Pharmacology Biochemistry & Behavior, Vol. 6, pp. 197--202. Copyright © 1977 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Acute Stress and the Brain Norepinephrine Uptake Mechanism in the Rat E. D. HENDLEY, G. H. BURROWS, E, S. ROBINSON, K. A. H E I D E N R E I C H A N D C. A. B U L M A N Department o f Physiology and Biophysics, University o f Vermont College o f Medicine Burlington, V T 05401 (Received 15 S e p t e m b e r 1976) HENDLEY, E. D., G. H. BURROWS, E. S. ROBINSON, K. A. HEIDENREICH AND C. A. BULMAN. Acute stress and the brain norepinephrine uptake mechanism in the rat. PHARMAC. BIOCHEM. BEHAV. 6(2) 197-202, 1977. - The kinetic constants for norepinephrine uptake in cerebral cortical homogenates were determined in vitro immediately following an acute stress consisting of either forced immobilization, cold-wet exposure, combined cold-plus-restraint, swim stress, or electric footshock in the rat. The kinetic constants apparent K and V for uptake of 3H-l-norepinephrine were . . . . . . ' o m max' significantly increased only following 10 min swim at 22 C or following 5 min electric footshock. When severe hypothermia accompanied the stress, the findings suggested that a profound reduction in body temperature was associated with depressed responsiveness of brain noradrenergic mechanisms to stress including decreased uptake kinetic constants. In a series in which the duration of electric footshock was varied from 2 to 30 rain, it was noted that the NE uptake kinetic constants were increased at 5 rain, but were similar to paired controls at 2, 10 and 30 rain following the onset of footshock. It was concluded that various acute stresses did not elicit a generalized response of the cortical NE uptake mechanism to stress in the rat. Furthermore, when uptake kinetic constants did change with stress, the values were often within the range of normal values seen in the rat. Norepinephrine Uptake Cold stress Swim stress
Stress Brain
Kinetic constants Cerebral cortex
Footshock
THE a p p l i c a t i o n o f various aversive t r e a t m e n t s , or stresses, in e x p e r i m e n t a l animals has long been k n o w n to decrease steady state levels o f brain n o r e p i n e p h r i n e (NE) and to increase its t u r n o v e r [1, 2, 5, 6, 15, 2 4 ] . More recently, K o r f et al. [13] e s t a b l i s h e d t h a t the locus coeruleus was essential for m e d i a t i n g the e n h a n c e d NE t u r n o v e r rate in rat cerebral c o r t e x following electric f o o t s h o c k , and Palkovits et al. [17] r e p o r t e d that only the arcuate nucleus a m o n g several h y p o t h a l a m i c nuclei in the rat brain was similarly activated in various acute stresses. The p r e s e n t s t u d y q u e s t i o n e d w h e t h e r the brain n o r a d r e n e r g i c n e u r o n a l m e m brane NE u p t a k e m e c h a n i s m was also altered by acute stress, in view o f the r e c e n t findings t h a t the a p p a r e n t K m (Michaelis c o n s t a n t ) for u p t a k e o f 3 H-NE was increased following such stressful stimuli as i s o l a t i o n - i n d u c e d fighting in mice and e l e c t r o c o n v u l s i v e s h o c k in rats or mice [ 7 - 1 0 , 28]. In the p r e s e n t s t u d y s h o r t - t e r m (acute) stress paradigms were used in which rats were restrained, or e x p o s e d to cold, or s u b j e c t e d to s h o r t swim p e r i o d s at varying water t e m p e r a t u r e s , or to electric f o o t s h o c k . The rats were killed immediately following each stress p r o c e d u r e , and Michaelis-Menten kinetics were used to d e t e r m i n e the a p p a r e n t Km and Vma x for u p t a k e o f NE in crude s y n a p t o s o m e - r i c h h o m o g e n a t e s o f the rat cerebral cortex.
Hypothermia
Restraint stress
and alternate 12 hr periods of light and dark. Rats were sacrificed by cervical dislocation b e t w e e n 10 a.m. and 1 p.m. Body weights ranged from 150 3 0 0 g but within a given series the mean weights were identical for c o n t r o l and e x p e r i m e n t a l rats. Restraint stress consisted of i m m o b i l i z i n g the rats in a wire m e s h restrainer in a p r o n e p o s i t i o n at r o o m t e m p e r a ture for 1 hr, t h e n killing i m m e d i a t e l y following the stress. C o n t r o l rats were u n d i s t u r b e d cage mates. Cold e x p o s u r e was carried out by i m m e r s i n g the rat m o m e n t a r i l y in a weak solution of a non-ionic d e t e r g e n t (Triton X-100, 0.05% in tap water) which soaked its fur so t h a t it r e m a i n e d wet t h r o u g h o u t the 45 min e x p o s u r e to cold. The wet rat was placed in a plastic cage w i t h o u t b e d d i n g in an e m p t y refrigerator at 2 - 4 °C for 45 rain, after which the rat was sacrificed. C o n t r o l s were u n d i s t u r b e d cage mates. C o m b i n e d cold-plus-retraint stress was a c o m b i n a t i o n o f the above two p r o c e d u r e s . Thus rats were doused with d e t e r g e n t s o l u t i o n and i m m o b i l i z e d in the wire mesh restrainer in a p r o n e p o s i t i o n and placed in 2 - 4 ~ C for 45 min t h e n killed. An i m p o r t a n t feature o f this stress was that the rat was n o t free to curl up, nor to shake excess liquid f r o m its coat, c o n s e q u e n t l y rectal t e m p e r a t u r e fell precipitously to a b o u t 17°C after 45 min cold exposure. Of 13 rats s u b j e c t e d to this p r o c e d u r e , one fatality was observed. Swim stress consisted of i m m e r s i n g rats in a plastic p i p e t cleaning jar filled with tap w a t e r at varying t e m p e r a t u r e s in each series f r o m 1 5 ° C - 4 2 ° C . The level of water was set so
