NeuroscienceandBiobehavioralReviews, Vol. 16, pp. 519-524, 1992
0149-7634/92 $5.00 + .00 Copyright ©1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
Decreased Reactivity to Sweetness Following Chronic Exposure to Mild Unpredictable Stress or Acute Administration of Pimozide DAVID
SAMPSON,
RICHARD
MUSCAT*,
GAVIN
PHILLIPSt
AND
PAUL
WILLNER I
D e p a r t m e n t o f P s y c h o l o g y , City o f L o n d o n Polytechnic, Old Castle St., L o n d o n E1 7NT, U K
R e c e i v e d 5 F e b r u a r y 1992 SAMPSON, D., R. MUSCAT, G. PHILLIPS AND P. WILLNER. Decreased reactivity to sweetness following chronic exposure to mild unpredictable stress or acute administration of pimozide. NEUROSCI BIOBEHAV REV 16(4)519-524, 1992.- Consumption of a palatable wet mash was examined in rats subjected chronically (4-10 weeks) to unpredictable mild stress. Intake of mash containing 0, 10°70, or 20°70 additional sucrose was normal in stressed animals. In control animals, the addition of 30070 or 40070 sucrose caused a decrease in the quantity of mash consumed, but increased the rate of eating. Both the increase in eating rate and the decrease in intake, at high sucrose concentration, were markedly attenuated in stressed animals (which therefore had higher intakes of very sweet mash and lower rates of eating, relative to control animals). Like chronic mild stress, the dopamine receptor antagonist pimozide (0.2 mg/kg) also increased the intake of a wet mash with 30070 added sucrose, while decreasing the rate of consumption. Stressed animals were relatively insensitive to pimozide, though there were significant additive effects on duration of eating (increased) and on postprandial resting (suppressed). The failure of stressed animals to adapt their intake to increases in sweetness, and the similarities between the effects of chronic mild stress and acute pimozide, are compatible with the hypothesis that animals exposed to chronic mild stress are anhedonic. Chronic mild stress
Food consumption
Sweetness
Behavioural satiety sequence
C H R O N I C sequential exposure to mild unpredictable stress has been found to depress the consumption of, and preference for, palatable sweet solutions; these deficits may be reversed by chronic administration of tricyclic antidepressants (13,14,20,32). The decreased intake of sucrose or saccharin has been hypothesized to reflect a generalized anhedonia: that is, a decreased response to rewards o f all kinds (8). This hypothesis is supported by studies using the place conditioning paradigm (see below), and by the recent demonstration that chronic mild stress causes an increase in the threshold for brain stimulation reward (10). However, some data are, at first sight, difficult to reconcile with this interpretation. In sucrose intake tests, a decrease in consumption is observed only when dilute solutions are used: At higher concentrations, sucrose intake is unaffected by chronic mild stress (27,30). Similarly, food consumption is also unaffected, or even increased, in chronically stressed animals (16). Nevertheless, there is evidence from place preference studies that chronic mild stress reduces the rewarding properties o f concentrated sucrose solutions and of solid food pellets, even though the quantity consumed is not decreased. In the place conditioning paradigm, animals are rewarded in a distinctive environment,
Pimozide
Rats
and the strength of the reward is assessed by the increase in preference for the reward-associated environment on a subsequent drug-free choice trial (5). Place preference conditioning, using sweet solutions or food pellets as the reward, was abolished or attenuated in animals exposed chronically to unpredictable mild stress, and these effects were independent of the amount of the reward consumed during the conditioning trials (16). The present study was carried out to examine the structure o f feeding behaviour in animals exposed to chronic mild stress, in order to understand better this discrepancy between, on the one hand, the effects o f chronic mild stress on food intake (unaffected or increased), and on the other, the rewarding effects of food (decreased). Like chronic mild stress (27,30), dopamine (DA) receptor antagonists also selectively suppress the consumption of weak sucrose solutions, while sparing intake at higher concentrations (11,12,17,25). At first sight, these data appear to be incompatible with the D A hypothesis of reward (33), but intake measures do not necessarily provide an accurate measure of reward value. At low concentrations of sucrose or saccharin, changes in sweetness are monotonically related to changes in reward value, as assessed in choice tests. However, if con-
* Present address: Department of Biomedical Sciences, University of Malta, Msida, Malta. t Present address: Department of Anatomy, Downing St, Cambridge, U.K. To whom requests for reprints should be addressed. 519
520
SAMPSON ET AL.
