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Role of the medial and lateral parabrachial nucleus in acquisition and retention of conditioned taste aversion in rats
Behavioural Brain Research 93 (1998) 63 – 70
Research report
Role of the medial and lateral parabrachial nucleus in acquisition and retention of conditioned taste aversion in rats Nobuyuki Sakai, Takashi Yamamoto * Department of Beha6ioral Physiology, Faculty of Human Sciences, Osaka Uni6ersity, 1 -2 Yamadaoka, Suita, Osaka, 565, Japan Received 17 March 1997; received in revised form 8 September 1997; accepted 8 September 1997
Abstract When ingestion of a taste stimulus is paired with internal malaise, the animal remembers the taste and rejects its ingestion thereafter. This learning is referred to as conditioned taste aversion (CTA). To establish CTA in adult male Wistar rats, 0.1% saccharin and an i.p. injection of 0.15 M LiCl were used as the conditioned and unconditioned stimuli, respectively. To elucidate the functional role of the medial part of the parabrachial nucleus (PBmed) which receives taste information and the lateral part (PBlat) which receives general visceral information, confined electrolytic lesions were made to either of these regions. Rats with bilateral lesions of the PBlat impaired the acquisition of CTA, but those lesions made after the acquisition of CTA had no effect on the retention of this learning. The bilateral lesions of the PBmed abolished the acquisition and retention of CTA. The PBlat-lesioned rats showed normal taste preference behavior, but PBmed-lesioned rats showed impaired sensibility to taste stimuli. These results suggest that both the PBlat and PBmed are essential for the acquisition of taste aversion learning, but the PBlat is not necessary for retrieval of CTA. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Conditioned taste aversion; Parabrachial nucleus; Electrolytic lesions; Taste preference; Rat
1. Introduction When the consumption of novel flavored food is followed by internal malaise, animals avoid ingesting the food on subsequent presentations [4,27,37]. It is well accepted that animals must learn to associate its taste with illness for this avoidance behavior. This type of association learning is referred to as conditioned taste aversion (CTA). Understanding of the neural mechanisms subserving the taste aversion learning has been a goal of a variety of animal experiments. In a typical experimental paradigm for CTA studies, saccharin is used as a conditioned stimulus (CS) and an intraperitoneal (i.p.) injection of lithium chloride (LiCl) is used as an unconditioned stimulus (US). In this * Corresponding author. Tel.: +81 6 8798047; fax: + 81 6 8798050; e-mail: [email protected] 0166-4328/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-4328(97)00133-2
simple model, neural information of the gustatory CS via the taste nerves is thought to be associated with that of the illness-inducing US via the visceral nerves or blood in the central nervous system. Unconditioned effects of LiCl are suggested to be mediated by the area postrema [22], and the area postrema is known to have large projections directly to the nucleus tractus solitarius (NTS) and the parabrachial nucleus (PBN) [28]. In general, both gustatory and visceral signals ascend side by side possibly by interacting with each other through the sensory relay stations and terminate in various brain areas [5,6,11,25]. Although a number of lesion studies have been performed to elucidate the neural substrates of CTA (Yamamoto et al. [37]), the role of the projection zone for each of the gustatory and visceral signals in CTA formation has not been clarified because almost all the previous lesions were large enough to invade the recipi-
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ent zones for the two sensory signals. The PBN is the second-order sensory relay station in the gustatory and general visceral pathways. It is generally accepted that gustatory inputs project to the medial part of the PBN (PBmed) and general visceral inputs project to the lateral part (PBlat) [12,19]. In previous lesion studies, researchers aimed to eliminate the function of the whole PBN including PBmed and PBlat, and demonstrated that the PBN was essential for the acquisition [2,16,21,26,31,38] and consolidation [17] of CTA. One could eliminate the function of either the gustatory zone or visceral zone of the PBN with the use of a confined lesion technique. In this way, Aguero et al. [1] showed that the rats with electrolytic lesions focusing to the external lateral subnucleus in the PBlat could not acquire CTA, and interpreted this impairment of learning as the disruption of transmission of visceral malaise information. Similarly, Nader et al. [18] showed that cytotoxic lesions of the PBlat blocked the acquisition of morphine-induced CTA to saccharin, and attributed this effect to the blockade of the aversive properties of morphine in the CTA paradigm. However, no reports have examined the effects of PBlat lesions made after the conditioning procedure on the retention of CTA. When the electrolytic lesion method was used to destroy the whole PBN in previous studies, the extents of the lesions were not always large enough to invade the entire PBN but were quite variable among the animals and depending on the researchers [7,9,21,31]. It is needed to correlate the lesion site in the PBN and the disruptive effects on CTA formation. The purpose of the present study was to examine the role of the PBlat, to which illness-inducing visceral information projects, and the PBmed, to which the gustatory CS information projects, in the acquisition and retention of CTA. Preliminary report on this work has appeared in abstract form [24].
2. Materials and methods
2.1. Subjects Adult male Wistar rats, weighing 250 – 300 g at the beginning of the experiment, were housed in individual home 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 were trained to drink distilled water for 20 min in the home
cages. Water was presented to the rats for the rest of the drinking time. They were allowed to free access to food throughout the experiments. Animal protocols were in accordance with the Guideline for the Care and Use of Laboratory Animals approved by National Institute of Health (1985).
2.3. Surgery Thirty rats were randomly divided into two classes of 15 each: one for the acquisition test and another for the retention test. Each of the classes consisted of three groups of five each: a sham control group which received only burr holes in the skull, an experimental group which consisted of rats with lesions of the PBmed (PBmedX group), and another experimental group which consisted of rats with lesions of the PBlat (PBlatX group). The animals 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.). After a scalp incision, two holes were drilled into the skull for the access to the bilateral PBN. An etched ‘elgiloy’ microelectrode [32] (50–80 mm tip diameter), insulated with glass except for the tip, was used as the DC reference electrode and positioned in the PBmed (10.2 mm caudal to bregma, 1.8 mm lateral to the midline) or PBlat (9.5 mm caudal to bregma, 2.5 mm lateral to the midline). Electrical activities of neurons were monitored during insertion of the electrode to facilitate locating the tip into the target area. DC cathodal current (0.1 mA for 30 s) was passed through the electrode against the indifferent electrode put on the skull to produce electrolytic lesions.
2.4. Acquisition test In this experiment, the rats received the PBN lesions before the conditioning procedure to examine lesion effects on the acquisition of CTA. After completion of the pre-experiment training to drink distilled water for 20 min in the home cages, the experimental rats received the PBN lesions. Following at least a week of postoperative recovery period in which all animals were offered water and food ad lib, the rats were again put on a water deprivation schedule, which permitted to drink distilled water for 20 min in home cages. During this period, each rat was examined for body weight and the amount of water drunk during the 20 min. After the amount of drinking was stable, each rat received an i.p. injection of 0.15 M LiCl (2% body weight) as an US soon after 20 min of free access to 0.1% (0.005 M) sodium saccharin instead of water as a CS. After 1 day of recovery from this conditioning procedure, the animals were then on the same water-de-
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privation 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 test days. The fluid bottles were weighed before and after testing to measure intake volume.
