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Unpredictable cold-immobilization stress effects on voluntary ethanol consumption in rats
Life Sciences, Vol. 40, pp. 1245-1251 Printed in the U.S.A.
Pergamon Journals
UNPREDICTABLE COLD-IMMOBILIZATION STRESS EFFECTS V O L U N T A R Y E T H A N O L C O N S U M P T I O N IN RATS Gary
E. R o c k m a n , *
Department
Arleen
Hall,**
Joe H o n g
and G a r y
ON
B. G l a v i n * *
of P s y c h o l o g y ,
U n i v e r s i t y of W i n n i p e g , W i n n i p e g , MB and + D e p a r t m e n t of P h a r m a c o l o g y and T h e r a p e u t i c s , F a c u l t y of M e d i c i n e , U n i v e r s i t y of M a n i t o b a , W i n n i p e g , MB (Received in final form December 18, 1986) Summary
The e f f e c t s of e x p o s u r e to a s c h e d u l e of u n p r e d i c t a b l e c o l d - i m m o b i l i z a t i o n stress on v o l u n t a r y e t h a n o l consumption were examined. F o l l o w i n g t e s t i n g for e t h a n o l p r e f e r e n c e , rats w e r e d i v i d e d into high, m e d i u m and low e t h a n o l c o n s u m i n g g r o u p s on the b a s i s of d a i l y e t h a n o l i n t a k e (g/kg/day) and e x p o s e d to i m m o b i l i z a t i o n stress over an 18 day period. Voluntary ethanol consumption w a s m o n i t o r e d d u r i n g the stress p e r i o d and for an a d d i t i o n a l 36 days p o s t - s t r e s s . Results indicated a d i f f e r e n t i a l e f f e c t of s t r e s s on e t h a n o l i n t a k e in that low e t h a n o l c o n s u m i n g rats i n c r e a s e d t h e i r e t h a n o l i n t a k e d u r i n g the s t r e s s p e r i o d and m a i n t a i n e d this i n c r e a s e t h r o u g h o u t the e n t i r e p o s t - s t r e s s p e r i o d as c o m p a r e d to n o n - s t r e s s e d c o n t r o l s . High ethanol consuming g r o u p s d e m o n s t r a t e d a small ( m a r g i n a l l y s i g n i f i c a n t ) d e c r e a s e in e t h a n o l intake d u r i n g the stress p e r i o d as c o m p a r e d to b a s e l i n e levels. No c h a n g e in e t h a n o l intake was o b s e r v e d for the m e d i u m e t h a n o l c o n s u m i n g groups. The r e s u l t s s u g g e s t that u n p r e d i c t a b l e i m m o b i l i z a t i o n s t r e s s has a d i f f e r e n t i a l e f f e c t on e t h a n o l i n t a k e d e p e n d i n g u p o n p r e - s t r e s s levels of ethanol consumption. C o n s i d e r a b l e r e s e a r c h has b e e n c o n d u c t e d e x a m i n i n g the i n t e r a c t i o n b e t w e e n stress and e t h a n o l c o n s u m p t i o n . Unfortunately, m u c h of the i n f o r m a t i o n e m a n a t i n g from t h e s e s t u d i e s is c o n t r a d i c t o r y (1). One area of i n v e s t i g a t i o n c o n c e r n s the e f f e c t s of e t h a n o l on the p a t h o p h y s i o l o g i c a l c o n s e q u e n c e s of e x p o s u r e to stress. For example, w h i l e e t h a n o l has been s h o w n to have a p r e v e n t a t i v e e f f e c t a g a i n s t s t r e s s - i n d u c e d a l t e r a t i o n s in p l a s m a c o r t i c o s t e r o n e level (2), b r a i n m o n o a m i n e a c t i v i t y (3), and p l a s m a n o n - e s t e r i f i e d fatty acid level (4), o t h e r s t u d i e s have d e m o n s t r a t e d e i t h e r a p o t e n t i a t i o n or no e f f e c t of e t h a n o l on stress-induced gastric mucosal injury (5,6,7,8,9,10). Another a s p e c t of the s t r e s s - e t h a n o l i n t e r a c t i o n c o n c e r n s the e f f e c t of v a r i o u s s t r e s s o r s on e t h a n o l c o n s u m p t i o n . For example, w h i l e it was o r i g i n a l l y d e m o n s t r a t e d that stress i n c r e a s e d e t h a n o l to w h o m r e p r i n t r e q u e s t s s h o u l d be sent, s u p p o r t e d A2605 ** NSERC summer undergraduate research scholar *** s u p p o r t e d by N S E R C 1020 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
by N S E R C
12&6
Stress and Ethanol Consumption
Vol. 40, No. 13, 1987
c o n s u m p t i o n in cats (ii), few s u b s e q u e n t s t u d i e s have found s i m i l a r r e s u l t s (12,13). In r e c e n t studies, c o n t r a s t i n g e f f e c t s of s t r e s s on e t h a n o l i n t a k e w e r e o b s e r v e d . R o c k m a n and G l a v i n (8) d e m o n s t r a t e d that a c t i v i t y stress i n d u c e d a d e c r e a s e in e t h a n o l c o n s u m p t i o n a m o n g h i g h e t h a n o l p r e f e r r i n g rats d u r i n g the stress period. M e d i u m and low e t h a n o l c o n s u m i n g g r o u p s did not a l t e r their e t h a n o l intake. In c o n t r a s t , R o c k m a n et al. (9), o b s e r v e d a t e m p o r a r y i n c r e a s e ~ e t h a n o l c o n s u m p t i o n in m e d i u m e t h a n o l c o n s u m i n g rats and a t e