Brain Research, 556 (1991) 95-100 ~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391168704

95

BRES 16870

Effects of conditioned taste aversion on extracellular serotonin in the lateral hypothalamus and hippocampus of freely moving rats Howard L. West, Gregory P. Mark and Bartley G. Hoebel Department of Psychology, Princeton University, Princeton, NJ 08544-1010 (U.S.A.) (Accepted 19 March 1991) Key words: Conditioned taste aversion; Serotonin; Lateral hypothalamus; Hippocampus; Microdialysis

This study used microdialysis to monitor extracellular levels of 5-HT and its metabofite, 5-hydroxyindole acetic acid (5-HIAA) in the lateral hypothalamus (LH) and hiplxx:zmlpusof freely moving rats that had developed a CTA to a 2.5 mM saccharin solution (CS) following its pairing with illness induced by lithium chloride (US). Results showed that oral infusion of the saccharin CS significantly enhanced extracellular LH 5-HT in animals that had developed a taste aversion compared with control groups, including unconditioned (CS-no US) and pseudoconditioned (no CS-US) subjects. As an anatomical control, the hippocampus was identified based on previous research suggesting that it is not integrally involved in CTA learning or retrieval and that 5-HT in this brain site does not directly mediate feeding behavior but is closely correlated with arousal. In contrast with the results obtained in the LH, hippocampal 5-HT was not preferentially elevated in subjects in the CTA group but rather was increased to the same extend in both CTA and control groups after saccharin infusion. Moreover, the increase in LH 5-HT for the CTA group was nearly twice that observed in the hippocampus for any group. Acute administration of LiCl elevated extracellular 5-HT to similar levels in both sites, well above the changes observed following conditioning. 5-HIAA was unaffected in either brain site by oral infusion of saccharin solution or injection of LiCl. These results are consistent with the hypothesis that increased 5-HT transmission in the LH plays a central role in the inhibition of ingestion during expression of a CTA that may not be entirely attributable to a global activation of the serotonergic system in arousal.

INTRODUCTION A large body of research indicates a primary role for serotonin (5-HT) in the neurochemical mechanisms of conditioned taste aversion, although the exact nature of this role remains controversial. Depletion of forebrain 5-HT following lesions of the raphe nuclei 22 or administration of the synthesis inhibitor P C P A 1° enhances CTA learning, whereas pretreatment with the 5-HT precursor 5-HTP 24 or the re-uptake blocker fluoxetine 2a attenuates subsequent LiCl-induced C T A development. While these results suggest that 5-HT exerts an inhibitory influence on C T A learning, other studies have supported the opposite effect for 5-HT. Increases in 5-HT transmission by administration of 5°HTP 40'41, fenfluramine a, the reuptake blocker zimeldine n , and monoamine oxidase inhibitors 2 all enhance or induce conditioned aversions. Taken together, these investigations all support a role for 5-HT in taste aversion, but its function remains inconclusive. Given the wide variability in results of experiments manipulating serotonergic transmission and assessing the effects on C T A learning and expression, a direct means

of assessing 5-HT levels at specific brain sites in the conditioned animal would be of considerable benefit. Microdialysis has become a useful technique for monitoring neurotransmitters and their metabolites in the extracellular space of isolated brain regions in freely moving animals 6,7,13,14,2s,32,37. Local concentrations of 5-HT and its metabolite, 5-hydroxyindole acetic acid ( 5 - H I A A ) can be determined from brain dialysates using high-performance liquid chromatography with electrochemical detection ( H P L C - E C ) . Thus, the microdialysis technique provides a means by which extraceUular levels of 5-HT can be directly evaluated as a dependent measure of the conditioning procedure, in contrast to previous studies in which behavioral measures were determined as a function of changes in serotonergic activity. In the present study, microdialysis was used to monitor changes in extracellular levels of 5-HT and 5 - H I A A in both the lateral hypothalamus (LH) and hippocampus of unanesthetized rats during oral infusion of a saccharin solution to which subjects had developed a C T A or following injection of the illness-inducing US (LiCl). The L H was chosen as a test site based on prior observations

Correspondence: B.G. Hoebel, Department of Psychology, Princeton University, Princeton, NJ 08544-1010, U.S.A.

