for an involvement of visceral nociceptive processes. Journal of Neurophysiology, 71, 1646-1660. Bernard, J.F., Peschanski, M., & Besson, J.M. (1989). A possible spino (trigemino)-ponto-amygdaloid pathway for pain. Neuroscience Letters, 100, 83-88. Bernardis, L.L. (1975). The dorsomedial hypothalamic nucleus in autonomic and neuroendocrine homeostasis. Canadian Journal of Neurological Sciences, 2, 4560. Bernstein, I.L. (1978). Learned taste aversions in children receiving chemotherapy. Science, 200, 1302-1303. Bernstein, I.L., (1985). Learned food aversion in the progression of cancer and its treatment. Annals of the New York Academy of Sciences, 443, 365-380. Bernstein, I.L. (1986). Etiology of anorexia in cancer. Cancer, 58, 1881-1886. Bernstein, I.L. (1996). Neural mediation of food aversions and anorexia induced by tumor necrosis factor and tumors. Neuroscience & Biobehavioral Reviews, 20, 177-181. 305

Bernstein, I.L. & Borson, S. (1986). Learned food aversion: a component of anorexia syndromes. Psychological Review, 93, 462-472. Bernstein, I.L., Courtney, L., & Braget, D.J. (1986). Estrogens and the Leydig LTW(m) tumor syndrome: anorexia and diet aversions attenuated by area postrema lesions. Physiology & Behavior, 38, 159-163. Bernstein, I.L., Chavez, M., Allen, D., & Taylor, E.M. (1992). Area postrema mediation of physiological and behavioral effects of lithium chloride in the rat. Brain Research, 575, 132-137. Berthoud, H.R. (2002). Multiple neural systems controlling food intake and body weight. Neuroscience & Biobehavioral Reviews, 26, 393-428. Bester, H., Menendez, L., Besson, J.M., & Bernard, J.F. (1995). Spino (trigemino) parabrachiohypothalic pathway: Electrophysiological evidence for an involvement in pain processes. Journal of Neurophysiology, 73, 568-585. Beutler, B., & Cerami, A. (1987). Cachectin: more than a tumor necrosis factor. New England Journal of Medicine, 316, 379-385. Bhavsar, S., Watkins, J., & Young, A. (1997). Comparison of central and peripheral administration of amylin on reduction of food intake in rats. Diabetologia, 40(suppl 1), A302. Bhavsar, S., Watkins, J., & Young, A. (1998). Synergy between amylin and cholecystokinin for inhibition of food intake in mice. Physiology & Behavior, 64, 557-561. Bianchi, R., Corsetti, G., Rodella, L., Tredici, G., & Gioia, M. (1998). Supraspinal connections and termination patterns of the parabrachial complex determined by the biocytin anterograde tract-tracing technique in the rat. Journal of Anatomy, 193, 417-430. Bibbo, M., Al-Naqeeb, M., Baccarini, I., Gill, W., Newton, M., et al. (1975). Follow-up study of male and female offspring of DES-treated mothers: a preliminary report. Journal of Reproductive Medicine, 15, 20-32. Bielavska, E., & Bures, J. (1994). Universality of parabrachial mediation of conditioned taste aversion. Behavioural Brain Research, 60, 35-42.

306

Bielavska, E., & Roldan, G. (1996). Ipsilateral connections between the gustatory cortex, amygdala and parabrachial nucleus are necessary for acquisition and retrieval of conditioned taste aversion in rats. Behavioural Brain Research, 81, 2531. Billington, C.J., Briggs, J.E., Grace, M., & Levine, A.S. (1991). Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. American Journal of Physiology, 260, R321-R327. Billington, C.J., Briggs, J.E., Harker, M., Grace, M., Levine, A.S. (1994). Neuropeptide Y in hypothalamic paraventricular nucleus: a center coordinating energy metabolism. American Journal of Physiology, 266, R1765-R1770. Bird, E., Cardone, C.C., & Contreras, R.J. (1983). Area postrema lesions disrupt food intake induced by cerebroventricular infusion of 5-thioglucose in the rat. Brain Research, 270, 193-196. Bittencourt, J.C., & Elias, C.F. (1998). Melanin-concentrating hormone and neuropeptide EI projections from the lateral hypothalamic area and zona incerta to the medial septal nucleus and spinal cord: a study using multiple neuronal tracers. Brain Research, 805, 1-19. Bittencourt, J.C., Presse, F., Arias, C., Peto, C., Vaughan, J., Nahon, J.L., Vale, W., & Sawchenko, P.E. (1992). The melanin-concentrating hormone system of the rat brain: an immuno- and hybridization histochemical characterization. Journal of Comparative Neurology, 319, 218-245. Blaustein, J.D., Gentry, R.T., Roy, E.J., & Wade, G.N. (1976). Effects of ovariectomy and estradiol on body weight and food intake in gold thioglucosetreated mice. Physiology & Behavior, 17, 1027-1030. Blevins, J.E., Hamel, F.G., Fairbairn, E., Stanley, B.G., & Reidelberger, R.D. (2000). Effects of paraventricular nucleus injection of CCK-8 on plasma CCK-8 levels in rats. Brain Research, 860, 11-20. Bliss, T.V., & Collingridge, G.L. (1993). A synaptic model of memory: long-term potentiation in the hippocampus. Nature, 361, 31-39. Bliss, T.V., & Lømo, T. (1973). Long-lasting Potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. Journal of Physiology, 232, 331-356.

307

Blizard, D.A., Lippman, H.R., & Chen, J.J. (1975). Sex differences in open-field behavior in the rat: the inductive and activational role of gonadal hormones. Physiology & Behavior, 14, 601-608. Blundell, J. Hunger, appetite, and satiety—Constructs in search of identities. In: Turner, M. (Ed.). Proceedings of the British Nutrition Foundation, First Annual Conference. London: Applied Science Pub. Ltd., 1979, p.21-42. Bonavera, J.J., Dube, M.G., Karla, P.S., & Karla, S.P. (1994). Anorectic effects of estrogen may be mediated by decreased neuropeptide Y release in the hypothalamic paraventricular nucleus. Endocrinology, 134, 2367-2370. Bonner, T.I., Buckley, N.J., Young, A.C., & Brann, M.R. (1987). Identification of a family of muscarinic acetylcholine receptor genes. Science, 237, 527-532. Bonner, T.I., Young, A.C., Brann, M.R., & Buckley, N.J. (1988). Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes. Neuron, 1, 403-410. Booth, D.A. (1974). Acquired sensory preference for protein in diabetic and normal rats. Physiological Psychology, 2, 344-348. Booth, D.A. (1977). Satiety and appetite are conditioned reactions. Psychosomatic Medicine, 39, 76-81. Booth, D.A. Food-conditioned eating preferences and aversions with interoceptive elements: conditioned appetites and satieties. In: N.S. Braveman and P. Bronstein (Eds.). Annals of the New York Academy of Sciences: experimental assessments and clinical applications of conditioned food aversions, vol. 443. New York: New York Academy of Sciences; 1985. p. 22-41. Booth, D.A., & Thibault, L. Macronutrient-specific hungers and satieties and their neural bases, learnt from pre- and postingestional effects of eating particular foodstuffs. In: H.R., Berthoud, & R.J. Seeley (Eds.). Neural and metabolic control of macronutrient intake. Boca Raton: CRC Press, 2000. p. 61-91. Borison, H.L., & Brizzee, K.R. (1951). Morphology of emetic chemoreceptor trigger zone in the cat medulla. Proceedings of the Society for Experimental Biology & Medicine, 77, 38-42. Borison, H.L., & Wang, S.C. (1953). Physiology and pharmacology of vomiting. Pharmacological Review, 5, 193-200.

308

Bouton, M.E. (1993). Context, time, and memory retrieval in the interference paradigms of Pavlovian learning. Psychological Bulletin, 114, 80-99. Brand, J.J., & Perry, W.L.M. (1966). Drugs used in motion sickness. Pharmacological Reviews, 18, 895-924. Braun, J.J., Slick, T.B., & Lorden, J.F. (1972). Involvement of the gustatory cortex in the learning of taste aversion. Physiology & Behavior, 9, 637-641. Braun, J.J., Lasiter, P.S., & Kiefer, S.W. (1982). The gustatory neocortex of the rat. Physiological Psychology, 10, 13-45. Bray, G.A., & York, D.A. (1979). Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiological Review, 59, 719-809. Breen, T.L., Conwell, I.M., & Wardlaw, S.L. (2005). Effects of fasting, leptin, and insulin on AGRP and POMC peptide release in the hypothalamus. Brain Research, 1032, 141-148. Broadwell, R.D., & Brightman, M.W. (1978). Entry of peroxidase into neurons of the central and peripheral nervous systems from extracerebral and cerebral blood. Journal of Comparative Neurology, 166, 257-283. Brobeck, J.R., Tepperman, J., & Long, C.N. (1943). Experimental hyperphagia in the albino rat. Yale Journal of Biology & Medicine, 15, 831-853. Broberger, C., Landry, M., Wong, H., Walsh, J.N., & Hökfelt, T. (1997). Subtype of Y1 and Y2 of the neuropeptide Y receptor are respectively expressed in proopiomelanocortin and neuropeptide Y-containing neurons of the rat hypothalamic arcuate nucleus. Neuroendocrinology, 66, 393-408. Broberger, C., de Lecea, L., Sutcliffe, J.G., & Hokfelt, T. (1998). Hypocretin/orexin- and melanin-concentrating hormone-expressing cells form distinct populations in the rodent lateral hypothalamus: relationship to the neuropeptide Y and agouti gene-related protein systems. Journal of Comparative Neurology, 402, 460-474. Broberger, C., Johansen, J., Johansson, C., Schalling, M., & Hökfelt, T. (1998). The neuropeptide Y/agouti gene-related protein (AgRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proceedings of the National Academy of Sciences, 95, 15043-15048.

309

Broberger, C. (1999). Hypothalamic cocaine- and amphetamine-regulated transcript (CART) neurons: histochemical relationship to thyrotropin-releasing hormone, melanin-concentrating hormone, orexin/hypocretin and neuropeptide Y. Brain Research, 848, 101-113. Brooks, C.McC., Lockwood, R.A., & Wiggins, M.L. (1946). A study of the effects of hypothalamic lesions on the eating habits of the albino rat. American Journal of Physiology, 147, 735-741. Browning, K.N., & Travagli, R.A. (2003). Neuropeptide Y and peptide YY inhibit excitatory synaptic transmission in the rat dorsal motor nucleus of the vagus. Journal of Physiology, 549, 775-785. Buckley, N.J., Bonner, T.I., & Brann, M.R. (1988). Localization of a family of muscarinic receptor mRNAs n rat brain. Journal of Neuroscience, 8, 4646-4652. Bucley, N.J., Bonner, T.I., Buckley, C.M., & Brann, M.R. (1989). Antagonist binding properties of five cloned muscarinic receptors expressed in CHO-K1 cells. Molecular Pharmacology, 35, 469-476. Bures, J., & Buresova, O. (1989a). Conditioned taste aversion to injected flavor: differential effect of anesthesia on the formation of the gustatory trace and on its association with poisoning in rats. Neuroscience Letters, 98, 305-309. Bures, J., & Buresova, O. (1989b). Conditioned taste aversion elicited by intracerebral administration of drugs. Acta Physiologica Hungarica, 74, 77-93. Bures, J., Yamamoto, T., & Bermudez-Rattoni, F. Conditioned Taste Aversion: memory of a special kind. Oxford University Press, New York, 1999. Buresova, O., & Bures, J. (1973). Cortical and subcortical components of the conditioned saccharin aversion. Physiology & Behavior, 11, 435-439. Buresova, O., Bures, J., Bohdanecky, Z., & Weiss, T. (1964). Effect of atropine on learning, extinction, retention and retrieval in rats. Psychopharmacologia, 5, 255263. Butcher, R.L., Collins, W.E., & Fugo, N.W. (1974). Plasma concentration of LH, FSH, prolactin, progesterone, and estradiol 17-beta throughout the 4-day estrous cycle of the rat. Endocrinology, 94, 1704-1708. Butera, P.C., & Beikirch, R.J. (1989). Central implants of diluted estradiol: independent effects on ingestive and reproductive behaviors of ovariectomized rats. Brain Research, 491, 226-273. 310

Butera, P.C., Bradway, D.M., & Cataldo, N.J. (1993). Modulation of the satiety effect of cholecystokinin by estradiol. Physiology & Behavior, 53, 1235-1238. Butera, P.C., & Czaja, J.A. (1984). Intracranial estradiol in ovariectomized guinea pigs: effects on ingestive behaviors and body weight. Brain Research, 322, 41-48. Butera, P.C., Willard, D.M., & Raymond, S.A. (1992). Effects of PVN lesions on the responsiveness of female rats to estradiol. Brain Research, 576, 304-310. Butler, P.C., Chou, J., Carter, W.B., Wang, Y.N., Bu, B.H., Chang, D. et al. (1990). Effects of meal ingestion on plasma amylin concentration in NIDDM and nondiabetic humans. Diabetes, 39, 752. Cabanac, M., & Lafrance, L. (1991). Facial consummatory responses in rats support the ponderostat hypothesis. Physiology & Behavior, 50, 179-183. Cairnie, A.B., & Leach, K.E. (1982). Dexamethasone: a potent blocker for radiation-induced taste aversion in rats. Pharmacology, Biochemistry & Behavior, 17, 305-311. Calingasan, N.Y., & Ritter, S. (1993). Lateral parabrachial subnucleus lesions abolish feeding induced by mercaptoacetate but not by 2-deoxy-D-glucose. American Journal of Physiology, 265, R1168-1178. Campbell, B.A., & Alberts, J.R. (1979). Ontogeny of long term memory for learned taste aversions. Behavioural & Neural Biology, 29, 139-156. Campfield, L.A., Smith, F.J., Gulsez, Y., Deos, R., & Burn, P. (1995). Mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science, 269, 546-549. Cannon, D.S., Baker, T.B., & Berman, R.F. (1977). Taste aversion disruption by drug pretreatment: dissociative and drug-specific effects. Pharmacology, Biochemistry & Behavior, 6, 93-100. Canteras, N.S., Simerly, R.B., & Swanson, L.W. (1994). Organization of projections from the ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. Journal of Comparative Neurology, 348, 41-79. Capaldi, E.D., & Sheffer, J.D. (1992). Contrast and reinforcement in consumption. Learning & Motivation, 23, 63-79.

311

Capuano, C.A., Barr, G.A., & Leibowitz, S.F. (1986). Neuropeptide Y and cholecystokinin: effects on independent feeding in preweanling rats via hypothalamic stimulation. Eastern Psychological Association Abstracts, 57, 57. Carlson, S.H., & Osborn, J.W. (1998). Splanchnic and vagal denervation attenuate central Fos but not AVP responses to intragastric salt in rats. American Journal of Physiology, 274, 1243-1252. Clark, J.T, Karla, P.S., Crowley, W.R., & Karla, S.P. (1984). Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 115, 427-429. Castellanos, C., Salas, J.A., Gonzalez, H., Roldan, G., & Garcia, D. (2000). Muscarinic blockade but not acetylcholine depletion impairs taste aversion learning in rats. Society of Neuroscience Abstracts. Cecchini, D.J., Chattoraj, S.C., Fanous, A.S., Panda, S.K., Brennan, T.F., & Edelin, K.C. (1983). Radioimmunoassay of 2-hydroxyestrone in plasma during the estrous cycle of the rat: Interrelationships with estradiol, progesterone, and the gonadotropins. Endocrinology, 112, 1122-1126. Cechetto, D.F. (1987). Central representation of visceral function. Federation Proceedings, 46, 17-230. Cechetto, D.F., & Saper, C.B. (1987). Evidence for a viscerotopic sensory representation in the cortex and thalamus in the rat. Journal of Comparative Neurology, 262, 27-45. Chalmers, D.T., Lovenberg, T.W., & de Souza, E.B. (1995). Localization of novel corticotropin-releasing factor receptor (CRF2) mRNA expression to specific subcortical nuclei in the brain: comparison with CRF1 receptor mRNA expression. Journal of Neuroscience, 15, 6340-6350. Chamberlin, N.L., & Saper, C.B. (1992). Topographic organization of cardiovascular responses to electrical and glutamate microstimulation of the parabrachial nucleus in the rat. Journal of Comparative Neurology, 326, 245-262. Chambers, K.C., & Bernstein, I.L. Conditioned flavor aversions. In: R.L. Doty (Ed.). Handbook of olfaction and gestation, 2nd ed. New York: Marcel Dekker; 2003.

312

Chambers, K.C., & Hayes, U.L. (2002). Exposure to estradiol before but not during acquisition of LiCl-induced conditioned taste avoidance accelerates extinction. Hormones & Behavior, 41, 297-305. Chambers, K. C., Thornton, J. E., & Roselli, C. E. 1991. Age-related deficits in brain androgen binding and metabolism, testosterone, and sexual behavior of male rats. Neurobiology of Aging, 12, 123-130. Chambers, K.C., & Wang, Y. (2004). Role of the lateral parabrachial nucleus in apomorphine-induced conditioned consumption reduction: cooling lesions and relationship of c-Fos-like immunoreactivity to strength of conditioning. Behavioral Neuroscience, 118, 199-213. Chance, W.T., Balasubramaniam, A., Zhang, F.S., Wimalawansa, S.J., & Fischer, J.E. (1991). Anorexia following the intrahypothalamic administration of amylin. Brain Research, 539, 352-354. Chang, F.-C. T., & Scott, T.R. (1984). Conditioned taste aversions modify neural responses in the rat nucleus tractus solitarius. Journal of Neuroscience, 4, 18501862. Chemelli, R.M., Willie, J.T., Sinton, C.M., Elmquist, J.K., Scammell, T., et al. (1999). Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation. Cell, 98, 437-451. Chen, H., Charlat, O., Tartaglia, L.A, Woolf, E.A., Weng, X., Ellis, S.J. et al. (1996). Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell, 84, 491-495. Cheung, C.C., Clifton, D.K., & Steiner, R.A. (1996). Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology, 138, 4489-4492. Choate, J.V.A., & Resko, J.A. (1994). Prenatal inhibition of aromatase activity affects lutenizing hormone feedback mechanisms and reproductive behaviors of adult guinea pigs. Biology of Reproduction, 51, 1273-1278. Christie, M.J., & North, R.A. (1988). Agonists at mu-opioid, M2-muscarinic and GABAB-receptors increase the same potassium conductance in rat lateral parabrachial neurones. British Journal of Pharmacology, 95, 896-902. Chua, S.C., Chung, W.K., Wu-Peng, X.S., Zhang, Y., Liu, S.M., Tartaglia, L., & Leibel, R.L. (1996). Phenotypes of mouse diabetes and rat fatty due to mutations in the OB (leptin). receptor. Science, 271, 994-996. 313

Chou, T.C., Scammell, T.E., Gooley, J.J., Gaus, S.E., Saper, C.B. , & Lu, J. (2003). Critical role of dorsomedial hypothalamic nucleus in a wide range of behavioral circadian rhythms. The Journal of Neuroscience, 23, 10691-10702. Ciccocioppo, R., Fedeli, A., Economidou, D, Policani, F., Weiss, F., & Massi, M. (2003). The bed nucleus is a neuroanatomical substrate for the anorectic effect of corticotropin-releasing factor and for its reversal by nociceptin/orphanin FQ. Journal of Neuroscience, 23, 9445-9451. Ciccocioppo, R., Cippitelli, A., Economidou, D., Fedeli, A., & Massi, M. (2004). Nociceptin/orphanin FQ acts as a functional antagonist of corticotropin-releasing factor to inhibit its anorectic effect. Physiology & Behavior, 82, 63-68. Ciriello, J., & Calaresu, F. (1981). Projections from buffer nerves to the nucleus of the solitary tract: an anatomical and electrophysiological study in the cat. Journal of Autonomic Nervous System, 3, 299-310, Clark, J.T, Karla, P.S., Crowley, W.R., & Karla, S.P. (1984). Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 115, 427-429. Clement, K., Vaisse, C., Lahlou, N., Cabrol, S., Pelloux, V., Cassuto, D. et al. (1998). A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature, 392, 398-401. Clineschmidt, B.V., & Lotti, V.J. (1973). Histamine: intraventricular injection suppresses ingestive behavior of the cat. Archives Internationales de Pharmacodynamie et de Therapie, 206, 288-298. Cochet, M., Chang, A.C., & Cohen, S.N. (1982). Characterization of the structural gene and putative 5′-regulatory sequences for human melanocortin. Nature, 297, 335-339. Cohen, I.T., Sherwin, B.B., & Fleming, A.S. (1987). Food cravings, mood, and the menstrual cycle. Hormones & Behavior, 21, 457-470. Coil, J.D., Rogers, R.C., Garcia, J., & Novin, D. (1978). Conditioned taste aversions: Vagal and circulatory mediation of the toxic unconditioned stimulus. Behavioral Biology, 24, 509-519. Coleman, S.R., & Gormezano, I. (1971). Classical conditioning of the rabbit’s (Oryctolagus cuniculus) nictitating membrane response under symmetrical CS-US interval shifts. Journal of Comparative & Physiological Psychology, 77, 447-455. 314

