Ind. J. Physio!. Pharmac., Volume 33, Number 3, 1989
INVESTIGATION OF INFLUENCE OF DIAZEPAM, VALPROATE, CYPROHEPTADINE AND CORTISOL ON THE REWARDING VENTRAL TEGMENTAL SELF-STIMULATION BEHAVIOUR
SONTI V. RAMANA AND T. DESIRAJU·
Department ofNeurophysiology, National Institute of Mental Health arzd Neuro Sciences, Bangalore - 560 029
( Received on June IS, 1989 )
Abstract: Experiments were carried on in the Wistar rats having self-stimulation (88) electrodes implanted chronically in substantia nigra-ventral tegmental area (SN-VTA) to examine whether modulations of GABAergic, serotonergic, histaminergic, dopaminergic, and glucocorticoid neuronal receptor functions will affect or not the brain reward system and the 88 behaviour. The modulalon are the wellknown drugs: diazepam which is a facilitator ohome of the GABA recepton, and used clinically (or its tranquilising, anxiolytic, sedative-hypnotic and anti-convulsant properties: sodium valproate which is known to enhance the GABA synapse function, and used clinically for its anti convulsant property; haloperidol which is a dopaminergic receptor (D2) blocker, and clinically used for its anti-psychotic property; cyproheptadine which is both anti-histaminic and anti-serotonergic (blocka 5-HT2 receptor), used clinically for its antihistaminic and other beneficial properties; and hydrocortisone which is the stress-resisting glucocorticoid having direct effects on both brain and body cells, used clinically for the wide-ranging glucocorticoid therapeutic effects. The results revealed that systemic administration of these drugs, except haloperidol, caused no significant influenee on the 88 behaviour, thereby indicating that these nondopaminergic drugs have no effect on brain-reward system and also these categories of synaptic actions are not likely to be involved in the primary organization of the mechanisms of the brain-reward system.
Key words : brain-reward self-stimulation diazepam haloperidol cyproheptadine cortisol drug effects
INTRODUCTION self-stimulation (SS) reward system, and also to understand actions of drugs that have therapeutic Since a long time, it is known that subjects self advantages, studies using drugs have been going on simulate electrically certain regions of brain without since several years (3-5). As the neuroleptic drugs Iling satiated (I). Although the great deal of have been found to block primarily the dopaminergic rt sea.rch of brain-reward system has not yet synapse (receptors) and as they block self-stimulation unra\ elled the rewarding neuronal mechanism, it has behaviour, the hypothesis arose that dopaminergic generated the hypothesis on hedonia, in drug synapse or neuron might form the basis of SS addiclion, psychosis, and of anhedonic actions of reward. However, it is also possible that the system neuroleptic drugs (2). In order to understand the may be very ccmplex, and additional roies of n lure uf neurons and synapses involved in the cholinergic, opioid, serotonerric, steroid, GABA, ·Corresponding Author 180 Ramana and Desiraju Ind. J. Physiol. Pharmac., Volume 33, Number 3, 1989 and other important types of neuronal involvements from bregma: 1.2 mm lateral; and 8 5 depth from have also to be investigated to delineate the neural surface (25). After a few days of post-operative substrate. recovery, the rats were trained in the Skinner box to learn the operant behaviour to pedal press to obtain GABA neurons contribute the important vcJitionally the electrical self-stimulation. With each inhibitory synapses (6). GABA synapses constitute pedal press, a train of sine waves of 50 Hz passed the majority of synapses in substantia nigra compacta for r.25 sec. The current was set in each rat at an (7) which contains dopaminergic neurons and also is optimal level that eV0ked the maximum possible one of the best areas of self-stimulation. Beno pedal press rate, while causing no other incapicitating diazepines ,,,,hich are well-known anti-anxiety and motor or aversive behavioural side effects. On