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Reversal of a feeding-reward system by dexfenfluramine: Neurochemical involvement
Physiology& Behavior,Vol. 48, pp. 887-892. ©PergamonPress plc, 1990.Printedin the U.S.A.
0031-9384/90$3.00 + .00
Reversal of a Feeding-Reward System by Dexfenfluramine: Neurochemical Involvement M. OROSCO,I j. j, ROBERT, C. ROUCH, C. JACQUOT AND Y. COHEN
Laboratoire de Pharmacologie, URA-CNRS 594, Facult( de Pharmacie, 92290 Chatenay-Malabry, France
OROSCO, M., J. J. ROBERT, C. ROUCH, C. JACQUOT AND Y. COHEN. Reversal of a feeding-reward system by dexfenfluramine: Neurochemicalinvolvement. 48(6) 887-892, 1990.--In addition to its anorecticproperties, dexfenfluraminemay inhibit some manifestationsof feeding-related reward. We attempted to verify this effect by measuringpaw-lick latency on the hot plate test in rats conditionedto expect a palatable food. The involvementof variationsin I~-endorphinergic,dopaminergicand serotonergic systems was assessed. Despite an inherenteffect of increasingpaw-lick latency, dexfenfluramine(1.5 mg/kg IP) partly reversed the expectancy-inducedincrease in this latency. Saline-treated"expectant" rats displayedelevated plasma 13-endorphinlevels and reduced hypothalamic 5-HIAA/5-HTand DOPAC/DA ratios. Only the decrease in the DOPAC/DA ratio was reversed by dexfenfluramine, suggestingan involvementof the dopaminergicsystem in this dexfenfluramine-sensitivereward system. Dexfenfluramine
Reward
Food motivation
[~-Endorphin Dopamine
REWARD systems may include paradigms such as intracranial self-stimulation (39,43), self-administration of drugs, place-preference conditioning procedures (52) and food-related reward. In this latter case, the reward manifestations may be visualized by behavioural changes, generally obtained after a training period. These include direct feeding responses (46) and running for food (36), but also increases in nociceptive thresholds and in rearing (12). Opioids have often been implicated in reward systems, as demonstrated by classical opiate agonist self-administrationin rats (5). Morphine and opiate agonists facilitate intracranial self-stimulation behaviour (7, 40, 57), while opiate antagonists inhibit it (49). In addition, reward activates hypothalamic 13-endorphin release (11). Food-related reward should particularly involve opioids since these substances also participate in the control of feeding behaviour (20, 28, 37, 38). Dexfenfluramine, in addition to its known anorectic properties (1, 27, 48), has been shown to act on different feeding reward paradigms. These include food-rewarded runway behaviour (25, 53, 54), food-rewarded motivational conflict (8) and lateral hypothalamus self-stimulation (32). This last paradigm may be related to feeding rewards since it mimics the reward of food ingestion (22). The aim of this study was first to confirm this effect of dexfenfluramine in another reward paradigm, the increase in nociceptive thresholds induced by the expectancy of a palatable food (12), and then to seek an involvement of neurochemical systems
Serotonin
in the case of an observable behavioural effect. We first sought related changes in the 13-endorphinergic system, since 13-endorphin has been shown to participate in this feeding reward (11,12) and has been implicated in fenfluramine anorexia (34). Other neurotransmittersystems are classically involved in feeding behaviour, reward, the mechanism of action of fenfluramine or all three. Dopamine plays a role in opiate reward (4, 5, 14) and food intake (29). Serotonin is particularly involved in feeding (30) and in the mechanism of action of dexfenfluramine (2, 18, 47). Furthermore, more general interactions between opioids and monoaminergic systems have been described either on a functional basis (6,58) or in relation to feeding effects (44). We thus sought to relate the effect of dexfenfluramine on a reward paradigm to changes in [~-endorphin, dopamine and serotonin metabolism. METHOD Animals Female Sprague-Dawley rats (250_ 20 g at the time of testing) were used. In an effort to eliminate extraneous stimuli and stress to the animals in this experiment, the rats were cared for exclusively by the experimenter in a separate climatized room, where the experiment was also performed. Rats were kept four in a box to avoid "isolation stress" and on a reversed light-dark cycle (light on at 0200 h, off at 1400 h). Lab chow and water were always available.
tRequests for reprints should be addressed to Dr. Martine Orosco, Neurobiologiedes Regulations, College de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05 France.
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Training Pieces of chocolate were used as preferred food. To familiarize the rats with it, several pieces of chocolate were placed in the home cages for four consecutive days. Thereafter, the rats were familiarized with the experimental situation every afternoon at 1400 h for 50 days by placing them on a disconnected (cold) hot plate. In order to condition the expectancy of chocolate, two pieces (1 g/piece) were dropped onto the cold plate for half the animals. The other half were given pieces of lab chow. Control rats were given the same amount of chocolate every day at 1500 h in their home cages during the same period. During the last week, the rats were injected with saline every day one hour before the training session. On the completion of conditioning, experimental animals ate the chocolate in less than one minute.
