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Ventral tegmental lesions reduce overconsumption of normally preferred taste fluid in rats
Behavioural Brain Research 134 (2002) 123 /130 www.elsevier.com/locate/bbr
Research report
Ventral tegmental lesions reduce overconsumption of normally preferred taste fluid in rats Tsuyoshi Shimura *, Yoko Kamada 1, Takashi Yamamoto Laboratory of Behavioral Physiology, Graduate School of Human Sciences, Osaka University, 1-2 Yamadaoka, Suita, Osaka 565-0871, Japan Received 17 September 2001; received in revised form 23 November 2001; accepted 23 November 2001
Abstract Previous studies have suggested that the brain regions along the taste pathway and its anatomical interfacing with the brain reward system are concerned with palatability-induced consumption. To clarify whether the ventral tegmental area (VTA) is involved in the behavioral expression induced by taste pleasantness, we examined the effects of lesions to the VTA on the consumption of taste stimuli in rats. (1) Bilateral extensive electrolytic lesions to the VTA selectively reduced the consumption of a normally preferred taste fluid (0.1 M sucrose) compared to that of sham-operated animals during a 24-h two-bottle choice test. The consumption of other fluids, including non-preferred taste fluids (HCl and quinine hydrochloride) was not different between the lesioned and sham animals. (2) The injection of midazolam (3 mg/kg), a benzodiazepine agonist, or morphine (2 mg/kg) significantly increased the consumption of 0.1 M sucrose fluids in the sham animals. The same injections, however, failed to increase intake of the 0.1 M sucrose in rats with 6-hydroxydopamine lesions of the VTA. Neither midazolam nor morphine modified the intake of nonpreferred quinine (0.0003 M) solution in both the lesioned and sham animals. These results suggest that dopaminergic mediation in the VTA is required to enhance the consumption of normally preferred fluids exclusively. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ventral tegmental area; Taste; Motivation; Dopamine; Benzodiazepine; Opiate; Rat
1. Introduction The pleasantness of taste powerfully influences the preference for food and fluids, and the level of consumption [32]. Particularly rewarding tastes can lead to considerable overconsumption. For example, non-deprived rats consumed large quantities of sweet solutions of sucrose, glucose and sodium saccharin [8]. Previous studies have suggested that brain regions along the taste pathway [11,23] and its anatomical interfacing with the brain reward system [19,20,37] are involved in palatability-induced consumption. In addition, it has been reported that ingestion of a palatable meal increased Fos-like immunoreactivity in several regions
* Corresponding author. Tel.: 81-6-6879-8048; fax: 81-6-68798010 E-mail address: [email protected] (T. Shimura). 1 Present address: Research Institute for New Product Development, Suntory Ltd., 1-1 Wakayama-dai, Shimamoto-cho, Mishima-gun, Osaka 618-8503 Japan.
of limbic projections of the gustatory pathway and the mesoaccumbens dopamine pathway, viewed as a final common reward path [27]. Thus, the mesolimbic dopamine system originating in the ventral tegmental area (VTA) seems to mediate some reward or motivational processes of food and fluid intake. While it was suggested that palatability-induced consumption was less likely mediated by the nigrostriatal dopamine system [3], the role of the VTA in the behavioral expression induced by taste pleasantness is still unclear. Recent evidence [13] has revealed that the extracellular levels of dopamine in the nucleus accumbens, the major projection site of VTA dopaminergic neurons, increased in response to licking of highly preferred sucrose but not to water. Furthermore, reverse microdialysis of nomifensine, the dopamine reuptake blocker, into the nucleus accumbens led to an increase of sucrose intake [13]. These findings strongly suggest that the mesoaccumbens dopamine system is concerned with the ingestion of preferred tastes. Therefore, we examined the effects of
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lesions to the VTA on the consumption of normally preferred and non-preferred taste fluids in rats.
2. Experiment 1 An earlier study reported that lowered intake of preferred stimuli after injection of a dopamine antagonist is due to the reduction of reward value [41]. If the mesolimbic dopamine system was critically involved in palatability-induced ingestion of food and fluids, lesions to the VTA would disrupt the normal intake pattern of preferred taste stimuli. Therefore, we examined the effects of electrolytic lesions of the VTA on preference/ aversion responses to four basic taste stimuli using the conventional two-bottle preference test. 2.1. Materials and methods 2.1.1. Subjects The subjects were 12 male Wistar rats (Nihon Dobutsu, Osaka, Japan) housed individually in polycarbonate cages in a temperature-controlled room with a 12:12 h light:dark cycle (light on at 07:00 h). All experimental manipulations and treatments were conducted during the light phase of the cycle. The rats were maintained with free access to rat chow (Oriental Yeast, MF) and water was available as noted below for experimental purposes. On the day of surgery, their body weights ranged from 265 to 310 g. Testing occurred during the light hours of a 12-h light:dark cycle. The study was approved by the animal welfare committee of our institute and carried out in accordance with the guidelines of the National Institute of Health of the USA. 2.1.2. Surgery Six experimental subjects (Group VTAx) were randomly selected and received bilateral electrolytic lesions to the VTA. The other six subjects (Sham Group) received sham lesions. Under Nembutal anesthesia (50 mg/kg, i.p.), the VTA lesions were made on each side of the brains by passing a 1-mA current for 20 s through a stainless steel electrode (0.25 mm in diameter) insulated with Epoxylite except at the tip. For the Sham rats, the electrodes were bilaterally lowered 2 mm dorsal to the VTA, but no current was passed. The coordinates for the electrode placements were 6.0 mm posterior to the bregma, 90.6 mm from the midline and 8.2 mm ventral to the surface of the skull. After surgery, all subjects were maintained on ad libitum food and water. Because aphagia and adipsia were observed after surgery in some VTAx rats, they were given wet mash (a 4:7 mixture of powdered rat chow and water) until they recovered.