2.5. Retention test In this experiment, the rats received the PBN lesions after the conditioning procedure to examine lesion effects on the retention of acquired taste aversion. After the amount of water drinking was stable in the pre-experiment training session, the rats received an i.p. injection of 0.15 M LiCl (2% body weight) soon after 20 min of free access to 0.1% sodium saccharin instead of water. After this conditioning procedure was repeated on subsequent 2 days (one procedure per day), the experimental rats received the PBN lesions. Following at least a week of postoperative recovery period in which all animals were offered water and food ad lib, the rats were again put on the same water deprivation schedule which permitted them to drink distilled water for 20 min in home cages. During this postoperative period, each rat was examined for body weight and the amount of water drunk during the 20 min. After the postoperative recovery, the animals were put on the test session, 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.
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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 mm throughout the PBN with a freezing microtome. Every two sections were mounted and stained with cresyl violet. The extent of the lesions was examined under a light microscope. Some sections were photographed and drawings were also made for representative sections with the aid of a camera lucida.
2.8. Data analysis On the basis of volumes of preconditioning water intake and of saccharin intake on the following 4 test days, we calculated a CTA index as an indicator of the strength of CTA formation. The larger this index, the stronger the acquisition of CTA: CTA index= 1− (total saccharin intake on the 1st, 2nd, 3rd and 4th days/4 times mean water intake) For the two-bottle preference test, the preference score was calculated. The larger this score, the more the solution was preferred: Preference score= (intake of taste solution/total intake of taste solution and distilled water)× 100 Data were analyzed using the ANOVA with post hoc comparison (Fisher’s least significant difference; LSD) test.
2.6. Two-bottle preference test
3. Results
After the completion of the CTA experiment, the rats were examined for their taste sensitivity to hedonically positive and negative taste stimuli with the conventional brief exposure two-bottle method. The taste stimuli used were 0.002 M quinine hydrochloride, 0.01 M HCl, 0.1 M NaCl and 0.5 M sucrose made up with distilled water. The animals were deprived of water for 20 h, and were presented with two drinking bottles, one for distilled water and the other for either one of the taste stimuli. They were allowed free access to the two bottles for 20 min. The positions of the water and solution bottles were exchanged every 5 min. The fluid bottles were weighed before and after testing to measure intake volume.
One rat in each of the three groups in the acquisition test and one rat of the PBmedX group in the retention test were discarded from the data analysis because three of them received large lesions invading both PBmed and PBlat and the rest did not recover well from the operation judging from the water intake and body weight, and the other did not show stable water intake in their daily sessions. Fig. 1 shows the extent of PBmed and PBlat lesions in subjects used for the acquisition test and retention test. The PBmed lesions invaded the medial PBN with the caudal half predominance including the dorsal lateral, central lateral and central medial subnuclei on both sides. The PBlat invaded the lateral PBN with the rostral half predominance including the external lateral and external medial subnuclei on both sides. Essentially the similar lesions were placed in rats for the acquisition test and for the retention test. The overall mean lesion size was 0.64 mm ranging from 0.25 to 1.0 mm mediolaterally, and 0.78 mm ranging from 0.5 to 1.2 mm rostrocaudally.
2.7. Histology At the completion of all the experiment, the rats were perfused intracardially with physiological saline followed by 10% Formalin under deep anesthesia with an overdose (80 mg/kg i.p.) of sodium pentobarbital. The
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3.1. Acquisition test The sham and lesion operations were made before the conditioning procedure in this experiment. The preconditioning (or postoperative) mean water intake 9 SE was 12.49 0.7, 5.3091.5 and 16.99 1.1 for sham control, PBmedX and PBlatX groups, respectively. The PBlatX rats ingested more water than control rats, whereas the PBmedX rats drank less than control rats (P B0.01). To facilitate a better comparison of the results, the volume of fluid consumption was normalized by taking the preconditioning mean water intake as standard (100%). Fig. 2 shows the mean relative amount of 0.1% saccharin (CS) drunk for 20 min on the conditioning day, and the amount of saccharin and water drunk for the succeeding 20 min on the subsequent 4 test days in the 3 groups. As shown in Fig. 2A, control rats showed decreased saccharin intake during the 4 test days (20 min/day), although the volume of intake increased gradually from the first to the fourth test day. Accordingly, the volume
Fig. 2. Saccharin and water intakes after conditioned taste aversions to 0.1% sodium saccharin in control and experimental groups. The relative values are shown when the preconditioning water intake is taken as 100. The groups consist of sham control rats (n = 5) (A), rats with lesions of the medial parabrachial nucleus (PBmedX, n= 4) (B), and the rats with lesions of the lateral parabrachial nucleus (PBlatX, n =4) (C). Sham (S) and lesion (L) operations were made before the conditioning procedure (Cond). The solid and open column with vertical line shows the mean intake 9SE for saccharin and water, respectively. *PB0.05, **PB0.01, ***PB0.001.
Fig. 1. Extent of the lesions in the parabrachial nucleus (PBN). Lesions of the medial PBN for eight rats used in acquisition and retention tests are superimposed on camera-lucida drawings of the representative sections taken from the rostral (AP = − 9.30 mm, Paxinos and Watson Atlas [20]) and caudal (AP = −9.80 mm) levels. Lesions of the lateral PBN for eight rats are similarly shown. Shaded area, lesion size for each rat; solid area, the area overlapped by more than four lesions; BC, brachium conjunctivum; MeV, mesencephalic nucleus of the trigeminal nerve. The bar indicates 1 mm.
of companion water intake for 20 min following the 20-min saccharin presentation decreased gradually. In both PBmedX rats (Fig. 2B) and PBlatX rats (Fig. 2C), the intake of saccharin on the first test day tended to be suppressed, but on the following 3 days it was similar to the preconditioning water intake level. For a quantitative comparison of lesion effects on CTA formation among the three groups, we calculated CTA indices for the three groups, and analyzed them with one-way ANOVA. The main effect of the group was significant (F(2,9)= 5.45, PB 0.05), i.e. the mean CTA index 9 SE was 0.63 90.09 for control rats, and was statistically significantly (PB 0.05, post hoc LSD test) larger than that (0.089 0.21) for PBmedX rats and that (0.15 9 0.08) for PBlatX rats. These results suggest that the lesions in the PBN severely damaged the acquisition of CTA.
