P,~armacology Biochemistry & Behavior, Vol. 3, pp. 1073--1076. Copyright © 1975 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

Neurochemical Correlates of Alcohol Preference in Inbred Strains of Mice A. K. S. HO A N D C. S. T S A I

Laboratory o f Neurochemistry and Neuropharmacology, Division o f Pharmacology Department o f Basic Sciences, Peoria School o f Medicine, University o f Illinois Peoria, Illinois 61606 AND B. KISSIN

Division o f Alcoholism and Drug Abuse, Department o f Psychiatry Downstate Medical Center, Brooklyn, New York ( R e c e i v e d 16 J u n e 1 9 7 5 ) HO, A. K. S., C. S. TSAI AND B. KISSIN. Neurochemical correlates of alcohol preference in inbred strains of mice. PHARMAC. BIOCHEM. BEHAV. 3(6) 1073-1076, 1975. - C s 7 B 1/6J, a specific inbred strain of mice with high alcohol preference and DBA/2J, a specific inbred strain with poor preference for alcohol were studied. Brain content of acetylcholine, uptake of 14C.Choline by whole brain hom ogenate were significantly higher in the C s 7 B 1/6J mice whereas brain acetylcholinesterase was higher in the DBA/2J mice. No significant difference was found for the level of brain serotonin, uptake of 3H.norepinephrine or 3H_dopamine. Treatment with a specific inhibitor of choline transferase, 4-(1-napthylvinyl) pyridine salt (10 mg/kg, twice daily) shifted the selection of alcohol to water in the C 57 B1/6J mice. These findings suggest a direct involvement of central cholinergic mechanism in alcohol preference. Ethanol preference Strain differences 4-(1-napthylvinyl) pyridium salt (NVP)

Central cholinergic mechanism

R E C E N T i n t e r e s t in t h e n e u r o c h e m i c a l correlates o f alcohol p r e f e r e n c e has been focused o n t h e role o f various p u t a t i v e n e u r o t r a n s m i t t e r s , p a r t i c u l a r l y , s e r o t o n i n (5-HT). Myers a n d co-workers [ 1 2 , 1 5 ] r e p o r t e d a decrease in t h e volitional c o n s u m p t i o n of e t h a n o l in rats a f t e r c h r o n i c t r e a t m e n t w i t h D L - p a r a c h l o r o p h e n y l a l a n i n e (PCPA), an i n h i b i t o r o f t r y p t o p h a n h y d r o x y l a s e , whereas a l p h a - m e t h y l p a r a - t y r o s i n e (a-MT), an i n h i b i t o r of t y r o s i n e h y d r o x y l a s e , h a d n o effect. However, t h e decrease in e t h a n o l c o n s u m p t i o n p r o d u c e d b y PCPA was n o t o b s e r v e d b y Geller [5] w h o s h o w e d t h a t PCPA increased e t h a n o l selection. R e c e n t studies in o u r l a b o r a t o r y using 5, 6 - d i h y d r o x y t r y p t a m i n e (5,6-DHT), a long-acting d e p l e t o r o f brain 5-HT, [3] slhowed an increase in t h e volitional c o n s u m p t i o n o f e t h a n o l . In a d d i t i o n , f o l l o w i n g 5,6-DHT t r e a t m e n t t h e r e was a significant increase in t h e b r a i n level of a c e t y l c h o l i n e ( A C h ) parallel to t h e a p p e a r a n c e of t h e increase in a l c o h o l p r e f e r e n c e [ 8 ] . It was suggested t h a t the 5,6-DHT i n d u c e d a l c o h o l p r e f e r e n c e m i g h t be a t t r i b u t e d t o an increase in c e n t r a l cholinergic activities. In o r d e r to f u r t h e r e l u c i d a t e the role of c e n t r a l cholinergic s y s t e m ( s ) in t h e selection o f e t h a n o l , we s t u d i e d t h e n e u r o c h e m i c a l correlates in t w o specific i n b r e d strains of mice with m a r k e d l y d i f f e r e n t a l c o h o l p r e f e r e n c e status, viz C 5 7 B 1 / 6 J , an alcohol p r e f e r r i n g strain, a n d DBA, an alcohol n o n - p r e f e r r i n g strain. T h u s far, the m a j o r b i o c h e m -

