Neuropharmacology Vol. 31, No. 1 I, pp. 1089-1094, Printed in Great Britain. All rights reserved
1992
0028-3908/92
$5.00 + 0.00
Copyright 0 1992Pergamon Press Ltd
EFFECTS OF REPEATED ELECTROCONVULSIVE SHOCK ON SEROTONIN,, RECEPTOR BINDING AND RECEPTOR-MEDIATED HYPOTHERMIA IN THE RAT C. A. STOCKMEIER,*PATRICIA WINGENFELDand G. A. GUDELSKY Laboratory of Biological Psychiatry, Departments of Psychiatry and Neuroscience, Case Western Reserve University, Cleveland, OH 44106, U.S.A. (Accepted 23 April 1992) Summary-Chronic treatment with electroconvulsive shock or antidepressant drugs has been reported to attenuate the hypothermia induced by 8-hydroxy-2-(di-n-propyl)aminotetralin (8-OH-DPAT), a serotonm,, receptor agonist. In the present study, the binding of [‘HII-OH-DPAT to serotonin,, receptors was assessed after treatment of rats with electroconvulsive shock. The effect of electroconvulsive shock on I-OH-DPAT-induced hypothermia also was evaluated. Male rats were handled or received electroconvulsive shock for either 1 or 10 days and were killed 2 days later. Ten days of electroconvulsive shock decreased /I-adrenergic receptor binding in the cerebral cortex, as previously reported. However, the binding of [‘H]8-OH-DPAT to serotonin,, receptors in the cortex or hippocampus was not affected by repeated electroconvulsive shock. In the hypothalamus, 10 days (but not 1day) of electroconvulsive shock significantly decreased the binding of [3H]8-OH-DPAT. In addition, 10 days of electroconvulsive shock resulted in an attenuation of the hypothermic response to I-OH-DPAT, when compared to the response in handled controls. The electroconvulsive shock-induced suppression of the hypothermic response to I-OH-DPAT was no longer evident 2 weeks after the last of 10 daily treatments. A single shock did not affect the hypothermic response to 8-OH-DPAT. The electroconvulsive shock-induced decrease in the binding of [3H]8-OH-DPAT in the hypothalamus may be related tot the electroconvulsive shock-induced attenuation of the hypothermic response to I-OH-DPAT. Key wordr-electroconvulsive
shock, body temperature, I-OH-DPAT,
serotonin,,
receptor, hypothala-
mus, hippocampus, cerebral cortex.
A subtype of serotonin receptor in brain, the serotomn,, receptor, has been identified and studied intensely during the past eight years (El Mestikaway, Fargin, Raymond, Gozlan and Hnatowich, 1991). The serotonin,, receptor may be involved in the mechanism of action of antidepressant and anxiolytic treatments and the pathophysiology of anxiety, schizophrenia, depression and suicide. For example, a selective serotonin,, receptor agonist, 8-hydroxy-2(di-n-propyl)aminotetralin(8-OH-DPAT) mimics the behavioral effect of antidepressant drugs in the forced swimming test (Cervo and Samanin, 1987) and the learned helplessness paradigm (Martin, Beninger, Hamon and Puech, 1990). In recent post-mortem studies, serotonin,, receptor binding was increased in the frontal cortex of suicide victims or patients with chronic schizophrenia (Matsubara, Arora and Meltzer, 1991; Hashimoto, Nishino, Nakai and Tanaka, 1990). An in vim method for investigating the sensitivity of serotonin,, receptors in the brain is to study the temperature response to agonists at serotonin,, re*Address correspondence and reprint requests to: Dr Craig A. Stockmeier, Department of Psychiatry, University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
ceptors. Systemic administration of agonists, like 8-OH-DPAT or ipsapirone in rats or humans, results in a dose-dependent decrease in body temperature (Hjorth, 1985; Goodwin and Green, 1985; Gudelsky, Koenig and Meltzer, 1986a; Koenig, Meltzer and Gudelsky, 1988; Lesch, Disselkamp-Tietze and Schmidtke, 1990). The hypothermic response to 8OH-DPAT in the rat is antagonized by serotonin receptor antagonists with a high affinity for the serotonin,, subtype of receptor (Goodwin, DeSouza, Green and Heal, 1987b; Gudelsky et al., 1986a; Moser, 1991). A role for serotonin,, receptors in the mechanism of action of antidepressant treatments has been suggested by studies demonstrating that chronic treatment of rats with electroconvulsive shock (ECS) diminished behavioral and biochemical responses elicited by activating the serotonin,, receptor. Repeated treatment with electroconvulsive shock attenuates the hypothermia and motor behavioral syndrome produced by 8-OH-DPAT (Goodwin, De Souza and Green, 1987a). In addition, chronic treatment with electroconvulsive shock likewise attenuates the ability of serotonin to inhibit forskolin-stimulated adenylate cyclase in hippocampus through serotonin,, receptors (Newman and Lerer, 1988; Varrault, Leviel and Bockaert, 1991).
1089
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A. STOCKMEIERet al.