m p o r a r y d e c r e a s e in high e t h a n o l c o n s u m i n g rats f o l l o w i n g a p e r i o d of p r e d i c t a b l e r e s t r a i n t stress. O t h e r r e s e a r c h e r s have r e p o r t e d large i n c r e a s e s in e t h a n o l c o n s u m p t i o n f o l l o w i n g e x p o s u r e to a s c h e d u l e of u n p r e d i c t a b l e i m m o b i l i z a t i o n stress (14). However, in this latter study, e t h a n o l c o n s u m i n g rats w e r e not g r o u p e d a c c o r d i n g to d i f f e r e n t levels of e t h a n o l intake. T h e s e s t u d i e s serve to h i g h l i g h t the c o n t r a d i c t o r y and c o n f u s i n g r e s u l t s in this area as w e l l as the i m p o r t a n c e of a s s e s s i n g d i f f e r e n t i a l e t h a n o l p r e f e r e n c e (low, m e d i u m and h i g h e t h a n o l p r e f e r r i n g rats) d u r i n g the stress and p o s t - s t r e s s periods. The p r e s e n t s t u d y was d e s i g n e d to i n v e s t i g a t e the e f f e c t of e x p o s u r e to an u n p r e d i c t a b l e s c h e d u l e of i m m o b i l i z a t i o n stress on l o n g - t e r m v o l u n t a r y e t h a n o l c o n s u m p t i o n in low, m e d i u m and high e t h a n o l c o n s u m i n g rats d u r i n g stress and p o s t - s t r e s s p e r i o d ~ Materials
and M e t h o d s
M a l e W i s t a r rats ( H o l t z m a n Co., M a d i s o n , WI.) w e i g h i n g 190-210 g on d e l i v e r y w e r e used. Rats w e r e h o u s e d i n d i v i d u a l l y in s t a n d a r d lab c a g e s w i t h food and w a t e r a v a i l a b l e ad l i b i t u m and w i t h a 12/12 L/D c y c l e (lights on 0700 h). Ethanol Screening: A f t e r 5 days of a d a p t a t i o n , the e t h a n o l s c r e e n i n g p r o c e d u r e began. T w o c a l i b r a t e d d r i n k i n g tubes w e r e a t t a c h e d to the left front of e a c h rat's cage. One c o n t a i n e d tap w a t e r w h i l e the o t h e r i n i t i a l l y c o n t a i n e d a 3% (v/v) s o l u t i o n of e t h a n o l . T h i s c o n c e n t r a t i o n was p r e s e n t e d e v e r y o t h e r day for 8 days; that is, e v e r y o t h e r day the rats r e c e i v e d two tubes of w a t e r and on a l t e r n a t e days, they r e c e i v e d one tube of w a t e r and o n e tube of ethanol. The p o s i t i o n of the e t h a n o l tube was c h a n ~ d upon e a c h p r e s e n t a t i o n to e l i m i n a t e the p o s s i b i l i t y of a p o s i t i o n p r e f e r e n c e by the rats. T h e same a l t e r n a t e day p r e s e n t a t i o n was c o n t i n u e d for e t h a n o l in i n c r e a s i n g c o n c e n t r a t i o n s of 5%, 7% and f i n a l l y 9% (v/v). At the end of the s c r e e n i n g p r o c e d u r e , all rats r e c e i v e d the 9% (v/v) e t h a n o l in a e v e r y d a y f r e e - c h o i c e w i t h w a t e r for 20 c o n s e c u t i v e days. T h e r e f o r e , the rats w e r e e x p o s e d to e t h a n o l for a total of 36 days p r i o r to i m m o b i l i z a t i o n . D a i l y fluid c o n s u m p t i o n (both e t h a n o l and water) and b o d y w e i g h t w e r e m o n i t o r e d and d a i l y e t h a n o l i n t a k e in grams per k i l o g r a m per day w a s c a l c u l a t e d . In this m a n n e r t h r e e g r o u p s of rats w e r e selected. Low, m e d i u m and high e t h a n o l p r e f e r r i n g g r o u p s w e r e d e f i n e d by t h e i r m e a n d a i l y e t h a n o l c o n s u m p t i o n (1.5-2.5; 2.5-4.5; 4 . 5 - 6 . 0 g / k g / d a y , r e s p e c t i v e l y ) w h i c h r e m a i n e d stable t h r o u g h o u t the e n t i r e 36 d a y s c r e e n i n g p r o c e d u r e . I m m o b i l i z a t i o n Stress: I m m e d i a t e l y f o l l o w i n g the last day of the e t h a n o l s c r e e n i n g , half of the rats ( s t r e s s e d group) from e a c h of the 3 g r o u p s w e r e r a n d o m l y s e l e c t e d and e x p o s e d to a
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t o t a l of 28 h o u r s of c o n f i n e m e n t in i m m o b i l i z a t i o n t u b e s (15) at 4 - 6 ° C o v e r an 18 d a y p e r i o d . S t r e s s w a s a d m i n i s t e r e d on an u n p r e d i c t a b l e b a s i s a c c o r d i n g to the s c h e d u l e s h o w n in T a b l e i. T h e o t h e r h a l f of the r a t s in e a c h g r o u p ( n o n - s t r e s s e d g r o u p s ) w e r e m o v e d i n t o an a d j o i n i n g r o o m a n d h a n d l e d at the b e g i n n i n g a n d at the e n d of e a c h s t r e s s s e s s i o n a n d w e r e n o t r e s t r a i n e d . This procedure ensured that both stressed and control rats r e c e i v e d e q u a l a m o u n t s of h a n d l i n g p e r se. Both stressed and n o n - s t r e s s e d r a t s w e r e d e n i e d a c c e s s to e t h a n o l , w a t e