2.1.3. Procedure Preference testing began after a minimum of 14 postoperative recovery days. At this time, each rat was given 24-h access to two plastic syringes equipped with stainless steel drinking spouts: one containing distilled water, the other a taste stimulus (reagent grade chemical dissolved in distilled water). The initial location (left or right) of the stimulus syringe varied randomly across tastants. The syringes were weighed before and after testing to determine intake volume. The four basic taste stimuli and the order of presentation were as follows: sucrose (0.01, 0.1, 1.0 M); NaCl (0.01, 0.1, 1.0 M); quinine hydrochloride (quinine, 0.00003, 0.0003, 0.003 M); and hydrochloric acid (HCl, 0.001, 0.01, 0.1 M). The amounts consumed during each 24-h period were recorded. 2.1.4. Data analysis The data were analyzed using a three-way (Group Fluid Concentration) analysis of variance (ANOVA) for repeated-measures. Post hoc comparisons between means were carried out using the Scheffe´’s multiple comparison’s test. Statistical tests were performed using Statistica (StatSoft Inc., Tulsa, OK) and a result was considered significant if P B 0.05. 2.1.5. Histology At the end of the experiments, the rats received an overdose of Nembutal (80 mg/kg) and were perfused intracardially with physiological saline followed by 10% formalin. The brains were removed and stored in a solution of 30% sucrose until they sank. After the brains were sectioned coronally at 60 mm on a freezing microtome, consecutive sections through the midbrain were stained for cell bodies with Cresyl violet. Lesion reconstructions were made on drawings derived from the Paxinos and Watson’s atlas [29], with the use of a microscope and a personal computer. 2.2. Results The mean 24-h intake of water during the day before the two-bottle test in both groups was not significantly different (26.391.9 g (mean9S.E.) for VTAx and 32.192.5 g for Sham; F (1, 10) 3.49, P 0.05). The body weight gain during the 12 days of the two-bottle test was 39.295.4 g (mean9S.E.) in VTAx and 48.394.4 g in Sham. The difference, however, was not significant. Fig. 1 illustrates the mean 24-h intake of water and taste solutions in VTAx and Sham rats. For sucrose, both groups of subjects clearly consumed more taste solution at 0.1 and 1.0 M than water. At 0.01 M, however, both groups equally consumed sucrose and water. A three-way ANOVA revealed significant main effects of Group (F (1, 20) 5.81, P B 0.05), Fluid
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Fig. 1. Mean (9S.E.) 24-h fluid intake during the sucrose, NaCl (sodium chloride), quinine (quinine hydrochloride) and HCl (hydrochloric acid) concentration series for control (Sham) and rats with lesions of the ventral tegmental area (VTAx) in the 24-h, two-bottle tests of Experiment 1. *P B 0.05 from the corresponding solution of Sham.
(F (1, 20) 38.57, P B 0.001) and Concentration (F (2, 40) 17.23, P B0.001). Fluid Concentration (F (2, 40) 45.43, P B0.001) and Group Fluid Concentration interactions (F (2, 40) 4.42, P B0.05) were also significant. Post hoc comparisons showed that the Sham rats consumed significantly more of the 0.1 M sucrose than the VTAx rats (P B0.01). There was no difference in the intake of the 1.0 M sucrose between the VTAx and Sham rats. For NaCl, there were significant main effects of Fluid (F (1, 20) 11.97, P B 0.01) and Concentration (F (2, 40) 4.73, P B 0.05). Significant Fluid Concentration (F (2, 40) 13.91, P B 0.001) and Group Fluid Concentration interactions (F (2, 40) 6.64, P B 0.01) were also detected. Post hoc comparisons, however, revealed that there were no significant differences in the intake of NaCl at each concentration and water in both groups. Nevertheless, as seen in Fig. 1, it was noted that the Sham animals tended to consume more of the 0.1 M NaCl than the VTAx rats. There were only significant main effects of Fluid for quinine (F (1, 20) 361.46, P B 0.001) and HCl (F (1, 20) 499.74, P B0.001). Thus, both groups seldom selected quinine and HCl solutions to ingest at any concentration. Fig. 2 shows the location of the lesions in the ventromedial region of the midbrain marked on sections of brain from Paxinos and Watson’s rat stereotaxic atlas [29]. From rostral to caudal, the VTA was almost fully destroyed in all six VTAx rats. Electrolytic lesions, however, sometimes invaded structures such as the
Fig. 2. (A) A schematic representation of the size and extent of the electrolytic lesions to the VTA. The portion shaded in black indicates the area common to all lesions and the black outline indicates the largest extent of all lesions. The number to the right indicates the distance caudal from the bregma in millimeters. (B) A photomicrograph of an example of electrolytic lesions in the ventral midbrain. Scale bar 2 mm.
substantia nigra, red nucleus, medial lemniscus, superior cerebellar peduncle, paranigral nucleus, mammillary peduncle, interfascicular nucleus, interpeduncular nucleus, retrorubral field and cerebral peduncle.
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2.3. Discussion The VTAx rats displayed almost normal preferred or aversive responses to all the taste stimuli except for 0.1 M sucrose. This finding suggests that their taste sensations remained intact. Some investigators have reported that lesions of the VTA disturb oral movements [17,24]. However, since the intake of water during the day before the two-bottle test in both groups was not significantly different, the VTAx rats in the present study seemingly showed few drinking disorders during the 24-h intake test. It is widely accepted that 0.1 M sucrose is highly preferred by rats [32,36]. A brief 15-min intake test [34] has demonstrated that 0.1 M NaCl is also a preferred solution for rats. Significantly less intake of 0.1 M sucrose and marginally less intake of 0.1 M NaCl in VTAx, therefore, indicate that the VTA might be exclusively involved in the consumption of highly preferred solutions. In contrast, responses to nonpreferred HCl and quinine were not different between both groups, suggesting that the VTA was not concerned with the intake of aversive tastes. It was noted, however, that the low consumption of HCl and quinine at the concentrations in the present experiment obscured a possible modification in consumption of these fluids at lower concentrations. Future studies will be necessary to determine the effects of VTA lesions on responses to normally aversive taste fluids at lower concentrations. Injections of pimozide, a dopamine antagonist, have been reported to block the reward value of food and fluids [10,40,41]. These observations support the anhedonia hypothesis [39] that dopamine systems amplify the hedonic impact of positive reinforcers. However, because of the extensive non-selective electrolytic lesions of the VTA in the present experiment, we cannot determine whether the dopamine cells in the VTA critically contributed to these behavioral results. The examination of this issue was included in the next experiment.
3. Experiment 2 The results of Experiment 1 demonstrate that the VTA is related to the consumption of highly preferred fluids under the conventional two-choice intake test. Increasing evidence suggests that benzodiazepine agonists facilitate feeding in animals due to a specific enhancement of the perceived palatability of food and fluids [2,5,6,38]. In Experiment 2, therefore, we compared the effects of benzodiazepine on the consumption of taste solutions between VTA-lesioned and shamoperated animals. We used a relatively shorter intake test in Experiment 2 because evidence indicates that post-ingestive factors strongly affect the taste preference [21,33]. Furthermore, since lesions aimed to the VTA in
the Experiment 1 were extensive and non-specific, we tried to make more specific lesions by injection of 6hydroxydopamine (6-OHDA), which is known as a catecholamine-specific neurotoxin. 3.1. Materials and methods 3.1.1. Subjects The subjects were 16 male Wistar rats (Nihon Dobutsu, Osaka, Japan). They were maintained as described in Experiment 1. On the day of surgery, their body weights ranged from 240 to 320 g. 3.1.2. Surgery Eight experimental subjects were randomly selected and received bilateral injections of 6-OHDA into the VTA under Nembutal anesthesia (50 mg/kg i.p.). The stereotaxic coordinates were 6.0 mm posterior to the bregma, 90.6 mm from the midline, and 8.2 mm ventral to the surface of the skull. 6-OHDA (8 mg) was dissolved in 4 ml of an isotonic saline containing 0.2 mg/ml of ascorbic acid and injected into the structures using a Hamilton syringe (10 ml). The injection solution was prepared immediately before administration and was infused at a rate of 1 ml/2 min. Eight sham animals received vehicle (isotonic saline with an appropriate amount of ascorbic acid). In order to protect noradrenergic neurons from the toxic effect of 6-OHDA, the rats were pretreated with desipramine (Sigma; 25 mg/kg i.p.) 30 min before the injection of 6-OHDA. 3.1.3. Procedure Each rat was deprived of water and given access to water for 2 h in the morning (09.00/11.00 h) and 1 h in the afternoon (16.00 /17.00 h) in a plastic syringe equipped with a stainless steel spout for 5 days. On the test day at 08.30 h, the rats were injected intraperitoneally with 0.3, 1.0 or 3.0 mg/kg of midazolam, a benzodiazepine agonist (in solution with physiological saline) or physiological saline solution, in a volume of 1 mg/kg. Later (30 min), they were presented with a syringe containing 0.1 M sucrose or 0.0003 M quinine solution for 2 h. The amounts consumed from each syringe were measured. A within-subjects design was used and each animal was tested at each dose and solution following injection of the drug vehicle. The order of injection was counterbalanced across the subjects, with 48 h between consecutive injections. 3.1.4. Data analysis The data were analyzed using a two-way (Group Dose) ANOVA. Post hoc comparisons between means were conducted, when appropriate, using the Scheffe´’s multiple comparison’s test.