Repeated treatment with electroconvulsive shock also decreases serotonin,, receptor binding in the cerebral cortex (Pandey, Isaac, Davis and Pandey, 1991). Thus, the therapeutic effect of chronic treatment with electroconvulsive shock may be partially mediated by diminishing the sensitivity of serotonin,, receptors. The purpose of this study was to determine whether electroconvulsive shock altered serotonin,, receptor binding in the cerebral cortex, hippocampus or hypothalamus and whether electroconvulsive shock-induced changes in serotonin,, receptor binding correlated with electroconvulsive shock-induced changes in hypothermia elicited by 8-OH-DPAT. METHODS
Male Sprague-Dawley rats (20&250 g) were purchased from Zivic Miller and housed in a light- and temperature-controlled room and had free access to food and water. Groups of rats were either handled (controls) or received electroconvulsive shock for either 1 or 10 days. No anesthesia was used. Rats were shocked once daily in the morning through earclip electrodes. Current (25 mA, 0.5 set) was delivered by a Medcraft Electroshock Generator and all rats experienced a generalized tonic-clonic seizure. The earclip electrodes were placed on control rats but no current was applied. The rats were killed by decapitation 2 days after the last shock. In one experiment, the rats were killed 14 days after the last of 10 daily shocks. Following decapitation, the brains were quickly dissected on an ice-chilled plate. Fronto-parietal cerebral cortex, hippocampus and hypothalamus were frozen on dry-ice and stored at -60°C until used for the binding assays. Serotonin,, receptor binding was measured using [3H]8-OH-DPAT (127.9 Ci/mmol; New England Nuclear), according to the method of Peroutka (1986), as described by Stockmeier and Kellar (1989). Briefly, the tissues were homogenized with a Polytron in ice-cold 50 mM Tris-HCl (pH 7.7 at 25°C) washed by centrifugation and preincubated with homogenization buffer for 10 min at 37°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.7 at 25°C) 4 mM CaCl,, 10 PM pargyline and 0.1% ascorbic acid. Aliquots of tissue homogenates, containing 4 mg (cortex or hypothalamus) or 3 mg (hippocampus) of tissue, were incubated in duplicate or triplicate with [3H]8-OH-DPAT (0.1-15 nM) at 25°C for 30 min, in a final volume of 0.5 ml. Nonspecific binding was determined in the presence of 10pM serotonin (HCl salt, Sigma). Incubations were terminated by vacuum filtration through glass fiber filters (Schleicher and Schuell, No. 34). The filters were washed 3 times with 4 ml aliquots of cold incubation buffer, transferred to vials to which scintillation fluid was added and counted by liquid scintillation spectrometry. Specific binding, defined as the difference between total binding and nonspecific binding, was 9565% of total binding in the cortex or hippo-
campus, depending on the concentration of [)H]8OH-DPAT. In the hypothalamus, specific binding was 60-70% of total binding. Binding of [‘251]pindolol was also measured in the cortex, using the method of O’Donnell and Frazer (1985). The concentrations of [‘ZSI]pindolol used were 40 and 140 pM; nonspecific binding was measured in the presence of 25 PM ( - )isoproterenol. Measurements of temperature were made, as outlined by Gudelsky et al. (1986a), in groups of control and electroconvulsive shock-treated rats not used for radioligand binding. Rectal temperatures were measured with a telethermometer and thermistor probe (Yellow Springs Instrument Co.). The probe was inserted approximately 5 cm into the rectum. Two days after the last shock, the rats were moved to an observation room (23-25°C) and a period of 2 hr was allowed for stabilization of the temperature. Body temperatures were recorded 30, 15 min and immediately prior to the administration of 8-OHDPAT (0.05 or 0.1 mg/kg, s.c.). A final measurement of body temperature was made 30min after the injection of 8-OH-DPAT. The doses of 8-OH-DPAT were chosen, based on a previously reported dose-response study (Gudelsky et al., 1986a). Data from saturation binding experiments were analyzed by nonlinear regression analysis, using the McPherson adaptation (Elsevier, Cambridge) of the LIGAND computer program (Munson and Rodbard, 1980). The data files selected for analysis by LIGAND had experimentally-measured nonspecific binding subtracted from them. Binding to a single receptor site best explained the experimental data. Reported values are the mean + SEM. One-way or two-way analysis of variance (ANOVA) was used to compare controls with electroconvulsive shocktreated rats. Pos: hoc testing, between related means, was performed with the Student-Newman-Keuls test. A P value of less than 0.05 was considered statistically significant. RESULTS
Treatment with electroconvulsive shock for 10 days resulted in a significant decrease in binding to badrenergic receptors in the cerebral cortex (ANOVA, P < 0.005; data not shown). A significant decrease (15-18%) was detected at the two different concentrations of [‘251]pindolol examined. In the cerebral cortex and hippocampus, [3H]8-OHDPAT bound in a saturable manner to one class of binding sites with a Kd of about 1.6 nM (Table 1). Repeated electroconvulsive shock for 10 days did not significantly affect the number (B,,,) or affinity (Kd) of [3H]8-OH-DPAT binding sites in the cerebral cortex or hippocampus (Table 1). However, in hypothalamus, the binding of [‘H]8-OH-DPAT was significantly decreased after 10 days of daily electroconvulsive shock (Table 2). A single administration of electroconvulsive shock did not significantly affect
Repeated ECS and serotonin, receptors Table I. Effect of electroconvulsive shock (ECS) on Treatment Handled ECS Handled ECS
Brain region Cortex Hippocampus
1091
[‘IQ-OH-DPATbindingin the brain of the rat
N
Treatment duration (days)
(fmol/mYtissue)
12 12 6 8
10 10 10 10
11.38 f 0.52 11.72 + 0.66 27.87 f. 2.38 29.44 + 2.95
B
1.66+0.15 1.68 + 0.10 1.59 f 0.21 1.50 + 0.19
Rats were killed 2 days after the final ECS. Binding values are the means k SEM.
the binding of [3H]8-OH-DPAT in the hypothalamus (Table 2). Basal body temperatures of rats, treated for 10 da,ys with electroconvulsive shock, did not differ significantly from those of handled controls (handled controls: 38.4 + O.l”C vs ECS: 38.2 f O.l”C). A hypothermic response was produced by both doses of 8-OH-DPAT (0.05 or 0.1 mg/kg, s.c.) in controls and rats treated with electroconvulsive shock for 10 days (Fig. 1). However, at both doses of 8-OH-DPAT, the hypothermic response was significantly less in chronic electroconvulsive shock-treated rats compared to handled controls (Fig. 1). The electroconvulsive shock-induced attenuation by the hypothermic response to 8-OH-DPAT was no longer evident 14 days after the last of 10 treatments with electroconvulsive shock (Fig. 2). A single treatment with electroconvulsive shock did not significantly affect the hypothermic response to 8-OH-DPAT (Fig. 2). DISCUSSION
Chronic treatment with electroconvulsive shock resulted in a significant decrease in /I-adrenergic receptor binding in the cerebral cortex, as previously reported (Bergstrom and Kellar, 1979; Nimgaonkar, Heal, Davies and Green, 1986; Stockmeier and Kellar, 1987). /I?-Adrenergic receptor binding was measured to serve as a control, with studies using other methods of applying electroconvulsive shock (e.g. with or without anesthesia, cornea1 versus earclip electrodes and administering electroconvulsive shock every day versus alternate days). In this study, repeated treatment with electroconvulsive shock resulted in a decrease in [3H]8-OHDPAT binding in the hypothalamus, as well as an attenuation of the hypothermia induced by 8-OHDPAT. The effect of electroconvulsive shock on
I-OH-DPAT-induced hypothermia was present at two days after 10 daily shocks but had dissipated by 14 days after chronic electroconvulsive shock. The attenuation of 8-OH-DPAT-induced hypothermia following chronic electroconvulsive shock was in accord with the finding of Goodwin et al. (1987a) with one notable exception. Goodwin et al. (1987a) observed that repeated electroconvulsive shock significantly attenuated the hypothermic response to 8-OH-DPAT only at 2 or 3 weeks after the final shock but not at 1 day after the final shock. Methodological differences between Goodwin et al. (1987a) and the present study may explain the different onset and duration of the effect of electroconvulsive shock on 8-OH-DPAT-induced hypothermia. Goodwin et al. (1987a) used halothane anesthesia and 5 shocks every other day for 10 days, versus treatment with no anesthesia and 10 daily shocks in the present study. The time-course of the altered hypothermic response following electroconvulsive shock in the present study is consistent with other reports of changes
clControl 2.0
6 $ 4
Treatment
N
Treatment duration (days)
Specific binding (fmol/mg tissue)
17 10 1.95 * 0.09 ECI 18 10 1.57 + 0.09. Handled 13 1 1.86&0.14 1 1.85+0.13 ECS’ 16 Rats were killed 2 days after the final ECS. Binding values are the neans k SEM. The concentration of [‘HIS-OH-DPAT was 2 nM. The values for the hypothalamus for 10 days and 1 day Jf ECS are the pooled results of 2-3 replicated experiments, with n = 5-8 rats per treatment group for each experiment. Two-way .ANOVA indicated a significant effect of treatment, *P -c 0.001 vs handled controls.