an tranquili5ing drugs have receptor sites complexed to average the charge was 30 IkC per train of some of the GABA receptors (8, 9) and act by stimulus. facilitating the GABA synapses. The anti-convulsant drug sodium valproate has al80 been considered to Diazepam pharmacwtical brand (Calmpose. exert facilitatory action in an unknown manner on Ranbaxy) was administered intra-peritoneally in two the GABA synapses (10-15). Hence it was felt doses: 1.25 mgjkg body weight, 2.5 mg/kg. In each interesting to examine the effect of both diazepam experimental session, a 15 min control data of self (a benzodiazepine) and sodium valproate (an anti stimulation was obtained, a saline injection control convulsant) that have targets of influence on GABA data was again obtained for 15 min, before the synapses, on the brain-reward system of self diazepam injection. A post-injection time of 15 min stimulation behaviour. Serotonergic system synapses for the diazepam to act was allowed and then the are present in all regions of the brain (3, 16, 17) and self-stimulation data for 15 min obtained. the system is known to be involved also in tbe regulation of antidepressive mood states (18). Hence Sodium valpronte commercial product (Valparin the effect of cyproheptadine which blocks seroto 200, Torrent Labs) was administered orally in a dose nergic receptors (19) on the rewarding SS behaviour of 90 mg/kg body weight. The post-drug self has been aimed to be examined. This drug is also stimulation was tested once at i hr, and second time an antagonist of histamine receptors, hence it is at I hr after the administration of drug. Before the additionally interesting as histamine is a neuro drug administration the two types of control data regulator (20, 21). Steroid hormones have been were also obtained as mentioned above. recognised in recent years to act on neurons of a number of limbic brain regions (22, 23). Hence, Cyproheptadine chloride commercial preparation whether the glucocorticoid cortisol which has a (Periactin syrup, Merck, Sharpe and Dohme) was major role in the relief of stress of the subject has administered orally in a dose of 80 ",g/kg body any effect also on the positive brain-reward system weight. The centro] data obtained as mentioned has been examined in this study. above, and the post-drug data was obtained at t hr, 1 hr and 2 hrs after the administration of the drug. METHODS Hydrocortisone sodium succinate commercial Experiments were done on Wistar rats with self preparation (Efcorlin, Allen-burry's Pharmaceuticals) stimulation bipolar stainless-steel electrodes implant was dissolved in distilled water and administered ed in substantia nigra-ventral tegmental area (SN intraperiton~allyin the dose of 1 mg/kg body weight. VTA) (24) at stereotaxic coordinates: - 3.5 mm The control data were obtained as mentioned above, Ind. ]. Physiol Pharmac., Volume 33, Number 3, 1989 Drug Influences on Brain Reward Function 181 and post-drug self-stimulation data was obtained the high dose, and these re~ults also confirmed lack at 15 min, 45 min, 75 min, and 105 min after of consistent effect of diazepam on the SS. In administra1ion. summary, SS rate data of the control and drug conditions in the 34 experimental sessions condu~ted Haloperidol commercial injectable preparat~on for the diazepam study at two different doses, no (Serenace, Searle) was administered intra-peritoneally significant effect on the brain reward system and self in a dose of 250 flog/kg body weight. The control stimulation behaviour was observed. data were obtained as above, and the post-drug data obtained 15 min after administration of the drug. 2000
mI!II CONTROL At least 24 hours were alIowed before testing the ~ CONTROL SALINE leoo Q DIAZEPAM drug a second time on the same rat, to provide n =4 enough time to allow metabolisation and lowering of c E the level of the previous dose. ~1600 .., 4 The electrode sites of SS were confirmed post ex ~ mortem in hand-cut sections of the brains fixed in ~ 1400 formalin.
