Psychology & Neuroscience

Psychology & Neuroscience Muscimol Infusions to Infralimbic Cortex Impair Extinction but Not Acquisition of a Trace Eyeblink Conditioned Response in Rabbits Barbara B. Oswald, Stephanie A. Maddox, Matthew R. Herbst, and Donald A. Powell Online First Publication, January 26, 2015. http://dx.doi.org/10.1037/h0100355

CITATION Oswald, B. B., Maddox, S. A., Herbst, M. R., & Powell, D. A. (2015, January 26). Muscimol Infusions to Infralimbic Cortex Impair Extinction but Not Acquisition of a Trace Eyeblink Conditioned Response in Rabbits. Psychology & Neuroscience. Advance online publication. http://dx.doi.org/10.1037/h0100355

Psychology & Neuroscience 2015, Vol. 8, No. 1, 000

In the public domain http://dx.doi.org/10.1037/h0100355

Muscimol Infusions to Infralimbic Cortex Impair Extinction but Not Acquisition of a Trace Eyeblink Conditioned Response in Rabbits Barbara B. Oswald and Stephanie A. Maddox

Matthew R. Herbst Miami University

Dorn VA Medical Center, Columbia, South Carolina and University of South Carolina

Donald A. Powell Dorn VA Medical Center, Columbia, South Carolina and University of South Carolina School of Medicine This study assessed the effects of reversible lesions with microinfusions of the GABAA agonist muscimol (MUSC) to infralimbic cortex (IL; Brodmann’s area 25) of the medial prefrontal cortex (mPFC) on trace eyeblink conditioning and extinction in rabbits (Oryctolagus cuniculus). Four groups were tested: rabbits receiving MUSC infusions 5 min before acquisition and extinction sessions (MUSC/MUSC group), rabbits receiving vehicle (VEH) 5 min before acquisition and extinction sessions (VEH/VEH group), rabbits receiving MUSC before acquisition and VEH before extinction sessions (MUSC/VEH group), and rabbits receiving VEH before acquisition and MUSC before extinction sessions (VEH/MUSC). Results revealed that MUSC infusions to IL had no effects on acquisition but retarded extinction when injected before either acquisition or extinction sessions. These findings are among the first to demonstrate that MUSC infusions to the IL disrupt extinction of an EB CR in rabbits, and are in agreement with data from rodent studies noting the critical role of IL for the extinction of aversive fear-conditioned responses. Keywords: medial prefrontal cortex, Pavlovian conditioning, temporary lesion

There is great interest in understanding the brain and behavioral mechanisms underlying extinction, as they pertain to disorders of re-

sponse inhibition. A variety of psychological disturbances including attention deficit/hyperactivity disorder (ADHD), posttraumatic stress

Barbara B. Oswald, Shirley L. Buchanan Neuroscience Laboratory, Dorn VA Medical Center, Columbia, South Carolina, and Department of Continuing Education Credit Programs, University of South Carolina; Stephanie A. Maddox, Shirley L. Buchanan Neuroscience Laboratory, Dorn VA Medical Center, and Department of Psychology, University of South Carolina; Matthew R. Herbst, Department of Psychology, Miami University; Donald A. Powell, Shirley L. Buchanan Neuroscience Laboratory, Dorn VA Medical Center, and Department of Neuropsychiatry and Behavioral Science, University of South Carolina School of Medicine. Barbara B. Oswald is now at the Department of Psychology, Miami University. Stephanie A. Maddox is now at the Department of Behavioral Neuroscience & Psychiatric Disorders, Emory University. This paper is dedicated to the fond memory of Donnie A. Powell, who devoted more than 40 years to careful

research that has greatly enhanced our knowledge of brain areas regulating autonomic and somatomotor conditioning. This work was supported by Department of Veterans’ Affairs Medical Research Funds to Barbara B. Oswald and Donald A. Powell. We thank Samuel Durrett, Cynthia Krafft, and Nikeya Tisdale for assistance with behavioral training and tissue histology, and Andrew Pringle for assistance with graphics preparation, Dr. Richard A. Edelmann and Matthew L. Duley in the Center for Advanced Microscopy & Imaging at Miami University for assistance with photomicrographs, and Dr. Stephen D. Berry for helpful comments on a draft. Correspondence concerning this article should be addressed to Barbara B. Oswald, Department of Psychology, Miami University, 90 North Patterson Avenue, Oxford, OH 45056. E-mail: oswaldbb@miamiOH .edu 1

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OSWALD, MADDOX, HERBST, AND POWELL

disorder (PTSD), schizophrenia, and drug addiction are characterized by the inability to inhibit inappropriate behaviors or thoughts. Elucidating the neural mechanisms underlying response inhibition will aid in the treatment of these and other disorders of inhibition. Although competing theories exist (e.g., see Larrauri & Schmajuk, 2008 for review), strong evidence suggests that extinction of previously conditioned responses is learned inhibition, and not “forgetting,” or the loss of a memory trace (Konorski, 1967; Pavlov, 1927). Evidence that extinction is learned inhibition is demonstrated by several phenomena including spontaneous recovery, renewal, and reinstatement, each of which leads to the demonstration of a previously extinguished conditioned response (e.g., see Bouton, 2004 and Larrauri & Schmajuk, 2008). For example, a smoker who was once conditioned to have a cigarette at the end of every meal may quit smoking and experience many years of meals without cigarettes (extinction). Yet even after years of abstinence, the end of a meal may suddenly induce craving in the former smoker. This recovery of the desire to smoke may be attributable to either the passage of time since extinction (spontaneous recovery), exposure to a different context for the meal (renewal), or subtle exposure to nicotine itself perhaps via second-hand smoke or other source (reinstatement). The return of extinguished responses demonstrates clearly that the connection between a learned stimulus and response remains intact. Extinction, then, rather than “erasing” a previously learned neural connection, instead creates a new neural connection in inhibitory brain structures that is generally stronger than the previously learned excitatory response. A rich literature exists regarding the brain structures involved in the acquisition of conditioned responses, yet much less is known regarding the structures mediating the extinction of conditioned responses. Research from the fear conditioning literature in rodents demonstrates that one area of the mPFC, the infralimbic cortex (IL) mediates the memory of extinguished responses, likely via connections to the amygdala (Burgos-Robles, Vidal-Gonzalez, & Quirk, 2009; Maren & Quirk, 2004; Milad & Quirk, 2002; Quirk, Russo, Barron, & Lebron, 2000; Quirk, Garcia, & González-Lima, 2006; Santini, Quirk, & Porter, 2008). However, fear