Handled
r
0.5 0.0
Table 2. Effect ofelectroconvulsive shock (ECS) on [‘H]B-OH-DPAT bindina in the hvoothalamus of the rat
Ia ECS
0.1
Dose of 8-OH-DPAT (mg/kgl Fig. 1. Effect of electroconvulsive shock (ECS) on hypothermia induced by 8-OH-DPAT. Rats were handled (controls) or received electroconvulsive shock administered daily for 10 days and the hypothermic response was measured 2 days after the last shock. Changes in body temperature (ABT) were measured between 0 and 30 min after the subcutaneous administration of 8-OH-DPAT (0.05 or 0.1 mg/kg). Temperature values represent the mean + SEM of 5-9 rats per group for the 0.05 mg/kg dose and 15-16 rats per group for the 0.1 mg/kg dose.. The experiment at 0.1 mg/kg was repeated twice. Two-way ANOVA indicated a significant treatment effect; *P < 0.001 vs handled controls.
1092
C.
A. STOCKMEER
Post-ECS Interval .4 Days A. 2 Days 8. 2.0 G O_ F
1.5 1.0
z 0.5 a :I 0.0 CON ECS Treat. Duration 1 Day
!
CON
ECS IO Days
Fig. 2. Effect of electroconvulsive shock (ECS) on hypothermia induced by 8-OH-DPAT. Rats were handled (controls, CON) or received electroconvulsive shock administered daily for a treatment duration of 1 (A) or 10 (B) days. The hypothermic response was measured 2 or 14 days after the last shock. Changes in body temperature (ABT) were measured between 0 and 30 min after the subcutaneous administration of 8-OH-DPAT (0.1 mg/kg). Temperature
values represent the mean + SEM of 68 rats per treatment group. The experiment with one electroconvulsive shock was repeated.
Treatment groups were not significantly different.
in monoamine receptors in response to electroconvulsive shock. For example, the effects of chronic electroconvulsive shock on serotonin, and fl-adrenergic receptor binding were found to be maximum at l-2 days after the last of several daily shocks and were diminished 1 week after the last shock (Kellar, Cascio, Butler and Kurtzke, 198la; Kellar, Cascio, Bergstrom, Butler and Iadarola, 1981b). Baseline body temperature was not affected by chronic treatment with electroconvulsive shock. In addition to serotonin, other neurotransmitters play a role in regulation of body temperature. Norepinephrine, prostaglandins and neuropeptides, such as melanotropin, thyrotropin releasing hormone and neurotensin are also involved in temperature regulation (Myers, 1984). The serotonin,, receptor may actually be involved in regulating baseline body temperature but complex effects of electroconvulsive shock on these other neurotransmitter systems could negate the influence of serotonin,, receptors on baseline temperature. There may be a homeostatic compensation when one system is perturbed (e.g. by ECS), so that baseline temperature is maintained. Repeated treatment with either electroconvulsive shock or antidepressant drugs has a similar effect on the hypothermic response to 8-OH-DPAT. As with electroconvulsive shock, repeated administration of monoamine oxidase inhibitors or selective serotonin reuptake inhibitors, blunted the hypothermic response to 8-OH-DPAT (Gudelsky, Koenig, Jackman and Meltzer, 1986b; Goodwin et al., 1987a; Hensler, Kovachich and Frazer, 1991). Thus, antidepressant
et al.
