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Shock induced ethanol consumption in rats
Pharmacology Biochemistry & Behavior, Vol. 6, pp. 107--115. Copyright © 1977 by AN KHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.
Shock Induced Ethanol Consumption in Rats K E N N E T H C. M I L L S 1 , J. W I L L I A M B E A N 1 A N D J. S T A N F O R D H U T C H E S O N 2
Center f o r Alcohol Studies 1 and Department o f Psychiatry 2 University o f North Carolina, Division o f Health Sciences Chapel Hill, North Carolina 27514 ( R e c e i v e d 29 S e p t e m b e r 1 9 7 5 )
MILLS, K. C., J. W. BEAN AND J. S. HUTCHESON. Shock induced ethanol consumption in rats. PHARMAC. BIOCHEM. BEHAV. 6(1) 107-115, 1 9 7 7 . - Two experiments are presented which describe the temporal and volumetric changes in ethanol consumption by rats exposed to recurring schedules of inescapable random shock. The animals in Experiment 1, which had a choice between ethanol and water, increased their voluntary ethanol consumption immediately after the shock schedule. The postshock changes occurred with both 5% and 10% V/V ethanol, were specific to the presence of shock and were not reflected by measures of total daily ethanol intake. Experiment 2 exposed rats to extended 2 2 h r stress sessions, during which each animal had four simultaneous fluid choices available: water, saccharin 0.1% W/V, ethanol 5% V/V, and ethanol 10% V/V. Temporal intake patterns for both 5% and 10% ethanol showed pronounced peaks for the interval immediately following the shock schedule. A shift of intake from 5% to 10% ethanol was also demonstrated with increasing time under shock, while saccharin and water intake decreased. The results are interpreted as a relationship between voluntary ethanol intake and escape from the consequences of stress. Stress and drinking Voluntary ethanol consumption Temporal patterns of fluid intake Escape drinking
Animal models of alcoholism Shock induced ethanol consumption
THIS p a p e r describes a t e m p o r a l r e l a t i o n s h i p b e t w e e n stress and free-choice alcohol c o n s u m p t i o n in rats. T h e focus is u p o n the s h o r t t e r m changes in fluid i n t a k e w h i c h o c c u r as a f u n c t i o n of p e r i o d i c s h o c k stress. Previous e x p e r i m e n t s have a t t e m p t e d to isolate t h e variables involved w i t h stress and d r i n k i n g and the designs have usually c e n t e r e d o n the type or a m o u n t of stressor e f f e c t i n g a c h a n g e in m e a s u r e s of daily h o m e cage e t h a n o l c o n s u m p t i o n [4, 10, 12, 26, 27, 3 5 ] . The range of stress m a n i p u l a t i o n s has b e e n extensive, and this l i t e r a t u r e has received review in several r e p o r t s [8, 23,241. We assumed t h a t m e a s u r e s of daily e t h a n o l i n t a k e m i g h t n o t reflect changes of s h o r t e r d u r a t i o n w h i c h could be occurring as the result of stress. R a t h e r t h a n e x p o s e t h e animals to stress in o n e e n v i r o n m e n t a n d m e a s u r e daily c o n s u m p t i o n in t h e h o m e cage, o u r animals were h o u s e d in the test e n v i r o n m e n t 24 hr daily. This a r r a n g e m e n t allowed us to q u a n t i f y fluid selection over smaller units of t i m e b y m e a s u r i n g licking w i t h d r i n k o m e t e r s as described b y a n u m b e r of investigators [1, 2, 20, 29, 3 1 ] . F r e u n d [18] and Eriksson [13] have also used d r i n k o m e t e r records t o describe the diurnal d i s t r i b u t i o n of e t h a n o l i n t a k e in rats and mice, and t h e c o n t i n u o u s m o n i t o r i n g p r o c e d u r e s provide an ideal m e t h o d to m e a s u r e the t e m p o r a l comp o n e n t s of e t h a n o l i n t a k e u n d e r stress. E x p e r i m e n t 1 describes e t h a n o l i n t a k e relative to recurring stress for rats h o u s e d c o n t i n u o u s l y in t h e test e n v i r o n m e n t for 50 days w i t h a t w o fluid choice of e t h a n o l
and water. E x p o s u r e to d i f f e r e n t c o n c e n t r a t i o n s of e t h a n o l was sequential. E x p e r i m e n t 2 describes the t e m p o r a l p a t t e r n s of fluid i n t a k e for a g r o u p o f animals given s i m u l t a n e o u s m u l t i p l e fluid choices a n d e t h a n o l conc e n t r a t i o n s d u r i n g e x t e n d e d stress and n o n stress exposure. EXPERIMENT 1
Method Animals. T h e animals were 12 male Long-Evans h o o d e d rats f r o m Blue Spruce Farms, A l t a m o n t , New York. T h e y . h a d a m e a n weight of 533.8 g w i t h a range f r o m 4 5 5 - 6 4 4 g at t h e start of t h e Basline I. Apparatus. The animals were individually h o u s e d in s o u n d a t t e n u a t e d c h a m b e r s w h i c h also allowed c o n t r o l o f diurnal light cycles (12 hr light, 12 hr dark). E a c h cage had a grid floor, a lever and t w o d r i n k o m e t e r s , spaced 5 cm apart. The living space m e a s u r e d 30 × 24 x 26 cm high. The d r i n k o m e t e r s , described in detail elsewhere, used a R i c h t e r d r i n k i n g s p o u t a n d were designed a r o u n d an infrared light e m i t t i n g diode so t h a t t h e possibility of t o n g u e s h o c k and visual cue p r o b l e m s in c o n t a c t a n d p h o t o c e l l designs could be e l i m i n a t e d [ 2 1 ] . F o o t s h o c k was p r o v i d e d by c o n s t a n t c u r r e n t s c r a m b l e d s h o c k sources and was set at 1.5 ma f o r all s h o c k procedures. T e m p o r a l schedules were c o n t r o l l e d w i t h solid state logic. Procedure. A l c o h o l s o l u t i o n s were p r e p a r e d f r o m 95% 107
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e t h a n o l in distilled water [ 11 }. The animals were h o u s e d in the closed e n v i r o n m e n t s 24 hr daily, w i t h one 20 min i n t e r r u p t i o n for weighing and r e c o r d i n g of daily fluid intake. F o o d was p r o v i d e d ad lib in h o p p e r s s u s p e n d e d from the wall of the cage. Baselines were defined as intervals in w h i c h licking d i s t r i b u t i o n s over t i m e were s a m p l e d for e t h a n o l and water. The p o s i t i o n of each fluid c h a n g e d every 48 hr. E s t i m a t e s of fluid i n t a k e for a c u m u l a t i v e t i m e interval were derived from the lick c o u n t for t h a t interval relative to the t o t a l c u m u l a t i v e n u m b e r of licks in a 24 h r session• T h e r a t i o was then m u l t i p l i e d by t h e t o t a l q u a n t i t y of fluid delivered to provide an e s t i m a t e o f the fluid c o n s u m e d in the interval [see 211. Licking data for E x p e r i m e n t 1 were o b t a i n e d b y a c c u m u l a t i n g lick c o u n t s f r o m five s e p a r a t e 12 m i n s e g m e n t s of each h o u r over 24 hr. A f t e r baseline, the same 12 m i n s e g m e n t in e a c h h r was d e s i g n a t e d as the s h o c k interval a n d the a s s i g n m e n t of pre- a n d p o s t s h o c k intervals was arbitrary. Using a digital p r i n t e r , lick c o u n t s o n b o t h e t h a n o l a n d w a t e r were also sampled to a 3 m i n r e s o l u t i o n d u r i n g selected sessions f r o m individual animals. S h o c k sessions were d e f i n e d b y delivering r a n d o m l y spaced 1 sec f o o t s h o c k s for the same 12 rain o f each hr a r o u n d the clock. T h e s c h e d u l e d u r i n g the 12 rain was VI 1 min, so t h a t each a n i m a l received o n t h e average 12 i n e s c a p a b l e s h o c k s p e r hr, or 288 s h o c k s p e r 24 h r session. W h e n s h o c k occurs for 12 rain of each hr, a r a n d o m l y d i s t r i b u t e d d r i n k i n g p a t t e r n w o u l d s h o w 40% of d r i n k i n g during the p r e s h o c k interval (24 min), 20% during the shock interval and 40% d u r i n g the p o s t s h o c k interval (24). A post interval ratio o f 0 . 4 0 would reflect this p a t t e r n and is typically very close to t h a t observed u n d e r baseline c o n d i t i o n s . Changes in the post interval r a t i o are used as one m e a s u r e of the effects of shock. However, a large change in a r a t i o m a y m e a n little on a daily basis if the a m o u n t of fluid c o n s u m e d is n o t t a k e n i n t o a c c o u n t . A shift of a p o s t interval ratio f r o m 0 . 4 0 - 0 . 8 0 could be applied to a daily decrease in fluid c o n s u m p t i o n f r o m 2 0 - 1 0 ml resulting in n o change in post interval fluid intake. D r i n k o m e t e r e s t i m a t e s of p o s t i n t e r v a l v o l u m e t r i c i n t a k e are also t a k e n as an i n d e x o f t h e effects of shock. F o l l o w i n g a 20 session (24 hr) baseline (Baseline 1), all animals were e x p o s e d to the 12 m i n recurring h o u r l y r a n d o m s h o c k s c h e d u l e for five daily sessions ( 2 1 - - 2 5 Shock I) and a five session after s h o c k baseline (sessions 2 6 - 3 0 ) . The e n t i r e cycle was r e p e a t e d w i t h a d i f f e r e n t c o n c e n t r a t i o n of e t h a n o l as a fluid choice for a 10 day baseline (sessions 3 1 - 4 0 Baseline II), 5 days o f r a n d o m s h o c k (sessions 4 1 - 4 5 S h o c k II), and 5 days of a f t e r s h o c k baseline (sessions 4 6 - 5 0 ) . Half of t h e a n i m a l s (N = 6) received a 5% V / V e t h a n o l choice and w a t e r for t h e first b a s e l i n e - s h o c k cycle and a 10% V / V e t h a n o l choice for the second cycle. T h e o t h e r half received the 10% V / V e t h a n o l as t h e i r initial c h o i c e and 5% V / V w i t h t h e second cycle. In essence, two i n d e p e n d e n t g r o u p s of six rats received a l t e r n a t e orders of e x p o s u r e to 5 and 10% e t h a n o l a n d f o u r t r e a t m e n t c o m b i n a t i o n s o c c u r r e d u n d e r each BaselineShock-Baseline cycle: S h o c k I-5%, S h o c k II-10%, S h o c k 1-10%, and S h o c k II-5%. T h e n u m b e r o f animals in G r o u p I1-5% was five due to e q u i p m e n t failure. Results Figure 1 displays t h e overall results o f r e p e a t e d baseline
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FIG. l. Mean daily ethanol and water intake expressed as cumulative hourly intake (total interval criterion) and as cumulative intake during a 24 min schedule segment (postinterval criterion). During the five shock sessions (24 hr) the shock schedule recurs hourly for 12 min followed by the 24 min postinterval.