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3.2. Results Fig. 3 shows the 2-h intake of 0.1 M sucrose and 0.0003 M quinine after systemic injection of midazolam or vehicle. For sucrose, the intake of Sham rats increased dose-dependently. A two-way ANOVA confirmed significant main effects of Group (F (1, 14) 18.06, P B0.001) and Dose (F (3, 42) 6.61, P B0.001). A Group Dose interaction was also significant (F (3, 42) 3.57, P B 0.05). The lesioned rats did not show any increase in the intake of sucrose after injection of midazolam, viz., Sham rats consumed significantly more sucrose than the lesioned rats at the dose of 3 mg/kg (P B0.001). For quinine, the 2-h intake of both groups did not differ at any dose. As clearly shown in Fig. 3, intakes of sucrose were higher than those of quinine at any dose in both groups. 3.3. Discussion It is well-documented that benzodiazepine-induced feeding is due to a specific enhancement of the perceived palatability of food and fluids [2,5,6]. In line with this notion, the Sham animals in the present experiment showed an increased intake of sucrose after an injection of 3 mg/kg midazolam. The same injection of midazolam, however, failed to facilitate the consumption of non-preferred quinine. If it was midazolam that enhanced the consumption of sucrose in the Sham animals due to an increased pleasantness, lesions of the VTA might disrupt the perceived palatability. However, the following evidence may rule out this possibility. First, if lesions to the VTA reduced the perceived palatability, the intake of sucrose in VTAx would be lower than that of Sham animals after injection of saline. However, the lesioned rats consumed almost an equal amount of sucrose as the Sham animals after saline injection (see Fig. 3). Second, lesions of the mesotelencephalic dopamine systems did not change the mimetic responses to taste stimuli [1,3] when assessed with the objective index
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of palatability developed by Grill and Norgren [12]. Preliminary observations in our laboratory also revealed no change in taste reactivity to preferred fluids in 6OHDA lesioned rats. Third, recent evidence suggests that receptors in the lower brainstem, such as the parabrachial nucleus (a second taste relay), are the primary substrate for the benzodiazepine-induced enhancement of taste palatability [14,15,31].
4. Experiment 3 Opioid agonists, as well as benzodiazepine agonists, facilitate feeding in animals presumably because of enhancing the perceived pleasantness of taste [5,7,28,35]. In the final experiment, we investigated the effects of systemic injection of an opioid agonist, morphine, on the consumption of taste solutions in the VTA-lesioned and Sham animals. 4.1. Materials and methods 4.1.1. Subjects The subjects were 16 male Wistar rats from Experiment 2. They were maintained as described above. 4.1.2. Procedure Each rat was deprived of water and given access to water for 2 h in the morning (09.00/11.00 h) and 1 h in the afternoon (16.00 /17.00 h) in a plastic syringe equipped with a stainless steel spout for 5 days. On the test day at 08:30 h, the rats were subcutaneously injected with either 2 mg/kg of morphine hydrochloride (Takeda, Japan) or the saline vehicle at a volume of 1 ml/kg. The dose of morphine has been shown to be effective in facilitating the consumption of food and fluids in non-deprived rats [4]. Later (30 min), they were presented with a syringe containing 0.1 M sucrose or 0.0003 M quinine solution for 2 h. The amounts consumed from each syringe were measured. A withinsubjects design was used. On each test day, each rat received either morphine or vehicle. The order of injection was counterbalanced across the subjects, with 48 h between consecutive injections. 4.1.3. Data analysis The data were analyzed using a two-way (Group Injection) ANOVA. Post hoc comparisons between means were conducted, when appropriate, using the Scheffe´’s multiple comparison’s test.
Fig. 3. Mean (9S.E.) 2-h intake of 0.1 M sucrose and 0.0003 M quinine after injections of midazolam (0.3 /3.0 mg/kg) or of saline in Sham and rats with 6-OHDA lesions of the VTA. *P B 0.001 from the corresponding solution of Sham.
4.1.4. Histology At the end of the experiments, the rats received an overdose of Nembutal (80 mg/kg) and were perfused intracardially with 0.02 M phosphate-buffered saline followed by 4% paraformaldehyde. The brains were
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removed and stored in a solution of 30% sucrose until they sank. After the brains were sectioned coronally at 60 mm on a freezing microtome, consecutive sections through the midbrain were stained for tyrosine hydroxylase immunoreactivity using the avidin-biotin-peroxidase technique [16]. 4.2. Results Fig. 4 shows the 2-h intake of 0.1 M sucrose and 0.0003 M quinine after subcutaneous injection of 2 mg/ kg of morphine or saline. A two-way ANOVA indicated significant main effects of Group (F (1, 14) 9.09, P B0.01) and Injection (F (1, 14) 14.02, P B0.01). Group Injection interaction was also significant (F (1, 14) 6.74, P B 0.05). The Sham animals consumed more sucrose after morphine injection than after saline injection (P B 0.001). However, the intake of sucrose after injections of morphine in the lesioned animals did not differ from that after saline injection (P 0.05). There were no significant differences in the intake of quinine across groups and injections. Fig. 5 shows the photomicrographs of the ventral midbrain stained for tyrosine hydroxylase immunoreactivity. In the Sham animals, immunoreactive neurons were extensively located in the ventral midbrain. The number of immunoreactive neurons in 6-OHDA-lesioned rats was much less compared with the Sham animals, as shown in Fig. 5. Immunoreactive neurons, thus, almost disappeared in the VTA of the lesioned animals. However, the 6-OHDA lesions were not restricted in the VTA, but invaded the medial part of the substantia nigra in most cases. 4.3. Discussion Morphine selectively enhanced the sucrose intake in the Sham animals, but not in the lesioned rats. This effect is similar to that of midazolam in Experiment 2. It is suggested that opioid receptors in the forebrain, not in the hindbrain, are mainly responsible for increased
Fig. 4. Mean (9S.E.) 2-h intake of 0.1 M sucrose and 0.0003 M quinine after injections of morphine (2 mg/kg) or of saline in Sham and rats with 6-OHDA lesions of the VTA. *P B 0.05 versus saline injection of Sham.
feeding due to a selective facilitation of the perceived palatability [30]. According to this suggestion, similar to midazolam, it is less likely that morphine directly acts on the VTA neurons to facilitate taste palatability. Histological examinations in the present study revealed that dopamine cells in the VTA were almost completely destroyed. Thus, it is reasonable to consider that dopaminergic projections from the ventral midbrain, especially from the VTA, are responsible for overconsumption of highly preferred fluids. However, because destroyed areas tended to invade beyond the VTA, both in the electrolytic and 6-OHDA lesions in the present study, it is possible that the areas around the VTA, such as the substantia nigra, also function in overconsumption of highly preferred fluids. In fact, a radiofrequency lesion study has shown that both dopaminergic and non-dopaminergic neurons in an intermediate zone between the VTA and substantia nigra are critically involved in producing aphagia and adipsia [22].
