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Stress reverses the anorexia induced by amphetamine and methylphenidate but not fenfluramine
580
Brain Research, 143 (1978) 580-585 ~) Elsevier/North-Holland Biomedical Press
Stress reverses the anorexia induced by amphetamine ,and methylphenidate but not fenfluramine
SEYMOUR M. ANTELMAN, ANTHONY R. CAGGIULA, CYNTHIA A. BLACK and DAVID J. EDWARDS
Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic and (S.M.A. and A.R.C.) Psychobiology Program, Department of Psychology, University of Pittsburgh, Pittsburgh, Pa. 15260. (U.S.A.) (Accepted October 6th, 1977)
The human clinical literature suggests that the causes of overeating and consequent obesity are varied is. Moreover, recent data indicate that anorectic agents may not be equally effective in their ability to suppress different types of overeating4. S;nce a large subgroup of obese individuals appears to overeat in response to stressful circumstances 5,9,15, this raises the question of whether appetite suppressant drugs are also likely to vary in the extent to which they influence stress-related overeating. Our laboratory has recently discovered that tail-pinch stress in rats can reliably induce eating when applied acutely1, and hyperphagia and obesity when administered chronically 17. Since tail-pinch-induced overeating appears to have much in common with stress-related overeating in humans 2,17, we used the tail-pinch paradigm to examine the effects of different types of anorectic agents. We now report that amphetamine and its congener, methylphenidate, fail to suppress stress-induced feeding or, to put it another way, stress overcomes the anorectic effects of amphetamine and methylphenidate. In contrast to these comparisons, however, fenfluramine, a different type of appetite suppressant, retains its anorectic properties even under stressful circumstances. Male albino rats of the Sprague-Dawley strain (Zivic-Miller, Pittsburgh) weighing 500-600 g, were used in the amphetamine and methylphenidate study. All animals were screened for tail-pinch-induced feeding by applying minimal pressure (which did not elicit vocalization) approximately 2.5 cm from the tip of the tail through a 25 cm sponge forceps insulated at the tips with foam rubber. Testing was done in shallow solution bowls 63.75 cm in diameter containing pellets of Purina rat chowL Several days following this screening procedure, all animals were food deprived for 22 h and, at the end of that time, injected with either D-amphetamine sulfate (1 mg/kg; N = 6) or isotonic saline (N = 6). Fifteen minutes following injection, both groups of animals were allowed access to food for 15 min in order to determine the degree of anorexia produced by amphetamine in the absence of tail-pinch. At the end of this time, tail-pinch was applied for 10 min. Pressure was gradually increased until an
581
r--t A-Amphetamine BWI M-Methylphenidate k ~ V-Ve;ie~l~roup
4.20 E3.40 ¢,D
2.60 "E 1.80 E < 1.00 0.20 V A 0.5
V A V A V M 1.0 2.0 5.6 mg/kg 15 Minute Pretest
V A V A V A V M 0.5 1.0 2.0 5.6 mg/kg 10 Minute Toil Pinch Test
Fig. 1. The mean (4- S.E.) amount of food eaten during a 15 min pretest and a 10 min tail-pinch period by 22-28 h, food-deprived rats that had been pretreated with amphetamine, methylphenidate or the saline vehicle. All drug-vehicle differenceswere statistically significant (smallest 't' = P < 0.01, twotail) for the pretest but not significant (P > 0.05) for the tail-pinch period. animal either ate or became frantic. The range of threshold pressures required to induce feeding in our laboratory, when measured by means of a tail cuff connected to a piston-driven, closed hydraulic system, is 80-120 psi. In addition to measuring the amount eaten during the 15 min prepinch and the subsequent 10 min tail-pinch periods, food intake was also determined during a 15 rain postpinch period. This precaution was taken in order to insure that the anorectic effects of amphetamine had not worn off prior to the conclusion of the tail-pinch test. The general procedure just outlined was repeated using the same animals, on