and s h o c k e x p o s u r e s w i t h 12 animals. T h e data are p l o t t e d separately for each g r o u p r e p r e s e n t i n g a t r e a t m e n t comb i n a t i o n u n d e r each of the t w o criteria for evaluating fluid intake. T h e t o t a l interval c r i t e r i o n is the c u m u l a t i v e daily intake of e t h a n o l and w a t e r in the 24 hr session. T h e post interval criterion is the c u m u l a t i v e daily i n t a k e of e t h a n o l and w a t e r for the 24 m i n following s h o c k and the same 24 rain interval during p r e s h o c k and a f t e r s h o c k baseline. To reiterate, s h o c k schedules are n o t in effect d u r i n g baselines and the selection of a similar 24 m i n s e g m e n t is a r b i t r a r y u n d e r n o n s h o c k c o n d i t i o n s . It should be n o t e d t h a t the b o d y weights for t h e animals d r o p p e d an average of 5.5% f r o m m e a n baseline weights with the i n t r o d u c t i o n of shock sessions, a n d fluid i n t a k e s are expressed relative to the observed weights for each p r o c e d u r e . It is a p p a r e n t from
SHOCK A N D E T H A N O L C O N S U M P T I O N Fig. 1 t h a t m o d u l a t i o n of e t h a n o l i n t a k e in t e r m s of t o t a l daily fluid i n t a k e was typical of m a n y studies l o o k i n g at the effects of s h o c k on drinking. With t h e e x c e p t i o n of t h e 5% animals o n t h e i r first s h o c k e x p o s u r e , all groups s e e m e d to show a decrease in t h e i r e t h a n o l i n t a k e following shock. The post interval criterion, h o w e v e r , i n d i c a t e d t h a t t h e intake of e t h a n o l increased for all f o u r g r o u p s d u r i n g the s h o c k s c h e d u l e e x p o s u r e . M o d u l a t i o n of w a t e r i n t a k e as reflected by b o t h criteria was n o t a p p a r e n t . In o r d e r to evaluate t h e effects of s h o c k o n e t h a n o l c o n s u m p t i o n s e p a r a t e b u t identical analyses of variance were carried o u t for b o t h t o t a l interval and post interval scores. Water scores were n o t i n c l u d e d in t h e analyses because o f t h e d i s p a r a t e i n t a k e v o l u m e s i n d i c a t e d in Fig. 1. Each analysis was a t w o b y two by t w o factorial design as described by Winer [ 3 6 ] . T h e t w o levels of F a c t o r A were O r d e r l ( 5 % - 1 0 % ) a n d O r d e r II ( 1 0 % - 5 % ) . This was the f a c t o r on w h i c h r e p e a t e d m e a s u r e s were n o t taken. Rep e a t e d m e a s u r e s were t a k e n o n t h e t w o levels of F a c t o r B w h i c h were 5% a n d 10% e t h a n o l i n t a k e a n d t h e two levels of F a c t o r C w h i c h were defined by baseline and s h o c k procedures. T h e c o n s u m p t i o n scores for each a f t e r s h o c k baseline are s h o w n in Fig. 1, b u t were n o t i n c l u d e d in the analyses. T h e analysis of variance of t h e t o t a l interval scores s h o w e d n o significant m a i n effects for e i t h e r the baseline to s h o c k m e a n d i f f e r e n c e s or t h e o r d e r of e x p o s u r e to 5 and 10% e t h a n o l . A significant m a i n effect was d e t e c t e d for t h e difference b e t w e e n 5 and 10% i n t a k e , F ( 1 , 1 0 ) = 2 8 . 6 6 , p < 0 . 0 5 . A significant i n t e r a c t i o n was also o b s e r v e d bet w e e n the c o n c e n t r a t i o n f a c t o r and o r d e r of e x p o s u r e , F ( 1 , 1 0 ) = 10.07, p < 0 . 0 5 . T h e analysis of t h e p o s t interval scores, h o w e v e r , yielded significant m a i n effects for b o t h baseline to s h o c k differences, F ( 1 , 1 0 ) = 16.24, p < 0 . 0 5 , a n d 5 and 10% e t h a n o l i n t a k e scores, F ( 1 , 1 0 ) = 18.80, p < 0 . 0 5 . Again, significant main effects for t h e o r d e r of e x p o s u r e f a c t o r were n o t evident. All i n t e r a c t i o n s for the p o s t i n t e r v a l analysis were nonsignificant. T h e statistical analyses implied t h a t e t h a n o l i n t a k e i m m e d i a t e l y a f t e r the s h o c k was a m o r e sensitive i n d e x of c o n s u m p t i o n t h a n the a m o u n t of e t h a n o l selected d u r i n g the t o t a l interval w h i c h is o f t e n expressed as daily e t h a n o l intake. U p o n i n s p e c t i o n of t h e data it was e v i d e n t t h a t t h e last t w o s h o c k sessions c o n t a i n e d p r o p o r t i o n a t e l y m o r e p o s t i n t e r v a l increases in e t h a n o l i n t a k e t h a n the first t w o shock sessions, suggesting a gradually emerging effect of shock o n drinking. Figure 2 s h o w s the p o s t i n t e r v a l r a t i o changing as a f u n c t i o n of s h o c k e x p o s u r e over all animals at b o t h c o n c e n t r a t i o n s for the first a n d s e c o n d s h o c k cycles. The increasing p e a k w i t h increasing t i m e in s h o c k a n d t h e a b r u p t r e t u r n to baseline levels i n d i c a t e t h a t e t h a n o l i n t a k e was specific to the s h o c k schedule. T h e p o s t i n t e r v a l r a t i o as o p p o s e d to p o s t i n t e r v a l i n t a k e (ml × ratio) does n o t take i n t o a c c o u n t t h e a b s o l u t e a m o u n t of fluid c o n s u m e d , and t h e m e a s u r e does n o t d i f f e r e n t i a t e t h o s e animals w h i c h s h o w e d a b s o l u t e increases in e t h a n o l i n t a k e w i t h s h o c k f r o m t h o s e w h i c h did n o t . T h e c o n s i s t e n c y of the data across b o t h t r e a t m e n t s and animals implies t h a t t h e p o s t s h o c k r a t i o is sensitive to s h o c k effects regardless of w h e t h e r or n o t this leads to increases in p o s t s h o c k e t h a n o l i n t a k e . Because of t h e high i n c i d e n c e of s h o c k sessions in w h i c h w a t e r i n t a k e was zero, c o m p a r a b l e p o s t i n t e r v a l data for w a t e r i n t a k e were n o t reliable. To illustrate an individual t e m p o r a l p a t t e r n of s h o c k
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FIG. 2. Postinterval ethanol licking as a proportion of total ethanol licking for shock and nonshock conditions on consecutive exposures. Each vertical hash mark represents the standard error of the mean. i n d u c e d c o n s u m p t i o n , Fig. 3 p r e s e n t s the records of the animal s h o w i n g t h e largest increases in e t h a n o l i n t a k e following shock. Because of e q u i p m e n t restrictions, i n t a k e records resolved to 3 rain r e s o l u t i o n a r o u n d t h e clock were n o t available for all sessions. A n i m a l 8 s h o w e d b o t h overall and p o s t i n t e r v a l increases in e t h a n o l i n t a k e at b o t h c o n c e n t r a t i o n s . This is illustrated by i n t a k e p e a k s for e t h a n o l i m m e d i a t e l y following s h o c k and b y t o t a l increases in e t h a n o l i n t a k e over baseline of 62.5 m l / k g / 2 4 hr for S h o c k I and 14 m l / k g / 2 4 h r for S h o c k II. P o s t i n t e r v a l increases reflected the peak i n t a k e s m o r e closely a n d were respectively 60 m l / k g / 2 4 hr for S h o c k 1 and 48.4 m l / k g / 2 4 hr for S h o c k II. T o t a l interval w a t e r intake for b o t h s h o c k e x p o s u r e s was 7.2 m l / k g / 2 4 hr for S h o c k I and 0.7 m l / k g / 2 4 hr for S h o c k II.
Discussion The animals in E x p e r i m e n t 1 displayed t e m p o r a l shifts in e t h a n o l i n t a k e relative to recurring i n e s c a p a b l e shock. Specifically, the d i s t r i b u t i o n of licking reflected fluid intake increases specific to e t h a n o l in t h e interval imm e d i a t e l y following the shock. T h e data d e m o n s t r a t e d t h a t the daily i n t a k e c r i t e r i o n was i n a d e q u a t e to describe the changes in e t h a n o l c o n s u m p t i o n , and t h a t w i t h some animals the p o s t s h o c k increases in e t h a n o l i n t a k e were quite d r a m a t i c . T h e central issue, h o w e v e r , is w h e t h e r or n o t t h e animals were using the i n t o x i c a t i n g p r o p e r t i e s of the e t h a n o l to s o m e h o w alter the c o n s e q u e n c e s of shock. T h e c o n t i n u o u s isolation p r o c e d u r e s m a k e it difficult to e x t r a c t b l o o d e t h a n o l samples w i t h o u t d i s t u r b i n g the animals t h e r e f o r e b l o o d samples were n o t taken. Nevertheless, it w o u l d seem necessary to display circulating b l o o d e t h a n o l levels w h i c h are in excess of t h e rat's m e t a b o l i c c a p a c i t y in o r d e r to
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m i g h t be due to s o m e p r o p e r t y of the e t h a n o l o t h e r t h a n the i n t o x i c a t i n g effect, such as a simple taste p r e f e r e n c e , w h i c h is amplified b y the s h o c k schedule. If t h e animal is sampling a n y t h i n g novel as an a t t e m p t to alter t h e shock, t h e n m o m e n t a r y i n t o x i c a t i o n f r o m e t h a n o l m i g h t be a b y - p r o d u c t of d r i n k i n g i n d u c e d b y the n o v e l taste of e t h a n o l relative to water. The animals in E x p e r i m e n t 1 also show an unusally high p r e f e r e n c e for e t h a n o l in t h e test cages as reflected by Fig. 1. These animals were n o t given h o m e cage p r e f e r e n c e tests so t h a t the d i s t r i b u t i o n o b t a i n e d d u r i n g t h e test cage baseline w o u l d n o t be c o n f o u n d e d w i t h e x p o s u r e and a c c l i m a t i o n effects [9, 25, 34, 3 7 ] . The test c h a m b e r s were isolated in c o m p a r i s o n with h o m e cages and this r e s t r i c t i o n m i g h t also have b e e n s t r o n g e n o u g h to i n d u c e a high e t h a n o l p r e f e r e n c e over w a t e r [ 2 8 ] . T a k e n t o g e t h e r the taste, p r e f e r e n c e a n d isolation factors could be o p e r a t i n g to p r o d u c e the exaggerated e t h a n o l c o n s u m p t i o n p a t t e r n s a t t r i b u t e d to the s h o c k stress and r e p l i c a t i o n with add i t i o n a l c o n t r o l s was w a r r a n t e d .
r 57 60
5 MINUTE INTERVALS
FIG. 3. Cumulative ethanol and water intake for subject 8 summarized over grouped and single sample sessions (24 hr) of baseline and shock exposure at 5 and 10% ethanol-water choice. The value in parentheses adjacent to the procedure represents the number of 24 hr sessions sampled to a 3 min resolution. state t h a t the animals are using the i n t o x i c a t i n g p r o p e r t i e s of e t h a n o l to achieve s o m e m e a s u r e of stress relief. Collateral studies in o u r lab o n rats e x p o s e d to the same s h o c k p a r a d i g m have i n d i c a t e d t h a t w h e n b l o o d samples are t a k e n at r a n d o m t i m e s d u r i n g the 24 h r session, circulating b l o o d e t h a n o l levels are o f t e n in excess of 150 r a g / 1 0 0 ml. Baseline b l o o d e t h a n o l levels average a r o u n d 10 m g / 1 0 0 ml w i t h a range f r o m 1 - 2 5 m g / 1 0 0 ml and s h o c k b l o o d levels average 35 m g / 1 0 0 m l ranging f r o m 5 - 1 6 0 m g / 1 0 0 ml. A l t h o u g h the increase f r o m baseline to s h o c k is statistically significant, it is difficult to i n t e r p r e t the d a t a in t h a t t h e m e t a b o l i c rate is c o n f o u n d e d w i t h t h e a n i m a l having c o n t i n u o u s access to t h e e t h a n o l and a t e m p o r a r y e l e v a t i o n of b l o o d e t h a n o l w o u l d n o t necessarily be evidence for c o n t i n u o u s i n t o x i c a t i o n . One p r o b l e m m a y be t h a t in o r d e r to c o n s i s t e n t l y p r o d u c e a d i s t r i b u t i o n of b l o o d levels above 50 m g / 1 0 0 ml, w h i c h w o u l d i n d i c a t e sustained i n t o x i c a t i o n during a 24 h r session, the a n i m a l w o u l d have to c o n s u m e at least 8.4 g/kg of e t h a n o l / d a y . This w o u l d be a p p r o x i m a t e l y 212 m l / k g of 5% e t h a n o l or 106 m l / k g of 10%. If we assume t h a t t h e a n i m a l is a c c u m u l a t i n g some b l o o d e t h a n o l f r o m h o u r to h o u r the i n t a k e r e q u i r e m e n t m i g h t be r e d u c e d s o m e w h a t to m a i n t a i n the b l o o d level at or above 50 m g / 1 0 0 ml, b u t the r e q u i r e m e n t is high in t h a t t h e t o t a l daily fluid i n t a k e for o u r animals in the test e n v i r o n m e n t is typically a r o u n d 80 m l / k g over all p r o c e d u r e s . With this i n f o r m a t i o n in m i n d , it can be argued t h a t the t e m p o r a l c o r r e l a t i o n of s h o c k and increased e t h a n o l i n t a k e
2
E x p e r i m e n t 2 was an a t t e m p t to f u r t h e r define the f u n c t i o n a l r e l a t i o n s h i p b e t w e e n stress and d r i n k i n g by describing the t e m p o r a l p r o p e r t i e s of fluid i n t a k e in rats. The animals were offered four fluid choices of water, s o d i u m saccharin, e t h a n o l at 5% V / V ( p r e f e r r e d ) and 10% V / V ( n o n p r e f e r r e d ) to c o n t r o l for taste and p r e f e r e n c e variables. S i m u l t a n e o u s r a t h e r t h a n s e q u e n t i a l e x p o s u r e to m u l t i p l e c o n c e n t r a t i o n s of e t h a n o l allowed e x a m i n a t i o n o f the shifting p a t t e r n s of e t h a n o l i n t a k e in response to stress. In a d d i t i o n , h o m e cage p r e f e r e n c e s were t a k e n p r i o r to test cage e x p o s u r e to d e t e r m i n e the e x t e n t to w h i c h test cage isolation altered fluid intake. E x p e r i m e n t i limited the animals to five days of stress e x p o s u r e at each e t h a n o l c o n c e n t r a t i o n , and a longer s h o c k e x p o s u r e was used in the s e c o n d study. An e x t e n d e d s h o c k e x p o s u r e p e r m i t t e d us to e x a m i n e w h e t h e r the animals a d a p t e d to the effects of s h o c k and increased t h e i r selection of n o n i n t o x i c a t i n g fluids or w h e t h e r persisting s h o c k w o u l d c o n t i n u e to i n f l u e n c e e t h a n o l i n t a k e as suggested by E x p e r i m e n t 1.