5. General discussion The consumption of sucrose after saline injection is not different between the lesioned and Sham groups in Experiments 2 and 3. In contrast, the 24-h two-bottle choice test clearly revealed the decreased preference for sucrose in the lesioned group in Experiment 1. Although these results seem to be inconsistent, several methodological differences between these experiments may explain the discrepancy; electrolytic versus 6-OHDA lesions, water-replete versus water-deplete, one-bottle versus two-bottle presentations and 24-h versus 2-h intake. Under the water-replete condition in Experiment 1, the preference for a particular fluid could be detected by the 24-h free consumption of the fluid from the two bottles. On the other hand, under the water-deprived conditions in Experiments 2 and 3, both groups of rats were so motivated to drink water that the consumption of sucrose seemed to be maximum during the 2-h intake test after saline injection. Accordingly, the preference for sucrose could not be assessed precisely under waterdeprived conditions. Rather, the important point is that the injection of midazolam or morphine induced overconsumption of sucrose in the Sham but not in the lesioned animals. Thus, throughout these three experiments, lesions to the VTA selectively disturbed overconsumption of highly preferred fluids. There are several explanations which may account for these results. First, it is possible that lesions of the VTA disrupt sensory and/or motor aspects of drinking behavior. Although VTA-lesioned rats were reported to show locomotor hyperactivity and hypoexploration [9], these motor disturbances seem to have had little effect, if any, on drinking behavior under
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Fig. 5. Photomicrographs of the ventral midbrain stained for tyrosine hydroxylase immunoreactivity of a rat that received 6-OHDA lesions to the VTA (A) and a Sham-lesioned rat (B). The number of immunoreactive neurons is much less in the 6-OHDA lesioned rat than in the Sham animal. IP, interpeduncular nucleus; SN, substantia nigra. Scale bar in B 500 mm.
the relatively long intake tests in the present study. In line with this explanation, lesions of the VTA were found to have little effect on water intake elicited by the microinjection of angiotensin II into the preoptic area [18]. Because VTAx rats displayed almost normal preferred or aversive responses to all the taste stimuli except for 0.1 M sucrose, as revealed in the present study, the present results cannot be attributed to general sensory or motor disturbances. Second, it is possible that lesions of the VTA reduce the perceived palatability. Although both midazolam and morphine similarly facilitate the consumption of sucrose in Sham animals, presumably due to an increased taste pleasantness, their action sites in the brain are suggested to be quite different [14,15,30,31]. These results favor the notion that the VTA is a common path for reward including preferred tastes [27]. It is unlikely, however, for reasons described in the earlier discussion of Experiment 2. In fact, it was shown that the mesotelencephalic dopamine systems are not concerned with taste pleasantness using the taste reactivity test [2]. Third, lesions of the VTA may disrupt the motivation to drink more of naturally preferred fluids. This notion is supported by the results that VTA-lesioned rats became insensitive to motivational thirst and hunger stimuli [25,26]. Under such decreased motivation, intake of the fluid in lesioned rats would be lower than those of the Sham animals, even if the perceived palatability of these rats remained normal. In this regard, Berridge [1] recently introduced a hypothesis that natural reward
contains distinguishable psychological or functional components, i.e. ‘liking’ and ‘wanting’. According to the hypothesis, ‘liking’ corresponds closely to the concept of palatability and ‘wanting’ corresponds more closely to appetite or craving. Because the perceived palatability of VTAx rats seems to be normal, as determined by the results of Experiments 2 and 3, it is unlikely that the VTA is critically concerned with a ‘liking’ process. Rather, the behavioral deficits in the VTAx rats in the present study may be ascribed to some disturbances of a ‘wanting’ process. Therefore, the present results also favor the suggestion that ‘liking’ and ‘wanting’ have separable neural substrates [1]. Thus, the VTAx rats appear to be unable to utilize adequately the information of taste pleasantness in consumption of fluids. Taking all the present and previous results together, it may be relevant to say that dopaminergic mediation in the VTA is required to enhance the consumption of normally preferred fluids exclusively.
Acknowledgements This work was supported by Grants-in-Aid for scientific research (Nos. 11680787 to TS and 11557135 to TY) from the Ministry of Education, Science, Sports and Culture of Japan and by the Research for the Future Program (JSPS-RFTF97L00906 to TY) of the Japan Society for the Promotion of Science.