treatments, which enhance serotoninergic neurotransmission (Blier, de Montigney and Chaput, 1990), all attenuate the hypothermic response to 8-OH-DPAT. Repeated treatment with electroconvulsive shock decreased the binding of [3H]8-OH-DPAT in the hypothalamus, in addition to attenuating the hypothermic response to 8-OH-DPAT. These two effects of electroconvulsive shock may be related and further experiments are required to establish this relationship. If 8-OH-DPAT elicits the hypothermic response by acting postsynaptically at serotonin,, receptors in the hypothalamus, the electroconvulsive shock-induced decrease in [3H]8-OH-DPAT binding to postsynaptic serotonin,, receptors in the hypothalamus may underlie the attenuation of the hypothermic response to 8-OH-DPAT. Experiments involving direct injection of 8-OH-DPAT into the hypothalamus compared to the midbrain raphe may help resolve the issue of the site of action of 8-OH-DPAT on temperature regulation and the ability of electroconvulsive shock to modulate that response. Hensler et al. (1991) have demonstrated that treatment with phenelzine and clorgyline attenuated the hypothermic response to 8-OH-DPAT and was accompanied by diminished binding of [3H]8-OH-DPAT in the ventro- and dorsomedial nuclei of the hypothalamus. However, other drugs, such as tranylcypromine, citalopram or sertraline, diminished the hypothermic response but did not alter serotonin,, receptor binding in these hypothalamic nuclei (Hensler et al., 1991), suggesting that antidepressant drugs may not attenuate the hypothermic response by acting directly at the hypothalamus. The exact site of action in the brain that mediates 8-OH-DPAT-induced hypothermia and the site at which electroconvulsive shock alters this hypothermic response is unknown. Goodwin et al. (1987b) have suggested that 8-OH-DPAT elicits hypothermia by acting at serotonin,, receptors, located presynaptitally on serotoninergic neurons. However, other groups report that depletion of serotonin with pchlorophenylalanine or lesions with 5,7-dihydroxytryptamine do not diminish the magnitude of 8-OH-DPAT-induced hypothermia (Hjorth, 1985; Gudelsky, Koenig and Meltzer, 1988). Repeated treatment with electroconvulsive shock did not significantly change serotonin,, receptor binding in the cortex in this study, in contrast to other reports noting a decrease (Pandey et al., 1991) or an increase (Nowak and Dulinski, 1991) in the number of receptors in the cortex. In these two studies, the rats were killed 24 hr after the last shock, while in the current study, the rats were killed 48 hr after the last shock. It is otherwise unclear why these three reports are not in agreement. There are conflicting reports on the effects of antidepressant drugs on serotonin,, receptor binding in the cerebral cortex. Chronic treatment with
IO93
Repeated ECS and serotonin,, receptors
imipramine (Mizuta and Sagawa, 198& 1989) or desipramine (Pandy et at., 1991) was reported to decrease serotonin,, receptor binding in the cortex. In contrast, neither monoamine oxidase inhibitors nor selective inhibitors of the reuptake of serotonin affected serotonin!, receptor binding in the cortex or hippocampus (Hensler et al., 1991). Thus, neither antidepressant drugs nor electroconvulsive shock produced consistent changes in serotonin,, receptor binding in the cerebral cortex and the therapeutic effect of these treatments does not appear to be determined by alterations in serotonin,, receptor binding in the cortex. Radioligand binding to serotonin,, receptors in the hippocampus, as well as the cortex, was unaffected by chronic treatment with electroconvulsive shock. Chronic e~e~tr~onvu~iv~ shock results in an increase in responsiveness of hippocampal neurons to serotonin or 8-OH-DPAT through postsynaptic serotonin,, receptors (de Montigny, 1984; Andrade and Nicoll, 1987; Chaput, de Montigney and Blier, 1991) and, conversely, in an attenuated ability of serotonin to inhibit forskolin-stimulated adenylate cyclase in the hippocampus (Newman and Lerer, 1988; Varrault, Leviel and Bockaert, 1991). However, electroconvufsive shock did not alter the binding of f3H]8-OH-DPAT to serotonin,, receptors in the hippocampus in this study or that ofpandy ef al. (1991). The electroconvulsive shock-induced increase in responsiveness of hippocampal neurons to &OHDI”AT, observed by Cbaput et al. (1991), may involve enhanced intracellular mechanisms, downstream from the serotonin,, receptor and does not appear to be the result of a simple increase in number of receptors. In summary, repeated treatment with electroconvulsive shock resulted in a diminished hypothermic response to 8-OH-DPAT, that was associated with a decrease in the binding of I-OH-DPAT to serotonm,, receptors in the hypothalamus. However, repeated treatment with electroconvulsive shock did net appear to elicit changes in the number or affinity of serotonin,, receptors in the cerebral cortex or hippocampus, as determined in radioligand binding assays. Thus, it does not appear that the therapeutic eflect of electroconvulsive shock in depression invclves an action on these binding sites in the frontal cortex or hippocampus but perhaps involves an action on intracellular processes, beyond the serotonin,, recognition site. The recent observation of increased numbers of serotonin,, receptors in the frontal cortex of nonviolent suicide victims (Matsubara et al., 1991), however, emphasizes the importance of continued research on mechanisms of action of artidepressant treatments on the serotonin,, receptor in animal models of depression. Atlnotr~ie~gements-This work was supported by United States Public Health Service Grant NS24523 and Clinical Research Center Grant MH41484. The technical assistance NJ’.
31/l 1-B
ofYing
Zhangand the typing and editorial assistance of Ms Lee Mason are gratefully a~~ow~~g~. We also thank Dr Paul Thompson, for helpful discussions on statistical analyses of the data. REFERENCES Andrade R. and Nicoll R. A. (1987) Pharmacologically distinct actions of serotonin on single pyramidal neurons ofthe rat bippocampus recorded in vitro. J. .P@&& to& 39& 95-124. Bergstrom D. A. and Kellar K. J. (1979) Effect of eIectroconvulsive shock on monoamineergic receptor binding sites in rat brain. Nature 278: 464-465. _ Blier P.. de Montianev C. and Chaaut Y. (19901 A role for the serotonin s&em in the mechanism of action of antidepressant treatments: preclinical evidence. J. clin. Psychiat. 51: Suppl. 4, 14-20. Cervo L. and Samanin R. (1987) Potential antidepressant properties of 8-hydroxy-2-(~-n-propyla~no)tetmlin, a sekctive serotonin,, receptor agonist. Eur. .& Fharmac. 144: 223-229. Chaput Y., de Montigney C. and Blier P. (1991) Presynaptic and postsynaptic modifications of the serotonin system by long-term administration of antidepressant treatments. An in vivo electraphysiologic study in the rat. Neuropsychopharmacolagy 5: 2 19-229.