Me tho d Animals. T h e animals for E x p e r i m e n t 2 were 12 male Long-Evans h o o d e d rats f r o m Blue Spruce Farms, A l t a m o n t , New York. The m e a n weight for all animals d u r i n g the first test cage baseline was 522 g w i t h a range from 4 5 4 - 5 8 9 g. The final m e a n weight for the animals during the last 10 sessions of the s t u d y was 519 g with a range of 426 6 1 4 g. One animal s h o w e d several indices of p o o r h e a l t h d u r i n g the initial phases of the s t u d y and was r e m o v e d from the test e n v i r o n m e n t w i t h t h e o n s e t of s h o c k stress. His data are n o t i n c l u d e d in the g r o u p data for test cage preferences. Apparatus. The a n i m a l s ' e n v i r o n m e n t was similar to the system used in E x p e r i m e n t 1, b u t the test cages and data facilities were redesigned to a c c o m m o d a t e i n p u t s from four s i m u l t a n e o u s fluid choices w i t h a 2-rain resolution. The animals were h o u s e d in similar s o u n d a t t e n u a t e d c h a m b e r s , b u t each cage (28 x 21 x 21 cm) h a d four b o t t l e s m o u n t e d b e h i n d o n e wall of the cage. Each b o t t l e was calibrated to 2 ml and was m o u n t e d b e h i n d an 8 m m d i a m e t e r access hole. The cages h a d grid floors, an i n o p e r a t i v e lever and i n t e r n a l
SHOCK AND E T H A N O L C O N S U M P T I O N house lights. The diurnal cycle was 11 hr light and 11 hr dark with 2 hr daily for a service break. F o o t s h o c k was provided by constant current shock sources, and the shock level was set at 1.5 ma for all procedures. The d r i n k o m e t e r s in each cage consisted of four optical couplers with an infrared light emitting diode ( L E D ) and photo-transistor. The infrared beam of light passed directly across the access hole to the drinking tube. The data collection system consisted of an 8K, PDP-8F (Digital E q u i p m e n t C o r p o r a t i o n ) c o m p u t e r . The software was written in PAL Ill and was designed to process and count licks occurring at each of the 48 individual inputs over a specified block of time (2 min). Following each time block, the software switched to another bank of 48 counters for the duration of the next block. The system continued this o p e r a t i o n for the total n u m b e r of blocks specified, or 30 for the 1 hr timebase of E x p e r i m e n t 2. After t w e n t y - t w o 1 hr cycles the cumulative data was outputed. Procedure. H o m e cage fluid preferences were d e t e r m i n e d for the 12 rats during a 24 day pretest exposure. The animals were housed in wire mesh h o m e cages with ad lib rat c h o w and two fluid choices c o n t i n u o u s l y available t h r o u g h o u t the day. Each cage had one drinking bottle for water and one bottle for a choice sloution of ethanol at 5% V/V, 7.5% V/V, 10%V/V 12.5% V / V or sodium saccharin at 0.1% W/V. The animals were given eight days of ethanol exposure or two days at each choice. As part of another study, the animals were also given sucrose solutions and water for an additional eight days. The order of presentation of the four ethanol c o n c e n t r a t i o n s was r a n d o m across animals. Bottle positions were reversed daily and solution concentrations were changed daily. The animals were weighed every o t h e r day. After a two week break w i t h o u t alcohol in the h o m e cage, the subjects were transferred to the test environments. They were housed continuously in the test environments for 55 consecutive 22 hr sessions. Baseline and shock session schedules were defined as in E x p e r i m e n t 1 with the e x c e p t i o n that the session length was shortened to 22 hr to allow for cage servicing and data collection. F o u r drinking solutions were always available in the test environment: water, 0.1% W/V sodium saccharin, 5% V / V ethanol, and 10% V / V ethanol. Ethanol solutions were prepared from 95% ethanol and distilled water. Saccharin solutions were prepared from c o m m e r c i a l saccharin tablets and distilled water. Ad lib rat c h o w was provided in hoppers suspended from the opposite wall of the cage on which the drinkers were m o u n t e d . During the service interval the animals were transferred to individual holding cages w i t h o u t access to f o o d or water and the positions of the drinking bottles were rotated in a cyclical fashion. The sequence of the f o u r bottles never changed and their positions repeated every four days. The drinking tubes were rinsed and drinking solutions were replenished with fresh solutions once daily. The baseline was e x t e n d e d to 25 sessions for the first exposure in E x p e r i m e n t 2 to allow the animals to accomm o d a t e to the four drinker situation and produce stable drinking patterns. Baseline 1 refers to the last 10 sessions of the 25 session baseline exposure. Following Baseline I, all animals were given 20 consecutive sesions of shock. As in the first study, shock occurred for the same 12 min of each hr on a VI one min schedule. F o r convenience in looking at the e x t e n d e d effects of
111 shock, the first 10 shock sessions are called Shock I, and the second 10 sessions are called Shock II. Ten 22 hr sessions of postshock baseline (Baseline II) followed the shock sessions. Results The total mean daily fluid intake during the h o m e cage pretest exposure was 126.3 ml/kg with the two bottle choice. When the rats were transferred to the test environments with the four bottle choice, the total mean volume of daily fluid decreased in Baseline I to 80.4 ml/kg. The reasons for the decrease were not clear, but the changes were reflected in measures of water intake rather than intake on saccharin, 5% or 10% ethanol. A t-test for correlated means indicated that daily water intake dropped significantly from 48 ml/kg to 21.4 ml/kg (t = 6.81; p < 0 . 0 5 ) . Saccharin intake decreased from 48.2 ml/kg to 37.3 m l / k g but the difference was not significant (t = 1.1 l ; p > 0 . 0 5 ) . Ethanol intake at b o t h concentrations dropped slightly but h o m e cage to test cage mean differences were not significant (5%, t = 1.03; 10%, t = 0.92). Five percent h o m e cage intake was 21.4 ml/kg and test cage intake was 14.8 ml/kg. Similarly 10% ethanol intake showed a mean daily level of 8.7 ml/kg in the h o m e cages and 6.9 ml/kg in the test cages. Both the h o m e cage and test cage data showed m o d e r a t e c o n s u m p t i o n of 5% and low c o n s u m p t i o n of 10% ethanol. These results were more consistent than the data of E x p e r i m e n t 1 with previous reports of the rats' aversion for higher concentrations of ethanol. The test cage data for the 11 animals in E x p e r i m e n t 2 are shown in Figs. 4, 5, and 6. The mean intake records during each of the 10 sessions of Shock I, Shock II and postshock Baseline II are expressed as departures from the intake records for the last 10 sessions of Baseline I. The net change in daily fluid intake ( m l / k g / 2 2 hr) is plotted for each of the fluid choices of 5% V / V ethanol, 10% V / V ethanol, distilled water and saccharin. The X-axis shows each hour of the 22 hr session broken into successive two min intervals. Each data point represents the mean of 2420 samples of licking during a specific two min interval (11 animals x 22 hr x 10 sessions = 2420). For example, the sum of all points along one line would represent the total change from baseline intake on a specific fluid, and the plot represents the t e m p o r a l and volumetric shift in drinking relative to the recurring one hr cycle. It can be seen in Fig. 4 that ethanol intake at b o t h 5 and 10% increases during shock sessions and the peaks occur 8 - 1 2 min after shock, with c o n t i n u e d intake for up to 26 min after shock. A peak is also n o t e d for saccharin intake corresponding to the initial segments of b o t h ethanol peaks, but c o n s u m p t i o n does not c o n t i n u e after the initial burst. Water intake shows negligible changes during and after shock. Figure 5 shows the baseline to shock difference for the second 10 sessions of shock (Shock II) and also indicates a shift away from 5% ethanol intake to increased peaking on 10%. Post shock saccharin intake dropped off sharply with increased shock exposure during Shock II. These data are consistent with E x p e r i m e n t 1 in that fluid intake under shock was suppressed and post shock drinking was characterized by sharp peaks in ethanol intake. Figure 6 shows the four fluid intake patterns for the 10 sessions of postshock Baseline II as a difference from the 10 sessions of preshock Baseline I. Shock was not in effect and peak intake patterns were not observed. However, a
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p r e f e r e n c e for b o t h 5 and 10% e t h a n o l above w a t e r and saccharin is reflected by the f o u r curves. The p a t t e r n o f saccharin intake s h o w s a m i r r o r like negative p a t t e r n to that observed u n d e r s h o c k and could suggest t h a t the animals had learned to r e s p o n d to the h o u r l y t e m p o r a l p r o p e r t i e s o f the s h o c k schedule. Whereas Figs. 4, 5 and 6 illustrate the cumulative h o u r l y fluid intake relative to the first baseline, the n e t change
FIG. 6. The net change in daily fluid intake tot 11 animals as a difference from Baseline I to the l 0 sessions of baseline (Baseline II) which followed the 20 sessions of shock exposure. Shock was not in effect for Baseline 1 or Baseline II. scores could be sensitive to d i s t o r t i o n f r o m the initial high p r e f e r e n c e s for saccharin and low p r e f e r e n c e s for e t h a n o l at 10% V/V. The d i s t o r t i o n could be reflected by a t e m p o r a l m o d u l a t i o n o f all fluid c o n s u m p t i o n to the p o s t s h o c k interval without c o r r e s p o n d i n g absolute volumetric changes. Figure 7 t h e r e f o r e r e p r e s e n t s the absolute changes in b o t h total interval and p o s t i n t e r v a l (24 min) fluid intake over baseline and shock sessions. The e t h a n o l intake is c o m b i n e d for the 5 and 10% solutions and expressed as g/kg intake. The absolute increase in e t h a n o l intake as a f u n c t i o n o f s h o c k is seen for b o t h the total and post interval criteria and is c o n s i s t e n t with the t e m p o r a l data. T w o o n e - w a y r e p e a t e d measures analyses o f variance were carried out to evaluate the changes in e t h a n o l intake observed for b o t h the total and postinterval measures. When e t h a n o l intakes (g/kg) were collapsed over sessions and s u m m a r i z e d for Baseline l, Shock I, Shock II and Baseline II, significant main effects were evident for the total interval scores, F(3,30) = 5.57, p < 0 . 0 5 , and the postinterval scores, F ( 3 , 3 0 ) = 4.98, p < 0 . 0 5 . P o s t h o c c o m p a r i s o n s were o b t a i n e d with D u n n e t s t statistic and c o m p a r e d each t r e a t m e n t with the Baseline 1 c o n t r o l [ 3 6 ] . Using the total interval scores, e t h a n o l intake for Shock I (t = 2.74, p < 0 . 0 5 ) , Shock II (t = 3.70, p < 0 . 0 5 ) and Baseline II (t = 3.33, p < 0 . 0 5 ) was significantly increased over Baseline I intake levels. With the postinterval scores, e t h a n o l intake for S h o c k I (t = 3.06, p < 0 . 0 5 ) and Shock 11 (t = 3.44, p < 0 . 0 5 ) increased significantly over Baseline I. As m i g h t be e x p e c t e d f r o m the data in Fig. 7, postinterva] e t h a n o l intake for Baseline II did n o t differ significantly from Baseline I (t = 1.47, p < 0 . 0 5 ) . The a b s o l u t e changes in the v o l u m e o f e t h a n o l c o n s u m e d c o i n c i d e n t with shock sessions indicate that the relative t e m p o r a l peaks s h o w n in Figs. 4 and 5 also r e p r e s e n t a v o l u m e t r i c m o d u l a t i o n of e t h a n o l intake. The total interval saccharin and water intake changes from Baseline to shock were also evaluated with D u n n e t s t-test. As might be e x p e c t e d from the
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FIG. 7. Absolute daily fluid intake of ethanol, saccharin and water across sessions and procedures for the 11 animals in Experiment 2. Ethanol intakes on 5 and 10% V/V are combined to give the total ethanol intake in g/k/22 hr. The postinerval criterion represents the cumulative amount of fluid consumed daily in the 24 min interval during the nonshock baselines. The total interval criterion represents the total daily fluid intake cumulated over twenty-four 1 hr segments.
