Pharmacology Biochemistry & Behavior, Vol. 6, pp. 79--85. Copyright © 1977 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

Plasma Corticosterone and Brain Catecholamines in Stress: Effect of Psychotropic Drugs K E V I N L. K E I M A N D E R N E S T B. S I G G

Roche Research Division, Nutley, N J 07110 ( R e c e i v e d 30 O c t o b e r 1 9 7 6 ) KEIM, K. L. AND E. B. SIGG. Plasma eorticosterone and brain eatecholamines in stress: effect of psychotropic drugs. PHARMAC. BIOCHEM. BEHAV. 6(1) 7 9 - 8 5 , 1977. - - In nonstressed rats, subcutaneous administration of haloperidol (HAL) and large doses of diazepam (DZ) increased plasma corticosterone (CS). Hypothalamic norepinephrine (NE) was lowered significantly by desmethylimipramine (DMI), HAL and, to a lesser extent, by DZ and phenobarbital (PHB). In rats pretreated with either DZ, DMI, HAL or PHB the restraint-induced rise of CS was diminished, DZ being most potent. CPZ had a variable effect, slightly increasing or decreasing the CS response. Pretreatment (16 hr) with pargyline (PA) did not affect the CS rise to stress. The reduction of hypothalamic NE evoked by restraint was attenuated by DZ, and to a lesser extent, by PHB and HAL. Restraint of PA-treated rats did not lower the PA-elevated hypothalamic NE. The stress-induced increase in hypothalamic dopamine was prevented by CPZ and, partially, by PHB. It is emphasized that the net plasma CS and brain catecholamine changes in response to stress are dependent on the drug-induced neuroendocrine feedback state prevalent immediately before commencement of the stress procedure. Stress Corticosterone Brain catecholamines Tranquilizers Hypothalamus

Antidepressants

Neuroleptics

association b e t w e e n c e n t r a l c a t e c h o l a m i n e s a n d ACTH s e c r e t i o n in c o n t i n u a t i o n of o u r earlier w o r k [ 1 7 , 3 3 ] .

IT HAS b e e n p r o p o s e d t h a t t h e resting s e c r e t i o n of a d r e n o c o r t i c o t r o p i n h o r m o n e ( A C T H ) is m o d u l a t e d b y central biogenic amines influencing those hypothalamic cells w h i c h p r o d u c e c o r t i c o t r o p i n releasing h o r m o n e . Catec h o l a m i n e s as well as i n d o l a m i n e s have b e e n i m p l i c a t e d , a n d it is still d e b a t e d w h e t h e r o n e or m o r e a m i n e s are i n v o l v e d in t h e basal, circadian a n d stress-evoked s e c r e t i o n of ACTH. Moreover, it r e m a i n s c o n t r o v e r s i a l w h e t h e r t h e a m i n e s e x e r t an i n h i b i t o r y [ 1 2 , 3 7 ] or f a c i l i t a t o r y [5,27] a c t i o n o n basal A C T H secretion. It is also claimed t h a t b r a i n a m i n e s play n o significant role in t h e c o n t r o l of A C T H s e c r e t i o n [ 1,8]. In c o n t r a s t to t h e u n c e r t a i n t y of a biogenic a m i n e c o n t r o l over resting A C T H secretion, the s t r e s s - i n d u c e d release of A C T H is c o n s i s t e n t l y associated with a decrease in c e r e b r a l n o r e p i n e p h r i n e (NE) a n d an increase in its t u r n o v e r (see [37] for references). M i n o r t r a n q u i l i z e r s n o t only p r e v e n t t h e stress-evoked increase in p l a s m a cortic o s t e r o n e (CS) [ 19] b u t also i n h i b i t t h e a c c e l e r a t i o n of NE t u r n o v e r [ 2 2 , 2 8 1 . However, studies w i t h o t h e r p s y c h o active agents k n o w n t o a f f e c t a m i n e r g i c m e c h a n i s m s , e.g. c h l o r p r o m a z i n e , have r e s u l t e d in c o n t r o v e r s i a l data concerning t h e i r effects o n t h e p i t u i t a r y - a d r e n a l axis [ 9 ] . T h e aim of this s t u d y was to e x a m i n e the effects of r e p r e s e n t a t i v e p s y c h o a c t i v e drugs o n s t r e s s - i n d u c e d changes in p r o s e n c e p h a l i c n o r e p i n e p h r i n e a n d d o p a m i n e , a n d on p l a s m a CS as an i n d e x o f A C T H secretion. In part, we also s o u g h t to f u r t h e r e l u c i d a t e t h e possible physiological