El Mestikawy S., Fargin A., Raymond J. R., Gozlan H. and Hnatowich M. (1991) The S-HT,, receptor: an overview of recent advances. ~~~~chern. Rex. I& l-10. Goodtin G. M. and Green A. R. ff985f A behavioural and biochemical study in mice and rats cf putative selective agonists and antagonists for 5-HT, and 5-HT, receptors. Br. J. Phurmuc. 84: 743-753. Goodwin G. M., DeSouza R. J. and Green A. R. (1987a) Attenuation by electroconvulsive shock and antidepressant drugs of the 5-HT,, receptor-mediated hypothermia and serotonin syndrome produced by 8-OH-DPAT in the rat. Psychaphwmacology 91: 50&505. Goodwin G. M., DeSouza R. J., Green A. R, and Heal D. J. (1987b) The ph~a~lo~ of the ~ba~o~~ and h~o~e~ic responses of rats to 8-hydroxy-b(di-np~pylam~no) tetralin (S-OH-DPAT). P~~c~op~r~~cology 91: 506-5
1I I
Gudelsky G. A., Koenig J. I. and Meltzer H. Y. (1986a) Thermoregulatory responses to serotonin (5HT) receptor stimulation in the rat. Evidence for opposing roles of 5-HT, and S-HT,, receptors. Neuropharmacology 25: 1307-1313. Gudelsky G. A., Koenig J. 1. and Meltzer H. Y. (1988) Invoivement of serotoninreceptor subtypes in thermoreguhtory responses. In: s--Hf Agonist~~us F~~~h~~~~~e Ikqp (Rech H. R. and Gudelsky 0. A., Eds), pp. 127-142. NPP Books, Ann Arbor. Gudelsky G. A., Koenig J. I., Jackman H. and Meltzer H. Y. (1986133Suooression of the hvno-. and hvoerthermic responses to 5HT8gonists following the repeated administration of monoamine oxidase inhibitors. Psychophar
1992
0028-3908/92
$5.00 + 0.00
Copyright 0 1992Pergamon Press Ltd
EFFECTS OF REPEATED ELECTROCONVULSIVE SHOCK ON SEROTONIN,, RECEPTOR BINDING AND RECEPTOR-MEDIATED HYPOTHERMIA IN THE RAT C. A. STOCKMEIER,*PATRICIA WINGENFELDand G. A. GUDELSKY Laboratory of Biological Psychiatry, Departments of Psychiatry and Neuroscience, Case Western Reserve University, Cleveland, OH 44106, U.S.A. (Accepted 23 April 1992) Summary-Chronic treatment with electroconvulsive shock or antidepressant drugs has been reported to attenuate the hypothermia induced by 8-hydroxy-2-(di-n-propyl)aminotetralin (8-OH-DPAT), a serotonm,, receptor agonist. In the present study, the binding of [‘HII-OH-DPAT to serotonin,, receptors was assessed after treatment of rats with electroconvulsive shock. The effect of electroconvulsive shock on I-OH-DPAT-induced hypothermia also was evaluated. Male rats were handled or received electroconvulsive shock for either 1 or 10 days and were killed 2 days later. Ten days of electroconvulsive shock decreased /I-adrenergic receptor binding in the cerebral cortex, as previously reported. However, the binding of [‘H]8-OH-DPAT to serotonin,, receptors in the cortex or hippocampus was not affected by repeated electroconvulsive shock. In the hypothalamus, 10 days (but not 1day) of electroconvulsive shock significantly decreased the binding of [3H]8-OH-DPAT. In addition, 10 days of electroconvulsive shock resulted in an attenuation of the hypothermic response to I-OH-DPAT, when compared to the response in handled controls. The electroconvulsive shock-induced suppression of the hypothermic response to I-OH-DPAT was no longer evident 2 weeks after the last of 10 daily treatments. A single shock did not affect the hypothermic response to 8-OH-DPAT. The electroconvulsive shock-induced decrease in the binding of [3H]8-OH-DPAT in the hypothalamus may be related tot the electroconvulsive shock-induced attenuation of the hypothermic response to I-OH-DPAT. Key wordr-electroconvulsive
shock, body temperature, I-OH-DPAT,
serotonin,,
receptor, hypothala-
mus, hippocampus, cerebral cortex.
A subtype of serotonin receptor in brain, the serotomn,, receptor, has been identified and studied intensely during the past eight years (El Mestikaway, Fargin, Raymond, Gozlan and Hnatowich, 1991). The serotonin,, receptor may be involved in the mechanism of action of antidepressant and anxiolytic treatments and the pathophysiology of anxiety, schizophrenia, depression and suicide. For example, a selective serotonin,, receptor agonist, 8-hydroxy-2(di-n-propyl)aminotetralin(8-OH-DPAT) mimics the behavioral effect of antidepressant drugs in the forced swimming test (Cervo and Samanin, 1987) and the learned helplessness paradigm (Martin, Beninger, Hamon and Puech, 1990). In recent post-mortem studies, serotonin,, receptor binding was increased in the frontal cortex of suicide victims or patients with chronic schizophrenia (Matsubara, Arora and Meltzer, 1991; Hashimoto, Nishino, Nakai and Tanaka, 1990). An in vim method for investigating the sensitivity of serotonin,, receptors in the brain is to study the temperature response to agonists at serotonin,, re*Address correspondence and reprint requests to: Dr Craig A. Stockmeier, Department of Psychiatry, University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
ceptors. Systemic administration of agonists, like 8-OH-DPAT or ipsapirone in rats or humans, results in a dose-dependent decrease in body temperature (Hjorth, 1985; Goodwin and Green, 1985; Gudelsky, Koenig and Meltzer, 1986a; Koenig, Meltzer and Gudelsky, 1988; Lesch, Disselkamp-Tietze and Schmidtke, 1990). The hypothermic response to 8OH-DPAT in the rat is antagonized by serotonin receptor antagonists with a high affinity for the serotonin,, subtype of receptor (Goodwin, DeSouza, Green and Heal, 1987b; Gudelsky et al., 1986a; Moser, 1991). A role for serotonin,, receptors in the mechanism of action of antidepressant treatments has been suggested by studies demonstrating that chronic treatment of rats with electroconvulsive shock (ECS) diminished behavioral and biochemical responses elicited by activating the serotonin,, receptor. Repeated treatment with electroconvulsive shock attenuates the hypothermia and motor behavioral syndrome produced by 8-OH-DPAT (Goodwin, De Souza and Green, 1987a). In addition, chronic treatment with electroconvulsive shock likewise attenuates the ability of serotonin to inhibit forskolin-stimulated adenylate cyclase in hippocampus through serotonin,, receptors (Newman and Lerer, 1988; Varrault, Leviel and Bockaert, 1991).
1089
1090
C.