Experimental Design On the day of the experiment, the plate was hot (51.5°C) and pieces of chocolate or lab chow were not given. Half the rats conditioned to expect chocolate and half the controls were injected with dexfenfluramine (Servier, France, 1.5 mg/kg IP) one hour before the test. The remaining animals received an equal volume of saline (1 ml/kg). Four groups of animals were thus obtained: saline controls (SC), dexfenfluramine controls (FC), saline "expectant" rats (SE) and dexfenfluramine "expectant" rats (FE). The nociceptive thresholds were measured by placing the animals with all four paws on the surface of the hot plate. The time taken to lick a paw (paw-lick latency) was measured with a stop watch. The animals were immediately decapitated upon removal from the plate in a separate room.
Tissue Collection and Preparation After sacrifice, the brains were quickly removed and dissected on a chilled plate for removal of the hypothalamus and pituitary. The hypothalamus was divided into two symmetrical parts and stored at - 8 0 ° C until use. 13-Endorphin was assayed in one half and monoamines in the other. Trunk blood was collected into chilled polypropylene tubes containing EDTA (5 mg/ml). Following centrifugation, plasma was stored at -30°C until assay of 13-endorphin.
I 3O
J
.-'3 2o
-I-
CS
Cdf
ES
EdF
FIG. 1. Effect of dexfenfluramine on paw-lick latencies in control and "expectant" rats. C--Control, E="Expectant," S=Saline, dF= dexfenfluramine, 1.5 mg/kg, 1 h prior to the test. *p<0.05 vs. CS; ***p<0.001 vs. CS;ffp<0.01 vs. CdF.
The results are expressed as fmol/mg wet weight for hypothalamic 13-endorphin. To the remaining acidified pellets of the individual pituitaries, 190 vd of 0.5 N NaOH was added prior to storage at 4°C until protein analysis according to Lowry et al. (31). Pituitary 13-endorphin levels are expressed as pmol/mg proteins. Plasma samples were assayed for 13-endorphin using a specific kit obtained from Immunonuclear Corporation. Nonspecific binding of 12sI-13-endorphin to high molecular weight plasma components at alkaline pH was avoided by affinity gel extraction of plasma by coupling anti-13-endorphin antiserum to Sepharose for 4 h. The extract was eluted with acid and neutralized and 200-p,1 aliquots were incubated with rabbit anti-13-endorphin antiserum and 1251-13-endorphin for 24 h. Bound label was precipitated by a 2-h incubation with 500 ixl goat anti-rabbit precipitating reagent, then centrifuged and the supernatant was aspirated. The minimum detectable concentration of 13-endorphin was 2 fmol/ml. The antiserum has the same crossreactivity as above, but cross-reacts to less than 5% with 13-1ipotropin using this assay procedure. Results are expressed as fmol/ml.
Monoamine and Metabolite Assay f3-Endorphin Assay Concentrations of 13-endorphin in the pituitary and hypothalamus were measured using a radioimmunoassay kit from Immunonuclear Corporation specific for tissues. Tissue samples were sonicated in 10 volumes of ice cold 0.2 N HC1 and then centrifuged. The supernatants were frozen and lyophilized. The lyophilisates were diluted with assay buffer to obtain concentrations within the range of the assay method and 100-vd aliquots were incubated in duplicate with rabbit anti-13-endorphin antisera for 24 h before addition of lz*I-13-endorphin. Bound label was precipitated by a 2-h incubation with 500 I~1 of goat anti-rabbit precipitating complex; following centrifugation the supernatant was aspirated. The minimum detectable concentration of 13-endorphin was 0.6 pmol/tube. The antiserum cross-reacts 100% with human 13-endorphin, [Des-Tyrl] human 13-endorphin, [2-Me-Ala 2] 13endorphin and alpha-acetyl-13-endorphin, 50% with human 13-1ipotropin and less than 0.1% with aipha-endorphin, Leu-enkephalin, ACTH and dynorphin, on a molar basis.
The remaining halves of hypothalamus were homogenized in 0.4 N perchloric acid containing 0.1% EDTA, N~S205 and cystein and then centrifuged. The supernatant was analyzed by means of liquid chromatography with electrochemical detection as previously described (42) for monoamine and metabolite assays. Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were separated and quantified. RESULTS
Paw-Lick Latencies As shown in Fig. I, dexfenfluramine increased paw-lick latency in the control group (34.2-3.1 s) over saline controls 0 5 . 6 - - 1 . 5 s). The saline "expectant" rats also showed an increase in the nociceptive threshold (22.9 ± 2.2 s), while dexfenfluramine-treated "expectant" rats did not ( 2 0 . 7 - 1.8 s).
