Articles in PresS. J Neurophysiol (February 18, 2004). 10.1152/jn.01090.2003 1
Centrifugal inputs modulate taste aversion learning associated parabrachial neuronal activities
Ken’ichi Tokita1, Zoltán Karádi2, Tsuyoshi Shimura1 and Takashi Yamamoto1 1
Department of Behavioral Physiology, Graduate School of Human Sciences, Osaka University, 1-2 Yamadaoka, Suita, Osaka 565-0871, Japan 2
Institute of Physiology, Neurophysiology Research Group of the Hungarian Academy of Sciences, Pécs University, Medical School, Szigeti út 12, H-7643 Pécs, Hungary
Running Head: Conditioned taste aversion and parabrachial gustatory activity Corresponding author: Takashi Yamamoto Department of Behavioral Physiology Graduate School of Human Sciences Osaka University 1-2 Yamadaoka, Suita, Osaka 565-0871, Japan E-mail:
[email protected] Phone: +81-6-6879-8047 Fax: +81-6-6879-8050
Copyright (c) 2004 by the American Physiological Society.
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ABSTRACT Our previous studies have demonstrated that gustatory neurons in the parabrachial nucleus (PBN) show altered responses after the acquisition of conditioned taste aversion (CTA) to NaCl (Shimura et al. 1997c).
The present study was conducted 1) to
examine centrifugal influences on the altered gustatory activity of CTA-trained rats, and 2) to evaluate the role of amiloride-sensitive (ASN) and -insensitive NaCl (AIN) best units in coding the taste of NaCl. Animals were separated into two groups: a CTA group that had acquired taste aversion to 0.1 M NaCl and a control group that underwent pseudo-conditioning before the recording experiment. Single neuron activity, in two separate series of experiments, was extracellularly recorded in anesthetized rats.
In the
stimulation studies, the effects of electrical stimulation of the gustatory cortex (GC) or the central nucleus of amygdala (CeA) were examined on firing of PBN taste units. CeA stimulation produced excitatory effect in significantly more neurons in the CTA group (n = 8) than in the control group (n = 1).
Furthermore, ASN-best units in the
CTA group showed larger responses to NaCl than similar units in the control group.
In
the decerebration experiment, there was no statistical difference among the taste responses between the two groups in any best-stimulus category.
These results suggest
that CTA conditioning utilizes an effective central amygdaloid input to modulate
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activity of gustatory neurons in the PBN. Data also substantiate that amiloride-sensitive components of NaCl-best neurons play a critical role in the recognition of distinctive taste of NaCl.
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INTRODUCTION Conditioned taste aversion (CTA) occurs when animals experience a novel taste (conditioned stimulus, CS) with a gastrointestinal illness-inducing toxin (unconditioned stimulus, US), and then they show a robust rejection to that taste.
CTA helps animals
to survive by preventing the repeated ingestion of food or fluid that contains toxic substances (Bures et al. 1998). It has been well established that the pontine parabrachial nucleus (PBN), the second central relay for taste in rodents, is critically involved in the formation of CTA. Lesion-behavior studies have repeatedly shown that bilateral electrolytic (Di Lorenzo 1988a; Reilly et al. 1993; Sakai and Yamamoto 1998; Spector et al. 1992) or ibotenic acid (Grigson et al. 1998; Scalera et al. 1995; Yamamoto et al. 1995) lesions of the PBN severely disturb the acquisition of CTA. Because bilateral lesions of the nucleus of the solitary tract (NTS), the first central gustatory relay, fail to disturb the acquisition of CTA (Grigson et al. 1997; Shimura et al. 1997b), the critical region for CTA appears to be located rostral to the NTS. Several factors including learning have been reported to alter the responsiveness of gustatory neurons in the PBN (Baird et al. 2001; Hajnal et al. 1999; Shimura et al. 1997a; Shimura et al. 1997c; Shimura et al. 2002).
In the case of CTA, aversive
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conditioning resulted in an increase in the magnitude of response to the conditioned taste when NaCl served as the CS (Shimura et al. 1997c), suggesting that neural coding of the CS taste changes by learning.
However, it is not clear whether the alteration of
taste responses in the PBN occurs at the brainstem level, or reflects the altered responsiveness of the forebrain areas that send descending fibers to the PBN. Anatomical studies show that the cells in the PBN project to the gustatory cortex (GC) via the ventroposteromedial nucleus of the thalamus (Norgren and Leonard 1971, 1973), and to the limbic structures including the central nucleus of the amygdala (CeA), the bed nucleus of the stria terminalis, the substantia innominata, and the lateral hypothalamic area /LHA/ (Fulwiler and Saper 1984; Norgren 1974, 1976; Saper and Loewy 1980).
The PBN, in turn, receives descending fibers from several higher
gustatory relays such as the GC, CeA and LHA (Halsell 1992; Krettek and Price 1978; Lasiter et al. 1982; Moga et al. 1990; Saper 1982; van der Kooy et al. 1984; Veening et al. 1984; Whitehead et al. 2000; Zahm et al. 1999). Recent electrophysiological studies revealed that some of these forebrain areas are functionally as well as anatomically connected with the PBN.
Electrical stimulation or
reversible chemical lesions of the GC (Di Lorenzo 1990; Di Lorenzo and Monroe 1992) and electrical stimulation of the CeA (Lundy and Norgren 2001) altered gustatory
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activities of parabrachial neurons. Apart from the PBN, the NTS also receives descending inputs from the GC, CeA, and LHA (Cho et al. 2002, 2003; Di Lorenzo and Monroe 1995; Li et al. 2002; Matsuo and Kusano 1984a; Matsuo et al. 1984b; Smith and Li, 2000).
These studies suggest that in these two brainstem taste relays gustatory
information is processed via close interactions with various forebrain structures. Among the forebrain gustatory structures mentioned above, both the GC and CeA are known to be involved in CTA (Berman et al. 1998; Lamprecht and Dudai 1996; Navarro et al. 2000; Ramirez-Lugo et al. 2003; Yamamoto et al. 1995, 1997; Yasoshima et al. 1995, 1997, 2000; Yasoshima and Yamamoto 1998) and thus can be considered as being good candidates for the brain sites that modulate the parabrachial gustatory activities during or after the acquisition of CTA.
In fact, several electrophysiological studies
report that neurons in both areas showed altered responses to the CS after the acquisition of CTA (Yasoshima et al. 1995; Yasoshima and Yamamoto, 1998).
