Effect of restraint stress on rat brain serotonin

Transcription

1 Biosci., Vol. 4, Number 3, September 1982, pp Printed in India. Effect of restraint stress on rat brain serotonin S. K. BHATTACHARYA and D. BHATTACHARYA Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi MS received 4 January 1982; revised 24 March 1982 Abstract. Restraint-induced stress in rats was found to enhance steady state concentrations of whole brain and hypothalamic serotonin, at 1,2 and 4 h after immobilization. The increase was maximal at 1 h and tended to decline thereafter. The rate of accumulation of rat brain serotonin, in pargyline pretreated animals, was significantly enhanced after restraint stress. Bilateral adrenalectomy and metyrapone, an endogenous corticoid synthesis inhibitor, failed to affect restraint stress (1h)-induced increase in rat brain serotonin levels. Thus restraint stress-induced autoanalgesia and potentiation of the pharmacological actions of several centrally acting drugs, in rats, are serotonin-mediated responses. The results also indicate that restraint stress-induced effects on rat brain serotonin are not dependent on endogenous corticoid activity. Keywords. Restraint stress; serotonin; adrenalectomy; metyrapone. Introduction Serotonin has been reported to be essential for maintaining physiological and psychological homeostasis, during exposure of animals to stressors (Saito, 1976). However, the effects of different stress situations, including immobilization, on brain serotonin are equivocal (Selye, 1976). Thus, restraint stress has been reported to enhance (Rosecrans and De Feo, 1965; Culman et al., 1978), have no effect (Curzon et al., 1972) or decrease (Curzon and Green, 1969,197 1) rat brain serotonin concentrations. In recent communications from this laboratory, restraint stress-induced autoanalgesia (Bhattacharya et al., 1978), and hypothermia (Amar and Sanyal, 1981) were shown to be serotonin-mediated responses. Furthermore, restraint stress-induced potentiation of morphine analgesia, anticonvulsant actions of phenobarbitone and diphenylhydantoin, hexobarbitone hypnosis and haloperidol catalepsy, in rats, were reported to be serotonin-mediated effects, since they were attenuated after pretreatment with pharmacological agents known to inhibit central serotonergic activity (Bhattacharya and Bhattacharya, 1979, 1982; Bhattacharya, 1980). In view of these pharmacological data, it was considered worthwhile to investigate the effect of restraint stress on rat brain serotonin concentrations and turnover using the same strain of rats, environmental conditions and method of restraint as used in earlier studies. The above factors are known to affect brain serotonin concentrations and may be held responsible, 269

2 270 Bhattacharya and Bhattacharya atleast partly, for the equivocal data on stress-induced changes in the levels of this neurotransmitter published from different laboratories (Green and Grahame- Smith, 1975). The effect of metyrapone, an inhibitor of endogenous corticoid synthesis (Temple and Liddle, 1970), and bilateral adrenalectomy on rat brain serotonin changes induced by restraint stress, was also investigated because of the reports indicating serotonin-mediated regulation of stress induced activation of the hypothalamo-hypophyseal-adrenal system (Vermes et al., 1972, 1973) and adrenocortical regulation of brain serotonin (Scapagnini et al., 1969). Materials and methods Male Wistar, litter-mate, albino rats ( g) were used. The rats were maintained on standard diet (Hindustan Lever pellet) and housed in an air cooled room (25 C) in colony cages. Animals were fasted overnight but water was allowed ad lib before experimentation. Experiments were always conducted between h and h. The rats were immobilised in adjustable metallic restraint chambers after tying fore and hind limbs, separately and then together (Singh, 1971). Groups of rats were immobilised for 1,2 and 4 h, removed from the restraint chamber and killed by decapitation. For estimation of hypothalamic serotonin concentrations, the hypothalamus, the region lying between the rostral borders of the optic chiasma and mammillary bodies, and medial to the area lateralis, was dissected out as quickly as possible and the hypothalami of three rats were pooled. Brain serotonin was estimated by the fluorometric method of Snyder et al. (1965). The rate of accumulation of rat brain serotonin, after administration of pargyline hydrochloride (75 mg/kg, i.p.), was estimated by the method of Neff and Tozer (1968). Pargyline was administered just prior to immobilisation and rats were sacrificed at 0, 1, 2 and 4 h and the rate of accumulation (µg/g/h) calculated. In a separate set of experiments, metyrapone (20 mg/kg, i.p.) was administered twice at four hourly intervals before immobilisation. In another group of rats, bilateral adrenalectomy was performed under ether anaesthesia, a day prior to restraint. In both these groups restraint was restricted to 1 h because the peak effect of restraint stress on brain serotonin levels was achieved at this time period. Furthermore, there was a high incidence of mortality when immobilisation was extended for more than 1 h in adrenalectomized rats.adequate controls, including sham-operated rats, were maintained in all experiments. Statistical evaluation was done by the Student's t test. Results Restraint stress for 1, 2 and 4 h, significantly enhanced whole brain and hypothalamic serotonin concentrations, the increase being maximal at 1 h in both tissues. Thereafter, there was a steady decline, though the serotonin concentrations of whole brain and hypothalamus at 4 h were significantly higher than control levels. Though hypothalamic serotonin concentrations were higher than that of whole brain, there was no qualitative difference in the stress-induced change in this parameter (figure 1).

