Conditioned taste aversion in rats with excitotoxic brain lesions

ELSEVIER

NEUROSCIENCE RESEARCH Neuroscience Research 22 (1995) 31-49

Conditioned taste aversion in rats with excitotoxic brain lesions Takashi Yamamoto *a, Yoshiyuki Fujimoto b, Tsuyoshi Shimura a, Nobuyuki Sakai a aDepartment of Behavioral Physiology. Faculty of Human Sciences, Osaka University, 1-2 Yamadaoka, Suita. Osaka 565. Japan bDepartment of Oral and Maxillofacial Surgery, Faculty of Dentistry, Osaka University, 1-2 Yamadaoka, Suita, Osaka 565, Japan

Received 10 November 1994; accepted 26 December 1994

Abstract

Conditioned taste aversion (CTA) is well known to be a robust and long-lasting learning after a single conditioned stimulus (CS) (taste) - - unconditioned stimulus (US) (malaise) pairing. The neural mechanisms of this taste aversion learning still remain to be resolved. To elucidate the basic brain mechanisms of the taste aversion learning, we examined the effects of lesions of various sites of the rat brain on the acquisition and retention of CTAs. Confined brain lesions were made by injections of a small amount of excitotoxic drug, ibotenic acid. CTAs were established to saccharin (CS) by pairing its ingestion with an i.p. injection of LiCI (US). Rats lacking the parabrachial nucleus (PBN) almost completely failed to acquire CTAs. The second most effective lesion was in the medial thalamus including the parvocellular part of the ventral posteromedial nucleus of the thalamus (VPMpc) and the midline part, followed by the damage of the lateral nuclear group of the amygdala including the basolateral amygdaloid nucleus. Lesions of the gustatory cortex (GC) and hippocampus induced moderate effects, but lesions in the other subnuclei of the amygdala, such as the medial and central amygdaloid nuclei, entorhinal cortex, lateral hypothalamic area, and ventromedial hypothalamic nucleus induced slight or no effects. On the other hand, paired lesions among the amygdala, medial thalamus and GC caused severe impairment of CTAs; in particular, lesions of amygdala and VPMpc completely disrupted acquisition of CTAs. These results suggest that the PBN, medial thalamus and the lateral nuclear group of the amygdala play an essential role in the formation of taste aversion learning. Keywords: Conditioned taste aversion; Learning; Brain; Ibotenic acid; Saccharin; LiCI

1. Introduction

When ingestion of a substance is followed by malaise manifested by gastrointestinal distress and nausea, an association between the taste of the ingested substance and internal consequences of its ingestion is quickly established, maintained in a long-term manner, so that animals reject ingestion of the substance at subsequent exposures. This feeding-related learning is referred to as a conditioned taste aversion (CTA) or taste aversion learning, and is known to have the following characteristics which are not found in classical conditioning (Bures et al., 1988; Bernstein, 1991): (1) Strong CTAs to novel taste stimuli can be established rapidly in a single learning procedure, i.e., after a single pairing of condiAbbreviations: see Appendix. * Corresponding author. Tel.: +81 6 879 8047; Fax: +81 6 879 8050.

tioned stimulus (CS) and unconditioned stimulus (US), which is preceded by the CS; (2) Successful CTAs can develop to the CS even after delays of as long as several hours between exposure to the CS and delivery of the US; (3) The association between the CS and the US can proceed under deep pentobarbital anesthesia. Since the pioneering scientific manipulation of this learning by Garcia and his colleagues (Garcia et al., 1955, 1966), CTAs have been studied and documented very well in terms of the unique nature of this learning (Ashe and Nachman, 1980; Bures et al., 1988; Bures and Buresova, 1990). However, brain mechanisms of this kind of learning, especially neural substrates for acquisition and retention of CTAs, are still unclear. One basic approach to elucidate the neural substrate of taste aversion learning is the behavioral lesion experiment. A number of investigators have examined the effects of confined lesions of various parts of the brain on

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T. Yamamoto et al. / Neuroscience Research 22 (1995) 31-49

acquisition and retention of CTAs. However, mainly due to differences in the experimental procedures, results are not always consistent among researchers. For example, some investigators mention that the gustatory cortex (GC) (Bermudez-Rattoni and McGaugh, 1991), thalamic taste area (parvocellular part of the ventral posteromedial nucleus of the thalamus; VPMpc) (Loullis et al., 1978), and the amygdala (Nachman and Ashe, 1974) are important in formation of CTAs, but others have opposite opinions with respect to the importance of these structures; e.g., lesions of the GC only attenuate, but not disrupt, CTA formation (Braun et al., 1972), no disruption of CTAs occurs after lesions of the VPMpc (Flynn et al., 1991b), and amygdaloid neurons have nothing to do with the establishment of CTAs (Dunn and Everitt, 1988). In the present article, we employed one and the same experimental procedure on rats with combined as well as single lesions of a number of brain sites, and the effects of lesions on CTA formation were compared among groups of rats with different brain lesions. The possible neural substrate for this taste aversion learning was discussed on the basis of present experimental results together with previous findings from other laboratories. 2. Methods

2.1. Subjects Adult male Wistar rats, weighing 250-300 g at the beginning of the experiment, were housed in individual wire mesh cages in a temperature (23°C) and humidity (60%)-controlled room on a 12:12 light/dark cycle. They had free access to food (dry pellets, MF, Oriental Yeast, Osaka) and tap water except when deprived for training and testing as described below.

2.2. Training The rats were deprived of water for 20 h and trained to drink distilled water for 40 rain in a test box of 21 × 21 x 30 cm to which a single drinking tube was attached. Water was presented to the rats for the rest of the drinking time in the home cages. No food was presented in the test box.

2.3. Surgery The rats were randomly grouped into 19 groups which consisted of an unconditioned control group, a conditioned control group, and 17 experimental groups with lesions of different brain sites as noted below. After one week training session, the rats were held in a Narishige stereotaxic instrument (Type SR-6N) so that the bregma and lambda were on the horizontal line while they were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and additional urethane (700 mg/kg, i.p.) when necessary. After a scalp incision, holes were drilled into each rat's skull. Only rats in the experimental