DEXFENFLURAMINE AND REWARD: NEUROCHEMISTRY
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TABLE 1 HYPOTHALAMIC,PITUITARYAND PLASMATICI3-ENDORPHINCONCENTRATIONS IN RATS EXPEC'rINGCHOCOLATEAND CONTROLRATS I N ~ WITH SALINE OR DF.J~FENFLURAMINE(1.5 mg/kg) Control Rats
Hypothalamus (fmol/mg tissue) Pituitary (pmol/mg protein) Plasma (fmol/ml)
Expectant Rats
Saline
Dexfenfluramine
524.3 ± 32.4
559.9 --- 30.1
608.8 ± 34.7
548.9 __- 23.0
197.9 -+ 21.5
230.0 -+ 32.1
203.0 _+ 24.2
172.9 +- 19.8
21.2 -+ 4.8
12.6 ±
2.8
Saline
46.3 _+ 6.3t§
Dexfenfluramine
67.5 _+ 16.2"~
Means ± SEM. n = 8 - 9 . *p<0.05; tp<0.01 versus control-saline; :~p<0.01, §p<0.001 versus control-dexfenfluramine.
f3-Endorphin Levels (Table 1) 13-Endorphin levels were not significantly changed in the hypothalamus or pituitary either by expectancy or by dexfenfluramine in either group. Plasma concentrations were significantly enhanced in "expectant" rats (p<0.01). This increase was not modified by dexfenfluramine.
Monoamine Levels and Metabolism Dexfenfluramine increased DOPAC levels in the "expectant" group (p<0.01). DA levels were not modified whatever the conditions. The DOPAC/DA ratio was reduced in "expectant" rats (p<0.05), but did not remain significandy lower after dexfenfluramine treatment in this group (Table 2). Neither expectancy nor dexfenfluramine modified hypothalamic 5-HIAA levels. Serotonin levels were increased only in dexfenfluramine-treated "expectant" rats versus saline controls (p<0.05). The 5-HIAA/5-HT ratio was decreased by dexfenfluramine in both groups (p<0.05 in controls and p < 0 . 0 1 in "expectant" rats). The basal value in saline "expectant" rats was lower than in saline controls (p<0.01) (Table 3). DISCUSSION In agreement with the results of Dum and Herz (12), the expectancy of a palatable food increased nociceptive thresholds in our study, as measured by paw-lick latencies on the hot plate test.
Dexfenfluramine also increased paw-lick latency in the control group. This kind of effect had previously been described on jumping and paw-licking (45), as well as on flinch-jump and tailflick latencies (3), but was not expected at the low dose used in this study. However, dexfenfluramine failed to produce such an effect in the "expectant" group. Indeed, on the one hand, the increased latency of the saline "expectant" versus saline control rats was no longer significant after dexfenfluramine treatment, while on the other hand, the significant increase observed between dexfenfluramine-treated controls versus saline-treated controis was no longer found in the "expectant" group. Indeed, a slight tendency towards a decrease was observed. Thus, despite its inherent effect, dexfenfluramine reduced the behavioural manifestation related to the expectancy of a reward. This result is in agreement with previous work using different reward paradigms such as food-rewarded runway behaviour (25, 53, 54), conflict of motivation (8,15) and lateral hypothalamus self-stimulation (32). The "expectancy" state was accompanied by an increase in plasma 13-endorphin concentrations which may be related to an increase in 13-endorphin release reflected in peripheral concentrations. A slight increase in hypothalamic 13-endorphin levels was observed in the present study, while Dum et al. (11) found a decrease in hypothalamic levels of the peptide, which they also ascribed to an enhanced release from the brain. It should be noted that in their experiment, this variation was observed after a shorter conditioning period than in our study where a regulatory process of 13-endorphin synthesis had time to occur.
TABLE 2 HYPOTHALAMICDOPAMINERGICACTIVITYIN RATS E X P ~ G CHOCOLATEAND CONTROL RATS INJECTEDWITH SALINEOR DEXFENFLURAMINE(1,5 mg/kg) Control Rats
DOPAC DA DOPAC/DA
Expectant Rats
Saline
Dexfenfluramine
Saline
Dexfenfluramine
113.3 ± 8.8 254.6 ± 30.6 0.508 ± 0.043
127.7 ± 5.9 262.1 ± 15.2 0.518_ 0.23
103.7 __. 8.5:~ 263.6 __. 24.3 0.401 __. 0.025"t
149.0 ± 11.6"§ 289.6 --- 23.4 0.455 __- 0.032
Means ± SEM. n-- 8 - 9 . *p<0.05 versus control-saline; #p<0.01 versus control-
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OROSCO ET AL.
TABLE 3 HYPOTHALAMICSEROTONERGICACTIVITYIN RATS EXPECTINGCHOCOLATEAND CONTROLRATS INJECTEDWITH SALINEOR DEXFENFLURAMINE(1.5 mg/kg) Control Rats
5-HIAA 5-HT 5-HIAA/5-HT
Expectant Rats
Saline
Dexfenfluramine
Saline
Dexfenfluramine
681.6 --- 30.6 500.3 +-- 30.5 1.433 ± 0.053
628.7 --- 21.2 518.3 ± 17.7 1.183 ± 0.080*
665.7 - 31.7 580.9 ± 25.0 1.222 ± 0.034t
630.3 ± 26.5 598.6 --- 30.7*§ 0.995 --- 0.050:~¶
Means _ SEM. n= 8-9. *p<0.05; tp<0.01; :~p<0.001 versus control-saline; §p<0.05 versus control-dexfenfluramine. ¶p<0.01 versus expectant-saline.