subsequent weeks, with 0.5 and 2.0 mg/kg of amphetamine. An additional 6 animals were treated with 5.6 mg/kg of methylphenidate (all doses are given as the salt). The results of this experiment are presented in Fig. 1. Although both amphetamine and methylphenidate produced their customary anorectic influence during the prepinch feeding test, this effect was completely overcome by tail-pinch. There was no difference in the amount eaten by amphetamine-treated animals during tail-pinch and vehicle-treated animals during either tail-pinch or prepinch periods. Examination of eating rates during tail-pinch indicated that methylphenidate-treated animals actually ate faster (0.38 d: 0.04 g/min) than vehicle-injected controls (0.27 :k 0.02 g/min; P < 0.05, t-test). A similar effect was obtained over the two amphetamine conditions for which time spent eating was recorded (0.5 mg/kg amphetamine 0.77 :k 0.17; vehicle 0.47 ~ 0.18 g/min; 2.0 mg/kg amphetamine 0.85 :k 0.17; vehicle 0.41 -4- 0.15 g/rain; t = 2,20, df = 20, P < 0.05 two-tailed test). During the postpinch test, food intake was depressed in both drug-treated and vehicle animals. Since the reduced intake in control animals was probably due to the fact that they had already consumed a good deal of food during the prepinch and pinch periods, we retested them following a subsequent deprivation. This time, however, in order to avoid satiation, we substituted a 10 min period during which no food was available in lieu of the tail-pinch period. When this was done, controls ate significantly more than the drug
582 4.20 -
~3.40 (.9
~] ~ F V NI
Group I ( n = 4 ) Group II ( n = 5 ) Fenfluramine ( 2.5 mg/kg Vehicle No Injection
)
2.60
1.80 0
E 1,00 < 0.20
ij n
NI V NI F 15 Minute Pretest
n
NI V NI F 10 Minute T,P. Period
Fig. 2. The mean ( ± S.E.) amount of food eaten during 15 min pretest and 10 rain tail-pinch period by two groups of 22-28 h, food deprived rats on two successive weeks. On the first week, both groups were tested without any drug treatment. On the second week, Group I received a saline vehicle injection while Group II received fenfluramine 15 min before the pretest. P < 0.05, two-tail for fenfluramine vs. no injection on both the pretest and tail-pinch comparisons.
treated animals during the subsequent "postpinch" period, indicating that both amphetamine and methylphenidate had still been potentially effective anorectic agents during tail pinch. Preliminary studies in our laboratory indicate that although tail pinch also induces a significant amount of eating in non-food deprived animals receiving amphetamine, it is somewhat less effective under this condition than in deprived rats. Although fenfluramine, like amphetamine, is an effective anorectic agent both in lower animals and in humans s, it differs from amphetamine in many respects, including the presumed mechanism underlying its appetite suppressant effectss. For instance, whereas amphetamine in low doses has been reported to facilitate both electrical self-stimulation of the brain and "stimulation-escape", (i.e. escape from the presumed aversive consequences of prolonged brain stimulation), fenfluramine depresses these behaviors 11. Moreover, although inhibition of catecholamine synthesis, lesioning of dopamine(DA)-containing neurons, or treatment with DA receptor antagonists have been shown to counteract the anorectic effects of amphetamine, they actually enhance the appetite suppressant influence of fenfluraminea,S,tL Conversely, interference with brain serotonin systems, either by pharmacological or surgical means, has been reported to prevent the anorectic effect of fenfluramine, while leaving that of amphetamine unaffected s. Taken collectively, these results have been interpreted to suggest that while the appetite suppressant effects of amphetamine may be related to increased DA activityS,S, 1~, the influence of fenfluramine on food intake is more likely to be due to its effect on serotonin s (and perhaps, at high doses, secondarily to its presumed DA-receptor blocking properties) 7. Since tail-pinch-induced feeding is also attenuated by interfering with DAt, z or by mainipulations which enhance brain serotonergic activity2, we predicted that, in contrast to amphetamine, fenfluramine would continue