conditioning studies typically involve highly aversive or exceptionally salient stimuli (painful footshock), and the initial memory is acquired in just one or perhaps a few conditioned stimulus (CS)– unconditioned stimulus (US) pairings. This type of aversive, rapid learning may not be akin to many types of human learning, such as anxiety-, or addiction-related behaviors that are often learned following multiple presentations of far less salient stimuli. Furthermore, diverse human behaviors including fear, anxiety, and addiction are often learned following the association of cues that are temporally distinct (Gallistel & Balsam, 2014). One model of learning commonly used to investigate the neural circuitry involved in the association of temporally distinct stimuli is trace classical conditioning. In trace conditioning, a neutral CS, for example a tone, is presented some time before the onset of a biologically meaningful US, for example, a puff or air or mild shock near the eye (see Figure 1, bottom panel). The target response is an eyeblink (EB), and in the rabbit, nictitating membrane extension. After learning, a conditioned response (CR) similar in form to the unconditioned EB reflex (UR) but with a decreased amplitude is emitted in response to the CS alone (see Figure 2). Separating the CS and US in time, even briefly, requires short-term or working memory to hold the memory for the CS through the trace period, and recruits a distributed network of brain areas, in particular, structures along the midline of the prefrontal cortex (mPFC). For example, in rabbits, permanent lesions to mPFC retard acquisition of a trace EB CR without affecting simple-delay conditioning, conditioning in which the CS and US are presented together in time (Figure 1, top panel); Kronforst-Collins & Disterhoft, 1998; McLaughlin, Powell, & White, 2002; Oswald, Knuckley, Mahan, Sanders, & Powell, 2009; Weible, McEchron, & Disterhoft, 2000). Although a number of studies demonstrate the engagement of fronto-thalamic-cerebellar circuitry to promote associative learning under noncontiguous trace conditioning parameters, that is, when stimuli are separated in time (Kalmbach, Ohyama, Kreider, Riusech, & Mauk, 2009; Powell & Churchwell, 2002; Weiss, Bouwmeester, Power, & Disterhoft, 1999), the neural correlates mediating the ex-

IL AND EXTINCTION OF TRACE EB CRS

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Figure 1. Diagram of simple-delay versus trace conditioning. Top lines show simple delay conditioning, in which a 500-ms tone CS is presented and coterminates with a 50-ms 2.5-mAmp mild periorbital shock CS. The bottom lines show a typical trace conditioning paradigm, in which the 500-ms CS is presented, followed by a 500-ms stimulus-free “trace” period, before the onset of the US. Lesions to mPFC typically disrupt trace, but not simple delay conditioning.

tinction of trace conditioned responses are not well-understood, and no studies have assessed the role of the IL in extinction of the conditioned EB CR in rabbits. In addition, virtually all of the aforementioned studies have assessed the effects of permanent lesions. Permanent lesion models have a distinct experimental disadvantage as they prevent the within-subject assessment of learning ability. Indeed, there is great variability in the rate at which healthy rabbits acquire trace EB CRs. To increase experimental rigor, it is useful to determine whether individual animals can acquire and extinguish a conditioned response independent of brain lesion effects, which is obviously impossible following permanent brain damage. Instead, temporarily deactivating brain structures via discrete microinfusion of drugs that block neural firing permits the assessment of learning both with the brain structure “offline” and following reactivation, that is, after the drug is cleared from the brain. Muscimol (MUSC) is a GABAA agonist that binds with high affinity to receptors, to hyperpolarize cells for 2 to 12 hours following intracranial injections (Edeline, Hars, Hennevin, & Cotillon, 2002; Martin &

Ghez, 1999; Martin, 1991; Wheal & Kerkut, 1976). Direct intracranial microinfusions of MUSC diffuse maximally within 1 to 2 hours within a radius of 1.5 to 2.5 mm from the injection site. MUSC is more selective than other methods of neuronal deactivation such as tetrodotoxin or local anesthetics because MUSC affects only postsynaptic cell membranes, and not axonal fibers of passage (Edeline et al., 2002; Majchrzak & Di Scala, 2000; Wheal & Kerkut, 1976). A number of studies have assessed the effects of temporary deactivation on delay EB conditioning in subcortical structures including the interpositus nucleus of the cerebellum (Bracha, Webster, Winters, Irwin, & Bloedel, 1994; Bracha et al., 1998; Christian & Thompson, 2005; Garcia & Mauk, 1998; Hardiman, Ramnani, & Yeo, 1996; Krupa & Thompson, 1997; Mojtahedian, Kogan, Kanzawa, Thompson, & Lavond, 2007), the inferior olive (Zbarska, Holland, Bloedel, & Bracha, 2007), brainstem motor nuclei (Krupa & Thompson, 2003; Krupa, Weng, & Thompson, 1996), and cerebellar cortex (Steinmetz & Freeman, 2014; see also Lavond, 2002 for review). Yet only a few studies have assessed effects on trace con-