A. STOCKMEIERet al.
Repeated treatment with electroconvulsive shock also decreases serotonin,, receptor binding in the cerebral cortex (Pandey, Isaac, Davis and Pandey, 1991). Thus, the therapeutic effect of chronic treatment with electroconvulsive shock may be partially mediated by diminishing the sensitivity of serotonin,, receptors. The purpose of this study was to determine whether electroconvulsive shock altered serotonin,, receptor binding in the cerebral cortex, hippocampus or hypothalamus and whether electroconvulsive shock-induced changes in serotonin,, receptor binding correlated with electroconvulsive shock-induced changes in hypothermia elicited by 8-OH-DPAT. METHODS
Male Sprague-Dawley rats (20&250 g) were purchased from Zivic Miller and housed in a light- and temperature-controlled room and had free access to food and water. Groups of rats were either handled (controls) or received electroconvulsive shock for either 1 or 10 days. No anesthesia was used. Rats were shocked once daily in the morning through earclip electrodes. Current (25 mA, 0.5 set) was delivered by a Medcraft Electroshock Generator and all rats experienced a generalized tonic-clonic seizure. The earclip electrodes were placed on control rats but no current was applied. The rats were killed by decapitation 2 days after the last shock. In one experiment, the rats were killed 14 days after the last of 10 daily shocks. Following decapitation, the brains were quickly dissected on an ice-chilled plate. Fronto-parietal cerebral cortex, hippocampus and hypothalamus were frozen on dry-ice and stored at -60°C until used for the binding assays. Serotonin,, receptor binding was measured using [3H]8-OH-DPAT (127.9 Ci/mmol; New England Nuclear), according to the method of Peroutka (1986), as described by Stockmeier and Kellar (1989). Briefly, the tissues were homogenized with a Polytron in ice-cold 50 mM Tris-HCl (pH 7.7 at 25°C) washed by centrifugation and preincubated with homogenization buffer for 10 min at 37°C. The incubation buffer contained 50 mM Tris-HCl (pH 7.7 at 25°C) 4 mM CaCl,, 10 PM pargyline and 0.1% ascorbic acid. Aliquots of tissue homogenates, containing 4 mg (cortex or hypothalamus) or 3 mg (hippocampus) of tissue, were incubated in duplicate or triplicate with [3H]8-OH-DPAT (0.1-15 nM) at 25°C for 30 min, in a final volume of 0.5 ml. Nonspecific binding was determined in the presence of 10pM serotonin (HCl salt, Sigma). Incubations were terminated by vacuum filtration through glass fiber filters (Schleicher and Schuell, No. 34). The filters were washed 3 times with 4 ml aliquots of cold incubation buffer, transferred to vials to which scintillation fluid was added and counted by liquid scintillation spectrometry. Specific binding, defined as the difference between total binding and nonspecific binding, was 9565% of total binding in the cortex or hippo-
campus, depending on the concentration of [)H]8OH-DPAT. In the hypothalamus, specific binding was 60-70% of total binding. Binding of [‘251]pindolol was also measured in the cortex, using the method of O’Donnell and Frazer (1985). The concentrations of [‘ZSI]pindolol used were 40 and 140 pM; nonspecific binding was measured in the presence of 25 PM ( - )isoproterenol. Measurements of temperature were made, as outlined by Gudelsky et al. (1986a), in groups of control and electroconvulsive shock-treated rats not used for radioligand binding. Rectal temperatures were measured with a telethermometer and thermistor probe (Yellow Springs Instrument Co.). The probe was inserted approximately 5 cm into the rectum. Two days after the last shock, the rats were moved to an observation room (23-25°C) and a period of 2 hr was allowed for stabilization of the temperature. Body temperatures were recorded 30, 15 min and immediately prior to the administration of 8-OHDPAT (0.05 or 0.1 mg/kg, s.c.). A final measurement of body temperature was made 30min after the injection of 8-OH-DPAT. The doses of 8-OH-DPAT were chosen, based on a previously reported dose-response study (Gudelsky et al., 1986a). Data from saturation binding experiments were analyzed by nonlinear regression analysis, using the McPherson adaptation (Elsevier, Cambridge) of the LIGAND computer program (Munson and Rodbard, 1980). The data files selected for analysis by LIGAND had experimentally-measured nonspecific binding subtracted from them. Binding to a single receptor site best explained the experimental data. Reported values are the mean + SEM. One-way or two-way analysis of variance (ANOVA) was used to compare controls with electroconvulsive shocktreated rats. Pos: hoc testing, between related means, was performed with the Student-Newman-Keuls test. A P value of less than 0.05 was considered statistically significant. RESULTS
Treatment with electroconvulsive shock for 10 days resulted in a significant decrease in binding to badrenergic receptors in the cerebral cortex (ANOVA, P < 0.005; data not shown). A significant decrease (15-18%) was detected at the two different concentrations of [‘251]pindolol examined. In the cerebral cortex and hippocampus, [3H]8-OHDPAT bound in a saturable manner to one class of binding sites with a Kd of about 1.6 nM (Table 1). Repeated electroconvulsive shock for 10 days did not significantly affect the number (B,,,) or affinity (Kd) of [3H]8-OH-DPAT binding sites in the cerebral cortex or hippocampus (Table 1). However, in hypothalamus, the binding of [‘H]8-OH-DPAT was significantly decreased after 10 days of daily electroconvulsive shock (Table 2). A single administration of electroconvulsive shock did not significantly affect
Repeated ECS and serotonin, receptors Table I. Effect of electroconvulsive shock (ECS) on Treatment Handled ECS Handled ECS
Brain region Cortex Hippocampus
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[‘IQ-OH-DPATbindingin the brain of the rat
N
Treatment duration (days)
(fmol/mYtissue)
12 12 6 8
10 10 10 10
11.38 f 0.52 11.72 + 0.66 27.87 f. 2.38 29.44 + 2.95
B
1.66+0.15 1.68 + 0.10 1.59 f 0.21 1.50 + 0.19
Rats were killed 2 days after the final ECS. Binding values are the means k SEM.