METHOD Male adult Sprague-Dawley rats (Canadian Breeding Labs., subsidiary o f Charles River Breeding Labs, Inc.) were h o u s e d in groups o f 6 per cage with f o o d and w a t e r ad lib, 197
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HENDLEY, BURROWS, ROBINSON, HEIDENREICH AND BULMAN
t h a t the rat c o u l d n o t c l i m b o u t of the w a t e r to the rim of the j a r and t h u s the rat swam c o n t i n u o u s l y or t r e a d e d w a t e r d u r i n g the full 10 rain period, a f t e r w h i c h it was sacrificed. C o n t r o l rats were u n d i s t u r b e d cage mates. F o l l o w i n g b o t h cold e x p o s u r e a n d swim stress the rectal t e m p e r a t u r e was m e a s u r e d i m m e d i a t e l y after d e c a p i t a t i o n using a l a b o r a t o r y t h e r m o m e t e r i n s e r t e d 10 cm i n t o the colon. Electric f o o t s h o c k was delivered to the paws of t h e rat placed in a lucite-walled cage, 20 x 24 x 19 cm high, e q u i p p e d with a grid floor of electrifiable rods set at 1.5 cm apart. S h o c k s were of 1 sec d u r a t i o n , 1.6 m A , 7 per rain, r a n d o m i z e d w i t h i n each rain by intervals of 3 - 1 5 sec b e t w e e n shocks. T h e t o t a l s h o c k p e r i o d was varied in each series f r o m 2 - 3 0 min. The rats were sacrificed i m m e d i a t e l y following the stress. C o n t r o l rats were u n d i s t u r b e d cage mates. The k i n e t i c c o n s t a n t s for the u p t a k e of 3 H-1n o r e p i n e p h r i n e (3H-1-NE) were d e t e r m i n e d as rapidly as possible a f t e r sacrificing a pair of rats, one stressed and one c o n t r o l , in a l t e r n a t i n g o r d e r in separate e x p e r i m e n t s w i t h i n a given series. The cerebral c o r t e x was rapidly dissected f r o m the w h o l e brain o n a chilled surface a n d s t o r e d briefly in chilled 0.25 M sucrose u n t i l b l o t t e d lightly, weighed, t h e n h o m o g e n i z e d g e n t l y in 10 v o l u m e s of 0.25 M sucrose, using 6 up a n d d o w n s t r o k e s of a Teflon pestle in a s m o o t h glass h o m o g e n i z e r ( K o n t e s Glass, 0 . 0 0 4 0 . 0 0 6 in. clearance). H o m o g e n a t e s were c e n t r i f u g e d in the cold ( D u p o n t / Sorvall I n s t r u m e n t s , RC2-B) for 10 m i n at 1000 g, and the s u p e r n a t a n t , a c r u d e s y n a p t o s o m e - r i c h fraction, was used w i t h o u t f u r t h e r p u r i f i c a t i o n for the u p t a k e studies. Aliq u o t s of 100 ul, e q u i v a l e n t to 10 mg of original cerebral cortex, were a d d e d in the cold to 2 ml of Krebs p h o s p h a t e Ringer [26] m o d i f i e d to c o n t a i n half the usual CaC12 c o n c e n t r a t i o n , d i s o d i u m e t h y l e n e - d i a m i n e t e t r a a c e t i c acid ( E D T A , 0.05 m g / m l ) , ascorbic acid (0.2 m g / m l ) , glucose (2 m g / m l ) , N i a l a m i d e ( 1 0 uM, Pfizer Corp.) to i n h i b i t m o n o a m i n e oxidase a n d 3 H-1-NE (New E n g l a n d Nuclear Corp., 3.7 Ci/mM, 0.04 uM). N o n r a d i o a c t i v e 1 - n o r e p i n e p h r i n e h y d r o c h l o r i d e ( C a l b i o c h e m Corp.), dissolved in 1 m M HC1 c o n t a i n i n g E D T A (0.05 m g / m l ) , a d d e d to the m e d i a p r o v i d e d a range of 5 or 6 d i f f e r e n t 3H-1-NE c o n c e n t r a tions f r o m 0 . 0 4 - 0 . 2 or 0.4 uM, while the acid vehicle was k e p t c o n s t a n t in all t u b e s at less t h a n 0.5% of t h e i n c u b a t i o n media. I n c u b a t i o n s were carried o u t in d u p l i c a t e at 37 ° C, w i t h shaking, in n y l o n or p o l y p r o p y l e n e c e n t r i f u g e t u b e s ( D u p o n t / S o r v a l l I n s t r u m e n t s ) for 5 min, using r o o m air as the gas phase. The u p t a k e process was rapidly t e r m i n a t e d by i m m e r s i o n of the t u b e s s i m u l t a n e o u s l y in an ethanol-ice w a t e r b a t h , t h e n c e n t r i f u g i n g for 20 m i n at 2 0 , 0 0 0 g in the cold. The surfaces of the pellets were rinsed with 10 ml of cold i s o t o n i c NaC1 (0.15 M) a n d c e n t r i f u g e d again for 10 m i n at 2 0 , 0 0 0 g. The washings were discarded and t h e t u b e s d r a i n e d over a b s o r b e n t p a p e r for at least 45 min. Pellets were dissolved in 10 ml of T r i t o n - X 100 in t o l u e n e ( 1 : 4 ) c o n t a i n i n g PPO and POPOP and 0.2 ml distilled water. T r i t i u m was c o u n t e d in a Packard Tricarb Model 3385 ( P a c k a r d I n s t r u m e n t s Corp.) liquid scintillation s p e c t r o m e t e r at 45% efficiency. Since all samples i n c l u d i n g b l a n k s p r e p a r e d in a similar m a n n e r b u t w i t h o u t a d d e d tissue, as well as samples of 0.2 ml of i n c u b a t i o n m e d i u m , were f o u n d to have t h e same c o u n t i n g efficiency, m o n i tored by sample c h a n n e l s ratio or by a u t o m a t i c e x t e r n a l s t a n d a r d i z a t i o n , n o c o r r e c t i o n s were m a d e for c o u n t i n g efficiency.