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Figure 2. Diagram of typical acquisition of an eyeblink conditioned response. In the model of trace classical conditioning employed in the present experiments, a 1200-Hz, 75-dB tone served as a conditioned stimulus (CS) that signaled the onset of a 50-ms 2.5-mAmp periorbital eye shock unconditioned stimulus (US). The CS and US were separated in time by a stimulus-free 500-ms trace period. The US naturally elicits an unconditioned response (UR) of reflexive eyeblink (EB) and nictitating membrane extension. The line tracings illustrate the amplitude and latency of onset of the EB response. On Day 1, before conditioning, the CS does not elicit a response, but the US does. Acquisition usually requires several hundred CS-US pairings, and on Day 3, a conditioned response (CR) similar in form but with decreased amplitude compared to the UR begins to emerge in response to the CS alone. During trace conditioning, animals learn to time the onset of the CR to coincide with the end of the trace period and close to the onset of the US, rather than during the presentation of the CS.

ditioning. For example, Pakaprot, Kim, and Thompson (2009) assessed the effects of muscimol infusions to the interpositus nucleus during trace EB conditioning in rabbits, and found that pretraining infusions prevented acquisition, and posttraining infusions prevented expression of the trace EB CR. Few studies have assessed the effects of temporary deactivation in mPFC on trace conditioning. Mauk and colleagues found that MUSC infusions to caudal mPFC that included the prelimbic (PL) and anterior cingulate (AC) cortices disrupted trace but not delay conditioning, but they did not assess effects in IL (Kalmbach et al., 2009). Therefore, in an effort to further research on the involvement of the IL on trace EB conditioning and extinction, we assessed the effects of microinfusions of MUSC into the IL of rabbits during acquisition and extinction of a trace EB CR. It is

our hope that elucidating the neural circuitry underlying the extinction of temporally distinct, moderately salient associations will facilitate the development of treatments for neuropsychological disorders of inhibition including PTSD, ADHD, schizophrenia, and drug addiction. Method Subjects and Groups Subjects were 32 adult male and female New Zealand rabbits (aged 2–3 months at the beginning of testing), obtained from a local USDA-licensed supplier (Robinson Services Inc., Monksville, NC). After a 1-week acclimation period to our colony, animals were housed singly and maintained in a facility accredited by the Association for the Accred-


itation and Assessment of Laboratory Animal Care International, and managed by the University of South Carolina Animal Resources Department. Rooms were environmentally controlled with a 12-h/12-h light– dark cycle (lights on 8am). Behavioral tests were performed in the morning portion of the cycle. All U.S. Department of Agriculture and Department of Veterans’ Affairs guidelines regarding the ethical treatment of animals were followed. Rabbits were randomly assigned to one of four drug treatment groups (n ⫽ 8 per group): (a) Rabbits in the vehicle/vehicle (VEH/VEH) group received bilateral microinfusions of vehicle (7.4 pH sterile phosphate buffered saline) to IL five minutes before all conditioning and extinction sessions; (b) Rabbits in the MUSC/ MUSC group received bilateral MUSC infusions (3.5 ␮mol dissolved in VEH) five minutes before all conditioning and extinction sessions; (c) Rabbits in the VEH/MUSC group received VEH infusions before conditioning sessions and MUSC infusions before extinction sessions; and (d) Rabbits in the MUSC/VEH group received MUSC before conditioning sessions and VEH before the extinction sessions. Cannulas and Accessories Stainless steel double-barreled guide cannula (26 g, 11 mm length, with a 2-mm center-tocenter distance, and held together in a 4-mm pedestal) were used (Plastics One Inc., Roanoke, VA). Double-barreled obturators (33 g, 11 mm length) were fitted flush with the tip of the guide cannulas, and remained in place chronically, except during infusions, to prevent occlusion. Double-barreled injector cannulas (33 g, 12 mm length, so that a 1-mm projection extended beyond the tip of the guide cannulas), were inserted into the flat face of the connector mold of a polyethylene double-cannulas connector tube (40 cm length). Injector cannulas were placed acutely into the guide cannulas for infusions, and fixed in place with a screw captive collar fitted around the flat face of the tubing. The other ends of the double tubing were attached to two 10-␮l Hamilton syringes, filled with either MUSC or VEH. Syringes were placed into two automatic syringe pumps (Harvard Apparatus, No. 22), fitted with multiunit

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devices so four animals could be infused simultaneously. Surgery A Kopf stereotaxic instrument equipped with a rabbit head holder (Kopf Instruments, Tujunga, CA) was used to surgically implant guide cannulas. Surgery was performed under aseptic conditions, under general anesthesia, using a combination of ketamine hydrochloride (55 mg/ kg, i.m.), acepromazine maleate (2.2 mg/kg, i.m.) and xylazine (4.4 mg/kg, i.m.). The head was leveled with bregma 1.5 mm higher than lambda and a small hole (3 ⫻ 2 mm) was made in the skull bilaterally above mPFC with a dentist drill and dura was removed from the area with a sterile 20 g needle. Small holes (4 – 6) were drilled in the surface of the skull around the insertion hole, and small stainless steel screws (3/16“, Small Parts, Inc., Miami Lakes, FL) were inserted to anchor the acrylic to the skull. For implantation, two double-barreled guide cannulas were positioned on either side of the midline sinus, directed at coordinates AP ⫹ 4.0 and ⫹ 6.0, L ⫾ 1.0, and V ⫺6 mm relative to begma, midline sinus, and the surface of the brain according to the atlas of Shek (1986). Dental acrylic secured the cannulas to the anchor screws and skull. Exposed scalp was closed with 1–2 sterile wound clips, obturators were inserted and covered with dust caps, and rabbits were injected subcutaneously with 0.03 mg/kg buprenorphine to prevent pain and 40cc’s lactated Ringers solution to prevent dehydration. Animals were observed until awake and returned to their homecage, where they were monitored daily for signs of pain or anorexia. Behavioral testing began two weeks after surgery. Drugs and Infusion Procedures Muscimol hydrate (Acros Organis N. V., Fair Lawn, NJ) was dissolved in 7.4pH sterile phosphate buffered saline at a concentration of 3.5 ␮mol/0.5 ␮L. Rabbits were placed in Plexiglas restrainers (Gormezano, 1966) near the infusion pump. A total volume of 1.0 ␮L per side (0.5 ␮L per cannula) was injected at a rate of 0.25 ␮L/min and allowed to diffuse for 2 min. Animals were carried in the restrainers a short distance across the room to the soundproof testing