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For a more precise comparison, the relative intake of saccharin on each day among the three groups was statistically analyzed using two-way ANOVA with repeated measures. The analysis showed reliable main effects of the group (F(2,9) = 9.04, P B 0.01) and the trial (F(4,36)= 11.00, P B0.01), but the group×trial was not significant (F(8,36) = 0.99, P \ 0.05). Post hoc LSD test showed that the relative intake of saccharin on the first test day in the control group was significantly (PB 0.05) smaller than the comparative value in both PBmedX and PBlatX groups. To compare the saccharin intake with the postoperative water intake, the raw data were analyzed using one-way ANOVA within each group. The main effect of the trial in the control and PBlatX group was significant (F(5,19)= 8.94, PB0.01 and F(5,19) = 3.50, P B0.05, respectively), but there was no effect in PBmedX group (F(5,15) =2.11, P\0.1). Post hoc analysis showed that the saccharin intake on the conditioning day and on the 4 test days in the control group were significantly (P B0.05) smaller them the postoperative water intake, while the saccharin intake only on the first test day was significantly (P B0.05) smaller than the postoperative water intake in the PBlatX group. In the PBmedX group, no significant difference was detected between the saccharin intake and the postoperative water intake. All the analyses in the present acquisition experiment suggest that lesions of PBmed or PBlat abolished neophobic responses to novel saccharin and also severely attenuated the acquisition of CTA, viz., while CTA can be weakly acquired, it is quickly extinguished in the lesioned rats.
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second conditioning day (P B 0.01) because of the acquired taste aversion to saccharin. The effects of lesions are also shown in Fig. 3. The control rats after the sham operation showed decreased saccharin intake during the 4 test days with gradual increase from the first to the fourth test day, and the volume of companion water intake was similar to the preconditioning water intake level throughout the 4 test days (Fig. 3A). The similar ingestion pattern was observed for the PBlatX rats as shown in Fig. 3C. In contrast, when the medial part of the PBN was destroyed after conditioning, those PBmedX rats drank saccharin more than 50% of the preconditioning water intake level during the 4 test days (Fig. 3B). The lesion effects on the retention of CTA were statistically analyzed using two-way ANOVA with repeated measures. Analysis of relative intake of saccharin (CS) showed reliable main effects of the group (F(2,11)= 8.52, PB 0.01) and the trial (F(5,55)= 5.57, PB 0.01), but the group× trial was not significant (F(10,55)= 1.88, P\ 0.05).
3.2. Retention test The sham and lesion operations were made after conditioning procedure in this experiment. The mean preconditioning (or preoperative) water intake9 SE was 12.6 90.8, 11.8 90.2 and 12.2 90.6, for control, PBmedX and PBlatX groups, respectively, which were very similar with each other (F(2,11) = 0.41, P \ 0.1). Fig. 3 shows the mean relative amount of saccharin for 20 min throughout the conditioning and test days and distilled water for the succeeding 20 min on the 4 test days in the three groups, when the preoperative mean water intake was taken as standard (100%). The three groups of rats showed similar intake of saccharin before surgery. The ANOVA showed the main effect of the trial (F(2,22) = 62.8, P B0.01), but no effects of the group (F(2,11) =0.34, P\ 0.1) or interaction of group × trial (F(4,22) =0.42, P \0.1) was found. The volume of saccharin consumption on the first conditioning day was significantly (P B 0.01) smaller than the preconditioning water intake in all the three groups, indicating neophobia to the novel CS. The animals avoided the saccharin CS more strongly on the
Fig. 3. Saccharin and water intakes after conditioned taste aversions to 0.1% sodium saccharin in control and experimental groups. Sham (S) and lesion (L) operations were made after the two conditioning procedures (C1 and C2). The solid and open column with vertical line shows the mean intake 9SE for saccharin and water, respectively. *P B0.05, **PB 0.01, ***PB 0.001
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(PB0.01) more than the control rats. These results suggest that the rats with lesions in the PBmed have insufficient ability to discriminate sapid solutions from water.
4. Discussion
Fig. 4. Preference scores for the four taste stimuli in the three groups of rats. Each column with vertical line shows the mean preference score9 SE.
To compare the saccharin intake with the preoperative water intake, the raw data were analyzed using one-way ANOVA in each group. The main effect of the trial in control and PBlatX group was significant (F(4,21)= 122.28, P B 0.01 and F(4,16) =15.00, P B 0.01, respectively), but there was no effect in PBmedX group (F(4,21)= 0.67, P \0.1). Post hoc analysis showed that the saccharin intake on the 4 test days in the control and PBlatX groups were significantly (PB 0.05) smaller them the postoperative water intake. In the PBmedX group, no significant difference was detected between the saccharin intake and the preoperative water intake. When the CTA indices were calculated and compared with one-way ANOVA, the main effect of the group on retention of CTA was reliable (F(2,11)= 16.55, PB 0.01). The mean CTA index 9SE was 0.349 0.12 for PBmedX rats, and was significantly (P B 0.01) smaller than that (0.9490.02) for control rats and that (0.82 9 0.07) for PBlatX rats. The PBlat lesions were not effective (P \0.1). These results suggest that the lesions in the PBmed severely disrupted the retention of CTA.