Cooper, G.J.S., Willis, A.C., Clark, A., Turner, R.C., Sim, R.B. , & Reid, K.B.M. (1987). Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proceedings of the National Academy of Sciences, 84, 8628. Corwin, R.L., Robinson, J.K., & Crawley, J.N. (1993). Galanin antagonists block galanin-induced feeding in the hypothalamus and amygdala of the rat. European Journal of Neuroscience, 5, 1528-1533. Cowley, M.A., Smart, J.L., Rubinstein, M., Cerdan, M.G., Diano, S., Horvath, T.L., et al. (2001). Leptin activates the anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature, 411, 480-484. Cowley, M.A., Smith, R.G., Diano, S., Tschöpp, M., Pronchuk, N., Grove, K.L., et al. (2003). The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regarding energy homeostasis. Neuron, 37, 649-661. Cox, J.E. (1998). Cholecystokinin satiety involves CCKA receptors perfused by the superior pancreaticoduodenal artery. American Journal of Physiology, 274, R1390-R1396. Cox, J.E., Kakolewski, J., & Valenstein, E. (1969). Ventromedial hypothalamic lesions and changes in body weight and food consumption in male and female rats. Journal of Comparative & Physiological Psychology, 67, 320-326. Cox, J.E., & Powley, T.L. (1981). Intragastric pair feeding fails to prevent VMH obesity or hyperinsulinemia. American Journal of Physiology, 240, E566-572. Crawley, J.N., & Kiss, J.Z. (1985). Paraventricular nucleus lesions abolish the inhibition of feeding induced by systemic cholecystokinin. Peptides Fayetteville, 6, 927-935. Crawley, J.N. (1995). Biological actions of galanin. Regulatory Peptides, 59, 116. Cubero, I., & Puerto, A. (2000). Lateral parabrachial lesions impair intraperitoneal but not intraventricular methylscopolamine-induced taste aversion learning. Brain Research, 871, 113-119. Cubero, I., Lopez, M., Navarro, M., & Puerto, A. (2001). Lateral parabrachial lesions impair taste aversion learning induced by blood-borne visceral stimuli. Pharmacology, Biochemistry & Behavior, 69, 157-163. 315

Culbert, K.M., Breedlove, M.S., Burt, A.S., & Klump, K.L. (2008). Prenatal horomone exposure and risk for eating disorders. Archives of General Psychiatry, 65, 329-336. Cummings, D.E., Purnell, J.Q., Frayo, R.S., Schmidova, K., Wisse, B.E., & Weigle, D.S. (2001). A preprandial ris in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes, 50, 1714-1719. Cummings, D.E., Weighle, D.S., Frayo, R.S., Breen, P.A., Ma, M.K., Dellinger, E.P., & Purnell, J.Q. (2002). Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. New England Journal of Medicine, 346, 1623-1630. Czaja, J.A. (1975). Food rejection by female rhesus monkeys during the menstrual cycle and early pregnancy. Physiology & Behavior, 14, 579-587. Czaja, J.A., & Goy, R.W. (1975). Ovarian hormones and food intake in female guinea pigs and rhesus monkeys. Hormones & Behavior, 6, 329-349. Dacey, D.M., & Grossman, S.P. (1977). Aphagia, adipisia, and sensory-motor deficits produced by amygdala lesions: a function of extra-amygdaloid damage. Physiology & Behavior, 19, 389-395. Dagnault, A., & Richard, D. (1994). Lesions of the hypothalamic paraventricular nuclei do not prevent the effect of estradiol on energy and fat balance. American Journal of Physiology, 267, E32-E38. Date, Y., Ueta, Y., Yamashita, H., Yamaguchi, H., Matsukura, S., Kangawa, K. et al. (1999). Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. Proceedings of the National Academy of Sciences, 96, 748-753. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., & Suganuma, T. et al. (2000). Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology, 141, 4255-4261. Date, Y., Murakami, N., Toshinai, K., Matsukura, S., Niijima, A., Matsuo, H., et al. (2002). The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology, 123, 1120-1128. Dawson, R., Pelleymounter, M., Millard, W., Liu, S., & Eppler, B. (1997). Attenuation of leptin-mediated effects by monosodium glutamate-induced arcuate nucleus damage. American Journal of Physiology, 273, E202-E206. 316

De Beun, R., Jansen, E., Smeets, M.A.M., Niesing, J., Slangen, J.L., & Van de Poll, N.E. (1991). Estradiol-induced conditioned taste aversion and place aversion in rats: sex- and dose-dependent effects. Physiology & Behavior, 50, 995-1000. De Beun, R., Peeters, B.W., & Broekkamp, C.L. (1993). Stimulus characterization of estradiol applying a crossfamiliarization taste aversion procedure in female mice. Physiology & Behavior, 53, 715-719. Decker, M.W., & McGaugh, J.L. (1991). The role of interaction between the cholinergic system and other neuromodulatory systems in learning and memory. Synapse, 7, 151-168. De Jong, F.H., Hey, A.H., & van der Molen, H.J. (1973). Effect of gonadotrophins on the secretion of oestradiol- and testosterone by the rat testis. Journal of Endocrinology, 57, 277-284. Delamater, A.R. (1996). Effects of several extinction treatments upon the integrity of Pavlovian stimulus-outcome associations. Animal Learning & Behavior, 24, 437-449. Della-Zuana, O., Presse, F., Ortola, C., Duhault, J., Nahon, J.L., & Levens, N. (2002). Acute and chronic administration of melanin-concentrating hormone enhances food intake and body weight in Wistar and Sprague-Dawley rats. International Journal of Obesity & Related Metabolic Disorders, 26, 1289-1295. De Lecea, L., Kilduff, T.S., Peyron, C., Gao, X.-B., Foye, P.E., Danielson, P.E., et al. (1998). The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity. Proceedings of the National Academy of Sciences, 95, 322-327. Delgado, J.M.R , & Anand, B.K. (1953). Increase of food intake induced by electrical stimulation of the lateral hypothalamus. American Journal of Physiology, 172, 162-168. De Pedro, N., Céspedes, M.V., Delgado, M.J., & Alonso-Bedate, M. (1995). The galanin-induced feeding stimulation is mediated via α2-adrenergic receptors in goldfish. Regulatory Peptides, 57, 77-84. Deutch, R. (1978). Effects of atropine on conditioned taste aversion. Pharmacology, Biochemistry & Behavior, 8, 685-694. Devine, E., & Young, A.A. (1998). Weight gain in male and female mice with amylin gene knockout. Diabetes, 47(suppl), A317.

317

Diano, S., Horvarth, B., Urbanski, H.F., Sotonyi, P., & Horvarth, T.L. (1993). Fasting activates the nonhuman primate hypocretin (orexin) system and its postsynaptic targets. Endocrinology, 144, 3774-3778. DiLorenzo, P.M. (1988). Long-delay learning in rats with parabrachial pontine lesions. Chemical Senses, 13, 219-229. DiMeo, A.N., & Wood, R.I. (2006). Self administration of estrogen and dihydrotestosterone in male hamsters. Hormones & Behavior, 49, 519-526. Dohler, K.D., Coquelin, A., Davis, F., Hines, M., Shryne, J.E., & Gorski, R.A. (1984). Pre- and postnatal influence of testosterone propionate and diethylstilbesterol differentiation of the sexually dimorphic nucleus of the preoptic area in male and female rats. Brain Research, 302, 291-295. Domjan, M., & Best, M.R. (1977). Paradoxical effects of proximal unconditioned stimulus preexposure: interference with and conditioning of a taste aversion. Journal of Experimental Psychology, Animal Behavior Processes, 3, 310-321. Domjan, M., Foster, K., & Gillan, D.J. (1979). Effects of distribution of the drug unconditioned stimulus on taste-aversion learning. Physiology & Behavior, 23, 931-938. Donohoe, T.P., & Stevens, R. (1981). Modulation of food intake by amygdaloid estradiol benzoate implants in female rats. Physiology & Behavior, 27, 105-114. Downs, D., Cardozo, C., Schneiderman, N., Yehle, A.L., Vandercar, D.H., & Zwilling, G. (1972). Central effects of atropine upon aversive classical conditioning in rabbits. Psychopharmacologia, 23, 319-322. Drewett, R.F. (1973). Oestrous and dioestrous components of the ovarian inhibition on hunger in the rat. Animal Behaviour, 21, 772-780. Drewett, R.F. (1974). The meal patterns of the oestrous cycle and their motivational significance. Quarterly Journal of Experimental Psychology, 26, 489494. Drucker, D.J. (1998). Glucagon-like peptides. Diabetes, 47, 159-169. Dubac, G.R., Phinney, S.D., Stern, J.S., & Havel, P.J. (1998). Changes of serum leptin and endocrine and metabolic parameters after 7 days of energy restriction in men and women. Metabolism, 47, 429-434.

318

Dube, M.G., Horvath, T.L., Leranth, C., Kalra, P.S., Kalra, S.P. (1994). Naloxone reduces the feeding evoked by intracerebroventricular galanin injection. Physiology & Behavior, 56, 811-813. Dumont, Y., Fournier, A., St-Pierre, S., Schwartz, T.W., & Quirion, R. (1990). Differential distribution of neuropeptide Y1 and Y2 receptors in the rat brain. European Journal of Pharmacology, 191, 501-503. Dunbar, J., Lapanowski, K., Barnes, M., & Rafols, J. (2005). Hypothalamic agouti-related protein immunoreactivity in food-restricted, obese, and insulintreated animals: evidence for glia cell localization. Experimental Neurology, 191, 184-192. Dunn-Meynell, A.A., Govek, E., Levin, B.E. (1997). Intracarotid glucose infusions selectively increase Fos-like immunoreactivity in paraventricular, ventromedial and dorsomedial nuclei neurons. Brain Research, 748, 100-106. Eberlein, G.A., Eysselein, V.E, Schaeffer, M., Layer, P., Grandt, D., Goebell, H., Niebel, W., Davis, M., Lee, T.D., & Shively, J.E. (1989). A new molecular form of PYY: structural characterization of human PYY(3-36) and PYY(1-36). Peptides, 10, 797-803. Ebert, U., Oertel, R., Wesnes, K.A., & Kirch, W. (1998). Effects of physostigmine on scopolamine induced changes in quantitative electroencephalogram and cognitive performance. Human Psychopharmacology, 13, 199-210. Eckel, L.A., & Geary, N. (2001). Estradiol treatment increases feeding-induced cfos expression in the brains of ovariectomized rats. American Journal of Physiology, 281, R738-746. Eckel, L.A., Houpt, T.A., & Geary, N. (1998). Chronic estradiol treatment increases cholecystokinin-induced c-fos expression in ovariectomized rats. Appetite, 31, 273-274. Eckel, L.A., Langhans, W., Kahler, A., Campfield, L.A., Smith, F.J., & Geary, N. (1998). Chronic administration of OB protein decreased food intake by selectively reducing meal size in female rats. American Journal of Physiology, 275, R186R193. Edholm, O.G. (1977). Energy balance in man. Journal of Human Nutrition, 31, 413-431.

319

Edwards, C.M., Abbott, C.R., Sunter, D., Kim, M., Dakin, C.L., & Murphy, K.G. (2000). Cocaine- and amphetamine-regulated transcript, glucagons-like peptide-1 and corticotrophin releasing factor inhibit feeding via agouti-related protein via independent pathways in the rat. Brain Research, 866, 128-134. Edwards, G.L., Ladenheim, E.E., & Ritter, R.C. (1986). Dorsomedial hindbrain participation in cholecystokinin-induced satiety. American Journal of Physiology, 251, 971-977. Edwards, G.L., & Johnson, A.K. (1991). Enhanced drinking after excitotoxic lesions of the parabrachial nucleus in the rat. American Journal of Physiology, 261, R1039-R1044. Edwards, G.L., Power, J.D., & Young, A.A. (1998). Attenuation of the satiogenic effects of amylin by lesions of the area postrema, but not by intraperitoneal capsaicin. Proceedings of Neuroscience Abstracts, 24, 406.3. Ekblad, E., Rokaeus, A., Hakanson, R., & Sundler, F. (1985). Galanin nerve fibers in the rat gut: distribution, origin and projections. Neuroscience, 16, 355-363. Elias, C.F., Aschkenasi, C., Lee, C., Kelly, J., Ahima, R.S., Bjorbaek, C. et al. (1999). Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron, 23, 775-786. Elias, C.F., Lee, C., Kelly, J., Aschkenasi, C., Ahima, R.S., Couceyro, P.R. et al. (1998a). Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron, 21, 1375-1385. Elias, C.F., Saper, C.B., Maratos-Flier, E., Tritos, N.A., Lee, C., Kelly, J. et al. (1998b). Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area. Journal of Comparative Neurology, 402, 442-459. Elmquist, J.K., Ahima, R.S., Maratos-Flier, E., Flier, J.S., & Saper, C.B. (1997). Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology, 138, 839-842. Elmquist, J.K., Bjorbaek, C., Ahima, R.S., Flier, J.S., & Saper, C.B. (1998a). Distributions of leptin receptor mRNA isoforms in the rat brain. Journal of Comparative Neurology, 395, 535-547. Elmquist, J.K., Ahima, R.S., Elias, C.F., Flier, J.S., & Saper, C.B. (1998b). Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei. Proceedings of the National Academy of Sciences, 95, 741-746. 320

Elmquist, J.K., Elias, C.F., & Saper, C.B. (1999). From lesions to leptin: hypothalamic control of food intake and body weight. Neuron, 22, 221-232. Emond, M., Schwartz, G.J., Ladenheim, E.E., & Moran, T.H. (1999). Central leptin modulates behavioral and neural responsivity to CCK. American Journal of Physiology, 276, R1545-R1549. Erickson, J.C., Clegg, K.E., & Palmiter, R.D. (1996). Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature, 381, 415-418. Escobar, M.L., & Bermudez-Rattoni, F. (2000). Long-term Potentiation in the insular cortex enhances conditioned taste aversion retention. Brain Research, 852, 208-212. Escobar, M.L., Chao, V., & Bermudez-Rattoni, F. (1998). In vivo long-term Potentiation in the insular cortex: NMDA receptor dependence. Brain Research, 779, 314-319. Evenden J.L., Lavis, L., Iversen, S.D. (1992). Blockade of conditioned taste aversion by scopolamine and N-methyl scopolamine: associative conditioning, not amnesia. Psychopharmacology, 106, 179-188. Eyre, L.J., Bland, R., Bujalska, I.J., Sheppard, M.C., Stewart, P.M., & Hewison, M. (1998). Characterization of aromatase and 17 beta hydroxysteroid dehydrogenase expression in rat osteoblastic cells. Journal of Bone & Mineral Research, 13, 9961004. Fahrbach, S.E., Meisel, R.L., & Pfaff, D.W. (1985). Preoptic implants of estradiol increase wheel running but not open field activity of female rats. Physiology & Behavior, 35, 985-992. Fan, W., Boston, B.A., Kesterson, R.A., Hruby, V.J., & Cone, R.D. (1997). Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature, 385, 165-168. Faulconbridge, L.F., Cummings, D.E., Kaplan, J.M., & Grill H.J. (2003). Hyperphagic effects of brainstem ghrelin administration. Diabetes, 52, 948-956. Fedorchak, P.M., & Bolles, R.C. (1988). Nutritive expectancies mediate cholecystokinin’s suppression-of-intake effect. Behavioral Neuroscience, 102, 451455.

321

Fellmann, D., Risold, P.Y., Bahjaoui, M., Compagnone, N., Bresson, J.L., Clavequin, M.C., Cardot, J., Goutet, A., Lenys, D., & Bugnon, C. (1993). Morphofunctional studies on the neurons producing melanin-concentrating hormone. Annals of the New York Academy of Science, 680, 511-516. Ferreira, G., Gutierrez, R., De la Cruz, V., & Bermudez-Rattoni, F. (2002). Differential involvement of cortical muscarinic and NMDA receptors in short- and long-term taste aversion memory. European Journal of Neuroscience, 16, 11391145. Ferry, B., Jarrard, L.E., Oberling, P., & DiScala, G. (1996). Facilitation of conditioned odor aversion by entorhinal cortex lesions in the rat. Behavioral Neuroscience, 110, 443-450. Fibiger, H.C. (1991). Cholinergic mechanisms in learning, memory, and dementia: a review of recent evidence. Trends in Neuroscience, 14, 220-223. Figlewicz, D.P., Stein, L.J., Woods, S.C., & Porte, D.Jr. (1985). Acute and chronic gastrin-releasing peptide decreases food intake in baboons. American Journal of Physiology, 248, R578-R583. Figlewicz, D.P., Stein, L.J., West, D., Porte, D. Jr. , & Woods, S.C. (1986). Intracisternal insulin alters sensitivity to CCK-induced meal suppression in baboons. American Journal of Physiology, 250, R856-R860. Figlewicz, D.P., Sipols, A.J., Seeley, R.J., Chavez, M., Woods, S.C., Porte, D. Jr. (1995). Intraventricular cholecystokinin octapeptide in the baboon. Behavioral Neuroscience, 109, 567-569. Filer, C.N., Lacy, J.M., & Peng, C.T. (2005). Ibotenic acid decarboxylation to muscimol: dramatic solvent and radiolytic rate acceleration. Synthetic Communications, 35, 967-970. Fischer, A.E., & Coury, J.N. (1962). Cholinergic tracing of a central neural circuit underlying the thirst drive. Science, 138, 691-693. Flaherty, C.F., & Checke, S. (1982). Anticipation of incentive gain. Animal Learning & Behavior, 10, 177-182. Flaherty, C.F., & Rowan, G.A. (1985). Anticipatory contrast: within-subjects analysis. Animal Learning & Behavior, 13, 2-5.