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[22] Nadaud D, Simon H, Herman JP, Le Moal M. Contributions of the mesencephalic dopaminergic system and the trigeminal sensory pathway to the ventral tegmental aphagia syndrome in rats. Physiol Behav 1984;33:879 /87. [23] Nakamura K, Ono T, Tamura R, Indo M, Takashima Y, Kawasaki M. Characteristics of rat lateral hypothalamic neuron responses to smell and taste in emotional behavior. Brain Res 1989;491:15 /32. [24] Numan M, Smith HG. Maternal behavior in rats: evidence for the involvement of preoptic projections to the ventral tegmental area. Behav Neurosci 1984;98:712 /27. [25] Papp M, Bal A. Motivational versus motor impairment after haloperidol injection or 6-OHDA lesions in the ventral tegmental area or substantia nigra in rats. Physiol Behav 1986;38:773 /9. [26] Papp M, Bal A. Separation of the motivational and motor consequences of 6-hydroxydopamine lesions of the mesolimbic or nigrostriatal system in rats. Behav Brain Res 1987;23:221 /9. [27] Park TH, Carr KD. Neuroanatomical patterns of Fos-like immunoreactivity induced by a palatable meal and meal-paired environment in saline- and naltrexone-treated rats. Brain Res 1998;805:169 /80. [28] Parker LA, Maier S, Rennie M, Crebolder J. Morphine- and naltrexone-induced modification of palatability: analysis by the taste reactivity test. Behav Neurosci 1992;106:999 /1010. [29] Paxinos G, Watson C. The rat brain in stereotaxic coordinates, 2nd ed. San Diego: Academic Press, 1986. [30] Pecin˜a S, Berridge KC. Central enhancement of taste pleasure by intraventricular morphine. Neurobiology (Bp) 1995;3:269 /80. [31] Pecin˜a S, Berridge KC. Brainstem mediates diazepam enhancement of palatability and feeding: microinjections into fourth ventricle versus lateral ventricle. Brain Res 1996;727:22 /30. [32] Pfaffmann C. Taste mechanisms in preference behavior. Am J Clin Nutr 1957;5:142 /7. [33] Rabe EF, Corbit JD. Postingestional control of sodium chloride solution drinking in the rat. J Comp Physiol Psychol 1973;84:268 /74. [34] Reilly S, Pritchard TC. Gustatory thalamus lesions in the rat. I. Innate taste preferences and aversions. Behav Neurosci 1996;110:737 /45. [35] Rideout HJ, Parker LA. Morphine enhancement of sucrose palatability: analysis by the taste reactivity test. Pharmacol Biochem Behav 1996;53:731 /4. [36] Shimura T, Grigson PS, Norgren R. Brainstem lesions and gustatory function. I. The role of the nucleus of the solitary tract during a brief intake test in rats. Behav Neurosci 1997;111:155 / 68. [37] Tanda G, Di Chiara G. A dopamine-m1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and nonpsychostimulant drugs of abuse. Eur J Neurosci 1998;10:1179 /87. [38] Treit D, Berridge KC. A comparison of benzodiazepine, serotonin, and dopamine agents in the taste-reactivity paradigm. Pharmacol Biochem Behav 1990;37:451 /6. [39] Wise RA. Neuroleptics and operant behavior: the anhedonia hypothesis. Behav Brain Sci 1982;5:39 /87. [40] Wise RA, Schwartz HV. Pimozide attenuates acquisition of leverpressing for food in rats. Pharmacol Biochem Behav 1981;15:655 /6. [41] Wise RA, Spindler J, deWit H, Gerber GJ. Neuroleptic-induced ‘anhedonia’ in rats: pimozide blocks reward quality of food. Science 1978;201:262 /4.
Research report
Ventral tegmental lesions reduce overconsumption of normally preferred taste fluid in rats Tsuyoshi Shimura *, Yoko Kamada 1, Takashi Yamamoto Laboratory of Behavioral Physiology, Graduate School of Human Sciences, Osaka University, 1-2 Yamadaoka, Suita, Osaka 565-0871, Japan Received 17 September 2001; received in revised form 23 November 2001; accepted 23 November 2001
Abstract Previous studies have suggested that the brain regions along the taste pathway and its anatomical interfacing with the brain reward system are concerned with palatability-induced consumption. To clarify whether the ventral tegmental area (VTA) is involved in the behavioral expression induced by taste pleasantness, we examined the effects of lesions to the VTA on the consumption of taste stimuli in rats. (1) Bilateral extensive electrolytic lesions to the VTA selectively reduced the consumption of a normally preferred taste fluid (0.1 M sucrose) compared to that of sham-operated animals during a 24-h two-bottle choice test. The consumption of other fluids, including non-preferred taste fluids (HCl and quinine hydrochloride) was not different between the lesioned and sham animals. (2) The injection of midazolam (3 mg/kg), a benzodiazepine agonist, or morphine (2 mg/kg) significantly increased the consumption of 0.1 M sucrose fluids in the sham animals. The same injections, however, failed to increase intake of the 0.1 M sucrose in rats with 6-hydroxydopamine lesions of the VTA. Neither midazolam nor morphine modified the intake of nonpreferred quinine (0.0003 M) solution in both the lesioned and sham animals. These results suggest that dopaminergic mediation in the VTA is required to enhance the consumption of normally preferred fluids exclusively. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ventral tegmental area; Taste; Motivation; Dopamine; Benzodiazepine; Opiate; Rat
1. Introduction The pleasantness of taste powerfully influences the preference for food and fluids, and the level of consumption [32]. Particularly rewarding tastes can lead to considerable overconsumption. For example, non-deprived rats consumed large quantities of sweet solutions of sucrose, glucose and sodium saccharin [8]. Previous studies have suggested that brain regions along the taste pathway [11,23] and its anatomical interfacing with the brain reward system [19,20,37] are involved in palatability-induced consumption. In addition, it has been reported that ingestion of a palatable meal increased Fos-like immunoreactivity in several regions
* Corresponding author. Tel.: 81-6-6879-8048; fax: 81-6-68798010 E-mail address: [email protected] (T. Shimura). 1 Present address: Research Institute for New Product Development, Suntory Ltd., 1-1 Wakayama-dai, Shimamoto-cho, Mishima-gun, Osaka 618-8503 Japan.
of limbic projections of the gustatory pathway and the mesoaccumbens dopamine pathway, viewed as a final common reward path [27]. Thus, the mesolimbic dopamine system originating in the ventral tegmental area (VTA) seems to mediate some reward or motivational processes of food and fluid intake. While it was suggested that palatability-induced consumption was less likely mediated by the nigrostriatal dopamine system [3], the role of the VTA in the behavioral expression induced by taste pleasantness is still unclear. Recent evidence [13] has revealed that the extracellular levels of dopamine in the nucleus accumbens, the major projection site of VTA dopaminergic neurons, increased in response to licking of highly preferred sucrose but not to water. Furthermore, reverse microdialysis of nomifensine, the dopamine reuptake blocker, into the nucleus accumbens led to an increase of sucrose intake [13]. These findings strongly suggest that the mesoaccumbens dopamine system is concerned with the ingestion of preferred tastes. Therefore, we examined the effects of
0166-4328/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 1 ) 0 0 4 6 1 - 2
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lesions to the VTA on the consumption of normally preferred and non-preferred taste fluids in rats.
2. Experiment 1 An earlier study reported that lowered intake of preferred stimuli after injection of a dopamine antagonist is due to the reduction of reward value [41]. If the mesolimbic dopamine system was critically involved in palatability-induced ingestion of food and fluids, lesions to the VTA would disrupt the normal intake pattern of preferred taste stimuli. Therefore, we examined the effects of electrolytic lesions of the VTA on preference/ aversion responses to four basic taste stimuli using the conventional two-bottle preference test. 2.1. Materials and methods 2.1.1. Subjects The subjects were 12 male Wistar rats (Nihon Dobutsu, Osaka, Japan) housed individually in polycarbonate cages in a temperature-controlled room with a 12:12 h light:dark cycle (light on at 07:00 h). All experimental manipulations and treatments were conducted during the light phase of the cycle. The rats were maintained with free access to rat chow (Oriental Yeast, MF) and water was available as noted below for experimental purposes. On the day of surgery, their body weights ranged from 265 to 310 g. Testing occurred during the light hours of a 12-h light:dark cycle. The study was approved by the animal welfare committee of our institute and carried out in accordance with the guidelines of the National Institute of Health of the USA. 2.1.2. Surgery Six experimental subjects (Group VTAx) were randomly selected and received bilateral electrolytic lesions to the VTA. The other six subjects (Sham Group) received sham lesions. Under Nembutal anesthesia (50 mg/kg, i.p.), the VTA lesions were made on each side of the brains by passing a 1-mA current for 20 s through a stainless steel electrode (0.25 mm in diameter) insulated with Epoxylite except at the tip. For the Sham rats, the electrodes were bilaterally lowered 2 mm dorsal to the VTA, but no current was passed. The coordinates for the electrode placements were 6.0 mm posterior to the bregma, 90.6 mm from the midline and 8.2 mm ventral to the surface of the skull. After surgery, all subjects were maintained on ad libitum food and water. Because aphagia and adipsia were observed after surgery in some VTAx rats, they were given wet mash (a 4:7 mixture of powdered rat chow and water) until they recovered.