METHOD Male Sprague-Dawley rats were h o u s e d individually for t w o weeks a f t e r t h e y h a d b e e n received at 4 4 - 5 5 days of age. F o o d a n d w a t e r were available ad lib, and the animals were k e p t in an isolated r o o m w i t h regulated t e m p e r a t u r e (23°C), h u m i d i t y and i l l u m i n a t i o n . The light p e r i o d was f r o m 0 6 0 0 to 1800 hr. The b o d y weight of the rats was a p p r o x i m a t e l y 295 g at t h e time of e x p e r i m e n t a t i o n . R e s t r a i n t stress was e f f e c t e d by placing a rat in a plastic c y l i n d e r o f 7 cm dia. and of variable length. A d e q u a t e v e n t i l a t i o n was p r o v i d e d by m e a n s of holes in the sides and f r o n t e n d o f the t u b e . T h e r e s t r a i n t d u r a t i o n was 30 min. T h e e x p e r i m e n t a l design was as follows: each experim e n t utilized 56 rats. Each drug was evaluated at t h r e e doses a n d c o m p a r e d to a p p r o p r i a t e c o n t r o l s . A basal c o n t r o l g r o u p (n = 8) was i n c l u d e d in each a n d was killed at 0 8 0 0 hr, w h i c h was zero t i m e for the following s u b s e q u e n t e x p e r i m e n t a l p r o c e d u r e s . The r e m a i n i n g 48 e x p e r i m e n t a l rats were divided i n t o a n o n s t r e s s a n d a stress group. Each of these t w o groups was f u r t h e r divided i n t o f o u r s u b g r o u p s of 6 rats each. One s u b g r o u p was i n j e c t e d w i t h a n a p p r o p r i a t e placebo, a n d t h e r e m a i n i n g t h r e e s u b g r o u p s received d i f f e r e n t doses of the test drug. F o l l o w i n g injection of e i t h e r p l a c e b o or drug, rats were r e t u r n e d to t h e i r h o m e cages a n d left u n d i s t u r b e d for 1 hr. At this time, the 79