3.3. Two-bottle preference test Fig. 4 shows the preference scores for the sham control and two PBN-lesioned groups. The control rats preferred sucrose solutions to water, but showed less preference for quinine, HCl and NaCl solutions than water. ANOVA showed that the main effect of the solution was reliable (F(3,30) =8.97, P B0.05) but the main effect of the group was not significant. The group ×solution interaction was significant (F(6,30)= 3.85, PB 0.01). The PBlatX rats showed similar preference and aversion to these four stimuli as in the control rats. The marked difference was observed for PBmedX rats, i.e. they drank quinine and HCl solutions significantly
In the present study, we made lesions with the electrolytic method because our technique allowed this method to be more suitable to make smaller and more confined lesions than the neurotoxic method. Since electrolytic lesions would also destroy fibers of passage, both ascending and descending, there is a possibility that the effects of lesions on CTA acquisition and expression might be due to the destruction of passing fibers rather than cell bodies in the PBN. However, the pivotal role of the PBN in CTA formation as well as transmission of taste information has been demonstrated by several behavioral experiments in rats. Dysfunction of the whole PBN by reversible lesions with topical application of tetrodotoxin [16,17] or irreversible lesions with excitotoxic ibotenic acid [26,38] disrupted the acquisition, consolidation, retrieval and retention of CTA. Rats with various degrees of electrolytic lesions in the PBN exhibited lowered taste sensitivity as revealed by two-bottle preference test [14] or taste-guided behaviors [30]. The role of the general visceral inputs to the PBN was not studied well because of the difficulty in evaluating general visceral functions. No systematic study has been performed so far to assess the functional role of taste signal recipient zone and visceral signal recipient zone within the PBN in different aspects of CTA formation. In the present study, therefore, we made lesions confined either to the medial part of the PBN to which gustatory CS information projects or to the lateral part to which LiCl US information projects on the basis of previous neuroanatomical studies [34,35]. It was technically difficult to make smaller lesions restricted to only one or a few subnuclei within the PBN, which we first intended since c-fos studies [35,37] suggested that gustatory information of saccharin and sodium ions projects to the central and medial subnuclei, respectively, and i.p. injection of LiCl activates neurons in the external lateral subnucleus. The present study has demonstrated the differential functional roles of PBmed and PBlat in CTA formation and taste function, i.e. both PBmed and PBlat are crucial for the acquisition and maintenance of CTA, the retention of CTA is impaired severely by PBmed lesions but only slightly by PBlat lesions, and PBmed but not PBlat is important in taste preference behavior. The PBmed lesions included the caudal half of the central lateral subnucleus and the central medial subnucleus to which gustatory information from the rostral
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part of the NTS projects. Accordingly, the present two-bottle preference study showed that the PBmedlesioned rats could not show normal aversion to quinine or HCl as well as normal preference to sucrose. In accordance with the present results, Hill and Almli [14] showed that the rats received bilateral electrolytic lesions of PBN in 10 days old displayed attenuated quinine aversions. Spector [30] examined the effects of electrolytic lesions in PBN on the rat’s taste-guided licking of quinine, and found that the concentration-response functions shifted to the right. Taken these findings together, the impairment of the acquisitions and retentions of CTA by the PBmed lesions may be due to interference with transmission and retrieval of the gustatory information. C-fos studies [36] from our laboratory have suggested that taste information of saccharin and NaCl projects mainly to the PBmed, while that of quinine and HCl projects mainly to the external medial and lateral subnuclei in the PBlat which were not damaged in the PBmed-lesioned rats. One explanation for the discrepancy that the PBmedX rats impaired aversions to quinine and HCl is that the solitarioparabrachial gustatory route passes the PBmed to reach the PBlat, and the electrolytic lesions to the PBmed damaged this route. Alternatively, neurons in the medial part of the caudal PBN around the waist area, which are activated by quinine and HCl as well as by sucrose and NaCl as revealed by c-fos study (Yamamoto et al., unpublished), may be important for taste preference behavior. It is noted that effects of lesions of PBN depend on the difference in the experimental procedures: electrolytic or excitotoxic lesions, reversible or irreversible blockade, one-bottle or two-bottle test, brief or long exposure, and intake or immediate responses. Ivanova and Bures [17] showed that the rats injected with tetrodotoxin into the PBN showed normal aversion behavior to quinine solution. Flynn et al. [8] found that PBN-lesioned rats exhibited only minor deficits in the single-bottle preference test and taste reactivity test. These results suggest that normal taste-elicited behaviors are maintained in rats with NTS intact. Electrolytic PBN lesions inevitably destroy ascending fibers from the NTS and induce retrograde degeneration of NTS neurons. Since it is known that NTS lesions elicit severe impairment of taste sensitivity [3,8,29], it is plausible that the impairment of taste function after electrolytic PBN lesions are attributable to a functional loss of NTS neurons. The PBlat-lesioned rats showed the normal preference behavior to the four taste stimuli similarly as the control rats did. This finding indicates that the impairment of the acquisition of CTA seen in these rats may not be due to the interference with transmission
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of the gustatory information, but may be due to the interruption of transmission of the visceral information elicited by LiCl injection. Our results and interpretation are consistent with those reported by Aguero et al. [1] who used LiCl as the US and Nader et al. [18] who used morphine as the US. Recently, we found that neurons in the external lateral subnucleus of the PBN were activated by various US stimuli effectively used in the CTA paradigm such as radiation, rotation, i.p. injection of ethanol, and subcutaneous injection of cocaine [23]. Another important finding in the present study is that PBlat lesions did not impair the retention of CTA suggests that PBlat is not responsible for retrieval of CTA on the basis of recalling of CS taste or that vagal inputs are not necessary for the retrieval phase [15]. Impairment of the acquisition of CTA in PBmedor PBlat-lesioned rats might be due to the impairment of association between the gustatory CS and the visceral US information. The present PBlat lesions included a possible site for the CS-US association, i.e. the external lateral subnucleus as suggested by cfos studies from our laboratory [33,36]. The PBmed lesions included an interstitial zone of the medial part to the caudal PBN where neurons responding to both taste and vagal (visceral) stimulation are found [13]. Decerebrated rats, even though they have intact PBN, cannot acquire CTA [10]. This finding suggests that the functions of the forebrain structures possibly including the amygdala or gustatory cortex [37] play essential roles in association learning between taste and visceral information, and/or descending influences from the forebrain to PBN are important. Further study is needed to disclose the neural substrates for taste-visceral association in the taste aversion learning.
5. Conclusion In conclusion, taste quality information of saccharin transmitted through the PBmed is important for acquisition, retention and/or retrieval of CTA, but the PBlat responsible for processing of illness-inducing visceral signals is crucial only for the acquisition phase of taste aversion learning.
Acknowledgements Dr. Tsuyoshi Shimura was helpful in statistical analysis. This study was supported by Grants-in-Aid (Nos. 08877275 and 09470401) for Science Research from the Ministry of Education, Science, Sports and Culture of Japan.