centrated solutions are used, the relationship between intake in single bottle tests and preference in 2-bottle tests breaks down: At high concentrations, intake in a single bottle test falls as concentration rises, but the higher concentration of sucrose is nonetheless preferred to a lower concentration in a choice test (11,12,17,25). The decrease in intake at high sucrose concentrations is usually thought to result from postingestive satiety effects (9), though recent data cast doubt on the generality of this explanation (12,17). However, for present purposes, the issue is not the interpretation of the paradoxical decrease in intake of concentrated sucrose solutions, but rather, the recognition that at higher concentrations, intake may provide a misleading measure of reward value. In addition to their similar effects on the intake of sweet solutions, chronic mild stress and acute administration of DA receptor antagonists are similar in other respects. For example, both chronic mild stress (16) and DA receptor antagonists (22,23) attenuate both food- and amphetamine-induced place preference conditioning. Animals exposed chronically to mild stress also resemble DA receptor antagonist-treated animals in being subsensitive to DA agonists (14,15). In view of these similarities in the effects of chronic mild stress and DA receptor antagonists, we have also compared the effects of chronic mild stress on the structure of feeding behaviour with those of a DA receptor antagonist, pimozide. METHOD
Subjects 48 male Lister hooded rats, weighing approximately 290 g at the start of the experiment, were obtained from the National Institute for Medical Research (U.K.). The animals were singly housed, except where pair-housed as part of the stress procedure (see below).
Procedure Rats were first trained to consume a palatable wet mash, made by powdering 100 g chow (Dixons, Ware, UK) and mixing with 200 ml tap water. The sucrose content of the chow was approximately 10070 (manufacturers' figure). Mash was made up immediately before use, and provided in spillproof jars, in the home cage. Animals were not deprived of food or water prior to these sessions, which were 30 min in duration. When all animals consumed more than 5 g wet mash in the 30-min session, the animals were divided into two groups matched for intake (n = 24/group), one of which was subjected chronically to unpredictable mild stress, for a total of l0 weeks; a variety of mild stressors were applied, each for a period of between 0.5 and 20 h. The stress regime was similar to that used previously [e.g., (32)] and consisted of: two 20-h periods of food and water deprivation, one followed by 2 h of restricted access to food (scattering of a few 45 mg precision pellets in the cage); one additional 16-h period of water deprivation; two periods of continuous overnight illumination; two periods (7 and 17 h) of 45 ° cage tilt; one 17 h period of paired housing; one 17-h period in a soiled cage (100 ml water in sawdust bedding); two periods (3 and 5 h) of intermittent white noise (85 dB); three periods (7,9, and 17 h) of low intensity stroboscopic illumination (300 flashes/minute). All of the individual stressors used were classified as being, at worst, mildly stressful, under the terms of the relevant (U.K.) legislation, the Animals (Scientific Procedures) Act of 1986. Wet mash was provided, as described above, at weekly
intervals throughout the experiment. In weeks 5-8, additional sucrose was added to the mash, rising from 10°70 additional sucrose in week 5, through 20070 and 30°70 in weeks 6 and 7, to 40°70 in week 8. In week 9, the animals were tested with wet mash containing 30°7o additional sucrose. 2 h earlier, subgroups of animals (n = 12/group), matched for their performance in week 8, received an i.p. injection of pimozide (0.2 mg/kg, in a volume of 1 ml/kg) or vehicle; pimozide (Janssen, Wantage) was dissolved in a drop of glacial acetic acid and made up to volume with distilled water. Earlier studies have indicated that at this dose, pimozide impairs reinforcer efficacy without causing motor impairment (25,29,31). In each of weeks 4-9, in addition to measuring wet mash consumption, the animals were observed for the duration of the test, and every 15 s behaviour was scored, using a BBC microcomputer, in one of five mutually exclusive categories: eating, active, grooming, standing motionless, or lying down. For the purposes of analysis and presentation, the two latter categories were considered together as "resting behaviour"; instances of "standing" were infrequent, and excluding them from the "resting" category did not alter the pattern of results obtained. The category "active," which includes sniffing, rearing, and locomotion, was used when none of the other categories were applicable; thus, the use of this category does not carry any implication of a stimulant effect. Eating rate (g/observation) was calculated by dividing the weight of food consumed by the number of observations of eating. Eating bouts were defined as continuous runs of at least 3 consecutive eating observations; bout duration was calculated by dividing the number of eating observations by the number of bouts and multiplying by 15 to convert this figure to seconds.