r a n d f o o d d u r i n g the s t r e s s s e s s i o n s . F o l l o w i n g s t r e s s s e s s i o n s , all r a t s w e r e r e t u r n e d to t h e i r h o m e c a g e s w i t h food, w a t e r a n d e t h a n o l (9%) a v a i l a b l e ad l i b i t u m . Body weight, water, and ethanol c o n s u m p t i o n w e r e m o n i t o r e d d a i l y d u r i n g all p h a s e s of the experiment which included baseline, stress and post-stress periods. E t h a n o l d a t a w e r e g r o u p e d i n t o b l o c k s of 6 d a y s e a c h a n d c a l c u l a t e d in t e r m s of m e a n g r a m s p e r k i l o g r a m per d a y (gm/kg/day). T h e r e f o r e t h e b a s e l i n e p e r i o d c o n s i s t e d of one b l o c k of 6 days, s t r e s s p e r i o d s c o m p r i s e d 4 b l o c k s a n d the p o s t s t r e s s p e r i o d c o n s i s t e d of 6 b l o c k s of 6 d a y s each. F o r the s a k e of c l a r i t y , the low, m e d i u m a n d h i g h e t h a n o l consuming groups are presented separately, with the appropriate control groups. In a d d i t i o n , s i n c e e t h a n o l c o n s u m p t i o n for a l l g r o u p s d u r i n g the p o s t - s t r e s s p e r i o d s d i d not c h a n g e o v e r time, o n l y the f i r s t b l o c k of 6 d a y s of t h e p o s t - s t r e s s p e r i o d is p r e s e n t e d in F i g u r e s i, 2, a n d 3 a l o n g w i t h the a p p r o p r i a t e statistical analysis. A l l d a t a w e r e a n a l y z e d by r e p e a t e d m e a s u r e s a n a l y s i s of v a r i a n c e [ g r o u p s ( s t r e s s vs. n o - s t r e s s ) x t i m e p e r i o d ( b a s e l i n e , s t r e s s 1-3, p o s t s t r e s s ) ], a p p r o p r i a t e p o s t hoc (Tukey) t e s t s , and s i m p l e m a i n e f f e c t s a n a l y s i s w h e n interactions were significant. TABLE Unpredictable Day
Time
1 2 3 4 5 6 7 8 9 10 ii 12 13 14 15 16 17 18
1500 - 1630 No S t r e s s 1230 - 1430 1400 - 1 5 3 0 0 9 3 0 - 1230 No S t r e s s No S t r e s s 1 0 0 0 - 1100 1430 - 1730 1000 - 1230 1400 - 1700 No S t r e s s 1030 - 1130 No S t r e s s 0930 - 1130 1400 - 1500 1100 - 1330 0 9 3 0 - 1330
This
schedule
has
I
Cold-Immobilization (hours)
an a v e r a g e
Schedule
Duration
(hours)
1.5 2.0 1.5 3.0
1.0 3.0 2.5 3.0 1.0 2.0 1.0 2.5 4.0 stress
density
of
1.55
hrs/day.
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Results F i g u r e s i, 2, and 3 i l l u s t r a t e e t h a n o l c o n s u m p t i o n for low, m e d i u m and high e t h a n o l d r i n k i n g g r o u p s r e s p e c t i v e l y . Ethanol i n t a k e for low e t h a n o l g r o u p s (Figure i) y i e l d e d a s i g n i f i c a n t e f f e c t of g r o u p [ F ( I , 3 7 ) = 5 . 2 6 , p<0.05] p e r i o d [ F ( 4 , 1 4 8 ) = 3 . 0 1 , p<0.05] and g r o u p x time p e r i o d i n t e r a c t i o n [ F ( 4 , 1 4 8 ) = 2 . 5 4 , p<0.05]. M o r e s p e c i f i c a l l y , p o s t - h o c T u k e y tests r e v e a l e d that s t r e s s e d rats s i g n i f i c a n t l y i n c r e a s e d t h e i r e t h a n o l c o n s u m p t i o n d u r i n g the s e c o n d stress p e r i o d (p<0.05) as w e l l as d u r i n g the t h i r d stress and p o s t - s t r e s s p e r i o d s as c o m p a r e d to b a s e l i n e and c o n t r o l levels (p<0.01). In contrast, e t h a n o l c o n s u m p t i o n for the m e d i u m c o n s u m i n g g r o u p s (Figure 2) showed a s i g n i f i c a n t main e f f e c t of time p e r i o d [ F ( 4 , 1 3 6 ) = I I . 0 2 , p<0.01] but not s i g n i f i cant e f f e c t s of g r o u p or g r o u p x time p e r i o d i n t e r a c t i o n [group x time p e r i o d : F ) 4 , 1 3 6 ) = 0 . 5 1 , p > 0 . 0 5 ] .
8.0
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,
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i
BASEUNE
STRESS I
STRESS 2
STRESS 3
STRESS
PERIO~
i
POSTSTRES5
NO STRESS
F i g u r e i. E t h a n o l c o n s u m p t i o n in terms of m e a n grams per k i l o g r a m for l o w - e t h a n o l s t r e s s e d (n=20) and n o n - s t r e s s e d groups.
(n=19)
Vol. 40, No. 13, 1987
Stress and Ethanol Consumption
1249
8.0
S.O O
T
4.0
O W
,
/
/
/// 3.0
g,
Z ~
/// / / /
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2.0
/ / / / / /
YZ
1.0
/ / /
//i / / /
0.0
i
BASELINE
,
I
u
STRESS 1
STRESS 2
STRESS 3
STRESS
PERIO~
POSTSTRESS
NO STRESS
F i g u r e 2. E t h a n o l c o n s u m p t i o n in terms of m e a n grams per k i l o g r a m for m e d i u m - e t h a n o l s t r e s s e d (n=18) and n o n - s t r e s s e d (n=18 groups). 6.0
5.0
/ / 0 •:
4..0
o 3.0
g~
Z ~.,
2.0
1.0
0.0 BASEUNE
STRESS 1 [7--/1
STRESS
STRESS 2 PERIOI~
STRESS 3
POSTSTRESS
NO STRESS
F i g u r e 3. E t h a n o l c o n s u m p t i o n in terms of m e a n g r a m s per k i l o g r a m for h i g h - e t h a n o l s t r e s s e d (n=14) and n o n - s t r e s s e d groups.