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discriminative properties. Behav Neurosci 1996;110:1496–502. [19] Norgren R. Projections from the nucleus of the solitary tract in the rat. Neuroscience 1978;3:207 – 18. [20] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press, 1986; 263 pp. [21] Reilly S, Grigson PS, Norgren R. Parabrachial nucleus lesions and conditioned taste aversion: evidence supporting an associative deficit. Behav Neurosci 1993;107:1005– 7. [22] Ritter S, McGlone JJ, Kelley KW. Absence of lithium-induced taste aversion after area postrema lesion. Brain Res 1980;201:501 – 6. [23] Sakai N, Yamamoto T. Conditioned taste aversion and c-fos expression in the rat brainstem after administration of various USs. Neuroreport 1997;8:2215 – 20. [24] Sakai N, Tanimizu T, Sako N, Shimura T, Yamamoto T. Effects of lesions of the medial and lateral parabrachial nuclei on acquisition and retention of conditioned taste aversion. In: Kurihara K, Suzuki N, Ogawa H, editors. Olfaction and Taste XI. Tokyo: Springer-Verlag, 1994:495 – 496. [25] Saper CB, Loewy AD. Efferent connections of the parabrachial nucleus in the rat. Brain Res 1980;197:291 – 317. [26] Scalera G, Spector AC, Norgren R. Excitotoxic lesions of the parabrachial nuclei prevent conditioned taste aversions and sodium appetite in rats. Behav Neurosci 1995;109:997 –1008. [27] Schafe GE, Bernstein IL. Taste aversion learning, In: Capaldi ED, editor. Why We Eat What We Eat. Washington DC: American Psychological Association, 1996; pp. 31 – 51. [28] Shapiro RE, Miselis RR. The central neural connections of the area postrema of the rat. J Comp Neurol 1985;234:344–64. [29] Shimura T, Grigson PS, Norgren R. Brainstem lesions and gustatory function: I. The role of the NST when assessed during a brief intake test in rats. Behav Neurosci 1997;111:155–68. [30] Spector AC. Gustatory parabrachial lesions disrupt taste-guided quinine responsiveness in rats. Behav Neurosci 1995;109:79–90. [31] Spector AC, Norgren R, Grill HJ. Parabrachial gustatory lesions impair taste aversion learning in rats. Behav Neurosci 1992;106:147 – 61. [32] Suzuki H, Azuma M. A glass-insulated ‘‘elgiloy’’ microelectrode for recording unit activity in chronic monkey experiments. Electroencephalogr Clin Neurophysiol 1976;41:93 – 5. [33] Yamamoto T. Neural mechanisms of taste aversion learning. Neurosci Res 1993;16:181 – 1859. [34] Yamamoto T, Shimura T, Sako N, Azuma S, Bai W-Zh, Wakisaka S. C-fos expression in the rat brain after intraperitoneal injection of lithium chloride. Neuroreport 1992;3:1049 –52. [35] Yamamoto T, Shimura T, Sako N, Sakai N, Tanimizu T, Wakisaka S. C-fos expression in the parabrachial nucleus after ingestion of sodium chloride in the rat. Neuroreport 1993;4:1223 – 6. [36] Yamamoto T, Shimura T, Sakai N, Ozaki N. Representation of hedonics and quality of taste stimuli in the parabrachial nucleus of the rat. Physiol Behav 1994;56:1197 – 202. [37] Yamamoto T, Shimura T, Sako N, Yasoshima Y, Sakai N. Neural substrates for conditioned taste aversion in the rat. Behav Brain Res 1994;65:123 – 37. [38] Yamamoto T, Fujimoto Y, Shimura T, Sakai N. Conditioned taste aversion in rats with excitotoxic brain lesions. Neurosci Res 1995;22:31 – 49.
Research report
Role of the medial and lateral parabrachial nucleus in acquisition and retention of conditioned taste aversion in rats Nobuyuki Sakai, Takashi Yamamoto * Department of Beha6ioral Physiology, Faculty of Human Sciences, Osaka Uni6ersity, 1 -2 Yamadaoka, Suita, Osaka, 565, Japan Received 17 March 1997; received in revised form 8 September 1997; accepted 8 September 1997
Abstract When ingestion of a taste stimulus is paired with internal malaise, the animal remembers the taste and rejects its ingestion thereafter. This learning is referred to as conditioned taste aversion (CTA). To establish CTA in adult male Wistar rats, 0.1% saccharin and an i.p. injection of 0.15 M LiCl were used as the conditioned and unconditioned stimuli, respectively. To elucidate the functional role of the medial part of the parabrachial nucleus (PBmed) which receives taste information and the lateral part (PBlat) which receives general visceral information, confined electrolytic lesions were made to either of these regions. Rats with bilateral lesions of the PBlat impaired the acquisition of CTA, but those lesions made after the acquisition of CTA had no effect on the retention of this learning. The bilateral lesions of the PBmed abolished the acquisition and retention of CTA. The PBlat-lesioned rats showed normal taste preference behavior, but PBmed-lesioned rats showed impaired sensibility to taste stimuli. These results suggest that both the PBlat and PBmed are essential for the acquisition of taste aversion learning, but the PBlat is not necessary for retrieval of CTA. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Conditioned taste aversion; Parabrachial nucleus; Electrolytic lesions; Taste preference; Rat
1. Introduction When the consumption of novel flavored food is followed by internal malaise, animals avoid ingesting the food on subsequent presentations [4,27,37]. It is well accepted that animals must learn to associate its taste with illness for this avoidance behavior. This type of association learning is referred to as conditioned taste aversion (CTA). Understanding of the neural mechanisms subserving the taste aversion learning has been a goal of a variety of animal experiments. In a typical experimental paradigm for CTA studies, saccharin is used as a conditioned stimulus (CS) and an intraperitoneal (i.p.) injection of lithium chloride (LiCl) is used as an unconditioned stimulus (US). In this * Corresponding author. Tel.: +81 6 8798047; fax: + 81 6 8798050; e-mail: [email protected] 0166-4328/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-4328(97)00133-2
simple model, neural information of the gustatory CS via the taste nerves is thought to be associated with that of the illness-inducing US via the visceral nerves or blood in the central nervous system. Unconditioned effects of LiCl are suggested to be mediated by the area postrema [22], and the area postrema is known to have large projections directly to the nucleus tractus solitarius (NTS) and the parabrachial nucleus (PBN) [28]. In general, both gustatory and visceral signals ascend side by side possibly by interacting with each other through the sensory relay stations and terminate in various brain areas [5,6,11,25]. Although a number of lesion studies have been performed to elucidate the neural substrates of CTA (Yamamoto et al. [37]), the role of the projection zone for each of the gustatory and visceral signals in CTA formation has not been clarified because almost all the previous lesions were large enough to invade the recipi-
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ent zones for the two sensory signals. The PBN is the second-order sensory relay station in the gustatory and general visceral pathways. It is generally accepted that gustatory inputs project to the medial part of the PBN (PBmed) and general visceral inputs project to the lateral part (PBlat) [12,19]. In previous lesion studies, researchers aimed to eliminate the function of the whole PBN including PBmed and PBlat, and demonstrated that the PBN was essential for the acquisition [2,16,21,26,31,38] and consolidation [17] of CTA. One could eliminate the function of either the gustatory zone or visceral zone of the PBN with the use of a confined lesion technique. In this way, Aguero et al. [1] showed that the rats with electrolytic lesions focusing to the external lateral subnucleus in the PBlat could not acquire CTA, and interpreted this impairment of learning as the disruption of transmission of visceral malaise information. Similarly, Nader et al. [18] showed that cytotoxic lesions of the PBlat blocked the acquisition of morphine-induced CTA to saccharin, and attributed this effect to the blockade of the aversive properties of morphine in the CTA paradigm. However, no reports have examined the effects of PBlat lesions made after the conditioning procedure on the retention of CTA. When the electrolytic lesion method was used to destroy the whole PBN in previous studies, the extents of the lesions were not always large enough to invade the entire PBN but were quite variable among the animals and depending on the researchers [7,9,21,31]. It is needed to correlate the lesion site in the PBN and the disruptive effects on CTA formation. The purpose of the present study was to examine the role of the PBlat, to which illness-inducing visceral information projects, and the PBmed, to which the gustatory CS information projects, in the acquisition and retention of CTA. Preliminary report on this work has appeared in abstract form [24].
2. Materials and methods
2.1. Subjects Adult male Wistar rats, weighing 250 – 300 g at the beginning of the experiment, were housed in individual home 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 were trained to drink distilled water for 20 min in the home
cages. Water was presented to the rats for the rest of the drinking time. They were allowed to free access to food throughout the experiments. Animal protocols were in accordance with the Guideline for the Care and Use of Laboratory Animals approved by National Institute of Health (1985).