The serotonergic system was little affected by expectancy: only a decrease in the 5-HIAA/5-HT ratio was observed. This variation is difficult to relate to a role of 5-HT in reward, particularly as, according to the experimental procedure, a facilitatory (35) or an inhibitory (55) role may be ascribed to serotonin. Early and Leonard (13) implicated serotonin rather in nonreward and punishment. The dopaminergic system was modified by expectancy in terms of a decrease in the DOPAC/DA ratio. This seems surprising since reward is usually related to an increase in DA transmission (4). However, dopamine is not involved in all reward systems (56). These latter authors failed to block heroin self-administration by haloperidol, while others found that neuroleptics reduce it (14). In addition, here again, the experimental conditions of the present study are quite different from those used in self-administration or self-stimulation paradigms. Furthermore, when an increase in DA transmission is evoked, this concerns DA release, both in studies of opiate action (10,24), and in fixed-ratio reinforcement schedules (16). Thus a decrease in intraneuronal metabolism, as reflected by the lower DOPAC/DA ratio, is not inconsistent with increased release. Dexfenfluramine did not modify 13-endorphin levels in the control group. This is not surprising since studies showing an effect involved the use of very high doses, i.e., 20--40 mg/kg (33) or chronic treatment (21,32). Dexfenfluramine was weakly active on the serotonergic system at the dose (1.5 mg/kg) used here. This is in agreement with studies in which the classically reported depletion in 5-HT and 5-HIAA levels appears with doses higher than 2.5 mg/kg (23). Only the 5-HIAA/5-HT ratio was reduced by dexfenfluramine. This ratio is usually found to be elevated with higher doses and longer times of action (9, 17, 19, 41). A decrease has, however, been observed in different experimental conditions (47) and with lower doses (unpublished results). In the "expectant" group, dexfenfluramine did not modify the expectancy-induced increase in plasma 13-endorphin levels. The increase in hypothalamic 13-endorphin levels in "expectant" rats, although not significant, was reversed by dexfenfluramine. However, the low level of these variations makes it difficult to implicate 13-endorphin system in the effect of dexfenfluramine. Similarly, dexfenfluramine did not reverse the expectancy-induced decrease
in the 5-HIAA/5-HT ratio. Its effect was similar in the control and "expectant" groups, with a similar degree of difference between treated and untreated rats. This suggests that the serotonergic system is not involved in this reward paradigm or in its reversal by dexfenfluramine. However, McClelland et al. (32) ascribed a role to serotonin in the reversal by dexfenfluramine of lateral hypothalamus self-stimulation. They argued that, in a previous study, d-l fenfluramine reversed both self-stimulation and stimulationescape (26), while dexfenfluramine, which is more selectively serotonergic in its mechanism, only reversed self-stimulation. The decrease in the DOPAC/DA ratio observed in "expectant" rats was reversed by dexfenfluramine via an increase in DOPAC levels. Thus, among the biochemical parameters assayed in our study, this would appear to be the only one directly involved in the reversal by dexfenfluramine of the manifestation of expectancy. This seems surprising at first sight, since the d-enantiomer of fenfluramine has been described as inactive on the dopaminergic system, contrary to the 1-form (23). However, a dopaminergic mechanism should not be ruled out. These latter authors, although claiming no effect of dexfenfluramine on the DA system, showed DA metabolite values which varied according to the dose. Recent microdialysis studies have shown that dexfenfluramine not only increases extracellular serotonin, but also leads to a release of dopamine in the lateral hypothalamus (50,51). In a previous study we also observed slight increases in DA metabolite levels after low doses of dexfenfluramine (unpublished results). Furthermore, the role of hypothalamic dopamine in the control of feeding (29) favours the hypothesis of a dopaminergic mechanism in the effect of dexfenfluramine. In conclusion, dexfenfluramine can reverse a feeding reward system by a mechanism which, while possibly involving an endorphinergic component, would appear to be principally dopaminergic. At all events, this property presents a supplementary advantage in the therapeutic use of dexfenfluramine. ACKNOWLEDGEMENTS The authors wish to thank Laboratoires Servier for the gift of dexfenfluramine and J.J.R. would like to thank the Institut de Formation Sup6deure Biom6dicale (IFSBM, Villejuif, France) for its financial support.
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DEXFENFLURAMINE AND REWARD: NEUROCHEMISTRY
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53. Thurlby, P. L.; Samanin, R. Effects of anorectic drugs and prior feeding on food-rewarded runway behavior. Pharmacol. Biochem. Behav. 14:799-804; 1981. 54. Thurlby, P. L.; Grimon, V. E.; Samanin, R. Feeding and satiation observed in the runway: The effects of d-amphetamine and d-fenfluramine compared. Pharmacol. Biochem. Behav. 18:841-846; 1983. 55. Tye, N. C.; Everitt, B. J.; Iversen, S. D. 5-hydroxytryptamine and punishment. Nature 268:741-743; 1977.