(n=15)
1250
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A n a l y s i s of the h i g h e t h a n o l g r o u p s y i e l d e d a s i g n i f i c a n t e f f e c t of time p e r i o d [ F ( 4 , 1 0 8 ) = 9 . 2 5 , p<0.05] as w e l l as a g r o u p x time period interaction which approached significance [F(4,108)=2.20, p<0.07]. This i n d i c a t e s that s t r e s s e d h i g h e t h a n o l c o n s u m i n g rats i n i t i a l l y r e d u c e d t h e i r e t h a n o l i n t a k e d u r i n g the first two stress p e r i o d s and then r e t u r n e d to b a s e l i n e levels. It is i m p o r t a n t to note that total fluid intake for all g r o u p s d u r i n g the e n t i r e e x p e r i m e n t did not s i g n i f i c a n t l y differ. In a d d i t i o n , no b e h a v i o r a l signs of o v e r t i n t o x i c a t i o n or w i t h d r a w a l w e r e o b s e r v e d at any time d u r i n g the day in this experiment. Discussion The r e s u l t s of the p r e s e n t study r e v e a l s e v e r a l i n t e r e s t i n g observations. First, it was o b s e r v e d that the s t r e s s e d low e t h a n o l c o n s u m i n g rats d e m o n s t r a e d an i n c r e a s e in e t h a n o l c o n s u m p t i o n d u r i n g the s e c o n d s t r e s s p e r i o d w h i c h was m a i n t a i n e d t h r o u g h o u t the r e m a i n d e r of the e x p e r i m e n t . This i n c r e a s e in e t h a n o l c o n s u m p t i o n is c o n s i s t e n t w i t h the r e c e n t report of N a s h and M a i c k e l (14). In c o n t r a s t , however, we failed to find any i n c r e a s e in e t h a n o l i n t a k e in the m e d i u m and high c o n s u m i n g groups. In fact, d a t a from the high e t h a n o l rats, w h i l e a p p r o a c h i n g s i g n i f i c a n c e , s u g g e s t a d e c r e a s e in e t h a n o l i n t a k e d u r i n g the s t r e s s periods. This o b s e r v a t i o n , w h i l e not cons i s t e n t w i t h d a t a from the low and m e d i u m g r o u p s and w i t h o t h e r r e p o r t s (11,13,14), is c o n s i s t e n t w i t h data from our l a b o r a t o r y d e m o n s t r a t i n g a d e c r e a s e in e t h a n o l c o n s u m p t i o n a m o n g high e t h a n o l c o n s u m i n g rats as a c o n s e q u e n c e of c h r o n i c stress (8,9). T h e s e d a t a c o n s i d e r e d t o g e t h e r s u g g e s t that the e f f e c t of c h r o n i c stress ( p r e d i c t a b l e and u n p r e d i c t a b l e ) on e t h a n o l c o n s u m p t i o n m a y d i f f e r d e p e n d i n g upon the level of e t h a n o l i n t a k e d u r i n g b a s e l i n e (pre-stress) periods. It is also i n t e r e s t i n g to note that b o t h s t r e s s e d and n o n - s t r e s s e d high e t h a n o l - c o n s u m i n g rats s h o w e d a r e d u c e d e t h a n o l i n t a k e c o m p a r e d to b a s e l i n e levels. This r e d u c t i o n in d r i n k i n g w o u l d a p p e a r to be the result of i n c r e a s e d h a n d l i n g r a t h e r than due to stress per se. We are i n v e s t i g a t i n g h a n d l i n g e f f e c t s on stress and a l c o h o l d r i n k i n g responses. T h e s e r e s u l t s serve to h i g h l i g h t the i m p o r t a n c e of a s s e s s i n g the e f f e c t s of stress on v a r i o u s levels of e t h a n o l i n t a k e (i.e. low, m e d i u m and high), since d i f f e r e n t i a l e f f e c t s of stress on e t h a n o l i n t a k e m a y be o b s c u r e d by c o m b i n i n g data from rats c o n s u m i n g d i f f e r e n t levels of ethanol. A mechanism for this o b s e r v a t i o n is not c l e a r at the p r e s e n t time, but r e s e a r c h in this l a b o r a t o r y is b e i n g c o n d u c t e d to i n v e s t i g a t e p o s s i b l e n e u r o c h e m i c a l a l t e r a t i o n s u n d e r l y i n g the e f f e c t s of stress on e t h a n o l c o n s u m p t i o n . In summary, the p r e s e n t r e s u t l s s u g g e s t that e x p o s u r e to u n p r e d i c t a b l e c o l d - i m m o b i l i z a t i o n stress p r o d u c e s d i f f e r e n t i a l e f f e c t s on e t h a n o l c o n s u m p t i o n d e p e n d i n g upon the rat's prestress level of e t h a n o l intake. References i. 2.
L.A. P o h o r e c k y , N e u r o s c i . B i o b e h a v . Rev. 5 208-229 (1981). J. B r i c k and L.A. P o h o r e c k y , P s y c h o p h a r m a c o l . 7 7 81-84 (1982).
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3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.
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K. K u r i y a m a , K. K a n m o r i a n d Y. Y o p e d a , N e u r o p h a r m a c o l . 23 6 4 9 - 6 5 4 (1984). L.A. P o h o r e c k y , E. R a s s i , J. W e i s s a n d V. M i c h a l a k , A l c o h o l . C l i n . Exp. Res. 4 4 2 3 - 4 2 6 (1980). R. Lev, K. K a w a s h i m a , and G.B. G l n s s , A r c h . P a t h o l . Lab. Med. i00 5 5 ~ - 5 5 8 (1976). G.B. G l a v i n and G.E. R o c k m a n , A l c o h o l 2 6 5 1 - 6 5 3 (1985). G.E. R o c k m a n and G.B. G l a v i n , A l c o h o l 1 2 9 3 - 2 9 5 (1984). G.E. R o c k m a n a n d G.B. G l a v i n , Pha~:macol. B i o c h e m . B e h a v . 24 8 6 9 - 8 7 3 (1986). G.E. R o c k m ~ n , A. H a l l and G.B. G l a v i n . P h a r m a c o l . B i o c h e m . B e h a v . in p r e s s (1986). K.M. Schmiclt and F.D. K l o p f e r , Q u a r t . J. Stud. Alc. 29 5 5 8 - 5 6 5 (1568). J.H. M a s s e r m a n and K.S. Yum, P s y c h o s o m . Med. 8 3 6 - 5 2 (1946). M.A. C a p l a n and K. P u g l i s i , P h a r m a c o l . B i o c h e m . B e h a v . 24 2 7 1 - 2 8 0 (1986). R. D e r r a n d S. L i n d b l a d , L i f e Sci. 27 2 1 8 3 - 2 1 8 6 (1980). J . ~ N a s h and R.P. M a i c k e l , L i f e Sci. 37 7 5 7 - 7 6 5 (1985). I.G. L u t h e r , G.T. H e i s t a d , and S.B. S p a r b e r , P s y c h o s o m . Med. 31, 4 5 - 5 6 (1969).