temporal data in Figs. 4 and 5, saccharin intake during Shock I did not show a significant decrease from baseline (t = 1.40, p>O.OS), but did show a significant mean decrease during the second ten sessions of Shock II (t = 2.21, pO.OS). The mean daily ethanol intake for all animals in Baseline I was 1.14 g/kg with a range from 0.14-2.49 g/kg. With the first 10 shock sessions, the mean increased to 2.18 g/kg and the range was from 0.15-6.81 g/kg. The second 10 shock sessions showed an additional mean increase to 2.54 g/kg with the range extending from 0.17-4.96 g/kg. Finally, Baseline II intake was relatively constant at 2.41 g/kg, and the range was from 0.19-5.78 g/kg. The largest individual daily intake under shock was 9.20 g/kg. Interestingly, consumption of 10% ethanol accounted for 47.5% of the total ethanol intake (g/kg) during Baseline I, but 62.3% of the total intake under Shock I, 73.2% under Shock II and 61.2% under Baseline II. The shift of preference toward 10% corresponds to the temporal peaks shown in Fig. 5 and may suggest that 10% intake is a more sensitive indicator of the prolonged effects of stress than
Discussion A simple explanation of the data is that some animals in Experiment 1 were capable of learning to drink alcohol as a specific response to short duration random shock stress. In effect, ethanol intake seemed to become a quickly acquired escape response. The escape may be functionally similar to that provided by moving the animals to the home other designs, but this comparison was not directly Following extended stress exposure, the animals
cage in tested. in Ex-
periment 2 continued to increase their quantity of absolute ethanol intake and continued to restrict their consumption primarily to the interval immediately after shock. The increasing dose intake could indicate that the stress became move aversive over time or that the animals showed some low dose tolerance to the initial choice of 5% ethanol. Both possibilities are consistent with the explanation that ethanol selection somehow modified the consequences of shock stress. Several experiments have pointed to a possible relationship between extended shock stress and drinking. An increase in ethanol consumption specific to the onset of inescapable shock was reported by Anisman and Wailer [ 5 I. Their animals were housed in the shock compartment with ethano! and water choices available throughout the daily cycle and animals receiving shock on both alternating six and twelve hr schedules increased their alcohol intake
114 a p p r o x i m a t e l y d o u b l e the c o n t r o l animals receiving n o shock. It is possible t h a t c u m u l a t i v e t e m p o r a l p a t t e r n s o f e t h a n o l c o n s u m p t i o n were o c c u r r i n g as a specific r e s p o n s e to shock. Similarly, H a t t o n and V i e t h [19] r e p o r t pilot data for t h r e e a n i m a l s w h i c h increased t h e i r e t h a n o l i n t a k e following, b u t n o t b e f o r e or d u r i n g s h o c k a v o i d a n c e sessions. These d a t a again suggest t h a t p a t t e r n s of posts h o c k e t h a n o l c o n s u m p t i o n m a y have b e e n emerging b u t t e m p o r a l data were n o t g a t h e r e d . In b o t h E x p e r i m e n t s 1 and 2 we a r b i t r a r i l y used t h e recurring h o u r l y s h o c k s c h e d u l e and s h o c k t e r m i n a t i o n always p r e d i c t e d 48 rain w h i c h were free o f shock, or an interval of relative safety. In t h a t d r i n k i n g was suppressed during the s h o c k schedule, licking in t h e p o s t s h o c k interval c o u l d r e p r e s e n t c o n s u m p t i o n in t h e m o s t stress free interval. With this view s h o c k b e c o m e s a d i s c r i m i n a t i v e s t i m u l u s for t h e o n s e t of t h e stress free interval and serves to signal d r i n k i n g ; a c o n t r a s t to a stress r e d u c t i o n interp r e t a t i o n of the data. A l t h o u g h o u r design did n o t test for this possibility, several factors argue against a p u r e l y n o n a v e r s i v e d i s c r i m i n a t i v e f u n c t i o n for t h e shock. First, some animals s h o w p o s t s h o c k p a t t e r n s a l m o s t i m m e d i a t e l y after s h o c k e x p o s u r e a n d t h e s h o r t e x p o s u r e time involved would n o t seem a d e q u a t e for t h e a n i m a l s to learn a b o u t the t e m p o r a l n a t u r e of t h e s h o c k schedule. In a d d i t i o n , p i l o t data f r o m a s e p a r a t e set of 18 rats w h i c h were given s h o c k only o n c e daily clearly i n d i c a t e d t h a t increasing p o s t s h o c k e t h a n o l ratios were a p p e a r i n g as a f u n c t i o n of s h o c k b u t n o t in t h e c u m u l a t i v e p r o p o r t i o n s e v i d e n c e d w i t h the r e c u r r i n g 24 h r schedule. With t h e s h o c k o c c u r r i n g only once daily, it is unlikely t h a t the animals were learning to use the s h o c k as a d i s c r i m i n a t i v e s t i m u l u s w h i c h p r e d i c t e d periods of relative safety. We do n o t k n o w t h e h o u r l y d i s t r i b u t i o n of t h e b l o o d e t h a n o l c o n c e n t r a t i o n s in the p r e s e n t e x p e r i m e n t s and t h e r e is a possibility t h a t t h e animals were achieving the highest levels of i n t o x i c a t i o n j u s t p r i o r to, or even during, the 12 m i n of shock. T h e p o s t s h o c k d r i n k i n g could, t h e r e f o r e , be c o n c e i v e d as an a v o i d a n c e response in a n t i c i p a t i o n of u p c o m i n g s h o c k in the n e x t h o u r . This seems unlikely because the rats w o u l d have to n o t o n l y learn the t e m p o r a l d i s c r i m i n a t i o n w h i c h e n a b l e d t h e m to p r e d i c t s h o c k , b u t also adjust t h e i r d r i n k i n g p a t t e r n s to a c c o m m o d a t e for the delays in achieving i n t o x i c a t i n g b l o o d e t h a n o l levels w h i c h w o u l d be c o i n c i d e n t w i t h s h o c k onset. One a n i m a l in the s e c o n d s t u d y , h o w e v e r , s h o w e d some m o d e r a t e p e a k i n g o n 10% e t h a n o l j u s t p r i o r to t h e o n s e t o f s h o c k ; an i n d i c a t i o n t h a t learning to use the e t h a n o l in an a v o i d a n c e r a t h e r t h a n an escape f a s h i o n m a y n o t be an i m p o s s i b l e task for the rat. T h e r e is also a possibility t h a t the c o n s u m p t i o n p a t t e r n s observed were g e n e r a t e d by the r e c u r r i n g n a t u r e of t h e s h o c k s c h e d u l e and t h a t any recurring event, stressful or n o n s t r e s s f u l , w o u l d p r o d u c e a similar p a t t e r n of drinking. A l t h o u g h the data do n o t test this, we w o u l d e x p e c t the e t h a n o l p a t t e r n s to persist w i t h saccharin or w a t e r if t h e p h e n o m e n o n were due to s c h e d u l e i n d u c t i o n [14, 15, 17]. T h e p r e s e n t studies have n o t ruled o u t the possibility t h a t the a n i m a l s were c o n s u m i n g g r e a t e r a m o u n t s o f e t h a n o l , a n d lesser a m o u n t s of saccharin, because of t h e calories w h i c h the alcohol p r o v i d e d d u r i n g e x t e n d e d stress. The weight loss o b s e r v e d u n d e r stress c o u l d i n d i c a t e t h a t the a n i m a l s were a t t e m p t i n g caloric r e p l a c e m e n t t h r o u g h
MILLS, B E A N A N D H U T C H E S O N e t h a n o l intake. T h e data f r o m E x p e r i m e n t 1 i n d i c a t e d that the overall daily i n t a k e of e t h a n o l s h o w e d a decrease while the d i s t r i b u t i o n of e t h a n o l c o n s u m p t i o n shifted to the p o s t s h o c k p a t t e r n . The n e t result, relative to baseline, is s h o c k i n d u c e d c o n s u m p t i o n w i t h l o w e r daily caloric i n t a k e from e t h a n o l . However, the animals w h i c h received ext e n d e d stress in E x p e r i m e n t 2 increased t h e i r daily e t h a n o l intake and could have b e e n d r i n k i n g e t h a n o l for its caloric p r o p e r t i e s as a m e a n s to alleviate the stress [6,17] A l c o h o l is high in calories (7 Kcal/g) and increased e t h a n o l i n t a k e is always likely to be c o n f o u n d e d with increased caloric intake. The ideal free choice e x p e r i m e n t a l design w o u l d be to p r o v i d e a n o n i n t o x i c a t i n g choice solution, such as sucrose or d e x t r o s e , as a caloric e q u i v a l e n t to e t h a n o l . However, w h e n o u r rats are given isocaloric s o l u t i o n s of d e x t r o s e or even a m i n i m a l l y sweet c o m m e r c i a l p r o d u c t at a p p r o x i m a t e l y 4 Kcal/g (Polycose, Ross Laboratories) e q u i v a l e n t to 5% e t h a n o l as a free choice, virtually all fluid selection is restricted to the sweet-calorie choice. Several o t h e r i n v e s t i g a t i o n s have a t t e s t e d to the rat's o v e r w h e l m i n g p r e f e r e n c e for sweet-calorie comb i n a t i o n s [7, 16, 22, 30, 3 3 ] . Our a n i m a l s will select sugar solutions p r e d o m i n a n t l y over water, saccharin and e t h a n o l b o t h during baseline and s h o c k p r o c e d u r e s . T h e r e is a need for research w h i c h will offer the a n i m a l an isocaloric and n o n i n t o x i c a t i n g c o n t r o l s o l u t i o n w h i c h w o u l d also p r o d u c e similar baseline selection rates to t h a t observed with e t h a n o l . The e x t r e m e sugar p r e f e r e n c e s p r e v e n t m e a n i n g f u l c o m p a r i s o n s to be m a d e b e t w e e n the separate caloric and i n t o x i c a t i n g c o m p o n e n t s of fluid selection as t h e y f u n c t i o n relative to stress. One p e r s i s t e n t f e a t u r e of the data is the relatively rapid a p p e a r a n c e of the p o s t s h o c k d r i n k i n g in r e s p o n s e to shock. A p p a r e n t l y an a n i m a l will try a n u m b e r o f responses w h e n faced with an inescapable stressor [3, 4, 3 2 ] , and one h y p o t h e s i s c o n c e r n i n g the existing data would be t h a t p o s t s h o c k e t h a n o l and saccharin c o m s u m p t i o n are b o t h subsets of p o s t s h o c k novel fluid intake. In the p r e s e n t study, the distinctive taste p r o p e r t i e s of the fluid m a y be the m o r e i m p o r t a n t variable w h e n a t t e m p t i n g to explain the origin of the a n i m a l ' s response. The data i n d i c a t e t h a t the animals progressively shifted from saccharin to 5 ~ e t h a n o l to 10% e t h a n o l with increasing e x p o s u r e to shock. This w o u l d argue t o w a r d a t w o c o m p o n e n t process based on taste and the s e c o n d a r y or m o r e delayed p r o p e r t i e s of fluid i n t a k e to respectively i n t i t i a t e and m a i n t a i n e t h a n o l i n t a k e in r e s p o n s e to stress. In c o n c l u s i o n , s h o c k i n d u c e d e t h a n o l c o n s u m p t i o n is a c u m u l a t i v e effect and the data d e m o n s t r a t e t h a t the r e l a t i o n s h i p b e t w e e n stress and d r i n k i n g is a m e n a b l e to t e m p o r a l m e a s u r e m e n t . The relation a p p a r e n t l y d e p e n d s to a large e x t e n t on the c o n t i n u o u s n a t u r e of the i n e s c a p a b l e stress and the cont i n u o u s l y available alcohol supply a p p e a r i n g t o g e t h e r . ACKNOWLEDGEMENTS The authors wish to express appreciation to John A. Ewing for his helpful suggestions in all phases of the research and Elaine Woody, Lois Boone, Richard Lutz and the Claude Gaebelein for their technical assistance. This investigation was supported by research grants 7405 from the North Carolina Alcoholism Research Authority and MH-21667 from the National Institute on Alcohol Abuse and Alcoholism.
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REFERENCES 1. Allison, J. Microbehavioral features of nutritive and nonnutritive drinking in rats. J. cornp, physiol. PsychoL 76: 4 0 8 - 4 1 7 , 1971. 2. Allison, J. and N. J. Castellan. Temporal characteristics of nutritive drinking in rats and humans. J. comp. physiol. Psychol. 70: 1 1 6 - 1 2 5 , 1970. 3. Anisman, H. Time dependent variations in aversively motivated behaviors: Nonassociative effects of cholinergic and catecholaminergic activity. Psychol. Rev. 82: 3 5 9 - 3 8 5 , 1975. 4. Anisman, H. and T. G. Waller. Effects of inescapable shock on subsequent avoidance performance: Role of response repertoire changes. Behav. Biol. 9: 3 3 1 - 3 3 5 , 1973. 5. Anisman, H. and T. G. Waller. Effects of inescapable shock and shock produced conflict on self selection of alcohol in rats. Pharrnac. Biochem. Behav. 2: 2 7 - 3 3 , 1974. 6. Antelman, S. M. and H. Szechtman. Tail pinch induces eating in sated rats which appears to depend on nigrostriatal dopamine. Science 189: 7 3 1 - 7 3 3 , 1975. 7. Booth, D. A., D. Lovett and G. M. McSherry. Postingestive modulation of the sweetness preference gradient in the rat. J. comp. physiol. Psychol. 78: 4 8 5 - 5 1 2 , 1972. 8. Cappell, H. and P. Herman. Alcohol and tension reduction: a review. Q. J. Stud. Alcohol 33: 3 3 - 6 4 , 1972. 9. Carey, R. J. Brief communication: a decrease in ethanol preference in rats resulting from forced ethanol drinking under fluid deprivation. Physiol. Behav. 8: 3 7 3 - 3 7 5 , 1972. 10. Casey, A. The effect of stress on the consumption of alcohol and reserpine. Q. Z Stud. Alcohol 21: 3 0 8 - 3 1 6 , 1960. 11. Cicero, T. and S. Hill. Ethanol self-selection in rats: a distinction between absolute and 95% ethanol. Physiol. Behav. 5: 7 8 7 - 7 9 1 , 1970. 12. Cicero, T., R. D. Myers and W. Black. Increase in volitional ethanol consumption following interference with a learned avoidance response. Physiol. Behav. 3: 6 5 7 - 6 6 0 , 1968. 13. Eriksson, K. Alcohol consumption and blood alcohol in rat strains selected for their behavior towards alcohol. Finnish Foundation Alcohol Stud. 20: 1 2 1 - 1 2 5 , 1972. 14. Falk, J. L. Production of polydipsia in normal rats by an intermittent food schedule. Science 133: 1 9 5 - 1 9 6 , 1961. 15. Falk, J. L. The motivational properties of schedule-induced polydipsia. J. exp. Analysis Behav. 9: 1 9 - 2 6 , 1966. 16. Falk, J. L. and H. H. Samson. Schedule-induced physical dependence on ethanol. Pharrnac. Rev. 27: 4 4 9 - 4 6 4 , 1976. 17. Freed, E. X. and D. Lester. Schedule induced consumption of ethanol: calories or chemotherapy? Physiol. Behav. 5: 555 560, 1970. 18. Freund, G. Alcohol consumption and its circadian distribution in mice. J. Nut. 100: 3 0 - 3 6 , 1970. 19. Hatton, G. I. and A. Vieth. Stress related and diurnal alcohol drinking in rats. Bull. Psychon. Soe. 4 : 1 9 5 196, 1974.
20.
21.
22.
23.
24. 25. 26.
27.
28.
29. 30.
31.
32. 33.
34. 35.
36. 37.
Hulse, S. Licking behavior of rats in relation to saccharin concentration and shifts in fixed ratio reinforcement. J. cornp. physiol. Psychol. 64: 4 7 8 - 4 8 4 , 1967. Hutcheson, J. and K. C. Mills. A compact and inexpensive drinkometer for use with small animals. Physiol. Behav. 13: 1 7 9 - 1 8 1 , 1974. Jacobs, H. L. Studies on sugar preference: 1) The preference for glucose solutions and its modification by injections of insulin. J. comp. physiol. Psychol. 51: 3 0 4 - 3 1 0 , 1958. Lester, D. Self selection of alcohol by animals, human variation and the etiology of alcoholism. Q. J. Stud. Alcohol 27: 3 9 5 - 4 3 8 , 1966. Lester, D. and E. Freed. Criteria for an animal model of alcoholism. Pharrnae. Biochem. Behav. 1: 1 0 3 - 1 0 7 , 1973. Myers, R. D. and R. Carey. Preference factors in experimental alcoholism. Science 134: 4 6 9 - 4 7 0 , 1961. Myers, R. D., R. Holman. Failure of stress of electric shock to increase ethanol in rats. Q. J. Stud. Alcohol 28: 132-137, 1967. Opsahl, C. and C. Hatton. Volitional ethanol increases during acquisition and extinction of avoidance responding. Physiol. Behav. 8: 8 7 - 9 3 , 1972. Parker, L. F. and B. L. Radow. Isolation stress and volitional ethanol consumption in the rat. Physiol. Behav. 12: 1 - 3 , 1974. Quinsey, V. Lick-shock contingencies in the rat. J. exp. Analysis Behav. 17: 119-125. 1972. Samson, H. H. and J. L. Falk. Alteration of fluid preference in ethanol-dependent animals. J. Pharmac. exp. Ther. 190: 3 6 5 - 3 7 6 , 1974. Stellar, E. and J. Hill. The rat's rate of drinking as a function of water deprivation. J. comp. physiol. Psychol. 45: 9 6 - 1 0 2 , 1952. Ulrich, R. E. and N. H. Azrin. Elimination of undesired escape from footshock. J. exp. Analysis Behav. 5: 72, 1962. Valenstein, E. S. Selection of nutritive and nonnutritive solutions under different conditions of need. Z comp. physiol. Psychol. 6 3 : 4 2 9 433, 1967. Veale, W. L. Ethanol selection in the rat following forced acclimation. Pharmac. Biochern. Behav. 1:233 235, 1973. von Wright, J., L. Pekanmaki and S. Malin. Effects of conflict and stress on alcohol intake in rats. Q. J. Stud. Alcohol 32: 4 2 0 - 4 3 3 , 1971. Winer, B. J. Statistical Principles in Experimental Design. McGraw-Hill Book Co., New York, 1962. Wise, R. A. Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia 29: 2 0 3 - 2 1 0 , 1973.