583 to exert its anorectic influence on the stress-induced overeating produced by tailpinch. The fenfluramine experiment was conducted on male albino rats weighing between 200 and 250 g. The procedure employed in this study was the same as that used with amphetamine and methylphenidate, except that it was repeated on two successive weeks. On the first week, the food intake of two groups of animals was measured in a 15-min prepinch period and in a subsequent 10-minute tail-pinch period. No drug was given during this test. A week later, the same testing paradigm was preceded by fenfluramine treatment for one group and the vehicle injection in the other. As is illustrated in Fig. 2, our prediction was borne out. Tail-pinch failed completely to overcome fenfluramine (2 mg/kg) anorexia. Higher doses of fenfluramine (5 and 7.5 mg/kg) were also tested. Since these produced total anorexia during both prepinch and pinch periods, they are not illustrated. The above findings clearly show that tail-pinch stress reverses both amphetamine- and methylphenidate-induced anorexia, but not fenfluramine-induced anorexia in food deprived animals. Although it is quite likely that higher doses of amphetamine and methylphenidate which produce severe stereotypy would have interfered with tail-pinch-induced eating, the failure of the ones used in the present study to block this phenomenon is probably not simply the consequence of an insufficient dosage since (1) the range of doses used in this produced a suppression of food intake of from 50 to almost 100 % without any obvious stereotypy in deprived, non-pinched animals (Fig. 1), (2) this range seemed most appropriate to those used to control appetite in humans, where stereotypy is also not a necessary, or even acceptable accompaniment and (3) these drugs actually increased the rate of eating in tail-pinched animals. It should be pointed out that our comparisons of the relative effectiveness of tail-pinch on amphetamine and fenfluramine anorexia are based on doses of these two drugs which were matched with respect to their anorectic effects. That is, while lower doses of fenfluramine might have also been ineffective in suppressing tail-pinch-induced eating, the smallest dose employed in this study (2 mg/kg), resulted in a reduction in eating of only 50 % in deprived, non-pinched controls, a degree of suppression almost identical to that achieved by the lowest dose of amphetamine. As mentioned above, brain DA has been implicated in both amphetamine anorexiaa,s,12 and tail-pinch-induced eating1,2. This presents an apparent paradox since the release of DA from neurons in the nigrostriatal bundle is believed by many to be responsible for amphetamine anorexia3,S,le, while, at the same time, tail-pinch-induced feeding appears to be dependent on the activity of this same system~,2,22. These considerations led us to collect information bearing on the level of activity in the nigrostriatal DA system, as reflected by the concentration of the DA metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), following amphetamine (1.0 mg/kg) and/or tail-pinch conditions. DOPAC has been thought to be an especially good reflection of the short term functional activity of DA- containing neurons in the nigrostriatal DA system16. These metabolites were measured according to the sensitive gas chromatographic method of Watson et al.20. There was a statistically significant decrease in striatal DOPAC, but not in HVA
584 TABLE I
The effects of D-amphetamine sulfate (1 mg/kg) with and without tail-pinch on striatal levels' of the DA metabolites, HVA and DOPAC Rats were deprived 22-28 h. M a n n - W h i t n e y U comparisons were made with vehicle-treated controls.
Group
N
H V A (#g/g)
DOP,4 C (ttg/g)
Vehicle Vehicle + tail-pinch Amphetamine Amphetamine + tail-pinch
7 7 7 7
0.73 0.64 0.64 0.65
1.28 1.09 0.88 0.96
± 4Jc ±
0.03 0.05 0.06 0.06
± ± ± ±
0.06 0.08* 0.06** 0.09§
* P = 0.07. ** P < 0.001. § P = 0.01, two-tail.