groups received microinjections of ibotenic acid (Sigma Chemical, St Louis, MO, I% dissolved in 0.1 M phosphate buffer). The injections were made with a 1 /xl Hamilton syringe mounted on the stereotaxic instrument. The standard lesion coordinates and the standard volumes of ibotenic acid injected were -12.5 mm posterior to the bregma, 1.8 mm lateral from the midsagittal sinus, 5.6 mm deep from the dural surface, and 0.35 t~l of ibotenic acid for the parabrachial nucleus (PBN), -3.7 mm, 1.2 mm, 6.4 mm and 0.2 t~l for the VPMpc, -3.7 mm, 0.6 mm, 6.0 mm and 0.3/zl for the midline and intralaminar complex (MITC), 1.8 mm, 5.0 mm, 4.5 mm and 0.4 t~l for the GC, -5.0 mm, 5.0 mm, 7.0 mm and 0.4/~1 for the entorhinal cortex (EC), -4.0 mm, 3.0 mm, 3.0 mm and 0.3 #1 for the dorsal part of the hippocampus (dorsal hippo), -5.3 mm, 5.2 mm, 6.0 mm and 0.6/~1 for the ventral part of the hippocampus (ventral hippo), -3.6 mm, 1.5 mm, 8.5 mm and 0.35 #1 for the lateral hypothalamic area (LH), -2.8 mm, 0.5 mm, 8.6 mm and 0.3 ttl for the ventromedial hypothalamic nucleus (VMH), -2.6 mm, 3.2 mm, 8.5 mm and 0.3/~1 for the medial amygdaloid nucleus (MeA), -2.6 mm, 4.0 mm, 7.5 mm and 0.3 ~1 for the central amygdaloid nucleus (CeA), and -2.8 mm, 5.0 mm, 7.7 mm and 0.4/xl for the basolateral amygdaloid nucleus (BLA). Since the PBN is ventral to the transverse sinuses, the injection needle was oriented 20 degrees off vertical in the antero-posterior plane with the tip anterior. The needle was left in place for 3 min before and after an injection of 0.1 /~1 per every 2 min or at a speed of 0.05 #l/min with a microinfusion system (Type XF-320J, Nihonkohden, Tokyo).

2.4. Behavioral analysis Following at least a week of postoperative recovery period in which all animals were offered water and food ad lib, the rats again underwent a water deprivation regimen, which permitted 40 min access to water in the test box. During this postoperative recovery period, each rat was examined for body weight and the amount of water drinking for the first 20 min. After 4 days of measurement of postoperative preconditioning water intake, the rats received an i.p. injection of 0.15 M LiC1 (2% of body weight) as an US soon after 20 min of drinking 0.2% (0.01 M, MW = 205.18) sodium saccharin (CS). The animals were then put on the same water-deprivation regime, and the volumes of intake of the CS for 20 min and of distilled water for the following 20 min were recorded on the successive 4 days. The fluid bottles were weighed before and after testing to measure intake volume. To attenuate the possible odour effects of the CS (Hankins et al., 1976), we familiarized the rats with the odour of 0.2% saccharin by putting a glass tube containing the same solution within each cage throughout the experiment.

T. Yamamoto et al./Neuroscience Research 22 (1995) 31-49

33

Besides control group rats, a few rats from every shipment were served for the same control treatments to check similar consumption and conditionability to the CS as in control rats.

and the following 4 test days, we calculated the following four kinds of indices as the parameters indicating some behavioral aspects concerning the taste aversion learning:

2.5. Two-bottle preference test After the completion of the CTA test, some rats were examined for their taste sensitivity to hedonically aversive tastes with the conventional brief exposure twobottle method. The taste stimuli used were 0.01 M HCI and 0.0001 M quinine hydrochloride made up with distilled water. The animals were deprived of water for 20 h, and put in the test box with two drinking bottles, one for distilled water and one for either one of the taste stimuli. They were first allowed free access to the two liquids at 10:00 am for 20 min, and second at 18:00 for another 20 min, after which water only was presented for the following 200 min. The positions of the water and solution bottles were exchanged at the second exposure. The fluid bottles were weighed before and after testing to measure intake volume.

Neophobia index ( N I ) = (1 -(saccharin intake on the conditioning day/preconditioning mean water intake)) x 100 Acquisition index (AI) = (1 - (saccharin intake on the 1st test day/preconditioning mean water intake)) x 100 Retention index (RI) = (1 - (total saccharin intakes on the 2nd, 3rd and 4th days/3 times preconditioning mean water intake)) × 100 Conditioned taste aversion index (CTAI) = (1 - (total saccharin intakes on the 1st, 2nd, 3rd and 4th days/4 times preconditioning mean water intake)) × 100

2.6. Histology At the completion of the testing, the rats were perfused intracardiaUy with physiological saline followed by 10% Formalin under deep anesthesia with an overdose (80 mg/kg i.p.) of sodium pentobarbital. The brains were removed, stored in the same fixative for several hours, and in 30% sucrose for at least 1 day. The brains were then blocked, and serial coronal sections were cut at 50 t~m with a freezing microtome. Sections were mounted and stained with cresyl violet. Drawings were made for representative sections with the aid of a camera lucida. Reconstruction of the lesions of the amygdala were made on the standard drawings derived from the Paxinos and Watson (1986) atlas. The lesions of the amygdala were measured following the method noted below. In reference to the drawings of Paxinos and Watson (1986) atlas, sections at the levels 1.8, 2.3, 2.8, 3.3, 3.8 and 4.3 mm posterior to the bregma were picked up. The area of each amygdaloid nucleus and the damaged area within each nucleus were measured with the aid of the microscopic image analyzer (XL-10, Olympus, Tokyo). The area of each amygdaloid nucleus and the damaged area within each nucleus on both sides in these 6 sections were summed separately, then the ratios between the two areas were calculated in each amygdala-lesioned rat. When the areas of amygdaloid nuclei could not be measured accurately because of large lesions, the size of each nucleus was measured in 8 NaCl-control rats and the averaged size was used as the standard area of each nucleus. 2. 7. Data analysis On the basis of volumes of preconditioning water intake and of saccharin intake on the conditioning day

For the two bottle preference test, the preference score was calculated: Preference score = 100 × (intake of taste solution/total intake of taste solution and distilled water) Data were analyzed using the two-tailed Student's ttest or two-tailed paired t-test with the level of statistical significance set at P < 0.05. 3. Results We intended to provide each experimental group with 8 rats, but we could not supply some groups with the intended number of animals because of the failure of successful lesions. Since we used many rats for amygdaloid lesioned groups, 3 additional groups, which had not been intended before the start of the experiment, were classified on the basis of histological examination and behavioral results. Fig. 1 shows photomicrographs of representative ibotenic acid lesions of the PBN (Fig. I A), GC (Fig. 1B), VPMpc and MITC (Fig. 1C), ventral hippocampus (Fig. 1D), and dorsal hippocampus (Fig. I E and F). The behavioral data in each group were obtained from the rats whose lesions were verified to be sufficient from histological examination of lesion extents. Fig. 2A shows the mean volume of 0.2% saccharin (CS) ingested on conditioning day and subsequent 4 test days in sham-operated unconditioned rats and in shamoperated conditioned rats. The former rats are designated as NaCl-control rats since they received an i.p. injection of 0.15 M NaCI instead of 0.15 M LiCI on the conditioning day. On the other hand, the latter rats are designated as LiCl-control rats. The volume of saccharin solution ingested on the conditioning day was highly significantly smaller (P < 0.001) than the postoperative preconditioning water intake level for both control groups. The intake of the saccharin solution increased gradually over the water intake level in NaCl-control