2.3. Surgery Thirty rats were randomly divided into two classes of 15 each: one for the acquisition test and another for the retention test. Each of the classes consisted of three groups of five each: a sham control group which received only burr holes in the skull, an experimental group which consisted of rats with lesions of the PBmed (PBmedX group), and another experimental group which consisted of rats with lesions of the PBlat (PBlatX group). The animals 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.). After a scalp incision, two holes were drilled into the skull for the access to the bilateral PBN. An etched ‘elgiloy’ microelectrode [32] (50–80 mm tip diameter), insulated with glass except for the tip, was used as the DC reference electrode and positioned in the PBmed (10.2 mm caudal to bregma, 1.8 mm lateral to the midline) or PBlat (9.5 mm caudal to bregma, 2.5 mm lateral to the midline). Electrical activities of neurons were monitored during insertion of the electrode to facilitate locating the tip into the target area. DC cathodal current (0.1 mA for 30 s) was passed through the electrode against the indifferent electrode put on the skull to produce electrolytic lesions.
2.4. Acquisition test In this experiment, the rats received the PBN lesions before the conditioning procedure to examine lesion effects on the acquisition of CTA. After completion of the pre-experiment training to drink distilled water for 20 min in the home cages, the experimental rats received the PBN lesions. Following at least a week of postoperative recovery period in which all animals were offered water and food ad lib, the rats were again put on a water deprivation schedule, which permitted to drink distilled water for 20 min in home cages. During this period, each rat was examined for body weight and the amount of water drunk during the 20 min. After the amount of drinking was stable, each rat received an i.p. injection of 0.15 M LiCl (2% body weight) as an US soon after 20 min of free access to 0.1% (0.005 M) sodium saccharin instead of water as a CS. After 1 day of recovery from this conditioning procedure, the animals were then on the same water-de-
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privation 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 test days. The fluid bottles were weighed before and after testing to measure intake volume.
2.5. Retention test In this experiment, the rats received the PBN lesions after the conditioning procedure to examine lesion effects on the retention of acquired taste aversion. After the amount of water drinking was stable in the pre-experiment training session, the rats received an i.p. injection of 0.15 M LiCl (2% body weight) soon after 20 min of free access to 0.1% sodium saccharin instead of water. After this conditioning procedure was repeated on subsequent 2 days (one procedure per day), the experimental rats received the PBN lesions. Following at least a week of postoperative recovery period in which all animals were offered water and food ad lib, the rats were again put on the same water deprivation schedule which permitted them to drink distilled water for 20 min in home cages. During this postoperative period, each rat was examined for body weight and the amount of water drunk during the 20 min. After the postoperative recovery, the animals were put on the test session, 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.
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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 mm throughout the PBN with a freezing microtome. Every two sections were mounted and stained with cresyl violet. The extent of the lesions was examined under a light microscope. Some sections were photographed and drawings were also made for representative sections with the aid of a camera lucida.
2.8. Data analysis On the basis of volumes of preconditioning water intake and of saccharin intake on the following 4 test days, we calculated a CTA index as an indicator of the strength of CTA formation. The larger this index, the stronger the acquisition of CTA: CTA index= 1− (total saccharin intake on the 1st, 2nd, 3rd and 4th days/4 times mean water intake) For the two-bottle preference test, the preference score was calculated. The larger this score, the more the solution was preferred: Preference score= (intake of taste solution/total intake of taste solution and distilled water)× 100 Data were analyzed using the ANOVA with post hoc comparison (Fisher’s least significant difference; LSD) test.
2.6. Two-bottle preference test
3. Results
After the completion of the CTA experiment, the rats were examined for their taste sensitivity to hedonically positive and negative taste stimuli with the conventional brief exposure two-bottle method. The taste stimuli used were 0.002 M quinine hydrochloride, 0.01 M HCl, 0.1 M NaCl and 0.5 M sucrose made up with distilled water. The animals were deprived of water for 20 h, and were presented with two drinking bottles, one for distilled water and the other for either one of the taste stimuli. They were allowed free access to the two bottles for 20 min. The positions of the water and solution bottles were exchanged every 5 min. The fluid bottles were weighed before and after testing to measure intake volume.
One rat in each of the three groups in the acquisition test and one rat of the PBmedX group in the retention test were discarded from the data analysis because three of them received large lesions invading both PBmed and PBlat and the rest did not recover well from the operation judging from the water intake and body weight, and the other did not show stable water intake in their daily sessions. Fig. 1 shows the extent of PBmed and PBlat lesions in subjects used for the acquisition test and retention test. The PBmed lesions invaded the medial PBN with the caudal half predominance including the dorsal lateral, central lateral and central medial subnuclei on both sides. The PBlat invaded the lateral PBN with the rostral half predominance including the external lateral and external medial subnuclei on both sides. Essentially the similar lesions were placed in rats for the acquisition test and for the retention test. The overall mean lesion size was 0.64 mm ranging from 0.25 to 1.0 mm mediolaterally, and 0.78 mm ranging from 0.5 to 1.2 mm rostrocaudally.
2.7. Histology At the completion of all the experiment, the rats were perfused intracardially with physiological saline followed by 10% Formalin under deep anesthesia with an overdose (80 mg/kg i.p.) of sodium pentobarbital. The
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3.1. Acquisition test The sham and lesion operations were made before the conditioning procedure in this experiment. The preconditioning (or postoperative) mean water intake 9 SE was 12.49 0.7, 5.3091.5 and 16.99 1.1 for sham control, PBmedX and PBlatX groups, respectively. The PBlatX rats ingested more water than control rats, whereas the PBmedX rats drank less than control rats (P B0.01). To facilitate a better comparison of the results, the volume of fluid consumption was normalized by taking the preconditioning mean water intake as standard (100%). Fig. 2 shows the mean relative amount of 0.1% saccharin (CS) drunk for 20 min on the conditioning day, and the amount of saccharin and water drunk for the succeeding 20 min on the subsequent 4 test days in the 3 groups. As shown in Fig. 2A, control rats showed decreased saccharin intake during the 4 test days (20 min/day), although the volume of intake increased gradually from the first to the fourth test day. Accordingly, the volume
Fig. 2. Saccharin and water intakes after conditioned taste aversions to 0.1% sodium saccharin in control and experimental groups. The relative values are shown when the preconditioning water intake is taken as 100. The groups consist of sham control rats (n = 5) (A), rats with lesions of the medial parabrachial nucleus (PBmedX, n= 4) (B), and the rats with lesions of the lateral parabrachial nucleus (PBlatX, n =4) (C). Sham (S) and lesion (L) operations were made before the conditioning procedure (Cond). The solid and open column with vertical line shows the mean intake 9SE for saccharin and water, respectively. *PB0.05, **PB0.01, ***PB0.001.