On the
basis of these anatomical and physiological studies, it is plausible to suppose that altered neuronal activity in the GC and/or CeA has some effect on neuronal activity in the PBN. It has been known that NaCl-best neurons (or NaCl-best fibers in the peripheral taste nerves) can be classified into amiloride-sensitive and -insensitive NaCl-best (ASN- and
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AIN-best) categories by application of an epithelial sodium transport blocker amiloride (Hettinger and Frank 1990; Ninomiya and Funakoshi 1988; Scott and Giza 1990; Yasumatsu et al. 2003).
Responses of ASN-best units to NaCl are substantially
reduced by amiloride, whereas AIN-best units remain unaffected.
These differences
are attributable to separate transduction mechanisms (DeSimone et al. 1981). Amiloride mixed with NaCl interferes with the ability of rats to discriminate sodium from nonsodium salts both behaviorally and neurally (Bernstein and Hennessy 1987; Hill et al. 1990; McCutcheon 1991; Ninomiya and Funakoshi 1988; Scott and Giza 1990; Spector et al. 1996; Yasumatsu et al. 2003). Taken these findings together, there is a possibility that the forebrain modulates gustatory activities in the PBN during and/or after the acquisition of CTA, and that taste responses of ASN-best units change when NaCl serves as the CS.
Nevertheless, to
date, there has been no such electrophysiological approach that tested this hypothesis in a single study.
The present experiments were conducted to elucidate descending
influence from forebrain structures on NaCl CTA associated activity of PBN gustatory neurons, as well as to unravel the role of ASN-best units in recognizing distinctive taste of NaCl.
In a series of stimulation experiments, we recorded PBN taste responses of
anesthetized rats in the presence of electrical stimulation of either the GC or CeA to
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mimic the effects of descending inputs from these forebrain areas.
In a decerebration
experiment, when the possibility of centrifugal influences was ruled out, PBN taste responses of decerebrate rats were also recorded among similar circumstances.
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METHODS Subjects Fifty-five male Wistar rats (Nihon Dobutsu, Osaka, Japan) for the stimulation experiment and 34 for the decerebration experiment were used. between 240 and 340 g at the start of the experiments.
They weighed
The animals were individually
caged with 12 hr light/dark cycle (lights on at 7:00 A.M., off at 7:00 P.M.) and constant temperature (23 ± 2°C) and humidity (60%).
They were arbitrarily divided into
control (Control; n = 27 for the stimulation, and 18 for the decerebration experiment) and conditioned (CTA; n = 28 for the stimulation, and 16 for the decerebration experiment) groups. All animals were cared for in accordance with institutional, national and international regulations.
Conditioning procedure Before conditioning procedure, the animals were acclimated to an 18-h water deprivation schedule, and trained to lick distilled water from a spout for 20 min in the morning (10:00) and 60 min in the afternoon (15:00).
The 20 min morning period
served as the training and testing sessions during which all behavioral manipulations were performed.
The afternoon session was always a distilled water presentation to
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provide for adequate hydration of the animals.
They were allowed free access to
normal rat chow (dry pellets, MF, Oriental Yeast, Osaka) except from 10:00 (morning session starts) to 16:00 (afternoon session ends).
Once baseline water intake was
established in the morning session, CTA animals were presented with 0.1 M NaCl, which served as a CS.
Immediately after the drinking period animals were injected
intraperitoneally with 0.15 M LiCl (2% of body weight) as a US. This procedure was repeated three times on consecutive days.
The control group was composed of the two
subgroups; the first control group in which animals were similarly treated as CTA animals except that they were given physiological saline instead of LiCl after the CS, and the second control group in which animals were injected LiCl 3h prior to the CS presentation.
In the control groups, as in case of the conditioned animals, the CS-US
pairing was also repeated three times on consecutive days.
Because the preliminary
analyses revealed that there was no fundamental difference in behavioral and neural data, i.e., the mean intake of the CS, proportion of each best-stimulus unit, spontaneous firing rates, magnitude of responsiveness, breadth of tuning were similar between the first and second control groups, the data were pooled together in the subsequent analyses.
Overnight food and water deprivation was employed after the final
afternoon session to ensure the stable induction of anesthesia.
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Surgery At least 24 h after the last injection of LiCl or saline, each rat was anesthetized with urethan (1.3 g/kg body weight, i.p.). Supplemental anesthetic (0.3 g/kg) was administered as necessary throughout the experiment to maintain the animals under deep anesthesia. Bilateral hypoglossal nerves were cut to prevent undesirable movement of the tongue during the recording session. After tracheotomy was performed to permit respiration, each rat was fixed in a stereotaxic instrument equipped with nontraumatic ear bars. The scalp was opened with a midline incision, and the skull was leveled between bregma and lambda by adjusting the bite bar. The body temperature was monitored and maintained at 36.8°C using an infrared lamp.
A hole
(3.0–4.0 mm in diameter, centered approximately 12.0 mm posterior to the bregma), serving access to the PBN, was unilaterally drilled through the skull. In the stimulation experiments, bipolar stimulating stainless steel electrodes (80 µm in diameter, insulated except at the edge of the tip) were placed in the ipsilateral (to recording) GC (anteroposterior = +1.5 mm, mediolateral = 5.4 mm, dorsoventral = –6.3 mm from bregma) and CeA (anteroposterior = –2.5 mm, mediolateral = 4.4 mm, dorsoventral = –7.8 mm from bregma).
Coordinates for both structures were chosen
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according to the brain atlas of the rat (Paxinos and Watson, 1998).
Each electrode was
fixed firmly to the skull with dental acrylic. In decerebration experiment, a 1.0 mm thick groove was made across the skull. After removing the dura, decerebration at the supra- or intercollicular level was made using an L-shaped spatula. The skull and brain surface were kept moist throughout the experiment with physiological saline.
Test solutions In both experiments, the taste stimuli used were 0.1 M NaCl, 0.5 M sucrose, 0.01 M HCl, 0.02 M quinine-HCl, 0.1 M KCl and 0.1M NaCl mixed with 10-4 M amiloride. All stimuli were prepared from reagent-grade chemicals and dissolved in distilled water. Taste solutions were maintained at room temperature during testing.
Electrophysiological recording Extracellular single neuron recordings from gustatory neurons in the PBN were made using dura-piercing, glass-insulated tungsten microelectrodes (Z = 1.5–3.0 M at 1 kHz).
With the skull flat, the PBN was ventral to the transverse sinuses and therefore
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the electrode was inserted in the parasagittal plane in a rostrocaudal direction at an angle of 20° off vertical (tip pointing rostrally).