2.1.3. Procedure Preference testing began after a minimum of 14 postoperative recovery days. At this time, each rat was given 24-h access to two plastic syringes equipped with stainless steel drinking spouts: one containing distilled water, the other a taste stimulus (reagent grade chemical dissolved in distilled water). The initial location (left or right) of the stimulus syringe varied randomly across tastants. The syringes were weighed before and after testing to determine intake volume. The four basic taste stimuli and the order of presentation were as follows: sucrose (0.01, 0.1, 1.0 M); NaCl (0.01, 0.1, 1.0 M); quinine hydrochloride (quinine, 0.00003, 0.0003, 0.003 M); and hydrochloric acid (HCl, 0.001, 0.01, 0.1 M). The amounts consumed during each 24-h period were recorded. 2.1.4. Data analysis The data were analyzed using a three-way (Group Fluid Concentration) analysis of variance (ANOVA) for repeated-measures. Post hoc comparisons between means were carried out using the Scheffe´’s multiple comparison’s test. Statistical tests were performed using Statistica (StatSoft Inc., Tulsa, OK) and a result was considered significant if P B 0.05. 2.1.5. Histology At the end of the experiments, the rats received an overdose of Nembutal (80 mg/kg) and were perfused intracardially with physiological saline followed by 10% formalin. The brains were removed and stored in a solution of 30% sucrose until they sank. After the brains were sectioned coronally at 60 mm on a freezing microtome, consecutive sections through the midbrain were stained for cell bodies with Cresyl violet. Lesion reconstructions were made on drawings derived from the Paxinos and Watson’s atlas [29], with the use of a microscope and a personal computer. 2.2. Results The mean 24-h intake of water during the day before the two-bottle test in both groups was not significantly different (26.391.9 g (mean9S.E.) for VTAx and 32.192.5 g for Sham; F (1, 10) 3.49, P 0.05). The body weight gain during the 12 days of the two-bottle test was 39.295.4 g (mean9S.E.) in VTAx and 48.394.4 g in Sham. The difference, however, was not significant. Fig. 1 illustrates the mean 24-h intake of water and taste solutions in VTAx and Sham rats. For sucrose, both groups of subjects clearly consumed more taste solution at 0.1 and 1.0 M than water. At 0.01 M, however, both groups equally consumed sucrose and water. A three-way ANOVA revealed significant main effects of Group (F (1, 20) 5.81, P B 0.05), Fluid
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Fig. 1. Mean (9S.E.) 24-h fluid intake during the sucrose, NaCl (sodium chloride), quinine (quinine hydrochloride) and HCl (hydrochloric acid) concentration series for control (Sham) and rats with lesions of the ventral tegmental area (VTAx) in the 24-h, two-bottle tests of Experiment 1. *P B 0.05 from the corresponding solution of Sham.
(F (1, 20) 38.57, P B 0.001) and Concentration (F (2, 40) 17.23, P B0.001). Fluid Concentration (F (2, 40) 45.43, P B0.001) and Group Fluid Concentration interactions (F (2, 40) 4.42, P B0.05) were also significant. Post hoc comparisons showed that the Sham rats consumed significantly more of the 0.1 M sucrose than the VTAx rats (P B0.01). There was no difference in the intake of the 1.0 M sucrose between the VTAx and Sham rats. For NaCl, there were significant main effects of Fluid (F (1, 20) 11.97, P B 0.01) and Concentration (F (2, 40) 4.73, P B 0.05). Significant Fluid Concentration (F (2, 40) 13.91, P B 0.001) and Group Fluid Concentration interactions (F (2, 40) 6.64, P B 0.01) were also detected. Post hoc comparisons, however, revealed that there were no significant differences in the intake of NaCl at each concentration and water in both groups. Nevertheless, as seen in Fig. 1, it was noted that the Sham animals tended to consume more of the 0.1 M NaCl than the VTAx rats. There were only significant main effects of Fluid for quinine (F (1, 20) 361.46, P B 0.001) and HCl (F (1, 20) 499.74, P B0.001). Thus, both groups seldom selected quinine and HCl solutions to ingest at any concentration. Fig. 2 shows the location of the lesions in the ventromedial region of the midbrain marked on sections of brain from Paxinos and Watson’s rat stereotaxic atlas [29]. From rostral to caudal, the VTA was almost fully destroyed in all six VTAx rats. Electrolytic lesions, however, sometimes invaded structures such as the
Fig. 2. (A) A schematic representation of the size and extent of the electrolytic lesions to the VTA. The portion shaded in black indicates the area common to all lesions and the black outline indicates the largest extent of all lesions. The number to the right indicates the distance caudal from the bregma in millimeters. (B) A photomicrograph of an example of electrolytic lesions in the ventral midbrain. Scale bar 2 mm.
substantia nigra, red nucleus, medial lemniscus, superior cerebellar peduncle, paranigral nucleus, mammillary peduncle, interfascicular nucleus, interpeduncular nucleus, retrorubral field and cerebral peduncle.
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2.3. Discussion The VTAx rats displayed almost normal preferred or aversive responses to all the taste stimuli except for 0.1 M sucrose. This finding suggests that their taste sensations remained intact. Some investigators have reported that lesions of the VTA disturb oral movements [17,24]. However, since the intake of water during the day before the two-bottle test in both groups was not significantly different, the VTAx rats in the present study seemingly showed few drinking disorders during the 24-h intake test. It is widely accepted that 0.1 M sucrose is highly preferred by rats [32,36]. A brief 15-min intake test [34] has demonstrated that 0.1 M NaCl is also a preferred solution for rats. Significantly less intake of 0.1 M sucrose and marginally less intake of 0.1 M NaCl in VTAx, therefore, indicate that the VTA might be exclusively involved in the consumption of highly preferred solutions. In contrast, responses to nonpreferred HCl and quinine were not different between both groups, suggesting that the VTA was not concerned with the intake of aversive tastes. It was noted, however, that the low consumption of HCl and quinine at the concentrations in the present experiment obscured a possible modification in consumption of these fluids at lower concentrations. Future studies will be necessary to determine the effects of VTA lesions on responses to normally aversive taste fluids at lower concentrations. Injections of pimozide, a dopamine antagonist, have been reported to block the reward value of food and fluids [10,40,41]. These observations support the anhedonia hypothesis [39] that dopamine systems amplify the hedonic impact of positive reinforcers. However, because of the extensive non-selective electrolytic lesions of the VTA in the present experiment, we cannot determine whether the dopamine cells in the VTA critically contributed to these behavioral results. The examination of this issue was included in the next experiment.