The test period of SS, under each of the control 1200 and the drug conditions of the session, was of 15 min duration. At least 3 rats were used for each drug study, three experimental sessions repeated on 1000 each rat. The mean SS rate (per 15 min) of the control periods of the three experiments of each rat DRAIN SfiMULATK>N REWARO DRIVE. EfFECT OF OIAIEAl.W ( 2.Stft9IkO; I.P.) was tested against the mean rate of the three periods under the drug cr.ndition, to find significance of Fig. : Effect of diazepam on self-stimulation of substantia difference, using Student's t-test. In addition, group nigra ventral tegmental area. Self-stimulation was tested 15 min afler intraperitoneal administration averages also were similarly assessed of the drug Each bar in the figure represents the mean and standard deviation of data of four sessions repeated on different days on each rat. RESULTS Sodium valproate : Experiments were conducted on Diazepam: 13 experimental sessions were made four rats, 3 experimental sessions made on each rat. on four rats with the low dose (1.25 mg/kg), and 16 Each sessions had the control and the drug test data. experimental sessions were made on four rats with The results showed that sodium valproate (90 mg/kg the high dose (2.5 mg/kg). The SS rates under the body' weight) had no significant influence on the drug condition were compared with the rates under brain stimulation reward behaviour (Fig. 2.) control condition. No statistically significant change in SS was caused by either the low dose or the high Halopnidol: Nine experimental sessions done on dose of the drug (Fig. l). The lack of influence of 3 rats, under the haloperidol (250 flog/kg), and diazepam was observed in relatively low SS rate as control conditions showed that haloperidol caused a well as high SS rate rats. The study was also repeated highly significant effect of reducing self-stimulation. in five experimental sessions on five more rats using behaviour by about 96% of control. Under the dOSe 182 Ramana and Desiraju Ind.]. Physiol. Pharm"c., Volume 33, Number 3, 1989'
t800
n.J
'- CONTROL ~ 1600 ~;. SESSION p.HEf~ 1/2 H~ ..... ~A.t'SESSiON Anf:~ l HR n;; 1 w -'::i IX ~ 1400. w ex: a.. ex: S 1100
Rat numbers _
BRAIN STIMULATiON. REWARD MYE ; EFFECT CF CYPRO.H E?TADlHE (SO pg' kg .Body w{ Orat ' Fig. 3 : Effect of cyproheptadine on self-stimulation of substantia nigra ventral tegmental area. Self stimulation data was obtained after 30 min (Pd, 60 min (P.), and 120 min (P3 ) of oral administration of the drug. Each bar represents the mean and BRAIN sTa.':l!lJ,.lI
Fig. 2 : Effect of sodium valproatc on self-stimulation of substantia nigra-ventral tegmental area. Self stimulation was tested half an-hour and Qne hour after oral administration of the drug. Each bar in ~~~ COllTf$O< , the figure represents the mean and standard H, _lOj r.,lr> .,..15-1 deviation of data of three sessions repeated on 1800 "1-45 min 1 different days on each rat. I4J -, Hr I~;;'T~ H" '" I Ht ~!.fl\l" I n • l of haloperidol used, the rat would initially try to self-stimulate but soon would give up in a few VI seconds, like in the non·rewarded extinction type ~ C< 0. behaviour. ex l~OO « CO
Cyrproheptadine : 9 experimental sessions 0D 3 rats, including the control and drug conditions were conducted. The data revealed that cyproheptadine (80 /Jog/kg) caused no significant alteration of the self-stimulation behaviour (Fig. 3}.