The electrode was advanced through the
cerebellum into the PBN with a micromanipulator (MO-81, Narishige, Japan). Neuronal activity was amplified with a conventional method, monitored with a computer aided data acquisition and analysis system (CED 1401, Spike2; Cambridge Electronic Design, Cambridge, UK), and stored on a DAT recorder for offline analysis. A mixture of the four basic tastants (0.5 M sucrose, 0.3 M NaCl, 0.02 M HCl and 0.02 M quinine-HCl) was used as a search stimulus.
After isolating a single unit in the
PBN, taste stimuli were presented at room temperature (23–24°C).
To ensure
stimulating the entire oral cavity, we used a similar method to that described by Chang and Scott (1984).
Briefly, fluid stimuli were delivered through a length of intraorally
inserted slender tubing, closed at the end and extensively perforated along its final 2 cm. The perforated end was slipped into the mouth, held just slightly agape, so that tastants, delivered under mild pressure from a 5 ml syringe, could irrigate the entire oral cavity. Each stimulus trial consisted of a 10 s rinse of distilled water, 10 s stimulus, and 10 s rinse of distilled water.
The flow rate was 0.5 ml/s for all stimuli, including rinse.
When taste responses persisted after the 10 s post-stimulus rinse of distilled water, we continued water rinse until the neural activity returned to the prestimulus level. The
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gustatory stimuli and the distilled water were cleared from the tubing by air pressure. At least ninety seconds were allowed to elapse between stimuli to avoid the effects of adaptation. In the stimulation experiment, recording sessions composed of taste only, taste + GC stimulation, and taste + CeA stimulation trials. After taste stimulation trials using the 6 taste stimuli were over, electrical stimulations of the GC or CeA (200 µs duration, rectangular pulses, 10Hz, 0.3 mA, generated from an electronic stimulator, SEN-3201, Nihon Kohden, Tokyo, Japan) were applied during the period of 10 s taste stimulation and spontaneous activity.
Effects of electrical stimulation of either GC or CeA on taste
responses were examined using four basic taste stimuli (0.5 M sucrose, 0.1 M NaCl, 0.01 M HCl and 0.02 M quinine-HCl).
The parameters of effective electrical
stimulation were determined in a preliminary experiment.
The intensity of electrical
stimulation of the present experiment is comparable with that (0.4 mA) of Lundy and Norgren (2001).
Data analysis Neuronal activity was counted using a computer aided data acquisition and analysis system (CED 1401, Spike2; Cambridge Electronic Design, Cambridge, UK).
All data
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analyses were based on neural activity in 10 s samples.
Spontaneous activity and
responses to pre-stimulus water were calculated from multiple samples.
The
spontaneous rate was determined during the periods just before the pre-stimulus water rinse.
Water and taste responses were calculated during the first 10 s period after the
onset of stimulation with pre-stimulus water or a taste solution.
An adjusted score (a
net response rate), obtained by subtracting the immediately preceding raw water responses from the raw taste responses, was employed for data analyses.
A response
to taste stimulus was considered to be significant if the neuronal activity increased or decreased at least 2 SD from the mean of the spontaneous activity of the neuron. With the use of adjusted response data, a breadth-of-response measure was derived for each neuron from the formula for entropy (Smith and Travers, 1979) H= K
4 i =1
pi log pi ,
where pi represents the response to each of the four basic taste stimuli. = 1.661 for four stimuli.
The constant K
Values of entropy (H) close to zero indicate sensitivity to a
single stimulus (narrow tuning); values close to 1.0 indicate sensitivity to all four stimuli (broad tuning). The similarity among taste stimuli, based on responses of each neuron to the 6 stimuli, was further analyzed by the use of hierarchical cluster analysis.
The primary cluster
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analysis utilized the Pearson product-moment correlation coefficients and the Ward method (Statistica version 5.5, StatSoft, OK).
Units were classified as excited or
inhibited if their neuronal activities (taste responses to the four basic stimuli and spontaneous activity) in electrical stimulation trials were different by ± 25% from those in taste stimulation trials.
Histology At the end of the last experimental session, a small electrolytic lesion (20 µA for 20 s, electrode positive) was made at the final recording site in the PBN.
Marking lesions in
the GC and CeA were also made at the tip of one electrode by passing anodal current (20 µA for 20 s).
Then, rats were given a lethal dose of urethan and perfused
transcardially with phosphate-buffered saline and 10% formalin. The brains were removed and placed in 10% formalin and then transferred to a 30% buffered sucrose solution and stored at 4°C at least for a week.
Coronal sections of 60 µm thickness
were serially cut using a freezing microtome, then stained with cresyl violet. The location of each recording and stimulation site was histologically verified and reconstructed.
In the present study, electrophysiological data of the stimulation
experiment were exclusively processed from those animals in which the tips of the
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stimulating electrodes were confirmed to be located in the targeted stimulating sites (GC or CeA).
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RESULTS Stimulation experiment
Behavioral results Figure 1 shows mean fluid intake of animals used in the stimulation experiment during the last water training and subsequent three conditioning sessions.
The mean
intake of the CS (0.1M NaCl) in the CTA group progressively decreased by repeated pairings of the CS and US.
A two-way (Group × Session) analysis of variance
(ANOVA) with repeated measures revealed a significant main effect of group (F[1,53] = 87.161, p < 0.01) and session (F[3,159] = 89.613, p < 0.01), and a significant group × session interaction (F[3,159] = 127.532, p < 0.01).
Post hoc analyses using Tukey’s
test showed that there were significant differences between intake of the CTA and control groups on the 2nd and 3rd acquisition day (p < 0.01).
Histology The location of stimulating and recording electrodes in animals of both groups (n = 55) was reconstructed in Figure 2.
Filled and open symbols represent stimulating and
recording sites for the CTA and control groups, respectively.
Distribution of
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stimulating sites and effects of electrical stimulation of the GC were similar in both groups (A).
On the other hand, CeA stimulation produced excitatory responses in only
1 neuron in the control group while it produced excitatory responses in 5 neurons in the CTA group (B).
In the present study, a given site in the GC or CeA was classified as
effective (satisfying the ± 25% criteria described in the Method) if stimulating that site facilitated or suppressed at least 1 neuron in the PBN. Therefore, stimulating a site could be effective for a neuron but not for another.
However, there was no site that
produced opposite effects on different neurons, i.e., the effects of electrical stimulation of a given site were uniform across neurons.
In the PBN (C), recording sites of 5
neurons (3 from CTA and 2 from control group) were not reconstructed because they could not be histologically identified.
However, the data of these neurons were
included in the subsequent analyses.