Analysis Results were analyzed by analysis of variance, supplemented by tests of simple main effects. Intake data from weeks 5-8 were subjected to two-way analyses (Groups, Weeks), which were also applied to data on eating rate and eating time. Data from week 9 were also subjected to two-way
16
14
o--o CON
12
0.1]. However, the addition of 30% or 40% sucrose to the mash caused a substantial decrease in intake in the control group; this decrease was greatly attenuated in the stressed animals [Groups, F(1, 46) = 9.5, p < 0.005; Weeks, F(4, 184) = 30.0, p < 0.001; interaction, F(4, 184) = 5.1,p < 0.001]. Examination of eating behaviour showed that stressed animals failed to adapt their behaviour to changes in sucrose concentration (Fig. 2). Eating times were higher in stressed animals at all levels of sweetness [F(1, 46) = 66.3, p < 0.001]. However, while control animals showed a substantial decrease in eating time at the three highest sucrose concentrations, accompanied by a greatly increased rate of eating [simple main effect of Weeks: time, F(4, 184) = 21.4, p < 0.001; rate, F(4, 184) = 37.4, p < 0.001], no changes were apparent in the behaviour of stressed animals, on either measure [time, F(4, 184) = 2.1; rate, F(4, 184) = 0.9; both n.s.]. In both cases, the Groups X Weeks interaction terms were highly significant [time, F(4, 184) = 6.3; rate, F(4, 184) = 13.7; both p < 0.001]. Like stress, pimozide also increased the consumption of wet mash sweetened with 30% additional sucrose [simple main effect in non-stressed animals, F(1,44) = 6.2, p < 0.05], and eating time [F(1, 44) = 7.7, p < 0.01], but significantly decreased the rate of eating [F(I, 44) = 10.3, p < 0.01]. These effects of pimozide were seen primarily in non-stressed animals. In stressed animals, further increases in intake and decreases in eating rate were small and non-significant (Fig. 3), though the increase in eating time was marginally significant [F(1, 44) = 4.1, p < 0.05]. Bout duration was increased to a similar extent by both pimozide and stress, with a small further increase in the pimozide-treated stressed group. [Means (s): control vehicle, 137; control pimozide, 187; stressed vehicle, 189; pimozide vehicle, 214; Drug: F(l, 44) = 4.5, p < 0.05; Stress: F(1, 44) = 5.0, p < 0.05; interaction: F(l, 44) = 0.5, N.S.]. Untreated control animals displayed a typical behavioural satiety sequence (1): Most eating took place early in the session, followed by a period in which active behaviours were prominent, which was superceded in the second half of the session by resting (Fig. 4). In stressed animals, and following pimozide-treatment in non-stressed controls, this normal pattern of postprandial behaviour was preserved; however, the appearance of the behavioural satiety sequence was delayed. In both cases, significantly more time was devoted to eating during the early part of the session and as a result, the peak of active behaviour was shifted to the right. These changes were even more exaggerated in pimozide-treated stressed animals, which continued eating almost until the end of the session, with the result that the peak of active behaviour occurred in the final 10 min, and very little resting was possible. DISCUSSION
Although small amounts (10-20%) of sucrose added to the wet mash appeared to cause a small increase in intake, it should be noted that consumption of the unsweetened mash was not stable at the start of the experiment; thus, these increases could represent practice effects rather than responses to sweetness. However, when the concentration of added sucrose was increased to 30 or 40%, intake and eating time fell sharply in control animals. We have no evidence from the
EATING
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FIG. 4. Observations of eating (above) and resting (below) over the course of the 30 min session. A total of 20 observations were made in each 5 rain period; the figure does not show grooming and other active behaviours. VEH: vehicle; PIM: pimozide, 0.2 mg/kg; CON: control; STR: chronic mild stress. Values are means; error bars have been omitted for clarity. *,p < 0.05; *% p < 0.01; **% p < 0.001, relative to vehicle-treated nonstressed animals: +, p < 0.05; + + +, p < 0.001, relative to vehicle-treated stressed animals.
present study to support or refute any of the several potential explanations of this observation (increased caloric density, increased osmolality, increased sweetness, etc.). However, we note that a similar decrease in intake of very sweet liquids has been widely reported (l l, 12,15,17,2 l). Despite the decrease in intake, the sweeter mash diets supported a substantial increase in the rate of eating. Eating rate is usually considered to be closely related to palatability, as assessed, for example in preference studies (2,6,7,21,26). Similarly, very sweet sucrose so-
STRESS A N D P I M O Z I D E D E C R E A S E R E A C T I V I T Y T O S W E E T N E S S lutions are under-consumed relative to lower sucrose concentrations, but nevertheless, the sweeter solution is preferred in a choice test (17,35). The present data are consistent with the view that the quantity o f food consumed may not provide an accurate reflection o f its rewarding properties as revealed in choice tests. In keeping with this conclusion, we have previously observed that chronic mild stress did not decrease chow intake, but did decrease the rewarding effect of chow as assessed in the place conditioning procedure (16). Similarly, chronic mild stress decreased the intake o f dilute but not of concentrated sucrose solutions (27,30), but decreased the rewarding properties o f both dilute and concentrated solutions as assessed by place preference conditioning (16). In the present study, stressed animals were less reactive to the effects of changes in sucrose concentration on both the quantity o f mash eaten and the rate of eating; in other words, stressed animals behaved towards the sweet mash in a manner appropriate to a less sweet diet. While changes (or the absence o f changes) in intake may not be readily interpretable, the failure of stressed animals to increase eating rate is compatible with a decrease in the rewarding properties o f the very sweet diets. These data may, therefore, be compatible with the hypothesis that chronic mild stress causes anhedonia, a generalized decrease in sensitivity to rewards (15,16,30). D A receptor antagonists usually suppress rewarded behaviour (4,28); these effects are usually interpreted as a decrease in the rewarding properties of the reinforcer. This interpretation is supported by the many parallels between the effects of D A receptor antagonists and those o f dilution o f the reward (9,11,12,17,25,28,33,34). However, D A receptor antagonists can also cause a paradoxical increase in behaviours main-
523
tained by very sweet rewards, that fall on the descending limb of the inverted-U-shaped sweetness-performance function. Experiments carried out to exclude a number of alternative explanations have led us to conclude that these data most likely reflect a reward-attenuating action of D A antagonists (I 1,17-19). The effects of pimozide in the present study appear to provide another instance of this paradoxical performance-enhancing effect of DA antagonists. Although the effects of pimozide were rather small when averaged across the session, a prolongation of eating is clearly apparent from a more detailed examination of its time course [cf. (3)]. The time course analysis (Fig. 4) also shows clear additive effects of stress and pimozide. The similarities between the effects of chronic mild stress and those of D A antagonists may be more than coincidental. We have recently reported that chronic exposure to mild stress caused a decrease in sensitivity to the rewarding and locomotor activating effects of the D2 agonist, quinpirole, administered systemically or within the nucleus accumbens (15). Chronic mild stress causes abnormalities of D A neurotransmission in the nucleus accumbens (24,27), which may include a decrease in D2 receptor number (27). As D A receptor activation in the nucleus accumbens is crucial for the expression of rewarding effects in a variety of hehavioural paradigms (33), the anhedonic effects of chronic mild stress may result from a functional antagonism of D2 receptors within this structure. ACKNOWLEDGEMENTS This study was supported in part by the Medical Research Council of Great Britain. We also thank Janssen for the generous gift of pimozide.