3 Restraint stress and rat brain serotonin 271 Figure 1. Effect of restraint stress (1, 2 and 4 h) on rat whole brain and hypothalamic serotonin concentrations, controls (C) were unrestrained rats. Each bar represents Mean ±S.E. of six experiments. Three hypothalami have been pooled for one estimation. * Represents P<0.001 in comparison to control. Metyrapone, sham adrenalectomy and bilateral adrenalectomy had no effect on rat brain serotonin as compared to untreated controls, nor did they affect restraint stress-induced increase in serotonin concentration (at 1 h) (table 1). Table 1. Effect of bilateral adrenalectomy and metyrapone on restraint stress (1h)- induced increase in rat brain serotonin concentrations. Values represent Mean ±S.E. Figures in parenthesis indicate number of animals in each group. The rate of accumulation of rat brain serotonin was enhanced by restraint stress, in pargyline-pretreated animals. The mean rate of serotonin accumulation (µg/g/ h) in pargyline-treated and stressed-pargyline-treated rats was 0.76 and 1.25 respectively. The increase in the rate of accumulation (µg/g/h) in stressed rats was thus 0.49 (figure 2).

4 272 Bhattacharya and Bhattacharya Figure 2. Effect of restraint stress (1,2 and 4 h) on the rate of accumulation of rat brain serotonin in pargyline hydrochloride (75 mg/kg, i.p.) pretreated rats. Each point represents Mean ±S.E. of six experiments. Note the significant (* P<0.001) increase in rate of accumulation at 1 and 2 h of restraint in comparison to control. Discussion A number of manipulations have been used in the laboratory which have some degree of analogy to physiological stress. Forced immobilisation is one of the best explored models of stress in rats. This model combines emotional stress (escape reaction) and physical stress (muscle work), resulting in both restricted mobility and aggression. Furthermore, painful stimuli are not directly involved in restraint stress and hence this form of stress is probably more akin to physiological stress (Selye, 1976). Restraint stress was found to enhance serotonin concentrations in both whole brain and hypothalamus with maximal increase found at 1 h after immobilization. Earlier studies on restraint stress and brain serotonin in rats have reported an increase (De Schaepdryver et al., 1969; Culman et al., 1978), no effect (Curzon et al., 1972) or a decrease (Curzon and Green, 1969, 1971) in rat brain serotonin concentration following immobilisation. Repeated restraint has also been shown to enhance rat brain serotonin concentrations, indicating absence of tolerance (Rosecrans and De Feo, 1965). These apparently discrepant data may be due to the different strains of rats used, difference in environmental conditions, including ambient temperature, and difference in the methodology of restraint, all of which are known to affect serotonin concentrations (Green and Grahame-Smith, 1975). The paradox becomes even more evident when it is noted that discrepant results on restraint stress-induced changes on rat brain serotonin have been reported from the same laboratory (Curzon and Green, 1969, 1971; Curzon et al., 1972). Restraint stress-induced increase in hypothalamic serotonin levels observed in the present study are in confirmity with the observations of Mueller et al. (1976), who have reported significant increase of hypothalamic serotonin concentrations at 45 and 150 min of restraint stress. However, the data of Telegdy and Vermes (1976) contradict the present observations and that of Mueller et al. (1976).