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test revealed that there was no difference in
the proportion of recording sites anterior (a+b) and posterior (c+d) level between the groups (p > 0.1). There was also no difference in the dorsoventral and mediolateral distribution of recording sites between the groups.
There was also no difference in the
dorsoventral and mediolateral distribution of recording sites between the CTA and control groups (p > 0.4 and p > 0.9, respectively).
However, significantly more
HCl-best neurons in both groups were located in the dorsal region (n = 11) than in the
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ventral region (n = 2) of the brachium conjunctivum (p < 0.05).
Basic characteristics A total of 84 taste-responsive neurons were recorded from the PBN: 41 from the CTA and 43 from the control group. All the neurons showed excitatory responses to at least one of the four basic taste stimuli (sucrose, NaCl, HCl or QHCl).
The mean
spontaneous firing rates (spikes/s) were 2.75 ± 0.44 for the CTA group and 1.96 ± 0.38 for the control group, respectively.
There was no significant difference between the
spontaneous firing rates of the two groups (t-test, p > 0.1).
Response profiles and classification Based on their largest response to the four standard taste stimuli, we classified PBN neurons as follows: 7 S-best, 24 N-best and 10 H-best in the CTA group; 8 S-best, 29 N-best and 6 H-best in the control group. in the stimulation experiment.
In both groups, no Q-best unit was recorded
N-best units were further classified by their sensitivity
to amiloride. Figure 3A indicates examples of taste responses of ASN- and AIN-best units to NaCl with and without amiloride.
Amiloride substantially suppressed the
NaCl elicited taste responses of the ASN-best unit while the responses of the AIN-best
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unit remained unaffected.
Figure 3B shows distribution of all ASN- and AIN-best
units from both groups in terms of their amiloride-induced response magnitude inhibition ratio.
We classified a neuron as amiloride-sensitive if its responses to NaCl
were inhibited by amiloride by more than 40% compared to the response magnitude without amiloride.
Forty units (18 from the CTA and 22 from the control group) were
classified as ASN-best and 13 (6 from the CTA and 7 from the control group) as AIN-best.
The bimodal distribution of salt sensitive neurons without any intermediate
type is apparent.
The total gustatory response profiles of PBN neurons of both groups
are shown in Figure 4.
Taste neurons are ordered by best-stimulus category (S,
sucrose; N, salt; H, acid; Q, quinine) and, within categories, by response magnitude. S-best neurons are on the left, followed by N-best units, then H-best units. N-best category, ASN- and AIN-best units are separately shown.
In the
Fisher’s exact
probability test revealed that the proportion of units responding to any of the standard taste stimuli was not significantly different between the CTA and control groups (p > 0.6).
Breadth of responsiveness Because there were some inhibitory responses in our data, the entropy measure was
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calculated from the absolute value of the responses to the four standard taste stimuli. There was no significant difference between the mean entropy of the two groups: 0.68 ± 0.03 for the CTA, 0.70 ± 0.03 for the control groups (t-test, p > 0.5).
Comparison of taste responses Figure 5 presents a comparison of mean response profiles of best-stimulus categories of units in both groups.
Taste responses to NaCl were significantly higher in the CTA
group than in the control group in the ASN-best category while taste responses in the AIN- and other best-stimulus categories were almost identical between the groups.
A
two-way ANOVA with repeated measures (Group × Stimulus) indicated a significant main effect of stimulus in the ASN-best category (F[6,228] = 60.325, p < 0.01). group × stimulus interaction was also significant (F[6,228] = 4.578, p < 0.01).
A Post
hoc analyses using Tukey’s test showed that the NaCl elicited response to NaCl of ASN-best units was significantly higher in the CTA group than in the control group (p < 0.01).
Hierarchical cluster analysis Figure 6 shows the results of a hierarchical cluster analysis derived from
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Pearson-product moment correlations. A cluster distance of 0 on the abscissa indicates that stimuli or groups of stimuli joined at this level had identical response profiles. Larger values of distance represent more dissimilar relationships. types of the neurons.
Labels indicate the
In the dendrogram of the control group, ASN- and AIN-best
units were nearly clustered.
In contrast, in the CTA group, ASN-best units formed a
distinct cluster obviously apart from the cluster of AIN-best units.
Effects of electrical stimulation An example of taste responses to the four basic stimuli and spontaneous activity of a neuron with and without CeA stimulation are shown in Figure 7.
The responses to all
of the taste stimuli and the spontaneous activity itself were similarly inhibited in this unit.
In general, the effects of electrical stimulations in the present study were
non-specific; i.e., electrical stimulation produced similar effects on taste responses to different stimuli and on spontaneous firing rates. Antidromic activation was observed in 2 neurons in the CTA group and 1 neuron in the control group by GC stimulation, and 2 neurons in the CTA group and 3 neurons in the control group by CeA stimulation. Responses induced by antidromic activation were subtracted from the 10s responses and none of these 8 neurons met the criteria to
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be classified as excited or inhibited. Figure 8 summarizes the effect of electrical stimulation on the taste responses to the basic four tastes and spontaneous firing activity. Nine units (2 S-best, 5 ASN-best and 2 H-best) were excited and 4 units (3 ASN-best and 1 H-best) were inhibited by GC stimulation in the CTA group.
In the control
group, GC stimulation resulted in excitation of 10 units (2 S-best, 6 ASN-best and 2 H-best) and inhibition of 6 units (3 ASN-best, 1 AIN-best and 2 H-best).
There was no
significant difference in the number of neurons affected by GC stimulation between the groups (Fishter’s exact probability test, p > 0.5). (Table 1) In case of CeA stimulations, 8 neurons (6 ASN-best, 2 H-best) were excited and 13 neurons (3 S-best, 5 ASN-best, 2 AIN-best, 3 H-best) were inhibited in the CTA group. On the other hand, 1 ASN-best unit was excited and 15 units (2 S-best, 5 ASN-best, 5AIN-best, 3 H-best) were inhibited by CeA stimulation in the control group.
Fisher’s
exact probability test revealed that the number of neurons affected by CeA stimulation was significantly different between the two groups (p < 0.05). (Table 2) Figure 9 shows mean response profiles of each neuron type that were affected by GC or CeA stimulation. We adopted an analysis that classifies all units into different neuron types because the effects of CTA training on the PBN neuronal activity were
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specific for ASN-best units. Only the best-stimulus categories whose number of samples was 5 or more are shown. A two-way ANOVA with repeated measures (Taste × Electrical stimulation) revealed that there was a significant main effect of taste in all of the neuron types that were affected by GC or CeA stimulation (p < 0.05 for E, p < 0.01 for A, B, C, D and F).