Net u p t a k e velocity was calculated as n m o l e s of NE a c c u m u l a t e d in 5 m i n per gram of original cerebral cortex at 37°C m i n u s t h a t a c c u m u l a t e d at 0°C (17% of the a c c u m u l a t i o n s at 37°C). NE a c c u m u l a t e d in 5 rain u n d e r similar c o n d i t i o n s was previously f o u n d to be a l m o s t c o m p l e t e l y u n m e t a b o l i z e d c a t e c h o l a m i n e [ 19]. The kinetic c o n s t a n t s , a p p a r e n t K m and V m a x , were e s t i m a t e d w i t h o u t w e i g h t i n g f r o m Cleland's [4] F o r t r a n p r o g r a m for n o n - l i n e a r regression (best fit to a h y p e r b o l a ) using a PDP-8/e c o m p u t e r (Digital E q u i p m e n t Corp.) and a 4010-1 graphics t e r m i n a l ( T e k t r o n i c s Corp.) c o n n e c t e d to a Sigma 6 ( X e r o x Corp.) c o m p u t e r . Special care was t a k e n to t r e a t c o n t r o l and stress tissues c o n c o m i t a n t l y and identically w i t h respect to time, t e m p e r a t u r e of i n c u b a t i o n and c e n t r i f u g a t i o n c o n d i t i o n s , thus ensuring a legitimate statistical c o m p a r i s o n b e t w e e n pairs of c o n t r o l a n d stressed animals (paired t-test). The kinetic c o n s t a n t s are derived values at best, and would n o t be c o m p a r a b l e e x c e p t u n d e r identical c o n d i t i o n s of measurement. RESULTS E x p o s u r e of rats, while wet, to 2 4°C for 45 m i n ( c o l d stress) failed to alter the k i n e t i c c o n s t a n t s for ~ I t - I - N E u p t a k e ( T a b l e 1). These rats h a d significantly r e d u c e d rectal t e m p e r a t u r e (30°C). I m m o b i l i z a t i o n of rats for one hr at r o o m t e m p e r a t u r e ( r e s t r a i n t stress) was also ineffective in altering t h e k i n e t i c c o n s t a n t s for 3 H-1-NE u p t a k e (Table 1). During c o m b i n e d cold-plus-restraint, rats were i m m o b i lized in the restrainer, t h e n placed in the refrigerator (2 4°C) for 45 min. F o l l o w i n g this stress t h e i r snouts, paws, tails a n d genitalia were bright red in a p p e a r a n c e , and t h e y were grossly inactive prior to sacrificing at the end of t h e stress period. T h e y failed to right t h e m s e l v e s w h e n released f r o m the restrainer and gave n o resistance to h a n d l i n g a n d sacrificing. T h e i r rectal t e m p e r a t u r e s fell m a r k e d l y to an average of 17°C (Table 1) as a result of being d e p r i v e d of two i m p o r t a n t m e a n s of conserving b o d y heat, n a m e l y shaking off excess m o i s t u r e and curling up to lower t o t a l b o d y surface area. More i m p o r t a n t l y , p e r i p h e r a l v a s o d i l a t i o n in t h e skin of these rats was evident, und o u b t e d l y c o n t r i b u t i n g significantly to the p r o f o u n d heat loss a n d i n d i c a t i n g a failure of the n o r a d r e n e r g i c a l l y m e d i a t e d r e s p o n s e to h e a t loss. in these rats the a p p a r e n t K m fell to an average of 17% b e l o w paired c o n t r o l values and this decrease was of b o r d e r l i n e significance (Table 1). T h e r e was n o significant a l t e r a t i o n in the V m a x . In rats s u b j e c t e d to swim stress (10 rain swim in tap w a t e r of varying t e m p e r a t u r e s ) the results o b t a i n e d varied w i t h the t e m p e r a t u r e of the w a t e r (Table 2). When rats swam in w a t e r at b o d y t e m p e r a t u r e (38°C), rectal t e m p e r a t u r e increased slightly b u t c o n s i s t e n t l y (p < 0 . 