96 that e n h a n c e d

5 - H T transmission

in t h e L H

inhibits

f e e d i n g 38 and t h a t the L H is i n v o l v e d in C T A b e h a v i o r TM 34

The

hippocampus

TABLE I Saccharin and water intake in CTA subjects and control animals

was s e l e c t e d as an a n a t o m i c a l

c o n t r o l site b a s e d o n the results o f investigations which

Condition

Sacc. intake (ml)

Water intake (ml)

Ratio (sacc./water)

CTA Uncond. Pseudocond.

2.59 + 0.32 10.67 + 0.81 10.48 + 0.34

10.54 + 0.72 6.03 + 0.44 5.62 + 0.73

0.25 + 0.03** 1.83 + 0.15 2.11 + 0.26

suggest that t h e h i p p o c a m p u s d o e s n o t play a m a j o r role in e i t h e r s e r o t o n e r g i c m o d u l a t i o n of f e e d i n g o r C T A b e h a v i o r 1,26,2s. T h e results p r e s e n t e d

here demonstrate

that while

p r e s e n t a t i o n o f an a v e r s i v e stimulus i n c r e a s e d 5 - H T in both

sites,

o n l y in t h e

LH

did this increase

occur

p r e f e r e n t i a l l y in the c o n d i t i o n e d g r o u p . MATERIALS AND METHODS Sixty adult, male, Sprague-Dawley rats weighing 325-375 g were housed individually on a 12-12 h reversed light-dark schedule (9.00-21.00) with food and water available ad libitum. Subjects were anesthetized with a combination of pentobarbital (25 mg/kg, i.p.) and ketamine (50 mg/kg, i.p.). An intra-oral catheter made of PE 20 polyethylene tubing with a flange at one end was inserted through the cheek and threaded subcutaneously to the top of the skull. Bilateral guide shafts aimed at either the LH (n = 30) or hippocampus (n = 30) were stereotaxically implanted as foUows3°: AP 6.5 mm, L 1.6 ram, V 3.0 for the LH; AP 3.2, L 4.5, V 2.0 for the hippocampus, with reference to the interaural line, midsaggital suture, and the surface of the level skull, respectively. CTA conditioning After a one-week recovery period, subjects were placed in Plexiglas chambers and allowed to habituate for a minimum of 1 h before receiving a 20-min oral infusion of the CS (2.5 mM saccharin, 250 /A/rain). One hour after the infusion, subjects received an injection of the US (LiCI, 130 mg/kg, i.p.). Subjects remained in the test chambers for a minimum of 3 h before being returned to their home cages. This procedure was repeated after 48 h. For each brain site, 3 different conditions were established with 6 subjects per group: (A) CTA (CS-US), saccharin infusion followed by LiCl injection; (B) Unconditioned (CS-no US), saccharin infusion followed by saline injection (0.1 ml/kg, i.p.); (C) Pseudoconditioned (no CS-US), water infusion followed by LiCl injection. Microdialysis sessions were conducted in the same chambers in which subjects were conditioned and began approximately 48 h after the second LiCI or saline injection. In order to behaviorally confirm the effect of the conditioning procedure, all subjects were water-deprived for 20 h immediately following microdialysis sessions. Rats were then allowed access for 20 min to pre-weighed bottles containing 2.5 mM saccharin solution followed by 20 min access to distilled water. Fluid intake was expressed as the ratio of saccharin solution to water during 20 min access for each subject. Data were analyzed by a one-way analysis of variance followed by post-hoc Neuman-Keuls tests when justified. Microdialysis procedure At least 12 h before each experiment, subjects were implanted with mierodialysis probes which extended 5 mm beyond the guide shaft. Probes were constructed with silica glass tubing inside a 26-gauge stainless-steel tube with a microdialysis tip 200/~m o.d. by 3 mm long for LH or 4 mm long for hippocampus, using 6000 molecular-weight-cutoff cellulose tubing (Spectrum Medical, CA). Technical details of the probes are given elsewhere 13. Probes were perfused at a flow rate of 1/A/rain with a Ringer solution (146 mM NaCI, 3.9 mM KCI, 1.20 mM CaCI 2, degassed, pH 5.6) with fluoxetine (10/~M, Sigma) to block 5-HT re-uptake in experimental sessions in which basal levels were initially undetectable. The outlet branch of the probe led to a 400-jul vial clipped to a flexible cable