Brain monoamines

ical d i f f e r e n t i a t i n g f a c t o r b e t w e e n the t w o strain has b e e n in t h e rate of a c e t a l d e h y d e m e t a b o l i s m , where the DBA strain has been s h o w n to m e t a b o l i z e e t h a n o l less rapidly a n d t h u s s h o w s a greater a c c u m u l a t i o n o f a c e t a l d e h y d e a f t e r e t h a n o l ingestion w i t h s u b s e q u e n t greater associated signs o f t o x i c i t y [ 1 6 ] . In a d d i t i o n , the effect of 4-(1n a p t h y l v i n y l ) p y r i d i u m salt (NVP), a choline a c e t y l t r a n s f e r ase i n h i b i t o r [ 1 7 ] , on e t h a n o l selection in the Cs 7B1 mice was studied. METHOD

Measurement of Alcohol Preference A d u l t male C s T B 1 / 6 J a n d D B A / 2 J mice, aged 6 - 8 weeks a n d weighing b e t w e e n 20 to 25 g were used (supplied b y J a c k s o n L a b o r a t o r y , Bar Harbor, Maine). T h e animals were h o u s e d individually in wire m e s h cages in a c o n s t a n t t e m p e r a t u r e r o o m ( 7 0 F ) wath ad h b access to food a n d water. To test for a l c o h o l p r e f e r e n c e , t w o small g r a d u a t e d 25 m l glass d r i n k i n g t u b e s ( R i c h t e r t y p e ) were used. One was filled w i t h w a t e r a n d the o t h e r w i t h e t h a n o l ( d i l u t e d fresh each day f r o m 95 p e r c e n t e t h a n o l w i t h tap w a t e r on a v o l u m e t o v o l u m e basis to t h e r e q u i r e d c o n c e n t r a t i o n ) . T h e t u b e s were r o t a t e d r a n d o m l y e a c h day to p r e v e n t t h e d e v e l o p m e n t of a p o s i t i o n h a b i t [ 1 3 ] . M e a s u r e m e n t s o f w a t e r a n d e t h a n o l c o n s u m p t i o n were t a k e n at 10 a.m. e a c h O

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1074 day. Alcohol preference was tested in each animal for 7 days and the base-line c o n s u m p t i o n was established using data obtained during the last 4 days. In a subgroup of 32 mice, NVP was injected IP twice daily at 10 a.m. and 10 p.m. using 2 mg/kg and 10 mg/kg doses respectively, and the daily selections of either 5 or 10 percent ethanol were recorded.