34

T. Yamamoto et aL / Neuroscience Research 22 (1995) 31-49

Fig. 1. Photomicrographs showing ibotenic acid lesion sites in coronal sections with cresyl violet stain. Panel A shows the extensive loss of cells in the parabrachial nucleus on the left (rat 300). Neurons in the trigeminal mesencephalic nucleus (MeV) are spared. Panel B shows the extent of lesions in the gustatory cortex on both sides (rat 654). The lesions are confined to the insular cortices. Panel C shows lesions in the medial part of the thalamus including the thalamic taste area (rat 701). The lesioned area is densely stained. Panel D shows lesions in the ventral hippocampus on both sides (rat 713). Most of the lesioned structures were lost during histological processing. Panel E shows lesions in the dorsal hippocampus on both sides (rat 712). Note the extensive loss of hippocampal cells compared with the normal appearance of the uninjected hippocampus (Panel C). Panel F is a magnified photomicrograph of the left side of the dorsal hippocampus shown in Panel E. Note the complete loss of hippocampal cells. The scale is 0.1 mm for A, and 1 mm for B, C, D, E, and F. Arrowheads indicate lesion sites.

rats, while L i C l - c o n t r o l rats showed decreased saccharin intake d u r i n g the subsequent 4 test days, a l t h o u g h the volume o f intake increased g r a d u a l l y f r o m the first to fourth days. The m e a n N I , A I , R I , a n d C T A I were 44, - 1 9 , - 4 3 , a n d - 3 7 , respectively, for 8 N a C l - c o n t r o l rats, a n d 54, 98, 81, a n d 86, respectively, for 8 L i C l - c o n t r o l rats.

3.1. P o n t i n e lesions

Fig. 2B shows the m e a n volume o f saccharin solution ingested for a 20 min p r e s e n t a t i o n p e r i o d on the conditioning d a y a n d the subsequent 4 test days in 4 rats with P B N lesions (PBN group). As shown by the p h o t o m i c r o g r a p h (Fig. 1A), ibotenic acid lesions invaded almost all the subnuclei o f the P B N r o s t r o c a u d a l l y and

T. Yamamoto et aL / Neuroscience Research 22 (1995) 31-49

35

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Fig. 2. Acquisition and retention of conditioned taste aversion to 0.2% sodium saccharin in control and experimental groups. The groups consist of NaCl-control (n = 8) and LiCI-control (n = 8) rats, and rats with lesions of parabrachial nucleus (PBN, n = 4), gustatory cortex (GC, n = 7), parvocellular part of the ventral posteromedial thalamic nucleus (VPMpc, n = 6), midline and intralaminar thalamic complex (MITC, n = 6), both VPMpc and MITC (VPMpc + MITC, n = 6), dorsal hippocampus (dorsal hippo, n = 6), ventral hippocampus (ventral hippo, n = 6), whole hippocampus (whole hippo, n = 6), entorhinal cortex (EC, n = 6), lateral hypothalamic area (LH, n = 6), and ventromedial hypothalamic nucleus (VMH, n = 4). Each column shows the mean -4- S.E.M. intake of 0.2% saccharin solution for 20 min on conditioning day (C, indicated by striped column) and subsequent 4 days (indicated by open column for NaCl-control rats and by solid column for LiCl-control rats). The open and solid squares with vertical lines in graph A, and the horizontal lines in other graphs show the mean :t: S.E.M. of postoperative preconditioning water intake. The shaded column indicates that the mean volume of intake is significantly smaller (P < 0.05) than that of NaCI-control rats and larger (P < 0.05) than that of LiCl-control rats on the comparative days. The open column indicates that the intake is significantly larger (P < 0.05) than that of LiCl-control rats, but is not different (P > 0.05) from that of NaCl-control rats. The solid column indicates that the intake is significantly smaller (P < 0.05) than that of NaCl-control rats, but is not significantly different (P > 0.05) from that of LiCl-control rats. The asterisk indicates that saccharin intake on the conditioning day is significantly smaller (P < 0.01) than the water intake level.

36

T. Yamamoto et al./ Neurosc&nce Research 22 (1995) 31-49

mediolaterally to which taste and general visceral inputs project as shown by our recent c-fos immunohistochemical studies (Yamamoto et al., 1992, 1993, 1994). The largest lesions invaded the supratrigeminal region and dorsal parts of the motor and principal sensory trigeminal nuclei, and the smallest lesions left one side of the medial or lateral parts of the PBN spared. On the conditioning day, these PBN-lesioned rats took the saccharin CS 1.3 times more than the preconditioning water intake level. On the first test day, or the acquisition test day, the rats showed a mean intake of 6.9 g of 0.2% saccharin solution, which was about 75% of the preconditioning water intake level. On the second to fourth retention test days, the volumes of saccharin intake were over the water intake level and were similar to those in NaCl-control rats. The mean NI, AI, RI, and CTAI were -25, 25, -33, and -18, respectively. 3.2. Thalamic lesions

Fig. 2D shows the results obtained from 6 rats with VPMpc lesions (VPMpc group). The largest lesions included a part of the midline thalamic nuclei and invaded slightly into the VPM and posterior thalamic nucleus lateral to VPMpc, and the smallest lesions were confined to the VPMpc with modest invasion to the surrounding structures. On the conditioning day, these rats took the saccharin CS slightly over (P > 0.05) the preconditioning water intake level. On the first test day, intake of saccharin was suppressed, but the volume ingested was significantly larger (P < 0.01) than that shown by LiC1control rats. On the second to fourth retention test days, the volume of saccharin intake increased steeply, and the saccharin intake on the fourth day was not different (P > 0.05) from that of NaCl-control rats. The mean NI, AI, RI, and CTAI were -20, 91, 12, and 32, respectively. Fig. 2E shows the results from 6 rats with MITC lesions (MITC group). All the lesions in this group invaded the MITC including whole or parts of the central medial, parafascicular, paraventricular, mediodorsal, and intermediodorsal, posteromedian, rhomboid, and reuniens nuclei. On the conditioning day, these rats took the saccharin CS as much as the preconditioning water intake level. From the first to fourth test days, the volume of saccharin intake was smaller than the preconditioning water intake level, but significantly larger (P < 0.05) than that of LiCI control rats. The mean NI, AI, RI, and CTAI were 2, 94, 61 and 70, respectively. Fig. 2F shows the results from 6 rats with combined lesions of VPMpc and MITC (VPMpc + MITC group). As shown by the photomicrograph (Fig. 1C), the lesions included both sides of VPMpc and MITC, and invaded more or less the medial part of the VPM as well. On the conditioning day, these rats took a similar amount of the saccharin CS to the preconditioning water intake level. On the first test day, the volume of saccharin in-

take was about 1/3 of the water intake level, but it was significantly larger (P < 0.01) than that of VPMpclesioned rats or MITC-lesioned rats as well as LiC1control rats. During the retention sessions, the volume of saccharin intake increased steeply, and the saccharin intake on the third and fourth days was within a level similar to that of NaCl-control rats. The mean NI, AI, RI, and CTAI were 7, 65, -6 and 12, respectively. 3.3. Cortical lesions