above the head of the rat. Samples were collected in the rats' early to middle dark period at 20 min intervals for 1 h prior to and 3 h after an intra-oral infusion of 2.5 mM sodium saccharin. Dialysates were analyzed by reverse-phase HPLC-EC. Samples were injected directly into the HPLC (EG&G/PAR Co. Model 400 detector with SSI Co. Model 200 pump) and were oxidized at 2 potentials. The dual potentiostat produced a ratio of peak heights which was compared to a ratio characteristic of a 5-HT standard (Sigma). Only sample peaks within 10% of this ratio were considered as representing brain 5-HT. In addition, the 5-HT1A autoreceptor agonist 8-OH-DPAT, which inhibits the firing of serotonergic cells9'15'36, was administered to one-third of the subjects in order to further confirm the identity of the measured peak as neuronal 5-HT. The HPLC system used a 20/~1 sample loop leading to a 10 cm column with 3.2 mm bore and 3/~m, C-18 packing (Brownlee Co. Model 6213). The mobile phase contained 116.8 mM NaOH, 144.7 mM monochloroacetic acid, 1.38 mM 1-octane sulfonic acid, 1.38 mM EDTA, and 6.5% v/v acetonitrile at pH 3.1.5-HIAA and 5-HT were eluted in that order and oxidized on glassy carbon electrodes at 0.55 V and 0.59 V, with retention times of 3.1 and 6.5 rain, respectively. The minimum detectable amount of 5-HT was 0.5 pg/20/~1 (signal-to-noise ratio 3:1). In cases in which baseline levels of 5-HT were undetectable, the 5-HT reuptake blocker fluoxetine (10/~M) was added to the perfusate 2-3 h before the experiment in order to elevate recoverable 5-HT levels. This occurred in approximately half of the LH and half of the hippocampal subjects. Absolute basal recovery of 5-HT and 5-HIAA varied considerably between subjects. This is a typical finding in dialysis experiments and is most likely due to differences in probe recovery, local terminal density and diffusion barriers. Consequently, data from each subject were normalized by converting peak heights to a percent of the mean of 3 baseline samples. Data were analyzed by a two-way analysis of variance (condition x time) for each brain site and were followed by post-hoc Neuman-Keuls tests when justified. Histologies were performed on all subjects in order the verify probe placement in the LH or hippocampus. Subjects received a lethal injection of sodium pentobarbital and were then perfused with 0.9% saline solution followed by formalin. Brains were removed frozen and cut in 40/~m serial sections. LiCI injection For each brain site, 2 groups of 6 subjects established the effect of the US alone. These animals underwent no behavioral training and received injections of either LiC1 (130 mg/kg, i.p.) or 0.9% saline (0.1 ml/kg, i.p.) on the day of testing. RESULTS T h e c o n d i t i o n i n g p r o c e d u r e p r o d u c e d a r o b u s t aversion in C T A subjects w h i c h was not e v i d e n t in c o n t r o l animals (Table I). O n e - w a y A N O V A

demonstrated

a

significant d i f f e r e n c e in the ratio of saccharin i n t a k e to w a t e r i n t a k e a m o n g t h e 3 g r o u p s (F2,33 = 35.753, P < 0.001), with p o s t - h o c N e w m a n - K e u l s tests r e v e a l i n g that

97 TABLE II Average absolute baseline recovery levels of both 5-HT and 5-HIAA from both brain sites

Neurochemical

Site

Mean recovery: pg/20/~l

S.E.M.

Range

5-HT

Ln Hippocampus LH Hippocampus

4.67 (2.55) 4.39 (1.58) 908 786

0.66 (0.37) 0.52 (0.24) 38 40

1.5-8.6 (0.5-4.8) 1.5-7.7 (0.6-4.1) 531-1373 510-1270

5-HIAA

the ratio was significantly lower in the CTA group than in the unconditioned and pseudoconditioned control groups (**P < 0.01). In both sites, approximately half of the subjects in each group were run with fluoxetine added to the perfusate, with nearly equal distribution across all individual conditions (2-4 out of 6 per group). In accordance with

differences in the effects of manipulations due to addition of fluoxetine for any group in either site. Because results were similar in conditions with and without fluoxetine in the perfusate, data were collapsed across this variable. Average absolute baseline recovery levels of both 5 - H T a n d 5 - H I A A from both brain sites are listed in

Lateral Hypothalamus *

160

previous studies 3"17'19'32, the addition of a 5 - H T re-uptake blocker in the perfusate elevated extraceUular 5 - H T levels 5- to 10-fold and had no effect on 5 - H I A A levels. A series of t w o - w a y A N O V A s revealed no significant

