a mouse model of alcoholism

8/12/2019 A Mouse Model of Alcoholism

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Physiology Behavior,Vol. 29, pp. 833-839. Pergam on Press, 1982. Printed in the U .S.A.

M o u s e M o d e l o f l c o h o l i smHU U R I J K , J O H N C . C R A B B E 1 A N D H E N K R I G T E R a

CNS Pharmacology Department, Scientific Development GroupOrganon, Oss, The NetherlandsR e c e i v e d 2 0 M a r c h 1 98 2

R I J K , H . , J . C . C R A B B E A N D H . R I G T E R . A mouse model of alcoholism. PH YS IOL . BEHA V. 29(5) 833-839 , 198 2 . - -Amode l o f a lcoho l i sm shou ld demon s t ra te se l f -admin is t ra t ion o f a lcoho l by the an imal and a wi thdraw al syndrom e when thean imal no longer has access to the d rug . A m ouse m ode l i s desc r ibed tha t mee ts these c r i t e r i a and enab les the induc t ion o fa lco ho l i sm in a l a rge num ber o f an imals wi th in a shor t pe r iod o f t ime . Mice were in jec ted da i ly wi th pyrazo le , aninh ib i to r o f a lcoho l dehydro genase . T he an im als se l f -admin is te red e thano l by inha l ing vapour con ta in ing a re la t ive lycons tan t concen t ra t ion o f e thano l . Se r f -admin is t ra t ion was vo lun ta ry : the mice cou ld move to a cha mber wi thou t e thano lv a p o u r . D u r i n g t h e f i r s t s ix d a y s o f e a c h e x p e r i m e n t , t h e a n i m a l s w e r e p r o m p t e d t o s e l e c t t h e e t h a n o l c h a m b e r b ydarken ing tha t chambe r . M os t mice con t inued to se l f -admin is te r e thano l wi thou t any p rom pt ing fo r a t l eas t 3 days ;the rea f te r , p re fe rence fo r e than o l waned . Se l f -admin is t ra t ion l ed to m easurab le l eve l s o f e thano l in b lood and to thed e v e l o p m e n t o f w i t h d r a w a l w h e n i n f u s io n o f v a p o u r w a s s t o p p e d .Alcoho l i sm Anim al mode l Se r f -admin is t ra t ion o f e thano l Wi thdraw alA l c o h o l d e p e n d e n c e Inha la t ion o f e thano l

T H E R E i s a c o n t i n u i n g n e e d f o r p r a c t i c a l a n i m a l m o d e l s o fa l c o h o l i s m ( a l c o h o l d e p e n d e n c e ) . S u c h m o d e l s i d e a l l ys h o u l d m e e t t h e r i g o r o u s r e q u i r e m e n t s s e t b y C i c e r o [ I ] :( 1) A l c o h o l s h o u l d b e s e l f - a d m i n i s t e r e d in p h a r m a c o l o g i -c a l l y s ig n i f i ca n t a m o u n t s . P r e f e r a b l y , a l c o h o l s h o u l d b e c o n -s u m e d o r a l l y . T h e a m o u n t s o f a l c o h o l s e l f - a d m i n i s t e r e ds h o u l d e x c e e d t h e m e t a b o l i c c a p a c i t y o f t h e o r g a n i s m t os u c h a n e x t e n t t h a t p h a r m a c o l o g i c a l l y m e a n i n g f u l b l o o da l c o h o l l e v e l s a r e a c h i e v e d . S e l f - a d m i n i s t r a t i o n s h o u l d b ev o l u n t a r y .( 2) T o l e r a n c e s h o u l d d e v e l o p a f t e r a p e r i o d o f c h r o n i cs e l f - a d m i n i s t r a t i o n .( 3) A w i t h d r a w a l s y n d r o m e s h o u l d e n s u e u p o n c e s s a t i o no f a l c o h o l i n t a k e .( 4) F i n a l l y , t h e r e a r e a n u m b e r o f b i o m e d i c a l c o m p l i c a -t i o n s a s s o c i a t e d w i t h t h e c h r o n i c c o n s u m p t i o n o f a l c o h o l i nm a n w h i c h s h o u l d a l s o d e v e l o p i n th e a n i m a l m o d e l .I n a c r i t i c a l r e v i e w o f e x i s t i n g m o d e l s , C i c e r o [ 1 ] c o n -c l u d e d t h a t , a l t h o u g h m a n y a n i m a l m o d e l s c o m e c l o s e t om e e t i n g m a n y o f th e s e c r i t e r i a , a t p r e s e n t n o s i n g l e p a r a d i g mh a s s a t i s f i e d al l o f t h e m . W e b e l i e v e t h a t t h e p r i n c i p a l w e a k -n e s s o f m o s t , i f n o t a l l , m e t h o d s i s th e f a i l u r e t o e s t a b l i s hs e l f - a d m i n i s t r a t io n ra p i d l y a n d p r a c t i c a l l y . W e n o w d e s c r i b ea m o u s e m o d e l o f a l c o h o l i s m th a t m e e t s t h e f i r st th r e ec r i t e r i a o f C i c e r o i n a l m o s t a l l r e s p e c t s ; t h e f o u r t h o n e h a sn o t y e t b e e n p u t t o t e s t . I t is k n o w n t h a t f o r c e d i n h a l a t i o n o fe t h a n o l m a y p r o d u c e t o l e r a n c e i n m i c e [ 8 ] , a n d w i t h d r a w a lc o n v u l s i o n s w h e n a d m i n i s t r a t i o n o f e t h a n o l is s t o p p e d [ 7] .W e h a v e f o u n d t h a t m i c e w i l l v o l u n t a r i l y in h a l e th e v a p o u rw h e n g i v e n t h e c h o i c e b e t w e e n s t a y i n g in a n e th a n o lc h a m b e r o r i n a n a ir c h a m b e r . S u f f i c ie n t a m o u n t s o f e t h a n o la r e s e l f - a d m i n i s t e r e d t o p r o d u c e m e a s u r a b l e b l o o d l e v e l s o f