Pergamon Journals
UNPREDICTABLE COLD-IMMOBILIZATION STRESS EFFECTS V O L U N T A R Y E T H A N O L C O N S U M P T I O N IN RATS Gary
E. R o c k m a n , *
Department
Arleen
Hall,**
Joe H o n g
and G a r y
ON
B. G l a v i n * *
of P s y c h o l o g y ,
U n i v e r s i t y of W i n n i p e g , W i n n i p e g , MB and + D e p a r t m e n t of P h a r m a c o l o g y and T h e r a p e u t i c s , F a c u l t y of M e d i c i n e , U n i v e r s i t y of M a n i t o b a , W i n n i p e g , MB (Received in final form December 18, 1986) Summary
The e f f e c t s of e x p o s u r e to a s c h e d u l e of u n p r e d i c t a b l e c o l d - i m m o b i l i z a t i o n stress on v o l u n t a r y e t h a n o l consumption were examined. F o l l o w i n g t e s t i n g for e t h a n o l p r e f e r e n c e , rats w e r e d i v i d e d into high, m e d i u m and low e t h a n o l c o n s u m i n g g r o u p s on the b a s i s of d a i l y e t h a n o l i n t a k e (g/kg/day) and e x p o s e d to i m m o b i l i z a t i o n stress over an 18 day period. Voluntary ethanol consumption w a s m o n i t o r e d d u r i n g the stress p e r i o d and for an a d d i t i o n a l 36 days p o s t - s t r e s s . Results indicated a d i f f e r e n t i a l e f f e c t of s t r e s s on e t h a n o l i n t a k e in that low e t h a n o l c o n s u m i n g rats i n c r e a s e d t h e i r e t h a n o l i n t a k e d u r i n g the s t r e s s p e r i o d and m a i n t a i n e d this i n c r e a s e t h r o u g h o u t the e n t i r e p o s t - s t r e s s p e r i o d as c o m p a r e d to n o n - s t r e s s e d c o n t r o l s . High ethanol consuming g r o u p s d e m o n s t r a t e d a small ( m a r g i n a l l y s i g n i f i c a n t ) d e c r e a s e in e t h a n o l intake d u r i n g the stress p e r i o d as c o m p a r e d to b a s e l i n e levels. No c h a n g e in e t h a n o l intake was o b s e r v e d for the m e d i u m e t h a n o l c o n s u m i n g groups. The r e s u l t s s u g g e s t that u n p r e d i c t a b l e i m m o b i l i z a t i o n s t r e s s has a d i f f e r e n t i a l e f f e c t on e t h a n o l i n t a k e d e p e n d i n g u p o n p r e - s t r e s s levels of ethanol consumption. C o n s i d e r a b l e r e s e a r c h has b e e n c o n d u c t e d e x a m i n i n g the i n t e r a c t i o n b e t w e e n stress and e t h a n o l c o n s u m p t i o n . Unfortunately, m u c h of the i n f o r m a t i o n e m a n a t i n g from t h e s e s t u d i e s is c o n t r a d i c t o r y (1). One area of i n v e s t i g a t i o n c o n c e r n s the e f f e c t s of e t h a n o l on the p a t h o p h y s i o l o g i c a l c o n s e q u e n c e s of e x p o s u r e to stress. For example, w h i l e e t h a n o l has been s h o w n to have a p r e v e n t a t i v e e f f e c t a g a i n s t s t r e s s - i n d u c e d a l t e r a t i o n s in p l a s m a c o r t i c o s t e r o n e level (2), b r a i n m o n o a m i n e a c t i v i t y (3), and p l a s m a n o n - e s t e r i f i e d fatty acid level (4), o t h e r s t u d i e s have d e m o n s t r a t e d e i t h e r a p o t e n t i a t i o n or no e f f e c t of e t h a n o l on stress-induced gastric mucosal injury (5,6,7,8,9,10). Another a s p e c t of the s t r e s s - e t h a n o l i n t e r a c t i o n c o n c e r n s the e f f e c t of v a r i o u s s t r e s s o r s on e t h a n o l c o n s u m p t i o n . For example, w h i l e it was o r i g i n a l l y d e m o n s t r a t e d that stress i n c r e a s e d e t h a n o l to w h o m r e p r i n t r e q u e s t s s h o u l d be sent, s u p p o r t e d A2605 ** NSERC summer undergraduate research scholar *** s u p p o r t e d by N S E R C 1020 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
by N S E R C
12&6
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c o n s u m p t i o n in cats (ii), few s u b s e q u e n t s t u d i e s have found s i m i l a r r e s u l t s (12,13). In r e c e n t studies, c o n t r a s t i n g e f f e c t s of s t r e s s on e t h a n o l i n t a k e w e r e o b s e r v e d . R o c k m a n and G l a v i n (8) d e m o n s t r a t e d that a c t i v i t y stress i n d u c e d a d e c r e a s e in e t h a n o l c o n s u m p t i o n a m o n g h i g h e t h a n o l p r e f e r r i n g rats d u r i n g the stress period. M e d i u m and low e t h a n o l c o n s u m i n g g r o u p s did not a l t e r their e t h a n o l intake. In c o n t r a s t , R o c k m a n et al. (9), o b s e r v e d a t e m p o r a r y i n c r e a s e ~ e t h a n o l c o n s u m p t i o n in m e d i u m e t h a n o l c o n s u m i n g rats and a t e m p o r a r y d e c r e