However, main effect of electrical stimulation was
significant only in ASN-best units which were excited by CeA stimulation (Fig. 9B, F[1,10] = 5.100, p < 0.05) and in ASN-best units inhibited by CeA stimulation (Fig. 9C,F[1,8] = 8.035, p < 0.05) in the CTA group.
There was no significant interaction
between taste and electrical stimulation in all of the neuron types, which means that the effects of electrical stimulation were not stimulus selective.
Decerebration experiment
Behavioral results Behavioral results of the decerebration experiment proved to be very similar to those observed in the stimulation experiment. The mean intake of the CS (0.1M NaCl) in the CTA group severely decreased by repeated pairings of the CS and US (from 13.0 g ± 0.98 to 4.3 g ± 0.59 to 0.6 g ± 0.31 in the CTA group; from 12.3 g ± 1.56 to 12.8 g ±
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0.88 to 13.1 g ± 0.64 in the control group).
A two-way (Group × Session) ANOVA
with repeated measures revealed a significant main effect of group (F[1,32] = 52.349, p < 0.01) and session (F[3,96] = 39.021, p < 0.01), and a significant group × session interaction (F[3,96] = 51.357, p < 0.01).
Post hoc analyses using Tukey’s test showed
that the intake of the CS significantly decreased across trials in the CTA group (p < 0.01).
These results clearly show that the CTA animals have acquired a strong
aversion to NaCl.
Histology A photomicrograph of a representative sagittal section of a decebrated rat’s brain is shown in Figure 10.
A complete transection at the supracollicular level can be seen.
Reconstructed recording sites in the PBN were similarly located to those in the stimulation experiment (data not shown). There was no fundamental difference in the distribution of recording sites between the CTA and control groups.
Basic characteristics A total of 84 taste-responsive neurons were recorded from the PBN: 42 from each group.
All the neurons showed excitatory responses to at least one of the four basic
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taste stimuli (sucrose, NaCl, HCl or QHCl). The mean spontaneous firing rates (spikes/s) were 1.79 ± 0.36 for the CTA group and 2.19 ± 0.30 for the control group, respectively.
There was no significant difference between the spontaneous firing rates
of the two groups (t-test, p > 0.4).
Response profiles and classification Based on their largest response to the four standard taste stimuli, we classified PBN neurons as follows: 11 S-best, 17 ASN-best, 4 AIN-best, 8 H-best and 2 Q-best in the CTA group; 8 S-best, 21 ASN-best, 4 AIN-best, 7 H-best and 2 Q-best in the control group.
The criteria to classify N-best units into ASN- and AIN-best were as described
in the stimulation experiment. units distributed bimodally. shown in Figure 11.
As in the stimulation experiment, ASN- and AIN-best
The response profiles of PBN neurons of both groups are
Taste neurons are ordered by best-stimulus category and, within
each category, by response magnitude.
S-best neurons are on the left, followed by
N-best units, H-best units, then Q-best unit. AIN-best units are separately shown.
In the N-best category, ASN- and
Fisher’s exact probability test revealed that the
proportion of units responding to each standard taste stimulus was not significantly different between the two groups (p > 0.8).
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Breadth of responsiveness Since there were some inhibitory responses in our data set (as seen in Fig. 11), the entropy measure were calculated from the absolute value of the responses to the four standard taste stimuli. There was no significant difference between the mean entropies: 0.69 ± 0.03 for the CTA, 0.77 ± 0.04 for the control groups (t-test, p > 0.1).
Comparison of taste responses Figure 12 presents a comparison of mean response profiles of best-stimulus categories of units in both groups. The ASN-best panel includes data from the intact animals of the stimulation experiment because this is the group of neurons for which evidence for changes in neuronal activity after CTA training was found.
A two-way
ANOVA with repeated measures (Group × Stimulus) indicated no significant main effect of group and group × stimulus interaction in S-, AIN- and H-best-stimulus categories.
In the ASN-best category, ANOVA indicated a significant main effect of
stimulus (F[6,444] = 103.603, p < 0.01) but not of group (F[3,74] = 1.152, p > 0.3). group × stimulus interaction was also significant (F[18,444] = 2.7896, p < 0.01). Post-hoc Tukey’s test revealed that the NaCl elicited taste responses of the ASN-best
A
29
neurons in the stimulation experiment were significantly higher in the CTA group than in the other three groups (p < 0.01). The ANOVA revealed that spontaneous firing rates of the four groups in the decerebration and stimulation experiments (1.79 ± 0.36 for the CTA group and 2.19 ± 0.30 for the control group in the decerebration experiment; 2.75 ± 0.44 for the CTA group and 1.96 ± 0.38 for the control group in the stimulation experiment) were not statistically different (F[3,164] = 1.235, p > 0.2). There was also no significant difference in the values of entropy among the four groups (0.69 ± 0.03 for the CTA group and 0.77 ± 0.04 for the control group in the decerebration experiment; 0.68 ± 0.03 for the CTA group and 0.70 ± 0.03 for the control group in the stimulation experiment, F[3,164] = 1.584, p > 0.19).
30
DISCUSSION In the present study, for the first time, we have demonstrated that forebrain gustatory areas contribute to the CTA induced altered taste responses in the PBN.
Furthermore,
increased taste responses were observed only in ASN-best units after the acquisition of taste aversion to NaCl.
These results suggest that interactions between the PBN and
higher taste relays play an important role in processing information of a taste conditioned to be aversive, and that ASN-best units in the PBN are critically involved in the distinctive taste of NaCl.
Acquisition of conditioned taste aversion Animals in the CTA group in the both experiments showed significantly reduced intake of NaCl solution across trials. completely avoided ingesting the CS.
On the day of the last CS-US paring, they almost Increased orofacial aversive taste reactivity
(Grill and Norgren 1978) to the CS was also observed in the CTA group although it was not quantified. These results clearly show that rats in the CTA group had acquired a strong CTA to NaCl by the day of single neuron recording experiments.
Therefore, it
is reasonable to suppose that some plastic changes took place in neuronal activity reflecting taste aversion learning in CTA animals.
31
Spontaneous firing rate Mean spontaneous firing rates of the parabrachial gustatory neurons (2.75 spikes/s for the CTA and 1.96 spikes/s for the control groups in the stimulation experiment; 1.79 spikes/s for the CTA and 2.19 spikes/s for the control groups in the decerebration experiment) were relatively low compared to those reported in previous studies using rats under urethan anesthesia (e.g., over 5 spikes/s, Di Lorenzo 1990; Di Lorenzo and Monroe 1992).