Hydrocortisone : The data of 9 experimental Fig. 4 sessions, including the control and drug conditions. were conducted on three rats. Cortisol (I mg/kg, IP), caused no significant change in the self-stimulation behaviour (Fig. 4). Ind. J. Physio\. Pharmac., Volume 33, Number 3,1989 Drug Influences on Brain Reward Function 183
DISCUSSION Sodium valproate : Sodium valproate is known to be a broad-spectrum anti-convulsant, Its bio Diazepam: The site of action of diazepam in the availability on oral administration is 100% and peak brain is the benzodiazepine site of GABA receptor time is about 2 brs (31, 32). Its mode of therapeutic complex (8). GABA synapses are widely distributed action is not fully known, but is generally consic~ered in the brain, one of the richest regions being to be due to a facilitatory effect en the GABAergic substantia nigra (7). Substantia nigra is also the synapse (11- 14), but at higher doses (I5). It has dopaminergic neuronal region merging with the been found to increase the potassium conductance ventral tegmental area (3). This dopaminergic region (Ie). Since dopaminergic neurons are known to be is known to be among the highest SS rate giving important in self-stimulation reward, and as these areas (24). Benzodiazepine binding sites of GABA neurons bave GABAergic synapses (as cited above), receptors cause facilitation of GABA mediated post the self-stimulation behaviour could be affected by synaptic inhibition (5, 8,9). Hence, the dopaminergic sodium valproate facilitating the GABA synapses. neurons having the GABAergic synapses are But the present results with valproate also did not expected to be inhibited by diazepam administration, confirm such a hypothesis in the role of the GABA and thereby reduce the self.stimulation. However, synapses on the dopaminergic neurons of SN-\iTA. this type of an effect was not observed in the present Sodium valproate does not seem to significantly experiments, hence a different explanation has to be affect the brain reward system in the dose level (90 thought of. As the self-stimulation currents excite mg/kg) that is normally used in the clir ical situation. neurons of the SN-VTA region the inhibitory effects However, with higber doses (200 mg/kg and higher) of the GABA synapses might be of little impediment. of valproate, SS is depressed (29). Or some other type of synapse on the dopaminergic neurons may be more relevant than the GABA HalojJeridol: Haloperidol is widely used in the benzodiazepine for self-stimulation. treatment of major psychosis. Its higher affinity is In previous studies also, either marginal to block the D-2 receptors of dopaminergic synapses. facilitation or no consistent effects of other benzo The results with haloperidol are cemparable to the diazepine compounds were reported on SS (4,2 -28). results previously reported on neuroleptic drugs in Our study being on SN-VTA regiGn, differed from the literature (2), hence it is not discussed further the previous studies made c,n lateral hypothalamus. here. Further more, another study (29) reported depression effect of diazepines (high doses) on the SS behaviour. Cyproheptadine: Cyproheptadine blocks the The present results with diazepam on SS of SN-VTA 5- HT2 reccptcrs (I6, 17). It also antagonises the region showed no significant elIect on SS, hence action of histamine. 5-HT axons are present suggest that the benzodiazepines produce their effects throughout the brain including the cerebral cortex. of tranquility and well-being not through their However, little is known about the physiological and action on the brain-reward or hedonic systel1l, but synaptic functions of the 5-HT synapses. Among perhaps through action (inhibition) on anxiogenic functions known, 5-HT neurons have been implied neuronal systems of the brain. Chronic usage of to influence also the antidepressive state (18). benzncliazepines bring drug dependence behaviour However, the present results revealed that there is no (30), and in such a situation the influence of the effect of cyproheptadine on the brain-reward GAllA synapses on neurons of brain-reward system or SS bebavinur. The preliminar)I studies of the could have developed into a different and effective past reported contradicting results that scrotcllergic kind. system is both facilitatory and inhibitory to SS (33), 184 Ramana and Desiraju Ind. J. Physiol. Pharmac., Volume 33, Number 3, 1989
and only inhibitory to SS (34) The present work receptors have been found in high concentrations in with cyproh ptadine shewed clearly that there is no seplum, amygdala, and hippocampus (23) Since significant effect of 5-HT2 receptor mediated action these structures were known to play in mnemonic on SS The results also revealed that histaminergic and affective prccessess, and also in SS behaviour S) naptic role IS negligible in the brain· re\\ ard (septum), it was anticipated that cortisol might alter mechanisms and SS behaviour, because cyprohepta the brain stimulation reward behaviour. In the old dine in also a major antihistaminic (i 9). reports on adrenal steroid effects, one (35) indicated an increase in tht: SS, and the other (36) a decrease. Hydrocortisone: Steroids have a fast access to all The present results with cortisol clearly show that the parts of the nervous system and exert both the glucocorticoids do not modify significantly the genomic and non-genomic actions of both short brain-reward system and self-stimulation behaviour. latency as well as long latency (23). Glucocorticoid
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