0 1 ) b y 0.5°C, p r e s u m a b l y as a result of i n a b i l i t y to dissipate h e a t g e n e r a t e d by the m u s c u l a r e x e r t i o n of swimming. T h e r e were n o significant changes in the k i n e t i c c o n s t a n t s a p p a r e n t K m a n d V m a x in these rats. T h e y swam vigorously at the b e g i n n i n g of the 10 m i n period but a f t e r several m i n u t e s , spent m u c h of the time in t r e a d i n g water. They did n o t a p p e a r to search c o n t i n u o u s l y for escape r o u t e s as did rats t h a t swam in cold water. Rats t h a t swam in relatively h o t water ( 4 2 ° C ) were severely h y p e r t h e r m i c after 10 m i n ( 4 1 . 5 ° C , Table 2). T h e y a p p e a r e d to be s o m e w h a t inactive at the e n d of the swim p e r i o d a l t h o u g h t h e y were aggressive w h e n h a n d l e d . T h e r e were n o significant d i f f e r e n c e s in the NE u p t a k e kinetic
STRESS AND BRAIN NOREPINEPHRINE
UPTAKE
199
TABLE
1
KINETIC CONSTANTS FOR UPTAKE OF 3H-I-NOREPINEPHRINE IN HOMOGENATES OF RAT CEREBRAL CORTEX: EFFECTS OF COLD, RESTRAINT STRESS Rectal T e m p °C Control Stress
37.6 _+ 0.2 (16)
--
30.0 _+ 0.1 (16) p 0.05
0.74 _+ 0.10 (6)
C o m b i n e d Cold-Plus-Restraint for 45 min 0.18 + 0.01 0.15 _+ 0.01 (12) (12) 0.1 >p >0.05
0.63 __ 0.04 (12)
0.75 _+ 0.05 (16) p >0.05 0.77 _+ 0.04 (6) p >0.05 0.61 _+ 0.04 (12) p >0.05
Values are m e a n s _+ S E M , n u m b e r of rats in p a r e n t h e s e s . p values from paired t-test. TABLE
2
KINETIC CONSTANTS FOR UPTAKE OF 3H-I-NOREPINEPHRINE IN HOMOGENATES OF RAT CEREBRAL CORTEX: SWIM AT VARYING TEMPERATURES Rectal T e m p °C Control Stress
Apparent K m IzM Control Stress
Vmax nmol/g/5 min Control Stress
37.6 __- 0.2 23.1 + 0.2 (8) (8) p 0.05
0.86 _+ 0.07 0.94 _+ 0.15 (8) (8) p >0.05
37.5 + 0.2 27.4 -+ 0.3 (9) (9) p0.05 p >0.05 5 min Footshock 0.15 ± 0.01 0.20 ± 0.02 0.54 e 0.05 0.69 ± 0,05 (9) (9) (9) (9) p 0.05 30 rain Footshock 0.23 ± 0.01 0.22 ± 0.01 0.85 ± 0.04 0.82 ± 0.04 (16) (16) (16) (16) p>0.05 p>0.05 Values are means ± SEM, number of rats in parentheses. p values from paired t-test. *Shocks were of 1 sec duration, 1.6 mAmp, 7/rain.
c o r t e x and h y p o t h a l a m u s were m a r k e d l y r e d u c e d , plasma c o r t i c o s t e r o n e was elevated, t y r o s i n e h y d r o x y l a s e activity in the f o r e b r a i n was u n c h a n g e d , and n o change in ~H-NE u p t a k e was discernible in cerebral cortical slices [271. F r o m this c o m p o s i t e picture it appears t h a t the earliest n e u r o c h e m i c a l changes were d e t e c t a b l e n o t only in the brainstem, where the n o r a d r e n e r g i c cell bodies are l o c a t e d , b u t also in t h e cerebral c o r t e x , where n o r a d r e n e r g i c nerve t e r m i n a l s e x h i b i t e d increased a p p a r e n t K m and V m a x for NE u p t a k e . With increasing d u r a t i o n of f o o t s h o c k stress, NE u t i l i z a t i o n rate was elevated at a time when rate of NE synthesis could n o t keep pace with t u r n o v e r , resulting in m a r k e d d e p l e t i o n s of e n d o g e n o u s levels of NEI as originally p r o p o s e d by Bliss et al. [ 3 ] . I n t e r e s t i n g / y , with m o r e p r o l o n g e d d u r a t i o n of f o o t s h o c k (3 h o u r s ; [ 2 4 ] ) , or c h r o n i c f o o t s h o c k (15 days; [ 2 7 1 ) , brain NE u p t a k e m e c h a n i s m s again a p p e a r e d to be altered in response to stress. ACKNOWLEDGEMENTS We are grateful to David Karnes for help in computer programming, and to Pfizer, Inc. for a generous gift of Nialamide. This work was supported by USPHS 05429-13 and by USPHS MH25811.