Fig. 2C shows the results obtained from 7 rats with GC lesions (GC group). As shown by the photomicrograph (Fig. 1B), the lesions included the insular cortex, and invaded dorsally a part of the somatosensory cortex and ventrally a part of the piriform cortex. On the conditioning day, these rats took a similar amount of the saccharin CS to the preconditioning water intake level. On the first test day, the rats showed acquisition of CTAs to saccharin, but the volumes of intake on the second to fourth days were significantly larger (P < 0.05) than those of LiCl-control rats. The mean NI, AI, RI, and CTAI were -1, 97, 46, and 59, respectively. Fig. 2J shows the results from 6 rats with EC lesions (EC group). On the conditioning day, these rats took a similar amount of the saccharin CS to the preconditioning water intake level, but its amount was significantly larger (P < 0.05) than that in NaCl-control rats. These rats acquired and retained CTAs well to the saccharin CS: the volume of saccharin intake was not significantly different (P > 0.1) from that of LiCl-control rats on the corresponding days. The mean NI, AI, RI, and CTAI were 13, 98, 68, and 75, respectively. 3.4. Hippocampal lesions

Fig. 2I shows the results from 6 rats with hippocampal lesions (whole hippo group). As shown by the photomicrographs (Fig. 1D, E and F), ibotenic acid injections damaged almost the whole hippocampus. On the conditioning day, these rats took the saccharin CS as much as the preconditioning water intake level. On the first test day, the rats showed strong aversions to saccharin, similar to LiCl-control rats. However, during the second to fourth retention trials, the volume of saccharin ingested increased steeply over the level of LiCI control rats (P < 0.05). The mean NI, AI, RI, and CTAI were 4, 95, 48, and 60, respectively. On the other hand, if either the dorsal part (Fig. 2G) or ventral part (Fig. 2H) of the hippocampus were destroyed, no significant disruption (P > 0.1) of the acquisition or retention of CTAs were detected. As shown in the graphs, on the conditioning day, the rats with the partial hippocampal lesions took the saccharin CS below the preconditioning water intake level, although a significant difference (P < 0.01) was detected only for rats with ventral hippocampal lesions. On the first to fourth test days, the volume of saccharin ingested was

T. Yamamoto et al./Neuroscience Research 22 (1995) 31-49

not different (P > 0.05) from that in LiCl-control rats on the corresponding days. The mean NI, AI, RI, and CTAI of 6 rats with dorsal hippocampal lesions (dorsal hippo group) were 27, 74, 69, and 70, respectively. The mean NI, AI, RI, and CTAI of 6 rats with ventral hippocampal lesions (ventral hippo group) were 55, 98, 82, and 86, respectively. 3.5. Hypothalamic lesions

Fig. 2K shows the results from 6 rats with LH lesions (LH group). These rats generally showed decreased amounts of ingestion, i.e., the volumes of water intake before conditioning and the saccharin CS intake on the conditioning day were significantly smaller (P < 0.01) than those in NaCl-control or LiCl-control rats. These rats ingested significantly less (P < 0.001) of the saccharin CS than of the preconditioning water intake and showed the strong acquisition and retention of CTAs: the volumes of saccharin intake during the first to fourth days were smaller than those of LiCl-control rats, but the difference was not statistically significant (P > 0.05). The mean NI, AI, RI, and CTAI were 75, 98, 91, and 93, respectively.

37

Fig. 2L shows the results from 4 rats with VMH lesions (VMH group). These rats took a significantly smaller (P < 0.05) amount of the saccharin CS than the preconditioning water intake on the conditioning day, and acquired and retained strong CTAs to the CS. The volumes of preconditioning water intake and of saccharin on each test day for VMH-lesioned rats were quite similar to those for LiCl-control rats which were shown in Fig. 2A. The mean NI, AI, RI, and CTAI were 53, 98, 83 and 87, respectively. 3.6. Amygdaloid lesions

The amygdala is composed of several densely packed nuclei. Although we tried to localize ibotenic acid lesions into the individual nuclei, it was difficult to damage single nuclei separately and bilaterally symmetrically. The photomicrographs in Fig. 3 show successful cases in which the individual nuclei were separately destroyed by injections of ibotenic acid. Fig. 3A shows a case where the CeA was exclusively lesioned, i.e., as shown in the magnified picture (Fig. 3B), neurons in the CeA were replaced by glia ceils while neurons in the BLA were spared. On the other hand, Fig. 3C shows

Fig. 3. Photomicrographsshowingibotenicacid lesions in the amygdaloidcomplex. Panel A and its magnifiedpart (B) show a case (rat 319)where the central amygdaloidnucleus (CeA) is exclusivelydestroyed. Note that neurons in the CeA are replacedby gila cells. Panel C and its magnified part (D) show a case (rat 533) where the basolateral amygdaloidnucleus(BLA)and adjacent structures are destroyedleavingthe CeA intact. Note that neurons in the BLA are replaced by glia cells.

38

T. Yamamoto et al./ Neuroscience Research 22 (1995) 31-49

Table 1 Correlation matrix of lesion size among amygdaloid nuclei

MeA CcA BMA LaA BLA BLV

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-0.109 0.242 0.851"** 0.884***

-0.066 0.257 0.878*** 0.756*** 0.936'**

0.197 0.114 0.737*** 0.489*** 0.615*** 0.700***

The extent of lesions in each of the 6 amygdaloid nuclei was calculated for each amygdala-lesioned rat. Correlation coefficients of lesion sizes were then calculated among the amygdalaoid nuclei across 49 rats. *P < 0,05, ***P < 0.001.

a case where the BLA was exclusively destroyed, i.e., as shown in the magnified picture (Fig. 3D), neurons in the BLA were replaced by glia cells while neurons in the CeA were spared. When we examined the extent of lesions of each amygdaloid nucleus in 49 amygdala-lesioned rats, it was

A

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Fig. 4. Effects of lesions of medial amygdaloid nucleus (MeA) on taste aversion learning. A, acquisition and retention of conditioned taste aversion to 0.2% saccharin in 6 rats with MeA lesions. See the legend of Fig. 2 for explanation of the graph. B, mean ~- S.E.M lesion size of each amygdaloid nucleus. C, extent of the lesion is encircled for each rat. Shaded area represents the area overlapped by more than 3 lesions; solid area, the area overlapped by 6 lesions. The panel uses a drawing from Paxinos and Watson (1986).