3. Experiment 2 The results of Experiment 1 demonstrate that the VTA is related to the consumption of highly preferred fluids under the conventional two-choice intake test. Increasing evidence suggests that benzodiazepine agonists facilitate feeding in animals due to a specific enhancement of the perceived palatability of food and fluids [2,5,6,38]. In Experiment 2, therefore, we compared the effects of benzodiazepine on the consumption of taste solutions between VTA-lesioned and shamoperated animals. We used a relatively shorter intake test in Experiment 2 because evidence indicates that post-ingestive factors strongly affect the taste preference [21,33]. Furthermore, since lesions aimed to the VTA in
the Experiment 1 were extensive and non-specific, we tried to make more specific lesions by injection of 6hydroxydopamine (6-OHDA), which is known as a catecholamine-specific neurotoxin. 3.1. Materials and methods 3.1.1. Subjects The subjects were 16 male Wistar rats (Nihon Dobutsu, Osaka, Japan). They were maintained as described in Experiment 1. On the day of surgery, their body weights ranged from 240 to 320 g. 3.1.2. Surgery Eight experimental subjects were randomly selected and received bilateral injections of 6-OHDA into the VTA under Nembutal anesthesia (50 mg/kg i.p.). The stereotaxic coordinates were 6.0 mm posterior to the bregma, 90.6 mm from the midline, and 8.2 mm ventral to the surface of the skull. 6-OHDA (8 mg) was dissolved in 4 ml of an isotonic saline containing 0.2 mg/ml of ascorbic acid and injected into the structures using a Hamilton syringe (10 ml). The injection solution was prepared immediately before administration and was infused at a rate of 1 ml/2 min. Eight sham animals received vehicle (isotonic saline with an appropriate amount of ascorbic acid). In order to protect noradrenergic neurons from the toxic effect of 6-OHDA, the rats were pretreated with desipramine (Sigma; 25 mg/kg i.p.) 30 min before the injection of 6-OHDA. 3.1.3. Procedure Each rat was deprived of water and given access to water for 2 h in the morning (09.00/11.00 h) and 1 h in the afternoon (16.00 /17.00 h) in a plastic syringe equipped with a stainless steel spout for 5 days. On the test day at 08.30 h, the rats were injected intraperitoneally with 0.3, 1.0 or 3.0 mg/kg of midazolam, a benzodiazepine agonist (in solution with physiological saline) or physiological saline solution, in a volume of 1 mg/kg. Later (30 min), they were presented with a syringe containing 0.1 M sucrose or 0.0003 M quinine solution for 2 h. The amounts consumed from each syringe were measured. A within-subjects design was used and each animal was tested at each dose and solution following injection of the drug vehicle. The order of injection was counterbalanced across the subjects, with 48 h between consecutive injections. 3.1.4. Data analysis The data were analyzed using a two-way (Group Dose) ANOVA. Post hoc comparisons between means were conducted, when appropriate, using the Scheffe´’s multiple comparison’s test.
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3.2. Results Fig. 3 shows the 2-h intake of 0.1 M sucrose and 0.0003 M quinine after systemic injection of midazolam or vehicle. For sucrose, the intake of Sham rats increased dose-dependently. A two-way ANOVA confirmed significant main effects of Group (F (1, 14) 18.06, P B0.001) and Dose (F (3, 42) 6.61, P B0.001). A Group Dose interaction was also significant (F (3, 42) 3.57, P B 0.05). The lesioned rats did not show any increase in the intake of sucrose after injection of midazolam, viz., Sham rats consumed significantly more sucrose than the lesioned rats at the dose of 3 mg/kg (P B0.001). For quinine, the 2-h intake of both groups did not differ at any dose. As clearly shown in Fig. 3, intakes of sucrose were higher than those of quinine at any dose in both groups. 3.3. Discussion It is well-documented that benzodiazepine-induced feeding is due to a specific enhancement of the perceived palatability of food and fluids [2,5,6]. In line with this notion, the Sham animals in the present experiment showed an increased intake of sucrose after an injection of 3 mg/kg midazolam. The same injection of midazolam, however, failed to facilitate the consumption of non-preferred quinine. If it was midazolam that enhanced the consumption of sucrose in the Sham animals due to an increased pleasantness, lesions of the VTA might disrupt the perceived palatability. However, the following evidence may rule out this possibility. First, if lesions to the VTA reduced the perceived palatability, the intake of sucrose in VTAx would be lower than that of Sham animals after injection of saline. However, the lesioned rats consumed almost an equal amount of sucrose as the Sham animals after saline injection (see Fig. 3). Second, lesions of the mesotelencephalic dopamine systems did not change the mimetic responses to taste stimuli [1,3] when assessed with the objective index
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of palatability developed by Grill and Norgren [12]. Preliminary observations in our laboratory also revealed no change in taste reactivity to preferred fluids in 6OHDA lesioned rats. Third, recent evidence suggests that receptors in the lower brainstem, such as the parabrachial nucleus (a second taste relay), are the primary substrate for the benzodiazepine-induced enhancement of taste palatability [14,15,31].
4. Experiment 3 Opioid agonists, as well as benzodiazepine agonists, facilitate feeding in animals presumably because of enhancing the perceived pleasantness of taste [5,7,28,35]. In the final experiment, we investigated the effects of systemic injection of an opioid agonist, morphine, on the consumption of taste solutions in the VTA-lesioned and Sham animals. 4.1. Materials and methods 4.1.1. Subjects The subjects were 16 male Wistar rats from Experiment 2. They were maintained as described above. 4.1.2. Procedure Each rat was deprived of water and given access to water for 2 h in the morning (09.00/11.00 h) and 1 h in the afternoon (16.00 /17.00 h) in a plastic syringe equipped with a stainless steel spout for 5 days. On the test day at 08:30 h, the rats were subcutaneously injected with either 2 mg/kg of morphine hydrochloride (Takeda, Japan) or the saline vehicle at a volume of 1 ml/kg. The dose of morphine has been shown to be effective in facilitating the consumption of food and fluids in non-deprived rats [4]. Later (30 min), they were presented with a syringe containing 0.1 M sucrose or 0.0003 M quinine solution for 2 h. The amounts consumed from each syringe were measured. A withinsubjects design was used. On each test day, each rat received either morphine or vehicle. The order of injection was counterbalanced across the subjects, with 48 h between consecutive injections. 4.1.3. Data analysis The data were analyzed using a two-way (Group Injection) ANOVA. Post hoc comparisons between means were conducted, when appropriate, using the Scheffe´’s multiple comparison’s test.
Fig. 3. Mean (9S.E.) 2-h intake of 0.1 M sucrose and 0.0003 M quinine after injections of midazolam (0.3 /3.0 mg/kg) or of saline in Sham and rats with 6-OHDA lesions of the VTA. *P B 0.001 from the corresponding solution of Sham.