t h e d r u g , t o l e r a n c e a n d w i t h d r a w a l c o n v u l s i o n s . T h i s p a p e rw i l l e m p h a s i z e t h e m e t h o d o l o g i c a l a s p e c t s , w h i c h w i l l b ei l l u s t ra t e d w i t h tw o e x p e r i m e n t s . E x p e r i m e n t 2 w a s l a r g e l y ar e p l i c a t i o n o f E x p e r i m e n t 1 , w i t h t h e e x c e p t i o n t h a t s e l f-a d m i n i s t r a t i o n w a s s t u d i e d f o r a l o n g e r p e r i o d o f t i m e .

METHODSubjects

E i g h t y m a l e S w i s s r a n d o m l y - b r e d a l b i n o m i c e f r o mB r o e k m a n ( S t i p h o u t , th e N e t h e r l a n d s ) w e r e u s e d i n e a c h o ft h e t w o s t u d i e s . I n a d d i t i o n , f o u r g r o u p s o f 1 9 - 2 0 m i c e o f th es a m e s e x a n d s t r a i n s e r v e d a s c o n t r o l s f o r t h e a s s e s s m e n t o fw i t h d r a w a l r e a c t iv i t y an d f o r t h e m e a s u r e m e n t o f b l o o da l c o h o l l e v e ls . T h e a n i m a l s w e i g h e d 3 0 - 3 5 g a t t h e b e g i n n i n go f th e e x p e r i m e n t s .Apparatus

T h e a p p a r a t u s c o n s i s t e d o f t w o c h a m b e r s e a c h c o n t ai n i ng4 w i r e - m e s h s e g m e n t s , c o n n e c t e d b y a b r id g e ( F ig . 1 ). E a c hc h a m b e r w a s m a d e o f g r e y o p a q u e p l a s t i c (P V C ) w i t h at r a n s p a r e n t li d , a n d m e a s u r e d l l 0 x 6 0 x 4 0 e r a. T h e f e et o ft h e b r i d g e w e r e p l a c e d 1 00 c m a p a r t . T h e b r i d g e w a s c o n -s t r u c t e d o f P V C a n d w r a p p e d w i t h w i r e - m e s h t o a f f o r d af o o t h o l d f o r t h e m i c e . T h e l i d o f t h e c h a m b e r s p r o c e e d e di n t o a s i m i l a r ly t r a n s p a r e n t e n c a s e m e n t o f th e b r i d g e . T h es l o p e s o f t h e b r i d g e m a d e a n a n g l e o f 45 * r e l a t i v e to t h eh o r i z o n t a l p l a n e o f t h e c h a m b e r s . T h e t o p o f t h e b r i d g e ,w h e r e t h e t w o s l o p e s j o i n e d , w a s w e d g e - s h a p e d . A 9 c mh i g h , 1 m m t h i c k s t a i n l e s s s t e e l w a l l r o s e b e t w e e n t h e t o p o ft h e b r i d g e a n d t h e e n c a s e m e n t . A 7 x 6 c m o p e n i n g i n th e

1Present addres s : Resea rch S erv ice , V . A . M edica l Cen te r , Por t l and , OR 97201 .~Present address : Dutch Sc ience Counc i l , P .O . Box 18524 , 2502 EM The Hague , T he N e ther lands ( fo r r ep r in t r eques t s ).