T. Yamamoto et al. t Neuroscience Research 22 (1995) 31-49

the lateral amygdaloid nuclei consisting of the basomedial amygdaloid nucleus (BMA), lateral amygdaloid nucleus (LaA), BLA, and ventral basolateral amygdaloid nucleus (BLV). Although we aimed to inject ibotenic acid into the BLA, these data clearly indicate that it was essentially impossible to destroy the BLA selectively in the present experiment. Fig. 4A shows the behavioral data obtained from 6 rats which were judged to receive lesions relatively confined to the MeA (MeA group). These 6 rats received ibotenic acid injections in the medial part of the amygdala, and as indicated by solid areas, the overlapped area of the 6 lesions was restricted to the MeA (Fig. 4C). A quantitative representation of average lesion extents of 6 rats across 7 amygdaloid nuclei indicates that about 80% of MeA, 45% of CeA, and 40% of the BMA were destroyed, and that the LaA, BLA, BLV, and cortical amygdaloid nucleus (CoA) were destroyed to the extent less than 25% (Fig. 4B). On the conditioning day, the MeA-lesioned rats took a similar amount of the sac-

A

39

charin CS to the preconditioning water intake level. These rats acquired and maintained strong CTAs to the saccharin CS as LiCl-control rats did. The mean NI, AI, RI, and CTAI were 10, 99, 77, and 82, respectively. Fig. 5A shows the behavioral results from 8 rats which were judged to receive lesions relatively confined to the CeA (CeA group). These 8 rats received ibotenic acid injections restricted to the CeA as indicated by the overlapped area of 8 lesions (Fig. 5C). The quantitative expression of lesion extents among 7 amygdaloid nuclei indicates that about 75% of CeA was destroyed, while less than 25% of the other nuclei were destroyed (Fig. 5B). These rats acquired and maintained CTAs to saccharin similar to the MeA group. The mean NI, AI, RI, and CTAI were 22, 98, 75, and 80, respectively. Fig. 6A shows the behavioral data from 6 rats which were judged to receive lesions including the BLA (BLA group). Unfortunately, the lesions were not well restricted to the BLA and were variable in lesion size among the rats, as indicated by the encircled areas in

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Fig. 5. Effects of lesions of central amygdaloid nucleus (CeA) on taste aversion learning. A, acquisition and retention of conditioned taste aversion to 0.2% saccharin in 8 rats with CeA lesions. See the legend of Fig. 2 for explanation of the graph. B, mean ± S.E.M. lesion size of each amygdaloid nucleus. C, extent of the lesion is encircled for each rat. The shaded area represents the area overlapped by more than 4 lesions; solid area, the area overlapped by 8 lesions. The panel uses a drawing from Paxinos and Watson (1986).

40

T. Yamamoto et al. / Neuroscience Research 22 (1995) 31-49

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take level, but the difference was not significant (P > 0.05). The lesions impaired the acquisition and retention of CTAs to the saccharin CS. The volumes of intake on the first and second days were significantly larger (P < 0.01) than the corresponding values of LiCl-control rats.

Fig. 6C. More quantitatively, these rats received about 75% of lesions of BMA, LaA, BLV, as well as BLA, but less than 30% destruction was observed for MeA, CeA and CoA (Fig. 6B). On the conditioning day, these rats took the CS slightly above the preconditioning water in-

Table 2 Correlations between each behavioral parameter and lesion size of each amygdaloid nuclei

MeA CeA BMA LaA BLA BLV CoA

DW

NI

A1

R1

CTAI

0.182 0.294 0.466** 0.182 0.283 0.291 0.117

0,274 0.334 0.532** 0.282 0.396* 0.529** 0.461 *

0.022 0.134 0.553** 0.602*** 0.728*** 0.646*** 0.291

-0.123 -0.010 0.482** 0.664"** 0.730*** 0.668*** 0.426*

-0.107 0.009 0.502** 0.671 *** 0.747*** 0.681"** 0.418*

Correlation coefficients were calculated in 30 rats which belonged to MeA, CeA, BLA, Am small, and Am large-I groups. Rats in Am small group received small amygdaloid lesions and acquired strong CTAs, and rats in Am large-I group received large amygdaloid lesions and failed to acquire strong CTAs. *P < 0.05, **P < 0.01, ***P < 0.001.

T. Yamamoto et al./Neuroscience Research 22 (1995) 31-49

The volumes of saccharin intake on the third and fourth days were not different from the corresponding values of NaCl-control rats. The mean NI, AI, RI, and CTAI were 14, 70, 1, and 19, respectively. Another approach to evaluate the role of each amygdaloid nucleus in CTA formation is to examine the interrelations between the lesion extents of each nucleus and the behavioral parameters, i.e., postoperative preconditioning water intake (DW), NI, AI, RI, and

A

41

CTAI. The results are shown in Table 2, where the correlation coefficients show that the MeA and CeA have nothing to do with any of the behavioral parameters. The lesion size of each of the BMA, LaA, BLA and BLV is highly significantly correlated with the AI, RI and CTAI, indicating that these nuclei, especially the BLA, play an important role in CTA formation. Lesions of the BMA, BLA and BLV are also significantly correlated with NI, and lesions of only the BMA show a

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Fig. 7. Effects of lesions of amygdaloid complex on taste aversion learning. Rats were classified into 3 groups: Amygdala small (n = 6), amygdala large-I (n = 4) and amygdala large-2 (n = 7), on the basis of lesion size and lesion effects. A, C and E, acquisition and retention of conditioned taste aversion to 0.2% saccharin in each group. See the legend of Fig. 2 for explanation of the graphs. B, D and F, mean -4- S.E.M. lesion size of each amygdaloid nucleus for each group. Asterisk indicates that saccharin intake on the conditioning day is significantly smaller (P < 0.01) than the postoperative water intake level.

42

T. Yamamoto et al./Neuroscience Research 22 (1995) 31-49

into another 3 groups on the basis of lesion size and lesion effects. One group (Amygdala small group) consists of 6 rats which received only small lesions within the amygdala. As shown in Fig. 7B, about 25% of LaA and BLA were destroyed and less than 15% of lesions were found for the rest of the amygdaloid nuclei. These rats took a significantly smaller (P < 0.01) amount of the saccharin CS than the preconditioning water intake on the conditioning day, and acquired and retained CTAs

significantly high correlation with DW. Lesions of CoA are significantly correlated with NI, RI, and CTAI. Since lesions of CoA are highly correlated with those of the lateral group of amygdaloid nuclei as shown in Table 1, there is a possibility that the high correlations shown by the CoA simply reflect the disruptive effects of lesions of the lateral amygdaloid nuclei. Besides the above mentioned MeA-, CeA-, and BLAlesioned rats, we categorized amygdala-lesioned rats

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Fig. 8. Effects of combined brain lesions on taste aversion learning. Acquisition and retention of conditioned taste aversion to 0.2% saccharin in rats with combined brain lesions (A. C and E). A, the amygdala (Am) and parvocellular part of the ventral posteromedial thalamic nucleus (VPMpc) were damaged (Am + VPMpe, n = 6); C, the amygdala and gustatory cortex (GC) were damaged (Am + GC, n = 6); E, VPMpc and GC were damaged (VPMpe + GC, n = 7). See the legend of Fig. 2 for explanation of graphs A, C and E. The asterisk indicates that saccharin intake on the conditioning day is significantly larger (P < 0.01) than the postoperative water intake level. B and D, mean ± S.E.M. lesion size of each amygdaloid nucleus for Am + VPMpc group and Am + GC group.