322

Flaherty, C.F, & Grigson, P.S. (1988). From contrast to reinforcement: role of response contingency in anticipatory contrast. Journal of Experimental Psychology: Animal Behavior Processes, 14, 165-176. Flaherty, C.F., Grigson, P.S., Checke, S., & Hnat, K.C. (1991). Deprivation state and temporal horizons in anticipatory contrast. Journal of Experimental Psychology: Animal Behavior Processes, 17, 503-518. Flanagan-Cato, L.M., Grigson, P.S., & King, J.L. (2001). Estrogen-induced suppression of intake is not mediated by taste aversion in female rats. Physiology & Behavior, 72, 549-558. Flint, A., Raben, A., Astrup, A., & Holst, J.J. (1998). Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. Journal of Clinical Investigations, 101, 515-520. Flood, J.F., & Morley, J.E. (1991). Increased food intake by neuropeptide Y is due to an increased motivation to eat. Peptides, 12, 1329-1332. Flynn, D.D., & Mash, D.C. (1993). Distinct kinetic binding properties of N-[3H]methylscopolamine afford differential labeling and localization of M1, M2, and M3 muscarinic receptor subtypes in primate brain. Synapse, 14, 283-296. Flynn, F.W., Grill, H.J., Schulkin, J., & Norgren, R. (1991). Central gustatory lesions: II. Effects on sodium appetite, taste aversion learning, and feeding behavior. Behavioral Neuroscience, 105, 944-954. Flynn, M.C., Scott, T.R., Pritchard, T.C., & Plata-Salaman, C.R. (1998). Mode of action of OB protein (leptin) on feeding. American Journal of Physiology, 275, R174-R179. Frankfurt, M., & Azmitia, E. (1983). The effect of intracerebral injections of 5,7dihydroxytryptamine and 6-hydroxy-dopamine on the serotonin immunoreactive cell bodies and fibers in the adult rat hypothalamus. Brain Research, 261, 91-99. Frey, S., Morris, R., & Petrides, M. (1997). A neuroanatomical method to assess the integrity of fibers of passage following ibotenate-induced damage to the central nervous system. Neuroscience Research, 28, 285-288. Friedman, M., Harris, R., Ji, H., Ramirez, I., & Tordoff, M. (1999). Fatty acid oxidation affects food intake by altering hepatic energy status. American Journal of Physiology, 276, R1046-R1053. 323

Fukuda, M., Ono, T., Nishino, H., & Sasaki, K. (1984). Independent glucose effects on rat hypothalamic neurons: an in vivo study. Journal of Autonomic Nervous System, 10, 373-381. Fulwiler, E.E., & Saper, C.B. (1984). Subnuclear organization of the efferent connections of the parabrachial nucleus in the rat. Brain Research Reviews, 7, 229259. Fulwiler, C.E., & Saper, C.B. (1985). Cholecystokinin-immunoreactive innervation of the ventromedial hypothalamus in the rat: possible substrate for autonomic regulation of feeding. Neuroscience Letters, 53, 289-296. Funahashi, H., Takenoya, F., Guan, J.-L., Kageyama, H., Yada, T., & Shioda, S. (2003). Hypothalamic neuronal networks and feeding-related peptides involved in the regulation of feeding. Anatomical Science International, 78, 123-138. Funahashi, H., Yamada, S., Kageyama, H., Takenoya, F., Guan, J.L., & Shioda, S. (2003). Co-existence of leptin- and orexin-receptors in feeding-regulating neurons in the hypothalamic arcuate nucleus: a triple labeling study. Peptides (in press). Galef, B.G., & Sherry, D.F. (1973). Mother’s milk: A medium for transmission of cues reflecting the flavor of mother’s diet. Journal of Comparative & Physiological Psychology, 83, 374-378. Gallo, M., Arnedo, M., Agüero, A., & Puerto, A. (1988). Electrical intracerebral stimulation of the area postrema on taste aversion learning. Behavioural Brain Research, 30, 289-296. Gallo, M., Arnedo, M., Agüero, A., & Puerto, A. (1990). The functional relevance of the area postrema in drug induced aversion learning. Pharmacology, Biochemistry & Behavior, 35, 543-551. Gallo, M., & Bures, J. (1991). Acquisition of conditioned taste aversion in rats is mediated by ipsilateral interaction of cortical and mesencephalic mechanisms. Neuroscience Letters, 133, 187-190. Gallo, M., Roldan, G., & Bures, J. (1992). Differential involvement of gustatory insular cortex and amygdala in the acquisition and retrieval of conditioned taste aversion in rats. Behavioural Brain Research, 52, 91-97. Ganesan, R., & Simpkins, J.W. (1990). The role of conditioned taste aversion in the suppression of food intake by estradiol. Physiology & Behavior, 48, 647-652. 324

Ganesan, R., & Simpkins, J.W. (1991). Conditioned taste aversion induced by estradiol pellets. Physiology & Behavior, 50, 849-852. Gantz,, I., Munzert, G., Tashiro, T., Schäffer, M., Wang, L., DelValle, J., et al. (1991). Molecular cloning of the human histamine H2 receptor. Biochemical & Biophysical Research Communications, 178, 1386-1392. Garbarg, M., Barbin, G., Feger, J., & Schwartz, J.C. (1974). Histaminergic pathway in rat brain evidenced by lesions of the MFB. Science, 186, 833-835. Garcia, J., Ervin, F.R., & Koelling, R.A. (1966). Learning with prolonged delay of reinforcement. Psychonomic Science, 5, 121-122. Garcia, J., Ervin, F.R., & Koelling, R.A. (1967). Toxicity of serum from irradiated donors. Nature, 213, 682-683. Garcia, J., Hankins, W.G., & Rusinak, K.W. (1974). Behavioral regulation of the milieu interne in man and rat. Science, 185, 824-831. Garcia, J., Kimeldorf, D.J., & Koelling, R.A. (1955). Conditioned aversion to saccharin resulting from exposure to gamma radiation. Science, 122, 157-158. Garcia, J., Kimeldorf, J., and Hunt, E.L. (1956). Conditioned responses to manipulative procedures resulting from exposure to gamma radiation. Radiation Research, 5, 79-87. Garcia, J., & Koelling, R.A. (1966). The relation of cue to consequence in avoidance learning. Psychonomic Science, 4, 123-124. Garcia, J., McGowan, B.K., & Green, K.F. (1972). Biological constraints on conditioning. In: M.E.P. Seligman & J.L. Hager (Eds.). Biological boundaries of learning. New York: Appleton-Century-Crofts. Garey, J., Morgan, M.A., Frohlich, J., McEwen, B.S. & Pfaff, D.W. (2001). Effects of they phytoestrogen coumestrol on locomotor and fear-related behaviors in female mice. Hormones & Behavior, 40, 65-76. Geary, N. (2000). Estradiol and appetite. Appetite, 35, 273-274. Geary, N. (2001). Estradiol, CCK and satiation. Peptides, 22, 1251-1263.

325

Geary, N., & Asarian, L. (1999). Cyclic estradiol treatment normalizes body weight and test meal size in ovariectomized rats. Physiology & Behavior, 67, 141147. Geary, N., Asarian, L., Chan, J., Korach, K., Pfaff, D., & Ogawa, S. (1996). Estradiol (E) treatment does not affect food intake (FI) or body weight (BW) in ovariectomized (OVX) estrogen receptor-α knockout mice. Society for Neuroscience Abstracts, 25, 77. Geary, N., Trace, D., McEwen, B., & Smith, G.P. (1994). Cyclic estradiol replacement increases the satiety effect of CCK-8 in ovariectomized rats. Physiology & Behavior, 56, 281-289. Gebre-Medhin, S., Mulder, H., Pekny, M., Zhang, Y., Tornell, J., Westermark, P., Sundler, F., Abren, B., & Betsholtz, C. (1997). Altered glucose homeostasis, body weight and nociception in IAPP (amylin). null mutant mice. Diabetes, 40, 29A. Gemberling, G.A., Domjan, M., & Amsel, A. (1980). Aversion learning in 5-dayold rats: taste-toxicosis and texture-shock associations. Journal of Comparative & Physiological Psychology, 94, 734-745. Gentil, C.G., Stevenson, J.A.F., & Mogenson, G.J. (1971). Effect of scopolamine on drinking elicited by hypothalamic stimulation. Physiology & Behavior, 7, 639641. Gentry, R.T., & Wade, G.N. (1976). Sex differences in sensitivity of food intake, body weight, and running-wheel activity to ovarian steroids in rats. Journal of Comparative & Physiological Psychology, 90, 747-754. Gibbs, J., Falasco, J.D., & McHugh, P.R. (1976). Cholecystokinin-decreased food intake in rhesus monkeys. American Journal of Physiology, 230, 15-18. Gibbs, J., Young, R.C., & Smith, G.P. (1973). Cholecystokinin decreases food intake in rats. Journal of Comparative & Physiological Psychology, 84, 488-495. Gibbs, J., & Smith, G.P. (1977). Cholecystokinin and satiety in rats and rhesus monkeys. American Journal of Clinical Nutrition, 30, 758-761. Gilbert, C., & Gillman, J. (1956). The changing pattern of food intake and appetite during the menstrual cycle of baboon (Papio ursinus) with consideration of some of the controlling endocrine factors. South African Journal of Medical Sciences, 21, 75-88.

326

Giovannini, M.G., Bartolini, L., Bacciottini, L., Greco, L., & Blandina, P. (1999). Effects of histamine H3 receptor agonists and antagonists on cognitive performance and scopolamine-induced amnesia. Behavioural Brain Research, 104, 147-155. Giraudo, S.Q., Kotz, C.M., Billington, C.J., & Levine, A.S. (1998). Association between the amygdala and nucleus of the solitary tract in μ-opioid induced feeding in the rat. Brain Research, 802, 184-188. Gold, R.M., Jones, A.P., Sawchenko, P.E., & Kapatos, G. (1977). Paraventricular area: critical focus of a longitudinal neurocircuitry mediating food intake. Physiology & Behavior, 18, 1111-1119. Golden, P.L., Maccagnan, T.J., & Pardridge, W.M. (1997). Human blood-brain barrier leptin receptor: binding and endocytosis in isolated human brain microvessels. Journal of Clinical Investigations, 99, 14. Gosnell, B.A., & Lipton, J.M. (1986), Opioid peptide effects on feeding in rabbits. Peptides, 7, 745-747. Goehler, L.E., Busch, C.R., Tartaglia, N., Relton, J., Sisk, D., Maier, S.F., et al. (1995). Blockade of cytokine induced conditioned taste aversion by subdiaphragmatic Vagotomy: further evidence for vagal mediation of immunebrain communication. Neuroscience Letters, 185, 163-166. Gosnell, B.A. (1988). Involvement of mu opioid receptors in the amygdala in the control of feeding. Neuropharmacology, 27, 319-326. Gould, M.N., & Yatvin, M.B. (1972). Studies of the mechanisms involved in radiation-conditioned saccharin avoidance and radiation and actinomycin-Dinduced bizarre eating in rats. International Journal of Radiation Biology, 21, 215222. Gould, M.N., & Yatvin, M.B. (1973). Atropine-caused central nervous system interference with radiation-induced learned and unlearned behaviours. International Journal of Radiation Biology, 24, 463-468. Grabauskas, G., & Moises, H.C. (2003). Gastrointestinal-projecting neurons in the dorsal motor nucleus of the vagus exhibit direct and viscerotopically organized sensitivity to orexin. Journal of Physiology, 549, 37-56. Graham, M., Shutter, J.R., Sarmiento, U., Sarosi, I. , & Stark, K.L. (1997). Overexpression of Agrt leads to obesity in transgenic mice. Nature Genetics, 17, 273-274. 327

Grandt, D., Schimiczek, M., Beglinger, Ch., Layer, P., Goebell, H., Eysselein, V.E., & Reeve Jr., J.R. (1994). Two molecular forms of peptide YY (PYY) are abundant in human blood: characterization of a radioimmunoassay recognizing PYY1-36 and PYY3-36. Regulatory Peptides, 51, 151-159. Grant, V.L., & Borison, H.L. (1988). Comments on the model of emesis. Psychopharmacology, 96, 278-279. Gray, T.S., & Magnuson, D.J. (1987). Neuropeptide neuronal efferents from the bed nucleus of the stria terminalis and central amygdaloid nucleus to the dorsal vagal complex of the rat. Journal of Comparative Neurology, 262, 365-374. Graybiel, A., Wood, C.D., Knepton, J., Hoche, J.P., & Perkins, G.F. (1975). Human assay of anti-motion sickness drugs. Aviation and Space Environmental Medicine, 46, 1107-1118. Greenberg, D. (1998). Intestinal Satiety. In: Smith, GP (Ed.). The structure and mechanisms of satiation. New York: Oxford University Press, p. 40-70. Griffin, J.E., & Ojeda, S.R. (1996). Textbook of Endocrine Physiology. New York: Oxford University Press. Griffond, B., Deray, A, Nguyen, N.U., Colard, C., & Fellmann, D. (1995). The synthesis of melanin-concentrating hormone is stimulated by ventromedial hypothalamic lesions in the rat lateral hypothalamus: A time-course study. Neuropeptides, 28, 267-275. Griffond, B., Risold, P.Y., Jacquemard, C., Colard, C., & Fellmann, D. (1999). Insulin-induced hypoglycemia increases pre-prohypocretin (orexin) mRNA in the rat lateral hypothalamic area. Neuroscience Letters, 262, 77-80. Grigson, P.S. (1997). Conditioned taste aversions and drugs of abuse: A reinterpretation. Behavioral Neuroscience, 111, 129-136. Grigson, P.S., Reilly, S., Shimura, T., & Norgren, R. (1998). Ibotenic acid lesions of the parabrachial nucleus and conditioned taste aversion: further evidence for an associative deficit. Behavioral Neuroscience, 112, 160-171. Grigson, P.S., Shimura, T., & Norgren, R. (1997a). Brainstem lesions and gustatory function: II. The role of the nucleus of the solitary tract in Na+ appetite, conditioned taste aversion, and conditioned odor aversion in rats. Behavioral Neuroscience, 111, 169-179.

328

Grigson, P.S., Shimura, T., & Norgren, R. (1997b). Brainstem lesions and gustatory function: III. The role of the nucleus of the solitary tract and the parabrachial nucleus in retention of a conditioned taste aversion in rats. Behavioral Neuroscience, 111, 180-187. Grigson, P.S., Spector, A.C., & Norgren, R. (1994). Lesions of the pontine parabrachial nuclei eliminate successive negative contrast effects in rats. Behavioral Neuroscience, 108, 714-723. Grill, H.J. Production and regulation of ingestive consummatory behavior in the chronic decerebrate rat. Brain Research Bulletin, 5, 79-87. Grill, H.J., & Norgren, R. (1978). Chronically decerebrate rats demonstrate satiation but not bait shyness. Science, 201, 267-269. Grill, H.J., & Smith, G.P. (1988). Cholecystokinin decreases sucrose intake in chronic decerebrate rats. American Journal of Physiology, 254, R853-R856. Grill, H.J., Schwartz, M.W., Kaplan, J.M., Foxhall, J.S., Breininger, J., Baskin, D.G. (2002). Evidence that the caudal brainstem is a target for the inhibitory effect of leptin on food intake. Endocrinology, 143, 239-246. Grodin, J.M., Siiteri, P.K., & MacDonald, P.C. (1973). Source of estrogen production in postmenopausal women. Journal of Clinical Endocrinology & Metabolism, 36, 207-214. Groenewegen, H.J., & Berendse, H.W. (1994). The specificity of the ‘nonspecific’ midline and intralaminar thalamic nuclei. Trends in Neuroscience, 17, 52-57. Grollman, A., & Grollman, E.F. (1970). Pharmacology and Therapeutics. Philadelphia: Lea and Febinger. Gu, Y., Gonzalez, F.G., Chen, D.Y., & Deutsch, J.A. (1993). Expression of c-fos in brain subcortical structures in response to nauseant lithium chloride and osmotic pressure in rats. Neuroscience Letters, 157, 49-52. Guan, J.L., Saotome, T., Wang, Q.P., Funahashi, H., Hori, T., Tanaka, S. et al. (2001). Orexinergic innervation of POMC-containing neurons in the rat arcuate nucleus. NeuroReport, 12, 547-551. Guan, J.L, Uehara, K., Lu, S., Wang, Q.P., Funahashi, H., Sakurai, T. et al. (2002). Reciprocal synaptic relationships between orexin- and melanin-concentrating hormone-containing neurons in the rat lateral hypothalamus: a novel circuit implicated in feeding regulation. International Journal of Obesity, 26, 1523-1532. 329

Guan, X.M., Yu, H., Palyha, O.C., McKee, K.K., Geighner, S.D., Sirinathsinghji, D.J. et al. (1997). Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Research & Molecular Brain Research, 48, 23-29. Gubernick, D.J., & Alberts, J.R. (1984). A specialization of taste aversion learning during suckling and its weaning-associated transformation. Developmental Psychobiology, 17,, 613-628. Gustavson, C.R., Gustavson, J.C., Noller, K.L., Melton, L.J., III, O’Brien, P.C., & Pumariega, A.J. (1989a). Fetal diethylstilbestrol exposure: a possible risk factor in the development of anorexia nervosa. Society of Neuroscience Abstracts, 15, 1131. Gustavson, C.R., Gustavson, J.C., Young, J.K., Pumariega, A.J., & Nicolaus, L.K. (1989b). Estrogen induced malaise. In: J.M. Lakoski, J.R. Perez-Polo, & D.K. Rassin (Eds.). Neural Control of Reproductive Function, New York: Alan R. Liss. Gustavson, C.R., Gustavson, J.C., Noller, K.L., O’Brien, P.C., Melton, L.J., Pumariega, A.J., et al. (1991). Increased risk of profound weight loss among women exposed to diethylstilbestrol in utero. Behavioral Neural Biology, 55, 307312. Gutierrez, H., Hemandez-Echegaray, E, Ramirez-Amaya, V., & Bermudez-Rattoni, F. (1999). The blockade of N-methyl-D-aspartate in the insular cortex disrupts taste aversion and spatial memory. Neuroscience, 89, 751-758. Gutierrez, R., Tellez, L.A., & Bermudez-Rattoni, F. (2003). Blockade of cortical muscarinic but not NMDA receptors prevents a novel taste from becoming familiar. European Journal of Neuroscience, 17, 1556-1562. Gutzwiller, J.P., Drewe, J., Hildebrand, P., Rossi, L., Lauper, J.Z., & Beglinger, C. (1994). Effect of intravenous human gastrin-releasing peptide on food intake in humans. Gastroenterology, 106, 1168-1173. Hagan, M.M., Rushing, P.A., Pritchard, L.M., Schwartz, M.W., Strack, A.M., van der Ploeg, L.H. et al. (2000). Long-term orexigenic effects of AgRP-(83—132). involve mechanisms other than melanocortin receptor blockade. American Journal of Physiology, 279, R47-R52. Hahn, T.M., Breininger, J.F., Baskin, D.G., & Schwartz, M.W. (1998). Coexpression of AgRP and NPY in fasting-activated hypothalamic neurons. Nature Neuroscience, 1, 271-272.

330

Hales, C.N., & Kennedy, G.C. (1964). Plasma glucose, nonesterified fatty acid and insulin concentration in hypothalamic-hyperphagic rats. Biochemical Journal, 90, 620-624. Håkansson, M.L., Brown, H., Ghilardi, N., Skoda, R.C., & Meister, B. (1998). Leptin receptor immunoreactivity in chemically defined target neurons of the hypothalamus. Journal of Neuroscience, 18, 559-572. Hamilton, R.B., & Norgren, R. (1984). Central projections of gustatory nerves in the rat. Journal of Comparative Neurology, 222, 560-577. Han, PW. (1968). Energy metabolism of tube-fed hypophysecotmized rats bearing hypothalamic lesions. American Journal of Physiology, 215, 1343-1350. Hajnal, A., Sandor, P. Jando, G., Vida, I., Czurko, A., Karadi, Z. , & Lenard, L. (1992). Feeding disturbances and EEG activity changes after amygdaloid kainate lesions in the rat. Brain Research Bulletin, 29, 909-916. Halmi, K.A., Casper, R.C., Eckert, E.D., Goldberg, S.C., & Davis, J.M. (1979). Unique features associated with age and onset of anorexia nervosa. Psychiatric Research, 1, 209-215. Hamilton, R.B., & Norgren, R. (1984). Central projections of gustatory nerves in the rat. Journal of Comparative Neurology, 222, 560-577. Hammes, B., & Laitman, C.J. (2003). Dithylstilbesterol (DES) update: recommendations for the identification and management of DES-exposed individuals. Journal of Midwifery & Women’sHealth,48, 19-29. Hanamori, T., Kunitake, T., Kato, K., & Kannan, H. (1998). Neurons in the posterior insular cortex responsive to gustatory stimulation of the pharyngolarynx, baroreceptor and chemoreceptive stimulation, and tail pinch in rats. Brain Research, 785, 97-106. Hanamori, T., Kunitake, T., Kato, K., & Kannan, H. (1998). Responses of neurons in the insular cortex to gustatory, visceral and nociceptive stimuli in rats. Journal of Neurophysiology, 79, 2535-2545. Hasselmo, M.E., & Bower, J.M. (1993). Acetylcholine and memory. Trends in Neuroscience, 16, 218-222. Harrell, E.H., & Remley, N.R. (1973). The immediate development of behavioral and biochemical changes following ventromedial hypothalamic lesions in rats. Behavioral Biology, 9, 49-63. 331

Harris, R.B.S., Kasser, T.R., & Martin, R.J. (1986). Dynamics of recovery of body composition after overfeeding, food restriction or starvation of mature female rats. Journal of Nutrition, 116, 2536-2546. Hathout, E.H., Sharkey, J., Racine, M., Ahn, D., Mace, J.W., & Saad, M.F. (1999). Changes in plasma leptin during the treatment of diabetic ketoacidosis. Journal of Clinical Endocrinology & Metabolism, 84, 4545-4548. Haug, E., Aakvaag, A., Sand, T., & Torjesen, P.A. (1974). The gonadotropin response to synthetic gonadotropin-releasing hormone in males in relation to age, dose, and basal serum levels of testosterone, oestradiol-17beta and gonadotropins. Acta Endocrinologica, 77, 625-635. Havel, P.J., Kasim-Karakas, S., Mueller, W., Johnson, P.R., Gingerich, R.L. , & Stern, J.S. (1996). Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sustained weight loss. Journal of Clinical Endocrinology & Metabolism, 81, 4406-4413. Havel, P.J., Uriu-Hare, J.Y., Liu, T., Stanhope, K.L., Stern, J.S., Keen, C.L. et al. (1998). Marked and rapid decreases of circulating leptin in streptozotocin diabetic rats: reversal by insulin. American Journal of Physiology, 274, R1482-R1491. Hawkins, R.A., Freedman, B., Marshall, A., & Killn, E. (1975). Oestradiol 17-beta and prolactin levels in rat peripheral plasma. British Journal of Cancer, 32, 179185. Hayakawa, T., Takanaga, A., Tanaka, K., Maeda, S., & Seki, M. (2003). Cells of origin of vagal motor neurons projecting to different parts of the stomach in the rat: confocal laser scanning and electron microscopic study. Anatomy & Embryology, 207, 289-297. Haynes, A.C., Jackson, B., Chapman, H., Tadayyon, M., Johns, A., Porter, R.A. et al. (2000). A selective orexin-1 receptor antagonist reduces food consumption in male and female rats. Regulatory Peptides, 22, 45-51. Heinbecker, P., White, H.L., & Rolf, D. (1944). Experimental obesity in the dog. American Journal of Physiology, 141, 549-565. Heinrichs, S.C., Menzaghi, F., Pich, E.M., Hauger, R.L., & Koob, G.F. (1993). Corticotropin-releasing factor in the paraventricular nucleus modulates feeding induced by neuropeptide Y. Brain Research, 611, 18-24.