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chambers. Daily conditioning sessions began 5 min after infusions. Animals received two 1-hr adaptation sessions to the infusion and behavioral testing procedures. On day two of adaptation, all animals received 1 ␮L per side of VEH, at a rate of 0.25 ␮L per min. Conditioning Procedures As noted, animals received two 1-hr adaptation sessions to the infusion and behavioral testing procedures. During the first day of adaptation, animals were placed loosely in the restrainer inside the experimental chambers for 60 minutes with no stimuli delivered. On day two of adaptation, all animals received 0.5 ␮L of VEH delivered through each cannulas as described. Obturators and dust caps were replaced, and animals were placed in the experimental chambers loosely restrained with the eyeblink recording wires and shockleads placed around the right orbit, but with no stimuli delivered. Following adaptation, 6 consecutive daily trace conditioning sessions began in which animals were exposed to 100 trials of CS–US presentations, in which a 1216-Hz, 75-dB tone conditioned stimulus (CS) was presented for 500 msec, followed by a 500-msec stimulus-free period, that coterminated with a 50-msec periorbital shock (2.5 mAmp) US. After Day 6 of acquisition, animals received 5 consecutive days of extinction training, in which 100 presentations of a 500-ms tone CS, separated by 22 ⫾ 5 sec, were delivered alone, namely, not followed by the periorbital shock US. Acquisition and extinction sessions lasted 45 to 60 min. Experimental contingencies were controlled by a PC-based data acquisition system (MACRO, Inc., Columbia, SC), supplemented by solid state transistor-transistor logic (TTL) programming devices. The EB responses were recorded on a Grass Model 7 polygraph (AstroMed, Inc., West Warwick, RI) equipped with EMG preamplifiers. During conditioning, the output of the polygraph was connected to the computer where A-D conversion was performed in real time. The shock US was delivered by a Grass Model S88 stimulator (AstroMed, Inc., West Warwick, RI) equipped with constant current and stimulus isolation units. This US was delivered to the animals around the right orbit through previously implanted stain-

less steel wound clips. For recording the EB response, electrodes constructed of orthodontic wire were acutely inserted beneath the upper and lower right eyelids. These electrodes allowed for simultaneous recording of eyelid closure and nictitating membrane extension (VanDercar, Swadlow, Elster, & Schneiderman, 1969). The eyeleads were connected to a Grass preamplifier and integrator. The amplitude of the signal (integrated over the CS interval) served as the EB measure. An EB CR was defined as a potential change of 100 ␮v or greater, corresponding to approximately 0.5 mm of eyelid movement (Powell & LevineBryce, 1988). EB latency was defined as the time interval from CS onset until the CR exceeded 100 uv. The EB leads were connected to Grass EMG preamplifiers with integrators. Output of the drive amplifiers were input to the A-D card of the computer, which sampled at 1000 Hz, beginning 50msec before tone onset and continuing until after US offset. A-D recording was performed in real time. Control Procedures As in previous studies, control experiments were conducted to assess the effects of MUSC and VEH infusions on nonassociative processes that may impact learning. Pseudoconditioning tests consisted of 6 daily sessions of 100 explicitly unpaired CS–US presentations (separated by 11 ⫾ 5 sec) delivered over 45 to 60 minutes, followed by 5 daily sessions of 100 CS-alone presentations (22 ⫾ 5 sec ITI). Presentation of stimuli during the six explicitly unpaired sessions were pseudorandomly programmed so that a single stimulus occurred on no more than three consecutive trials, and the total number of CS and US presentations per session did not differ by more than two. Eight animals were used, four infused with MUSC and four infused with VEH 5 min before all 11 sessions. Histology Upon completion of behavioral testing, subjects were euthanized with intravenous pentobarbital and perfused intracardially with physiological saline and a fixation solution of 10% formalin in normal saline. In most animals, position of the cannulas tips were marked by inserting 33-g, 12-mm lesion electrodes (No.