after amphetamine treatment (Table I). A similar, though non-significant tendency was obtained after tail-pinch. Moreover, almost exactly the same pattern of results was obtained when both treatments were combined. This tendency for DOPAC to decline while HVA remains unchanged (except at extremely high dosage levels) has also been obtained for non-food deprived animals treated with amphetamine14,16 or subjected to tail-pinch2. The decrease in DOPAC may reflect a feedback inhibition of the presynaptic neuron by a non-dopaminergic striatonigral pathway activated as a consequence of an initial release of DA by amphetamine. Such a mechanism has also been invoked to explain the decrease in the firing rate of nigrostriatal neurons which accompanies amphetamine administration6. These metabolic data raise the question of why procedures which appear to have very similar effects on DA function produce opposite effects on feeding, and why one of these, tail pinch, can actually reverse the feeding effects produced by the other, amphetamine. Of course, it is altogether possible that the answers to this question may be found in a non-DA-related influence of tail-pinch. We would, however, like to suggest an alternative, and perhaps more parsimonious explanation. According to this view, tail-pinch, which is merely a moderately stressful stimulus to the organism, should exert its DA-releasing action in a regulated, biologically meaningful way. By contrast, amphetamine may produce its anorectic effects not by release of DA per se, but by the non-physiological and exaggerated nature of this releasing action ~1. When tail pinch is combined with amphetamine, it may impose a physiologically meaningful regulatory influence on the latter which might then manifest itself (as in the present study) as an actual potentiation of the eating seen with tail-pinch alone. Our main finding is that amphetamine fails to suppress tail-pinch-induced eating in rats. Since there is a substantial amount of evidence which suggests that the tailpinch paradigm can legitimately serve as an experimental model of emotionallyrelated overeating in humans, the present discovery raises the possibility that amphetamine may also be ineffective clinically in counteracting this form of eating disorder. Evidence suggests that this may, in fact, be the case la. On the other hand, the ability
585 of fenfluramine to m a i n t a i n its anorectic properties in the presence of tail-pinch predicts that it m a y be a more effective (although n o t necessarily safer 1°) anorectic in those cases of h u m a n overeating a n d obesity where stress or e m o t i o n a l factors are t h o u g h t to play a m a j o r role 19. Supported by U S P H S grants 24114 (SMA) a n d 16581 (ARC). We t h a n k C. R u p p for typing the manuscript.
1 Antelman, S. M. and Szechtman, H., Tail pinch induces eating in sated rats which appears to depend on nigrostriatal dopamine, Science, 189 (1975) 731-733. 2 Antelman, S. M. and Caggiula, A. R., Tails of stress-related behavior: A neuropharmacological analysis. In I. Hanin and E. Usdin (Eds.), Animal Models in Psychiatry and Neurology, Pergamon Press, in press. 3 Baez, L. A., Role of catecholamines in the anorectic effects of amphetamine inrats, Psychopharmacologia, 35 (1974) 91-98. 4 Blondell, J. E., Latham, C. J. and Leshem, M. B., Differences between the anorexic actions of amphetamine and fenfluramine - - possible effects on hunger and satiety, J. Pharm. Pharmacol., 28 (1976) 471-477. 5 Bruch, H., Eating Disorders: Obesity, Anorexia Nervosa and the Person Within, Basic Books, New York, 1973. 6 Bunney,B.S.andAghajanian,G. K.,Electrophysiologicaleffects ofamphetamineondopaminergic neurons. In E. Usdin and S. H. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, New York, 1973, pp. 957-962. 7 Fuller, R. W., Perry, K. W. and Love, G., Elevation of 3,4-dihydroxypbenylacetic acid concentrations in rat brain and stimulation of prolactin secretion by fenfluramine: Evidence for antagonism at dopamine receptor sites, J. Pharm. PharmacoL, 28 (1976) 643-644. 8 Garattini, S. and Samanin, R., Anorectic drugs and brain neurotransmitters. In T. Silverstone (Ed.), Appetite and Food Intake, Dahlem Konferenzen, Berlin, 1976, pp. 83-108. 