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56. Van Ree, J. M.; Ramsey, N. The dopamine hypothesis of opiate reward challenged. Eur. J. Pharmacol. 134:239-243; 1987. 57. Weibet, S. L.; Wolf, H. H. Opiate modification of intracranial selfstimulation in the rat. Pharmacol. Biochem. Behav. 10:71-78; 1979. 58. Wood, P. L. Opioid regulation of CNS dopaminergic pathways: A review of methodology, receptor types, regional variations and species differences. Peptides 4:595-601; 1983.
0031-9384/90$3.00 + .00
Reversal of a Feeding-Reward System by Dexfenfluramine: Neurochemical Involvement M. OROSCO,I j. j, ROBERT, C. ROUCH, C. JACQUOT AND Y. COHEN
Laboratoire de Pharmacologie, URA-CNRS 594, Facult( de Pharmacie, 92290 Chatenay-Malabry, France
OROSCO, M., J. J. ROBERT, C. ROUCH, C. JACQUOT AND Y. COHEN. Reversal of a feeding-reward system by dexfenfluramine: Neurochemicalinvolvement. 48(6) 887-892, 1990.--In addition to its anorecticproperties, dexfenfluraminemay inhibit some manifestationsof feeding-related reward. We attempted to verify this effect by measuringpaw-lick latency on the hot plate test in rats conditionedto expect a palatable food. The involvementof variationsin I~-endorphinergic,dopaminergicand serotonergic systems was assessed. Despite an inherenteffect of increasingpaw-lick latency, dexfenfluramine(1.5 mg/kg IP) partly reversed the expectancy-inducedincrease in this latency. Saline-treated"expectant" rats displayedelevated plasma 13-endorphinlevels and reduced hypothalamic 5-HIAA/5-HTand DOPAC/DA ratios. Only the decrease in the DOPAC/DA ratio was reversed by dexfenfluramine, suggestingan involvementof the dopaminergicsystem in this dexfenfluramine-sensitivereward system. Dexfenfluramine
Reward
Food motivation
[~-Endorphin Dopamine
REWARD systems may include paradigms such as intracranial self-stimulation (39,43), self-administration of drugs, place-preference conditioning procedures (52) and food-related reward. In this latter case, the reward manifestations may be visualized by behavioural changes, generally obtained after a training period. These include direct feeding responses (46) and running for food (36), but also increases in nociceptive thresholds and in rearing (12). Opioids have often been implicated in reward systems, as demonstrated by classical opiate agonist self-administrationin rats (5). Morphine and opiate agonists facilitate intracranial self-stimulation behaviour (7, 40, 57), while opiate antagonists inhibit it (49). In addition, reward activates hypothalamic 13-endorphin release (11). Food-related reward should particularly involve opioids since these substances also participate in the control of feeding behaviour (20, 28, 37, 38). Dexfenfluramine, in addition to its known anorectic properties (1, 27, 48), has been shown to act on different feeding reward paradigms. These include food-rewarded runway behaviour (25, 53, 54), food-rewarded motivational conflict (8) and lateral hypothalamus self-stimulation (32). This last paradigm may be related to feeding rewards since it mimics the reward of food ingestion (22). The aim of this study was first to confirm this effect of dexfenfluramine in another reward paradigm, the increase in nociceptive thresholds induced by the expectancy of a palatable food (12), and then to seek an involvement of neurochemical systems
Serotonin
in the case of an observable behavioural effect. We first sought related changes in the 13-endorphinergic system, since 13-endorphin has been shown to participate in this feeding reward (11,12) and has been implicated in fenfluramine anorexia (34). Other neurotransmittersystems are classically involved in feeding behaviour, reward, the mechanism of action of fenfluramine or all three. Dopamine plays a role in opiate reward (4, 5, 14) and food intake (29). Serotonin is particularly involved in feeding (30) and in the mechanism of action of dexfenfluramine (2, 18, 47). Furthermore, more general interactions between opioids and monoaminergic systems have been described either on a functional basis (6,58) or in relation to feeding effects (44). We thus sought to relate the effect of dexfenfluramine on a reward paradigm to changes in [~-endorphin, dopamine and serotonin metabolism. METHOD Animals Female Sprague-Dawley rats (250_ 20 g at the time of testing) were used. In an effort to eliminate extraneous stimuli and stress to the animals in this experiment, the rats were cared for exclusively by the experimenter in a separate climatized room, where the experiment was also performed. Rats were kept four in a box to avoid "isolation stress" and on a reversed light-dark cycle (light on at 0200 h, off at 1400 h). Lab chow and water were always available.
tRequests for reprints should be addressed to Dr. Martine Orosco, Neurobiologiedes Regulations, College de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05 France.
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Training Pieces of chocolate were used as preferred food. To familiarize the rats with it, several pieces of chocolate were placed in the home cages for four consecutive days. Thereafter, the rats were familiarized with the experimental situation every afternoon at 1400 h for 50 days by placing them on a disconnected (cold) hot plate. In order to condition the expectancy of chocolate, two pieces (1 g/piece) were dropped onto the cold plate for half the animals. The other half were given pieces of lab chow. Control rats were given the same amount of chocolate every day at 1500 h in their home cages during the same period. During the last week, the rats were injected with saline every day one hour before the training session. On the completion of conditioning, experimental animals ate the chocolate in less than one minute.