UE(BO1), Frederick Haer & Co) through the guide cannulas and stimulating at 1.5 mAmp for 20 sec. This placed the electrode tip 1 mm below the guide cannulas, and at the same position as the injector cannulas. In several animals (n ⫽ 2 in each of the conditioned and pseudoconditioned groups) electrodes were not inserted, but animals were perfused as described, and histology performed, to assess the extent of tissue damage caused by infusions. After perfusion, brains were removed and frozen so that 40-␮m sections could be taken on a cryostat. Sections were stained with thionin to visualize cannula tracts and area of damage produced by the electrode. All histological examination and reconstruction was performed using a Leitz drawing tube by reference to a millimeter grid structure superimposed on plates from the atlas of Urban and Richard (1972). Photomicrographs of representative sections were captured using an Olympus AX70 light microscope fitted with Nikon D300 digital SLR camera with Camera Control Pro 2 software (Center for Advanced Microscopy & Imaging, Miami University). Data Analysis All data were analyzed by mixed repeated measures analysis of variance (ANOVA), using group as a nonrepeated dimension, and session and trials as repeated dimensions. Significant effects were posttested using Duncan’s Multiple Range Test (Edwards, 1964). Sphericity was controlled by the Greenhouse-Geisser correction (Greenhouse & Geisser, 1959). Results Histology Results from histological verification of cannulas placement revealed misplaced cannulas in five animals: three in the VEH/VEH group and two in the VEH/MUSC group; their data were not included in the analyses. In the remaining 27 animals (n ⫽ 5 VEH/VEH, 8 MUSC/MUSC, 6 MUSC/VEH, 8 VEH/MUSC) both barrels of each bilateral cannulas were directed at IL (Brodmann’s area 25, as described by Vogt, Sikes, Swadlow, & Weyand, 1986; see Figure 3). Histological analyses revealed virtually no cell loss due to tissue displacement caused by

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repeated infusions of MUSC or VEH; see Figure 4. Pseudoconditioning Histology One animal in the VEH group died after surgery, leaving a total of four animals in the MUSC group and three in the VEH group. Posttraining histology revealed that all seven animals had cannulas directed at IL. Acquisition Data A 4 (drug group) ⫻ 6 (trace conditioning days) mixed ANOVA with group as a nonrepeated measure and conditioning day as a repeated measure compared the effects of MUSC and VEH infusions on acquisition of the trace conditioned EB CR. Results revealed no significant difference in acquisition rate among the four drug groups, F(3,23) ⫽ 1.91, p ⬎ .05. There was a significant day effect [, F(5,115) ⫽ 14.83, p ⬍ .0001, but no Day ⫻ Group interaction, suggesting that animals in the four groups acquired the EB CR to the same extent over conditioning days (see Figure 5, left panel). To evaluate the effects of MUSC infusions on general motor function, analyses compared EB amplitude and latency for URs and CRs across sessions. A 4 (group) ⫻ 6 (session) mixed ANOVA comparing UR amplitude across conditioning sessions indicated no significant effect of group on UR amplitude, F(3,13) ⫽ 0.47, p ⬎ .70, but a significant effect of session, F(5,13) ⫽ 3.27, p ⬍ .014], and a Group ⫻ Session interaction, F(3,13) ⫽ 2.14, p ⬍ .03; Figure 6A). Post hoc analyses indicated only CD 6 resulted in UR amplitude that was significantly lower than the other conditioning days. There were no effects on UR latency (data not shown) between groups, F(3,13) ⫽ 0.47, p ⬎ .70, but there was a significant session effect on UR latency, F(5,13) ⫽ 2.16, p ⬍ .07, and a Group ⫻ Session interaction, F(5,13) ⫽ 2.68, p ⬍ .006. Latency to respond to the UR decreased across sessions in all groups, although the MUSC/MUSC group latency did not decrease as much as the other groups. However, as noted via the nonsignificant effects revealed by the overall ANOVA, comparison of latency means between MUSC/ MUSC, VEH/MUSC, and MUSC/VEH found no significant differences in latency as a result of MUSC treatment. Therefore, any latency effects that may have occurred in our MUSC

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Figure 3. Histology line drawings of coronal sections of rabbit brain indicating cannulas placement in each of the 4 groups. Double-barreled infusion cannulas were directed at infralimbic cortex (IL; Brodmann’s area 25). Approximate stereotaxic coordinates are given anterior (A) to bregma ⫹ 0 to ⫹ 10. Brodmann numbers are based on anatomical analysis of rabbit prefrontal cortex by Vogt et al. (1986). Lower limits for the Brodmann areas are as follows: 8 ⫽ Brodmann Area 8, prefrontal eyefields; 24 ⫽ Brodmann Area 24, anterior cingulate cortex; 32 ⫽ Brodmann Area 32, prelimbic area; 25 ⫽ Brodmann Area 25, infralimbic area.

treated animals are likely the result of random error and not likely due to the effects of MUSC treatment. Indeed, as noted in our analyses of pseudoconditioned animals (described below), there were not effects of MUSC treatment on UR latency in animals receiving MUSC treatment for all 11 days, F(1,10) ⫽ 1.28, p ⬍ .28, further suggesting that repeated dosing with MUSC had no effects on latency to respond to the UR. A 4 (group) ⫻ 6 (session) mixed ANOVA comparing CR amplitude across conditioning sessions (Figure 6, Panel B, left side) revealed no significant differences between infusion groups, F(3,13) ⫽ 0.12, p ⬎ .94, but an expected significant effect of session, F(5,13) ⫽ 8.5, p ⬍ .0001, with no Group ⫻ Session interaction, F(3,5) ⫽ 0.77, p ⬎ .69l. These findings suggest that CR amplitude increased across training sessions, and that this increase was not impacted by MUSC infusions. Analyses of response la-

tency (data not shown) also revealed similar expected significant increases in CR latency across training sessions, F(5,13) ⫽ 5.81, p ⬍ .0002, that were not different across groups, F(3,13) ⫽ 0.99, p ⬎ .43, with no Group ⫻ Session interaction, F(3,5) ⫽ 1.57, p ⬎ .11. Extinction Data A separate 4 (group) ⫻ 5 (day) mixed ANOVA compared the effects of MUSC and VEH infusions on extinction of the trace conditioned EB CR, that is, the percentage of CRs emitted across extinction sessions. There was a significant effect of group, F(3,25) ⫽ 6.82, p ⬍ .002, but not day, F(4,96) ⫽ 1.61, p ⬎ .18, and no Day ⫻ Group interaction, F(12,96) ⫽ 0.77, p ⬎ .67. On the main effect of group, post hoc tests revealed a significantly greater percentage of EB CRs in MUSC-treated animals (see Figure