9 Hamburger, W. W., Psychological aspects of obesity, Bull. iV. Y. Acad. Med., 33 (1957) 771-782. 10 Harvey, J. A. and McMaster, S. E., Fenfluramine- - evidence for a neurotoxic action on midbrain and a long-term depletion of serotonin, Psychopharm. Comm., 1 (1975) 217-228. ~: 11 Hoebel, B. G., Satiety: hypothalamic stimulation, anorectic drugs, and neurochemical substrates. In D. Novin, W. Wirwicka and G. Bray (Eds.), Hunger: Basic Mechanisms and Clinical Implications, Raven Press, New York, 1975, pp. 33-50. 12 Hollister, A. S., Ervin, G. N., Cooper, B. R. and Breese, G. R., The role of monoamine neural systems in the anorexia induced by (+)-amphetamine and related compounds, Neuropharmacology, 14 (1975) 715-723. 13 Innes, I. and Nickerson, M., Drugs acting on postganglionic adrenergic nerve endings and structures innervated by them. In L. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Experimental Therapeutics, 4th Ed., Macmillan, London, 1970, p. 503. 14 Jori, A. and Bernardi, D., Effect of amphetamine and amphetamine-like drugs on homovanillic acid concentration:ih the brain, J. Pharm. PharmacoL, 21 (1969) 694--695. 15 LeMagnen, J., Stress et Obesit6, La Recherche, 7 (1976) 777-778. 16 Roth, R. H., Waiters, J. R. and Aghajanian, G., Effect of impulse flow on the release and synthesis of dopamine in the rat striatum. In E. Usdin and S. H. Snyder (Eds.), Frontiers in Catecholamine Research, Pergamon Press, New York, 1973, pp. 567-574. 17 Rowland, N. E. and Antelman, S. MI Stress-induced hyperphagia and obesity in rats: A possible model for understanding human obesity, Science, 191 (1976) 310-312. 18 Silverstone, T. (Ed.), Appetite an~lFoodlntake, Dahlem Konferenzen, Berlin, 1976. 19 Silverstone, T. Anorectic drugs. In T. J. Silverstone (Ed.), Obesity: Pathogenesis and Management, Publishing Sciences Group, Acton, Mass., 1975, pp. 193-227. 20 Watson, E., Travis, B. artd W!lk, S., Simultaneous determination of 3,4-dihydroxyphenylacetic acid homovanillic acid in milligram amounts of rat striatal tissue by gas-liquid chromatography, Life Sci., 15 (1974) 2167-2178. 21 Yehuda, S., Brain dopamine, D-amphetamine and thermoregulation in rats, lsraeli J. Med. ScL, 12 (1976) 1063-1064. 22 York, D. H., Lentz, S. and Love, D., Neural discharge associated with tail-pinch-inducedstereotyped behavior, Fed. Proc., 35 (1976) 668.
Brain Research, 143 (1978) 580-585 ~) Elsevier/North-Holland Biomedical Press
Stress reverses the anorexia induced by amphetamine ,and methylphenidate but not fenfluramine
SEYMOUR M. ANTELMAN, ANTHONY R. CAGGIULA, CYNTHIA A. BLACK and DAVID J. EDWARDS
Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic and (S.M.A. and A.R.C.) Psychobiology Program, Department of Psychology, University of Pittsburgh, Pittsburgh, Pa. 15260. (U.S.A.) (Accepted October 6th, 1977)
The human clinical literature suggests that the causes of overeating and consequent obesity are varied is. Moreover, recent data indicate that anorectic agents may not be equally effective in their ability to suppress different types of overeating4. S;nce a large subgroup of obese individuals appears to overeat in response to stressful circumstances 5,9,15, this raises the question of whether appetite suppressant drugs are also likely to vary in the extent to which they influence stress-related overeating. Our laboratory has recently discovered that tail-pinch stress in rats can reliably induce eating when applied acutely1, and hyperphagia and obesity when administered chronically 17. Since tail-pinch-induced overeating appears to have much in common with stress-related overeating in humans 2,17, we used the tail-pinch paradigm to examine the effects of different types of anorectic agents. We now report that amphetamine and its congener, methylphenidate, fail to suppress stress-induced feeding or, to put it another way, stress overcomes the anorectic effects of amphetamine and methylphenidate. In contrast to these comparisons, however, fenfluramine, a different type of appetite suppressant, retains its anorectic properties even under stressful circumstances. Male albino rats of the Sprague-Dawley strain (Zivic-Miller, Pittsburgh) weighing 500-600 g, were used in the amphetamine and methylphenidate study. All animals were screened for tail-pinch-induced feeding by