REFERENCES I. Antin, J.; Gibbs, J.; Holt, J.; Young, R. C.; Smith, G. P. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J. Comp. Physiol. Psychol. 89:784-790; 1975. 2. Bellisle, F. Quantifying palatability in humans. Ann. N.Y. Acad. Sci. 575:363-374; 1989. 3. Blundell, J. E.; Latham, C. J. Characteristics of adjustments to the structure of feeding behaviour following pharmacological treatment: Effects of amphetamine and fenfluramine and the antagonism produced by pimozide and methergoline. Pharmacol. Biochem. Behav. 12:717-722, 1980. 4. Bradshaw, C. M.; Szabadi, E. Central neurotransmitter systems and the control of operant behaviour by "natural" positive reinforcers. In: Liebman, J. M.; Cooper, S. J., eds. The neuropharmacological basis of reward. Oxford: Oxford University Press; 1978:320-376. 5. Cart, G. D.; Fibiger, H. C.; Phillips, A. G. Conditioned place preference as a measure of drug reward. In: Liebman, J. M.; Cooper, S. J., eds. The neuropharmacological basis of reward. Oxford: Oxford University Press; 1989:264-319. 6. Davis, J. D. The effectiveness of some sugars in stimulating licking behavior in the rat. Physiol. Behav. I 1:39-45; 1973. 7. Davis, J. D. The microstructure of ingestive behavior. Ann. N.Y. Acad. Sci. 575:106-119; 1989. 8. Fawcett, J.; Clark, D. C.; Scheftner, W. A.; Gibbons, R. D. Assessing anhedonia in psychiatric patients. The pleasure scale. Arch. Gen. Psychiatr. 40:79-84; 1983a. 9. Geary, N.; Smith, G. P. Pimozide decreases the reinforcing effect of sham-fed sucrose in the rat. Pharrnacol. Biochem. Behav. 22: 787-790; 1985. 10. Moreau, J.-L.; Jenck, F.; Martin, J. R.; Mortas, P.; Haefely, W. W. Antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmental
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17. 18. 19. 20.
self-stimulation behavior in rats. Europ. Neuropsychopharmacol. 2:43-49; 1992. Muscat, R.; Willner, P. Effects of selective dopamine receptor antagonists on sucrose consumption and preference. Psychopharmacology 99:98-102; 1989. Muscat, R.; Kyprianou, T.; Osman, M.; Phillips, G.; Willner, P. Sweetness-dependent facilitation of sucrose drinking by raclopride is unrelated to calorie content. Pharmacol. Biochem. Behay. 40:209-213; 1991. Muscat, R.; Sampson, D.; Willner, P. Dopaminergic mechanism of imipramine action in an animal model of depression. Biol. Psychiatr. 28:223-230; 1990. Muscat, R.; Towell, A.; Willner, P. Changes in dopamine autoreceptor sensitivity in an animal model of depression. Psychopharmacology 94:545-550; 1988. Papp, M.; Muscat, R.; Willner, P. Subsensitivity to rewarding and locomotor stimulant effects of a dopamine agonist following chronic mild stress. Psychopharmacology (Bed.) (in press). Papp, M.; Willner, P.; Muscat, R. An animal model of anhedonia: Attenuation of sucrose consumptions and place preference conditioning by chronic mild unpredictable stress. Psychopharmacology 104:255-259; 1991. Phillips, G.; Willner, P.; Muscat, R. Reward-dependent suppression or facilitation of consummatory behaviour by raclopride. Psychopharmacology 105:355-360; 1991. Phillips, G.; Willner, P.; Muscat, R. Suppression or facilitation of operant behaviour by raclopride dependent on concentration of sucrose reward. Psychopharmacology 105:239-246, 1991. Phillips, G.; Willner, P.; Muscat, R. Anatomical substrates for neuroleptic-induced reward attenuation and neuroleptic-induced response decrement. Behav. Pharmacol. 2:129-141; 1991. Sampson, D.; Muscat, R.; Willner, P. Reversal of antidepressant
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25. 26. 27. 28.