a s e in high e t h a n o l c o n s u m i n g rats f o l l o w i n g a p e r i o d of p r e d i c t a b l e r e s t r a i n t stress. O t h e r r e s e a r c h e r s have r e p o r t e d large i n c r e a s e s in e t h a n o l c o n s u m p t i o n f o l l o w i n g e x p o s u r e to a s c h e d u l e of u n p r e d i c t a b l e i m m o b i l i z a t i o n stress (14). However, in this latter study, e t h a n o l c o n s u m i n g rats w e r e not g r o u p e d a c c o r d i n g to d i f f e r e n t levels of e t h a n o l intake. T h e s e s t u d i e s serve to h i g h l i g h t the c o n t r a d i c t o r y and c o n f u s i n g r e s u l t s in this area as w e l l as the i m p o r t a n c e of a s s e s s i n g d i f f e r e n t i a l e t h a n o l p r e f e r e n c e (low, m e d i u m and h i g h e t h a n o l p r e f e r r i n g rats) d u r i n g the stress and p o s t - s t r e s s periods. The p r e s e n t s t u d y was d e s i g n e d to i n v e s t i g a t e the e f f e c t of e x p o s u r e to an u n p r e d i c t a b l e s c h e d u l e of i m m o b i l i z a t i o n stress on l o n g - t e r m v o l u n t a r y e t h a n o l c o n s u m p t i o n in low, m e d i u m and high e t h a n o l c o n s u m i n g rats d u r i n g stress and p o s t - s t r e s s p e r i o d ~ Materials
and M e t h o d s
M a l e W i s t a r rats ( H o l t z m a n Co., M a d i s o n , WI.) w e i g h i n g 190-210 g on d e l i v e r y w e r e used. Rats w e r e h o u s e d i n d i v i d u a l l y in s t a n d a r d lab c a g e s w i t h food and w a t e r a v a i l a b l e ad l i b i t u m and w i t h a 12/12 L/D c y c l e (lights on 0700 h). Ethanol Screening: A f t e r 5 days of a d a p t a t i o n , the e t h a n o l s c r e e n i n g p r o c e d u r e began. T w o c a l i b r a t e d d r i n k i n g tubes w e r e a t t a c h e d to the left front of e a c h rat's cage. One c o n t a i n e d tap w a t e r w h i l e the o t h e r i n i t i a l l y c o n t a i n e d a 3% (v/v) s o l u t i o n of e t h a n o l . T h i s c o n c e n t r a t i o n was p r e s e n t e d e v e r y o t h e r day for 8 days; that is, e v e r y o t h e r day the rats r e c e i v e d two tubes of w a t e r and on a l t e r n a t e days, they r e c e i v e d one tube of w a t e r and o n e tube of ethanol. The p o s i t i o n of the e t h a n o l tube was c h a n ~ d upon e a c h p r e s e n t a t i o n to e l i m i n a t e the p o s s i b i l i t y of a p o s i t i o n p r e f e r e n c e by the rats. T h e same a l t e r n a t e day p r e s e n t a t i o n was c o n t i n u e d for e t h a n o l in i n c r e a s i n g c o n c e n t r a t i o n s of 5%, 7% and f i n a l l y 9% (v/v). At the end of the s c r e e n i n g p r o c e d u r e , all rats r e c e i v e d the 9% (v/v) e t h a n o l in a e v e r y d a y f r e e - c h o i c e w i t h w a t e r for 20 c o n s e c u t i v e days. T h e r e f o r e , the rats w e r e e x p o s e d to e t h a n o l for a total of 36 days p r i o r to i m m o b i l i z a t i o n . D a i l y fluid c o n s u m p t i o n (both e t h a n o l and water) and b o d y w e i g h t w e r e m o n i t o r e d and d a i l y e t h a n o l i n t a k e in grams per k i l o g r a m per day w a s c a l c u l a t e d . In this m a n n e r t h r e e g r o u p s of rats w e r e selected. Low, m e d i u m and high e t h a n o l p r e f e r r i n g g r o u p s w e r e d e f i n e d by t h e i r m e a n d a i l y e t h a n o l c o n s u m p t i o n (1.5-2.5; 2.5-4.5; 4 . 5 - 6 . 0 g / k g / d a y , r e s p e c t i v e l y ) w h i c h r e m a i n e d stable t h r o u g h o u t the e n t i r e 36 d a y s c r e e n i n g p r o c e d u r e . I m m o b i l i z a t i o n Stress: I m m e d i a t e l y f o l l o w i n g the last day of the e t h a n o l s c r e e n i n g , half of the rats ( s t r e s s e d group) from e a c h of the 3 g r o u p s w e r e r a n d o m l y s e l e c t e d and e x p o s e d to a
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t o t a l of 28 h o u r s of c o n f i n e m e n t in i m m o b i l i z a t i o n t u b e s (15) at 4 - 6 ° C o v e r an 18 d a y p e r i o d . S t r e s s w a s a d m i n i s t e r e d on an u n p r e d i c t a b l e b a s i s a c c o r d i n g to the s c h e d u l e s h o w n in T a b l e i. T h e o t h e r h a l f of the r a t s in e a c h g r o u p ( n o n - s t r e s s e d g r o u p s ) w e r e m o v e d i n t o an a d j o i n i n g r o o m a n d h a n d l e d at the b e g i n n i n g a n d at the e n d of e a c h s t r e s s s e s s i o n a n d w e r e n o t r e s t r a i n e d . This procedure ensured that both stressed and control rats r e c e i v e d e q u