332

Henderson, S.R., Baker, C., & Fink, G. (1977). Oestradiol-17β and pituitary responsiveness to luteinizing hormone releasing factor in the rat: A study using rectangular pulses of oestradiol-17β monitored by non-chromatographic radioimmunoassay. Journal of Endocrinology, 73, 441-453. Herbert, H., & Saper, C.B. (1990). Cholecytokinin-, galanin-, and corticotropinreleasing factor-like immunoreactive projections from the nucleus of the solitary tract to the parabrachial nucleus in the rat. Journal of Comparative Neurology, 293, 581-598. Hermann, G.E., Kohlermann, N.J., & Rogers, R.C. (1983). Hepatic-vagal and gustatory afferent interactions in the brainstem of the rat. Journal of the Autonomic Nervous System, 9, 477-495. Hermann, G.E., & Rogers, R.C. (1985), Convergence of vagal and gustatory afferent input within the parabrachial nucleus of the rat. Journal of the Autonomic Nervous System, 13, 1-17. Herve, C., & Fellmann, D. (1997). Changes in the rat melanin-concentrating hormone and dynorphin messenger ribonucleic acids induced by food deprivation. Neuropeptides, 31, 237-242. Hetherington, A.W., & Ranson, S.W. (1940). Hypothalamic lesions and adiposity in the rat. Anatomical Record, 78, 149-172. Heyer, B.R., Taylor-Burds, C.C., Tran, L.H., & Delay, E.R. (2003). Monosodium glutamate and sweet taste: Generalization of conditioned taste aversion between monosodium glutamate and sweet stimuli in rats. Chemical Senses, 28, 631-641. Heymsfield, S.B., Greenberg, A.S., Fujioka, K., Dixon, R.M., Kushner, R., Hunt, T., Lubina, J.A., Patane, J., Self, B., Hunt, P., & McCamish, M. (1999). Recombinant leptin for weight loss in obese and lean adults: a randomized, controlled, dose-escalation trial. Jama, 282, 1568-1575. Hickey, M.S., Israel, R.G., Gardiner, S.N., Cosidine, R.V., McCammon, M.R., Tyndall, G.L., Houmard, J.A., Marks, R.H. , & Caro, J.R. (1996). Gender differences in serum leptin levels in humans. Biochemical & Molecular Medicine, 59, 1-6. Hill, D.L., & Almli, C.D. (1983). Parabrachial nuclei damage in infant rats produces residual deficits in gustatory preferences/aversions and sodium appetite. Developmental Psychobiology, 16, 519-533.

333

Hill, S.J., & Young, M. (1978). Antagonism of central histamine H1 receptors by antipsychotic drugs. European Journal of Pharmacology, 52, 397-399. Hines, M., Alsum, P., Roy, M., Gorski, R.A., & Goy, R.W. (1987). Estrogenic contributions to sexual differentiation in the female guinea pig influences of diethylstilbesterol and tamoxifen on neural, behavioral, and ovarian development. Hormones & Behavior, 21, 402-417. Hines, M., & Goy, R.W. (1985). Estrogens before birth and development of sexrelated reproductive traits in the female guinea pig. Hormones & Behavior, 19, 331-347. Hintiryan, H., Hayes, U.L., & Chambers, K.C. (2005). The role of histamine in estradiol-induced conditioned consumption reductions. Physiology & Behavior, 84, 117-128. Hintiryan, H. (2003) Reexamining the role of histamine in conditioned consumption reductions. Master’s thesis, University of Southern California. Holder, M.D., Garcia, J., Yirmiya, R., & Raizer, J. (1989). Conditioned taste aversions are not readily disrupted by external excitation. Behavioral Neuroscience, 103, 605-611. Hollopeter, G., Erickson, J.C., Seeley, R.J., Marsh, D.J., & Palmiter, R.D. (1998). Response of neuropeptide Y-deficient mice to feeding effectors. Regulatory Peptides, 785, 383-389. Horvath, T., Diano, S., Anthoney, N., van de Pol, A.N. (1999). Synaptic interaction between hypocretin (orexin) and neuropeptide Y cells in the rodent and primate hypothalamus: a novel circuit implicated in metabolic and endocrine regulations. Journal of Neuroscience, 19, 1072-1087. Hosoya, Y., Sugiura, Y., Okado, N., Loewy, A.D., & Kohno, K. (1991). Descending input from the hypothalamic paraventricular nucleus to sympathetic preganglionic neurons in the rat. Experimental Brain Research, 85, 10-20. Hough, L.B. (2001). Genomics meets histamine receptors: new subtypes, new receptors. Molecular Pharmacology, 59, 415-419. Houpt, T.A., & Berlin, R. (1999). Rapid, labile, and protein synthesis independent short-term memory in conditioned taste aversion. Learning and Memory, 6, 37-46.

334

Houpt, T.A., Philopena, J.M., Joh, T.H., Smith, G.P. (1996). C-fos expression in the rat nucleus of the solitary tract after conditioned taste aversion formation. Learning & Memory, 3, 25-30. Hulst, S.G. Th. (1972). Intracerebral implantation of carbachol in the rat: its effects on water intake and body temperature. Physiology & Behavior, 8, 865-872. Hunt, T., & Amit, Z. (1987). Conditioned taste aversion induced by selfadministered drugs: paradox revisited. Neuroscience and Biobehavioral Reviews, 11, 107-30. Huszar, D., Lynch, C.A., Fairchild-Huntress, V., Dunmore, J.H., Fang, Q., Berkemeier, L.R. et al. (1997). Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell, 88, 131-141. Iida, M., Murakami, T., Ishida, K., Mizuno, A., Kuwajima, M., , & Shima, K. (1996). Phenotype-linked amino acid alteration in leptin receptor cDNA from Zucker fatty (fa/fa) rat. Biochemical & Biophysical Research Communication, 222, 19-26. Inagaki, S., Shiotani, Y., Yamano, M., Shiosaka, S., Takagi, H., Tateishi, K., et al. (1984). Distribution, origin, and fine structures of cholecystokinin-8-like immunoreactive terminals in the nucleus ventromedialis hypothalami of the rat. Journal of Neuroscience, 4, 1289-1299. Ivanova, S.F., & Bures, J. (1990a). Conditioned taste aversion is disrupted by prolonged retrograde effects of intracerebral injection of tetrodotoxin in rats. Behavioral Neuroscience, 104, 948-954. Ivanova, S.F., & Bures, J. (1990b). Acquisition of conditioned taste aversion in rats is prevented by tetrodotoxin blockade of small midbrain region centered around the parabrachial nuclei. Physiology & Behavior, 48, 543-549. Jewett, D.C., Cleary, J., Levine, A.S., Schaal, D.W., & Thompson, T. (1992). Effects of neuropeptide Y on food-reinforced behavior in satiated rats. Pharmacology, Biochemistry & Behavior, 42, 207-212. Jhamandas, J.H., & Harris, K.H. (1992). Excitatory amino acids may mediate nucleus tractus solitarius input to rat parabrachial neurons. American Journal of Physiology, 263, R324-R330.

335

Jia, Hong-Ge, Rao, Zhi-Ren & Shi & Ji-Wu (1994). An indirect projection from the nucleus of the solitary tract to the central nucleus of the amygdala via the parabrachial nucleus in the rat: a light and electron microscopic study. Brain Research, 663, 181-190. Jin, S.L.C., Han, V.K.M., Simmons, J.G., Towle, A.C., Lauder, J.M., & Lund, P.K. (1988). Distribution of glucagon-like peptide-1 (GLP-1) glucagon, and glicentin in the rat brain. Journal of Comparative Neurology, 271, 519-532. Jones, D.J., Fox, M.M., Babigian, H.M., & Hutton, H.E. (1980). Epidemiology of anorexia nervosa in Monroe County, New York: 1960-1976. Psychosomatic Medicine, 42, 551-558. Jones, M.E.E., Thorburn, A.W., Britt, K.L., Hewitt, K.N., Wreford, N.G., Proietto, J., et al. (2000). Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proceedings of the National Academy of Sciences, 97, 12735-12740. Jones, M.W., French, P.J., Bliss, T.V., & Rosenblum, K. (1999). Molecular mechanisms of long-term Potentiation in the insular cortex in vivo. Journal of Neuroscience, 19, RC36. Joseph, S.A., Pilcher, W.H., & Knigge, K.M. (1985). Anatomy of corticotropin releasing factor and opiomlanocortin systems of the brain. Federation Proceedings, 44, 100-107. Kagotani, Y., Sakata, I., Yamazaki, M., Nakamura, K., Hayashi, Y., & Kangawa, K. (2001). Localization of ghrelin-immunopositive cells in the rat hypothalamus and intestinal tract. Proceedings of the 83rd Annual Meeting of the Endocrine Society, Denver, CO, p. 337. Kahler, A., Geary, N., Eckel, L.A., Campfield, L.A., Smith, F.J. , & Langhans, W. (1998). Chronic administration of OB protein decreases spontaneous food intake by reducing meal size in male rats. American Journal of Physiology, 275, R180-R186. Kaiyala, K.J., Woods, S.C., & Schwartz, M.W. (1995). New model for the regulation of energy balance by the central nervous system. American Journal of Clinical Nutrition, 62, 1123S. Kakolewski, J.W., Cox, V.C., & Valenstein, E.S. (1968). Sex differences in bodyweight change following Gonadectomy of rats. Psychological Report, 22, 547-554. Kalat, J.W., & Rozin, P. (1971). Role of interference in taste-aversion learning. Journal of Comparative and Physiological Psychology, 77, 53-58. 336

Kalia, M. & Sullivan, M.J. (1982). Brainstem projections of sensory and motor components of the vagus nerve in the rat. Journal of Comparative Neurology,211, 248-264. Kalucy RS. (1980). Drug-induced weight gain. Drugs. 19, 268-78. Kamei, C., Okumura, Y., & Tasaka, K. (1993). Influence of histamine depletion on learning and memory recollection in rats. Psychopharmacology, 111, 376-382. Kamei, C., & Tasaka, K. (1991). Participation of histamine in the step-through active avoidance response and its inhibition byH1-blockers. Japanese Journal of Pharmacology, 57, 473-482. Kamm, J.J., Ferullo, C.R., Miller, D., & van Loon, E.J. (1969). Metabolism of chlorpheniramine-3H by the rat and dog. Biochemistry & Pharmacology, 18, 659671. Kappauf, W.E., & Schlosberg, H. (1937). Conditioned responses in the white rat. III. Conditioning as a function of the length of the period of delay. Journal of Genetic Psychology, 50, 27-45. Karla, S.P., Dube, M.G., Pu, S., Xu, B., Horvarth, T.L., & Karla, P.S. (1999). Interacting appetite-regulating pathways in the hypothalamic regulation of body weight. Endocrine Reviews, 20, 68-100. Karla, S.P., Dube, M.G., Sahu, A., Phelps, C., & Karla, P.S. (1991). Neuropeptide Y secretion increases in the paraventricular nucleus in association with increase appetite with food. Proceedings of the National Academy of Sciences, 88, 1093110935. Kawakami, S., Bungo, T., Ohgushi, A., Ando, R., Shimojo, M., Masuda, Y. et al. (2000). Brain-derived mast cells could mediate histamine-induced inhibition of food intake in neonatal chicks. Brain Research, 857, 313-316. Kemnitz, J.W., Eisele, S.G., Lindsay, K.A., Engle, M.J., Perelman, R.H., & Farrell, P.M. (1984). Changes in food intake during menstrual cycles and pregnancy of normal and diabetic rhesus monkeys. Diabetes, 26, 60-64. Kennett, G.A., & Cruzon, G. (1988). Evidence that hypophagia induced by mCPP and TFMPP requires 5-HT1C and 5-HT1B receptors; hypophagia induced by RU 24969 only requires 5-HT1B receptors. Psychopharmacology, 96, 241-250.

337

Khachaturian, H., Lewis, M.E., Haber, S.N., Akil, H., & Watson, S.J. (1984). Proopiomelanocortin peptide immunocytochemistry in rhesus monkey brain. Brain Research Bulletin, 13, 785-800. Khavari, K.A., & Maickel, R.P. (1967). Atropine and atropine methyl bromide effects on behavior of rats. International Journal of Neuropharmacology, 6, 301307. Kiefer, S.W. (1985). Neural mediation of conditioned food aversions. Annals of the New York Academy of Sciences, 443, 100-109. Kiefer, S.W., Cabral, R.J., Rusinak, K.W., & Garcia, J. (1980). Ethanol-induced flavor aversions in rats with subdiaphragmatic vagotomies. Behavioural Neural Biology, 29, 246-254. Kiefer, S.W., Rusinak, K.W., & Garcia, J. (1981). Vagotomy facilitates extinction of conditioned taste aversions in rats. Journal of Comparative and Physiological Psychology, 95, 114-122. Kilduff, T.S., & de Lecea, L. (2001). Mapping of the mRNAs for the hypocretin/orexin and melanin-concentrating hormone receptors: networks of overlapping peptide systems. Journal of Comparative Neurology, 435, 1-5. Kim, M.H., Hosseinian, A.H., & Dupon, C. (1974). Plasma levels of estrogens, androgens, and progesterone during normal and dexamethasone-treated cycles. Journal of Clinical Endocrinology & Metabolism, 39, 706-712. Kimble, G.A. (1947). Eyelid conditioning as a function of the interval between conditioned and unconditioned stimuli. New York, NY: Irvington Publishers. King, B.M., Carpenter, R.G., Stamoutsos, B.A., Frohman, L.A., & Grossman, S.P. (1978). Hyperphagia and obesity following ventromedial hypothalamic lesions in rats with subdiaphragmatic vagotomy. Physiology & Behavior, 20, 643-651. King, B.M., & Frohman, L.A. (1986). Hypothalamic obesity: comparison of radiofrequency and electrolytic lesions in male and female rats. Brain Research Bulletin, 17, 409-413. King, J.M. (1979). Effects of lesions of the amygdala, preoptic area, and the hypothalamus on estradiol-induced activity in the female rat. Journal of Comparative Physiological Psychology, 93, 360-367.

338

Koh, M.T., & Berntein, I.L. (2005). Mapping conditioned taste aversion associations using c-fos reveals a dynamic role for insular cortex. Behavioral Neuroscience, 119, 388-398. Kohl, R.L., Homick, J.L., Cintron, N., & Calkins, D.S. (1987). Lack of effects of astemizole on vestibular ocular reflex, motion sickness and cognitive performance in man. Aviation Space & Environmental Medicine, 58, 1171-1174 Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H.,, & Kangawa, K. (2001). Ghrelin is a growth- hormone-releasing acylated peptide from stomach. Nature, 402, 656-660. Kosten, T., & Contreras, R. J. (1989). Deficits in conditioned heart rate and taste aversion in area postrema-lesioned rats. Behavioural Brain Research, 35, 9-21. Kotz, C.M., Billington, C.J. , & Levine, A.S. (1997). Opioids in the nucleus of the solitary tract are involved in feeding in the rat. American Journal of Physiology, 272, R1028-R1032. Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., & Gruters, A. (1998). Severe early-onset obesity, adrenal insufficiency and red hair pigmentation caused by POMC mutations in humans. Nature Genetics, 19, 155-157. Kow, L., & Pffaf, D. (1991). The effects of the TRH-metabolite cyclo (His-Pro) and its analogs on feeding. Pharmacology, Biochemistry & Behavior, 38, 359-364. Koylu, E.O., Couceyro, P.R., Lambert, P.D., Ling, N.C., DeSouza, E.B., Kuhar, M.J. (1997). Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland. Journal of Neuroendocrinology, 9, 823-833. Kristensen, P., Judge, M.E., Thim, L., Ribel, U., Christjansen, K.N., Wulff, B.S., Clausen, J.T., Jensen, P.B., Madsen, O.D., Vrang, N., Larsen, P.J., & Hastrup, S. (1998). Hypothalamic CART is a new anorectic peptide regulated by leptin. Nature, 393, 72-76. Krukoff, T.L, Harris, K.H., & Jhamandas, J.H. (1993). Efferent projections from the parabrachial nucleus demonstrated with the anterograde tracer phaseolus vulgaris leucoagglutinin. Brain Research Bulletin, 30, 163-172. Kuldosky, P.J., Glazner, G.W., Moore, H.D., Low, C.A., & Woods, S.C. (1988). Neuropeptide Y: behavioral effects in the golden hamster. Peptides, 9, 1389-1393.

339

Kyrkouli, S.E., Stanley, B.G., Leibowitz, S.F. (1986). Galanin: stimulation of feeding induced by medial hypothalamic injection of this novel peptide. European Journal of Pharmacology, 122, 159-160. Kyrkouli, S.E., Stanley, B.G., Hutchinson, R., Seirafi, R.D., Leibowitz, S.F. (1990). Peptide-amine interactions in the hypothalamic paraventricular nucleus: analysis of galanin and neuropeptide Y in relation to feeding. Brain Research, 521, 185-191. Kyrkouli, S.E., Stanley, B.G., Seirafi, R.D., Leibowitz, S.F. (1990). Stimulation of feeding by galanin: anatomical localization and behavioral specificity of this peptide’s effects in the brain. Peptides, 11, 995-1001. Kyrkouli, S.E., Stanley, B.G., Leibowitz, S.F. (1992). Differential effects of galanin and neuropeptide Y on extracellular norepinephrine levels in the paraventricular hypothalamic nucleus of the rat: a microdialysis study. Life Sciences, 51, 203-210. Lambert, P.D., Couceyro, P.R., McGirr, K.M., Dall Vechia, S.E., Smith, Y., & Kuhar, M.J. (1998). CART peptides in the central control of feeding and interactions with neuropeptide Y. Synapse, 29, 293-298. Lamprecht, R., & Dudai, Y. (1995). Differential modulation of brain immediate early genes by intraperitoneal LiCl. NeuroReport, 7, 289-293. Lanca, A.J., & Van der Kooy, D. (1985). A serotonin-containing pathway from the area postrema to the parabrachial nucleus in the rat. Neuroscience, 14, 1117-1126. Landt, M., Gingerich, R.L., Havel, P.J., Mueller, W.M., Schoner, B., Hale, J.E., & Heiman, M.L. (1998). Radioimmunoassay of rat leptin: sexual dimorphism reversed from humans. Clinical Chemistry, 44, 565-570. Lang, I.M., & Marvig, J. (1989). Functional localization of specific receptors mediating gastrointestinal motor correlates of vomiting. American Journal of Physiology, 19, 692-699. Langhans, W., Grossman, F., & Geary, N. (2001). Intrameal hepatic-portal infusion of glucose reduces spontaneous meal size in rats. Physiology & Behavior, 73, 499-507.