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Fig. 1. A: oral infusion of 2.5 mM saccharin solution significantly increased extracellular levels of lateral hypothalamic 5-HT in the CTA group (solid line) above unconditioned (dashed line) and pseudoconditioned (dotted line) control groups (*P < 0.01). LH serotonin was significantly increased in pseudoconditioned subjects (tP < 0.05) above saline controls during saccharin infusion but was nevertheless significantly below levels in the CTA group. Serotonin is expressed as the percent of 3 baseline samples. B: saccharin infusion induced no significant changes in extracellular levels of LH 5-HIAA in any group.

I

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Time (in min) 60

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Time (in min) Fig. 2. A: saccharin infusion significantly elevated hippocampal 5-HT levels to the same extent in CTA and control groups during and immediately following the manipulation (*P < 0.05). CTA group, solid line; unconditioned group, dashed line; pseudoconditioned group, dotted line. B: saccharin induced no significant changes in hippocampai 5-HIAA in any group.

98 Lateral

Hypothalamus

Hippoeampus

A

200 "

220 .~ 200

180"

"~ 180

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*

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Fig. 3. A and C: lithium chloride (130 mg/kg i.p., solid lines) significantly increased extracellular serotonin in the LH and hippocampus above saline controls (dashed lines; *P < 0.01). B and D: LiCI caused no significant changes in 5-HIAA levels at either site.

Table II. For 5-HT, numbers in parentheses indicate recovery in subjects which did not have fluoxetine added to the perfusion medium. The various training protocols were correlated with differences in extracellular levels of 5-HT during and following presentation of the CS. Two-way ANOVA revealed a significant interaction effect between condition and time on extracellular levels of LH 5-HT (F2o,150 = 1.953, P < 0.025; Fig. 1A). A simple effects analysis revealed that differences among groups were significant both during and immediately after oral infusions of saccharin solution (during 20 min saccharin: P < 0.01; 1st post 20 min saccharin: P < 0.05). Specifically, while the pseudoconditioned group was significantly elevated (20%) above unconditioned controls during the oral infusion (dotted line; Newman-Keuls: P < 0.05) but not immediately after it, subjects that had acquired an aversion to the taste demonstrated an enhancement of LH 5-HT levels (48%) significantly above both control groups (solid line; Newman-Keuls: P < 0.01) both during and after the saccharin infusion. In contrast to the changes observed in the LH, two-way ANOVA revealed no significant interaction between condition and time for hippocampal 5-HT (F2o,]50 = 0.776, ns; Fig. 2A), although there was a main effect of time in control and experimental groups (F10,15o

= 9.573, P < 0.0001). 5-HT was consistently elevated to 18-25% above baseline during and immediately following saccharin infusion in all of the groups tested (Newman-Keuls: P < 0.01 for both time points). In neither brain site did a two-way ANOVA reveal overall effects on extracellular levels of the 5-HT metabolite, 5-HIAA, for either time (LH: Flo,15o = 1.856, ns; Fig. 1B; hippocampus; Ft0,15o = 1.827, ns; Fig. 2B) or condition (LH: F2j 5 = 0.069, ns; hippocampus: /72.15 = 1.258, ns). Injection of LiCI produced a dramatic increase in 5-HT levels in both sites (LH: 86%; hippocampus: 80% above baseline; Fig. 3A and C) immediately following injection compared to saline controls. Two-way ANOVA revealed a significant interaction between condition and time for 5-HT levels in both LH and hippocampus (LH: Fg,9e = 9.512, P < 0.001; hippocampus: F9,9o =7.091, P < 0.001). A simple effects analysis demonstrated that this significant difference between experimental and control groups across time was limited to the sample immediately following injection, during which 5-HT levels in the experimental group were significantly elevated over those of the control group (Newman-Keuls: P < 0.01 for both sites). LiC1 injections produced no significant changes in extracellular levels of 5-HIAA in either site (Fig. 3B and