Experimental Design On the day of the experiment, the plate was hot (51.5°C) and pieces of chocolate or lab chow were not given. Half the rats conditioned to expect chocolate and half the controls were injected with dexfenfluramine (Servier, France, 1.5 mg/kg IP) one hour before the test. The remaining animals received an equal volume of saline (1 ml/kg). Four groups of animals were thus obtained: saline controls (SC), dexfenfluramine controls (FC), saline "expectant" rats (SE) and dexfenfluramine "expectant" rats (FE). The nociceptive thresholds were measured by placing the animals with all four paws on the surface of the hot plate. The time taken to lick a paw (paw-lick latency) was measured with a stop watch. The animals were immediately decapitated upon removal from the plate in a separate room.
Tissue Collection and Preparation After sacrifice, the brains were quickly removed and dissected on a chilled plate for removal of the hypothalamus and pituitary. The hypothalamus was divided into two symmetrical parts and stored at - 8 0 ° C until use. 13-Endorphin was assayed in one half and monoamines in the other. Trunk blood was collected into chilled polypropylene tubes containing EDTA (5 mg/ml). Following centrifugation, plasma was stored at -30°C until assay of 13-endorphin.
I 3O
J
.-'3 2o
-I-
CS
Cdf
ES
EdF
FIG. 1. Effect of dexfenfluramine on paw-lick latencies in control and "expectant" rats. C--Control, E="Expectant," S=Saline, dF= dexfenfluramine, 1.5 mg/kg, 1 h prior to the test. *p<0.05 vs. CS; ***p<0.001 vs. CS;ffp<0.01 vs. CdF.
The results are expressed as fmol/mg wet weight for hypothalamic 13-endorphin. To the remaining acidified pellets of the individual pituitaries, 190 vd of 0.5 N NaOH was added prior to storage at 4°C until protein analysis according to Lowry et al. (31). Pituitary 13-endorphin levels are expressed as pmol/mg proteins. Plasma samples were assayed for 13-endorphin using a specific kit obtained from Immunonuclear Corporation. Nonspecific binding of 12sI-13-endorphin to high molecular weight plasma components at alkaline pH was avoided by affinity gel extraction of plasma by coupling anti-13-endorphin antiserum to Sepharose for 4 h. The extract was eluted with acid and neutralized and 200-p,1 aliquots were incubated with rabbit anti-13-endorphin antiserum and 1251-13-endorphin for 24 h. Bound label was precipitated by a 2-h incubation with 500 ixl goat anti-rabbit precipitating reagent, then centrifuged and the supernatant was aspirated. The minimum detectable concentration of 13-endorphin was 2 fmol/ml. The antiserum has the same crossreactivity as above, but cross-reacts to less than 5% with 13-1ipotropin using this assay procedure. Results are expressed as fmol/ml.
Monoamine and Metabolite Assay f3-Endorphin Assay Concentrations of 13-endorphin in the pituitary and hypothalamus were measured using a radioimmunoassay kit from Immunonuclear Corporation specific for tissues. Tissue samples were sonicated in 10 volumes of ice cold 0.2 N HC1 and then centrifuged. The supernatants were frozen and lyophilized. The lyophilisates were diluted with assay buffer to obtain concentrations within the range of the assay method and 100-vd aliquots were incubated in duplicate with rabbit anti-13-endorphin antisera for 24 h before addition of lz*I-13-endorphin. Bound label was precipitated by a 2-h incubation with 500 I~1 of goat anti-rabbit precipitating complex; following centrifugation the supernatant was aspirated. The minimum detectable concentration of 13-endorphin was 0.6 pmol/tube. The antiserum cross-reacts 100% with human 13-endorphin, [Des-Tyrl] human 13-endorphin, [2-Me-Ala 2] 13endorphin and alpha-acetyl-13-endorphin, 50% with human 13-1ipotropin and less than 0.1% with aipha-endorphin, Leu-enkephalin, ACTH and dynorphin, on a molar basis.
The remaining halves of hypothalamus were homogenized in 0.4 N perchloric acid containing 0.1% EDTA, N~S205 and cystein and then centrifuged. The supernatant was analyzed by means of liquid chromatography with electrochemical detection as previously described (42) for monoamine and metabolite assays. Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were separated and quantified. RESULTS
Paw-Lick Latencies As shown in Fig. I, dexfenfluramine increased paw-lick latency in the control group (34.2-3.1 s) over saline controls 0 5 . 6 - - 1 . 5 s). The saline "expectant" rats also showed an increase in the nociceptive threshold (22.9 ± 2.2 s), while dexfenfluramine-treated "expectant" rats did not ( 2 0 . 7 - 1.8 s).