43

T. Yamamoto et al./ Neuroscience Research 22 (1995,) 31-49

Table 3 Correlations between preference scores for HCI and quinine with behavioral parameters Preference score

DW NI AI RI CTAI

HC1

Quinine

0.115 -0.392** -0.398** -0.343* -0.415"*

0.225 -0.441"* -0.399** -0.359* -0.398**

as LiCl-control rats did (Fig. 7E). However, the amygdala large-2 group rats took the CS as much as the preconditioning water intake level on the conditioning day. The mean NI, AI, RI, and CTAI of 7 rats in this group were 3.9, 95.9, 62.3, and 70.7, respectively. 3. 7. Combined lesions

*P < 0.05, **P < 0.01.

to saccharin as strong as those acquired by the LiC1control rats (Fig. 7A). The mean NI, AI, RI, and CTAI were 60, 96, 58, and 67, respectively. Two other groups (amygdala large-I and amygdala large-2) received quite large lesions in the amygdala, but the effects were very different. As shown in Fig. 7D, the rats in the amygdala large-I group lost almost all parts of the CeA, BMA, BLA and BLV, about 75% of LaA, about 40% of MeA, and about 25% of CoA. These lesions disrupted CTAs to the saccharin CS as shown in Fig. 7C. The mean NI, AI, RI and CTAI of 4 rats in this group were -25.9, 58.5, -0.3, and - 14.4, respectively. Fig. 7F shows that the rats in the amygdala large-2 group also lost BMA, LaA, BLA and BLV almost completely, about 50% of CeA, about 40% of CoA, and about 20% of MeA. In spite of such large lesions, these rats acquired and maintained strong CTAs to saccharin

When the amygdala was largely destroyed in addition to VPMpc lesions, as shown in Fig. 8A, 6 rats in this Am + VPMpc group took a significantly larger (P < 0.01) amount of the saccharin CS than the preconditioning water intake level on the conditioning day. The acquisition and retention of CTAs to saccharin were severely disrupted. The mean NI, AI, RI and CTAI were -30, 4, -29, and -21, respectively. The extents of amygdaloid lesions in these rats are shown in Fig. 8B as ratios of lesion size for each nucleus, indicating that almost whole parts of the BMA, LaA, BLA, and BLV were lost, nearly half of the CeA and CoA was destroyed, and about 40% of MeA was damaged. When the amygdala was lesioned in addition to GC lesions, as shown in Fig. 8C, 6 rats in this amygdala + GC group took the saccharin CS slightly over the preconditioning water intake level. The acquisition and retention of CTAs to saccharin were disrupted, i.e., the volumes of saccharin intake on the first acquisition test day was significantly larger (P < 0.01) than that in LiCIcontrol rats, and the volumes of intake on the second to fourth retention tests were similar to those of the NaCIcontrol rats. The mean NI, AI, RI, and CTAI were -27, 51, -38, and -16, respectively. As shown in Fig. 8D, the

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Fig. 9. Comparison between preference score for 0.01 M HCI and 0.0001 M quinine hydrochloride and conditioned taste aversion index (CTAI) among 7 groups with lesions of lateral hypothalamic area (LH), ventromedial hypothalamic nucleus (VMH), small areas of amygdala (Am small), basolateral amygdaloid nucleus (BLA), parvocellular part of the ventral posteromedial nucleus and gustatory cortex (VPMpc + GC), amygdala and GC (Am + GC), amygdala and VPMpc (Am + VPMpc), and one conditioned control (LiCl-control) group.

44

T. Yamamoto et aL / Neuroscience Research 22 (1995) 31-49

extents of amygdaloid lesions in these rats were generally smaller than those of amygdala + VPMpc group rats. However, more than half the size of each of the CeA, BMA, LaA, BLA, and BLV, and 20%-30% of MeA and CoA were destroyed. When both the VPMpc and GC were damaged bilaterally, as shown in Fig. 8E, 7 rats in this VPMpc + GC group showed the mean saccharin intake slightly over the preconditioning water intake level. The acquisition and retention of CTAs to saccharin were impaired, i.e., the volumes of saccharin intake on the first and second days were significantly larger (P < 0.05 and P < 0.001, respectively) than those of LiCl-control rats, and those on the third and fourth days were similar to those of NaCl-control rats. The mean NI, AI, RI, and CTAI were - 13, 62, - 16, and 4, respectively.

3.8. Two-bottle preference test The two-bottle preference test was performed on 47 rats: 1 in the VPMpc + MITC group, MeA group, amygdala large-1 group, and in amygdala large-2 group, 2 in the whole hippo group and GC group, 3 in the LiC1control group, 4 in the VMH group, BLA group, and amygdala small group, 5 in the LH group, 6 in the amygdala + VPMpc group and amygdala + GC group, and 7 in the VPMpc + GC group. When we examined the relationships between the preference score and each of the behavioral parameters obtained from these 47 rats, as shown in Table 3, the preference scores for aversive HC1 and quinine tastes were negatively correlated at a significant level with the NI, AI, RI, and CTAI. This result indicates that CTA-impaired rats tend to show lowered taste sensitivity to aversive tastes. In fact, as shown in Fig. 9, the rats in LH, LiCl-control, VMH, and amygdala small groups, which showed the larger CTAIs, exhibited the smaller preference scores to both 0.01 M HCI and 0.0001 M quinine, whereas the rats in the VPMpc + GC, amygdala + GC, and amygdala + VPMpc groups, which showed the smaller CTAIs, exhibited the larger preference scores. The mean CTAI in these 8 groups was significantly negatively correlated with the preference scores for HC1 (r = - 0 . 7 9 2 , P < 0.05) and quinine (r = -0.739, P < 0.05). 4, Discussion 4.1. Behavioral parameters In the present study, we examined the effects of various brain lesions on taste aversion learning with one and the same experimental procedure and the same CTA paradigm to disclose the brain sites responsible for CTA formation. To compare ingestive behaviors and strength of CTAs formed among different groups of rats, we have employed 4 indices as behavioral parameters: NI, AI, RI, and CTAI, as well as DW.

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Fig. 10. Comparison of values of 5 behavioral parameters among 2 control and 20 experimental groups. The groups are arranged in the order of magnitude of CTAI from the right. Solid column indicates that the value is significantly different (P < 0.05) from that of the leftmost NaCl-control group. DW, postoperative preconditioning water intake; NI, neophobia index; AI, acquisition index; RI, retention index; CTAI, conditioned taste aversion index.