S A M P S O N E T AL. action by dopamine antagonists in an animal model of depression. Psychopharmacology 104:491-495; 1990. Sclafani, A.; Clyne, A. E. Hedonic response of rats to polysaccharide and sugar solutions. Neurosci. Biobehav. Rev. 11:173180; 1987. Spyraki, C.; Fibiger, H. C.; Phillips, A. G. Attenuation by haloperidol of place preference conditioning using food reinforcement. Psychopharmacology 77:379-382; 1982. Spyraki, C.; Fibiger, H. C.; Phillips, A. G. Dopaminergic substrates of amphetamine-induced place preference conditioning. Brain Res. 253:185-193; 1982. Stamford, J. A.; Muscat, R.; O'Connor, J. J.; Patel, J.; Trout, S. J.; Wieczorek, W. J.; Kruk, Z. L.; Willner, P. Subsensitivity to reward following chronic mild stress is associated with increased release of mesolimbic dopamine. Psyehopharmacology 105:275282; 1991. Towell, A., Muscat, R.; Willner, P. Effects of pimozide on sucrose consumption and preference. Psychopharmacology 92:262264; 1987. Weinganen, I-I. P.; Watson, S. D. Sham feeding as a procedure for assessing the influence of diet palatability on food intake. Physiol. Behav. 28:401-407; 1982. Willner, P.; Klimek, V.; Golembiowska, K.; Muscat, R. Changes in mesolimbic dopamine may explain stress-induced anhedonia. Psychobiology 19:79-84; 1991. Willner, P.; Phillips, G.; Muscat, R. Suppression of rewarded
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33. 34. 35.
behaviour by neuroleptic drugs: Can't or won't, and why? In: Willner, P.; Scheel-Kruger, J., eds. The mesolimbic dopamine system: From motivation to action. Chichester: Wiley; 1991:249270. Willner, P.; Sampson, D.; Phillips, G.; Muscat, R. A matching law analysis of the effects of dopamine receptor antagonists. Psychopharmacology 101:560-567; 1990. Willner, P.; Sampson, D.; Papp, M.; Phillips, G.; Muscat, R. Animal models of anhedonia. In: Soubrie, P., ed. Animal models of psychiatric disorders, vol. 3. Basel: Karger; 1991:71-99. Willner, P.; Towell, A.; Muscat, R. Effects of amphetamine and pimozide on reinforcement and motor parameters in variable interval performance. J. Psychopharmacology 1:140-153; 1987. Willner, P.; Towell, A.; Sampson, D.; Muscat, R.; Sophokleous, S. Reduction of sucrose preference by chronic mild stress and its restoration by a tricyclic antidepressant. Psychopharmacology 93: 358-364; 1987. Wise, R. A.; Rompre, LP.-P. Brain dopamine and reward. Ann. Rev. Psychol. 191-225; 1989. Xenakis, S.; Sclafani, A. The effects of pimozide on the consumption of a palatable saccharin-glucose solution in the rat. Pharmacol. Biochem. Behav. 15:435-442; 1981. Young, P. T. Studies of food preference, appetite and dietary habit. IX. Palatability versus appetite as determinants of the critical concentrations of sucrose and sodium chloride. Comp. Psychol. Monogr. 19:1-44; 1949.
0149-7634/92 $5.00 + .00 Copyright ©1992 Pergamon Press Ltd.
Printed in the U.S.A. All rights reserved.