80 rats in the nonstress group were killed to d e t e r m i n e whether the drugs, per se, altered the prestress resting h o r m o n e levels. The rats in the stress group were then restrained for 30 min and decapitated i m m e d i a t e l y thereafter. Trunk blood was collected through siliconized funnels into tubes which contained 14 mg EDTA. Plasma was obtained by centrifugation (3000 rpm for 20 rain at 4°C) and was frozen. The brain was rapidly r e m o v e d from the skull and placed on ice. T w o parts were dissected from the ice-cooled brains: the telencephalon, which consisted of the frontal pole anterior to the c a u d a t e - p u t a m e n c o m p l e x (mean sample weight 191.2 -+ 7.6 mg), and the hypothalamus which was represented" by a tissue block extending from behind the chiasma to the mammillary nucleus, the lateral borders being a p p r o x i m a t e l y 2 m m from the midline and the dorsal border just above the arcuate nucleus (mean sample weight 102. 6 -+ 9.8 rag). Following weighing, the samples were h o m o g e n i z e d in 0.4 N perchloric acid and frozen. Norepinephrine (NE) and dopamine (DA) eluates were prepared according to the m e t h o d of Horst et al. [16]. Supernatants of the tissue h o m o g e n a t e s were obtained on the day of assay, and the specific catecholamines were differentially eluted from a Dowex AG50W-X4 (Na + form) column. A standard curve was determined with each assay. Recovery of b o t h catecholamines was consistently b e t w e e n 70 and 80%. NE and DA were determined fluorometrically according to the m e t h o d of Laverty and Sharman [ 21 ]. Plasma CS was assayed according to the spectrofluorometric m e t h o d of Mattingly [25] with minor modifications [17]. A CS standard curve was prepared on each assay day. Recovery was n o t less than 80%, and precision was greater than 95%. The in vitro reactivity of the adrenal to A C T H (Cortrosyn, Organon) was measured by assaying CS (ng CS/mg adrenal/2 hr) released from adrenal fragments as previously described [ 17]. The following drugs were administered subcutaneously 1 hr prior to the sacrifice of the nonstress group and the c o m m e n c e m e n t of restraint of the stress group: desmethylimipramine h y d r o c h l o r i d e (DMI), c h l o r p r o m a z i n e hydrochloride (CPZ), haloperidol (HAL), diazepam (DZ), and phenobarbital sodium (PHB). Pargyline h y d r o c h l o r i d e (PA) was administered intraperitoneally 16 hr before restraint. When appropriate, the drug dose was calculated in mg of its salt. DZ was used as the commercially available injectable. A 0.1% solution of H A L was prepared in a m i x t u r e of 0.1 N HC1, absolute ethanol, and water in a v o l u m e t r i c ratio of 1:2:100. The drugs were injected in a constant v o l u m e of 1 ml/kg body weight, except for 10 mg/kg DZ which was administered at 2 ml/kg. Saline served as the vehicle control except for the experiments with HAL and DZ in which acidified ethanol-saline and the c o m m e r c i a l vehicle were used respectively. (Commercial vehicle: each ml contains benzyl alcohol 0.015 ml, ethyl alcohol 0.1 ml, propylene glycol 0.4 ml, sodium benzoate 48.8 mg benzoic acid 1.2 rag, water for injection qs 1 ml, pH = 6,55.) Statistical differences in h o r m o n e or catecholamine c o n c e n t r a t i o n between the means of placebo-treated and drug-treated groups from nonstressed and stressed animals were determined by an unpaired Student's t-test. Sets of three t-tests (placebo vs each one of three different doses of each drug) were carried out so that the overall significance was set at 0.06. However, to provide an estimate of significance within any single t analysis, the probability level was

KEIM A N D SIGG adjusted to 0.02 according to Bonferroni t statistics for simultaneous statistical inference [26]. Thus, a difference with p < 0 . 0 2 was regarded as significant. RESULTS The subcutaneous administration of various placebos (saline, acidified ethanol-saline and DZ vehicle) elicited a small stress response in rats. CS often doubled 1 hr after administration from the basal range of 4.7 5.5 u g h . Simultaneously, h y p o t h a l a m i c NE decreased by an average of 11% from a basal range of 1 . 8 7 - 2 . 2 5 ug/g. In 4 of the 5 experiments in which DA was determined (DA was not measured in the DMI experiment), vehicle administration did not significantly alter the DA c o n t e n t when c o m p a r e d to the basal range of 0 . 4 1 - 0 . 5 5 ug/g; in one e x p e r i m e n t h y p o t h a l a m i c DA was significantly reduced. In 3 of 6 experiments telencephalic NE was unaffected by vehicle administration while it was reduced in the remaining 3 experiments. The magnitude or frequency of the observed changes did n o t depend upon a particular placebo. When placebo-treated rats were restrained for 30 min, marked changes in all biochemical parameters occurred. Pooling data from tile individual experiments, plasma CS increased by a p p r o x i m a t e l y 700%. H y p o t h a l a m i c NE and telencephalic NE decreased 33%, whereas h y p o t h a l a m i c DA rose 28%. The difference in values between the placebotreated, nonstressed and the placebo-treated, stressed rats represented the control response to restraint and was used in the evaluation of the psychoactive drugs. Since alterations in telencephalic NE were qualitatively similar to changes in h y p o t h a l a m i c NE, the telencephalic data are, though described in the text, not illustrated. A n tidepressan ts DMI, in doses of 3, 10 and 30 mg/kg SC, did not raise plasma CS in nonstressed rats more than that of a saline placebo. However, the restraint stress-induced increase in CS was significantly attenuated by 10 and 30 mg/kg DMI (p