340

Larsen, P.J., Moller, M., & Mikkelsen, J.D. (1991). Efferent projections from the periventricular and medial parvicellular subnuclei of the hypothalamic paraventricular nucleus to circumventricular organs of the rat: a Phaseolus vulgarisleucoagglutinin (PHA-L) tracing study. Journal of Comparative Neurology, 306, 462-479. Larsen, P.J., Hay-Schmidt, A., Vrang, N., & Mikkelsen, J.D. (1996). Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: a combined retrograde and anterograde tracing study. Neuroscience, 70, 963-988. Larsen, P.J., Tang-Christensen, M., Holst, J.J., & Ørskov, C. (1997). Distribution of glucagon-like peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem. Neuroscience, 77, 257-270. Larsen, P.J., Vrang, N., Tang-Christensen, M, Jensen, P.B., Hay-Schmidt, A., Rømer, J, Bjerre-Knudsen, L., & Kristensen, P. (2002). Ups and downs for neuropeptides in body weight homeostasis: pharmacological potential of cocaine amphetamine regulated transcript and pre-proglucagon-derived peptides. European Journal of Pharmacology, 440, 159-172. Lee, D. W., & Green, K.F. (1989.) Effects of antihistamines on centrifugal rotation-induced analgesia and conditioned flavor aversions. Physiology & Behavior, 45, 459-464. Lee, M.D., Aloyo, V.J., Fluharty, S.J., & Simansky, K.J. (1998). Infusion of serotonin1B (5-HT1B) agonist CP-93,129 into the parabrachial nucleus potently and selectively reduces food intake in rats. Psychopharmacology, 136, 304-307. Leedom, L., J., & Meehan, W.P. (1989). The psychoneuroendocrinology of diabetes mellitus in rodents. Psychoneuroendocrinology, 14, 275-294. Leibowitz, S.F. (1978). Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking. Pharmacology, Biochemistry & Behavior, 8, 163-175. Leibowitz, S.F., Hammer, N.J., & Chang, K. (1981). Hypothalamic paraventricular nucleus lesions produce overeating and obesity in the rat. Physiology & Behavior, 27, 1031-1040. Leibowitz, S.F. (1990). The role of serotonin in eating disorders. Drugs, 39, 3348.

341

Leshner, A.I., & Collier, G. (1973). The effects of gonadectomy on the sex differences in dietary self-selection patterns and carcass composition of rats. Physiology & Behavior, 11, 671-676. Leslie, R.A., & Gwyn, D.G. (1984). Neural connections of the area postrema. Federal Proceedings, 43, 2941-2943. Leslie, R.A., Gwyn, D.G., & Hopkins, D.A. (1982). The central distribution of the cervical vagus nerve and gastric afferent and efferent projections in the rat. Brain Research Bulletin, 8, 37-43. Levitt, R.A., & Boley, R.P. (1970). Drinking elicited by injection of esterine or carbachol into rat brain. Physiology & Behavior, 5, 693-695. Levy, C.J., Carrol, M.E., Smith, J.C., & Hofer, K.G. (1974). Antihistamines block radiation-induced taste aversions. Science, 186, 1044-1046. Levy, C.J., Carrol, M.E., & Smith, J.C. (1975). Histamine and radiation-induced taste aversion conditioning. Science, 190, 402-403. Lewis, D.E., Shellard, L., Koeslag, D.G., Boer, D.E., McCarthy, H.D., McKibbin, P.E., Russell, J.C., & Williams, G. (1993). Intense exercise and food restriction cause similar hypothalamic neuropeptide Y increases in rats. American Journal of Physiology, 264, E279-E284. Li, B.H., Spector, A.C., & Rowland, N.E. (1994). Reversal of dexfenfluramineinduced anorexia and c-Fos/c-Jun expression by lesion in the lateral parabrachial nucleus. Brain Research, 640, 255-267. Li, C., Chen, P., & Smith, M.S. (1998). The acute suckling stimulus induces expression of neuropeptide Y (NPY) in cells in the dorsomedial hypothalamus and increases NPY expression in the arcuate. Endocrinology, 139, 1645-1652. Li, C., Chen, P., & Smith, M.S. (1999). Identification of neuronal input to the arcuate nucleus (ARH). activated during lactation: implications in activation of neuropeptide Y neurons. Brain Research, 824, 267-276. Li, C.S., & Smith, D.V. (1997). Glutamate receptor antagonists block gustatory afferent input to the nucleus of the solitary tract. Journal of Neurophysiology, 77, 1514-1525. Lin, M.T., Chu, P.C., & Leu, S.Y. (1983). Effects of TSH, TRH, LH and LHRH on thermoregulation and food and water intake in the rat. Neuroendocrinology, 37, 206-211. 342

Lindén, A., Uvnäs-Moberg, K., Forsberg, G., Bednar, I., & Södersten, P. (1990). Involvement of cholecystokinin in food intake: III. Oestradiol potentiates the inhibitory effect of cholecystokinin octapeptide on food intake in ovariectomized rats. Journal of Neuroendocrinology, 2, 797-801. Lindstrom, P.A., & Brizzee, K.R. (1962). Relief from intractable vomiting from surgical lesions in the area postrema. Journal of Neuroscience, 19, 228-236. Lintunin, M., Sallmen, T., Karlstedt, K., Fkui, H., Eriksson, K.S., & Panula, P. (1998). Postnatal expression of H1 receptor mRNA in the rat brain: correlation to Lhistidine decarboxylase expression and local upregulation in limbic seizures. European Journal of Neuroscience, 10, 2287-2301. Lopez-Garcia, J.C., Bermudez-Rattoni, F., & Tapia, R. (1990). Release of acetylcholine, γ-aminobutyrate, dopamine and glutamate, and activity of some related enzymes, in rat gustatory neocortex. Brain Research, 523, 100-104. Lovenberg, T.W., Rowland, B.L., Wilson, S.J., Jiang, X, Pyati, J., Huvar, A., et al. (1999). Cloning and functional expression of the human histamine H3 receptor. Molecular Pharmacology, 55, 1101-1107. Lowey, A.D., & Burton, H. (1979). Nuclei of the solitary tract: efferent projections to the lower brain stem and spinal cord of the cat. Journal of Comparative Neurology, 18, 421-450. Lozeva, V., Tuomisto, L., Sola, D., Plumed, C., Hippeläninen, M., & Butterworth. (2001). Increased density of brain histamine H1 receptors in rats with portacaval anastomosis and in cirrhotic patients with chronic hepatic encephalopathy. Hepatology, 33, 1370-1376. Lu, J.K., LaPolt, P.S., Nass, T.E., Matt, D.W., & Judd, H.J. (1985). Relation of circulating estradiol and progesterone to gonadotropin secretion and estrous cyclicity in aging female rats. Endocrinology, 116, 1953-1959. Lu, S., Guan, J.L., Wang, Q.P., Uehara, K., Yamada, S., Goto, N. et al. (2002). Immunohistochemical observation of ghrelin-containing neurons in the rat arcuate nucleus. Neuroscience Letters, 321, 157-160. Lucas, G.A., Timberlake, W., Gawley, D.J., & Drew, J. (1990). Anticipation of future food: suppression and facilitation of saccharin intake depending on the delay and type of future food. Journal of Experimental Psychology: Animal Behavior Processes, 16, 169-177.

343

Ludwig, D.S., Tritos, N.A., Mastaitis, J.W., Kulkami, R., Kokkotou, E., Elmquist, J. et al. (2001). Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance. Journal of Clinical Investigations, 107, 379-386. Luiten, P.G., ter Horst, G.J. , & Steffens, A.B. (1987). The hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism. Progress in Neurobiology, 28, 1-54. Lutz, T.A., del Prete, E., & Scharrer, E. (1994). Reduction of food intake in rats by intraperitoneal injection of low doses of amylin. Physiology & Behavior, 55, 891895. Lutz, T.A., del Prete, E., & Scharrer, E. (1995). Subdiaphragmatic vagotomy does not influence the anorectic effect of amylin. Peptides, 16, 457-462. Lutz, T.A., Mollet, A., Rushing, P.A., Riediger, T., & Scharrer, E. (2001). The anorectic effect of a chronic peripheral infusion of amylin is abolished in area postrema/nucleus of the solitary tract (AP/NST) lesioned rats. International Journal of Obesity, 25, 1005-1011. Lutz, T.A., Senn, M., Althaus, J., del Prete, E., Ehrensperger, F., & Scharrer, E. (1998a). Lesions of the area postrema/nucleus of the solitary tract (AP/NST) attenuates the anorectic effects of amylin and calcitonin gene-related peptide (CGRP) in rats. Peptides, 19, 309-317. Lutz, T.A., Rossi, R., Althaus, J., del Prete, E., & Scharrer, E. (1998b). Amylin reduces food intake more potently than calcitonin gene-regulated peptide (CGRP) when injected into the lateral brain ventricle in rats. Peptides, 19, 1533-1540. Lutz, T.A., Tschudy, S., Rushing, P.A., & Scharrer, E. (2000). Attenuation of the anorectic effects of cholecystokinin and bombesin by the specific amylin antagonist AC253. Physiology & Behavior, 70, 533-536. Lynch, D.R., Walker, M.W., & Snyder, S.H. (1989). Neuropeptide Y receptor binding sites in rat brain: differential autoradiographic localizations with 125Ipeptide YY and 125I-neuropeptide Y imply receptor heterogeneity. Journal of Neuroscience, 9, 2607-2619. Machidori, H., Sakata, T., Yoshimatsu, H., Ookuma, K., Fujimoto, K., Kurokawa, M., et al. (1992). Zucker obese rats: defect in brain histamine control of feeding. Brain Research, 90, 180-186.

344

Mackintosh, N.J. (1974). The physiology of animal learning. Academic Press, New York, NY. MacLusky, N.J., & Naftolin, F. (1981). Sexual differentiation of the central nervous system. Science, 211, 1294-1303. Mallios, V.J., Lydic, R., & Baghdoyan, H.A. (1995). Muscarinic receptor subtypes are differentially distributed across brain stem respiratory nuclei. American Journal of Physiology, 268, L941-949. Download and read this article. Need to see which subnuclei. Mansour, A., Fox, C.A., Akil, H., & Watson, S.J. (1995). Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications. Trends in Neuroscience, 18, 22-29. Mantyh, P.W., & Hunt, S.P. (1984). Neuropeptides are present in projection neurons at all levels in visceral and taste pathways: from the periphery to the sensory cortex. Brain Research, 299, 297-311. Markowska, A., Bakke, H.K., Walther, B., & Ursin, H. (1985). Comparison of electrolytic and ibotenic acid lesions in the lateral hypothalamus. Brain Research, 328, 313-323. Marcus, J.N., Aschkenasi, C.J., Lee, C.E., Chemelli, R.M., Saper, C.B., Yanagisawa, M. et al. (2001). Differential expression of orexin receptors 1 and 2 in the rat brain. Journal of Comparative Neurology, 435, 6-15. Marsh, D.J., Miura, Y., Yagaloff, K.A., Schwartz, M.W., Barsh, G.S., & Palmiter, R.D. (1999). Effects of neuropeptide Y deficiency on hypothalamic agouti-related expression and responsiveness to melanocortin analogues. Brain Research, 848, 66-77. Martin, J.R., Cheng, F.Y., & Novin, D. (1978). Acquisition of learned taste aversion following bilateral subdiaphragmatic vagotomy in rats. Physiology & Behavior, 21, 13-17. Martin, J.R., Rogers, R.C., Novin, D., & VanderWeele, D.A. (1997). Excessive gastrointestinal retention by vagotomized rats and rabbits given a solid diet. Bulletin of the Psychonomic Society, 10; 291-294. Martin, L.T., & Alberts, J.R. (1979). Taste aversion to mother’s milk: The agerelated role of nursing in acquisition and expression of a learned association. Journal of Comparative & Physiological Psychology, 93, 430-445. 345

Martinez-Mir, M.I., Pollard, H., Moreau, J., Traiffort, E., Ruat, M., Schwartz, J.C., et al. (1993). Loss of striatal histamine H2 receptors in Huntington’s Chorea but not in Parkinson’s disease: comparative with animal models. Synapse, 15, 209220. Martyn, J.A.J., Greenblatt, D.J., & Abernethy, D.R. (1985). Increased cimetidine clearance in burned patients. Journal of American Medical Association, 253, 12881291. Masugi, M., Yokoi, M., Shigemoto R., Muguruma, K, Watanabe, Y., Sansig, G., van der Putten, H., & Nakanishi, S. (1999). Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion. Journal of Neuroscience, 19, 955-963. Mayer, J. (1955). Regulation of the energy intake and the body weight: the glucostatic theory and the lipostatic hypothesis. Annals of the New York Academy of Sciences, 63, 15-43. McDonald, T.J., Brooks, B.D., Rokaeus, A., Tinner, B., & Staines, W.A. (1992). Pancreatic galanin: molecular forms and anatomical locations. Pancreas, 7, 624635. McGowan, M.K., Andrews, K.M., Kelly, J., & Grossman, S.P. (1990). Effects of chronic intrahypothalamic infusion of insulin on food intake and diurnal meal patterning in the rat. Behavioral Neuroscience, 104, 373. Meguro, K.I., Yanai, K., Sakai, N., Sakurai, E., Maeyama, K., Sasaki, H., et al. (1995). Effects of thioperamide, a histamine H3 antagonist, on the step-through passive avoidance response and histidine decarboxylase activity in senescenceaccelerated mice. Pharmacology, Biochemistry, & Behavior, 50, 321-325. Melander, T., Hokfelt, T., Rokaeus, A., Fahrenkrug, J., Tatemoto, K. , & Mutt, V. (1985). Distribution of galanin-like immunoreactivity in the gastro-intestinal tract of several mammalian species. Cell Tissue Research, 239, 253-270. Mercer, J.G., Hoggard, N., Williams, L.M., Lawrence, Hannah, L.T., Morgan, P.J., & Trayburn, P. (1996). Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. Journal of Neuroendocrinology, 8, 733-735. Mercer, J.G., Hoggard, N., Williams, L.M., Lawrence, C.B., Hannah, L.T., & Trayburn, P. (1996). Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in sit hybridization. FEBS Letters, 387, 113-116. 346

Mercer, J.G., Moar, K.M., Findlay, P.A., Hoggard, N.,, & Adam, C.L. (1998). Association of leptin receptor (OB-Rb), NPY and GLP-1 gene expression in the ovine and murine brainstem. Regulatory Peptides, 75-76, 271-278. Mercer, J.G., & Speakman, J.R. (2001). Hypothalamic neuropeptide mechanisms for regulating energy balance: from rodent models to human obesity. Neuroscience Biobehavioral Reviews, 25, 101-116. Merchenthaler, I., Lopez, F.J., & Negro-Vilar, A. (1993). Anatomy and physiology of central galanin-containing pathways. Progress in Neurobiology, 40, 711-769. Merwin, A.A., & Doty, R.L. (1994). Early exposure to low levels of estradiol (E2) mitigates E2-induced conditioned taste aversions in prepubertally ovariectomized female rats. Physiology & Behavior, 55, 185-187. Meyers, B., Roberts, K.H., Riciputi, R.H., & Domino, E.F. (1964). Some effects of muscarinic cholinergic blocking drugs on behavior and the electrocorticogram. Psychopharmacologia, 5, 289-300. Mezey, E., Reisine, T.D., Skirboll, L., Beinfeld, M , & Kiss, J.Z. (1986). Role of cholecystokinin in corticotrophin release: coexistence with vasopressin and corticotropin-releasing factor in cells of the rat hypothalamic paraventricular nucleus. Proceedings of the National Academy of Sciences, 83, 3510-3512. Miceli, M.O., & Fleming, A.S. (1983). Variation of fat intake with estrous cycle, ovariectomy and estradiol replacement in hamsters (Mesocricetus auratus) eating a fractionated diet. Physiology & Behavior, 30, 415-420. Miceli, M.O., Post, C.A., & van der Kooy, D. (1987). Catecholamine and serotonin colocalization in projection neurons of the area postrema. Brain Research, 412, 391-385. Miele, J., Rosellini, R.A., & Svare, B. (1988). Estradiol benzoate can function as an unconditioned stimulus in a conditioned taste aversion paradigm. Hormones & Behavior, 22, 116-130. Milner, T.A., Joh, T.H., & Pickel, V.M. (1986). Tyrosine hydroxylase in the rat parabrachial region: Ultarstructural localization and extrinsic sources of immunoreactivity. Journal of Neuroscience, 6, 2585-2603.

347

Miranda, M.I., Ferreira, M.G., Ramirez-Lugo, L., & Bermudez-Rattoni, F. (2002.) Glutamatergic activity in the amygdala signals visceral input during taste memory formation. Proceedings from the National Academy of Sciences, USA, 99, 1141711422. Miranda, M.I., Ramirez-Lugo, L., & Bermudez-Rattoni, F. (2000). Cortical cholinergic activity is related to the novelty of stimulus. Brain Research, 882, 2305. Miyazaki, S., Imaizumi, M., & Onodera, K. (1995). Ameliorating effects of histidine on scopolamine-induced learning deficits using an elevated plus-maze test in mice. Life Sciences, 56, 1563-1570. Moga, M.M., Herbert, H., Hurley, K.M., Yasui, Y., Gray, T.S. & Saper, C.B. (1990). Organization of cortical, basal forebrain, and hypothalamic afferent to the parabrachial nucleus in the rat. Journal of Comparative Neurology, 295, 624-661. Moga, M.M., Saper, C.B., & Gray, T.S. (1990). Neuropeptide organization of the hypothalamic projection to the parabrachial nucleus in the rat. Journal of Comparative Neurology, 295, 662-682. Moore, K.E. Biochemical correlates of the behavioral effects of drugs. In: An Introduction to Psychopharmacology. R.H. Rech and K.E. Moore (Eds.) Raven Press: New York, 1971. Moran, T., & Schwartz, G. (1994). Neurobiology of cholecystokinin. Critical Reviews in Neurobiology, 9, 1-28. Mordes, J.P., & Rossini, A.A. (1981). Tumor-induced anorexia in the Wistar rat. Science, 213, 565-567. Mordes, J.P., Longscope, C., Flatt, J.P., MacLean, D.B., & Rossini, A.A. (1984). The rat LTW(m) Leydig cell tumor: cancer anorexia due to estrogen. Endocrinology, 115, 167-173. Morgan, M.A., & Pfaff, D.W. (2001). Effects of estrogen on activity and fearrelated behaviors in mice. Hormones & Behavior, 40, 472-482. Mori, K., Yoshimoto, A., Takaya, K., Hosoda, K., Ariyasu, H., Yahata, K. et al. (2000). Kidney produces a novel acylated peptide, ghrelin. FEBS Letters, 486, 213216.

348

Morin, L.P. & Fleming, A.S. (1978). Variation in food intake and body weight with estrous cycle, ovariectomy and estradiol benzoate treatment in hamsters (Mesocricetus auratus). Journal of Comparative & Physiological Psychology, 92, 1-6. Morley, J.E., Hernandez, E.N., & Flood, J.F. (1987). Neuropeptide Y increases food intake in mice. American Journal of Physiology, 253, R515-R522. Morley, J.E., Levine, A.S., Gosnell, B.A., Kneip, J., & Grace, M. (1987). Effect of neuropeptide Y on ingestive behaviors in the rat. American Journal of Physiology, 252, R599-R609. Morley, J.E., Levine, A.S., Yim, G.K., & Lowy, M.T. (1983). Opioid modulation of appetite. Neuroscience and Biobehavioral Reviews, 7, 281-305. Morrel, J.I., Krieger, M.S., & Pfaff, D.W. (1986). Quantitative autoradiographic analysis of estradiol retention by cells in the preoptic area, hypothalamus, and amygdala. Experimental Brain Research, 62, 343-354. Morris, R., Frey, S, Kasambira, T., & Petrides, M. (1999). Ibotenic acid lesions of the basolateral, but not the central amygdala interfere with conditioned taste aversion: Evidence from a combined behavioral and anatomical tract-tracing investigation. Behavioral Neuroscience, 113, 291-302. Moulton, R. (1942). A psychosomatic study of anorexia nervosa, including the use of vaginal smears. Psychosomatic Medicine, 4, 62-74. Muir, J.L, Page, K.J., Sirinathsinghji, D.J.S., Robbins, T.W., & Everitt, B.J. (1993). Excitotoxic lesions of basal forebrain cholinergic neurons: effects on learning, memory, and behavior. Behavioural Brain Research, 57, 123-131. Myers, R. (1966). Injection of solutions into cerebral tissue: relation between volume and diffusion. Physiology & Behavior, 1, 171-174. Myers, R.H., & de Castro, J.M. (1977). Learned aversions to intracerebral carbachol. Physiology & Behavior, 19, 467-472. Mystkowski, P., & Schwartz, M.W. (2000). Gonadal steroids and energy homeostasis in the leptin era. Nutrition, 16, 937-946. Nachman, M. (1970). Learned taste and temperature aversions due to lithium chloride sickness after temporal delays. JCPP, 73, 22-30.