a l a m o u n t s of h a n d l i n g p e r se. Both stressed and n o n - s t r e s s e d r a t s w e r e d e n i e d a c c e s s to e t h a n o l , w a t e r a n d f o o d d u r i n g the s t r e s s s e s s i o n s . F o l l o w i n g s t r e s s s e s s i o n s , all r a t s w e r e r e t u r n e d to t h e i r h o m e c a g e s w i t h food, w a t e r a n d e t h a n o l (9%) a v a i l a b l e ad l i b i t u m . Body weight, water, and ethanol c o n s u m p t i o n w e r e m o n i t o r e d d a i l y d u r i n g all p h a s e s of the experiment which included baseline, stress and post-stress periods. E t h a n o l d a t a w e r e g r o u p e d i n t o b l o c k s of 6 d a y s e a c h a n d c a l c u l a t e d in t e r m s of m e a n g r a m s p e r k i l o g r a m per d a y (gm/kg/day). T h e r e f o r e t h e b a s e l i n e p e r i o d c o n s i s t e d of one b l o c k of 6 days, s t r e s s p e r i o d s c o m p r i s e d 4 b l o c k s a n d the p o s t s t r e s s p e r i o d c o n s i s t e d of 6 b l o c k s of 6 d a y s each. F o r the s a k e of c l a r i t y , the low, m e d i u m a n d h i g h e t h a n o l consuming groups are presented separately, with the appropriate control groups. In a d d i t i o n , s i n c e e t h a n o l c o n s u m p t i o n for a l l g r o u p s d u r i n g the p o s t - s t r e s s p e r i o d s d i d not c h a n g e o v e r time, o n l y the f i r s t b l o c k of 6 d a y s of t h e p o s t - s t r e s s p e r i o d is p r e s e n t e d in F i g u r e s i, 2, a n d 3 a l o n g w i t h the a p p r o p r i a t e statistical analysis. A l l d a t a w e r e a n a l y z e d by r e p e a t e d m e a s u r e s a n a l y s i s of v a r i a n c e [ g r o u p s ( s t r e s s vs. n o - s t r e s s ) x t i m e p e r i o d ( b a s e l i n e , s t r e s s 1-3, p o s t s t r e s s ) ], a p p r o p r i a t e p o s t hoc (Tukey) t e s t s , and s i m p l e m a i n e f f e c t s a n a l y s i s w h e n interactions were significant. TABLE Unpredictable Day
Time
1 2 3 4 5 6 7 8 9 10 ii 12 13 14 15 16 17 18
1500 - 1630 No S t r e s s 1230 - 1430 1400 - 1 5 3 0 0 9 3 0 - 1230 No S t r e s s No S t r e s s 1 0 0 0 - 1100 1430 - 1730 1000 - 1230 1400 - 1700 No S t r e s s 1030 - 1130 No S t r e s s 0930 - 1130 1400 - 1500 1100 - 1330 0 9 3 0 - 1330
This
schedule
has
I
Cold-Immobilization (hours)
an a v e r a g e
Schedule
Duration
(hours)
1.5 2.0 1.5 3.0
1.0 3.0 2.5 3.0 1.0 2.0 1.0 2.5 4.0 stress
density
of
1.55
hrs/day.
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Vol. 40, No. 13, 1987
Results F i g u r e s i, 2, and 3 i l l u s t r a t e e t h a n o l c o n s u m p t i o n for low, m e d i u m and high e t h a n o l d r i n k i n g g r o u p s r e s p e c t i v e l y . Ethanol i n t a k e for low e t h a n o l g r o u p s (Figure i) y i e l d e d a s i g n i f i c a n t e f f e c t of g r o u p [ F ( I , 3 7 ) = 5 . 2 6 , p<0.05] p e r i o d [ F ( 4 , 1 4 8 ) = 3 . 0 1 , p<0.05] and g r o u p x time p e r i o d i n t e r a c t i o n [ F ( 4 , 1 4 8 ) = 2 . 5 4 , p<0.05]. M o r e s p e c i f i c a l l y , p o s t - h o c T u k e y tests r e v e a l e d that s t r e s s e d rats s i g n i f i c a n t l y i n c r e a s e d t h e i r e t h a n o l c o n s u m p t i o n d u r i n g the s e c o n d stress p e r i o d (p<0.05) as w e l l as d u r i n g the t h i r d stress and p o s t - s t r e s s p e r i o d s as c o m p a r e d to b a s e l i n e and c o n t r o l levels (p<0.01). In contrast, e t h a n o l c o n s u m p t i o n for the m e d i u m c o n s u m i n g g r o u p s (Figure 2) showed a s i g n i f i c a n t main e f f e c t of time p e r i o d [ F ( 4 , 1 3 6 ) = I I . 0 2 , p<0.01] but not s i g n i f i cant e f f e c t s of g r o u p or g r o u p x time p e r i o d i n t e r a c t i o n [group x time p e r i o d : F ) 4 , 1 3 6 ) = 0 . 5 1 , p > 0 . 0 5 ] .
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F i g u r e i. E t h a n o l c o n s u m p t i o n in terms of m e a n grams per k i l o g r a m for l o w - e t h a n o l s t r e s s e d (n=20) and n o n - s t r e s s e d groups.
(n=19)
Vol. 40, No. 13, 1987
Stress and Ethanol Consumption
1249
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NO STRESS
F i g u r e 2. E t h a n o l c o n s u m p t i o n in terms of m e a n grams per k i l o g r a m for m e d i u m - e t h a n o l s t r e s s e d (n=18) and n o n - s t r e s s e d (n=18 groups). 6.0
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NO STRESS
F i g u r e 3. E t h a n o l c o n s u m p t i o n in terms of m e a n g r a m s per k i l o g r a m for h i g h - e t h a n o l s t r e s s e d (n=14) and n o n - s t r e s s e d groups.