Decreased Reactivity to Sweetness Following Chronic Exposure to Mild Unpredictable Stress or Acute Administration of Pimozide DAVID
SAMPSON,
RICHARD
MUSCAT*,
GAVIN
PHILLIPSt
AND
PAUL
WILLNER I
D e p a r t m e n t o f P s y c h o l o g y , City o f L o n d o n Polytechnic, Old Castle St., L o n d o n E1 7NT, U K
R e c e i v e d 5 F e b r u a r y 1992 SAMPSON, D., R. MUSCAT, G. PHILLIPS AND P. WILLNER. Decreased reactivity to sweetness following chronic exposure to mild unpredictable stress or acute administration of pimozide. NEUROSCI BIOBEHAV REV 16(4)519-524, 1992.- Consumption of a palatable wet mash was examined in rats subjected chronically (4-10 weeks) to unpredictable mild stress. Intake of mash containing 0, 10°70, or 20°70 additional sucrose was normal in stressed animals. In control animals, the addition of 30070 or 40070 sucrose caused a decrease in the quantity of mash consumed, but increased the rate of eating. Both the increase in eating rate and the decrease in intake, at high sucrose concentration, were markedly attenuated in stressed animals (which therefore had higher intakes of very sweet mash and lower rates of eating, relative to control animals). Like chronic mild stress, the dopamine receptor antagonist pimozide (0.2 mg/kg) also increased the intake of a wet mash with 30070 added sucrose, while decreasing the rate of consumption. Stressed animals were relatively insensitive to pimozide, though there were significant additive effects on duration of eating (increased) and on postprandial resting (suppressed). The failure of stressed animals to adapt their intake to increases in sweetness, and the similarities between the effects of chronic mild stress and acute pimozide, are compatible with the hypothesis that animals exposed to chronic mild stress are anhedonic. Chronic mild stress
Food consumption
Sweetness
Behavioural satiety sequence
C H R O N I C sequential exposure to mild unpredictable stress has been found to depress the consumption of, and preference for, palatable sweet solutions; these deficits may be reversed by chronic administration of tricyclic antidepressants (13,14,20,32). The decreased intake of sucrose or saccharin has been hypothesized to reflect a generalized anhedonia: that is, a decreased response to rewards o f all kinds (8). This hypothesis is supported by studies using the place conditioning paradigm (see below), and by the recent demonstration that chronic mild stress causes an increase in the threshold for brain stimulation reward (10). However, some data are, at first sight, difficult to reconcile with this interpretation. In sucrose intake tests, a decrease in consumption is observed only when dilute solutions are used: At higher concentrations, sucrose intake is unaffected by chronic mild stress (27,30). Similarly, food consumption is also unaffected, or even increased, in chronically stressed animals (16). Nevertheless, there is evidence from place preference studies that chronic mild stress reduces the rewarding properties o f concentrated sucrose solutions and of solid food pellets, even though the quantity consumed is not decreased. In the place conditioning paradigm, animals are rewarded in a distinctive environment,
Pimozide
Rats
and the strength of the reward is assessed by the increase in preference for the reward-associated environment on a subsequent drug-free choice trial (5). Place preference conditioning, using sweet solutions or food pellets as the reward, was abolished or attenuated in animals exposed chronically to unpredictable mild stress, and these effects were independent of the amount of the reward consumed during the conditioning trials (16). The present study was carried out to examine the structure o f feeding behaviour in animals exposed to chronic mild stress, in order to understand better this discrepancy between, on the one hand, the effects o f chronic mild stress on food intake (unaffected or increased), and on the other, the rewarding effects of food (decreased). Like chronic mild stress (27,30), dopamine (DA) receptor antagonists also selectively suppress the consumption of weak sucrose solutions, while sparing intake at higher concentrations (11,12,17,25). At first sight, these data appear to be incompatible with the D A hypothesis of reward (33), but intake measures do not necessarily provide an accurate measure of reward value. At low concentrations of sucrose or saccharin, changes in sweetness are monotonically related to changes in reward value, as assessed in choice tests. However, if con-
* Present address: Department of Biomedical Sciences, University of Malta, Msida, Malta. t Present address: Department of Anatomy, Downing St, Cambridge, U.K. To whom requests for reprints should be addressed. 519
520
SAMPSON ET AL.
centrated solutions are used, the relationship between intake in single bottle tests and preference in 2-bottle tests breaks down: At high concentrations, intake in a single bottle test falls as concentration rises, but the higher concentration of sucrose is nonetheless preferred to a lower concentration in a choice test (11,12,17,25). The decrease in intake at high sucrose concentrations is usually thought to result from postingestive satiety effects (9), though recent data cast doubt on the generality of this explanation (12,17). However, for present purposes, the issue is not the interpretation of the paradoxical decrease in intake of concentrated sucrose solutions, but rather, the recognition that at higher concentrations, intake may provide a misleading measure of reward value. In addition to their similar effects on the intake of sweet solutions, chronic mild stress and acute administration of DA receptor antagonists are similar in other respects. For example, both chronic mild stress (16) and DA receptor antagonists (22,23) attenuate both food- and amphetamine-induced place preference conditioning. Animals exposed chronically to mild stress also resemble DA receptor antagonist-treated animals in being subsensitive to DA agonists (14,15). In view of these similarities in the effects of chronic mild stress and DA receptor antagonists, we have also compared the effects of chronic mild stress on the structure of feeding behaviour with those of a DA receptor antagonist, pimozide. METHOD
Subjects 48 male Lister hooded rats, weighing approximately 290 g at the start of the experiment, were obtained from the National Institute for Medical Research (U.K.). The animals were singly housed, except where pair-housed as part of the stress procedure (see below).