349

Nader, K., Bechara, A., & van der Kooy, D. (1996). Lesions of the lateral parabrachial block the aversive motivational effects of both morphine and morphine withdrawal but pare morphine’s discriminative properties. Behavioral Neuroscience, 110, 1496-1502. Nagai, K., Ino, H., Yamamoto, H., Nakagawa, H., Yamano, M., Tohyama, M., et al. (1987). Lesions in the lateral part of the dorsal parabrachial nucleus caused hyperphagia and obesity. Journal of Clinical and Biochemical Nutrition, 3, 103112. Nagy, Z.M., & Glaser, H.D. (1970). Open-field behavior of C57BL/6J mice: effects of illumination, age and number of test days. Psychonomic Science, 19, 143-145. Nakazato, M., Murakami, N., Date, Y., Kojima, M., Matsuo, H., Kangawa, K. et al. (2001). A role for ghrelin in the central regulation of feeding. Nature, 409, 194198. Naor, C., & Dudai, Y. (1996). Transient impairment of cholinergic function in the rat insular cortex disrupts the encoding of taste in conditioned taste aversion. Behavioural Brain Research, 79, 61-7. Näslund, E., Bogefors, J., Skogar, S., Brybäck, P., Jacosson, H., Holst, J.J. et al. (1999). Glucagon-like peptide-1 slows solid gastric emptying with inhibition of insulin, C-peptide, glucagon and YY peptide release in humans. American Journal of Physiology, 277, R910-916. Näslund, E., Gutniak, M., Skogar, S., Rossner, S., & Hellström, P.M. (1998). Glucagon-like peptide 1 increases the period of postprandial satiety and slows gastric emptying in obese men. American Journal of Clinical Nutrition, 68, 525530. Nelson, L.R., & Bulun, S.E. (2001). Estrogen production and action. Journal of the American Academy of Dermatology, 45, S116-S124. Nerad, L., Ramirez-Amaya, V., Ormsby, C.E., & Bermudez-Rattoni, F. (1996). Differential effects of anterior and posterior insular cortex lesions on the acquisition of conditioned taste aversion and spatial learning. Neurobiology of Learning & Memory, 66, 44-50. Nicholls, J.G., & Kuffler, S.W. (1964). Extracellular space as a pathway for exchange between blood and neurons in the central nervous system of the leech: ionic composition of glial cells and neurons. Journal of Neurophysiology, 27, 645671. 350

Nijima, A., & Yamamoto, T. (1994). The effects of LiCl on the activity of the afferent nerve fibers from the abdominal visceral organs in the rat. Brain Research Bulletin, 35, 141-145. Norgren, R. (1974). Gustatory afferents to ventral forebrain. Brain Research, 81, 285-295. Norgren, R. (1976). Taste pathways to hypothalamus and amygdala. Journal of Comparative Neurology, 166, 17-30. Norgren, R. (1978). Projections from the nucleus of the solitary tract in the rat. Neuroscience, 3, 207-218. Norgren, R. (1984). Central neural mechanisms of taste. In: Darian Smith, I. (Ed.). Handbook of Physiology, Section I, The Nervous System Sensory Processes Part 2, vol. III. American Physiological Society, Bethesda, pp. 1087-1128. Norgren, R., & Leonard, C.M. (1971). Taste pathways in rat brainstem. Science, 173, 1136-1139. Norgren, R., & Leonard, C.M. (1973). Ascending central gustatory pathways. Journal of Comparative Neurology, 150, 217-238. Novin, D., VanderWeele, D.A., & Zidek, L. (1973). Hepatic-portal 2-deoxy-Dglucose infusion causes eating: Evidence for peripheral glucoreceptors. Science, 181, 858-860. Ogawa, S., Chan, J., Gustafsson, J, Korach, K.S., & Pfaff, D.W. (2003). Estrogen increases locomotor activity in mice through estrogen receptor α: specificity for the type of activity. Endocrinology, 144, 230-239. Ollmann, M.M., Wilson, B.D., Yang, Y.K., Kerns, J.A., Chen, Y., Gantz, I. et al. (1997). Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein. Science, 278, 135-138. Olszewski, P.K., Grace, M.K., Billington, C.J., & Levine, A.S. (2003). Hypothalamic paraventricular injections of ghrelin: effect on feeding and c-Fos immunoreactivity. Peptide, 24, 919-923. Ørskov, C. (1992). Glucagon-like peptide-1, a new hormone of the enteroinsular axis. Diabetologia, 35, 701-711.

351

Ossenkopp, K.P. (1983). Taste aversions conditioned with gamma radiation: attenuation by area postrema lesions in rats. Behavioural Brain Research, 7, 295305. Ossenkopp, K.P., & Giugno, L. (1990). Nicotine-induced conditioned taste aversions are enhanced in rats with lesions of the area postrema. Pharmacology, Biochemistry & Behavior, 36, 625-630. Ossenkopp, K.P., Rabi, Y.J., & Eckel, L.A. (1996). Oestradiol-induced taste avoidance is the result of a conditioned palatability shift. Neuroreport, 7, 277727780. Otto, B., Cuntz, U., Fruehauf, E., Wawarta, R., Folwaczny, C., Riepl, R.L. et al. (2001). Weight gain decreases elevated plasma ghrelin concentrations of patients with anorexia nervosa. European Journal of Endocrinology, 145, 669-673. Overpeck, J.G., Colson, S.H., Hohmann, J.R., Applestine, M.S., & Reilly, J.F. (1978). Concentrations of circulating steroids in normal prepubertal and adult male and female humans, chimpanzees, rhesus monkeys, rats, mice, and hamsters: a literature survey. Journal of Toxicology & Environmental Health, 4, 785-803. Overton, D.A. (1974). Experimental methods for the study of state-dependent learning. Federal Proceedings, 33, 1800-1813. Palmer, K., & Gray, J.M. (1986). Central vs. peripheral effects of estrogen on food intake and lipoprotein lipase activity in ovariectomized rats. Physiology & Behavior, 37, 187-189. Panula, P., Yang, H.Y.T., & Costa, E. (1984). Histamine-containing neurons in the rat hypothalamus. Proceedings of the National Academy of Sciences, USA, 81, 25722576. Parker, L.A. (1988). Positively reinforcing drugs may produce a different kind of CTA than drugs which are not positively reinforcing. Learning & Motivation, 10, 207-220. Parker, L.A. (1995). Rewarding drugs produce taste avoidance, but not taste aversion. Neuroscience and Biobehavioral Reviews, 19, 143-151. Parker, L.A., & Brosseau, L. (1990). Apomorphine-induced flavor-drug associations: A dose-response analysis by the taste reactivity test and the conditioned taste avoidance test. Pharmacology, Biochemistry & Behavior, 35, 583-587. 352

Pau, M.Y., Pau, K.Y. , & Spies, H.G. (1985). Characterization of central actions of neuropeptide Y on food and water intake in rabbits. Physiology & Behavior, 44, 797-802. Pavlov, I.P. (1927). Conditioned reflexes. London: Oxford University Press. Pillot, C., Heron, A., Cochois, V., Tardivel-Lacombe, J., Ligneau, X., Schwartz, J.C., et al. (2002). A detailed mapping of the histamine H3 receptor and its gene transcripts in rat brain. Neuroscience, 14, 173-193. Pearson, J.A. (1973). Effect of scopolamine and atropine on habituation of the flexor withdrawal reflex. Pharmacology, Biochemistry & Behavior, 1, 155-157. Pedersen-Bjergaard, U., Host, U., Kelbaek, H., Schifter, S., Rehfeld, J.F., Faber, J. et al. (1996). Influence of meal composition on postprandial peripheral plasma concentrations of vasoactive peptides in man. Scandinavian Journal of Clinical & Laboratory Investigation. 56, 497-503. Peeters, B.W., Smets, R.J., & Broekkamp, C.L. (1992). Sex steroids possess distinct stimulus properties in female and male mice. Brain Research Bulletin, 28, 319-321. Perrotto, R.S., & Scott, T.R. (1976). Gustatory neural coding in the pons. Brain Research, 110, 283-300. Persinger, M.A. (1977). Mast cells in the brain: possibilities for physiological psychology. Physiological Psychology, 5, 166-176. Persinger, M.A., & Fiss, T.B. (1978). Mesenteric mast cell degranulation is not essential for conditioned taste aversion. Pharmacology, Biochemistry & Behavior, 9, 725730. Pert, A. (1975). The cholinergic system and nociception in the primate: interactions with morphine. Psychopharmacologia, 44, 131-137. Peterson, S.R., Saldanha, C. J., & Schlinger, B. A. (2001). Rapid Upregulation of Aromatase mRNA and Protein Following Neural Injury in the Zebra Finch (Taeniopygia guttata). Journal of Neuroendocrinology, 13, 317-323. Petrov, T., Jhamandas, J.H., & Krukoff, T.L. (1996). Connectivity between brainstem autonomic structures and expression of c-fos following electrical stimulation of the central nucleus of the amygdala in rat. Cell Tissue Research, 283, 367-374. 353

Peyron, C., Tighe, D.K., van den Pol, A.N., de Lecea, L., Heller, H.C., Sutcliffe, J.G. et al. (1998). Neurons containing hypocretin (orexin) project to multiple neuronal systems. Journal of Neuroscience, 18, 9996-10015. Pieber, T.R., Roitelman, J., Lee, Y., Luskey, K.L., & Stein, D.T. (1994). Direct plasma radioimmunoassay for rat amylin-(1-37) concentrations with acquired and genetic obesity. American Journal of Physiology, 267, E156. Pinch, M.E., Messori, B., Zoli, M., Ferraguti, F., Marrama, P., Biagini, G. et al. (1992). Feeding and drinking responses to neuropeptide Y injections in the paraventricular hypothalamic nucleus of aged rats. Brain Research, 575, 265-271. Placios, J.M., Wamsley, J.K., & Kuhar, M.J. (1981). The distribution of histamine H1-receptors in the rat brain: an autoradiographic study. Neuroscience, 6, 15-37. Plotnik, R., Molenauer, S., & Snyder, E. (1974). Fear reduction in the rat following central cholinergic blockade. Journal of Comparative & Physiological Psychology, 86, 1074-1082. Poggioli, R., Vergoni, A.V., & Bertolini, A. (1986). ACTH(1-24) and alpha-MSH antagonize feeding behavior stimulated by kappa opiate agonists. Peptides, 7, 843848. Polidori, C., de Caro, G., & Massi, M. (2000). The hyperphagic effect of nociception/orphanin FQ in rats. Peptides, 21, 1051-1062. Powley, T.L. (1977). The ventromedial hypothalamic syndrome, satiety, and a cephalic phase hypothesis. Psychological Reviews, 84, 89-126. Powley, T.L., & Opsahl, C.A. (1974). Ventromedial hypothalamic obesity abolished by subdiaphragmatic vagotomy. American Journal of Physiology, 226, 25-33. Presse, F., Sorokovsky, I., Max, J.P., Nicolaidis, S., & Nahon, J.L. (1996). Melanin-concentrating hormone is a potent anorectic peptide regulated by fooddeprivation and glucopenia in the rat. Neuroscience, 71, 735-745. Preston, K.L., & Schuster, C.R. (1981). Conditioned gustatory avoidance induced by three cholinergic agents. Pharmacology, Biochemistry & Behavior, 15, 827828. Quillan, J.M, Sadee, W., Wei, E.T., Jimenez, C., Ji, L., & Chang, J.K. (1998). A synthetic human Agouti-related protein-(83-132).-NH2 fragment is a potent inhibitor of melanocortin receptor function. FEBS Letters, 428, 59-62. 354

Rabin, B.M., Hunt, W.A., & Lee, J. (1982). Studies on the role of central histamine in the acquisition of radiation-induced conditioned taste aversion. Radiation Research, 90, 609-620. Rabin, B.M., Hunt, W.A., & Lee, J. (1983a). Attenuation of radiation- and druginduced conditioned taste aversions following area postrema lesions in the rat. Radiation Research, 93, 388-394. Rabin, B.M., Hunt, W.A., & Lee, J. (1983b). Recall of a previously acquired conditioned taste aversion in rats following lesions of the area postrema. Physiology & Behavior, 32, 119-122. Rabin, B.M., Hunt, W.A., & Lee, J. (1989). Attenuation and cross-attenuation in taste aversion learning in the rat: studies with ionizing radiation, lithium chloride and ethanol. Pharmacology, Biochemistry & Behavior, 31, 909-918. Ramirez-Lugo, L., Miranda, M.I., Escobar, M.L., Espinosa, E., & BermudezRattoni, F. (2003). The role of cortical cholinergic pre- and post-synaptic receptors in taste memory formation. Neurobiology of Learning & Memory, 79, 184-193. Ramsden, M., Berchtokd, N.C., Kesslak, J.P., Cotman, C.W., & Pike, C.J. (2003) Exercise increases the vulnerability of rat hippocampal neurons to kainate lesion. Brain Research, 971, 239-244. Rehfeld, J.F. (1978). Immunohistochemical studies on cholecystokinin. Journal of Biological Chemistry, 253, 4022-4030. Reicher, M.A., & Holman, E.W. (1977). Location preference and flavor aversion reinforced by amphetamine in rats. Animal Learning & Behavior, 5, 343-346. Reilly, S., Bornovalova, M., & Trifunovic, R. (2004). Excitotoxic lesions of the gustatory thalamus spare simultaneous contrast effects but eliminate anticipatory negative contrast: evidence against a memory deficit. Behavioral Neuroscience, 118, 365-376. Reilly, S., Grigson, P.S., & Norgren, R. (1993a). Parabrachial nucleus lesions and conditioned taste aversion: evidence supporting an associative deficit. Behavioral Neuroscience, 107, 1005-1007. Reilly, S., Harley, C., & Revusky, S. (1993b). Ibotenic lesions of the hippocampus enhance latent inhibition in conditioned taste preference. Behavioral Neuroscience, 107, 996-1004.

355

Reilly ,S., & Trifunovic, R. (2000a). Lateral parabrachial nucleus lesions in the rat: aversive and appetitive gustatory conditioning. Brain Research Bulletin, 52, 269278. Reilly, S., & Trifunovic, R. (2000b). Lateral parabrachial nucleus lesions in the rat: long- and short-duration gustatory preference tests. Brain Research Bulletin, 51, 177-186. Reilly, S., & Trifunovic, R. (2001). Lateral parabrachial nucleus lesions in the rat: neophobia and conditioned taste aversion. Brain Research Bulletin, 55, 359-366. Rescorla, R.A. (2001). Retraining of extinguished Pavlovian stimuli. Journal of Experimental Psychology: Animal Behavior Processes, 27, 115-124. Revusky, S.H. (1968). Aversion to sucrose produced by contingent X-irradiation: temporal and dosage parameters. Journal of Comparative & Physiological Psychology, 65, 17-22. Revusky, S.H., & Bedarf, E.W. (1967). Association of illness with prior ingestion of novel foods. Science, 155, 219-220. Reynolds, B. (1945). The acquisition of a trace conditioned response as a function of the magnitude of the stimulus trace. Journal of Experimental Psychology, 35, 15-30. Reynolds, R.W., & Simpson, C.W. (1969). Pulmonary edema induced by intracranial carbachol infusion in rabbits and rats. Physiology & Behavior, 4, 635639. Ricardo, J.A., & Koh, E.T. (1978). Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Brain Research, 153, 1-26. Rice, A.G., Lopez, A., Garcia, J. (1987). Estrogen produces conditioned taste aversions in rats which are blocked by antihistamines. Society for Neuroscience Abstract, 13, 556. Rice, A.G. (1989). Estrogen produced conditioned taste aversions. Unpublished Dissertation, University of California, Los Angeles, Los Angeles. Ridley, P.T., & Brooks, F.P. (1965). Alterations in gastric secretion following hypothalamic lesions producing hyperphagia. American Journal of Physiology, 209, 319-323. 356

Riediger, T., Zuend, D., Becskei, C., & Lutz, T.A. (2004). The anorectic hormone amylin contributes to feeding-related changes of neuronal activity in key structures of the gut-brain axis. American Journal of Physiology, 286, R114-R122. Ritter, R.C., Slusser, P.G., & Stone, S. (1981). Glucoreceptors controlling feeding and blood glucose: location in the hindbrain. Science, 213, 451-453. Roldan, G., & Bures, J. (1994). Tetrodotoxin blockade of amygdala overlapping with poisoning impairs acquisition of conditioned taste aversion in rats. Behavioural Brain Research, 65, 213-219. Romano, J.A., & King, J.M. (1987). Conditioned taste aversion and cholinergic drugs: pharmacological antagonism. Pharmacology, Biochemistry & Behavior, 27, 81-85. Roseberry, A.G., Liu, H., Jackson, A.C., Cai, X., & Friedman, J.M. (2004). Neuropeptide Y-mediated inhibition of proopiomelanocortin neurons in the arcuate nucleus shows enhanced desensitization in ob/ob mice. Neuron, 41, 711-722. Rossi, M., & Bloom, S.R. (1997). MCH acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology, 138, 351-355. Rossi, M., Choi, S.J., O’Shea, D., Miyoshi, T., Ghatel, M.A., & Bloom, S.R. (1997). Melanin-concentrating hormone acutely stimulates feeding, but chronic administration has no effect on body weight. Endocrinology, 138, 351-355. Rowland, N.E., Crews, E.C., & Gentry, R.M. (1997). Comparison of Fos induced in rat brain by GLP-1 and amylin. Regulatory Peptides, 71, 171-174. Rowland, N.E., & Richmond, R.M. (1999). Area postrema and the anorectic actions of dexfenfluramine and amylin. Brain Research, 820, 86-91. Roy, E.J., & Wade, G.N. (1977). Role of food intake in estradiol-induced body weight changes in female rats. Hormones & Behavior, 8, 265-274. Roy, E.J., & Wade, G.N. (1975). Role of estrogens in androgen-induced spontaneous activity in male rats. Journal of Comparative & Physiological Psychology, 89, 573-579. Roy, M.M. (1992). Effects of prenatal testosterone and ATD on reproductive behavior in guinea pigs. Physiology & Behavior, 51, 105-109.