However, there were also studies reporting low spontaneous firing
rates (Ogawa et al. 1987; Perrotto and Scott 1976; Scott and Perrotto 1980) compatible to those in the present study.
Anesthesia might have suppressed the spontaneous firing
rates by blocking the descending excitatory inputs to the PBN because spontaneous firing rates of the PBN neurons in conscious rats were found to be much higher (ranging from 6.1 to 11.2 spikes/s, Hajnal et al. 1999; Nishijo and Norgren 1997).
Taste response profiles The proportion of S-best units in the present study is somewhat larger, resulting in the smaller proportion of N-best units, than in our previous studies in which N-best units dominate the sample (Shimura et al. 1997c; Shimura et al. 2002).
This difference may
32
reflect the fact that the entire oral cavity was sufficiently stimulated and/or that a search stimulus consisted of a mixture of the four basic chemicals more successfully detected taste responses in the PBN (0.3M NaCl was used as a search stimulus in our previous studies). The proportion of each best-stimulus unit of rats used in the stimulation and decerebration experiments was not fundamentally different.
Although no Q-best unit
was recorded in the stimulation experiment, this result should not be overemphasized because the sample sizes were relatively small in both experiments. In the present study, ASN-best units comprised about 75–85% of the N-best units in the four groups in the both experiments.
These results are comparable with those of a
recent electrophysiological study which also classified N-best units into ASN- and AIN-best by their sensitivity to amiloride in the PBN (Lundy and Norgren 2001). The value of entropy of neurons in the PBN is not statistically different among the four groups used in the decerebration and stimulation experiments, which indicates that the breadth of tuning is unchanged by CTA or decerebration.
These results are also in
line with previous reports (Di Lorenzo 1988b; Shimura et al. 1997c).
Recording site
33
The location of recording sites was almost identical to that shown by previous electrophysiological experiments (Norgren and Pfaffmann 1975; Scott and Perrotto 1980; Shimura et al. 1997c; Shimura et al. 2002).
While there was no fundamental
difference in the distribution of ASN- and AIN-best neurons in the PBN, H-best units were preferentially located in the dorsal region of the brachium conjunctivum as it was previously reported by Ogawa et al. (1984, 1987).
Taste responses after conditioned taste aversion In the decerebration experiment, there was no significant difference in the magnitude of taste responses between the two groups in all best-stimulus categories.
Because the
experimental procedure was practically the same as that of our previous experiments which show that gustatory activities in the PBN are altered after the acquisition of CTA (Shimura et al. 1997c; Shimura et al. 2002), it is reasonable to suppose that the decerebration prevented the development of altered gustatory activities induced by CTA. This, furthermore, means that interconnections with forebrain structures are an indispensable requirement of adaptive taste information processing mechanisms. The same recording condition for the CTA and control groups assured that the different response characteristics between the two groups were primarily reflecting
34
some plastic changes that developed as a result of taste aversion learning.
In the
stimulation experiment, responses to NaCl significantly increased only in the ASN-best units in the CTA group. between the groups.
Furthermore, responses to other taste qualities did not differ
Therefore, aversive conditioning appears to increase the
responsiveness only to behaviorally relevant taste stimuli in the PBN, which may help the animals to avoid the ingestion of aversive, potentially harmful fluids.
Similar
results have been reported by a previous study in which CTA to sodium saccharin resulted in an increased responsiveness to the CS only among the sweet-sensitive subset of gustatory neurons in the NTS (Chang and Scott 1984). Cluster analyses also detected altered responsiveness in the CTA group in the stimulation experiment. ASN-best units in the CTA group formed a distinct cluster apart from the cluster of AIN-best units whereas in the control group ASN- and AIN-best units were nearly clustered. The shift of the cluster is thought to be reflecting the fact that in the CTA animals NaCl becomes a more salient stimulus for the ASN-best units. The above data suggest that specific activity changes of these ASN-best neurons of the PBN may represent the underlying neural substrate of plastic changes resulting in the taste of NaCl CS becoming more distinct and sharply discriminated from the other taste qualities.
35
It should be noted that taste quality of the CS may influence the effects of CTA training on gustatory responses in the brainstem.
When sweet saccharin was used as
the CS, the changes in taste activity in the NTS were relatively modest (Chang and Scott 1984) compared to those of our earlier (Shimura et al. 1997c) and the present studies in which the salty NaCl CS was used.
Moreover, when non-preferred sour HCl
was used as the CS, PBN neuronal responses to the CS decreased although the same conditioning procedures were used as in the present study (Shimura et al. 2002). These results suggest that neural mechanisms underlying the CTA are different for various tastes serving as the CS.
Effect of decerebration on the parabrachial neuronal activities In the present study, decerebration did not have clear effect on taste responses and spontaneous activities in the PBN. A similar study by Di Lorenzo (1988b) reported that decerebration, while leaving spontaneous firing rates in the PBN unchanged, produced smaller responses to NaCl and HCl and larger responses to saccharin sodium. This discrepancy may be explained by the different experimental procedures employed, i.e., animals were anesthetized with urethan in the present study whereas animals were paralyzed with Flaxedil during recording in Di Lorenzo’s study.
Deep anesthesia
36
induced by urethan in the present experiments might have reduced centrifugal influences and this could mask the effect of decerebration on neuronal activities. Nevertheless, the finding that taste responses to NaCl increase after CTA in intact but not in decerebrate animals suggests that the influence of forebrain structures is not completely suppressed even despite the deep anesthesia.
This notion is supported by
the recent finding showing that gustatory activities in the PBN of rats under urethan anesthesia were different before and after electrolytic lesions in the CeA (Huang et al. 2003).
Effects of electrical stimulation In the present study, CeA stimulation produced mainly inhibitory effects on the PBN neurons.
These results are in line with those of the previous reports which examined
descending influences from the CeA to the gustatory neurons in the PBN (Lundy and Norgren 2001; Huang et al. 2003).
However, the CeA stimulation did not produced a
taste-quality-specific effect in the present study, i.e., in our case the effect was similar for the spontaneous activities and for the responses to various taste stimuli. This non-specific effect may partly be due to relatively strong intensity of electrical pulses (0.3 mA) employed in our stimulation experiments.
It is, therefore, possible that a
37
centrifugal influence plays a more complex role in conscious rats than in anesthetized ones. The GC has been known to be implicated in several phases of CTA memory (Berman et al. 1998; Ramirez-Lugo et al. 2003; Yasoshima and Yamamoto 1998).
In the present
study, however, there was no significant effect produced by GC stimulation.