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BURROWS,
ROBINSON,
HEIDENREICH
AND BULMAN
19. Snyder, S. H. and J. T. Coyle. Regional differences in H3-norepinephrine and H3-dopamine uptake into rat brain homogenates. J. Pharmac. exp. Ther. s,165: 7 8 - 8 6 , 1969. 20. Stolk, J. M. and J. D. Barchas. Brain stem norepinephrine: Behavioural and biochemical differentiation of responses to footshock in rats. In: Frontiers in Catecholamine Research, edited by E. Usdin and S. H. Snyder. New York: Pergamon Press, 1973, pp. 719 722. 21. Stolk, J. M., J. D. Barchas, M. Goldstein, W. Boggan and D. X. Freedman. A comparison of p s y c h o m i m e t i c drug effects on rat brain norepinephrine metabolism. Z Pharmac. exp. Ther. 189: 4 2 - 5 0 , 1974. 22. Stone, E. A. Swimm-stress-induced inactivity: Relation to body temperature and brain norepinephrine, and effects o f d-amphetamine. Psychosom. Med. 3 2 : 5 1 - 5 9 , 1970. 23. Stone, E. A. Behavioral and neurochemical effects of acute swim stress are due to hypothermia. Life Sci. 9: 8 7 7 - 8 8 8 , 1970. 24. Thierry, A. M., F. Javoy, J. Glowinski and S. S. Kety. Effects of stress on the metabolism of norepinephrine, dopamine and serotonin in the central nervous system o f the rat. 1. Modifications of norepinephrine turnover. J. Pharmac. exp. Ther. 163: 1 6 3 - 1 7 1 , 1968. 25. Thierry, A. M., G. Blanc and J. Glowinski. Effect of stress on the disposition of catecholamines localized in various intraneuronal storage forms in the brain stem of the rat. Neurochemistry 18: 4 4 9 - 4 6 1 , 1971. 26. Umbreit, W. W., R. H. Burris and J. F. Stauffer. Manometric Techniques. Minneapolis: Burgess, 1957, pp. 1 4 9 - 1 5 0 . 27. Weiss, J. M., H. I. Glazer, L. A. Pohorecky, J. Brick and N. E. Miller. Effects of chronic exposure to stressors on avoidanceescape behavior and on brain norepinephrine. Psyehosom. Meal. 37: 5 2 2 - 5 3 4 , 1975. 28. Welch, B. L., E. D. Hendley and I. Turek. Norepinephrine uptake into cerebral cortical s y n a p t o s o m e s after one fight or electro-convulsive shock. Science 183: 2 2 0 - 2 2 1 , 1974. 29. Wilbrandt, W. and T. Rosenberg. The concept of carrier transport and its corollaries in pharmacology. Pharmac. Rev. 1 3 : 1 0 9 183, 1961.
Acute Stress and the Brain Norepinephrine Uptake Mechanism in the Rat E. D. HENDLEY, G. H. BURROWS, E, S. ROBINSON, K. A. H E I D E N R E I C H A N D C. A. B U L M A N Department o f Physiology and Biophysics, University o f Vermont College o f Medicine Burlington, V T 05401 (Received 15 S e p t e m b e r 1976) HENDLEY, E. D., G. H. BURROWS, E. S. ROBINSON, K. A. HEIDENREICH AND C. A. BULMAN. Acute stress and the brain norepinephrine uptake mechanism in the rat. PHARMAC. BIOCHEM. BEHAV. 6(2) 197-202, 1977. - The kinetic constants for norepinephrine uptake in cerebral cortical homogenates were determined in vitro immediately following an acute stress consisting of either forced immobilization, cold-wet exposure, combined cold-plus-restraint, swim stress, or electric footshock in the rat. The kinetic constants apparent K and V for uptake of 3H-l-norepinephrine were . . . . . . ' o m max' significantly increased only following 10 min swim at 22 C or following 5 min electric footshock. When severe hypothermia accompanied the stress, the findings suggested that a profound reduction in body temperature was associated with depressed responsiveness of brain noradrenergic mechanisms to stress including decreased uptake kinetic constants. In a series in which the duration of electric footshock was varied from 2 to 30 rain, it was noted that the NE uptake kinetic constants were increased at 5 rain, but were similar to paired controls at 2, 10 and 30 rain following the onset of footshock. It was concluded that various acute stresses did not elicit a generalized response of the cortical NE uptake mechanism to stress in the rat. Furthermore, when uptake kinetic constants did change with stress, the values were often within the range of normal values seen in the rat. Norepinephrine Uptake Cold stress Swim stress
Stress Brain
Kinetic constants Cerebral cortex
Footshock
THE a p p l i c a t i o n o f various aversive t r e a t m e n t s , or stresses, in e x p e r i m e n t a l animals has long been k n o w n to decrease steady state levels o f brain n o r e p i n e p h r i n e (NE) and to increase its t u r n o v e r [1, 2, 5, 6, 15, 2 4 ] . More recently, K o r f et al. [13] e s t a b l i s h e d t h a t the locus coeruleus was essential for m e d i a t i n g the e n h a n c e d NE t u r n o v e r rate in rat cerebral c o r t e x following electric f o o t s h o c k , and Palkovits et al. [17] r e p o r t e d that only the arcuate nucleus a m o n g several h y p o t h a l a m i c nuclei in the rat brain was similarly activated in various acute stresses. The p r e s e n t s t u d y q u e s t i o n e d w h e t h e r the brain n o r a d r e n e r g i c n e u r o n a l m e m brane NE u p t a k e m e c h a n i s m was also altered by acute stress, in view o f the r e c e n t findings t h a t the a p p a r e n t K m (Michaelis c o n s t a n t ) for u p t a k e o f 3 H-NE was increased following such stressful stimuli as i s o l a t i o n - i n d u c e d fighting in mice and e l e c t r o c o n v u l s i v e s h o c k in rats or mice [ 7 - 1 0 , 28]. In the p r e s e n t s t u d y s h o r t - t e r m (acute) stress paradigms were used in which rats were restrained, or e x p o s e d to cold, or s u b j e c t e d to s h o r t swim p e r i o d s at varying water t e m p e r a t u r e s , or to electric f o o t s h o c k . The rats were killed immediately following each stress p r o c e d u r e , and Michaelis-Menten kinetics were used to d e t e r m i n e the a p p a r e n t Km and Vma x for u p t a k e o f NE in crude s y n a p t o s o m e - r i c h h o m o g e n a t e s o f the rat cerebral cortex.