Shock Induced Ethanol Consumption in Rats K E N N E T H C. M I L L S 1 , J. W I L L I A M B E A N 1 A N D J. S T A N F O R D H U T C H E S O N 2
Center f o r Alcohol Studies 1 and Department o f Psychiatry 2 University o f North Carolina, Division o f Health Sciences Chapel Hill, North Carolina 27514 ( R e c e i v e d 29 S e p t e m b e r 1 9 7 5 )
MILLS, K. C., J. W. BEAN AND J. S. HUTCHESON. Shock induced ethanol consumption in rats. PHARMAC. BIOCHEM. BEHAV. 6(1) 107-115, 1 9 7 7 . - Two experiments are presented which describe the temporal and volumetric changes in ethanol consumption by rats exposed to recurring schedules of inescapable random shock. The animals in Experiment 1, which had a choice between ethanol and water, increased their voluntary ethanol consumption immediately after the shock schedule. The postshock changes occurred with both 5% and 10% V/V ethanol, were specific to the presence of shock and were not reflected by measures of total daily ethanol intake. Experiment 2 exposed rats to extended 2 2 h r stress sessions, during which each animal had four simultaneous fluid choices available: water, saccharin 0.1% W/V, ethanol 5% V/V, and ethanol 10% V/V. Temporal intake patterns for both 5% and 10% ethanol showed pronounced peaks for the interval immediately following the shock schedule. A shift of intake from 5% to 10% ethanol was also demonstrated with increasing time under shock, while saccharin and water intake decreased. The results are interpreted as a relationship between voluntary ethanol intake and escape from the consequences of stress. Stress and drinking Voluntary ethanol consumption Temporal patterns of fluid intake Escape drinking
Animal models of alcoholism Shock induced ethanol consumption
THIS p a p e r describes a t e m p o r a l r e l a t i o n s h i p b e t w e e n stress and free-choice alcohol c o n s u m p t i o n in rats. T h e focus is u p o n the s h o r t t e r m changes in fluid i n t a k e w h i c h o c c u r as a f u n c t i o n of p e r i o d i c s h o c k stress. Previous e x p e r i m e n t s have a t t e m p t e d to isolate t h e variables involved w i t h stress and d r i n k i n g and the designs have usually c e n t e r e d o n the type or a m o u n t of stressor e f f e c t i n g a c h a n g e in m e a s u r e s of daily h o m e cage e t h a n o l c o n s u m p t i o n [4, 10, 12, 26, 27, 3 5 ] . The range of stress m a n i p u l a t i o n s has b e e n extensive, and this l i t e r a t u r e has received review in several r e p o r t s [8, 23,241. We assumed t h a t m e a s u r e s of daily e t h a n o l i n t a k e m i g h t n o t reflect changes of s h o r t e r d u r a t i o n w h i c h could be occurring as the result of stress. R a t h e r t h a n e x p o s e t h e animals to stress in o n e e n v i r o n m e n t a n d m e a s u r e daily c o n s u m p t i o n in t h e h o m e cage, o u r animals were h o u s e d in the test e n v i r o n m e n t 24 hr daily. This a r r a n g e m e n t allowed us to q u a n t i f y fluid selection over smaller units of t i m e b y m e a s u r i n g licking w i t h d r i n k o m e t e r s as described b y a n u m b e r of investigators [1, 2, 20, 29, 3 1 ] . F r e u n d [18] and Eriksson [13] have also used d r i n k o m e t e r records t o describe the diurnal d i s t r i b u t i o n of e t h a n o l i n t a k e in rats and mice, and t h e c o n t i n u o u s m o n i t o r i n g p r o c e d u r e s provide an ideal m e t h o d to m e a s u r e the t e m p o r a l comp o n e n t s of e t h a n o l i n t a k e u n d e r stress. E x p e r i m e n t 1 describes e t h a n o l i n t a k e relative to recurring stress for rats h o u s e d c o n t i n u o u s l y in t h e test e n v i r o n m e n t for 50 days w i t h a t w o fluid choice of e t h a n o l
and water. E x p o s u r e to d i f f e r e n t c o n c e n t r a t i o n s of e t h a n o l was sequential. E x p e r i m e n t 2 describes the t e m p o r a l p a t t e r n s of fluid i n t a k e for a g r o u p o f animals given s i m u l t a n e o u s m u l t i p l e fluid choices a n d e t h a n o l conc e n t r a t i o n s d u r i n g e x t e n d e d stress and n o n stress exposure. EXPERIMENT 1
Method Animals. T h e animals were 12 male Long-Evans h o o d e d rats f r o m Blue Spruce Farms, A l t a m o n t , New York. T h e y . h a d a m e a n weight of 533.8 g w i t h a range f r o m 4 5 5 - 6 4 4 g at t h e start of t h e Basline I. Apparatus. The animals were individually h o u s e d in s o u n d a t t e n u a t e d c h a m b e r s w h i c h also allowed c o n t r o l o f diurnal light cycles (12 hr light, 12 hr dark). E a c h cage had a grid floor, a lever and t w o d r i n k o m e t e r s , spaced 5 cm apart. The living space m e a s u r e d 30 × 24 x 26 cm high. The d r i n k o m e t e r s , described in detail elsewhere, used a R i c h t e r d r i n k i n g s p o u t a n d were designed a r o u n d an infrared light e m i t t i n g diode so t h a t t h e possibility of t o n g u e s h o c k and visual cue p r o b l e m s in c o n t a c t a n d p h o t o c e l l designs could be e l i m i n a t e d [ 2 1 ] . F o o t s h o c k was p r o v i d e d by c o n s t a n t c u r r e n t s c r a m b l e d s h o c k sources and was set at 1.5 ma f o r all s h o c k procedures. T e m p o r a l schedules were c o n t r o l l e d w i t h solid state logic. Procedure. A l c o h o l s o l u t i o n s were p r e p a r e d f r o m 95% 107
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e t h a n o l in distilled water [ 11 }. The animals were h o u s e d in the closed e n v i r o n m e n t s 24 hr daily, w i t h one 20 min i n t e r r u p t i o n for weighing and r e c o r d i n g of daily fluid intake. F o o d was p r o v i d e d ad lib in h o p p e r s s u s p e n d e d from the wall of the cage. Baselines were defined as intervals in w h i c h licking d i s t r i b u t i o n s over t i m e were s a m p l e d for e t h a n o l and water. The p o s i t i o n of each fluid c h a n g e d every 48 hr. E s t i m a t e s of fluid i n t a k e for a c u m u l a t i v e t i m e interval were derived from the lick c o u n t for t h a t interval relative to the t o t a l c u m u l a t i v e n u m b e r of licks in a 24 h r session• T h e r a t i o was then m u l t i p l i e d by t h e t o t a l q u a n t i t y of fluid delivered to provide an e s t i m a t e o f the fluid c o n s u m e d in the interval [see 211. Licking data for E x p e r i m e n t 1 were o b t a i n e d b y a c c u m u l a t i n g lick c o u n t s f r o m five s e p a r a t e 12 m i n s e g m e n t s of each h o u r over 24 hr. A f t e r baseline, the same 12 m i n s e g m e n t in e a c h h r was d e s i g n a t e d as the s h o c k interval a n d the a s s i g n m e n t of pre- a n d p o s t s h o c k intervals was arbitrary. Using a digital p r i n t e r , lick c o u n t s o n b o t h e t h a n o l a n d w a t e r were also sampled to a 3 m i n r e s o l u t i o n d u r i n g selected sessions f r o m individual animals. S h o c k sessions were d e f i n e d b y delivering r a n d o m l y spaced 1 sec f o o t s h o c k s for the same 12 rain o f each hr a r o u n d the clock. T h e s c h e d u l e d u r i n g the 12 rain was VI 1 min, so t h a t each a n i m a l received o n t h e average 12 i n e s c a p a b l e s h o c k s p e r hr, or 288 s h o c k s p e r 24 h r session. W h e n s h o c k occurs for 12 rain of each hr, a r a n d o m l y d i s t r i b u t e d d r i n k i n g p a t t e r n w o u l d s h o w 40% of d r i n k i n g during the p r e s h o c k interval (24 min), 20% during the shock interval and 40% d u r i n g the p o s t s h o c k interval (24). A post interval ratio o f 0 . 4 0 would reflect this p a t t e r n and is typically very close to t h a t observed u n d e r baseline c o n d i t i o n s . Changes in the post interval r a t i o are used as one m e a s u r e of the effects of shock. However, a large change in a r a t i o m a y m e a n little on a daily basis if the a m o u n t of fluid c o n s u m e d is n o t t a k e n i n t o a c c o u n t . A shift of a p o s t interval ratio f r o m 0 . 4 0 - 0 . 8 0 could be applied to a daily decrease in fluid c o n s u m p t i o n f r o m 2 0 - 1 0 ml resulting in n o change in post interval fluid intake. D r i n k o m e t e r e s t i m a t e s of p o s t i n t e r v a l v o l u m e t r i c i n t a k e are also t a k e n as an i n d e x o f t h e effects of shock. F o l l o w i n g a 20 session (24 hr) baseline (Baseline 1), all animals were e x p o s e d to the 12 m i n recurring h o u r l y r a n d o m s h o c k s c h e d u l e for five daily sessions ( 2 1 - - 2 5 Shock I) and a five session after s h o c k baseline (sessions 2 6 - 3 0 ) . The e n t i r e cycle was r e p e a t e d w i t h a d i f f e r e n t c o n c e n t r a t i o n of e t h a n o l as a fluid choice for a 10 day baseline (sessions 3 1 - 4 0 Baseline II), 5 days o f r a n d o m s h o c k (sessions 4 1 - 4 5 S h o c k II), and 5 days of a f t e r s h o c k baseline (sessions 4 6 - 5 0 ) . Half of t h e a n i m a l s (N = 6) received a 5% V / V e t h a n o l choice and w a t e r for t h e first b a s e l i n e - s h o c k cycle and a 10% V / V e t h a n o l choice for the second cycle. T h e o t h e r half received the 10% V / V e t h a n o l as t h e i r initial c h o i c e and 5% V / V w i t h t h e second cycle. In essence, two i n d e p e n d e n t g r o u p s of six rats received a l t e r n a t e orders of e x p o s u r e to 5 and 10% e t h a n o l a n d f o u r t r e a t m e n t c o m b i n a t i o n s o c c u r r e d u n d e r each BaselineShock-Baseline cycle: S h o c k I-5%, S h o c k II-10%, S h o c k 1-10%, and S h o c k II-5%. T h e n u m b e r o f animals in G r o u p I1-5% was five due to e q u i p m e n t failure. Results Figure 1 displays t h e overall results o f r e p e a t e d baseline
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and s h o c k e x p o s u r e s w i t h 12 animals. T h e data are p l o t t e d separately for each g r o u p r e p r e s e n t i n g a t r e a t m e n t comb i n a t i o n u n d e r each of the t w o criteria for evaluating fluid intake. T h e t o t a l interval c r i t e r i o n is the c u m u l a t i v e daily intake of e t h a n o l and w a t e r in the 24 hr session. T h e post interval criterion is the c u m u l a t i v e daily i n t a k e of e t h a n o l and w a t e r for the 24 m i n following s h o c k and the same 24 rain interval during p r e s h o c k and a f t e r s h o c k baseline. To reiterate, s h o c k schedules are n o t in effect d u r i n g baselines and the selection of a similar 24 m i n s e g m e n t is a r b i t r a r y u n d e r n o n s h o c k c o n d i t i o n s . It should be n o t e d t h a t the b o d y weights for t h e animals d r o p p e d an average of 5.5% f r o m m e a n baseline weights with the i n t r o d u c t i o n of shock sessions, a n d fluid i n t a k e s are expressed relative to the observed weights for each p r o c e d u r e . It is a p p a r e n t from