Thus, it is
suggested that the GC is not the principal source of excitatory inputs which causes increased responses to the CS in the PBN after acquisition of a CTA.
These results of
course do not deny the functional involvement of the GC in CTA. Although the effect of electrical stimulation was, in general, non-specific, we found that CeA stimulation excited 8 neurons in the CTA group, whereas only 1 neuron was excited by CeA stimulation in the control group.
This increased excitatory effect may
partly reflect the plastic changes that had occurred in the PBN during the acquisition phase of CTA.
Possible mechanisms Deep anesthesia has already been suggested to block descending influences from the forebrain (Shimura et al. 1997b; Shimura et al. 2002), thus, we previously concluded that alteration of taste responses induced by CTA could occur within the PBN and/or at
38
lower gustatory representation levels below the PBN.
In the present study, however, it
has been demonstrated that the decerebration effectively prevents such alteration of taste responses after CTA acquisition. This obviously means that descending inputs from the forebrain, but not ascendingly wired information, induce increased taste responses to the CS at least in the retrieval phase of CTA.
Nevertheless, it should be
noted that this finding does not necessarily preclude the possibility that plastic changes occur within the PBN.
In addition to forebrain areas, such plastic changes may also
develop in the synapses formed by the PBN neurons and terminal buttons of neurons in the forebrain that convey descending information.
Although the present study cannot
conclusively rule this out, we found no evidence that the brainstem was sufficient to maintain changes in the taste responses properties of the neurons normally after CTA acquisition in intact rats.
This notion is consistent with the behavioral finding that
decerebrate rats can neither acquire nor retain CTA (Grill and Norgren 1978). Which site causes enhanced responses to NaCl CS in the PBN?
As already
mentioned, the effect of GC stimulation on neuronal activity in the PBN was not statistically significant.
By contrast, CeA stimulation produced more excitatory effects
in the CTA group than in the control group though such effects were not exclusively confined to the ASN-best units.
Thus, these results suggest that at least the CeA may
39
contribute to the altered responsiveness of PBN neurons in the CTA-trained rats. Plastic changes that occur between the CeA and the PBN might underlie increased responses to the CS. For example, direct measurement of long-term potentiation between the CeA-PBN projection is required to confirm this hypothesis.
Descending
inputs from the CeA to the PBN might also function as feedback systems that facilitate discrimination of the taste of the CS. There is some evidence in the literature that centrifugal influences from the GC and CeA play a role in CTA.
Schafe and Bernstein (1996, 1998) demonstrated that
electrolytic lesions of both GC and CeA reduce c-Fos-like immunoreactivity in the intermediate division of the NTS which is already well known to be correlated with the behavioral expression of CTA. Results of the present study along with these findings demonstrate that the gustatory relays in the brainstem and forebrain form a highly organized neural network that
- in order to contribute to adaptive behavioral outputs -
almost simultaneously operates with gustatory information, perceptual, motivational and learning determinants, as well as memory constituents of these processes.
Recent
views in the gustatory neural coding also emphasize interactions of neurons in spatially separate brain regions (Katz et al. 2002). It should be noted that effect of electrical stimulation in the present study can also be
40
attributed to activation of intrinsic neurons and fibers of passage as well as that of forebrain neurons connected with the PBN and/or lower gustatory levels below the PBN. Since many changes in gustatory neurons in the NTS have been shown by stimulation of GC (Di Lorenzo and Monre 1995; Smith and Li 2000) and CeA (Cho et al. 2003; Li et al. 2002), it is possible that the present results of electrical stimulation are simply reflections of changes in the NTS which are then transferred to the PBN.
Moreover,
forebrain areas that send fibers to the PBN are also connected with each other (Halsell 1992; van der Kooy et al. 1984; Yamamoto et al. 1984).
For example, gustatory
neurons in the GC respond to CeA stimulation and, in turn, gustatory neurons in the CeA also respond to GC stimulation (Yamamoto et al. 1984).
Interpretation of
stimulation data, therefore, must not be confined to the effects that were produced by monosynaptic connections.
Conclusions In summary, CTA to NaCl modified ASN-best units in the PBN so that sodium taste became more salient than other tastes in these rats. The present data substantiate that this neuronal modification requires active inputs from the forebrain to facilitate a highly sensitive, adaptive gustatory discrimination of conditioned animals.
41
ACKNOWLEDGEMENTS The present study was supported by the Grant-in-Aid for scientific research (no. 14370593 to T.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by the Grant-in-Aid for JPSP Fellows (no. 03901 to K.T.) from the Japan Society for the Promotion of Science, and by The Salt Science Research Foundation (no. 0244), and by The Mishima Kaiun Research Foundation.
This work
was a part of the 21st Century COE entitled ‘‘Origination of Frontier BioDentistry’’ at Osaka University Graduate School of Dentistry supported by the Ministry of Education, Culture, Sports, Science and Technology.
42
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FIGURE LEGENDS
Figure1.
Intake of distilled water (DW) on the last day of water training before
conditioning and 0.1 M NaCl across three acquisition (Acqu) trials in CTA (closed bars) and control (open bars) group. to 0.1 M NaCl.
Figure 2.
Animals in the CTA group acquired a robust aversion
*p < 0.01 compared with Aqcu 1 of the CTA group.
The distribution of stimulating and recording sites. A and B: the location of
stimulating electrodes in animals of both groups plotted in the standard atlas sections of the GC (A) and CeA (B) of rat (Paxinos and Watson, 1998).
Symbols indicate the
effects produced by electrical stimulation on the PBN gustatory neurons.
Triangles
indicate sites that evoked excitatory responses, inverted triangles indicate sites that produced inhibitory responses, and squares indicate sites that produced no effect. Filled symbols, the CTA group; open symbols, the control group. section show distances from the bregma. sites in the PBN.
Values under each
C: anatomical reconstruction of recording
Sections a–d are arranged rostral to caudal throughout the extent of
the taste-responsive region of the parabrachial pons separated by approximately 200 µm.
58
Filled symbols, the CTA group; open symbols, the control group; circles, S-best units; triangles, amiloride-sensitive N-best units; inverted triangles, amiloride-insensitive N-best units; squares, H-best units.
BC, brachium conjunctivum; MesV,
mesencephalic trigeminal nucleus. Scale bars = 0.5 mm.
Figure 3. PBN.
Effects of amiloride on taste responses to NaCl of N-best neurons in the A: examples of responses of ASN- (amiloride-sensitive) and AIN-
(amiloride-insensitive) best units to NaCl with and without amiloride.