applying minimal pressure (which did not elicit vocalization) approximately 2.5 cm from the tip of the tail through a 25 cm sponge forceps insulated at the tips with foam rubber. Testing was done in shallow solution bowls 63.75 cm in diameter containing pellets of Purina rat chowL Several days following this screening procedure, all animals were food deprived for 22 h and, at the end of that time, injected with either D-amphetamine sulfate (1 mg/kg; N = 6) or isotonic saline (N = 6). Fifteen minutes following injection, both groups of animals were allowed access to food for 15 min in order to determine the degree of anorexia produced by amphetamine in the absence of tail-pinch. At the end of this time, tail-pinch was applied for 10 min. Pressure was gradually increased until an
581
r--t A-Amphetamine BWI M-Methylphenidate k ~ V-Ve;ie~l~roup
4.20 E3.40 ¢,D
2.60 "E 1.80 E < 1.00 0.20 V A 0.5
V A V A V M 1.0 2.0 5.6 mg/kg 15 Minute Pretest
V A V A V A V M 0.5 1.0 2.0 5.6 mg/kg 10 Minute Toil Pinch Test
Fig. 1. The mean (4- S.E.) amount of food eaten during a 15 min pretest and a 10 min tail-pinch period by 22-28 h, food-deprived rats that had been pretreated with amphetamine, methylphenidate or the saline vehicle. All drug-vehicle differenceswere statistically significant (smallest 't' = P < 0.01, twotail) for the pretest but not significant (P > 0.05) for the tail-pinch period. animal either ate or became frantic. The range of threshold pressures required to induce feeding in our laboratory, when measured by means of a tail cuff connected to a piston-driven, closed hydraulic system, is 80-120 psi. In addition to measuring the amount eaten during the 15 min prepinch and the subsequent 10 min tail-pinch periods, food intake was also determined during a 15 rain postpinch period. This precaution was taken in order to insure that the anorectic effects of amphetamine had not worn off prior to the conclusion of the tail-pinch test. The general procedure just outlined was repeated using the same animals, on subsequent weeks, with 0.5 and 2.0 mg/kg of amphetamine. An additional 6 animals were treated with 5.6 mg/kg of methylphenidate (all doses are given as the salt). The results of this experiment are presented in Fig. 1. Although both amphetamine and methylphenidate produced their customary anorectic influence during the prepinch feeding test, this effect was completely overcome by tail-pinch. There was no difference in the amount eaten by amphetamine-treated animals during tail-pinch and vehicle-treated animals during either tail-pinch or prepinch periods. Examination of eating rates during tail-pinch indicated that methylphenidate-treated animals actually ate faster (0.38 d: 0.04 g/min) than vehicle-injected controls (0.27 :k 0.02 g/min; P < 0.05, t-test). A similar effect was obtained over the two amphetamine conditions for which time spent eating was recorded (0.5 mg/kg amphetamine 0.77 :k 0.17; vehicle 0.47 ~ 0.18 g/min; 2.0 mg/kg amphetamine 0.85 :k 0.17; vehicle 0.41 -4- 0.15 g/rain; t = 2,20, df = 20, P < 0.05 two-tailed test). During the postpinch test, food intake was depressed in both drug-treated and vehicle animals. Since the reduced intake in control animals was probably due to the fact that they had already consumed a good deal of food during the prepinch and pinch periods, we retested them following a subsequent deprivation. This time, however, in order to avoid satiation, we substituted a 10 min period during which no food was available in lieu of the tail-pinch period. When this was done, controls ate significantly more than the drug
582 4.20 -
~3.40 (.9
~] ~ F V NI
Group I ( n = 4 ) Group II ( n = 5 ) Fenfluramine ( 2.5 mg/kg Vehicle No Injection
)
2.60
1.80 0
E 1,00 < 0.20
ij n
NI V NI F 15 Minute Pretest
n
NI V NI F 10 Minute T,P. Period
Fig. 2. The mean ( ± S.E.) amount of food eaten during 15 min pretest and 10 rain tail-pinch period by two groups of 22-28 h, food deprived rats on two successive weeks. On the first week, both groups were tested without any drug treatment. On the second week, Group I received a saline vehicle injection while Group II received fenfluramine 15 min before the pretest. P < 0.05, two-tail for fenfluramine vs. no injection on both the pretest and tail-pinch comparisons.