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between groups across extinction sessions. Analyses revealed no significant changes in CR amplitude between groups, F(3,12) ⫽ 0.52, p ⬎ .67, or across sessions during extinction, F(4,12) ⫽ 2.06, p ⬎ .10 (see Figure 6, Panel B, right side). These findings are somewhat surprising based on the significant decrease in the percentage of EB CRs observed in the VEH/VEH group, but could be due to the high variability in performance of EB CRs observed across extinction. Pseudoconditioned Control Animals

Figure 4. Histology: Sample photomicrograph of a coronal section of rabbit brain with double-barreled bilateral infusion cannulas implanted, directed at infralimbic cortex (IL). Arrows indicate terminal ends of cannulas, and this representative slice indicates the most dorsal infusion points (i.e., those closest to PL). Muscimol or vehicle infusions would be expected to diffuse approximately 1 to 2 mm from these points. See the online article for the color version of this figure.

5, right panel). Of interest, animals that received MUSC during acquisition and vehicle during extinction (the MUSC/VEH group) did not extinguish faster than animals treated with MUSC during extinction; in fact, performance between these groups was essentially equal until the final day of extinction, when animals treated with VEH during extinction (but MUSC during acquisition) finally stopped expressing CRs, unlike animals receiving MUSC during extinction. These findings suggest that treatment with MUSC during acquisition may have had some carry over effect through extinction. Alternatively, it is possible that differential neural activity during acquisition and extinction created a different internal context, which could have disrupted learning in a state-dependent fashion (Taylor & Ivry, 2013). These hypotheses are considered more fully under Discussion. Additional separate two-way mixed ANOVAs compared the amplitude and latency of CRs

Separate mixed ANOVAs compared the percentage of EB responses in pseudoconditioned animals across Days 1– 6 (to correspond with conditioning Days 1– 6 in trained animals) and 7–11 (to correspond with extinction Days 1–5 in trained animals; data shown in Figure 5). Analyses revealed no significant difference between the groups over Days 1– 6, F(1,5) ⫽ 1.28, p ⬎ .30, or Days 7–11, F(1,4) ⫽ 1.12, p ⬎ .33. In addition, separate 2-way mixed ANOVAs compared performance between conditioned and pseudoconditioned animals (data collapsed across groups) over Sessions 1– 6 and 7–11. The ANOVA comparing percentage of EB CRs over Sessions 1– 6 revealed a significantly greater number of EB CRs emitted by conditioned animals, F(1,5) ⫽ 7.68, p ⬍ .04. The difference in performance across extinction days only approached significance, F(1,4) ⫽ 5.55, p ⬎ .06) likely attributable to the high degree of variability in performance. Separate mixed ANOVAs also compared the amplitude and latency of responses emitted to tones and eyeshocks in pseudoconditioned animals. Results revealed no significant differences between MUSC and VEH-treated pseudoconditioned animals in EB amplitude, F(1,5) ⫽ 0.38, p ⬎ .56 (Figure 6, Panel A), or latency, F(1,5) ⫽ 0.08, p ⬎ .79 (data not shown), in response to shocks or tones across Sessions 1– 6, F(1,5) ⫽ 0.19, p ⬎ .69 (Figure 6, Panel B, left side). There were also no significant effects on amplitude to tones, F(1,4) ⫽ 2.48, p ⬎ .19, latency to tones, F(1,4) ⫽ 0.14, p ⬎ .72, or amplitude to shocks, F(1,4) ⫽ 2.27, p ⬎ .20, or latency to shocks, F(1,4) ⫽ 0.42, p ⬎ 0.55, across Days 7–11 (amplitude data shown in Figure 6, Panel B, right side). Thus behavioral deficits in extinction rate observed in our exper-

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Figure 5. Results from behavioral tests and pseudoconditioned controls. Four groups were tested: MUSC/VEH received muscimol (MUSC; 1.0 ␮l per side of 3.5 ␮l/ml solution) infusions 5 min before daily acquisition sessions, and received vehicle (VEH; 7.4pH sterile phosphate buffered saline) before daily extinction sessions. VEH/MUSC received VEH before acquisition and MUSC before extinction sessions. VEH/VEH received vehicle infusions before all acquisition and extinction sessions, and MUSC/MUSC received MUSC infusions before acquisition and extinction sessions. Pseudoconditioned controls received either MUSC (PSEUDOMUSC) or VEH (PSEUDOVEH) before all sessions. The left panel shows the percentage of eyeblink (EB) conditioned responses (CRs) across 6 days of acquisition training. Each acquisition session consisted of trace conditioning with 100 pairings of a 500-ms, 75-dB tone CS with a 50-ms, 2.5-mAmp periorbital eye shock US to the right orbit, separated by a 500ms stimulus-free trace period. CS-US presentations were separated by a 22 ⫾ 5 sec ITI. Pseudoconditioned control animals received 100 explicitly unpaired CS–US presentations separated by 11 ⫾ 5 seconds. On training Day 7, all animals (including pseudoconditioned controls) began extinction training (right panel) for 5 consecutive days, in which they received 100 CS-alone presentations, separated by a 22 ⫾ 5 sec ITI. There were no significant differences in acquisition rates among the 4 training groups, indicating that inhibiting activity of the IL had no effect on acquisition of a trace EB CR. MUSC infusions before acquisition or extinction sessions significantly delayed extinction across the 5 sessions compared with the VEH/VEH group. ⴱ p ⬍ .05.