DEXFENFLURAMINE AND REWARD: NEUROCHEMISTRY
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TABLE 1 HYPOTHALAMIC,PITUITARYAND PLASMATICI3-ENDORPHINCONCENTRATIONS IN RATS EXPEC'rINGCHOCOLATEAND CONTROLRATS I N ~ WITH SALINE OR DF.J~FENFLURAMINE(1.5 mg/kg) Control Rats
Hypothalamus (fmol/mg tissue) Pituitary (pmol/mg protein) Plasma (fmol/ml)
Expectant Rats
Saline
Dexfenfluramine
524.3 ± 32.4
559.9 --- 30.1
608.8 ± 34.7
548.9 __- 23.0
197.9 -+ 21.5
230.0 -+ 32.1
203.0 _+ 24.2
172.9 +- 19.8
21.2 -+ 4.8
12.6 ±
2.8
Saline
46.3 _+ 6.3t§
Dexfenfluramine
67.5 _+ 16.2"~
Means ± SEM. n = 8 - 9 . *p<0.05; tp<0.01 versus control-saline; :~p<0.01, §p<0.001 versus control-dexfenfluramine.
f3-Endorphin Levels (Table 1) 13-Endorphin levels were not significantly changed in the hypothalamus or pituitary either by expectancy or by dexfenfluramine in either group. Plasma concentrations were significantly enhanced in "expectant" rats (p<0.01). This increase was not modified by dexfenfluramine.
Monoamine Levels and Metabolism Dexfenfluramine increased DOPAC levels in the "expectant" group (p<0.01). DA levels were not modified whatever the conditions. The DOPAC/DA ratio was reduced in "expectant" rats (p<0.05), but did not remain significandy lower after dexfenfluramine treatment in this group (Table 2). Neither expectancy nor dexfenfluramine modified hypothalamic 5-HIAA levels. Serotonin levels were increased only in dexfenfluramine-treated "expectant" rats versus saline controls (p<0.05). The 5-HIAA/5-HT ratio was decreased by dexfenfluramine in both groups (p<0.05 in controls and p < 0 . 0 1 in "expectant" rats). The basal value in saline "expectant" rats was lower than in saline controls (p<0.01) (Table 3). DISCUSSION In agreement with the results of Dum and Herz (12), the expectancy of a palatable food increased nociceptive thresholds in our study, as measured by paw-lick latencies on the hot plate test.
Dexfenfluramine also increased paw-lick latency in the control group. This kind of effect had previously been described on jumping and paw-licking (45), as well as on flinch-jump and tailflick latencies (3), but was not expected at the low dose used in this study. However, dexfenfluramine failed to produce such an effect in the "expectant" group. Indeed, on the one hand, the increased latency of the saline "expectant" versus saline control rats was no longer significant after dexfenfluramine treatment, while on the other hand, the significant increase observed between dexfenfluramine-treated controls versus saline-treated controis was no longer found in the "expectant" group. Indeed, a slight tendency towards a decrease was observed. Thus, despite its inherent effect, dexfenfluramine reduced the behavioural manifestation related to the expectancy of a reward. This result is in agreement with previous work using different reward paradigms such as food-rewarded runway behaviour (25, 53, 54), conflict of motivation (8,15) and lateral hypothalamus self-stimulation (32). The "expectancy" state was accompanied by an increase in plasma 13-endorphin concentrations which may be related to an increase in 13-endorphin release reflected in peripheral concentrations. A slight increase in hypothalamic 13-endorphin levels was observed in the present study, while Dum et al. (11) found a decrease in hypothalamic levels of the peptide, which they also ascribed to an enhanced release from the brain. It should be noted that in their experiment, this variation was observed after a shorter conditioning period than in our study where a regulatory process of 13-endorphin synthesis had time to occur.
TABLE 2 HYPOTHALAMICDOPAMINERGICACTIVITYIN RATS E X P ~ G CHOCOLATEAND CONTROL RATS INJECTEDWITH SALINEOR DEXFENFLURAMINE(1,5 mg/kg) Control Rats
DOPAC DA DOPAC/DA
Expectant Rats
Saline
Dexfenfluramine
Saline
Dexfenfluramine
113.3 ± 8.8 254.6 ± 30.6 0.508 ± 0.043
127.7 ± 5.9 262.1 ± 15.2 0.518_ 0.23
103.7 __. 8.5:~ 263.6 __. 24.3 0.401 __. 0.025"t
149.0 ± 11.6"§ 289.6 --- 23.4 0.455 __- 0.032
Means ± SEM. n-- 8 - 9 . *p<0.05 versus control-saline; #p<0.01 versus control-
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OROSCO ET AL.
TABLE 3 HYPOTHALAMICSEROTONERGICACTIVITYIN RATS EXPECTINGCHOCOLATEAND CONTROLRATS INJECTEDWITH SALINEOR DEXFENFLURAMINE(1.5 mg/kg) Control Rats
5-HIAA 5-HT 5-HIAA/5-HT
Expectant Rats
Saline
Dexfenfluramine
Saline
Dexfenfluramine
681.6 --- 30.6 500.3 +-- 30.5 1.433 ± 0.053
628.7 --- 21.2 518.3 ± 17.7 1.183 ± 0.080*
665.7 - 31.7 580.9 ± 25.0 1.222 ± 0.034t
630.3 ± 26.5 598.6 --- 30.7*§ 0.995 --- 0.050:~¶
Means _ SEM. n= 8-9. *p<0.05; tp<0.01; :~p<0.001 versus control-saline; §p<0.05 versus control-dexfenfluramine. ¶p<0.01 versus expectant-saline.