Hypothermia
Restraint stress
and alternate 12 hr periods of light and dark. Rats were sacrificed by cervical dislocation b e t w e e n 10 a.m. and 1 p.m. Body weights ranged from 150 3 0 0 g but within a given series the mean weights were identical for c o n t r o l and e x p e r i m e n t a l rats. Restraint stress consisted of i m m o b i l i z i n g the rats in a wire m e s h restrainer in a p r o n e p o s i t i o n at r o o m t e m p e r a ture for 1 hr, t h e n killing i m m e d i a t e l y following the stress. C o n t r o l rats were u n d i s t u r b e d cage mates. Cold e x p o s u r e was carried out by i m m e r s i n g the rat m o m e n t a r i l y in a weak solution of a non-ionic d e t e r g e n t (Triton X-100, 0.05% in tap water) which soaked its fur so t h a t it r e m a i n e d wet t h r o u g h o u t the 45 min e x p o s u r e to cold. The wet rat was placed in a plastic cage w i t h o u t b e d d i n g in an e m p t y refrigerator at 2 - 4 °C for 45 rain, after which the rat was sacrificed. C o n t r o l s were u n d i s t u r b e d cage mates. C o m b i n e d cold-plus-retraint stress was a c o m b i n a t i o n o f the above two p r o c e d u r e s . Thus rats were doused with d e t e r g e n t s o l u t i o n and i m m o b i l i z e d in the wire mesh restrainer in a p r o n e p o s i t i o n and placed in 2 - 4 ~ C for 45 min t h e n killed. An i m p o r t a n t feature o f this stress was that the rat was n o t free to curl up, nor to shake excess liquid f r o m its coat, c o n s e q u e n t l y rectal t e m p e r a t u r e fell precipitously to a b o u t 17°C after 45 min cold exposure. Of 13 rats s u b j e c t e d to this p r o c e d u r e , one fatality was observed. Swim stress consisted of i m m e r s i n g rats in a plastic p i p e t cleaning jar filled with tap w a t e r at varying t e m p e r a t u r e s in each series f r o m 1 5 ° C - 4 2 ° C . The level of water was set so
METHOD Male adult Sprague-Dawley rats (Canadian Breeding Labs., subsidiary o f Charles River Breeding Labs, Inc.) were h o u s e d in groups o f 6 per cage with f o o d and w a t e r ad lib, 197
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t h a t the rat c o u l d n o t c l i m b o u t of the w a t e r to the rim of the j a r and t h u s the rat swam c o n t i n u o u s l y or t r e a d e d w a t e r d u r i n g the full 10 rain period, a f t e r w h i c h it was sacrificed. C o n t r o l rats were u n d i s t u r b e d cage mates. F o l l o w i n g b o t h cold e x p o s u r e a n d swim stress the rectal t e m p e r a t u r e was m e a s u r e d i m m e d i a t e l y after d e c a p i t a t i o n using a l a b o r a t o r y t h e r m o m e t e r i n s e r t e d 10 cm i n t o the colon. Electric f o o t s h o c k was delivered to the paws of t h e rat placed in a lucite-walled cage, 20 x 24 x 19 cm high, e q u i p p e d with a grid floor of electrifiable rods set at 1.5 cm apart. S h o c k s were of 1 sec d u r a t i o n , 1.6 m A , 7 per rain, r a n d o m i z e d w i t h i n each rain by intervals of 3 - 1 5 sec b e t w e e n shocks. T h e t o t a l s h o c k p e r i o d was varied in each series f r o m 2 - 3 0 min. The rats were sacrificed i m m e d i a t e l y following the stress. C o n t r o l rats were u n d i s t u r b e d cage mates. The k i n e t i c c o n s t a n t s for the u p t a k e of 3 H-1n o r e p i n e p h r i n e (3H-1-NE) were d e t e r m i n e d as rapidly as possible a f t e r sacrificing a pair of rats, one stressed and one c o n t r o l , in a l t e r n a t i n g o r d e r in separate e x p e r i m e n t s w i t h i n a given series. The cerebral c o r t e x was rapidly dissected f r o m the w h o l e brain o n a chilled surface a n d s t o r e d briefly in chilled 0.25 M sucrose u n t i l b l o t t e d lightly, weighed, t h e n h o m o g e n i z e d g e n t l y in 10 v o l u m e s of 0.25 M sucrose, using 6 up a n d d o w n s t r o k e s of a Teflon pestle in a s m o o t h glass h o m o g e n i z e r ( K o n t e s Glass, 0 . 0 0 4 0 . 0 0 6 in. clearance). H o m o g e n a t e s were c e n t r i f u g e d in the cold ( D u p o n t / Sorvall I n s t r u m e n t s , RC2-B) for 10 m i n at 1000 g, and the s u p e r n a t a n t , a c r u d e s y n a p t o s o m e - r i c h fraction, was used w i t h o u t f u r t h e r p u r i f i c a t i o n for the u p t a k e studies. Aliq u o t s of 100 ul, e q u i v a l e n t to 10 mg of original cerebral cortex, were a d d e d in the cold to 2 ml of Krebs p h o s p h a t e Ringer [26] m o d i f i e d to c o n t a i n half the usual CaC12 c o n c e n t r a t i o n , d i s o d i u m e t h y l e n e - d i a m i n e t e t r a a c e t i c acid ( E D T A , 0.05 m g / m l ) , ascorbic acid (0.2 m g / m l ) , glucose (2 m g / m l ) , N i a l a m i d e ( 1 0 uM, Pfizer Corp.) to i n h i b i t m o n o a m i n e oxidase a n d 3 H-1-NE (New E n g l a n d Nuclear Corp., 3.7 Ci/mM, 0.04 uM). N o n r a d i o a c t i v e 1 - n o r e p i n e p h r i n e h y d r o c h l o r i d e ( C a l b i o c h e m Corp.), dissolved in 1 m M HC1 c o n t a i n i n g E D T A (0.05 m g / m l ) , a d d e d to the m e d i a p r o v i d e d a range of 5 or 6 d i f f e r e n t 3H-1-NE c o n c e n t r a tions f r o m 0 . 