4.1.4. Histology At the end of the experiments, the rats received an overdose of Nembutal (80 mg/kg) and were perfused intracardially with 0.02 M phosphate-buffered saline followed by 4% paraformaldehyde. The brains were
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removed and stored in a solution of 30% sucrose until they sank. After the brains were sectioned coronally at 60 mm on a freezing microtome, consecutive sections through the midbrain were stained for tyrosine hydroxylase immunoreactivity using the avidin-biotin-peroxidase technique [16]. 4.2. Results Fig. 4 shows the 2-h intake of 0.1 M sucrose and 0.0003 M quinine after subcutaneous injection of 2 mg/ kg of morphine or saline. A two-way ANOVA indicated significant main effects of Group (F (1, 14) 9.09, P B0.01) and Injection (F (1, 14) 14.02, P B0.01). Group Injection interaction was also significant (F (1, 14) 6.74, P B 0.05). The Sham animals consumed more sucrose after morphine injection than after saline injection (P B 0.001). However, the intake of sucrose after injections of morphine in the lesioned animals did not differ from that after saline injection (P 0.05). There were no significant differences in the intake of quinine across groups and injections. Fig. 5 shows the photomicrographs of the ventral midbrain stained for tyrosine hydroxylase immunoreactivity. In the Sham animals, immunoreactive neurons were extensively located in the ventral midbrain. The number of immunoreactive neurons in 6-OHDA-lesioned rats was much less compared with the Sham animals, as shown in Fig. 5. Immunoreactive neurons, thus, almost disappeared in the VTA of the lesioned animals. However, the 6-OHDA lesions were not restricted in the VTA, but invaded the medial part of the substantia nigra in most cases. 4.3. Discussion Morphine selectively enhanced the sucrose intake in the Sham animals, but not in the lesioned rats. This effect is similar to that of midazolam in Experiment 2. It is suggested that opioid receptors in the forebrain, not in the hindbrain, are mainly responsible for increased
Fig. 4. Mean (9S.E.) 2-h intake of 0.1 M sucrose and 0.0003 M quinine after injections of morphine (2 mg/kg) or of saline in Sham and rats with 6-OHDA lesions of the VTA. *P B 0.05 versus saline injection of Sham.
feeding due to a selective facilitation of the perceived palatability [30]. According to this suggestion, similar to midazolam, it is less likely that morphine directly acts on the VTA neurons to facilitate taste palatability. Histological examinations in the present study revealed that dopamine cells in the VTA were almost completely destroyed. Thus, it is reasonable to consider that dopaminergic projections from the ventral midbrain, especially from the VTA, are responsible for overconsumption of highly preferred fluids. However, because destroyed areas tended to invade beyond the VTA, both in the electrolytic and 6-OHDA lesions in the present study, it is possible that the areas around the VTA, such as the substantia nigra, also function in overconsumption of highly preferred fluids. In fact, a radiofrequency lesion study has shown that both dopaminergic and non-dopaminergic neurons in an intermediate zone between the VTA and substantia nigra are critically involved in producing aphagia and adipsia [22].
5. General discussion The consumption of sucrose after saline injection is not different between the lesioned and Sham groups in Experiments 2 and 3. In contrast, the 24-h two-bottle choice test clearly revealed the decreased preference for sucrose in the lesioned group in Experiment 1. Although these results seem to be inconsistent, several methodological differences between these experiments may explain the discrepancy; electrolytic versus 6-OHDA lesions, water-replete versus water-deplete, one-bottle versus two-bottle presentations and 24-h versus 2-h intake. Under the water-replete condition in Experiment 1, the preference for a particular fluid could be detected by the 24-h free consumption of the fluid from the two bottles. On the other hand, under the water-deprived conditions in Experiments 2 and 3, both groups of rats were so motivated to drink water that the consumption of sucrose seemed to be maximum during the 2-h intake test after saline injection. Accordingly, the preference for sucrose could not be assessed precisely under waterdeprived conditions. Rather, the important point is that the injection of midazolam or morphine induced overconsumption of sucrose in the Sham but not in the lesioned animals. Thus, throughout these three experiments, lesions to the VTA selectively disturbed overconsumption of highly preferred fluids. There are several explanations which may account for these results. First, it is possible that lesions of the VTA disrupt sensory and/or motor aspects of drinking behavior. Although VTA-lesioned rats were reported to show locomotor hyperactivity and hypoexploration [9], these motor disturbances seem to have had little effect, if any, on drinking behavior under
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Fig. 5. Photomicrographs of the ventral midbrain stained for tyrosine hydroxylase immunoreactivity of a rat that received 6-OHDA lesions to the VTA (A) and a Sham-lesioned rat (B). The number of immunoreactive neurons is much less in the 6-OHDA lesioned rat than in the Sham animal. IP, interpeduncular nucleus; SN, substantia nigra. Scale bar in B 500 mm.
the relatively long intake tests in the present study. In line with this explanation, lesions of the VTA were found to have little effect on water intake elicited by the microinjection of angiotensin II into the preoptic area [18]. Because VTAx rats displayed almost normal preferred or aversive responses to all the taste stimuli except for 0.1 M sucrose, as revealed in the present study, the present results cannot be attributed to general sensory or motor disturbances. Second, it is possible that lesions of the VTA reduce the perceived palatability. Although both midazolam and morphine similarly facilitate the consumption of sucrose in Sham animals, presumably due to an increased taste pleasantness, their action sites in the brain are suggested to be quite different [14,15,30,31]. These results favor the notion that the VTA is a common path for reward including preferred tastes [27]. It is unlikely, however, for reasons described in the earlier discussion of Experiment 2. In fact, it was shown that the mesotelencephalic dopamine systems are not concerned with taste pleasantness using the taste reactivity test [2]. Third, lesions of the VTA may disrupt the motivation to drink more of naturally preferred fluids. This notion is supported by the results that VTA-lesioned rats became insensitive to motivational thirst and hunger stimuli [25,26]. Under such decreased motivation, intake of the fluid in lesioned rats would be lower than those of the Sham animals, even if the perceived palatability of these rats remained normal. In this regard, Berridge [1] recently introduced a hypothesis that natural reward
contains distinguishable psychological or functional components, i.e. ‘liking’ and ‘wanting’. According to the hypothesis, ‘liking’ corresponds closely to the concept of palatability and ‘wanting’ corresponds more closely to appetite or craving. Because the perceived palatability of VTAx rats seems to be normal, as determined by the results of Experiments 2 and 3, it is unlikely that the VTA is critically concerned with a ‘liking’ process. Rather, the behavioral deficits in the VTAx rats in the present study may be ascribed to some disturbances of a ‘wanting’ process. Therefore, the present results also favor the suggestion that ‘liking’ and ‘wanting’ have separable neural substrates [1]. Thus, the VTAx rats appear to be unable to utilize adequately the information of taste pleasantness in consumption of fluids. Taking all the present and previous results together, it may be relevant to say that dopaminergic mediation in the VTA is required to enhance the consumption of normally preferred fluids exclusively.
Acknowledgements This work was supported by Grants-in-Aid for scientific research (Nos. 11680787 to TS and 11557135 to TY) from the Ministry of Education, Science, Sports and Culture of Japan and by the Research for the Future Program (JSPS-RFTF97L00906 to TY) of the Japan Society for the Promotion of Science.
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