Procedure Rats were first trained to consume a palatable wet mash, made by powdering 100 g chow (Dixons, Ware, UK) and mixing with 200 ml tap water. The sucrose content of the chow was approximately 10070 (manufacturers' figure). Mash was made up immediately before use, and provided in spillproof jars, in the home cage. Animals were not deprived of food or water prior to these sessions, which were 30 min in duration. When all animals consumed more than 5 g wet mash in the 30-min session, the animals were divided into two groups matched for intake (n = 24/group), one of which was subjected chronically to unpredictable mild stress, for a total of l0 weeks; a variety of mild stressors were applied, each for a period of between 0.5 and 20 h. The stress regime was similar to that used previously [e.g., (32)] and consisted of: two 20-h periods of food and water deprivation, one followed by 2 h of restricted access to food (scattering of a few 45 mg precision pellets in the cage); one additional 16-h period of water deprivation; two periods of continuous overnight illumination; two periods (7 and 17 h) of 45 ° cage tilt; one 17 h period of paired housing; one 17-h period in a soiled cage (100 ml water in sawdust bedding); two periods (3 and 5 h) of intermittent white noise (85 dB); three periods (7,9, and 17 h) of low intensity stroboscopic illumination (300 flashes/minute). All of the individual stressors used were classified as being, at worst, mildly stressful, under the terms of the relevant (U.K.) legislation, the Animals (Scientific Procedures) Act of 1986. Wet mash was provided, as described above, at weekly
intervals throughout the experiment. In weeks 5-8, additional sucrose was added to the mash, rising from 10°70 additional sucrose in week 5, through 20070 and 30°70 in weeks 6 and 7, to 40°70 in week 8. In week 9, the animals were tested with wet mash containing 30°7o additional sucrose. 2 h earlier, subgroups of animals (n = 12/group), matched for their performance in week 8, received an i.p. injection of pimozide (0.2 mg/kg, in a volume of 1 ml/kg) or vehicle; pimozide (Janssen, Wantage) was dissolved in a drop of glacial acetic acid and made up to volume with distilled water. Earlier studies have indicated that at this dose, pimozide impairs reinforcer efficacy without causing motor impairment (25,29,31). In each of weeks 4-9, in addition to measuring wet mash consumption, the animals were observed for the duration of the test, and every 15 s behaviour was scored, using a BBC microcomputer, in one of five mutually exclusive categories: eating, active, grooming, standing motionless, or lying down. For the purposes of analysis and presentation, the two latter categories were considered together as "resting behaviour"; instances of "standing" were infrequent, and excluding them from the "resting" category did not alter the pattern of results obtained. The category "active," which includes sniffing, rearing, and locomotion, was used when none of the other categories were applicable; thus, the use of this category does not carry any implication of a stimulant effect. Eating rate (g/observation) was calculated by dividing the weight of food consumed by the number of observations of eating. Eating bouts were defined as continuous runs of at least 3 consecutive eating observations; bout duration was calculated by dividing the number of eating observations by the number of bouts and multiplying by 15 to convert this figure to seconds.
Analysis Results were analyzed by analysis of variance, supplemented by tests of simple main effects. Intake data from weeks 5-8 were subjected to two-way analyses (Groups, Weeks), which were also applied to data on eating rate and eating time. Data from week 9 were also subjected to two-way
16
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0.1]. However, the addition of 30% or 40% sucrose to the mash caused a substantial decrease in intake in the control group; this decrease was greatly attenuated in the stressed animals [Groups, F(1, 46) = 9.5, p < 0.005; Weeks, F(4, 184) = 30.0, p < 0.001; interaction, F(4, 184) = 5.1,p < 0.001]. Examination of eating behaviour showed that stressed animals failed to adapt their behaviour to changes in sucrose concentration (Fig. 2). Eating times were higher in stressed animals at all levels of sweetness [F(1, 46) = 66.3, p < 0.001]. However, while control animals showed a substantial decrease in eating time at the three highest sucrose concentrations, accompanied by a greatly increased rate of eating [simple main effect of Weeks: time, F(4, 184) = 21.4, p < 0.001; rate, F(4, 184) = 37.4, p < 0.001], no changes were apparent in the behaviour of stressed animals, on either measure [time, F(4, 184) = 2.1; rate, F(4, 184) = 0.9; both n.s.]. In both cases, the Groups X Weeks interaction terms were highly significant [time, F(4, 184) = 6.3; rate, F(4, 184) = 13.7; both p < 0.001]. Like stress, pimozide also increased the consumption of wet mash sweetened with 30% additional sucrose [simple main effect in non-stressed animals, F(1,44) = 6.2, p < 0.05], and eating time [F(1, 44) = 7.7, p < 0.01], but significantly decreased the rate of eating [F(I, 44) = 10.3, p < 0.01]. These effects of pimozide were seen primarily in non-stressed animals. In stressed animals, further increases in intake and decreases in eating rate were small and non-significant (Fig. 3), though the increase in eating time was marginally significant [F(1, 44) = 4.1, p < 0.05]. Bout duration was increased to a similar extent by both pimozide and stress, with a small further increase in the pimozide-treated stressed group. [Means (s): control vehicle, 137; control pimozide, 187; stressed vehicle, 189; pimozide vehicle, 214; Drug: F(l, 44) = 4.5, p < 0.05; Stress: F(1, 44) = 5.0, p < 0.05; interaction: F(l, 44) = 0.5, N.S.]. Untreated control animals displayed a typical behavioural satiety sequence (1): Most eating took place early in the session, followed by a period in which active behaviours were prominent, which was superceded in the second half of the session by resting (Fig. 4). In stressed animals, and following pimozide-treatment in non-stressed controls, this normal pattern of postprandial behaviour was preserved; however, the appearance of the behavioural satiety sequence was delayed. In both cases, significantly more time was devoted to eating during the early part of the session and as a result, the peak of active behaviour was shifted to the right. These changes were even more exaggerated in pimozide-treated stressed animals, which continued eating almost until the end of the session, with the result that the peak of active behaviour occurred in the final 10 min, and very little resting was possible. DISCUSSION
Although small amounts (10-20%) of sucrose added to the wet mash appeared to cause a small increase in intake, it should be noted that consumption of the unsweetened mash was not stable at the start of the experiment; thus, these increases could represent practice effects rather than responses to sweetness. However, when the concentration of added sucrose was increased to 30 or 40%, intake and eating time fell sharply in control animals. We have no evidence from the
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FIG. 4. Observations of eating (above) and resting (below) over the course of the 30 min session. A total of 20 observations were made in each 5 rain period; the figure does not show grooming and other active behaviours. VEH: vehicle; PIM: pimozide, 0.2 mg/kg; CON: control; STR: chronic mild stress. Values are means; error bars have been omitted for clarity. *,p < 0.05; *% p < 0.01; **% p < 0.001, relative to vehicle-treated nonstressed animals: +, p < 0.05; + + +, p < 0.001, relative to vehicle-treated stressed animals.