C o p y r i g h t 1 98 2 P e r g a m o n P r e s s - -0 0 3 1 - 9 3 8 4 / 8 2 / 1 1 0 8 3 3 - 0 7 5 0 3 .0 0 / 0


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m i d d l e o f t h e w a l l , 2 c m f r o m t h e t o p o f t h e b r i d g e , a l l o w e dp a s s a g e f r o m o n e s l o p e to t h e o t h e r . T h u s , t h e m i c e c o u l du s e t h e b r i d g e t o m o v e f r o m o n e c h a m b e r t o t h e o t h e r b u t n o tt o e s c a p e f r o m t h e a p p a r a t u s .T h i s p a r t i c u l a r a p p a r a t u s w a s d e v e l o p e d a f t e r t e s t in g o f af e w l e s s s u c c e s s f u l p r o t o t y p e s . I n a f ir s t p r o t o t y p e , t h e t w oc h a m b e r s w e r e c o n n e c t e d a t f l o o r l e v e l b y a h o r i z o n t a lt r a n s p a r e n t t u b e ( 5 c m d i a m e t e r ) . T h e m i c e t e n d e d t o h u d d l ei n h e a p s i n t h e t u b e r a t h e r t h a n t o s e l e c t o n e o f th e c h a m -b e r s . T h e s l o p e s o f t h e b r i d g e u s e d in t h e p r e s e n t s t u d y w e r es u f f i c ie n t l y s t e e p t o p r e v e n t m i c e f r o m s e t t li n g d o w n o nt h e m , b u t n o t t o o d i f f ic u l t t o c l i m b f o r m o d e r a t e l y i n t o x i -c a t e d a n i m a l s . M o r e o v e r , w i t h t h e b r i d g e r i s in g 1 2 c m a b o v et h e l e v e l o f t h e l i d o f th e c h a m b e r s , e t h a n o l c o u l d n o t f l o wf r o m o n e c h a m b e r t o t h e o t h e r. T h e 2 c m r i d g e i n t h e o p e n i n go f t h e w a l l o n t o p o f t h e b r i d g e w a s n e c e s s a r y t o s t o p m i c ef r o m l y i n g w i th t h e i r b o d i e s f o l d e d a c r o s s t h e c u r v a t u r e o ft h e b r i d g e .T h e c h a m b e r s a n d b r i d g e w e r e d i v i d e d i n to f o u r o b l o n gs e g m e n t s o f 3 0 x 2 0 x 1 3 c m . T h e s e g m e n t s w e r e s e p a r a t e df r o m e a c h o t h e r b y d o u b l e w i r e - m e s h p a r t i t i o n s w i t h 2 c ms p a c e i n b e t w e e n . T h e w i r e - m e s h p a r t i ti o n s r a n a c r o s s t h eb r i d g e , s o t h a t e a c h s e g m e n t e x t e n d e d i n t o t h e o t h e rc h a m b e r . T h e s e g m e n t s h a d a l s o w i r e - m e s h f l o o r s. P V Cd r o p p a n s f i l l e d w i t h s a w d u s t w e r e i n s e r t e d u n d e r t h e f l o o r s .T h e p a n s w e r e r e m o v e d o n c e d a i l y a n d c l e a n e d . M i c e h a da c c e s s t o s t a n d a r d p e l l e t e d f o o d a n d t o w a t e r a t b o t h s i d e s o fe a c h s e g m e n t ( i . e ., in e a c h c h a m b e r ) . F o o d w a s a v a i l a b l ef r o m a t r o u g h a n d w a t e r f r o m b o t t l e s f ix e d t o t h e o u t s i d e o ft h e c h a m b e r s w i t h t h e s p o u t s p r o t r u d i n g t o t h e i n s i d e .