imental groups were not due to alterations in responsiveness to repeated presentations of tones following MUSC or VEH infusions to the IL. Discussion Results of the present experiment are among the first to demonstrate a role for the IL in extinction, but not acquisition, of the trace conditioned EB CR. Animals receiving infusions of the GABAA agonist MUSC to IL 5 min before one hour acquisition or extinction sessions demonstrated normal acquisition, but poor extinction, as indicated by a significantly greater number of EB CRs during CS-alone extinction trials. Indeed, rabbits that received MUSC in-

fusions to IL before extinction training sessions never extinguished EB CRs, even after 5 days of extinction training (500 CS-alone presentations). Further, animals exposed to MUSC during acquisition but VEH during extinction exhibited significantly delayed extinction compared to animals trained or extinguished under VEH, suggesting either that MUSC has a significant carryover effect, or that input from IL during acquisition may be necessary to learn to inhibit a previously learned response. This second explanation is more likely, based on findings from the fear conditioning literature noting the importance of IL for the retrieval of an extinguished response (discussed more fully below). Results from pseudoconditioning control tests following MUSC or VEH infusions to


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Figure 6. Amplitude of EB URs (A) and CRs (B) in conditioned and pseudoconditioned controls. Effects of muscimol on EB amplitude (in microvolts, uV) following 2.5-mAmp periorbital eye shock presented for 50 ms (A) and 500 ms, 75-dB tones paired with eye shock (B) in conditioned and pseudoconditioned control animals across conditioning (URs and CRs) and extinction sessions (CRs only). Dotted lines indicate the amplitude of EB URs and CRs in pseudoconditioned control animals, that is, animals who received an equal number of tone and shock presentations but in an explicitly unpaired fashion. Following acquisition across conditioning days, conditioned animals exhibited URs that significantly decreased in amplitude (A) and CRs that significantly increased in amplitude (B) across all groups (ⴱ ps ⬍ 0.05). Pseudoconditioned controls did not display many CRs, and when they did, their amplitude was low to suggest little more than reflexive eye movement. Amplitude of EB CRs was significantly higher for conditioned animals than pseudoconditioned controls across all conditioning but not extinction sessions (Panel B; ⴙ ps ⬍ 0.05). Muscimol treatment did not affect EB CR amplitude in any group (group comparison of means all p ⬎ .05).

IL revealed no changes in EB responsivity following repeated presentations of tones, eyeshocks, or MUSC or VEH infusions to IL. Together these findings suggest that the IL is necessary for extinction, but not acquisition, of a trace conditioned EB CR, independent of any motor processes necessary to perform the EB reflex in the rabbit.

Burgeoning evidence from the rodent fearconditioning literature suggests that IL plasticity is essential for extinction consolidation of a fearconditioned response (Burgos-Robles, VidalGonzalez, Santini, & Quirk, 2007; Fanselow & LeDoux, 1999; Maren & Quirk, 2004; Milad & Quirk, 2002; Peters, Kalivas, & Quirk, 2009; Quirk, Armony, & LeDoux, 1997; Quirk, Garcia,

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& Gonzalez-Lima, 2006; Santini et al., 2008; Senn et al., 2014; Sotres-Bayon & Quirk, 2010; Vidal-Gonzalez, Vidal-Gonzalez, Rauch, & Quirk, 2006). Blocking IL activity during extinction of a fear-conditioned freezing response generally has no effects on extinction learning, but impairs extinction recall 24 hours later (Laurent & Westbrook, 2009; Milad & Quirk, 2002; Quirk, Garcia, & Gonzalez-Lima, 2006). For example, Laurent and Westbrook (2009) found that MUSC infusions to IL during extinction sessions did not inhibit extinction learning, but did impair relearning and consolidation of the extinguished CR. In this study, rats were conditioned to fear a CS context (the fear conditioning chamber) by placing rats in the chamber and presenting a 0.8mAmp footshock for 1 sec. The next day, one half of the rats were returned to the chamber (“reextinction” groups) for 20 minutes with no footshock presentations (extinction training), and freezing behavior was measured over time. Within 6 minutes, these rats had extinguished, that is, none were exhibiting freezing CRs. On Day 3, all rats received a second fear conditioning session identical to Day 1. On Day 4, all rats received microinfusions of either VEH or MUSC to IL, and returned to the chamber for extinction training. This produced four groups of rats: rats that had been extinguished previously (a reextinction group) who received VEH before the second extinction session, rats that had been extinguished previously who had MUSC before the second extinction session (a second reextinction group), rats who had not been extinguished previously and received VEH before their first extinction session (a novel extinction group), and rats who had not been extinguished previously and received MUSC before their first extinction session (a second novel extinction group). Results revealed that animals receiving VEH or MUSC before their first extinction session extinguished normally. However, rats receiving MUSC before their second extinction session exhibited impaired extinction. Further, all animals who received MUSC infusions on Day 4 exhibited impaired memory for the extinguished response when tested on Day 5 (that is, freezing behavior returned). Other rats that received MUSC infusions to IL immediately after extinction training also exhibited impaired extinction. The authors concluded these results suggested that IL input is necessary for consolidation but not initial acquisition of an extinguished response.