the binding of [3H]8-OH-DPAT in the hypothalamus (Table 2). Basal body temperatures of rats, treated for 10 da,ys with electroconvulsive shock, did not differ significantly from those of handled controls (handled controls: 38.4 + O.l”C vs ECS: 38.2 f O.l”C). A hypothermic response was produced by both doses of 8-OH-DPAT (0.05 or 0.1 mg/kg, s.c.) in controls and rats treated with electroconvulsive shock for 10 days (Fig. 1). However, at both doses of 8-OH-DPAT, the hypothermic response was significantly less in chronic electroconvulsive shock-treated rats compared to handled controls (Fig. 1). The electroconvulsive shock-induced attenuation by the hypothermic response to 8-OH-DPAT was no longer evident 14 days after the last of 10 treatments with electroconvulsive shock (Fig. 2). A single treatment with electroconvulsive shock did not significantly affect the hypothermic response to 8-OH-DPAT (Fig. 2). DISCUSSION
Chronic treatment with electroconvulsive shock resulted in a significant decrease in /I-adrenergic receptor binding in the cerebral cortex, as previously reported (Bergstrom and Kellar, 1979; Nimgaonkar, Heal, Davies and Green, 1986; Stockmeier and Kellar, 1987). /I?-Adrenergic receptor binding was measured to serve as a control, with studies using other methods of applying electroconvulsive shock (e.g. with or without anesthesia, cornea1 versus earclip electrodes and administering electroconvulsive shock every day versus alternate days). In this study, repeated treatment with electroconvulsive shock resulted in a decrease in [3H]8-OHDPAT binding in the hypothalamus, as well as an attenuation of the hypothermia induced by 8-OHDPAT. The effect of electroconvulsive shock on
I-OH-DPAT-induced hypothermia was present at two days after 10 daily shocks but had dissipated by 14 days after chronic electroconvulsive shock. The attenuation of 8-OH-DPAT-induced hypothermia following chronic electroconvulsive shock was in accord with the finding of Goodwin et al. (1987a) with one notable exception. Goodwin et al. (1987a) observed that repeated electroconvulsive shock significantly attenuated the hypothermic response to 8-OH-DPAT only at 2 or 3 weeks after the final shock but not at 1 day after the final shock. Methodological differences between Goodwin et al. (1987a) and the present study may explain the different onset and duration of the effect of electroconvulsive shock on 8-OH-DPAT-induced hypothermia. Goodwin et al. (1987a) used halothane anesthesia and 5 shocks every other day for 10 days, versus treatment with no anesthesia and 10 daily shocks in the present study. The time-course of the altered hypothermic response following electroconvulsive shock in the present study is consistent with other reports of changes
clControl 2.0
6 $ 4
Treatment
N
Treatment duration (days)
Specific binding (fmol/mg tissue)
17 10 1.95 * 0.09 ECI 18 10 1.57 + 0.09. Handled 13 1 1.86&0.14 1 1.85+0.13 ECS’ 16 Rats were killed 2 days after the final ECS. Binding values are the neans k SEM. The concentration of [‘HIS-OH-DPAT was 2 nM. The values for the hypothalamus for 10 days and 1 day Jf ECS are the pooled results of 2-3 replicated experiments, with n = 5-8 rats per treatment group for each experiment. Two-way .ANOVA indicated a significant effect of treatment, *P -c 0.001 vs handled controls.
Handled
r
0.5 0.0
Table 2. Effect ofelectroconvulsive shock (ECS) on [‘H]B-OH-DPAT bindina in the hvoothalamus of the rat
Ia ECS
0.1
Dose of 8-OH-DPAT (mg/kgl Fig. 1. Effect of electroconvulsive shock (ECS) on hypothermia induced by 8-OH-DPAT. Rats were handled (controls) or received electroconvulsive shock administered daily for 10 days and the hypothermic response was measured 2 days after the last shock. Changes in body temperature (ABT) were measured between 0 and 30 min after the subcutaneous administration of 8-OH-DPAT (0.05 or 0.1 mg/kg). Temperature values represent the mean + SEM of 5-9 rats per group for the 0.05 mg/kg dose and 15-16 rats per group for the 0.1 mg/kg dose.. The experiment at 0.1 mg/kg was repeated twice. Two-way ANOVA indicated a significant treatment effect; *P < 0.001 vs handled controls.
1092
C.
A. STOCKMEER
Post-ECS Interval .4 Days A. 2 Days 8. 2.0 G O_ F
1.5 1.0
z 0.5 a :I 0.0 CON ECS Treat. Duration 1 Day
!
CON
ECS IO Days
Fig. 2. Effect of electroconvulsive shock (ECS) on hypothermia induced by 8-OH-DPAT. Rats were handled (controls, CON) or received electroconvulsive shock administered daily for a treatment duration of 1 (A) or 10 (B) days. The hypothermic response was measured 2 or 14 days after the last shock. Changes in body temperature (ABT) were measured between 0 and 30 min after the subcutaneous administration of 8-OH-DPAT (0.1 mg/kg). Temperature
values represent the mean + SEM of 68 rats per treatment group. The experiment with one electroconvulsive shock was repeated.
Treatment groups were not significantly different.
in monoamine receptors in response to electroconvulsive shock. For example, the effects of chronic electroconvulsive shock on serotonin, and fl-adrenergic receptor binding were found to be maximum at l-2 days after the last of several daily shocks and were diminished 1 week after the last shock (Kellar, Cascio, Butler and Kurtzke, 198la; Kellar, Cascio, Bergstrom, Butler and Iadarola, 1981b). Baseline body temperature was not affected by chronic treatment with electroconvulsive shock. In addition to serotonin, other neurotransmitters play a role in regulation of body temperature. Norepinephrine, prostaglandins and neuropeptides, such as melanotropin, thyrotropin releasing hormone and neurotensin are also involved in temperature regulation (Myers, 1984). The serotonin,, receptor may actually be involved in regulating baseline body temperature but complex effects of electroconvulsive shock on these other neurotransmitter systems could negate the influence of serotonin,, receptors on baseline temperature. There may be a homeostatic compensation when one system is perturbed (e.g. by ECS), so that baseline temperature is maintained. Repeated treatment with either electroconvulsive shock or antidepressant drugs has a similar effect on the hypothermic response to 8-OH-DPAT. As with electroconvulsive shock, repeated administration of monoamine oxidase inhibitors or selective serotonin reuptake inhibitors, blunted the hypothermic response to 8-OH-DPAT (Gudelsky, Koenig, Jackman and Meltzer, 1986b; Goodwin et al., 1987a; Hensler, Kovachich and Frazer, 1991). Thus, antidepressant
et al.