treated animals during the subsequent "postpinch" period, indicating that both amphetamine and methylphenidate had still been potentially effective anorectic agents during tail pinch. Preliminary studies in our laboratory indicate that although tail pinch also induces a significant amount of eating in non-food deprived animals receiving amphetamine, it is somewhat less effective under this condition than in deprived rats. Although fenfluramine, like amphetamine, is an effective anorectic agent both in lower animals and in humans s, it differs from amphetamine in many respects, including the presumed mechanism underlying its appetite suppressant effectss. For instance, whereas amphetamine in low doses has been reported to facilitate both electrical self-stimulation of the brain and "stimulation-escape", (i.e. escape from the presumed aversive consequences of prolonged brain stimulation), fenfluramine depresses these behaviors 11. Moreover, although inhibition of catecholamine synthesis, lesioning of dopamine(DA)-containing neurons, or treatment with DA receptor antagonists have been shown to counteract the anorectic effects of amphetamine, they actually enhance the appetite suppressant influence of fenfluraminea,S,tL Conversely, interference with brain serotonin systems, either by pharmacological or surgical means, has been reported to prevent the anorectic effect of fenfluramine, while leaving that of amphetamine unaffected s. Taken collectively, these results have been interpreted to suggest that while the appetite suppressant effects of amphetamine may be related to increased DA activityS,S, 1~, the influence of fenfluramine on food intake is more likely to be due to its effect on serotonin s (and perhaps, at high doses, secondarily to its presumed DA-receptor blocking properties) 7. Since tail-pinch-induced feeding is also attenuated by interfering with DAt, z or by mainipulations which enhance brain serotonergic activity2, we predicted that, in contrast to amphetamine, fenfluramine would continue
583 to exert its anorectic influence on the stress-induced overeating produced by tailpinch. The fenfluramine experiment was conducted on male albino rats weighing between 200 and 250 g. The procedure employed in this study was the same as that used with amphetamine and methylphenidate, except that it was repeated on two successive weeks. On the first week, the food intake of two groups of animals was measured in a 15-min prepinch period and in a subsequent 10-minute tail-pinch period. No drug was given during this test. A week later, the same testing paradigm was preceded by fenfluramine treatment for one group and the vehicle injection in the other. As is illustrated in Fig. 2, our prediction was borne out. Tail-pinch failed completely to overcome fenfluramine (2 mg/kg) anorexia. Higher doses of fenfluramine (5 and 7.5 mg/kg) were also tested. Since these produced total anorexia during both prepinch and pinch periods, they are not illustrated. The above findings clearly show that tail-pinch stress reverses both amphetamine- and methylphenidate-induced anorexia, but not fenfluramine-induced anorexia in food deprived animals. Although it is quite likely that higher doses of amphetamine and methylphenidate which produce severe stereotypy would have interfered with tail-pinch-induced eating, the failure of the ones used in the present study to block this phenomenon is probably not simply the consequence of an insufficient dosage since (1) the range of doses used in this produced a suppression of food intake of from 50 to almost 100 % without any obvious stereotypy in deprived, non-pinched animals (Fig. 1), (2) this range seemed most appropriate to those used to control appetite in humans, where stereotypy is also not a necessary, or even acceptable accompaniment and (3) these drugs actually increased the rate of eating in tail-pinched animals. It should be pointed out that our comparisons of the relative effectiveness of tail-pinch on amphetamine and fenfluramine anorexia are based on doses of these two drugs which were matched with respect to their anorectic effects. That is, while lower doses of fenfluramine might have also been ineffective in suppressing tail-pinch-induced eating, the smallest dose employed in this study (2 mg/kg), resulted in a reduction in eating of only 50 % in deprived, non-pinched controls, a degree of suppression almost identical to that achieved by the lowest dose of amphetamine. As mentioned above, brain DA has been implicated in both amphetamine anorexiaa,s,12 and tail-pinch-induced eating1,2. This presents an apparent paradox since the release of DA from neurons in the nigrostriatal bundle is believed by many to be responsible for amphetamine anorexia3,S,le, while, at the same time, tail-pinch-induced feeding appears to be dependent on the activity of this same system~,2,22. These considerations led us to collect information bearing on the level of activity in the nigrostriatal DA system, as reflected by the concentration of the DA metabolites, homovanillic acid (HVA) and dihydroxyphenylacetic acid (DOPAC), following amphetamine (1.0 mg/kg) and/or tail-pinch conditions. DOPAC has been thought to be an especially good reflection of the short term functional activity of DA- containing neurons in the nigrostriatal DA system16. These metabolites were measured according to the sensitive gas chromatographic method of Watson et al.20. There was a statistically significant decrease in striatal DOPAC, but not in HVA
584 TABLE I
The effects of D-amphetamine sulfate (1 mg/kg) with and without tail-pinch on striatal levels' of the DA metabolites, HVA and DOPAC Rats were deprived 22-28 h. M a n n - W h i t n e y U comparisons were made with vehicle-treated controls.