The serotonergic system was little affected by expectancy: only a decrease in the 5-HIAA/5-HT ratio was observed. This variation is difficult to relate to a role of 5-HT in reward, particularly as, according to the experimental procedure, a facilitatory (35) or an inhibitory (55) role may be ascribed to serotonin. Early and Leonard (13) implicated serotonin rather in nonreward and punishment. The dopaminergic system was modified by expectancy in terms of a decrease in the DOPAC/DA ratio. This seems surprising since reward is usually related to an increase in DA transmission (4). However, dopamine is not involved in all reward systems (56). These latter authors failed to block heroin self-administration by haloperidol, while others found that neuroleptics reduce it (14). In addition, here again, the experimental conditions of the present study are quite different from those used in self-administration or self-stimulation paradigms. Furthermore, when an increase in DA transmission is evoked, this concerns DA release, both in studies of opiate action (10,24), and in fixed-ratio reinforcement schedules (16). Thus a decrease in intraneuronal metabolism, as reflected by the lower DOPAC/DA ratio, is not inconsistent with increased release. Dexfenfluramine did not modify 13-endorphin levels in the control group. This is not surprising since studies showing an effect involved the use of very high doses, i.e., 20--40 mg/kg (33) or chronic treatment (21,32). Dexfenfluramine was weakly active on the serotonergic system at the dose (1.5 mg/kg) used here. This is in agreement with studies in which the classically reported depletion in 5-HT and 5-HIAA levels appears with doses higher than 2.5 mg/kg (23). Only the 5-HIAA/5-HT ratio was reduced by dexfenfluramine. This ratio is usually found to be elevated with higher doses and longer times of action (9, 17, 19, 41). A decrease has, however, been observed in different experimental conditions (47) and with lower doses (unpublished results). In the "expectant" group, dexfenfluramine did not modify the expectancy-induced increase in plasma 13-endorphin levels. The increase in hypothalamic 13-endorphin levels in "expectant" rats, although not significant, was reversed by dexfenfluramine. However, the low level of these variations makes it difficult to implicate 13-endorphin system in the effect of dexfenfluramine. Similarly, dexfenfluramine did not reverse the expectancy-induced decrease
in the 5-HIAA/5-HT ratio. Its effect was similar in the control and "expectant" groups, with a similar degree of difference between treated and untreated rats. This suggests that the serotonergic system is not involved in this reward paradigm or in its reversal by dexfenfluramine. However, McClelland et al. (32) ascribed a role to serotonin in the reversal by dexfenfluramine of lateral hypothalamus self-stimulation. They argued that, in a previous study, d-l fenfluramine reversed both self-stimulation and stimulationescape (26), while dexfenfluramine, which is more selectively serotonergic in its mechanism, only reversed self-stimulation. The decrease in the DOPAC/DA ratio observed in "expectant" rats was reversed by dexfenfluramine via an increase in DOPAC levels. Thus, among the biochemical parameters assayed in our study, this would appear to be the only one directly involved in the reversal by dexfenfluramine of the manifestation of expectancy. This seems surprising at first sight, since the d-enantiomer of fenfluramine has been described as inactive on the dopaminergic system, contrary to the 1-form (23). However, a dopaminergic mechanism should not be ruled out. These latter authors, although claiming no effect of dexfenfluramine on the DA system, showed DA metabolite values which varied according to the dose. Recent microdialysis studies have shown that dexfenfluramine not only increases extracellular serotonin, but also leads to a release of dopamine in the lateral hypothalamus (50,51). In a previous study we also observed slight increases in DA metabolite levels after low doses of dexfenfluramine (unpublished results). Furthermore, the role of hypothalamic dopamine in the control of feeding (29) favours the hypothesis of a dopaminergic mechanism in the effect of dexfenfluramine. In conclusion, dexfenfluramine can reverse a feeding reward system by a mechanism which, while possibly involving an endorphinergic component, would appear to be principally dopaminergic. At all events, this property presents a supplementary advantage in the therapeutic use of dexfenfluramine. ACKNOWLEDGEMENTS The authors wish to thank Laboratoires Servier for the gift of dexfenfluramine and J.J.R. would like to thank the Institut de Formation Sup6deure Biom6dicale (IFSBM, Villejuif, France) for its financial support.
REFERENCES 1. Bltmdell, J. E. Serotonin and appetite. Neuropharmacology 2t3: 1537-1551; 1984. 2. Blundell, J. E.; Latham, C. J.; Leshem, M. B. Differences between the anorexic actions of amphetamine and fenfluramine. Possible effects on hunger and satiety. J. Pharm. Pharmacol. 28:471-477; 1976. 3. Bodnar, R. J.; Simone, D.; Kelly, D. D.; Brutus, M.; Glusman, M.;
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