present study to support or refute any of the several potential explanations of this observation (increased caloric density, increased osmolality, increased sweetness, etc.). However, we note that a similar decrease in intake of very sweet liquids has been widely reported (l l, 12,15,17,2 l). Despite the decrease in intake, the sweeter mash diets supported a substantial increase in the rate of eating. Eating rate is usually considered to be closely related to palatability, as assessed, for example in preference studies (2,6,7,21,26). Similarly, very sweet sucrose so-
STRESS A N D P I M O Z I D E D E C R E A S E R E A C T I V I T Y T O S W E E T N E S S lutions are under-consumed relative to lower sucrose concentrations, but nevertheless, the sweeter solution is preferred in a choice test (17,35). The present data are consistent with the view that the quantity o f food consumed may not provide an accurate reflection o f its rewarding properties as revealed in choice tests. In keeping with this conclusion, we have previously observed that chronic mild stress did not decrease chow intake, but did decrease the rewarding effect of chow as assessed in the place conditioning procedure (16). Similarly, chronic mild stress decreased the intake o f dilute but not of concentrated sucrose solutions (27,30), but decreased the rewarding properties o f both dilute and concentrated solutions as assessed by place preference conditioning (16). In the present study, stressed animals were less reactive to the effects of changes in sucrose concentration on both the quantity o f mash eaten and the rate of eating; in other words, stressed animals behaved towards the sweet mash in a manner appropriate to a less sweet diet. While changes (or the absence o f changes) in intake may not be readily interpretable, the failure of stressed animals to increase eating rate is compatible with a decrease in the rewarding properties o f the very sweet diets. These data may, therefore, be compatible with the hypothesis that chronic mild stress causes anhedonia, a generalized decrease in sensitivity to rewards (15,16,30). D A receptor antagonists usually suppress rewarded behaviour (4,28); these effects are usually interpreted as a decrease in the rewarding properties of the reinforcer. This interpretation is supported by the many parallels between the effects of D A receptor antagonists and those o f dilution o f the reward (9,11,12,17,25,28,33,34). However, D A receptor antagonists can also cause a paradoxical increase in behaviours main-
523
tained by very sweet rewards, that fall on the descending limb of the inverted-U-shaped sweetness-performance function. Experiments carried out to exclude a number of alternative explanations have led us to conclude that these data most likely reflect a reward-attenuating action of D A antagonists (I 1,17-19). The effects of pimozide in the present study appear to provide another instance of this paradoxical performance-enhancing effect of DA antagonists. Although the effects of pimozide were rather small when averaged across the session, a prolongation of eating is clearly apparent from a more detailed examination of its time course [cf. (3)]. The time course analysis (Fig. 4) also shows clear additive effects of stress and pimozide. The similarities between the effects of chronic mild stress and those of D A antagonists may be more than coincidental. We have recently reported that chronic exposure to mild stress caused a decrease in sensitivity to the rewarding and locomotor activating effects of the D2 agonist, quinpirole, administered systemically or within the nucleus accumbens (15). Chronic mild stress causes abnormalities of D A neurotransmission in the nucleus accumbens (24,27), which may include a decrease in D2 receptor number (27). As D A receptor activation in the nucleus accumbens is crucial for the expression of rewarding effects in a variety of hehavioural paradigms (33), the anhedonic effects of chronic mild stress may result from a functional antagonism of D2 receptors within this structure. ACKNOWLEDGEMENTS This study was supported in part by the Medical Research Council of Great Britain. We also thank Janssen for the generous gift of pimozide.
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