SHOCK A N D E T H A N O L C O N S U M P T I O N Fig. 1 t h a t m o d u l a t i o n of e t h a n o l i n t a k e in t e r m s of t o t a l daily fluid i n t a k e was typical of m a n y studies l o o k i n g at the effects of s h o c k on drinking. With t h e e x c e p t i o n of t h e 5% animals o n t h e i r first s h o c k e x p o s u r e , all groups s e e m e d to show a decrease in t h e i r e t h a n o l i n t a k e following shock. The post interval criterion, h o w e v e r , i n d i c a t e d t h a t t h e intake of e t h a n o l increased for all f o u r g r o u p s d u r i n g the s h o c k s c h e d u l e e x p o s u r e . M o d u l a t i o n of w a t e r i n t a k e as reflected by b o t h criteria was n o t a p p a r e n t . In o r d e r to evaluate t h e effects of s h o c k o n e t h a n o l c o n s u m p t i o n s e p a r a t e b u t identical analyses of variance were carried o u t for b o t h t o t a l interval and post interval scores. Water scores were n o t i n c l u d e d in t h e analyses because o f t h e d i s p a r a t e i n t a k e v o l u m e s i n d i c a t e d in Fig. 1. Each analysis was a t w o b y two by t w o factorial design as described by Winer [ 3 6 ] . T h e t w o levels of F a c t o r A were O r d e r l ( 5 % - 1 0 % ) a n d O r d e r II ( 1 0 % - 5 % ) . This was the f a c t o r on w h i c h r e p e a t e d m e a s u r e s were n o t taken. Rep e a t e d m e a s u r e s were t a k e n o n t h e t w o levels of F a c t o r B w h i c h were 5% a n d 10% e t h a n o l i n t a k e a n d t h e two levels of F a c t o r C w h i c h were defined by baseline and s h o c k procedures. T h e c o n s u m p t i o n scores for each a f t e r s h o c k baseline are s h o w n in Fig. 1, b u t were n o t i n c l u d e d in the analyses. T h e analysis of variance of t h e t o t a l interval scores s h o w e d n o significant m a i n effects for e i t h e r the baseline to s h o c k m e a n d i f f e r e n c e s or t h e o r d e r of e x p o s u r e to 5 and 10% e t h a n o l . A significant m a i n effect was d e t e c t e d for t h e difference b e t w e e n 5 and 10% i n t a k e , F ( 1 , 1 0 ) = 2 8 . 6 6 , p < 0 . 0 5 . A significant i n t e r a c t i o n was also o b s e r v e d bet w e e n the c o n c e n t r a t i o n f a c t o r and o r d e r of e x p o s u r e , F ( 1 , 1 0 ) = 10.07, p < 0 . 0 5 . T h e analysis of t h e p o s t interval scores, h o w e v e r , yielded significant m a i n effects for b o t h baseline to s h o c k differences, F ( 1 , 1 0 ) = 16.24, p < 0 . 0 5 , a n d 5 and 10% e t h a n o l i n t a k e scores, F ( 1 , 1 0 ) = 18.80, p < 0 . 0 5 . Again, significant main effects for t h e o r d e r of e x p o s u r e f a c t o r were n o t evident. All i n t e r a c t i o n s for the p o s t i n t e r v a l analysis were nonsignificant. T h e statistical analyses implied t h a t e t h a n o l i n t a k e i m m e d i a t e l y a f t e r the s h o c k was a m o r e sensitive i n d e x of c o n s u m p t i o n t h a n the a m o u n t of e t h a n o l selected d u r i n g the t o t a l interval w h i c h is o f t e n expressed as daily e t h a n o l intake. U p o n i n s p e c t i o n of t h e data it was e v i d e n t t h a t t h e last t w o s h o c k sessions c o n t a i n e d p r o p o r t i o n a t e l y m o r e p o s t i n t e r v a l increases in e t h a n o l i n t a k e t h a n the first t w o shock sessions, suggesting a gradually emerging effect of shock o n drinking. Figure 2 s h o w s the p o s t i n t e r v a l r a t i o changing as a f u n c t i o n of s h o c k e x p o s u r e over all animals at b o t h c o n c e n t r a t i o n s for the first a n d s e c o n d s h o c k cycles. The increasing p e a k w i t h increasing t i m e in s h o c k a n d t h e a b r u p t r e t u r n to baseline levels i n d i c a t e t h a t e t h a n o l i n t a k e was specific to the s h o c k schedule. T h e p o s t i n t e r v a l r a t i o as o p p o s e d to p o s t i n t e r v a l i n t a k e (ml × ratio) does n o t take i n t o a c c o u n t t h e a b s o l u t e a m o u n t of fluid c o n s u m e d , and t h e m e a s u r e does n o t d i f f e r e n t i a t e t h o s e animals w h i c h s h o w e d a b s o l u t e increases in e t h a n o l i n t a k e w i t h s h o c k f r o m t h o s e w h i c h did n o t . T h e c o n s i s t e n c y of the data across b o t h t r e a t m e n t s and animals implies t h a t t h e p o s t s h o c k r a t i o is sensitive to s h o c k effects regardless of w h e t h e r or n o t this leads to increases in p o s t s h o c k e t h a n o l i n t a k e . Because of t h e high i n c i d e n c e of s h o c k sessions in w h i c h w a t e r i n t a k e was zero, c o m p a r a b l e p o s t i n t e r v a l data for w a t e r i n t a k e were n o t reliable. To illustrate an individual t e m p o r a l p a t t e r n of s h o c k
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FIG. 2. Postinterval ethanol licking as a proportion of total ethanol licking for shock and nonshock conditions on consecutive exposures. Each vertical hash mark represents the standard error of the mean. i n d u c e d c o n s u m p t i o n , Fig. 3 p r e s e n t s the records of the animal s h o w i n g t h e largest increases in e t h a n o l i n t a k e following shock. Because of e q u i p m e n t restrictions, i n t a k e records resolved to 3 rain r e s o l u t i o n a r o u n d t h e clock were n o t available for all sessions. A n i m a l 8 s h o w e d b o t h overall and p o s t i n t e r v a l increases in e t h a n o l i n t a k e at b o t h c o n c e n t r a t i o n s . This is illustrated by i n t a k e p e a k s for e t h a n o l i m m e d i a t e l y following s h o c k and b y t o t a l increases in e t h a n o l i n t a k e over baseline of 62.5 m l / k g / 2 4 hr for S h o c k I and 14 m l / k g / 2 4 h r for S h o c k II. P o s t i n t e r v a l increases reflected the peak i n t a k e s m o r e closely a n d were respectively 60 m l / k g / 2 4 hr for S h o c k 1 and 48.4 m l / k g / 2 4 hr for S h o c k II. T o t a l interval w a t e r intake for b o t h s h o c k e x p o s u r e s was 7.2 m l / k g / 2 4 hr for S h o c k I and 0.7 m l / k g / 2 4 hr for S h o c k II.
Discussion The animals in E x p e r i m e n t 1 displayed t e m p o r a l shifts in e t h a n o l i n t a k e relative to recurring i n e s c a p a b l e shock. Specifically, the d i s t r i b u t i o n of licking reflected fluid intake increases specific to e t h a n o l in t h e interval imm e d i a t e l y following the shock. T h e data d e m o n s t r a t e d t h a t the daily i n t a k e c r i t e r i o n was i n a d e q u a t e to describe the changes in e t h a n o l c o n s u m p t i o n , and t h a t w i t h some animals the p o s t s h o c k increases in e t h a n o l i n t a k e were quite d r a m a t i c . T h e central issue, h o w e v e r , is w h e t h e r or n o t t h e animals were using the i n t o x i c a t i n g p r o p e r t i e s of the e t h a n o l to s o m e h o w alter the c o n s e q u e n c e s of shock. T h e c o n t i n u o u s isolation p r o c e d u r e s m a k e it difficult to e x t r a c t b l o o d e t h a n o l samples w i t h o u t d i s t u r b i n g the animals t h e r e f o r e b l o o d samples were n o t taken. Nevertheless, it w o u l d seem necessary to display circulating b l o o d e t h a n o l levels w h i c h are in excess of t h e rat's m e t a b o l i c c a p a c i t y in o r d e r to
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m i g h t be due to s o m e p r o p e r t y of the e t h a n o l o t h e r t h a n the i n t o x i c a t i n g effect, such as a simple taste p r e f e r e n c e , w h i c h is amplified b y the s h o c k schedule. If t h e animal is sampling a n y t h i n g novel as an a t t e m p t to alter t h e shock, t h e n m o m e n t a r y i n t o x i c a t i o n f r o m e t h a n o l m i g h t be a b y - p r o d u c t of d r i n k i n g i n d u c e d b y the n o v e l taste of e t h a n o l relative to water. The animals in E x p e r i m e n t 1 also show an unusally high p r e f e r e n c e for e t h a n o l in t h e test cages as reflected by Fig. 1. These animals were n o t given h o m e cage p r e f e r e n c e tests so t h a t the d i s t r i b u t i o n o b t a i n e d d u r i n g t h e test cage baseline w o u l d n o t be c o n f o u n d e d w i t h e x p o s u r e and a c c l i m a t i o n effects [9, 25, 34, 3 7 ] . The test c h a m b e r s were isolated in c o m p a r i s o n with h o m e cages and this r e s t r i c t i o n m i g h t also have b e e n s t r o n g e n o u g h to i n d u c e a high e t h a n o l p r e f e r e n c e over w a t e r [ 2 8 ] . T a k e n t o g e t h e r the taste, p r e f e r e n c e a n d isolation factors could be o p e r a t i n g to p r o d u c e the exaggerated e t h a n o l c o n s u m p t i o n p a t t e r n s a t t r i b u t e d to the s h o c k stress and r e p l i c a t i o n with add i t i o n a l c o n t r o l s was w a r r a n t e d .
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FIG. 3. Cumulative ethanol and water intake for subject 8 summarized over grouped and single sample sessions (24 hr) of baseline and shock exposure at 5 and 10% ethanol-water choice. The value in parentheses adjacent to the procedure represents the number of 24 hr sessions sampled to a 3 min resolution. state t h a t the animals are using the i n t o x i c a t i n g p r o p e r t i e s of e t h a n o l to achieve s o m e m e a s u r e of stress relief. Collateral studies in o u r lab o n rats e x p o s e d to the same s h o c k p a r a d i g m have i n d i c a t e d t h a t w h e n b l o o d samples are t a k e n at r a n d o m t i m e s d u r i n g the 24 h r session, circulating b l o o d e t h a n o l levels are o f t e n in excess of 150 r a g / 1 0 0 ml. Baseline b l o o d e t h a n o l levels average a r o u n d 10 m g / 1 0 0 ml w i t h a range f r o m 1 - 2 5 m g / 1 0 0 ml and s h o c k b l o o d levels average 35 m g / 1 0 0 m l ranging f r o m 5 - 1 6 0 m g / 1 0 0 ml. A l t h o u g h the increase f r o m baseline to s h o c k is statistically significant, it is difficult to i n t e r p r e t the d a t a in t h a t t h e m e t a b o l i c rate is c o n f o u n d e d w i t h t h e a n i m a l having c o n t i n u o u s access to t h e e t h a n o l and a t e m p o r a r y e l e v a t i o n of b l o o d e t h a n o l w o u l d n o t necessarily be evidence for c o n t i n u o u s i n t o x i c a t i o n . One p r o b l e m m a y be t h a t in o r d e r to c o n s i s t e n t l y p r o d u c e a d i s t r i b u t i o n of b l o o d levels above 50 m g / 1 0 0 ml, w h i c h w o u l d i n d i c a t e sustained i n t o x i c a t i o n during a 24 h r session, the a n i m a l w o u l d have to c o n s u m e at least 8.4 g/kg of e t h a n o l / d a y . This w o u l d be a p p r o x i m a t e l y 212 m l / k g of 5% e t h a n o l or 106 m l / k g of 10%. If we assume t h a t t h e a n i m a l is a c c u m u l a t i n g some b l o o d e t h a n o l f r o m h o u r to h o u r the i n t a k e r e q u i r e m e n t m i g h t be r e d u c e d s o m e w h a t to m a i n t a i n the b l o o d level at or above 50 m g / 1 0 0 ml, b u t the r e q u i r e m e n t is high in t h a t t h e t o t a l daily fluid i n t a k e for o u r animals in the test e n v i r o n m e n t is typically a r o u n d 80 m l / k g over all p r o c e d u r e s . With this i n f o r m a t i o n in m i n d , it can be argued t h a t the t e m p o r a l c o r r e l a t i o n of s h o c k and increased e t h a n o l i n t a k e
2
E x p e r i m e n t 2 was an a t t e m p t to f u r t h e r define the f u n c t i o n a l r e l a t i o n s h i p b e t w e e n stress and d r i n k i n g by describing the t e m p o r a l p r o p e r t i e s of fluid i n t a k e in rats. The animals were offered four fluid choices of water, s o d i u m saccharin, e t h a n o l at 5% V / V ( p r e f e r r e d ) and 10% V / V ( n o n p r e f e r r e d ) to c o n t r o l for taste and p r e f e r e n c e variables. S i m u l t a n e o u s r a t h e r t h a n s e q u e n t i a l e x p o s u r e to m u l t i p l e c o n c e n t r a t i o n s of e t h a n o l allowed e x a m i n a t i o n o f the shifting p a t t e r n s of e t h a n o l i n t a k e in response to stress. In a d d i t i o n , h o m e cage p r e f e r e n c e s were t a k e n p r i o r to test cage e x p o s u r e to d e t e r m i n e the e x t e n t to w h i c h test cage isolation altered fluid intake. E x p e r i m e n t i limited the animals to five days of stress e x p o s u r e at each e t h a n o l c o n c e n t r a t i o n , and a longer s h o c k e x p o s u r e was used in the s e c o n d study. An e x t e n d e d s h o c k e x p o s u r e p e r m i t t e d us to e x a m i n e w h e t h e r the animals a d a p t e d to the effects of s h o c k and increased t h e i r selection of n o n i n t o x i c a t i n g fluids or w h e t h e r persisting s h o c k w o u l d c o n t i n u e to i n f l u e n c e e t h a n o l i n t a k e as suggested by E x p e r i m e n t 1.