Fig. 10 compares the values of these 5 parameters among a total of 2 control and 20 experimental lesion groups. The NI was calculated based on the DW and the intake volume of saccharin which was exposed to the rat for the first time on the conditioning day. The smaller amount of intake or the large value of NI means stronger neophobic responses to the CS. The AI indicates the strength of CTA acquisition to the saccharin CS, which was calculated on the basis of the volume of saccharin solution consumed on the first test day. The RI indicates how strong the aversive learning is retained after acquisition of CTAs, which was calculated on the basis of the saccharin intakes on the second, third, and fourth test days. The CTAI indicates general intensity of formation of CTAs, which includes both acquisition and retention of CTAs. Graphical presentation of these indices in Fig. 10 suggests that the magnitude of each index is correlated well

T. Yaraamoto et al./Neuroscience Research 22 (1995) 31-49 Table 4 Correlation matrix among behavioral parameters

DW NI AI R1

NI

AI

RI

CTAI

-0.491"

-0.191 0.638**

-0.266 0.814"** 0.847***

-0.259 0.801"** 0.893*** 0.996***

Correlation coefficients were calculated with the mean values of each parameter obtained from the 20 experimental groups. *P < 0.05, **P < 0.01, ***P < 0.001

with each other. Precise interrelations among the 5 behavioral parameters are shown in terms of correlation coefficients in Table 4. The NI is significantly negatively correlated with the DW, suggesting that an enhanced motivation to drink tends to overcome the neophobic responses. The NI was highly correlated with AI, RI and CTAI, indicating that the stronger the neophobia, the stronger the CTA formation. The fact that the AI is very highly correlated with the RI indicates that weakly acquired CTAs are quickly extinguished, while firmly acquired CTAs are slowly extinguished. Besides this general tendency, we could arbitrarily categorize the groups arranged on the abscissa into three parts as segregated by the vertical dotted lines in Fig. 10: the left 4 groups which have the negative values of CTAIs, the middle 5 groups which show positive CTAIs but below 50, and the right 13 groups which have CTAIs over 50. The left 4 groups can be regarded as having no ability of acquiring CTAs. The inadequate disruption of CTA acquisition shown by the amygdala + GC group may be due to the inadequate amygdaloid lesions (see Fig. 8D). The middle groups can acquire taste aversion learning with the remaining intact brain structures, but show a quick extinction of the learning because the brain sites necessary to retain the learning have been destroyed. The right groups shov~ the strong acquisition and retention of CTAs, indicating that the brain lesions in these groups have minor or essentially no roles in taste aversion learning, although GC and whole hippo groups do have some deficiency in retention of CTAs.

4.2. Brain sites responsible for CTAs In accordance to the present result, several investigators have reported that bilateral reversible (Ivanova and Bures, 1990) or irreversible (DiLorenzo, 1988a; Scalera et al., 1992) lesions of the whole PBN severely impair the ability of rats to acquire CTAs. It has been well accepted recently that the initial integration of gustatory and visceral signals takes place in the PBN, and that the behavioral expression of the acquisition and retention of CTAs require participation of the forebrain structures including the GC and amygdala (e.g., Bures and Buresova, 1990; Reilly et al., 1993b). We

45

(Yamamoto, 1993; Sakai et al., 1994; Yamamoto et al., 1994) have proposed that lesions of the medial part of the PBN impair taste quality transmission and lesions of the lateral part of the PBN (PBNIat) impair the function of the hedonically negative site where taste-visceral integration occurs. Thus, it is not surprising that lesions of the PBN caused severe disruptive effects on the CTA formation. Earlier thalamic lesion studies (Loullis et al., 1978; Lasiter, 1985, Flynn et al., 1991b) in the rat employed different CTA paradigms, which makes it difficult to compare the results with each other. The present study and our recent study (Yamamoto, 1994) have shown that cytotoxic lesions of the VPMpc markedly attenuated both acquisition and retention of CTAs, whereas lesions of the MITC attenuated acquisition, but had no effects on retention of CTAs which had been acquired before operation (Yamamoto, 1994). Moreover, we have found that lesions of both VPMpc and MITC caused more severe disruption of CTAs than isolated lesions of either structures. Neuroanatomical findings (Groenewegen and Berendse, 1994) indicating that the MITC receives projections from PBNlat (Saper and Loewy, 1980; Groenewegen, 1988) and send axons to the BLA (Ottersen and Ben-Ari, 1979; Groenewegen, 1988) suggest that general visceral and hedonically negative information are sent via the MITC to the amygdaloid nuclei other than the CeA. Previous studies have shown that insular lesions attenuated acquisition (Braun et al., 1972, 1982; Buresova and Bures, 1974; Kiefer and Braun, 1977; Kiefer et al., 1982; Lasiter and Glanzman, 1982; Gallo et al., 1992; Escobar et al., 1993) and retention (Yamamoto et al., 1980, 1981; Braun et al., 1981; Gallo et al., 1992) of CTAs. The essential role of the cerebral cortex in acquisition of CTAs has been pointed out by Bures and his colleagues (Bures et al., 1988, 1991; Gallo and Bures, 1991) and Bermudez-Rattoni and his colleagues (LopezGarcia et al., 1990; Fernandez-Ruiz et al., 1991; Escobar et al., 1993). In contrast to these results, previous behavioral lesion experiments (Yamamoto et al., 1980, 1981) together with the present study showed that the GC is not indispensable for acquisition of CTAs, but that it is rather important for retention of CTAs. The findings that 0.2% saccharin has olfactory qualities in rats (Hankins et al., 1976) and the taste potentiated odor aversion has nothing to do with the GC lesions (Kiefer et al., 1982) suggest that our rats might have used olfactory cues to mediate saccharin aversions. This possibility may not be plausible, since we familiarized the rats with the odor of 0.2% saccharin; moreover, although not shown in the present study, we have obtained similar results to 0.1% saccharin CS which has been used more commonly by other workers. Anyway, many earlier data suggest that elimination of the GC impairs CTAs less

46

T. Yamamoto et al./Neuroscience Research 22 (1995) 31-49

completely than subeortical lesions. It may be relevant to say that the insular cortex is involved, but not essential in the establishment of taste aversion learning (Braun et al., 1982). The EC has been given an important role in mediating cortical sensory information to the hippocampal formation (Room and Groenewegen, 1986), and may function as an integrating system related to modulation of cognition and memory. Norgren and Grill (1976) suggested using a tract tracing method that corticofugal efferents from the GC project to the EC. The present study showed that EC lesions resulted in reduced neophobia to a novel CS (saccharin). However, another finding that EC lesions exhibited essentially no disruptive effects on CTAs suggests that corticoentorhinal hippocampal pathways are not involved in formation of CTAs. The present study showed that lesions of the whole hippocampus had no effect on acquisition, but attenuated the neophobic response to the saccharin CS, and showed a significant disruptive effect on the retention of CTAs. Separate lesions of the dorsal or ventral parts of the hippocampus had no effects on neophobic responses, acquisition or retention of CTAs. These resuits are consistent with those of other researchers (Murphy and Brown, 1974; Miller et al., 1975). Recently, ReiUy et al. (1993a) showed that ibotenic acid lesions of the hippocampus had essentially no effect on the acquisition of CTAs, whereas maze learning was significantly impaired by these lesions. These results suggest that hippocampal neurons do not play an important role in the acquisition of CTAs or association of CS with US, but are concerned with previous experiences such that the stimulus is novel or familiar, and safe or dangerous. According to the present study, lesions of the hypothalamic nuclei concerning feeding behaviors, i.e., the LH known as the feeding center and the VMH as the satiety center, had essentially no or rather stronger (see Fig. 2K for LH group) effects on the formation of CTAs. These results are consistent with those by Gold and Proulx (1972), Weisman et al. (1972), and Kramer et al. (1983), but are not consistent with those by Roth et al. (1973) who demonstrated that the LH-lesioned rats failed to learn to avoid a preferred flavor that signaled impending electric shock to the tongue, and that the LH rats retained a taste aversion acquired prior to lesioning, but 70% of them failed to learn a new aversion. Although a further study is necessary, these discrepancies might be attributed to procedural differences. To our knowledge, no one else has ever examined the effect of excitotoxic lesions of the hypothalamus on taste aversion learning. In the present study, we could partly confirm the previous finding (Rolls and Rolls, 1973; Nachman and Ashe, 1974; Simbayi et al., 1986) that lesions of the BLA