Establishment of Self AdministrationA t 1 0 :0 0 a . m . o n D a y 1 , t h e m i c e w e r e i n j e c t e d I P w i t h ap r i m i n g d o s e o f e t h a n o l ( 1 .2 5 g / k g) , f o l l o w e d b y p y r a z o l e( 6 8. 1 m g / k g ) . A g r o u p o f 2 0 m i c e w a s t h e n p l a c e d i n e a c hs e g m e n t a t t h e s i d e o f t h e a i r c h a m b e r . T h e o t h e r c h a m b e r

w a s d e n o t e d t h e e t h a n o l c h a m b e r . B e g i n n i n g 0. 5 h r b e f o r ep l a c i n g th e a n i m a l s i n t o t h e a p p a r a t u s , e t h a n o l v a p o u r w a sd e l i v e r e d i n to t h e e t h a n o l c h a m b e r t h r o u g h o u t t h e e x p e r i -m e n t e x c e p t i n g d a i l y b r e a k s ( s e e b e l o w ) . A i r w a s l e d t h r o u g ha c o n s t a n t v o l u m e o f a b s o l u t e e t h a n o l a n d f r o m t h e r e t o t h ec h a m b e r [ 10 ]. T h e f l o w ra t e o f t h e v a p o u r r a n g e d b e t w e e n4 a n d 5 1 / m i n, a n d w a s m i x e d w i t h 1 2 . 5 l / r a i n f l o w o fp r e s s u r i z e d a i r b e f o r e b e i n g i n f u s e d i n t o th e c h a m b e r .P r e s s u r i z e d a i r o f th e s a m e f l o w r a t e ( 1 6 . 5 -1 7 . 5 l / m i n ) w a sl e d i n to t h e a i r c h a m b e r ; t h i s is a n i m p o r t a n t p r e c a u t i o n , n o to n l y t o p r o v i d e t h e a n i m a l s w i t h s u f f i c i e n t o x y g e n i n b o t hc h a m b e r s , b u t a l s o t o p r e v e n t d i f f e r e n c e s i n a i r f l o w i n b o t hc h a m b e r s w h i c h m i g h t i n f l u e n c e th e a n i m a l s i n s e l ec t i n g ac h a m b e r . I n o r d e r to a c h i e v e a n e v e n d i s t r i b u t i o n o f v a p o u rt h r o u g h o u t t h e c h a m b e r , t h e v a p o u r w a s l e d in t o a p u n c t u r e dt u b e ( 1 . 5 c m d i a . ) t h a t r a n a l o n g t h e i n s i d e p e r i m e t e r o f t h r e es i d e s o f th e c h a m b e r a t a h e i g h t o f 3 0 c m a n d u n d e r n e a t h t h eb r i d g e a t a h e ig h t o f 1 2 c m . E t h a n o l w a s s a m p l e d f r o m t h ec h a m b e r e v e r y 1 o r 2 h r d u r i n g d a y t i m e a n d o c c a s i o n a l l y atn i g h t; i ts c o n c e n t r a t i o n w a s a s s a y e d g a s c h r o m a t o g r a p h -i c a l ly . T h e s a m p l e s w e r e t a k e n h a l f - w a y u p t h e c h a m b e r ( 3 5c m ) , u s in g a v a c u u m p u m p . D e t e r m i n a t i o n s w e r e m a d e w i t ha P a c k a r d 4 2 7 g a s c h r o m a t o g r a p h w i t h a t h e r m a l c o n d u c t i v -i t y d e t e c t o r ( f i l am e n t t e m p e r a t u r e : 3 00 C) a n d h e l i u m a s t h ec a r r i e r g a s w i t h a f lo w r a t e o f 1 0 m l / m i n . T h e t e m p e r a t u r e o ft h e c o l u m n w a s 1 70 C ; d e t e c t o r a n d i n j e c t i o n p o r t t e m p e r a -t u r e w a s 2 0 0 C . A 2 m , 2 m m i . d . ~ la s s c o l u m n p a c k e d w i t hP o r a p a k Q , 8 0 - 1 00 m e s h ( W a t e r s ) w a s u s e d . E t h a n o l , 1

( 10 0 e t h a n o l i n a c e t o n e ) , w a s u s e d a s s t a n d a r d . C h a m b e re t h a n o l c o n c e n t r a t i o n s r a n g i n g b e t w e e n 7 a n d 9 m g /1 ai r w e r ed e e m e d a c c e p t a b l e . T h e s e c o n c e n t r a t i o n s p r o d u c e t o l e r a n c ea n d , u p o n c e s s a t i o n o f a l c o h o l in f l o w , w i t h d r a w a l c o n v u l -s i o n s [ 1 0 ] b u t a r e n o t s o h i g h , t h a t a t a x i a e n s u e s d u r i n g i n h a -l a t io n o f v a p o u r . I f a c o n c e n t r a t i o n a p p e a r e d t o b e l o w e rt h a n 7 m g/ 1 o r h i g h e r t h a n 9 m g/ 1, t h e r a t e o f f lo w o f v a p o u rw a s a d j u s t e d .