357

Ruat, M., Traiffort, E., Bouthenet, M.L., Schwartz, J.C., Hirschfeld, & Buschauer, A. (1990). Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using [125I]iodinated probes. Proceedings of the National Academy of Sciences, 87, 1658-1662. Rubio, S., Begega, A., Santin, L.J., Miranda, R., Arias, J.L. (2001). Effects of histamine precursor and (R)-α-methylhistamine on the avoidance response in rats. Behavioural Brain Research, 124, 177-181. Rushing, P.A., Lutz, T.A., Seeley, R.J., & Woods, S.C. (2000). Amylin and insulin interact to reduce food intake in rats. Hormone & Metabolic Research, 32, 62-65. Rushing, P.A., Hagan, M.M., Seeley, R.J., Lutz, T.A., D’Alessio, D.A., Air, E.L. et al. (2001). Inhibition of central amylin signaling increases food intake and body adiposity in rats. Endocrinology, 142, 5035-5038. Sahu, A. (1998). Leptin decreases food intake induced by melanin-concentrating hormone (MCH), galanin (GAL) and neuropeptide Y (NPY) in the rat. Endocrinology, 129, 4739-4742. Sahu, A., & Karla, S.P. (1993). Neuropeptidergic regulation of feeding behavior: Neuropeptide Y. Trends in Endocrinology & Metabolism, 4, 217-224. Sahu, A., Karla, P.S., & Karla, S.P. (1988). Food deprivation and ingestion induced reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus. Peptides, 9, 83-86. Sahu, A., Sninsky, C.A., Phelps, C.P., Dube, M.G., Karla, P.S., & Karla, S.P. (1992). Neuropeptide Y release from the paraventricular nucleus increases in association with hyperphagia in strptozotocin-induced diabetic rats. Endocrinology, 131, 2979-2985. Sainsbury, A., Cusin, I., Doyle, P., Rohner-Jeanrenaud, F., & Jeanrenaud, B. (1996). Intracerebroventricular administration of neuropeptide Y to normal rats increases obese gene expression in white adipose tissue. Diabetoogia, 39, 353-356. Sakai, N., & Yamamoto, T. (1997). Conditioned taste aversion and c-fos expression in the rat brainstem after administration of various USs. NeuroReport, 8, 2215-2220. Sakai, N., & Yamamoto, T. (1998). Role of the medial and lateral parabrachial nucleus in acquisition and retention of conditioned taste aversion in rats. Behavioural Brain Research, 93, 63-70. 358

Sakai, N., & Yamamoto, T. (1999). Possible routes of visceral information in the rat brain in formation of conditioned taste aversion. Neuroscience Research, 35, 53-61. Sakai, N., Tanimizu, T., Sako, N., Shimura, T., & Yamamoto, T. (1994). Effects of lesions of the medial and lateral parabrachial nuclei on acquisition and retention of conditioned taste aversion. In: K. Kurihara, N. Suzuki, and H. Ogawa (Eds.). Olfaction and taste, XI. Tokyo: Springer-Verlag, pp.495-496. Sakurai, T., Amemiya, A., Ishii, M., Matsuzaki, I., Chemelli, R.M., Tanaka, H. et al. (1998). Orexins and orexin receptors: a family of hypothalamic neuropeptides and G-protein-coupled receptors that regulate feeding behavior. Cell, 92, 573-585. Saladin, R., de Vos, P., Guerre-Millo, M., Leturque, A., Girard, J., Staels, B., et al. (1995). Transient increase in obese gene expression after food intake or insulin administration. Nature, 337, 527-529. Saleh, T.M., & Saleh, M.C. (2001). Inhibitory effect of 17 beta estradiol in the parabrachial is mediated by GABA. Brain Research, 911, 116-124. Saleh, T.M., Saleh, M.C., Deacon, C.L., & Chisholm, A. (2002). 17β-estradiol release in the parabrachial nucleus of the rat evoked by visceral afferent activation. Molecular & Cellular Endocrinology, 186, 101-110. Sand, W.A., Arruda Camargo, L.A., Silva Netto, C.R., Gentil, C.G., AntunesRodriguez, J., & Covian, M.R. (1975). Natriuresis, kaliuresis and diuresis in the rat following microinjections of carbachol into the sepatal area. Physiology & Behavior, 15, 985-992. Sandberg, D., David, S., & Stewart, J. (1982). Effects of estradiol benzoate on the pattern of eating and ethanol consumption. Physiology & Behavior, 29, 61-65. Sanders, M.K. Lakey, J.R., & Singh, D. (1973). Sex differences in hyperphagia and body weight gains following goldthioglucose-induced hypothalamic lesions in mice. Physiological Psychology, 1, 237-240. Saper, C.B. (1982). Convergence of autonomic and limbic connections in the insular cortex of the rat. Journal of Comparative Neurology, 210, 163-173. Saper, C.B., & Loewy, A.D. (1980). Efferent connections of the parabrachial nucleus in the rat. Brain Research, 197, 291-317. Saper, C.B. (2000). Hypothalamic connections with the cerebral cortex. Progress in Brain Research, 126, 39-48. 359

Saper, C.B. (2002). The central autonomic nervous system: conscious visceral perception and autonomic pattern generation. Annual Review of Neuroscience, 25, 433-469. Sar, M., & Stumpf, W.E. (1981). Central noradrenergic neurons concentrate 3Hoestradiol. Nature, 289, 500-502. Sar, M., Sahu, A., Crowley, W.R., & Karla, S.P. (1990). Localization of neuropeptide Y (NPY) immunoreactivity in estradiol concentrating cells in the hypothalamus. Endocrinology, 127, 2752-2756. Sato, M.A., Colombari, E., & Morrison, S.F. (2001). Inhibition of neurons in commissural nucleus of solitary tract reduces sympathetic nerve activity in SHR. American Journal of Physiology, 282, 1679-1684. Satoh, N., Ogawa, Y., Katsuura, G., Hayase, M., Tsuji, T., Imagawa, K. et al. (1997). The arcuate nucleus as a primary site of satiety effect of leptin in rats. Neuroscience Letters, 224, 149-152. Sawchenko, P.E., & Swanson, L.W. (1982). The organization of forebrain afferents to the paraventricular an supraoptic nuclei of the rat. Journal of Comparative Neurology, 218, 121-144. Sawchenko, P.E., Swanson, L.W., & Vale, W.W. (1984). Co-expression of corticotropin-releasing factor and vasopressin immunoreactivities in parvocellular neurosecretory neurons in the hypothalamus of adrenalectomized rats. Proceedings of the National Academy of Sciences, 81, 1883-1887. Sawchenko, P.E., Swanson, L.W., & Joseph, S.A. (1982). The distribution and cells of origin of ACTH(1-39).-stained varicosities in the paraventricular and supraoptic nuclei. Brain Research, 232, 365-374. Scalera, G., Grigson, P.S., Shimura, T., Reilly, S., & Norgren R. (1992). Excitotoxic parabrachial nucleus lesions disrupt conditioned taste aversion, conditioned odor aversion, and sodium appetite in rats. Society for Neuroscience Abstracts, 18, 1039. Scalera, G., Spector, A.C., & Norgren, R. (1995). Excitotoxic lesions of the parabrachial nuclei prevent conditioned taste aversions and sodium appetite in rats. Behavioral Neuroscience, 109, 997-1008. Schafe, G.E., Sollars, S.I., & Bernstein, I.L. (1995). The CS-US interval and taste aversion learning: a brief look. Behavioral Neuroscience, 109, 799-802. 360

Schick, R.R., Yaksh, T.L., & Go, V.L.W. (1986). An intragastric meal releases the putative satiety factor cholecystokinin from hypothalamic neurons in cats. Brain Research, 370, 349-353. Schick, R.R., Samsami, S., Zimmermann, J.P., Eberl, T., Endres, C., Schusdziarra, V. et al. (1993). Effect of galanin on food intake in rats: involvement of lateral and ventromedial hypothalamic sites. American Journal of Physiology, 264, R355R361. Schick, R.R., Schusdziarra, V., Yaksh, T.L., & Go, V.L.W. (1994). Brain regions where cholecystokinin exerts its effect on satiety. Annals of the New York Academy of Sciences, 713, 242-254. Schick, R.R., Zimmermann, J.P., vorm Walde, T., & Schusdziarra, V. (2003). Glucagon-like peptide 1-(7-36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats. American Journal of Physiology, 284, R1427-1435. Schirra, J., Leicht, P., Hildebrand, P., Beglinger, C., Arnold, R., Goke, B. et al. (1998). Mechanisms of the antidiabetic action of subcutaneous glucagon-like peptide-1(7-36). amide in non-insulin dependent diabetes mellitus. Journal of Endocrinology, 156, 177-186. Schoenfeld, T.A., & Hamilton, L.W. (1981). Disruption of appetite but not hunger or satiety following small lesions in the amygdala of rats. Journal of Comparative & Physiological Psychology, 95, 565-587. Schou, M. (1968). Lithium in psychiatry—A review. In D.H. Efron (Ed.), Pharmacology: A review of progress 1957–1967 (National Institute of Mental Health, Public Health Service Publication No. 1836, Chevy Chase, Maryland, pp. 701–718). Schwarcz, R., Kohler, C.H., Fuxe, Hokfelt, T., & Goldstein, M. (1979). On the mechanism of selective neuronal degeneration in the rat brain: Studies with ibotenic acid. In: T.N.Chase, N. Wexler, & A. Barbeau (Eds.), Advances in Neurology, vol. 23 (pp. 655-668). New York: Raven Press. Schwartz, M.W., Sipols, A.J., Kahn, S.E., Lattermann, D.F., Taborsky, G.J., Bergman, R.N. et al. (1990). Kinetics and specificity of insulin uptake from plasma into cerebrospinal fluid. American Journal of Physiology, 259, E378. Schwartz, M.W., Bergman, R.N., Kahn, S.E., Taborsky, G.J., Jr., Fisher, L.D., Sipols, A.J. et al. (1991). Evidence for entry of plasma insulin into cerebrospinal fluid through an intermediate compartment in dogs: quantitative aspects and implications for transport. Journal of Clinical Investigations, 88, 1272-1281. 361

Schwartz, M.W., Sipols, A.J., Marks, J.L., Sanacora, G., White, J.D., Scheurink, A. et al. (1992). Inhibition of hypothalamic neuropeptide Y gene expression by insulin. Endocrinology, 130, 3608-3616. Schwartz, M.W., Baskin, D.G., Bukowski, T.R., Kuijper, J.L., Foster, D., Lasser, G. et al. (1996). Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes, 45, 531535. Schwartz, M.W., Seeley, R.J., Campfield, L.A., Burn, P., & Baskin, D.G. (1996). Journal of Clinical Investigations, 98, 1101-1106. Schwartz, M.W., & Seeley, R.J. (1997). The new biology of body weight regulation. Journal of American Diet Association, 97, 54. Schwartz, M.W., Woods, S.C., Porte, D.J., Seeley, R.J., & Baskin, D.G. (2000). Central nervous system control of food intake. Nature, 404, 661. Schwartz, W.J., & Gainer, H. (1977). Suprachiasmatic nucleus: use of 14C-labeled deoxyglucose uptake as a functional marker. Science, 197, 1089-1091. Segerson, T.P., Kauer, J., Wolfe, H.C., Mobtaker, H., Wu, P., Jackson, I.M., & Lechan, R.M. (1987). Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus. Science, 238, 78-80. Sewards, T.V. (2004). Dual separate pathways for sensory and hedonic aspects of taste. Brain Research Bulletin, 62, 271-283. Seyle, H. The mast cells. Washington: Buttersworths; 1965. Shapiro, R.E., & Miselis, R.R. (1985a). The central neural connections of the area postrema of the rat. Journal of Comparative Neurology, 234, 344-364. Shapiro, R.E., & Miselis, R.R. (1985b). The central organization of the vagus nerve innervating the stomach of the rat. Journal of Comparative Neurology, 238, 473-488. Shimada, M., Tritos, N.A., Lowell, B.B., Flier, J.S., & Maratos-Flier, E. (1998). Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature, 396, 670-674. Shimizu, I., Hirota, M., Ohboshi, C., & Shima, K. (1987). Identification and localization of the glucagon-like peptide-1 and its receptor in rat brain. Endocrinology, 121, 1076-1082. 362

Shimura, T., Zuzuki, M., & Yamamoto, T. (1995). Aversive taste stimuli facilitate extracellular acetylcholine release in the insular gustatory cortex of the rat: A microdialysis study. Brain Research, 679, 221-226. Shimura, T., Grigson, P.S., & Norgren, R. (1997). Brainstem lesions and gustatory function: I. The role of the nucleus of the solitary tract during a brief intake test in rats. Behavioral Neuroscience, 111, 155-168. Shintani, M., Ogawa, Y., Ebihara, K., Aizawa-Abe, M., Miyanaga, F., Takaya, K., et al. (2001). Ghrelin, an endogenous growth hormone secretagogue, is a novel orexigenic peptide that antagonizes leptin action through the activation of hypothalamic neuropeptide Y/Y1 receptor pathway. Diabetes, 50, 227-232. Shioda, S., Funahashi, H., Nakajo, S., Yada, T., Maruta, O., & Nakai, Y. (1998). Immunohistochemical localization of leptin receptor in the rat brain. Neuroscience Letters, 243, 41-44. Shipley, M.T., & Sanders, M.S. (1982). Special senses are really special: Evidence for reciprocal bilateral pathway between insular cortex and nucleus parabrachialis. Brain Research Bulletin, 8, 493-501. Shoham, Z., & Schachter, M. (1996). Estrogen biosynthesis-regulation, action, remote effects, and value of monitoring in ovarian stimulation cycles. Fertility & Sterility, 65, 687-701. Shughrue, P.J., Lane, M.V., & Merchenthaler, I. (1997). Comparative distribution of estrogen receptor-α and -β mRNA in the rat central nervous system. The Journal of Comparative Neurology, 388, 507-525. Silva, R.M., Hadjimarkou, M.M., Rossi, G.C., Pasternak, G.W., & Bodnar, R.J. (2001). β-Endorphin-induced feeding: pharmacological characterization using selective opiod antagonists and anitsense probes in rats. Journal of Pharmacology & Experimental Therapeutics, 297, 590-596. Silver, W.L., & Finger, T.E. (1991). The trigeminal system. In T.V. Getchell, L.M. Bartoshuk, R.I. Doty, & J.B. Snow (Eds.), Smell and taste in health and disease (pp. 97-108). New York: Raven Press. Silver, I.A., & Erecinska, M. (1998). Glucose-induced intracellular ion change in sugar-sensitive hypothalamic neurons. Journal of Neurophysiology, 79, 17331745.

363

Silver, R., LeSauter, J., Tresco, P.A., & Lehman, M.N. (1996). A diffusible coupling signal from the transplanted suprachiasmatic nucleus controlling circadian locomotor rhythms. Nature, 382, 810-813. Simansky, K.J., & Nicklous, D.M. (2002). Parabrachial infusion of D-fenfluramine reduces food intake blockade by the 5-HT1B antagonist SB-216641. Pharmacology, Biochemistry & Behavior, 71, 681-690. Simerly, R.B. (1995b). Anatomical substrates of hypothalamic integration. The rat nervous system. In: Paxinos, G. (Ed.). San Diego, Academic Press, p. 353-376. Simons, K.J., Chen, X., Fraser, T.G., & Simons, F.E.R. (1996). Effect of cimetidine on the pharmacokinetics and pharmacodynamics of chlorpheniramine and diphenhydramine in rabbits. Pharmaceutical Research, 13, 301-304. Simpson, S.J., & Raubenheimer, D. Geometric models of macronutrient selection. In: H.R. Berthoud, R.J. Seeley (Eds.). Neural and metabolic control of macronutrient intake. Boca Raton: CRC Press, 2000. p.29-42. Sindelar, D.K., Havel, P.J., Seeley, R.J., Wilkinson, C.W., Woods, S.C., & Schwartz, M.W. (1999). Low plasma leptin levels contribute to diabetic hyperphagia in rats. Diabetes, 48, 1275-1280. Sinha, M.K., Channesian, J.P., Heiman, M.L., Kriauciunas, A., Stephens, T.W., Magosin, S., et al. (1996). Nocturnal rise of leptin in lean, obese, and non-insulindependent diabetes mellitus subjects. Journal of Clinical Investigations, 97, 13441347. Sipols, A.J., Baskin, D.G., & Schwartz, M.W. (1995). Effect of intracerebroventricular insulin infusion on diabetic hyperphagia and hypothalamic neuropeptide gene expression. Diabetes, 44, 147-151. Skinner, L.G., England, P.C., Cottrell, K.M., & Selwood, R.A. (1974). Proceedings: serum oestradiol 17-beta in normal premenopausal women and in patients with benign and malignant breast disease. British Journal of Cancer, 30, 176-177. Slonaker, J. (1924). The effect of pubescence, oestruation and menopause on the voluntary activity n the albino rat. American Journal of Physiology, 68, 294-315. Smith, B.K., York, D.A., & Bray, G.A. (1999). Activation of hypothalamic serotonin receptors reduced intake of dietary fat and protein but not carbohydrate. American Journal of Physiology, 277, R802-811. 364

Smith, D.E. (1958). Influence of antihistamines on mast cell disruption following x-irradiation. Proceedings of the Society for Experimental Biology & Medicine, 97, 872-874. Smith, G.P. Satiation from the gut to the brain. New York: Oxford University Press, 1998. Smith, K.E., Walker, M.W., Artymyshyn, R., Bard, J., Borowsky, B., Tamm, J.A. et al. (1998). Cloned human and rat galanin GALR3 receptors. Journal of Biological Chemistry, 23, 321-323, 326. Smith, J.C., & Roll, D.L. (1967). Trace conditioning with X-rays as the aversive stimulus. Psychonomic Science, 9, 11-12. Smith, M.S. (1993). Lactation alters neuropeptide-Y and proopiomelancortin gene expression in the arcuate nucleus of the rat. Endocrinology, 133, 1258-1265. Snyder, J.J., & Levitt, R.A. (1975). Neural activity changes correlated with central anticholinergic blockade of cholinergically-induced drinking. Pharmacology, Biochemistry & Behavior, 3, 75-79. Socher, S.H., Friedman, A., & Martinez, D. (1988). Recombinant human tumor necrosis factor induces acute reactions in food intake and body weight in mice. Journal of Experimental Medicine, 167, 1957-1962. Södersten, P., DeJong, F.H., Vreeburg, J.T.M., & Baum, M. J. (1974). Lordosis behavior in intact male rats: absence of correlation with mounting behavior or testicular secretion of estradiol 17-beta and testosterone. Physiology & Behavior, 13, 803-808. Spanswick, D., Smith, M.A., Groppi, V.E., Gogan, S.D., & Ashford, M.L. (1997). Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature, 390, 521-525. Spector, A.C., Breslin, P., & Grill, H.J. (1988). Taste reactivity as a dependent measure of the rapid formation of conditioned taste aversion: A tool for the neural analysis of taste-visceral associations. Behavioral Neuroscience, 102, 942-952. Spector, A.C., & Grill, H.J. (1988). Differences in the taste quality of maltose and sucrose in rats: issues involving the generalization of conditioned taste aversions. Chemical Senses, 13, 95-113.

365

Spector, A.C., Norgren, R., & Grill, H.J. (1992). Parabrachial gustatory lesions impair taste aversion learning in rats. Behavioral Neuroscience, 106, 147-161. Spencer, C.M., & Houpt, T.A. (2001). Dynamics of c-fos and ICER mRNA expression in rat forebrain following lithium chloride injection. Molecular Brain Research, 93, 113-126. Spencer, D.G.J., Horvarth, E., & Traber, J. (1986). Direct autoradiographic determination of M1 and M2 muscarinic acetylcholine receptor distribution in the rat brain: relation to cholinergic nuclei and projections. Brain Research, 380, 5968. Spyraki, C., Fibiger, H.C., & Phillips, A.G. (1982). Dopaminergic substrates of amphetamine- induced place preference conditioning. Brain Research, 253, 185193. Stallone, D.D., & Stunkard, A.J. (1991). The regulation of body weight: evidence and clinical implications. Annals of Behavioral Medicine, 13, 220. Stein, L. (1963). Anti-cholinergic drugs and the control of thirst. Science, 139, 4648. Stanley, B.G., Chin, A.S., & Leibowitz, S.F. (1985). Feeding and drinking elicited by central injection of neuropeptide Y: evidence for hypothalamic site(s) of action. Brain Research Bulletin, 14, 521-524. Stanley, B.G., Daniel, D.R., Chin, A.S., & Lebowitz, S.F. (1985). Paraventricular nucleus injections of peptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion. Peptides, 6, 1205-1211. Stanley, B.G., & Leibowitz, S.F. (1985). Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sciences, 35, 26352642. Stanley, B.G., Kyrkouli, S.E., Lampert, S., & Leibowitz, S.F. (1986). Neuropeptide Y chronically injected into the hypothalamus: a powerful neurochemical inducer of hyperphagia and obesity. Peptides, 7, 1189-1192. Stanley, B.G., Willet, V.L. III., Donias, H.W., Ha, L.H., & Spears, L.C. (1993). The lateral hypothalamus: a primary site mediating excitatory amino acid-elicited eating. Brain Research, 630, 41-49.