(n=15)
1250
Stress and Ethanol Consumption
Vol. 40, No. 13, 1987
A n a l y s i s of the h i g h e t h a n o l g r o u p s y i e l d e d a s i g n i f i c a n t e f f e c t of time p e r i o d [ F ( 4 , 1 0 8 ) = 9 . 2 5 , p<0.05] as w e l l as a g r o u p x time period interaction which approached significance [F(4,108)=2.20, p<0.07]. This i n d i c a t e s that s t r e s s e d h i g h e t h a n o l c o n s u m i n g rats i n i t i a l l y r e d u c e d t h e i r e t h a n o l i n t a k e d u r i n g the first two stress p e r i o d s and then r e t u r n e d to b a s e l i n e levels. It is i m p o r t a n t to note that total fluid intake for all g r o u p s d u r i n g the e n t i r e e x p e r i m e n t did not s i g n i f i c a n t l y differ. In a d d i t i o n , no b e h a v i o r a l signs of o v e r t i n t o x i c a t i o n or w i t h d r a w a l w e r e o b s e r v e d at any time d u r i n g the day in this experiment. Discussion The r e s u l t s of the p r e s e n t study r e v e a l s e v e r a l i n t e r e s t i n g observations. First, it was o b s e r v e d that the s t r e s s e d low e t h a n o l c o n s u m i n g rats d e m o n s t r a e d an i n c r e a s e in e t h a n o l c o n s u m p t i o n d u r i n g the s e c o n d s t r e s s p e r i o d w h i c h was m a i n t a i n e d t h r o u g h o u t the r e m a i n d e r of the e x p e r i m e n t . This i n c r e a s e in e t h a n o l c o n s u m p t i o n is c o n s i s t e n t w i t h the r e c e n t report of N a s h and M a i c k e l (14). In c o n t r a s t , however, we failed to find any i n c r e a s e in e t h a n o l i n t a k e in the m e d i u m and high c o n s u m i n g groups. In fact, d a t a from the high e t h a n o l rats, w h i l e a p p r o a c h i n g s i g n i f i c a n c e , s u g g e s t a d e c r e a s e in e t h a n o l i n t a k e d u r i n g the s t r e s s periods. This o b s e r v a t i o n , w h i l e not cons i s t e n t w i t h d a t a from the low and m e d i u m g r o u p s and w i t h o t h e r r e p o r t s (11,13,14), is c o n s i s t e n t w i t h data from our l a b o r a t o r y d e m o n s t r a t i n g a d e c r e a s e in e t h a n o l c o n s u m p t i o n a m o n g high e t h a n o l c o n s u m i n g rats as a c o n s e q u e n c e of c h r o n i c stress (8,9). T h e s e d a t a c o n s i d e r e d t o g e t h e r s u g g e s t that the e f f e c t of c h r o n i c stress ( p r e d i c t a b l e and u n p r e d i c t a b l e ) on e t h a n o l c o n s u m p t i o n m a y d i f f e r d e p e n d i n g upon the level of e t h a n o l i n t a k e d u r i n g b a s e l i n e (pre-stress) periods. It is also i n t e r e s t i n g to note that b o t h s t r e s s e d and n o n - s t r e s s e d high e t h a n o l - c o n s u m i n g rats s h o w e d a r e d u c e d e t h a n o l i n t a k e c o m p a r e d to b a s e l i n e levels. This r e d u c t i o n in d r i n k i n g w o u l d a p p e a r to be the result of i n c r e a s e d h a n d l i n g r a t h e r than due to stress per se. We are i n v e s t i g a t i n g h a n d l i n g e f f e c t s on stress and a l c o h o l d r i n k i n g responses. T h e s e r e s u l t s serve to h i g h l i g h t the i m p o r t a n c e of a s s e s s i n g the e f f e c t s of stress on v a r i o u s levels of e t h a n o l i n t a k e (i.e. low, m e d i u m and high), since d i f f e r e n t i a l e f f e c t s of stress on e t h a n o l i n t a k e m a y be o b s c u r e d by c o m b i n i n g data from rats c o n s u m i n g d i f f e r e n t levels of ethanol. A mechanism for this o b s e r v a t i o n is not c l e a r at the p r e s e n t time, but r e s e a r c h in this l a b o r a t o r y is b e i n g c o n d u c t e d to i n v e s t i g a t e p o s s i b l e n e u r o c h e m i c a l a l t e r a t i o n s u n d e r l y i n g the e f f e c t s of stress on e t h a n o l c o n s u m p t i o n . In summary, the p r e s e n t r e s u t l s s u g g e s t that e x p o s u r e to u n p r e d i c t a b l e c o l d - i m m o b i l i z a t i o n stress p r o d u c e s d i f f e r e n t i a l e f f e c t s on e t h a n o l c o n s u m p t i o n d e p e n d i n g upon the rat's prestress level of e t h a n o l intake. References i. 2.
L.A. P o h o r e c k y , N e u r o s c i . B i o b e h a v . Rev. 5 208-229 (1981). J. B r i c k and L.A. P o h o r e c k y , P s y c h o p h a r m a c o l . 7 7 81-84 (1982).
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3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.
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K. K u r i y a m a , K. K a n m o r i a n d Y. Y o p e d a , N e u r o p h a r m a c o l . 23 6 4 9 - 6 5 4 (1984). L.A. P o h o r e c k y , E. R a s s i , J. W e i s s a n d V. M i c h a l a k , A l c o h o l . C l i n . Exp. Res. 4 4 2 3 - 4 2 6 (1980). R. Lev, K. K a w a s h i m a , and G.B. G l n s s , A r c h . P a t h o l . Lab. Med. i00 5 5 ~ - 5 5 8 (1976). G.B. G l a v i n and G.E. R o c k m a n , A l c o h o l 2 6 5 1 - 6 5 3 (1985). G.E. R o c k m a n and G.B. G l a v i n , A l c o h o l 1 2 9 3 - 2 9 5 (1984). G.E. R o c k m a n a n d G.B. G l a v i n , Pha~:macol. B i o c h e m . B e h a v . 24 8 6 9 - 8 7 3 (1986). G.E. R o c k m ~ n , A. H a l l and G.B. G l a v i n . P h a r m a c o l . B i o c h e m . B e h a v . in p r e s s (1986). K.M. Schmiclt and F.D. K l o p f e r , Q u a r t . J. Stud. Alc. 29 5 5 8 - 5 6 5 (1568). J.H. M a s s e r m a n and K.S. Yum, P s y c h o s o m . Med. 8 3 6 - 5 2 (1946). M.A. C a p l a n and K. P u g l i s i , P h a r m a c o l . B i o c h e m . B e h a v . 24 2 7 1 - 2 8 0 (1986). R. D e r r a n d S. L i n d b l a d , L i f e Sci. 27 2 1 8 3 - 2 1 8 6 (1980). J . ~ N a s h and R.P. M a i c k e l , L i f e Sci. 37 7 5 7 - 7 6 5 (1985). I.G. L u t h e r , G.T. H e i s t a d , and S.B. S p a r b e r , P s y c h o s o m . Med. 31, 4 5 - 5 6 (1969).