disrupt the formation of CTAs, because the degree of disruption of CTAs is most highly correlated with the lesion size of the BLA (see Table 2). However, since selective lesions of only the BLA apart from the neighboring LaA, BMA, and BLV were technically difficult (see Table 1 and Fig. 6), a possibility still remains that the LaA, BMA or BLV is also crucial for the establishment of CTAs. It is at least possible to conclude from the present study that the CeA or MeA alone are not important for CTA formation. Neuroanatomical studies in the rat have shown that gustatory information from the PBN (Norgren, 1976) and VPMpc (Turner and Herkenham, 1991) projects exclusively to the CeA, and from the insular cortex including GC (Veening, 1978; Ottersen, 1982; Saper, 1982; van der Kooy et al., 1984; Turner and Herkenham, 1991) to the CeA, LaA, BLA, and other amygdaloid nuclei. General visceral inputs also project to the CeA from the NTS (Ricardo and Koh, 1978) and PBN (Saper and Loewy, 1980; Norgren, 1984; Cechetto, 1987). Thus, the main projection site for brainstem gustatory and visceral inputs to the amygdala is the CeA. However, the present study together with other studies (Kemble et al., 1979; Galaverna et al., 1993; Yamamoto, 1994) have shown that lesions of the CeA have essentially no or only minor disruptive effects on acquisition and weak effects on retention of CTAs. It is possible that the gustatory and visceral information necessary for CTAs projects directly to other nuclei than the CeA via thalamus or insular cortex. In the present study, we found that some animals (amygdala large-2 group) with large amygdaloid lesions including the BLA could acquire and maintain CTAs just like normal rats did, whereas the similarly large lesions impaired the formation of CTAs in the amygdala large-I group. We cannot give a reasonable explanation for this discrepant finding because the subtle differences of lesion extents can hardly explain the robust behavioral differences between the amygdala large-1 and large-2 groups (see Fig. 7). Moreover, the amygdala large-2 group received larger amygdaloid lesions than the BLA group, which exhibited severe impairment of CTA formation (compare Fig. 6 with Fig. 7E and F). Other sensory cues or other brain sites might compensate for the deficits of CTA formation in the amygdala. In accordance with such a finding of ours, other researchers (Fitzgerald and Burton, 1983; Dunn and Everitt, 1988; Chambers, 1990; Bermudez-Rattoni and McGaugh, 1991) also showed that amygdaloid lesions had essentially no effects on CTA formation, and assumed that the amygdala was less important than GC for the formation of CTAs. They suggest that the impairment of CTAs after lesions of the BLA is due to the destruction of the corticofugal fibers passing to the brainstem, but not to the destruction of cell bodies in the

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BLA, because electrolytic lesions, but not cytotoxic ibotenic acid lesions, disrupted CTA formation. Although it is true that corticofugal fibers affect taste responsiveness of neurons in the brainstem nuclei (Kiyomitsu et al., 1988; Di Lorenzo, 1988b; 1990) and that they might carry information whether the CS is novel or familiar and whether it is experienced as safe or dangerous, we do not think that these descending influences are indispensable for CTA formation because of the present finding that GC lesions had almost nothing to do with the acquisition of taste aversion learning.

4.3. Neural substrates of CTAs An interesting finding in the present study is that brain lesions which attenuated CTA formation also tended to attenuate aversive responses to HC1 and quinine (see Fig. 9). It is noted that PBN lesions (Flynn et al., 1991a), VPMpc lesions (Ables and Benjamin, 1960; Flynn et al., 1991a), and GC lesions (Ables and Benjamin, 1960; Braun et al., 1982; Kiefer and Orr, 1992) elicited severe, moderate, and little effects, respectively, on taste responses to aversive tastes, and in accordance with this order, the CTA acquisition was impaired severely, moderately, and negligibly. It is probable that the brain sites responsible for CTA formation are also important in transmission and/or processing of gustatory information in terms of hedonical as well as qualitative aspects. The failure of CTA acquisition after PBN lesions could easily be understood if one considers that the PBN stands for a pivot for the ascending gustatory and general visceral routes. The finding that combined lesions of the GC and VPMpc severely disrupted CTA acquisition indicates that sensory inputs to the amygdala from the PBN are not sufficient or very weak to establish CTAs. As already reported by Gallo et al. (1992), combined lesions of the amygdala and GC completely disrupted CTA acquisition, indicating that CTAs are not acquired within the VPMpc, MITC, or PBN. Taking these facts together with a slight disrupting effect by GC lesions and a large effect by VPMpc + MITC lesions, it is relevant to say that the sensory information necessary for CTA acquisition is conveyed mainly from the VPMpc and MITC to the lateral nuclear group of the amygdala including the BLA, but not directly from the PBN. Similar findings are reported in the fear conditioning to auditory stimuli by LeDoux et al. (1990), who suggest the importance of thalamoamygdaloid projection by showing that removal of cortical sensory areas did not interfere with the fear conditioning and that the medial geniculate body received inputs from the inferior colliculus and sent axons to the LaA. The GC and hippocampus may be more important in retention of acquired gustatory memory in short-term and/or longterm manners than in acquisition of taste aversion learning.

Appendix Abbreviations AI Am BC BLA BLV BMA CeA CG CM CoA CPu CS CTA CTAI dorsal hippo DW EC FR GC LaA LH MeA MeV MITC NI PBN PF RI US ventral hippo VMH VPM VPMpc

acquisition index amygdala brachium conjunctivum basolateral amygdaloid nucleus ventral basolateral amygdaloid nucleus basomedial amygdaloid nucleus central amygdaloid nucleus central gray central medial thalamic nucleus cortical amygdaloid nucleus caudate putamen conditioned stimulus conditioned taste aversion conditioned taste aversion index dorsal hippocampus postoperative preconditioning water intake entorhinal cortex faseiculus retroflexus gustatory cortex lateral amygdaloid nucleus lateral hypothalamic area medial amygdaloid nucleus trigeminal mesencephalic nucleus midline and intralaminar thalamic complex neophobia index parabrachial nucleus parafaseicular thalamic nucleus retention index unconditioned stimulus ventral hippoeampus ventromedial hypothalamic nucleus ventral posteromedial thalarnic nucleus parvoceilular part of VPM

Acknowledgements This study was supported by grants from the Japanese Ministry of Education (04454465, 05267101), and the Salt Science Foundation.

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