A marked
inhibition of taste responses to NaCl was observed in the ASN- but not in the AIN-best unit.
Arrows indicate stimulus onset. B: numbers of ASN- (amiloride-sensitive) and
AIN- (Amiloride-insensitive) best units of both groups.
ASN-best units consist of 38
(17 from CTA and 21 from control group) and AIN-best units, 8 units (4 from each group).
Bimodal distribution of ASN- and AIN-best neurons without any intermediate
type is apparent.
Figure 4.
Response profiles of PBN taste neurons to four standard stimuli.
Taste
neurons were grouped into best-stimulus categories and arranged within those categories in descending order of response magnitude to the best-stimulus.
Taste
59
responses were adjusted to water responses, i.e. responses to water were subtracted from raw responses to taste stimuli. Filled bars indicate significant responses: 2 S.D. above or below spontaneous rate. inhibitory ones.
Up bars indicate excitatory responses; down bars,
The CTA group (n = 41: S-best, 7; ASN-best, 18; AIN-best, 6; H-best,
10), the control group (n = 43: S-best, 8; ASN-best, 22; AIN-best 7; H-best, 6).
QHCl,
quinine hydrochloride.
Figure 5.
Mean response profiles of units in both groups classified into each
best-stimulus category.
Taste responses to NaCl of ASN-best units were significantly
higher in the CTA group than in the control group (p < 0.01).
Figure 6.
Cluster trees depicting relative response similarity of 84 neurons in the CTA
and control groups.
A cluster distance of 0 on the abscissa indicates that stimuli or
groups of stimuli joined at this level had identical response profiles. distance represent more dissimilar relationships. neuron.
Labels indicate the types of the
Note that ASN-best units in the CTA group form a distinct cluster apart from
the cluster of the AIN-best units. H-best.
Larger values of
S, S-best; ASN, ASN-best; AIN, AIN-best; H,
60
Figure 7.
Taste responses to the four basic tastes and spontaneous activity of a PBN
neuron with and without concurrent electrical stimulation in the CeA.
Note that CeA
stimulation similarly inhibits the responses to different taste stimuli and spontaneous activity.
The thin bar corresponds to 10 s taste stimulation and the thick bar
corresponds to concurrent electrical stimulation.
QHCl, quinine hydrochloride.
Figure 8.
Effects of electrical stimulation of the GC and CeA on the PBN gustatory
neurons.
Neurons whose activity was affected by electrical stimulation are shown.
The gray and black areas indicate taste responses and spontaneous discharge which were excited or inhibited by ± 25% by electrical stimulation compared with those without electrical stimulation.
The white areas indicate taste responses and
spontaneous discharge unaffected by electrical stimulation.
S, sucrose; N, NaCl; H,
HCl; Q, quinine hydrochloride.
Figure 9.
Mean response profiles of each neuron type that were affected by GC or
CeA stimulation.
Both GC and CeA stimulation produced similar effects on taste
responses to different tastes and spontaneous activity.
61
Figure 10.
A representative sagittal section of a decerebrate brain.
transected at the superacollicular level.
Figure 11.
The brain is
The section was stained with cresyl violet.
Response profiles of PBN taste neurons of decerebrate rats to the four
standard stimuli.
Taste neurons were grouped into best-stimulus categories and
arranged within those categories in descending order of response magnitude to the best-stimulus.
Taste responses were adjusted to water responses, i.e. responses to
water were subtracted from raw responses to taste stimuli. Up bars indicate excitatory responses; down bars, inhibitory ones. The CTA group (n = 42: S-best, 11; ASN-best, 17; AIN-best, 4; H-best, 8; Q-best 2), the control group (n = 42: S-best, 8; ASN-best, 21; AIN-best 4; H-best, 7; Q-best 2).
Figure 12.
QHCl, quinine hydrochloride.
Mean response profiles of units in both groups in the decerebration
experiment classified into each best-stimulus category.
The ASN-best panel includes
data from the intact animals of the stimulation experiment (I-CTA and I-Control).
A
two-way ANOVA with repeated measures (Group × Stimulus) indicated no statistical difference between the groups in S-, H- and AIN-best-stimulus categories.
In
62
ASN-best category, responses of ASN-best units to NaCl in the stimulation experiment were significantly higher in the CTA group than in the other three groups (p < 0.01).
63
Table 1.
Effects of GC stimulation on PBN neurons CTA
Neuron type
Control
Excitation Inhibition No effect
Total
Excitation Inhibition No effect
Total
S-best
2 (29)
0 (0)
5 (71)
7
2 (25)
0 (0)
6 (75)
8
ASN-best
5 (28)
3 (17)
10 (56)
18
6 (27)
3 (14)
13 (59)
22
AIN-best
0 (0)
0 (0)
6 (100)
6
2 (29)
1 (14)
4 (57)
7
H-best
2 (20)
1 (10)
7 (70)
10
0 (0)
2 (33)
4 (67)
6
Total
9 (22)
4 (10)
28 (68)
41
10 (21)
6 (14)
27 (63)
43
AIN-best, amiloride-insensitive NaCl-best; ASN-best, amiloride-sensitive NaCl-best; CTA, conditioned taste aversion; GC, gustatory cortex; H-best, HCl-best; PBN, parabrachial nucleus; S-best, sucrose-best.
Percentages are in parentheses.
64
Table 2.
Effects of CeA stimulation on PBN neurons CTA
Neuron type
Control
Excitation Inhibition No effect
Total
Excitation Inhibition No effect
Total
S-best
0 (0)
3 (43)
4 (57)
7
0 (0)
2 (25)
6 (75)
8
ASN-best
6 (33)
5 (28)
7 (39)
18
1 (0.5)
5 (23)
16 (73)
22
AIN-best
0 (0)
2 (33)
4 (67)
6
0 (0)
5 (71)
2 (29)
7
H-best
2 (20)
3 (30)
5 (50)
10
0 (0)
3 (50)
3 (50)
6
Total
8 (20)
13 (32)
20 (49)
41
1 (0.02)
15 (35)
27 (63)
43
AIN-best, amiloride-insensitive NaCl-best; ASN-best, amiloride-sensitive NaCl-best; CeA, central nucleus of the amygdala; CTA, conditioned taste aversion; H-best, HCl-best; PBN, parabrachial nucleus; S-best, sucrose-best. parentheses.
Percentages are in
65
Figure 1
66
Figure 2
67
Figure 3
68
Figure 4
69
Figure 5
70
Figure 6
71
Figure 7
72
Figure 8
73
Figure 9
74
Figure 10
75
Figure 11
76
Figure 12