Fig. 1. Extent of the lesions in the parabrachial nucleus (PBN). Lesions of the medial PBN for eight rats used in acquisition and retention tests are superimposed on camera-lucida drawings of the representative sections taken from the rostral (AP = − 9.30 mm, Paxinos and Watson Atlas [20]) and caudal (AP = −9.80 mm) levels. Lesions of the lateral PBN for eight rats are similarly shown. Shaded area, lesion size for each rat; solid area, the area overlapped by more than four lesions; BC, brachium conjunctivum; MeV, mesencephalic nucleus of the trigeminal nerve. The bar indicates 1 mm.
of companion water intake for 20 min following the 20-min saccharin presentation decreased gradually. In both PBmedX rats (Fig. 2B) and PBlatX rats (Fig. 2C), the intake of saccharin on the first test day tended to be suppressed, but on the following 3 days it was similar to the preconditioning water intake level. For a quantitative comparison of lesion effects on CTA formation among the three groups, we calculated CTA indices for the three groups, and analyzed them with one-way ANOVA. The main effect of the group was significant (F(2,9)= 5.45, PB 0.05), i.e. the mean CTA index 9 SE was 0.63 90.09 for control rats, and was statistically significantly (PB 0.05, post hoc LSD test) larger than that (0.089 0.21) for PBmedX rats and that (0.15 9 0.08) for PBlatX rats. These results suggest that the lesions in the PBN severely damaged the acquisition of CTA.
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For a more precise comparison, the relative intake of saccharin on each day among the three groups was statistically analyzed using two-way ANOVA with repeated measures. The analysis showed reliable main effects of the group (F(2,9) = 9.04, P B 0.01) and the trial (F(4,36)= 11.00, P B0.01), but the group×trial was not significant (F(8,36) = 0.99, P \ 0.05). Post hoc LSD test showed that the relative intake of saccharin on the first test day in the control group was significantly (PB 0.05) smaller than the comparative value in both PBmedX and PBlatX groups. To compare the saccharin intake with the postoperative water intake, the raw data were analyzed using one-way ANOVA within each group. The main effect of the trial in the control and PBlatX group was significant (F(5,19)= 8.94, PB0.01 and F(5,19) = 3.50, P B0.05, respectively), but there was no effect in PBmedX group (F(5,15) =2.11, P\0.1). Post hoc analysis showed that the saccharin intake on the conditioning day and on the 4 test days in the control group were significantly (P B0.05) smaller them the postoperative water intake, while the saccharin intake only on the first test day was significantly (P B0.05) smaller than the postoperative water intake in the PBlatX group. In the PBmedX group, no significant difference was detected between the saccharin intake and the postoperative water intake. All the analyses in the present acquisition experiment suggest that lesions of PBmed or PBlat abolished neophobic responses to novel saccharin and also severely attenuated the acquisition of CTA, viz., while CTA can be weakly acquired, it is quickly extinguished in the lesioned rats.
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second conditioning day (P B 0.01) because of the acquired taste aversion to saccharin. The effects of lesions are also shown in Fig. 3. The control rats after the sham operation showed decreased saccharin intake during the 4 test days with gradual increase from the first to the fourth test day, and the volume of companion water intake was similar to the preconditioning water intake level throughout the 4 test days (Fig. 3A). The similar ingestion pattern was observed for the PBlatX rats as shown in Fig. 3C. In contrast, when the medial part of the PBN was destroyed after conditioning, those PBmedX rats drank saccharin more than 50% of the preconditioning water intake level during the 4 test days (Fig. 3B). The lesion effects on the retention of CTA were statistically analyzed using two-way ANOVA with repeated measures. Analysis of relative intake of saccharin (CS) showed reliable main effects of the group (F(2,11)= 8.52, PB 0.01) and the trial (F(5,55)= 5.57, PB 0.01), but the group× trial was not significant (F(10,55)= 1.88, P\ 0.05).
3.2. Retention test The sham and lesion operations were made after conditioning procedure in this experiment. The mean preconditioning (or preoperative) water intake9 SE was 12.6 90.8, 11.8 90.2 and 12.2 90.6, for control, PBmedX and PBlatX groups, respectively, which were very similar with each other (F(2,11) = 0.41, P \ 0.1). Fig. 3 shows the mean relative amount of saccharin for 20 min throughout the conditioning and test days and distilled water for the succeeding 20 min on the 4 test days in the three groups, when the preoperative mean water intake was taken as standard (100%). The three groups of rats showed similar intake of saccharin before surgery. The ANOVA showed the main effect of the trial (F(2,22) = 62.8, P B0.01), but no effects of the group (F(2,11) =0.34, P\ 0.1) or interaction of group × trial (F(4,22) =0.42, P \0.1) was found. The volume of saccharin consumption on the first conditioning day was significantly (P B 0.01) smaller than the preconditioning water intake in all the three groups, indicating neophobia to the novel CS. The animals avoided the saccharin CS more strongly on the
Fig. 3. Saccharin and water intakes after conditioned taste aversions to 0.1% sodium saccharin in control and experimental groups. Sham (S) and lesion (L) operations were made after the two conditioning procedures (C1 and C2). The solid and open column with vertical line shows the mean intake 9SE for saccharin and water, respectively. *P B0.05, **PB 0.01, ***PB 0.001
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(PB0.01) more than the control rats. These results suggest that the rats with lesions in the PBmed have insufficient ability to discriminate sapid solutions from water.
4. Discussion
Fig. 4. Preference scores for the four taste stimuli in the three groups of rats. Each column with vertical line shows the mean preference score9 SE.
To compare the saccharin intake with the preoperative water intake, the raw data were analyzed using one-way ANOVA in each group. The main effect of the trial in control and PBlatX group was significant (F(4,21)= 122.28, P B 0.01 and F(4,16) =15.00, P B 0.01, respectively), but there was no effect in PBmedX group (F(4,21)= 0.67, P \0.1). Post hoc analysis showed that the saccharin intake on the 4 test days in the control and PBlatX groups were significantly (PB 0.05) smaller them the postoperative water intake. In the PBmedX group, no significant difference was detected between the saccharin intake and the preoperative water intake. When the CTA indices were calculated and compared with one-way ANOVA, the main effect of the group on retention of CTA was reliable (F(2,11)= 16.55, PB 0.01). The mean CTA index 9SE was 0.349 0.12 for PBmedX rats, and was significantly (P B 0.01) smaller than that (0.9490.02) for control rats and that (0.82 9 0.07) for PBlatX rats. The PBlat lesions were not effective (P \0.1). These results suggest that the lesions in the PBmed severely disrupted the retention of CTA.