99 D). Two-way A N O V A revealed no effects for either time (LH: F9,9o = 1.119, ns; hippocampus: F9,9o = 1.332, ns) or condition (LH: F~,10 = 0.364, ns; hippocampus: FL10 = 0.171, ns). Consistent with previous reports 33'39, injections of 8-OH-DPAT produced an average decrease of 61% in 5-HT levels in the 20 animals to which it was administered (data not shown). No differences in this reduction were detected between the 2 brain sites. DISCUSSION Although recent research suggests an important role for the serotonergic system in CTA behavior, these studies have implicated 5-HT by indirectly facilitating or suppressing its transmission and investigating the effects of these manipulations on subsequent development or expression of a conditioned aversion. In contrast, the purpose of the present experiment was to study changes in extracellular 5-HT as a dependent measure of conditioning. The results demonstrate that 5-HT levels in the LH were significantly increased during oral infusion of saccharin solution in animals which had developed a CTA to this stimulus. In contrast, hippocampal 5-HT increased in response to oral infusion of saccharin to a lesser extent in all groups, but no preferential increase was exhibited in the CTA group. In response to injection of LiCI alone, 5-HT levels were significantly enhanced in both the LH and hippocampus. Under no condition in either site did 5-HIAA change significantly during or after manipulations. The increase in L H serotonin reported here is consistent with the findings of several experiments which suggest that 5-HT exerts an inhibitory control over neurons in the LH which initiate feeding behavior. Intravenous administration of fenfluramine has been shown to reduce LH unit activity in both cats 2~ and monkeys 5, while the serotonergic post-synaptic receptor antagonist cyproheptadine has been observed to not only increase food intake and body weight in cats but also to selectively facilitate LH electrical activity4. More recently, Kai et al. 16 reported that activity of most glucose-sensitive neurons in the LH was suppressed by iontophoretic application of 5-HT or natural release of 5-HT via stimulation of the midbrain raphe nuclei, and that the observed inhibition was attenuated by serotonergic post-synaptic receptor blockers. Glucose-sensitive neurons in the LH have also been shown to be inhibited REFERENCES 1 Best, P. and Orr, J., Effects of hippocampal lesions on passive avoidance and taste aversion conditioning, Physiol. Behav., 10 (1973) 193-196.

by aversive stimuli which suppress feeding, an effect which may be mediated by endogenous 5-HT release 35. In contrast to the possible role of lateral hypothalamic 5-HT as an inhibitory influence on feeding or its reinforcement, the role of hippocampal 5-HT has yet to be clearly identified. The most commonly cited functions of hippocampal 5-HT, however, indicate an involvement in memory and arousal. Several recent investigations support its function in memory retrieval, thus explaining to some degree the findings of previous studies that hippocampal lesions abolish passive avoidance learning and retrieval 1'26'27. However, the lack of a significant effect on CTA development and retrieval following ablation of this area suggests that hippocampal 5-HT has relatively little role in taste aversion behavior in comparison to the amygdala 2°'29 and the LH 31'34. Kalen et al. 17 have demonstrated that extracellular levels of hippocampal 5-HT are positively correlated with arousal and are significantly enhanced by stress induced by tail-pinch or handling. The results of the present study support the general conclusion that hippocampal 5-HT is related to arousal. Although CTA and control groups exhibited very different behavioral responses to saccharin infusion, all of these animals were obviously aroused during the manipulation, often displaying the mouthing movements characterizing either ingestive approach or aversion ~2 in response to saccharin, depending on the training protocol. In addition to the effects of conditioning on 5-HT in the LH and hippocampus, LiC1 produced a robust increase in extracellular 5-HT in both sites immediately following injection. Because this finding may potentially be misinterpreted as confounding the results obtained in the conditioning paradigm, it is important to emphasize that no LiCI was administered to conditioned groups on the day of testing. Thus, the increases in 5-HT observed during saccharin infusions were attributed to the association between this taste and LiCl-induced illness. The lack of significant changes in extracellular 5H I A A following any manipulation is in marked contrast to the dramatic increases in 5-HT produced by saccharin infusions or LiCI injections. Similar results have been reported previously x7-~9, serving as an indicator that the functional recovery of the microdialysis probe remained stable over the sampling period. The lack of correlation between 5-HT and 5 - H I A A is consistent with the conclusion that extracellular levels of 5 - H I A A are a poor index of 5-HT activity within the synapse ~7'~9. 2 Buresova, O. and Bures, J., Conditioned taste aversion induced in rats by intracerebral or systemic administration of monoamine oxidase inhibitors, Psychopharmacology, 91 (1987) 209-212. 3 Carboni, E. and DiChiara, G., Serotonin release estimated by transcortical dialysis in freely-moving rats, Neuroscience, 32

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