Group
N
H V A (#g/g)
DOP,4 C (ttg/g)
Vehicle Vehicle + tail-pinch Amphetamine Amphetamine + tail-pinch
7 7 7 7
0.73 0.64 0.64 0.65
1.28 1.09 0.88 0.96
± 4Jc ±
0.03 0.05 0.06 0.06
± ± ± ±
0.06 0.08* 0.06** 0.09§
* P = 0.07. ** P < 0.001. § P = 0.01, two-tail.
after amphetamine treatment (Table I). A similar, though non-significant tendency was obtained after tail-pinch. Moreover, almost exactly the same pattern of results was obtained when both treatments were combined. This tendency for DOPAC to decline while HVA remains unchanged (except at extremely high dosage levels) has also been obtained for non-food deprived animals treated with amphetamine14,16 or subjected to tail-pinch2. The decrease in DOPAC may reflect a feedback inhibition of the presynaptic neuron by a non-dopaminergic striatonigral pathway activated as a consequence of an initial release of DA by amphetamine. Such a mechanism has also been invoked to explain the decrease in the firing rate of nigrostriatal neurons which accompanies amphetamine administration6. These metabolic data raise the question of why procedures which appear to have very similar effects on DA function produce opposite effects on feeding, and why one of these, tail pinch, can actually reverse the feeding effects produced by the other, amphetamine. Of course, it is altogether possible that the answers to this question may be found in a non-DA-related influence of tail-pinch. We would, however, like to suggest an alternative, and perhaps more parsimonious explanation. According to this view, tail-pinch, which is merely a moderately stressful stimulus to the organism, should exert its DA-releasing action in a regulated, biologically meaningful way. By contrast, amphetamine may produce its anorectic effects not by release of DA per se, but by the non-physiological and exaggerated nature of this releasing action ~1. When tail pinch is combined with amphetamine, it may impose a physiologically meaningful regulatory influence on the latter which might then manifest itself (as in the present study) as an actual potentiation of the eating seen with tail-pinch alone. Our main finding is that amphetamine fails to suppress tail-pinch-induced eating in rats. Since there is a substantial amount of evidence which suggests that the tailpinch paradigm can legitimately serve as an experimental model of emotionallyrelated overeating in humans, the present discovery raises the possibility that amphetamine may also be ineffective clinically in counteracting this form of eating disorder. Evidence suggests that this may, in fact, be the case la. On the other hand, the ability
585 of fenfluramine to m a i n t a i n its anorectic properties in the presence of tail-pinch predicts that it m a y be a more effective (although n o t necessarily safer 1°) anorectic in those cases of h u m a n overeating a n d obesity where stress or e m o t i o n a l factors are t h o u g h t to play a m a j o r role 19. Supported by U S P H S grants 24114 (SMA) a n d 16581 (ARC). We t h a n k C. R u p p for typing the manuscript.
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