3.3. Two-bottle preference test Fig. 4 shows the preference scores for the sham control and two PBN-lesioned groups. The control rats preferred sucrose solutions to water, but showed less preference for quinine, HCl and NaCl solutions than water. ANOVA showed that the main effect of the solution was reliable (F(3,30) =8.97, P B0.05) but the main effect of the group was not significant. The group ×solution interaction was significant (F(6,30)= 3.85, PB 0.01). The PBlatX rats showed similar preference and aversion to these four stimuli as in the control rats. The marked difference was observed for PBmedX rats, i.e. they drank quinine and HCl solutions significantly
In the present study, we made lesions with the electrolytic method because our technique allowed this method to be more suitable to make smaller and more confined lesions than the neurotoxic method. Since electrolytic lesions would also destroy fibers of passage, both ascending and descending, there is a possibility that the effects of lesions on CTA acquisition and expression might be due to the destruction of passing fibers rather than cell bodies in the PBN. However, the pivotal role of the PBN in CTA formation as well as transmission of taste information has been demonstrated by several behavioral experiments in rats. Dysfunction of the whole PBN by reversible lesions with topical application of tetrodotoxin [16,17] or irreversible lesions with excitotoxic ibotenic acid [26,38] disrupted the acquisition, consolidation, retrieval and retention of CTA. Rats with various degrees of electrolytic lesions in the PBN exhibited lowered taste sensitivity as revealed by two-bottle preference test [14] or taste-guided behaviors [30]. The role of the general visceral inputs to the PBN was not studied well because of the difficulty in evaluating general visceral functions. No systematic study has been performed so far to assess the functional role of taste signal recipient zone and visceral signal recipient zone within the PBN in different aspects of CTA formation. In the present study, therefore, we made lesions confined either to the medial part of the PBN to which gustatory CS information projects or to the lateral part to which LiCl US information projects on the basis of previous neuroanatomical studies [34,35]. It was technically difficult to make smaller lesions restricted to only one or a few subnuclei within the PBN, which we first intended since c-fos studies [35,37] suggested that gustatory information of saccharin and sodium ions projects to the central and medial subnuclei, respectively, and i.p. injection of LiCl activates neurons in the external lateral subnucleus. The present study has demonstrated the differential functional roles of PBmed and PBlat in CTA formation and taste function, i.e. both PBmed and PBlat are crucial for the acquisition and maintenance of CTA, the retention of CTA is impaired severely by PBmed lesions but only slightly by PBlat lesions, and PBmed but not PBlat is important in taste preference behavior. The PBmed lesions included the caudal half of the central lateral subnucleus and the central medial subnucleus to which gustatory information from the rostral
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part of the NTS projects. Accordingly, the present two-bottle preference study showed that the PBmedlesioned rats could not show normal aversion to quinine or HCl as well as normal preference to sucrose. In accordance with the present results, Hill and Almli [14] showed that the rats received bilateral electrolytic lesions of PBN in 10 days old displayed attenuated quinine aversions. Spector [30] examined the effects of electrolytic lesions in PBN on the rat’s taste-guided licking of quinine, and found that the concentration-response functions shifted to the right. Taken these findings together, the impairment of the acquisitions and retentions of CTA by the PBmed lesions may be due to interference with transmission and retrieval of the gustatory information. C-fos studies [36] from our laboratory have suggested that taste information of saccharin and NaCl projects mainly to the PBmed, while that of quinine and HCl projects mainly to the external medial and lateral subnuclei in the PBlat which were not damaged in the PBmed-lesioned rats. One explanation for the discrepancy that the PBmedX rats impaired aversions to quinine and HCl is that the solitarioparabrachial gustatory route passes the PBmed to reach the PBlat, and the electrolytic lesions to the PBmed damaged this route. Alternatively, neurons in the medial part of the caudal PBN around the waist area, which are activated by quinine and HCl as well as by sucrose and NaCl as revealed by c-fos study (Yamamoto et al., unpublished), may be important for taste preference behavior. It is noted that effects of lesions of PBN depend on the difference in the experimental procedures: electrolytic or excitotoxic lesions, reversible or irreversible blockade, one-bottle or two-bottle test, brief or long exposure, and intake or immediate responses. Ivanova and Bures [17] showed that the rats injected with tetrodotoxin into the PBN showed normal aversion behavior to quinine solution. Flynn et al. [8] found that PBN-lesioned rats exhibited only minor deficits in the single-bottle preference test and taste reactivity test. These results suggest that normal taste-elicited behaviors are maintained in rats with NTS intact. Electrolytic PBN lesions inevitably destroy ascending fibers from the NTS and induce retrograde degeneration of NTS neurons. Since it is known that NTS lesions elicit severe impairment of taste sensitivity [3,8,29], it is plausible that the impairment of taste function after electrolytic PBN lesions are attributable to a functional loss of NTS neurons. The PBlat-lesioned rats showed the normal preference behavior to the four taste stimuli similarly as the control rats did. This finding indicates that the impairment of the acquisition of CTA seen in these rats may not be due to the interference with transmission
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of the gustatory information, but may be due to the interruption of transmission of the visceral information elicited by LiCl injection. Our results and interpretation are consistent with those reported by Aguero et al. [1] who used LiCl as the US and Nader et al. [18] who used morphine as the US. Recently, we found that neurons in the external lateral subnucleus of the PBN were activated by various US stimuli effectively used in the CTA paradigm such as radiation, rotation, i.p. injection of ethanol, and subcutaneous injection of cocaine [23]. Another important finding in the present study is that PBlat lesions did not impair the retention of CTA suggests that PBlat is not responsible for retrieval of CTA on the basis of recalling of CS taste or that vagal inputs are not necessary for the retrieval phase [15]. Impairment of the acquisition of CTA in PBmedor PBlat-lesioned rats might be due to the impairment of association between the gustatory CS and the visceral US information. The present PBlat lesions included a possible site for the CS-US association, i.e. the external lateral subnucleus as suggested by cfos studies from our laboratory [33,36]. The PBmed lesions included an interstitial zone of the medial part to the caudal PBN where neurons responding to both taste and vagal (visceral) stimulation are found [13]. Decerebrated rats, even though they have intact PBN, cannot acquire CTA [10]. This finding suggests that the functions of the forebrain structures possibly including the amygdala or gustatory cortex [37] play essential roles in association learning between taste and visceral information, and/or descending influences from the forebrain to PBN are important. Further study is needed to disclose the neural substrates for taste-visceral association in the taste aversion learning.
5. Conclusion In conclusion, taste quality information of saccharin transmitted through the PBmed is important for acquisition, retention and/or retrieval of CTA, but the PBlat responsible for processing of illness-inducing visceral signals is crucial only for the acquisition phase of taste aversion learning.
Acknowledgements Dr. Tsuyoshi Shimura was helpful in statistical analysis. This study was supported by Grants-in-Aid (Nos. 08877275 and 09470401) for Science Research from the Ministry of Education, Science, Sports and Culture of Japan.
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