366

Steinbusch, H.W.M., & Mulder, A.H. (1984). Immunohistochemical localization of histamine in neurons and mast cells in the rat brain. In: A. Bjorklund, T. Hokfelt, & M.J. Kuhar, (Eds.)., Handbook of chemical neuroanatomy. Classical transmitters and transmitter receptors in the CNS (pp. 126-140). Amsterdam: Elsevier. Stellar, E.M. (1954) The physiology of motivation. Psychological Review, 154, 522. Stephens, T.W., Basinski, M., Bristow, P.K., Bue-Valleskey, J.M., Burgett, S.G., Craft, L. et al. (1995). The role neuropeptide Y in the antiobesity action of the obese gene product. Nature, 377, 530-534. Sullivan, E., Daniels, A., & Cameron, J. (2003). Ovariectomy leads to rapid changes in food intake, body weight, and metabolic regulation in female rhesus monkeys. Society for Neuroscience Abstracts, 803.19. Swank, M.W., & Bernstein, I.L. (1994). C-Fos induction in response to a conditioned stimulus after single trial taste aversion learning. Brain Research, 636, 202-208. Swank, M.W., Schafe, G.E., & Bernstein, I.L. (1995). C-fos induction in response to taste stimuli previously paired with amphetamine or LiCl during taste aversion learning. Brain Research, 673, 251-261. Takaki, A., Nagai, K., Takaki, S., Yanaihara, N., & Nakagawa, H. (1990). Satiety function of neurons containing a CCK-like substance in the dorsal parabrachial nucleus. Physiology & Behavior, 48, 865-871. Takeda, N., Morita, M., Kubo, T., Yamatodani, A., Watanabe, T., Wada, H., & Matsunaga, T. (1986). Histaminergic mechanisms of motion sickness: neurochemical and neuropharmacological studies in rats. Acta Oto-laryngologica, 101, 416-421. Takeo, T., & Sakuma, Y. (1995). Diametrically opposite effects of estrogen on the excitability of female rat medial and lateral peroptic neurons with axons to the midbrain locomotor region. Neuroscience Research, 22, 73-80. Takeuchi, Yoshiki, McLean, J.H., & Hopkins, D.A. (1982). Reciprocal connections between the amygdala and parabrachial nuclei: ultrastructural demonstration by degeneration and axonal transport of horseradish peroxidase in the cat. Brain Research, 239, 583-588.

367

Tang-Christensen, M., Holst, J., Hartmann, B., & Vrang, N. (1999). The arcuate nucleus is pivotal in mediating the anorectic effects of centrally administered leptin. NeuroReport, 10, 1183-1187/ Tang-Christensen, M., Larsen, P.J., Goke, R., Fink-Jensen,A., Jessop, D.S., Moller, M. et al. (1996). Central administration of GLP-1(7-36) amide inhibits food and water intake in rats. American Journal of Physiology, 271, R848-R856. Tarttelin, M.F., & Gorski, R.A. (1973). The effects of ovarian steroids on food and water intake and body weight in the female rat. Acta Endocrinologica, 72, 551568. Taylor, J.E., & Richelson, E. (1980). High affinity binding of tricyclic antidepressants to histamine H1-receptors: fact and artifact. European Journal of Pharmacology. 67, 41-46. Ter Horst, G., de Boer, Pl, Luiten, P., & van Wilingen, J. (1989). Ascending projections from the solitary tract nucleus to the hypothalamus. A Phaseolus vulgaris lectin tracing study in the rat. Neuroscience, 31, 785-797. Thammacharoen, S., Lutz, T.A., Geary, N., & Asarian, L. (2008). Hindbrain administration of estradiol inhibits feeding and activates estrogen receptor-αexpressing cells in the nucleus tractus solitarius of ovariectomized rats. Endocrinology, 149, 1609-1616. Thiele, T.E., Roitman, M.F., & Bernstein, I.L. (1996). c-Fos induction in rat brainstem in response to ethanol- and lithium chloride-induced conditioned taste aversions. Alcoholism: Clinical & Experimental Research, 20, 1023-1028. Thompson, R.F., & Krupa, D.A. (1994). Organization of memory traces in the mammalian brain. Annual Review of Neuroscience, 17, 519-549. Thompson, R.F., & Swanson, L.W. (1998). Organization of inputs to the dorsomedial nucleus of the hypothalamus: reexamination with Fluorogold and PHAL in the rat. Brain Research Reviews, 27, 89-118. Tordoff, M.G., & Friedman, M.I. (1986). Hepatic portal glucose infusions decrease food intake and increase food preference. American Journal of Physiology, 251, R192-R196. Torvik, A. (1956). Afferent connections to the sensory trigeminal nuclei, the nucleus of the solitary tract and adjacent structures, Journal of Comparative Neurology, 106, 51-132. 368

Toshinai, K., Date, Y., Murakami, N., Shimada, M., Mondal, M.S., Shimbara, T. et al. (2003). Ghrelin-induced food intake is mediated via the orexin pathway. Endocrinology, 144, 1506-1512. Travers, J.B., Urbanek, K., & Grill, H.J. (1999). Fos-like immunoreactivity in the brain stem following oral quinine stimulation in decerebrate rats. American Journal of Physiology, 277, R384-R394. Trifunovic, R., & Reilly, S. (2001). Medial versus lateral parabrachial nucleus lesions in the rat: effects on cholecystokinin- and D-fenfluramine-induced anorexia. Brain Research, 894, 288-296. Trifunovic, R., & Reilly, S. (2002). Medial versus lateral parabrachial nucleus lesions in the rat: Effects on mercaptoacetate-induced feeding and conditioned taste aversion. Brain Research Bulletin, 58, 107-113. Tritos, N.A., Segal-Lieberman, G., Vezeridis, P.S., & Maratos-Flier, E. (2004). Estradiol-induced anorexia is independent of leptin and melanin-concentrating hormone. Obesity Research, 12, 716-724. Tritos, N.A., Vicent, D., Gillette, J., Ludwig, D.S., Flier, E.S., & Maratos-Flier, E. (1998). Functional interactions between melanin-concentrating hormone, neuropeptide Y, and anorectic neuropeptides in the rat hypothalamus. Diabetes, 47, 1687-1692. Trivedi, P., Yu, H., MacNeil, D.J., van der Ploeg, L.H., & Guan, X.M. (1998). Distribution of orexin receptor mRNA in the rat brain. FEBS Letters, 438, 72-75. Tschöpp, M., Smiley, D.L., & Heiman, M.L. (2000). Ghrelin induces adiposity in rodents. Nature, 407, 908-913. Tucci, S., Rada, P., & Hernandez, L. (1998). Role of glutamate in the amygdala and lateral hypothalamus in conditioned taste aversion. Brain Research, 813, 4449. Turner, B.H., & Herkenham, M. (1991). Thalamoamygdaloid projections in the rat: a test of amygdala’s role in sensory processing. Journal of Comparative Neurology, 313, 295-325. Turton, M.D., O’Shea, D., Gunn, I., Beak, S.A., Edwards, C.M., Meeran, K. et al. (1996). A role for glucagon-like peptide 1 in the central regulation of feeding. Nature, 379, 69-72.

369

Vaisse, C., Clement, K., Durand, E., Hercberg, S., Guy-Grand, B., & Froguel, P. (2000). Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity. Journal of Clinical Investigations, 106, 253-262. van der Kooy, D. & Koda, L.Y. (1983). Organization of the projections of a circumventricular organ: the area postrema in the rat. Journal of Comparative Neurology, 219, 328-338. van der Kooy, D., Swerdlow, N.R., & Koob, G.F. (1983). Paradoxical reinforcing properties of apomorphine: Effects of nucleus accumbens and area postrema lesions. Brain Research, 259, 111-118. van Dijk, G., de Groote, C., Chavez, M., van der Werf, Y., Steffens, A.B., & Strubbe, J.H. (1997). Insulin in the arcuate nucleus reduces fat consumption in rats. Brain Research, 777, 147. van Dijk, G., Thiele, T.E., Donahey, J.C., Campfield, L.A., Smith, F.J., Burn, P. Bernstein, I.L, Woods, S.C., & Seeley, R.J. Central infusions of leptin and GLP-1(7-36) amide differentially stimulate c-FLI in the rat brain. (1996). American Journal of Physiology, 271, R1096-R1100. Verbalis, J., Blackburn, R., Hoffman, G., & Stricker, E. (1995). Establishing behavioral and physiological functions of central oxytocin: insights from studies of oxytocin and ingestive behaviors. Advances in Experimental Medicine & Biology, 395, 209-225. Vliagoftis, H., Dimitriadou, V., Boucher, W., Rozniecki, J., Correia, I., Raam, S. et al. (1992). Estradiol augments while tamoxifen inhibits rat mast cell secretion. International Archives of Allergy & Immunology, 98, 398-409. Vrang, N., Larsen, P.J., Clausen, J.T., & Kristensen, P. (1999). Neurochemical characterization of hypothalamic concaine-amphetamine-regulated transcript neurons. Journal of Neuroscience, 19, RC5. Wade, G.N. (1972). Gonadal hormones and behavioral regulation of body weight. Physiology & Behavior, 8, 523-534. Wade, G.N., & Gray, J.M. (1979). Gonadal effects on food intake and adiposity: a metabolic hypothesis. Physiology & Behavior, 22, 583-593. Waldrop, M.M. (1982). Astronaut’s can’t stomach zero gravity. Science, 218, 1106.

370

Walford, R.L., Mock, D., MacCallum, T. , & Laseter, J.L. (1999). Physiologic changes in humans subjected to severe, selective calorie restriction for two years in Biosphere 2: health, aging, and toxicological perspectives. Toxicological Sciences, 52, 61-65. Wall, S.J., Yasuda, R.P., Hory, F., Flagg, S., Martin, B.M., Ginns, E.I., & Wolfe, B.B. (1991). Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain. Molecular Pharmacology, 39, 643-649. Wamsley, J.K., Lewis, M.S., Young, W.S., & Kuhar, M.J. (1981). Autoradiographic localization of muscarinic receptors in rat brainstem. The Journal of Neuroscience, 1, 176-191. Wang, L., Martinez, V., Barrachina, M.D., & Tache, Y. (1998). C-Fos expression in the bran induced by peripheral injection of CCK or leptin plus CCK in fasted lean mice. Brian Research, 791, 157-166. Wang, J., & Leibowitz, K.L. (1997). Central insulin inhibits hypothalamic galanin and neuropeptide Y gene expression and peptide release in intact rats. Brain Research, 777, 231-236. Wang, J., Osaka, T., & Inoue, S. (2001). Energy expenditure by intracerebroventricular administration of orexin to anesthetized rats. Neuroscience Letters, 315, 49-52. Wang, Q., Bing, C., Al-Barazanji, K., Mossakowaska, D.E., Wang, X.M., McBay, D.L. et al. (1997). Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat. Diabetes, 46, 335-341. Wang, Y., & Chambers, K.C. (2002). Cooling lesions of the lateral parabrachial nucleus during LiCl activation block acquisition of conditioned taste avoidance in male rats. Brain Research, 934, 7-22. Wang, Y., Lavond, D.G., & Chambers, K.C. (1997a). The effects of cooling the area postrema of male rats on conditioned taste aversions induced by LiCl and apomorphine. Behavioural Brain Research, 82, 149-158. Wang, Y., Lavond, D.G., & Chambers, K.C. (1997b). Cooling the area postrema induces conditioned taste aversions in male rats and blocks acquisition of LiClinduced aversions. Behavioral Neuroscience, 111, 768-776.

371

Wang, S.C., & Borison, H.L. (1951). Copper sulphate emesis: a study of afferent pathways from the gastrointestinal tract. American Journal of Physiology, 164, 520-526. Watts, A.G. (2000). Understanding the neural control of ingestive behaviors: helping to separate cause from effect with dehydration-associated anorexia. Hormones & Behavior, 37, 261-283. Webb, M., Bond, N., & Stevens, R. (1975). Drinking and eating in rats followed by chronic atropine administration. Physiology & Behavior, 14, 669-672. Weigle, D.S., Duell, P.B., Connor, W.E., Steiner, R.A., Soules, MR. , & Kuijper, J.L. (1997). Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels. Journal of Clinical Endocrinology & Metabolism, 82, 561-565. Weingarten, H.P., & Powley, T.L. (1980). Ventromedial hypothalamic lesions elevate basal and cephalic phase gastric acid output. American Journal of Physiology, 239, G221-228. Westermark, P., Wernstedt, C., Wilander, E., & Sletten, K. (1986). A novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. Biochemical & Biophysical Research Communication, 140, 827. Wettergren, A., Schjøldager, B., Mortensen, P.E., Myhre, J., Christiansen, J., & Holst, J.J. (1993). Truncated GLP-1 (proglucagon 87-107-amide) inhibits gastric and pancreatic functions in man. Digestive Diseases & Sciences, 38, 665-673. Whitehouse, J.M. (1967). Cholinergic mechanisms in discrimination learning as a function of stimuli. Journal of Comparative & Physiological Psychology, 63, 448451. Wilcox, R.R. Fundamentals of modern statistical methods: substantially improving power and accuracy. New York: Singer-Verlag; 2003a. Wilcox, R.R. Applying contemporary statistical techniques. San Diego: Academic Press; 2003b. Wilcox, R.R., & Keselman, H.J. (2003). Modern robust data analysis methods: measures of central tendency. Psychological Methods, 8, 254-274. Willensen, M.G., Kristensen P., & Romer, J. (1999). Co-localization of growth hormone secretagogue receptor and NPY mRNA in the arcuate nucleus of the rat. Neuroendocrinology, 70, 306-316. 372

Williams, G., Gill, J.S., Lee, Y.C., Cardoso, H.M., Okpere, B.E., & Bloom, S.R. (1989). Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats. Diabetes, 38, 321-327. Wilson, B.E., Meyer, G.E., Cleveland, J.C., & Weigle, D.S. (1990). Identification of candidate genes for a factor regulating body weight in primates. American Journal of Physiology, 259, R1148-R1155. Wilson, J.D., Nicklous, D.M., Aloyo, V.J., & Simansky, K.J. (2003). An orexigenic role for μ-opioid receptors in the lateral parabrachial nucleus. American Journal of Physiology, 285, R1055-R1065. Wise, R.A., Yokel, B., & DeWitt, H. (1976). Both positive reinforcement and conditioned aversion from amphetamine and apomorphine in rats. Science, 191, 1273-1275. Wollnik, F., & Turek, F.W. (1988). Estrous correlated modulations of circadian and ultradian wheel-running activity rhythms in LEW/Ztm rats. Physiology & Behavior, 43, 389-396. Wood, C.D. (1979). Antimotion sickness and antiemetic drugs. Drugs, 17, 471479. Wood, C.D., & Graybiel, A. (1970). A theory of motion sickness based on pharmacological reactions. Clinical Pharmacology & Therapeutics, 11, 621-629. Woods, S., Lotter, E., McKay, L., & Porte, D.J. (1979). Chronic intracerebroventricular infusion of insulin reduces food intake and body weight of baboons. Nature, 282, 503-505. Woods, S., Seeley, R.J., Porte, D.J., & Schwartz, M.W. (1998). Signals that regulate food intake and energy homeostasis. Science, 280, 1378. Woolley, C.S. & McEwen, B.S. (1993). Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. Journal of Comparative Neurology, 336: 293-306. Woolf, N.J., & Butcher, L.L. (1982). Cholinergic projections to the basolateral amygdala: a combined evans blue and acetylcholinesterase analysis. Brain Research Bulletin, 8, 751-763. Wouterlood, F.G., & Steinbusch, H.W.M. (1991). Afferent and efferent fiber connections of histaminergic neurons in the rat brain: comparison with dopaminergic, noradrenergic and serotonergic systems. In T. Watanabe , & H. 373

Wada, (Eds.)., Histaminergic neurons: morphology and function (145-162). Boca Raton: CRC Press. Wren, A.M., Seal, L.J., Cohen, M.A., Brynes, A.E., Frost, G.S., Murphy, K.G. et al. (2001). Ghrelin enhances appetite and increases food intake in humans. Journal of Clinical Endocrinology & Metabolism, 86, 5992-5995. Wren, A.M., Small, C.J., Abbott, C.R., Dhillo, W.S., Seal, L.J., Cohen, M.A. et al. (2001). Ghrelin causes hyperphagia and obesity in rats. Diabetes, 50, 2540-2547. Wu, P.C. (2002). Central limit theorem comparing means, trimmed means, onestep M-estimators and modified one-step M-estimators under non-normality. Unpublished Dissertation, University of Southern California, Los Angeles, Los Angeles. Wu, X., Gao, J., Yan, J., Owyang, C., & Li, Y. (2004). Hypothalamus-brain stem circuitry responsible for vagal efferent signaling to the pancreas evoked by hypoglycemia in rat. Journal of Neurophysiology, 91, 1734-1747. Wynick D., Small C.J., Bloom, S.R., & Pachnis, V. (1998). Targeted disruption of the murine galanin gene. Annals of the New York Academy of Sciences. 863, 22-47. Yamamoto, T., Azuma, S., & Kawamura, Y. (1984). Functional relations between the cortical gustatory area and the amygdala: electrophysiological and behavioral studies in rats. Experimental Brain Research, 56, 23-31. Yamamoto, T., & Fujimoto, Y. (1991). Brain mechanisms of taste aversion learning in the rat. Brain Research Bulletin, 27, 403-406. Yamamoto, T., Shimura, T., Sako, N., Azuma, S., & Bai, W.-Zh. (1992). C-fos expression in the rat brain after intraperitoneal injection of lithium chloride. NeuroReport, 3, 1049-1052. Yamamoto, T., Fujimoto, Y., Shimura, T., & Sakai, N. (1995). Conditioned taste aversion in rats with excitotoxic brain lesions. Neuroscience Research, 22, 31-49. Yasoshima, Y., Morimoto, T., & Yamamoto, T. (2000). Different disruptive effects on the acquisition and expression of conditioned taste aversion by blockades of amygdalar ionotropic and metabotropic glutamatergic receptor subtypes in rats. Brain Research, 869, 15-24.

374

Yamamoto, T., & Sawa, K. (2000). C-Fos-like immunoreactivity in the brainstem following gastric loads of various chemical solutions in rats. Brain Research. 866(1-2),135-413. Yamamoto, T., Shimura, T., Sako, N., Yasoshima, Y., & Sakai, N. (1994). Neural substrates for conditioned taste aversion in the rat. Behavioural Brain Research, 65, 123-137. Yamamoto, T., Shimura, T., Sako, N., Azuma, S., Bai, W.-Zh., & Wakisaka, S. (1992). C-fos expression in the rat brain after intraperitoneal injection of lithium chloride. NeuroReport, 3, 1049-1052. Yamamoto, T., Sako, N., Sakai, N., & Iwafune, A. (1997). Gustatory and visceral inputs to the amygdala of the rat: conditioned taste aversion and induction of c-foslike immunoreactivity. Neuroscience Letters, 226, 127-130. Yamamoto, T., Shimura, T., Sako, N., Azuma, S., Bai, W.Z., & Wakisaka, S. (1992). c-Fos expression in the rat brain after intraperitoneal injection of lithium chloride. NeuroReport, 3, 1049-1052. Yamanaka, A., Sakurai, T., Katsumoto, T., Yanagisawa, M., & Goto, K. (1999). Chronic intracerebroventricular administration of orexin-A to rats increases food intake in the daytime, but has no effect on body weight. Brain Research, 849, 248252. Yamanaka, A., Kunii, K., Nambu, T., Tsujino, N., Sakai, A., & Matsuzaki, I. (2000). Orexin-induced food intake involves neuropeptide Y pathway. Brain Research, 859, 404-409. Yang, B., & Ferguson, A.V. (2003). Orexin-A depolarizes nucleus tractus solitarius neurons trough effects on nonselective cationic and K+ conductances. Journal of Neurophysiology, 89, 2167-2175. Yaswen, L., Diehl, N., Brennan, M.B., & Hochgeschwender, U. (1999). Obesity in the mouse model of pro-opiomelanocortin deficiency responds to peripheral melanocortin. Nature Medicine, 5, 1066-1070. Yettefti, K., Orsini, J.C., & Perrin, J. (1997). Characteristics of glycemia-sensitive neurons in the nucleus tractus solitarius: Possible involvement in nutritional regulation. Physiology & Behavior, 61, 93-100. Young, J. (1991). Estrogen and the etiology of anorexia nervosa. Neuroscience and biobehavioral reviews, 15, 327-331. 375

Yox, D.P., Brenner, L., & Ritter, R.C. (1992). CCK-receptor antagonists attenuate suppression of sham feeding by intestinal nutrients. American Journal of Physiology, 262, R554-R561. Yuan, D.L., & Chambers, K.C. (1999). Estradiol accelerates extinction of a conditioned taste aversion in female and male rats. Hormones & Behavior, 36, 116. Zalaquett, C.P., & Parker, L.A. (1989). Further evidence that CTAs produced by lithium and amphetamine are qualitatively different. Learning & Motivation, 20, 413-427. Zarjevski, N., Cusin, I., Vettor, R., Rohner-Jeanrenaud, F., & Jeanrenaud, B. (1993). Chronic intracerebroventricular neuropeptide-Y administration to normal rats mimics hormonal and metabolic changes of obesity. Endocrinology, 133, 1753-1758. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., & Friedman, J.M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature, 372, 425-432. Zucker, I. (1969). Hormonal determinants of sex differences in saccharin preference, food intake and body weight. Physiology & Behavior, 4, 595-602.

376