Neurochemical Studies To determine whether there were significant differences in the brain chemistry of these two strains, naive animals were used. Various parameters were compared, including the levels of brain ACh, norepinephrine (NE), dopamine (DA) and 5-HT; the activities of acetylcholinesterase (ACHE), choline acetyltransferase (ChAc) and the in vitro uptake of 14C-Choline, 3H-NE and 3H-DA by whole brain homogenates. The mice were killed by decapitation at about 3 to 5 p.m. and the brains were removed on ice. The brains were dissected into two halves along the mid-line; one half was used for ACh and the other half for the m o n o a m i n e assays. ACh was extracted according to the m e t h o d described by Hebb [ 6 ] . The brains were weighed and h o m o g e n i z e d in 4 volumes of 10 percent trichloroacetic acid, centrifuged, washed and the c o m b i n e d supernatant was further extracted with ether to remove the lipids. The last trace of ether was removed by aspiration. ACh was assayed using the guinea pig ileum preparation in the presence of morphine (2 rag/l) and d i p h e n h y d r a m i n e (2 rag/1 ) [4]. For catecholamines and 5-HT assays, the brains were weighed, h o m o g e n i z e d in 4 volumes of chilled 0.4 N perchloric acid, centrifuged and the supernatants were decanted into stoppered tubes containing washed alumina. NE, DA and 5-HT were assayed fluorometrically according to the m e t h o d of A n s e l l a n d Beeson [ 1 ]. In another subgroup of mice, whole brain homogenates were prepared and used for both e n z y m e and uptake experiments, in vitro uptake of 14C-Choline, 3H-NE and 3H-DA, whole brain homogenates were used. The brains were h o m o g e n i z e d in 8 volumes of ice-cold 0.32 M sucrose as described by Whittaker [ 18]. After centrifugation of the homogenate for 10 rain at 1000 x g and 4°C, the precipitate which consisted of nuclei and u n b r o k e n cells (PI) was discarded. The supernatant fraction ($1) was used for uptake experiments. Into 4.4 ml of an incubation medium containing 118 mM sodium chloride, 4.7 mM potassium chloride, 2.2 mM calcium chloride, 1.18 mM magnesium sulfate, 11 mM glucose and 25 mM sodium phosphate at pH 7.0, 0.25 ml of the brain h o m o g e n a t e was added. One tenth ml of incubation m e d i u m containing 50x concentrated solution of 14 C-Choline (specific activity 61 mCi/mM; final incubation concentration 0.01 raM), 3H-NE (specific activity, 3.7 Ci/mM; final concentration, 0.1 **M) or 3H-DA (specific activity, 11.0 Ci/mM; final concentration, 0.1 uM) was added and incubated at 37°C for 20 rain (All three isotopes were supplied by New England Nuclear Corp., Boston, Mass.). The incubated samples were centrifuged at 17,000 G and the pellets resuspended in 400 ul of 0.4 N perchloric acid in 50 percent ethanol. A 200 ul aliquot was then transferred into counting vials containing a 12 ml of phosphor ethanol counting mixture and the radioactivity was measured by liquid scintillation spectrometry. Crude extracts containing brain A C h E and ChAc were prepared by the m e t h o d previously described [7]. AChE

[tO, TSAI A N D KISSIN was determined by a modified radiometric m e t h o d according to McCamen and Hunt [ 11 ] using acetyl-l-14C-Choline iodide as substrate. The incubation mixture consisted of 0.2 mt potassium buffer, pH 7.2, 0.085 M NaC1, 0.15 M and 0.04 M MgC12; 0.1 ml of 14C-ACh, 0.t mM (containing 0.02 uCi acetyl-1-14-C-Choline diluted with acetylcholine iodide) and 0.1 ml of tissue extract containing 8 10 mg tissue wet weight. The incubations were carried out for 10 min in a D u b n o f f shaking i n c u b a t o r at 37°C. At the end of the incubation, the tubes were removed and immersed in ice. The reaction was stopped by addition of 1 g of D o w e x 50 resin, filtered, and 100 ul aliquot of the filtrate, containing hydrolysed acetic-1-14C acid, was transferred to counting vials containing 15 ml of Bray's PPO-POPOP liquid scintillation cocktail. Radioactivity was measured by liquid scintillation spectrometry in a LS-Beckman 100 counter and corrections were made for quenching. ChAc activity was determined using 1-( 14C)-acetylcoenzyme A as substrate according to the method described previously [7]. Protein was determined by the m e t h o d of Lowry et al [10]. RESULTS

Data obtained on the volitional c o n s u m p t i o n of ethanol showed that the C 5 7 B I / 6 J mice consumed significantly greater amounts of ethanol than the DBA/2J strain. In groups of 20 CsTB1/6J mice, mean daily values of c o n s u m p t i o n were 182 -+ 16 ml/kg and 230 + 18 ml/kg for 5 and 10 percent ethanol respectively. Mean value of consumption for DBA/2J mice was 59 -+ 6 ml/kg for 5 percent ethanol. The consumption of 10 percent ethanol was less than 10 ml/kg and in most mice, no c o n s u m p t i o n was recorded. Mean daily values for water and food c o n s u m p t i o n prior to testing for alcohol preference for C s T B 1 / 6 J mice were 253 -+ 3 ml/kg and 189 + [1 g/kg respectively; for the DBA/2J, the values were 213 +- 20 ml/kg and 176 -+ 19 g/kg respectively. The mean level of brain ACh was significantly higher in the C s T B 1 / 6 J than in the DBA/2J mice (p