Indeed, studies reveal that IL plasticity activates GABAergic intercalated (ITC) cell masses that lie between the lateral nucleus of the amygdala (LA) and the central nucleus of the amygdala (CE) (Amano, Unal, & Paré, 2010; Likhtik, Popa, Apergis-Schoute, Fidacaro, & Paré, 2008; Sotres-Bayon & Quirk, 2010). A number of elegant studies demonstrate the important connections between IL and ITC for mediating the extinction of conditioned fear (e.g., Cho, Deisseroth, & Bolshakov, 2013; Knapska & Maren, 2009; Senn et al., 2014). Activating inhibitory ITC cells blunts communication between the LA and the CE, which leads to diminished fear responding. During acquisition of a conditioned fear response, LA, CE, and basal amygdala (BA) are activated to promote rapid learning and expression of the CR, likely via reciprocal connections with prelimbic cortex (PL; e.g., see Sotres-Bayon & Quirk, 2010 for review). During extinction, ITC cells become activated (via connections with IL), which inhibits amygdala output neurons of the CE to BA and PL, to hinder the expression of a previously conditioned fear response. Research has verified that inhibiting ITC cells causes deficits in extinction consolidation in a matter similar to lesions to IL (Likhtik et al., 2008), suggesting that it is indeed the integration of signals from IL to ITC that mediates fear extinction. These findings are congruent with Pavlov’s early idea that extinction involves the activation of inhibitory brain pathways to suppress the expression of a previously learned excitatory response. Nonetheless, the fear conditioning literature generally does not report deficits in initial extinction learning following lesions to IL, and yet we found that MUSC infusions to IL significantly impaired extinction learning. Further, previous studies in our lab have not found deficits in extinction following permanent lesions to prefrontal cortex that included prelimbic cortex (Oswald, Knuckley, Mahan, Sanders, & Powell, 2006), an area implicated in the expression of the fear response in the rodent literature (Cho et al., 2013; Knapska & Maren, 2009; Sotres-Bayon & Quirk, 2010). However, important differences in training paradigms exist that can account for these differences. Our eyeblink conditioning paradigm employed multiple-trial acquisition sessions that included 100 CS–US pairings separated by a 22 ⫾ 5 sec intertrial


interval, followed by multiple-trial extinction sessions that included 100 CS-alone presentations; all sessions lasted roughly 60 minutes. In fear conditioning, animals experience far fewer trials (1–10) and much shorter sessions (e.g., 20 min; Amano et al., 2010). In the long multitrial sessions employed in the present experiments, it is likely that memory consolidation is occurring within a single session. It makes sense, then, that MUSC infusions to IL could inhibit the overall expression of extinction across a long, 100-trial session. This is because MUSC to IL in our experiment likely interfered with the within-session consolidation expressed by control animals. Future work should ascertain this hypothesis, by assessing extinction of an EB CR when sessions contain only 1 to 10 CS-alone presentations. Also of interest in the present research is the finding that animals receiving MUSC infusions to IL during acquisition, followed by VEH during extinction, acquired the trace EB CR normally, but exhibited delayed extinction. These findings suggest several interpretations. One explanation is that MUSC has a significant carryover effect. This explanation is unlikely as extinction training did not begin until at least 24 hours after the final MUSC infusion, and MUSC is known to be biologically active in brain tissue for less than 12 hours (Edeline et al., 2002; Wheal & Kerkut, 1976). A second explanation for the delayed extinction observed in the MUSC/VEH group is that increased activity of neurons from the adjacent PL during extinction relative to acquisition could have inhibited extinction of the CR. Studies demonstrate that PL activity is necessary for fear expression during extinction (BurgosRobles et al., 2009; Senn et al., 2014; SotresBayon & Quirk, 2010). We do not expect that MUSC infusions in our experiment spread more than 2 mm from the infusion sites and thus we do not expect that PL neurons were inhibited during acquisition. Nonetheless, because of reciprocal connections between PL and BA noted above, future recording experiments should verify the extent of IL and PL activity following MUSC infusions to the IL in rabbits using the present cannula coordinates. Alternatively, the importance of context on learning is well-established, and recent data support the notion that neural activity could create an internal state that when altered, can

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disrupt learning (Taylor & Ivry, 2013). In the present experiment, MUSC infusions during acquisition create a different neural state (e.g., inhibition) that is distinct from VEH during extinction. This change in internal context could explain disruptions in extinction learning, and appears to be a viable explanation. Future studies must determine that neural activity and inhibition during acquisition and extinction create state-dependent effects on learning and extinction. In summary, the present research provides evidence that not only can suppression of neural activity in IL disrupt recall of an extinguished fear response (e.g., Quirk et al., 2006; Santini et al., 2008; Senn et al., 2014), temporarily inhibiting cell firing in IL with the GABAA agonist MUSC can disrupt extinction learning, at least for a moderately salient EB conditioning task that required several hundred CS-US pairings to acquire. We conclude that IL may serve as one locus of inhibitory learning. Future studies will continue to examine the effects of IL blockade on inhibitory learning during both aversive and appetitive tasks, as learning to inhibit previously learned responses offers great hope for the treatment of addictive and stress-related disorders. References Amano, T., Unal, C. T., & Paré, D. (2010). Synaptic correlates of fear extinction in the amygdala. Nature Neuroscience, 13, 489 – 494. http://dx.doi.org/ 10.1038/nn.2499 Bouton, M. E. (2004). Context and behavioral processes in extinction. Learning & Memory, 11, 485– 494. http://dx.doi.org/10.1101/lm.78804 Bracha, V., Irwin, K. B., Webster, M. L., Wunderlich, D. A., Stachowiak, M. K., & Bloedel, J. R. (1998). Microinjections of anisomycin into the intermediate cerebellum during learning affect the acquisition of classically conditioned responses in the rabbit. Brain Research, 788(1–2), 169 –178. http://www.sciencedirect.com/science/article/ B6SYR-3SJVG9V-S/2/9ea3273fababcba9c1ff da839bf76728. http://dx.doi.org/10.1016/S00068993(97)01535-7 Bracha, V., Webster, M. L., Winters, N. K., Irwin, K. B., & Bloedel, J. R. (1994). Effects of muscimol inactivation of the cerebellar interposed-dentate nuclear complex on the performance of the nictitating membrane response in the rabbit. Experimental Brain Research, 100, 453– 468. http://dx .doi.org/10.1007/BF02738405

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