Me tho d Animals. T h e animals for E x p e r i m e n t 2 were 12 male Long-Evans h o o d e d rats f r o m Blue Spruce Farms, A l t a m o n t , New York. The m e a n weight for all animals d u r i n g the first test cage baseline was 522 g w i t h a range from 4 5 4 - 5 8 9 g. The final m e a n weight for the animals during the last 10 sessions of the s t u d y was 519 g with a range of 426 6 1 4 g. One animal s h o w e d several indices of p o o r h e a l t h d u r i n g the initial phases of the s t u d y and was r e m o v e d from the test e n v i r o n m e n t w i t h t h e o n s e t of s h o c k stress. His data are n o t i n c l u d e d in the g r o u p data for test cage preferences. Apparatus. The a n i m a l s ' e n v i r o n m e n t was similar to the system used in E x p e r i m e n t 1, b u t the test cages and data facilities were redesigned to a c c o m m o d a t e i n p u t s from four s i m u l t a n e o u s fluid choices w i t h a 2-rain resolution. The animals were h o u s e d in similar s o u n d a t t e n u a t e d c h a m b e r s , b u t each cage (28 x 21 x 21 cm) h a d four b o t t l e s m o u n t e d b e h i n d o n e wall of the cage. Each b o t t l e was calibrated to 2 ml and was m o u n t e d b e h i n d an 8 m m d i a m e t e r access hole. The cages h a d grid floors, an i n o p e r a t i v e lever and i n t e r n a l
SHOCK AND E T H A N O L C O N S U M P T I O N house lights. The diurnal cycle was 11 hr light and 11 hr dark with 2 hr daily for a service break. F o o t s h o c k was provided by constant current shock sources, and the shock level was set at 1.5 ma for all procedures. The d r i n k o m e t e r s in each cage consisted of four optical couplers with an infrared light emitting diode ( L E D ) and photo-transistor. The infrared beam of light passed directly across the access hole to the drinking tube. The data collection system consisted of an 8K, PDP-8F (Digital E q u i p m e n t C o r p o r a t i o n ) c o m p u t e r . The software was written in PAL Ill and was designed to process and count licks occurring at each of the 48 individual inputs over a specified block of time (2 min). Following each time block, the software switched to another bank of 48 counters for the duration of the next block. The system continued this o p e r a t i o n for the total n u m b e r of blocks specified, or 30 for the 1 hr timebase of E x p e r i m e n t 2. After t w e n t y - t w o 1 hr cycles the cumulative data was outputed. Procedure. H o m e cage fluid preferences were d e t e r m i n e d for the 12 rats during a 24 day pretest exposure. The animals were housed in wire mesh h o m e cages with ad lib rat c h o w and two fluid choices c o n t i n u o u s l y available t h r o u g h o u t the day. Each cage had one drinking bottle for water and one bottle for a choice sloution of ethanol at 5% V/V, 7.5% V/V, 10%V/V 12.5% V / V or sodium saccharin at 0.1% W/V. The animals were given eight days of ethanol exposure or two days at each choice. As part of another study, the animals were also given sucrose solutions and water for an additional eight days. The order of presentation of the four ethanol c o n c e n t r a t i o n s was r a n d o m across animals. Bottle positions were reversed daily and solution concentrations were changed daily. The animals were weighed every o t h e r day. After a two week break w i t h o u t alcohol in the h o m e cage, the subjects were transferred to the test environments. They were housed continuously in the test environments for 55 consecutive 22 hr sessions. Baseline and shock session schedules were defined as in E x p e r i m e n t 1 with the e x c e p t i o n that the session length was shortened to 22 hr to allow for cage servicing and data collection. F o u r drinking solutions were always available in the test environment: water, 0.1% W/V sodium saccharin, 5% V / V ethanol, and 10% V / V ethanol. Ethanol solutions were prepared from 95% ethanol and distilled water. Saccharin solutions were prepared from c o m m e r c i a l saccharin tablets and distilled water. Ad lib rat c h o w was provided in hoppers suspended from the opposite wall of the cage on which the drinkers were m o u n t e d . During the service interval the animals were transferred to individual holding cages w i t h o u t access to f o o d or water and the positions of the drinking bottles were rotated in a cyclical fashion. The sequence of the f o u r bottles never changed and their positions repeated every four days. The drinking tubes were rinsed and drinking solutions were replenished with fresh solutions once daily. The baseline was e x t e n d e d to 25 sessions for the first exposure in E x p e r i m e n t 2 to allow the animals to accomm o d a t e to the four drinker situation and produce stable drinking patterns. Baseline 1 refers to the last 10 sessions of the 25 session baseline exposure. Following Baseline I, all animals were given 20 consecutive sesions of shock. As in the first study, shock occurred for the same 12 min of each hr on a VI one min schedule. F o r convenience in looking at the e x t e n d e d effects of
111 shock, the first 10 shock sessions are called Shock I, and the second 10 sessions are called Shock II. Ten 22 hr sessions of postshock baseline (Baseline II) followed the shock sessions. Results The total mean daily fluid intake during the h o m e cage pretest exposure was 126.3 ml/kg with the two bottle choice. When the rats were transferred to the test environments with the four bottle choice, the total mean volume of daily fluid decreased in Baseline I to 80.4 ml/kg. The reasons for the decrease were not clear, but the changes were reflected in measures of water intake rather than intake on saccharin, 5% or 10% ethanol. A t-test for correlated means indicated that daily water intake dropped significantly from 48 ml/kg to 21.4 ml/kg (t = 6.81; p < 0 . 0 5 ) . Saccharin intake decreased from 48.2 ml/kg to 37.3 m l / k g but the difference was not significant (t = 1.1 l ; p > 0 . 0 5 ) . Ethanol intake at b o t h concentrations dropped slightly but h o m e cage to test cage mean differences were not significant (5%, t = 1.03; 10%, t = 0.92). Five percent h o m e cage intake was 21.4 ml/kg and test cage intake was 14.8 ml/kg. Similarly 10% ethanol intake showed a mean daily level of 8.7 ml/kg in the h o m e cages and 6.9 ml/kg in the test cages. Both the h o m e cage and test cage data showed m o d e r a t e c o n s u m p t i o n of 5% and low c o n s u m p t i o n of 10% ethanol. These results were more consistent than the data of E x p e r i m e n t 1 with previous reports of the rats' aversion for higher concentrations of ethanol. The test cage data for the 11 animals in E x p e r i m e n t 2 are shown in Figs. 4, 5, and 6. The mean intake records during each of the 10 sessions of Shock I, Shock II and postshock Baseline II are expressed as departures from the intake records for the last 10 sessions of Baseline I. The net change in daily fluid intake ( m l / k g / 2 2 hr) is plotted for each of the fluid choices of 5% V / V ethanol, 10% V / V ethanol, distilled water and saccharin. The X-axis shows each hour of the 22 hr session broken into successive two min intervals. Each data point represents the mean of 2420 samples of licking during a specific two min interval (11 animals x 22 hr x 10 sessions = 2420). For example, the sum of all points along one line would represent the total change from baseline intake on a specific fluid, and the plot represents the t e m p o r a l and volumetric shift in drinking relative to the recurring one hr cycle. It can be seen in Fig. 4 that ethanol intake at b o t h 5 and 10% increases during shock sessions and the peaks occur 8 - 1 2 min after shock, with c o n t i n u e d intake for up to 26 min after shock. A peak is also n o t e d for saccharin intake corresponding to the initial segments of b o t h ethanol peaks, but c o n s u m p t i o n does not c o n t i n u e after the initial burst. Water intake shows negligible changes during and after shock. Figure 5 shows the baseline to shock difference for the second 10 sessions of shock (Shock II) and also indicates a shift away from 5% ethanol intake to increased peaking on 10%. Post shock saccharin intake dropped off sharply with increased shock exposure during Shock II. These data are consistent with E x p e r i m e n t 1 in that fluid intake under shock was suppressed and post shock drinking was characterized by sharp peaks in ethanol intake. Figure 6 shows the four fluid intake patterns for the 10 sessions of postshock Baseline II as a difference from the 10 sessions of preshock Baseline I. Shock was not in effect and peak intake patterns were not observed. However, a
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p r e f e r e n c e for b o t h 5 and 10% e t h a n o l above w a t e r and saccharin is reflected by the f o u r curves. The p a t t e r n o f saccharin intake s h o w s a m i r r o r like negative p a t t e r n to that observed u n d e r s h o c k and could suggest t h a t the animals had learned to r e s p o n d to the h o u r l y t e m p o r a l p r o p e r t i e s o f the s h o c k schedule. Whereas Figs. 4, 5 and 6 illustrate the cumulative h o u r l y fluid intake relative to the first baseline, the n e t change
FIG. 6. The net change in daily fluid intake tot 11 animals as a difference from Baseline I to the l 0 sessions of baseline (Baseline II) which followed the 20 sessions of shock exposure. Shock was not in effect for Baseline 1 or Baseline II. scores could be sensitive to d i s t o r t i o n f r o m the initial high p r e f e r e n c e s for saccharin and low p r e f e r e n c e s for e t h a n o l at 10% V/V. The d i s t o r t i o n could be reflected by a t e m p o r a l m o d u l a t i o n o f all fluid c o n s u m p t i o n to the p o s t s h o c k interval without c o r r e s p o n d i n g absolute volumetric changes. Figure 7 t h e r e f o r e r e p r e s e n t s the absolute changes in b o t h total interval and p o s t i n t e r v a l (24 min) fluid intake over baseline and shock sessions. The e t h a n o l intake is c o m b i n e d for the 5 and 10% solutions and expressed as g/kg intake. The absolute increase in e t h a n o l intake as a f u n c t i o n o f s h o c k is seen for b o t h the total and post interval criteria and is c o n s i s t e n t with the t e m p o r a l data. T w o o n e - w a y r e p e a t e d measures analyses o f variance were carried out to evaluate the changes in e t h a n o l intake observed for b o t h the total and postinterval measures. When e t h a n o l intakes (g/kg) were collapsed over sessions and s u m m a r i z e d for Baseline l, Shock I, Shock II and Baseline II, significant main effects were evident for the total interval scores, F(3,30) = 5.57, p < 0 . 0 5 , and the postinterval scores, F ( 3 , 3 0 ) = 4.98, p < 0 . 0 5 . P o s t h o c c o m p a r i s o n s were o b t a i n e d with D u n n e t s t statistic and c o m p a r e d each t r e a t m e n t with the Baseline 1 c o n t r o l [ 3 6 ] . Using the total interval scores, e t h a n o l intake for Shock I (t = 2.74, p < 0 . 0 5 ) , Shock II (t = 3.70, p < 0 . 0 5 ) and Baseline II (t = 3.33, p < 0 . 0 5 ) was significantly increased over Baseline I intake levels. With the postinterval scores, e t h a n o l intake for S h o c k I (t = 3.06, p < 0 . 0 5 ) and Shock 11 (t = 3.44, p < 0 . 0 5 ) increased significantly over Baseline I. As m i g h t be e x p e c t e d f r o m the data in Fig. 7, postinterva] e t h a n o l intake for Baseline II did n o t differ significantly from Baseline I (t = 1.47, p < 0 . 0 5 ) . The a b s o l u t e changes in the v o l u m e o f e t h a n o l c o n s u m e d c o i n c i d e n t with shock sessions indicate that the relative t e m p o r a l peaks s h o w n in Figs. 4 and 5 also r e p r e s e n t a v o l u m e t r i c m o d u l a t i o n of e t h a n o l intake. The total interval saccharin and water intake changes from Baseline to shock were also evaluated with D u n n e t s t-test. As might be e x p e c t e d from the
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FIG. 7. Absolute daily fluid intake of ethanol, saccharin and water across sessions and procedures for the 11 animals in Experiment 2. Ethanol intakes on 5 and 10% V/V are combined to give the total ethanol intake in g/k/22 hr. The postinerval criterion represents the cumulative amount of fluid consumed daily in the 24 min interval during the nonshock baselines. The total interval criterion represents the total daily fluid intake cumulated over twenty-four 1 hr segments.