0 4 - 0 . 2 or 0.4 uM, while the acid vehicle was k e p t c o n s t a n t in all t u b e s at less t h a n 0.5% of t h e i n c u b a t i o n media. I n c u b a t i o n s were carried o u t in d u p l i c a t e at 37 ° C, w i t h shaking, in n y l o n or p o l y p r o p y l e n e c e n t r i f u g e t u b e s ( D u p o n t / S o r v a l l I n s t r u m e n t s ) for 5 min, using r o o m air as the gas phase. The u p t a k e process was rapidly t e r m i n a t e d by i m m e r s i o n of the t u b e s s i m u l t a n e o u s l y in an ethanol-ice w a t e r b a t h , t h e n c e n t r i f u g i n g for 20 m i n at 2 0 , 0 0 0 g in the cold. The surfaces of the pellets were rinsed with 10 ml of cold i s o t o n i c NaC1 (0.15 M) a n d c e n t r i f u g e d again for 10 m i n at 2 0 , 0 0 0 g. The washings were discarded and t h e t u b e s d r a i n e d over a b s o r b e n t p a p e r for at least 45 min. Pellets were dissolved in 10 ml of T r i t o n - X 100 in t o l u e n e ( 1 : 4 ) c o n t a i n i n g PPO and POPOP and 0.2 ml distilled water. T r i t i u m was c o u n t e d in a Packard Tricarb Model 3385 ( P a c k a r d I n s t r u m e n t s Corp.) liquid scintillation s p e c t r o m e t e r at 45% efficiency. Since all samples i n c l u d i n g b l a n k s p r e p a r e d in a similar m a n n e r b u t w i t h o u t a d d e d tissue, as well as samples of 0.2 ml of i n c u b a t i o n m e d i u m , were f o u n d to have t h e same c o u n t i n g efficiency, m o n i tored by sample c h a n n e l s ratio or by a u t o m a t i c e x t e r n a l s t a n d a r d i z a t i o n , n o c o r r e c t i o n s were m a d e for c o u n t i n g efficiency.
Net u p t a k e velocity was calculated as n m o l e s of NE a c c u m u l a t e d in 5 m i n per gram of original cerebral cortex at 37°C m i n u s t h a t a c c u m u l a t e d at 0°C (17% of the a c c u m u l a t i o n s at 37°C). NE a c c u m u l a t e d in 5 rain u n d e r similar c o n d i t i o n s was previously f o u n d to be a l m o s t c o m p l e t e l y u n m e t a b o l i z e d c a t e c h o l a m i n e [ 19]. The kinetic c o n s t a n t s , a p p a r e n t K m and V m a x , were e s t i m a t e d w i t h o u t w e i g h t i n g f r o m Cleland's [4] F o r t r a n p r o g r a m for n o n - l i n e a r regression (best fit to a h y p e r b o l a ) using a PDP-8/e c o m p u t e r (Digital E q u i p m e n t Corp.) and a 4010-1 graphics t e r m i n a l ( T e k t r o n i c s Corp.) c o n n e c t e d to a Sigma 6 ( X e r o x Corp.) c o m p u t e r . Special care was t a k e n to t r e a t c o n t r o l and stress tissues c o n c o m i t a n t l y and identically w i t h respect to time, t e m p e r a t u r e of i n c u b a t i o n and c e n t r i f u g a t i o n c o n d i t i o n s , thus ensuring a legitimate statistical c o m p a r i s o n b e t w e e n pairs of c o n t r o l a n d stressed animals (paired t-test). The kinetic c o n s t a n t s are derived values at best, and would n o t be c o m p a r a b l e e x c e p t u n d e r identical c o n d i t i o n s of measurement. RESULTS E x p o s u r e of rats, while wet, to 2 4°C for 45 m i n ( c o l d stress) failed to alter the k i n e t i c c o n s t a n t s for ~ I t - I - N E u p t a k e ( T a b l e 1). These rats h a d significantly r e d u c e d rectal t e m p e r a t u r e (30°C). I m m o b i l i z a t i o n of rats for one hr at r o o m t e m p e r a t u r e ( r e s t r a i n t stress) was also ineffective in altering t h e k i n e t i c c o n s t a n t s for 3 H-1-NE u p t a k e (Table 1). During c o m b i n e d cold-plus-restraint, rats were i m m o b i lized in the restrainer, t h e n placed in the refrigerator (2 4°C) for 45 min. F o l l o w i n g this stress t h e i r snouts, paws, tails a n d genitalia were bright red in a p p e a r a n c e , and t h e y were grossly inactive prior to sacrificing at the end of t h e stress period. T h e y failed to right t h e m s e l v e s w h e n released f r o m the restrainer and gave n o resistance to h a n d l i n g a n d sacrificing. T h e i r rectal t e m p e r a t u r e s fell m a r k e d l y to an average of 17°C (Table 1) as a result of being d e p r i v e d of two i m p o r t a n t m e a n s of conserving b o d y heat, n a m e l y shaking off