5 Restraint stress and rat brain serotonin 273 Restraint stress was found to enhance the rate of accumulation of rat brain serotonin in pargyline treated animals. The rate of accumulation of serotonin after inhibition of the major metabolic pathway by a monoamine oxidase inhibitor like pargyline, indicates the rate of synthesis of the amine which, in turn, gives an assessment of the rate of its turnover (Neff and Tozer, 1968). Our data are in conformity with that of some earlier reports (Nistico and Preziosi, 1969; Ladisich, 1974). Immobilisation has been reported to selectively enhance rat brain tryptophan levels (Knott et al.,1973), thus making available the precursor amino acid for serotonin synthesis. Elevation of adrenocortical secretion by stress has been reported to influence brain serotonergic activity, and hypothalamic serotonin is thought to have a role in stress-induced activation of the hypothalamo-hypophyseal-adrenal system (Vermes et al., 1972, 1973). Enhanced adrenocortical activity or treatment with cortisol, corticosterone and synthetic glucocorticoids have been reported to decrease brain serotonin levels, probably by inducing hepatic tryptophan pyrrolase activity, resulting in diversion of tryptophan from the pathway of brain serotonin synthesis (Curzon and Green, 1968; Scapagnini et al., 1969). In keeping with these observations bilateral adrenalectomy has been reported to enhance rat brain serotonin levels (De Schaepdryver et al., 1969). However, adrenalectomy has also been reported to inhibit trytophan hydroxylase activity, an effect reversed by corticosterone (Rastogi and Singhal, 1978). In the present study, bilateral adrenalectomy had no effect on rat brain serotonin, nor did the surgical manoeuvre affect restraint stress-induced increase of the amine levels. However, it should be pointed out that the latter effect was investigated only at 1 h restraint because mortality during extended immobilisation was high in these animals. Metyrapone, which inhibits the synthesis of corticosterone, the major endogenous corticoid in rats, by decreasing 11-β-hydroxylation (Temple and Liddle, 1970) failed to affect rat brain serotonin levels in unrestrained and restrained animals. These data suggest that peripheral corticoid activity is not related to stress-induced changes in brain serotonin concentrations. Curzon (1971) has inferred that many factors are likely to play a role in stress-induced effects on central serotonergic activity, and it cannot be said that the brain serotonin metabolism is entirely at the mercy of the adrenal gland. References Amar, Alka and Sanyal, A. K. (1981) Psychopharmacology, 73, 157. Bhattacharya, D. (1980) Neuropharmacological effects of restraint stress, M.D. (Pharmacology) thesis, Banaras Hindu University, Varanasi. Bhattacharya, D. and Bhattacharya, S. K. (1979) Indian Pharmacol., 11, 50. Bhattacharya, S. K. and Bhattacharya, D. (1982) Indian Exp. Biol., 20, 406. Bhattacharya, S. Κ., Keshary, P. R. and Sanyal, A. K. (1978) Eur. Pharmacol., 50, 83. Culman, J., Murgas, Κ., Torda, Τ. and Mitro, A. (1978) Bratis. Lek. Listy, 70, 465. Curzon, G. (1971) in Brain chemistry and mental disease, eds B. T. Ho and W. M. Mclsaac (New York: Plenum Press) p Curzon, G. and Green, A. R. (1968) Life Sci., 7, 657. Curzon, G. and Green, A. R. (1969) Br. Pharmacol., 37, 689. Curzon, G. and Green, A. R. (1971) Br. Pharmacol., 43, 39. Curzon, G., Joseph, M. H. and Knott, P. J. (1972). Neurochem., 19, De Schaepdryver, Α., Preziosi, P. and Scapagnini, U. (1969) Br. Pharmacol., 35, 460,

6 274 Bhattacharya and Bhattacharya Green, A. R. and Grahame-Smith, D. G. (1975) in Handbook of Psychopharmacology, eds. L. Iversen, S. D. Iversen and S. H. Snyder, (New York: Plenum Press) Vol. 3, p Knott, P. J. Joseph, M. H. and Curzon, G. (1973). Neurochem., 20, 249. Ladisich, W. (1974) Arzneim. Forsch., 24, Mueller, G. P., Towhy, C. P., Chen, H. T., Advis, J. P. and Meites, J. (1976) Life Sci., 18, 715. Neff, N. H. and Tozer, T. N. (1968) Adv. Pharmacol, 6A, 97. Nistico, G. and Presiosi, P. (1969) Pharmacol. Res. Commun., 1, 363. Rastogi, R. B. and Singhal, R. L. (1978) Neurol Trans., 42, 63. Rosecrans, J. A. and De Feo, J. J. (1965) Arch. Int. Pharmacodyn., 157, 487. Saito, H. (1976) in Catecholamines and stress, eds. R. KvetnanskyandI.J. Kopin (London: Pergamon Press) p. 95. Scapagnini, U., Preziosi, P. and De Schaepdryver, A. (1969) Pharmacol. Res. Commun., 1, 63. Selye, H. (1976) in Stress in health and disease, ed. H. Selye (London: Butterworths) p. 15. Singh, G. B. (1967) in Peptic Ulcer, eds. C.J. Pfeiffer and J. L. A. Roth (Copenhagen: Munksgaard) p. 98. Snyder, S. H., Axelrod, J. and Zweig, Μ. (1965) Biochem. Pharmacol., 14, 831. Temple, Τ. Ε. and Liddle, G. W. (1970) Ann. Rev. Pharmacol., 10,199. Telegdy, G. and Vermes, I. (1976) in Catecholamines and stress, eds. R. Kvetnansky and I.J. Kopin (London: Pergamon Press) p Vermes, I., Dull, G., Telegdy, G. and Lissak, K. (1972) Acta Physiol Acad. Sci. Hung., 42, 219. Vermes, I., Telegdy, G. and Lissak, K. (1973) Acta Physiol Acad. Sci. Hung., 43, 33.