temporal data in Figs. 4 and 5, saccharin intake during Shock I did not show a significant decrease from baseline (t = 1.40, p>O.OS), but did show a significant mean decrease during the second ten sessions of Shock II (t = 2.21, p
Discussion A simple explanation of the data is that some animals in Experiment 1 were capable of learning to drink alcohol as a specific response to short duration random shock stress. In effect, ethanol intake seemed to become a quickly acquired escape response. The escape may be functionally similar to that provided by moving the animals to the home other designs, but this comparison was not directly Following extended stress exposure, the animals
cage in tested. in Ex-
periment 2 continued to increase their quantity of absolute ethanol intake and continued to restrict their consumption primarily to the interval immediately after shock. The increasing dose intake could indicate that the stress became move aversive over time or that the animals showed some low dose tolerance to the initial choice of 5% ethanol. Both possibilities are consistent with the explanation that ethanol selection somehow modified the consequences of shock stress. Several experiments have pointed to a possible relationship between extended shock stress and drinking. An increase in ethanol consumption specific to the onset of inescapable shock was reported by Anisman and Wailer [ 5 I. Their animals were housed in the shock compartment with ethano! and water choices available throughout the daily cycle and animals receiving shock on both alternating six and twelve hr schedules increased their alcohol intake
114 a p p r o x i m a t e l y d o u b l e the c o n t r o l animals receiving n o shock. It is possible t h a t c u m u l a t i v e t e m p o r a l p a t t e r n s o f e t h a n o l c o n s u m p t i o n were o c c u r r i n g as a specific r e s p o n s e to shock. Similarly, H a t t o n and V i e t h [19] r e p o r t pilot data for t h r e e a n i m a l s w h i c h increased t h e i r e t h a n o l i n t a k e following, b u t n o t b e f o r e or d u r i n g s h o c k a v o i d a n c e sessions. These d a t a again suggest t h a t p a t t e r n s of posts h o c k e t h a n o l c o n s u m p t i o n m a y have b e e n emerging b u t t e m p o r a l data were n o t g a t h e r e d . In b o t h E x p e r i m e n t s 1 and 2 we a r b i t r a r i l y used t h e recurring h o u r l y s h o c k s c h e d u l e and s h o c k t e r m i n a t i o n always p r e d i c t e d 48 rain w h i c h were free o f shock, or an interval of relative safety. In t h a t d r i n k i n g was suppressed during the s h o c k schedule, licking in t h e p o s t s h o c k interval c o u l d r e p r e s e n t c o n s u m p t i o n in t h e m o s t stress free interval. With this view s h o c k b e c o m e s a d i s c r i m i n a t i v e s t i m u l u s for t h e o n s e t of t h e stress free interval and serves to signal d r i n k i n g ; a c o n t r a s t to a stress r e d u c t i o n interp r e t a t i o n of the data. A l t h o u g h o u r design did n o t test for this possibility, several factors argue against a p u r e l y n o n a v e r s i v e d i s c r i m i n a t i v e f u n c t i o n for t h e shock. First, some animals s h o w p o s t s h o c k p a t t e r n s a l m o s t i m m e d i a t e l y after s h o c k e x p o s u r e a n d t h e s h o r t e x p o s u r e time involved would n o t seem a d e q u a t e for t h e a n i m a l s to learn a b o u t the t e m p o r a l n a t u r e of t h e s h o c k schedule. In a d d i t i o n , p i l o t data f r o m a s e p a r a t e set of 18 rats w h i c h were given s h o c k only o n c e daily clearly i n d i c a t e d t h a t increasing p o s t s h o c k e t h a n o l ratios were a p p e a r i n g as a f u n c t i o n of s h o c k b u t n o t in t h e c u m u l a t i v e p r o p o r t i o n s e v i d e n c e d w i t h the r e c u r r i n g 24 h r schedule. With t h e s h o c k o c c u r r i n g only once daily, it is unlikely t h a t the animals were learning to use the s h o c k as a d i s c r i m i n a t i v e s t i m u l u s w h i c h p r e d i c t e d periods of relative safety. We do n o t k n o w t h e h o u r l y d i s t r i b u t i o n of t h e b l o o d e t h a n o l c o n c e n t r a t i o n s in the p r e s e n t e x p e r i m e n t s and t h e r e is a possibility t h a t t h e animals were achieving the highest levels of i n t o x i c a t i o n j u s t p r i o r to, or even during, the 12 m i n of shock. T h e p o s t s h o c k d r i n k i n g could, t h e r e f o r e , be c o n c e i v e d as an a v o i d a n c e response in a n t i c i p a t i o n of u p c o m i n g s h o c k in the n e x t h o u r . This seems unlikely because the rats w o u l d have to n o t o n l y learn the t e m p o r a l d i s c r i m i n a t i o n w h i c h e n a b l e d t h e m to p r e d i c t s h o c k , b u t also adjust t h e i r d r i n k i n g p a t t e r n s to a c c o m m o d a t e for the delays in achieving i n t o x i c a t i n g b l o o d e t h a n o l levels w h i c h w o u l d be c o i n c i d e n t w i t h s h o c k onset. One a n i m a l in the s e c o n d s t u d y , h o w e v e r , s h o w e d some m o d e r a t e p e a k i n g o n 10% e t h a n o l j u s t p r i o r to t h e o n s e t o f s h o c k ; an i n d i c a t i o n t h a t learning to use the e t h a n o l in an a v o i d a n c e r a t h e r t h a n an escape f a s h i o n m a y n o t be an i m p o s s i b l e task for the rat. T h e r e is also a possibility t h a t the c o n s u m p t i o n p a t t e r n s observed were g e n e r a t e d by the r e c u r r i n g n a t u r e of t h e s h o c k s c h e d u l e and t h a t any recurring event, stressful or n o n s t r e s s f u l , w o u l d p r o d u c e a similar p a t t e r n of drinking. A l t h o u g h the data do n o t test this, we w o u l d e x p e c t the e t h a n o l p a t t e r n s to persist w i t h saccharin or w a t e r if t h e p h e n o m e n o n were due to s c h e d u l e i n d u c t i o n [14, 15, 17]. T h e p r e s e n t studies have n o t ruled o u t the possibility t h a t the a n i m a l s were c o n s u m i n g g r e a t e r a m o u n t s o f e t h a n o l , a n d lesser a m o u n t s of saccharin, because of t h e calories w h i c h the alcohol p r o v i d e d d u r i n g e x t e n d e d stress. The weight loss o b s e r v e d u n d e r stress c o u l d i n d i c a t e t h a t the a n i m a l s were a t t e m p t i n g caloric r e p l a c e m e n t t h r o u g h
MILLS, B E A N A N D H U T C H E S O N e t h a n o l intake. T h e data f r o m E x p e r i m e n t 1 i n d i c a t e d that the overall daily i n t a k e of e t h a n o l s h o w e d a decrease while the d i s t r i b u t i o n of e t h a n o l c o n s u m p t i o n shifted to the p o s t s h o c k p a t t e r n . The n e t result, relative to baseline, is s h o c k i n d u c e d c o n s u m p t i o n w i t h l o w e r daily caloric i n t a k e from e t h a n o l . However, the animals w h i c h received ext e n d e d stress in E x p e r i m e n t 2 increased t h e i r daily e t h a n o l intake and could have b e e n d r i n k i n g e t h a n o l for its caloric p r o p e r t i e s as a m e a n s to alleviate the stress [6,17] A l c o h o l is high in calories (7 Kcal/g) and increased e t h a n o l i n t a k e is always likely to be c o n f o u n d e d with increased caloric intake. The ideal free choice e x p e r i m e n t a l design w o u l d be to p r o v i d e a n o n i n t o x i c a t i n g choice solution, such as sucrose or d e x t r o s e , as a caloric e q u i v a l e n t to e t h a n o l . However, w h e n o u r rats are given isocaloric s o l u t i o n s of d e x t r o s e or even a m i n i m a l l y sweet c o m m e r c i a l p r o d u c t at a p p r o x i m a t e l y 4 Kcal/g (Polycose, Ross Laboratories) e q u i v a l e n t to 5% e t h a n o l as a free choice, virtually all fluid selection is restricted to the sweet-calorie choice. Several o t h e r i n v e s t i g a t i o n s have a t t e s t e d to the rat's o v e r w h e l m i n g p r e f e r e n c e for sweet-calorie comb i n a t i o n s [7, 16, 22, 30, 3 3 ] . Our a n i m a l s will select sugar solutions p r e d o m i n a n t l y over water, saccharin and e t h a n o l b o t h during baseline and s h o c k p r o c e d u r e s . T h e r e is a need for research w h i c h will offer the a n i m a l an isocaloric and n o n i n t o x i c a t i n g c o n t r o l s o l u t i o n w h i c h w o u l d also p r o d u c e similar baseline selection rates to t h a t observed with e t h a n o l . The e x t r e m e sugar p r e f e r e n c e s p r e v e n t m e a n i n g f u l c o m p a r i s o n s to be m a d e b e t w e e n the separate caloric and i n t o x i c a t i n g c o m p o n e n t s of fluid selection as t h e y f u n c t i o n relative to stress. One p e r s i s t e n t f e a t u r e of the data is the relatively rapid a p p e a r a n c e of the p o s t s h o c k d r i n k i n g in r e s p o n s e to shock. A p p a r e n t l y an a n i m a l will try a n u m b e r o f responses w h e n faced with an inescapable stressor [3, 4, 3 2 ] , and one h y p o t h e s i s c o n c e r n i n g the existing data would be t h a t p o s t s h o c k e t h a n o l and saccharin c o m s u m p t i o n are b o t h subsets of p o s t s h o c k novel fluid intake. In the p r e s e n t study, the distinctive taste p r o p e r t i e s of the fluid m a y be the m o r e i m p o r t a n t variable w h e n a t t e m p t i n g to explain the origin of the a n i m a l ' s response. The data i n d i c a t e t h a t the animals progressively shifted from saccharin to 5 ~ e t h a n o l to 10% e t h a n o l with increasing e x p o s u r e to shock. This w o u l d argue t o w a r d a t w o c o m p o n e n t process based on taste and the s e c o n d a r y or m o r e delayed p r o p e r t i e s of fluid i n t a k e to respectively i n t i t i a t e and m a i n t a i n e t h a n o l i n t a k e in r e s p o n s e to stress. In c o n c l u s i o n , s h o c k i n d u c e d e t h a n o l c o n s u m p t i o n is a c u m u l a t i v e effect and the data d e m o n s t r a t e t h a t the r e l a t i o n s h i p b e t w e e n stress and d r i n k i n g is a m e n a b l e to t e m p o r a l m e a s u r e m e n t . The relation a p p a r e n t l y d e p e n d s to a large e x t e n t on the c o n t i n u o u s n a t u r e of the i n e s c a p a b l e stress and the cont i n u o u s l y available alcohol supply a p p e a r i n g t o g e t h e r . ACKNOWLEDGEMENTS The authors wish to express appreciation to John A. Ewing for his helpful suggestions in all phases of the research and Elaine Woody, Lois Boone, Richard Lutz and the Claude Gaebelein for their technical assistance. This investigation was supported by research grants 7405 from the North Carolina Alcoholism Research Authority and MH-21667 from the National Institute on Alcohol Abuse and Alcoholism.
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REFERENCES 1. Allison, J. Microbehavioral features of nutritive and nonnutritive drinking in rats. J. cornp, physiol. PsychoL 76: 4 0 8 - 4 1 7 , 1971. 2. Allison, J. and N. J. Castellan. Temporal characteristics of nutritive drinking in rats and humans. J. comp. physiol. Psychol. 70: 1 1 6 - 1 2 5 , 1970. 3. Anisman, H. Time dependent variations in aversively motivated behaviors: Nonassociative effects of cholinergic and catecholaminergic activity. Psychol. Rev. 82: 3 5 9 - 3 8 5 , 1975. 4. Anisman, H. and T. G. Waller. Effects of inescapable shock on subsequent avoidance performance: Role of response repertoire changes. Behav. Biol. 9: 3 3 1 - 3 3 5 , 1973. 5. Anisman, H. and T. G. Waller. Effects of inescapable shock and shock produced conflict on self selection of alcohol in rats. Pharrnac. Biochem. Behav. 2: 2 7 - 3 3 , 1974. 6. Antelman, S. M. and H. Szechtman. Tail pinch induces eating in sated rats which appears to depend on nigrostriatal dopamine. Science 189: 7 3 1 - 7 3 3 , 1975. 7. Booth, D. A., D. Lovett and G. M. McSherry. Postingestive modulation of the sweetness preference gradient in the rat. J. comp. physiol. Psychol. 78: 4 8 5 - 5 1 2 , 1972. 8. Cappell, H. and P. Herman. Alcohol and tension reduction: a review. Q. J. Stud. Alcohol 33: 3 3 - 6 4 , 1972. 9. Carey, R. J. Brief communication: a decrease in ethanol preference in rats resulting from forced ethanol drinking under fluid deprivation. Physiol. Behav. 8: 3 7 3 - 3 7 5 , 1972. 10. Casey, A. The effect of stress on the consumption of alcohol and reserpine. Q. Z Stud. Alcohol 21: 3 0 8 - 3 1 6 , 1960. 11. Cicero, T. and S. Hill. Ethanol self-selection in rats: a distinction between absolute and 95% ethanol. Physiol. Behav. 5: 7 8 7 - 7 9 1 , 1970. 12. Cicero, T., R. D. Myers and W. Black. Increase in volitional ethanol consumption following interference with a learned avoidance response. Physiol. Behav. 3: 6 5 7 - 6 6 0 , 1968. 13. Eriksson, K. Alcohol consumption and blood alcohol in rat strains selected for their behavior towards alcohol. Finnish Foundation Alcohol Stud. 20: 1 2 1 - 1 2 5 , 1972. 14. Falk, J. L. Production of polydipsia in normal rats by an intermittent food schedule. Science 133: 1 9 5 - 1 9 6 , 1961. 15. Falk, J. L. The motivational properties of schedule-induced polydipsia. J. exp. Analysis Behav. 9: 1 9 - 2 6 , 1966. 16. Falk, J. L. and H. H. Samson. Schedule-induced physical dependence on ethanol. Pharrnac. Rev. 27: 4 4 9 - 4 6 4 , 1976. 17. Freed, E. X. and D. Lester. Schedule induced consumption of ethanol: calories or chemotherapy? Physiol. Behav. 5: 555 560, 1970. 18. Freund, G. Alcohol consumption and its circadian distribution in mice. J. Nut. 100: 3 0 - 3 6 , 1970. 19. Hatton, G. I. and A. Vieth. Stress related and diurnal alcohol drinking in rats. Bull. Psychon. Soe. 4 : 1 9 5 196, 1974.
20.
21.
22.
23.
24. 25. 26.
27.
28.
29. 30.
31.
32. 33.
34. 35.
36. 37.
Hulse, S. Licking behavior of rats in relation to saccharin concentration and shifts in fixed ratio reinforcement. J. cornp. physiol. Psychol. 64: 4 7 8 - 4 8 4 , 1967. Hutcheson, J. and K. C. Mills. A compact and inexpensive drinkometer for use with small animals. Physiol. Behav. 13: 1 7 9 - 1 8 1 , 1974. Jacobs, H. L. Studies on sugar preference: 1) The preference for glucose solutions and its modification by injections of insulin. J. comp. physiol. Psychol. 51: 3 0 4 - 3 1 0 , 1958. Lester, D. Self selection of alcohol by animals, human variation and the etiology of alcoholism. Q. J. Stud. Alcohol 27: 3 9 5 - 4 3 8 , 1966. Lester, D. and E. Freed. Criteria for an animal model of alcoholism. Pharrnae. Biochem. Behav. 1: 1 0 3 - 1 0 7 , 1973. Myers, R. D. and R. Carey. Preference factors in experimental alcoholism. Science 134: 4 6 9 - 4 7 0 , 1961. Myers, R. D., R. Holman. Failure of stress of electric shock to increase ethanol in rats. Q. J. Stud. Alcohol 28: 132-137, 1967. Opsahl, C. and C. Hatton. Volitional ethanol increases during acquisition and extinction of avoidance responding. Physiol. Behav. 8: 8 7 - 9 3 , 1972. Parker, L. F. and B. L. Radow. Isolation stress and volitional ethanol consumption in the rat. Physiol. Behav. 12: 1 - 3 , 1974. Quinsey, V. Lick-shock contingencies in the rat. J. exp. Analysis Behav. 17: 119-125. 1972. Samson, H. H. and J. L. Falk. Alteration of fluid preference in ethanol-dependent animals. J. Pharmac. exp. Ther. 190: 3 6 5 - 3 7 6 , 1974. Stellar, E. and J. Hill. The rat's rate of drinking as a function of water deprivation. J. comp. physiol. Psychol. 45: 9 6 - 1 0 2 , 1952. Ulrich, R. E. and N. H. Azrin. Elimination of undesired escape from footshock. J. exp. Analysis Behav. 5: 72, 1962. Valenstein, E. S. Selection of nutritive and nonnutritive solutions under different conditions of need. Z comp. physiol. Psychol. 6 3 : 4 2 9 433, 1967. Veale, W. L. Ethanol selection in the rat following forced acclimation. Pharmac. Biochern. Behav. 1:233 235, 1973. von Wright, J., L. Pekanmaki and S. Malin. Effects of conflict and stress on alcohol intake in rats. Q. J. Stud. Alcohol 32: 4 2 0 - 4 3 3 , 1971. Winer, B. J. Statistical Principles in Experimental Design. McGraw-Hill Book Co., New York, 1962. Wise, R. A. Voluntary ethanol intake in rats following exposure to ethanol on various schedules. Psychopharmacologia 29: 2 0 3 - 2 1 0 , 1973.