excess m o i s t u r e and curling up to lower t o t a l b o d y surface area. More i m p o r t a n t l y , p e r i p h e r a l v a s o d i l a t i o n in t h e skin of these rats was evident, und o u b t e d l y c o n t r i b u t i n g significantly to the p r o f o u n d heat loss a n d i n d i c a t i n g a failure of the n o r a d r e n e r g i c a l l y m e d i a t e d r e s p o n s e to h e a t loss. in these rats the a p p a r e n t K m fell to an average of 17% b e l o w paired c o n t r o l values and this decrease was of b o r d e r l i n e significance (Table 1). T h e r e was n o significant a l t e r a t i o n in the V m a x . In rats s u b j e c t e d to swim stress (10 rain swim in tap w a t e r of varying t e m p e r a t u r e s ) the results o b t a i n e d varied w i t h the t e m p e r a t u r e of the w a t e r (Table 2). When rats swam in w a t e r at b o d y t e m p e r a t u r e (38°C), rectal t e m p e r a t u r e increased slightly b u t c o n s i s t e n t l y (p < 0 . 0 1 ) b y 0.5°C, p r e s u m a b l y as a result of i n a b i l i t y to dissipate h e a t g e n e r a t e d by the m u s c u l a r e x e r t i o n of swimming. T h e r e were n o significant changes in the k i n e t i c c o n s t a n t s a p p a r e n t K m a n d V m a x in these rats. T h e y swam vigorously at the b e g i n n i n g of the 10 m i n period but a f t e r several m i n u t e s , spent m u c h of the time in t r e a d i n g water. They did n o t a p p e a r to search c o n t i n u o u s l y for escape r o u t e s as did rats t h a t swam in cold water. Rats t h a t swam in relatively h o t water ( 4 2 ° C ) were severely h y p e r t h e r m i c after 10 m i n ( 4 1 . 5 ° C , Table 2). T h e y a p p e a r e d to be s o m e w h a t inactive at the e n d of the swim p e r i o d a l t h o u g h t h e y were aggressive w h e n h a n d l e d . T h e r e were n o significant d i f f e r e n c e s in the NE u p t a k e kinetic
STRESS AND BRAIN NOREPINEPHRINE
UPTAKE
199
TABLE
1
KINETIC CONSTANTS FOR UPTAKE OF 3H-I-NOREPINEPHRINE IN HOMOGENATES OF RAT CEREBRAL CORTEX: EFFECTS OF COLD, RESTRAINT STRESS Rectal T e m p °C Control Stress
37.6 _+ 0.2 (16)
--
30.0 _+ 0.1 (16) p 0.05
0.74 _+ 0.10 (6)
C o m b i n e d Cold-Plus-Restraint for 45 min 0.18 + 0.01 0.15 _+ 0.01 (12) (12) 0.1 >p >0.05
0.63 __ 0.04 (12)
0.75 _+ 0.05 (16) p >0.05 0.77 _+ 0.04 (6) p >0.05 0.61 _+ 0.04 (12) p >0.05
Values are m e a n s _+ S E M , n u m b e r of rats in p a r e n t h e s e s . p values from paired t-test. TABLE
2
KINETIC CONSTANTS FOR UPTAKE OF 3H-I-NOREPINEPHRINE IN HOMOGENATES OF RAT CEREBRAL CORTEX: SWIM AT VARYING TEMPERATURES Rectal T e m p °C Control Stress
Apparent K m IzM Control Stress
Vmax nmol/g/5 min Control Stress
37.6 __- 0.2 23.1 + 0.2 (8) (8) p 0.05
0.86 _+ 0.07 0.94 _+ 0.15 (8) (8) p >0.05
37.5 + 0.2 27.4 -+ 0.3 (9) (9) p0.05 p >0.05 5 min Footshock 0.15 ± 0.01 0.20 ± 0.02 0.54 e 0.05 0.69 ± 0,05 (9) (9) (9) (9) p 0.05 30 rain Footshock 0.23 ± 0.01 0.22 ± 0.01 0.85 ± 0.04 0.82 ± 0.04 (16) (16) (16) (16) p>0.05 p>0.05 Values are means ± SEM, number of rats in parentheses. p values from paired t-test. *Shocks were of 1 sec duration, 1.6 mAmp, 7/rain.
c o r t e x and h y p o t h a l a m u s were m a r k e d l y r e d u c e d , plasma c o r t i c o s t e r o n e was elevated, t y r o s i n e h y d r o x y l a s e activity in the f o r e b r a i n was u n c h a n g e d , and n o change in ~H-NE u p t a k e was discernible in cerebral cortical slices [271. F r o m this c o m p o s i t e picture it appears t h a t the earliest n e u r o c h e m i c a l changes were d e t e c t a b l e n o t only in the brainstem, where the n o r a d r e n e r g i c cell bodies are l o c a t e d , b u t also in t h e cerebral c o r t e x , where n o r a d r e n e r g i c nerve t e r m i n a l s e x h i b i t e d increased a p p a r e n t K m and V m a x for NE u p t a k e . With increasing d u r a t i o n of f o o t s h o c k stress, NE u t i l i z a t i o n rate was elevated at a time when rate of NE synthesis could n o t keep pace with t u r n o v e r , resulting in m a r k e d d e p l e t i o n s of e n d o g e n o u s levels of NEI as originally p r o p o s e d by Bliss et al. [ 3 ] . I n t e r e s t i n g / y , with m o r e p r o l o n g e d d u r a t i o n of f o o t s h o c k (3 h o u r s ; [ 2 4 ] ) , or c h r o n i c f o o t s h o c k (15 days; [ 2 7 1 ) , brain NE u p t a k e m e c h a n i s m s again a p p e a r e d to be altered in response to stress. ACKNOWLEDGEMENTS We are grateful to David Karnes for help in computer programming, and to Pfizer, Inc. for a generous gift of Nialamide. This work was supported by USPHS 05429-13 and by USPHS MH25811.
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