treatments, which enhance serotoninergic neurotransmission (Blier, de Montigney and Chaput, 1990), all attenuate the hypothermic response to 8-OH-DPAT. Repeated treatment with electroconvulsive shock decreased the binding of [3H]8-OH-DPAT in the hypothalamus, in addition to attenuating the hypothermic response to 8-OH-DPAT. These two effects of electroconvulsive shock may be related and further experiments are required to establish this relationship. If 8-OH-DPAT elicits the hypothermic response by acting postsynaptically at serotonin,, receptors in the hypothalamus, the electroconvulsive shock-induced decrease in [3H]8-OH-DPAT binding to postsynaptic serotonin,, receptors in the hypothalamus may underlie the attenuation of the hypothermic response to 8-OH-DPAT. Experiments involving direct injection of 8-OH-DPAT into the hypothalamus compared to the midbrain raphe may help resolve the issue of the site of action of 8-OH-DPAT on temperature regulation and the ability of electroconvulsive shock to modulate that response. Hensler et al. (1991) have demonstrated that treatment with phenelzine and clorgyline attenuated the hypothermic response to 8-OH-DPAT and was accompanied by diminished binding of [3H]8-OH-DPAT in the ventro- and dorsomedial nuclei of the hypothalamus. However, other drugs, such as tranylcypromine, citalopram or sertraline, diminished the hypothermic response but did not alter serotonin,, receptor binding in these hypothalamic nuclei (Hensler et al., 1991), suggesting that antidepressant drugs may not attenuate the hypothermic response by acting directly at the hypothalamus. The exact site of action in the brain that mediates 8-OH-DPAT-induced hypothermia and the site at which electroconvulsive shock alters this hypothermic response is unknown. Goodwin et al. (1987b) have suggested that 8-OH-DPAT elicits hypothermia by acting at serotonin,, receptors, located presynaptitally on serotoninergic neurons. However, other groups report that depletion of serotonin with pchlorophenylalanine or lesions with 5,7-dihydroxytryptamine do not diminish the magnitude of 8-OH-DPAT-induced hypothermia (Hjorth, 1985; Gudelsky, Koenig and Meltzer, 1988). Repeated treatment with electroconvulsive shock did not significantly change serotonin,, receptor binding in the cortex in this study, in contrast to other reports noting a decrease (Pandey et al., 1991) or an increase (Nowak and Dulinski, 1991) in the number of receptors in the cortex. In these two studies, the rats were killed 24 hr after the last shock, while in the current study, the rats were killed 48 hr after the last shock. It is otherwise unclear why these three reports are not in agreement. There are conflicting reports on the effects of antidepressant drugs on serotonin,, receptor binding in the cerebral cortex. Chronic treatment with
IO93
Repeated ECS and serotonin,, receptors
imipramine (Mizuta and Sagawa, 198& 1989) or desipramine (Pandy et at., 1991) was reported to decrease serotonin,, receptor binding in the cortex. In contrast, neither monoamine oxidase inhibitors nor selective inhibitors of the reuptake of serotonin affected serotonin!, receptor binding in the cortex or hippocampus (Hensler et al., 1991). Thus, neither antidepressant drugs nor electroconvulsive shock produced consistent changes in serotonin,, receptor binding in the cerebral cortex and the therapeutic effect of these treatments does not appear to be determined by alterations in serotonin,, receptor binding in the cortex. Radioligand binding to serotonin,, receptors in the hippocampus, as well as the cortex, was unaffected by chronic treatment with electroconvulsive shock. Chronic e~e~tr~onvu~iv~ shock results in an increase in responsiveness of hippocampal neurons to serotonin or 8-OH-DPAT through postsynaptic serotonin,, receptors (de Montigny, 1984; Andrade and Nicoll, 1987; Chaput, de Montigney and Blier, 1991) and, conversely, in an attenuated ability of serotonin to inhibit forskolin-stimulated adenylate cyclase in the hippocampus (Newman and Lerer, 1988; Varrault, Leviel and Bockaert, 1991). However, electroconvufsive shock did not alter the binding of f3H]8-OH-DPAT to serotonin,, receptors in the hippocampus in this study or that ofpandy ef al. (1991). The electroconvulsive shock-induced increase in responsiveness of hippocampal neurons to &OHDI”AT, observed by Cbaput et al. (1991), may involve enhanced intracellular mechanisms, downstream from the serotonin,, receptor and does not appear to be the result of a simple increase in number of receptors. In summary, repeated treatment with electroconvulsive shock resulted in a diminished hypothermic response to 8-OH-DPAT, that was associated with a decrease in the binding of I-OH-DPAT to serotonm,, receptors in the hypothalamus. However, repeated treatment with electroconvulsive shock did net appear to elicit changes in the number or affinity of serotonin,, receptors in the cerebral cortex or hippocampus, as determined in radioligand binding assays. Thus, it does not appear that the therapeutic eflect of electroconvulsive shock in depression invclves an action on these binding sites in the frontal cortex or hippocampus but perhaps involves an action on intracellular processes, beyond the serotonin,, recognition site. The recent observation of increased numbers of serotonin,, receptors in the frontal cortex of nonviolent suicide victims (Matsubara et al., 1991), however, emphasizes the importance of continued research on mechanisms of action of artidepressant treatments on the serotonin,, receptor in animal models of depression. Atlnotr~ie~gements-This work was supported by United States Public Health Service Grant NS24523 and Clinical Research Center Grant MH41484. The technical assistance NJ’.
31/l 1-B
ofYing
Zhangand the typing and editorial assistance of Ms Lee Mason are gratefully a~~ow~~g~. We also thank Dr Paul Thompson, for helpful discussions on statistical analyses of the data. REFERENCES Andrade R. and Nicoll R. A. (1987) Pharmacologically distinct actions of serotonin on single pyramidal neurons ofthe rat bippocampus recorded in vitro. J. .P@&& to& 39& 95-124. Bergstrom D. A. and Kellar K. J. (1979) Effect of eIectroconvulsive shock on monoamineergic receptor binding sites in rat brain. Nature 278: 464-465. _ Blier P.. de Montianev C. and Chaaut Y. (19901 A role for the serotonin s&em in the mechanism of action of antidepressant treatments: preclinical evidence. J. clin. Psychiat. 51: Suppl. 4, 14-20. Cervo L. and Samanin R. (1987) Potential antidepressant properties of 8-hydroxy-2-(~-n-propyla~no)tetmlin, a sekctive serotonin,, receptor agonist. Eur. .& Fharmac. 144: 223-229. Chaput Y., de Montigney C. and Blier P. (1991) Presynaptic and postsynaptic modifications of the serotonin system by long-term administration of antidepressant treatments. An in vivo electraphysiologic study in the rat. Neuropsychopharmacolagy 5: 2 19-229.
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Gudelsky G. A., Koenig J. I. and Meltzer H. Y. (1986a) Thermoregulatory responses to serotonin (5HT) receptor stimulation in the rat. Evidence for opposing roles of 5-HT, and S-HT,, receptors. Neuropharmacology 25: 1307-1313. Gudelsky G. A., Koenig J. 1. and Meltzer H. Y. (1988) Invoivement of serotoninreceptor subtypes in thermoreguhtory responses. In: s--Hf Agonist~~us F~~~h~~~~~e Ikqp (Rech H. R. and Gudelsky 0. A., Eds), pp. 127-142. NPP Books, Ann Arbor. Gudelsky G. A., Koenig J. I., Jackman H. and Meltzer H. Y. (1986133Suooression of the hvno-. and hvoerthermic responses to 5HT8gonists following the repeated administration of monoamine oxidase inhibitors. Psychophar