A t 8 : 0 0 a . m . e a c h d a y , f r o m D a y 2 o n w a r d s , t h e m i c ew e r e t a k e n f r o m t h e a p p a r a t u s f o r 2 h r . D u r i n g t h a t p e r io d ,t h e c h a m b e r s w e r e c l e a n e d , e t h a n o l v a p o u r w a s r e d i r e c t e di n t o t h e n e w e t h a n o l c h a m b e r , a n d t h e a n i m a l s w e r e i n j e c te dI P w i t h t h e s t a n d a r d d o s e o f p y r a z o l e b e f o r e b e i n g p l a c e d i nt h e a i r c h a m b e r . A r a n d o m s c h e d u l e w a s u s e d f o r d e te r m i n -i n g w h i c h c h a m b e r w a s t o b e f i l le d w i t h e t h a n o l v a p o u r f o r ap a r t i c u l a r d a y , w i t h t h e r e s t r i c t io n t h a t n o c h a m b e r w a s t o b ea l l o t t e d to t h e e t h a n o l c o n d i t i o n f o r m o r e t h a n 2 d a y s i ns u c c e s s i o n .W e h a v e n o t e d t h a t m i c e , i n t h e a b s e n c e o f e t h a n o l ,r e a d i l y d e v e l o p a p r e f e r e n c e f o r o n e o f t h e t w o c h a m b e r s .T h i s p o s i t i o n p r e f e r e n c e w a s q u i t e s t r o n g i n p il o t e x p e r i -m e n t s , a n d a p p e a r e d t o b e p a r t l y a s s o c i a t e d w i t h an u n e v e nl i g h t i n f l u x i n t h e t w o c h a m b e r s . T h e r e f o r e t h e l i g h t b u l b s i nt h e e x p e r i m e n t a l r o o m w e r e p l a c e d e x a c t l y a b o v e t h e r i d g eo f t h e b r i d g e , a s s u r i n g e q u a l i l l u m i n a t io n o f t h e c h a m b e r s .T h e d a y - n i g h t c y c l e in o u r e x p e r i m e n t s w a s 1 2 :1 2 h r ( li g h t so n a t 6 a . m . ) . T h e i n fl u x o f l ig h t i n t o b o t h c h a m b e r s , m e a s -u r e d a t t h e f o o t o f t h e b r i d g e , w a s 1 50 l u x o n t h e a v e r a g ed u r i n g d a y t i m e , a n d 1 0 l u x d u r i n g n i g h t.T h e t e n d e n c y o f m i c e t o a d o p t a p o s i t i o n p r e f e r e n c e m a yh i n d e r o r c o n f o u n d t h e d e v e l o p m e n t o f s e lf - a d m i n i s t r a ti o n .T o c o u n t e r a c t t h i s t e n d e n c y , w e e x p r e s s l y i n t r o d u c e d ad a r k - l i g h t d i f f e r e n c e b e t w e e n t h e t w o c h a m b e r s f o r t h e f i rs ts i x d a y s o f t h e e x p e r i m e n t . D u r i n g t h i s p e r i o d , t h e l i d o f t h ee t h a n o l c h a m b e r w a s c o v e r e d w i t h a n o p a q u e p l a s t ic p l a t e .T h e r e s u l t in g d a r k n e s s i n d u c e d t h e m i c e t o s e l e c t t h e e t h a n o lc h a m b e r , i r r e s p e c t i v e o f it s p o s i t i o n o n a p a r t i c u l a r d a y . O nD a y 7 , th e c o v e r w a s r e m o v e d . T h e u n p r o m p t e d s e l e c t i o n o ft h e e t h a n o l c h a m b e r b y t h e m i c e d u r i n g t h e s u b s e q u e n t p a r to f t h e e x p e r i m e n t ( D a y s 7 t h r o u g h I 0 f o r E x p e r i m e n t 1 D a y s7 t h r o u g h 13 f o r E x p e r i m e n t 2 ) w a s c o n s i d e r e d e v i d e n c e f o rv o l u n t a r y s e l f - a d m i n i s tr a t i o n . A t h o u r l y i n t e r v a l s , b e g in n i n ga t 1 1 :0 0 a . m . ( t h a t i s , 1 h r a f t e r p l a c e m e n t o f t h e m i c e i n t h ea p p a r a t u s ) t h r o u g h 1 6:0 0 p . m . , t h e n u m b e r o f m i c e f ro me a c h s e g m e n t s t a y i n g in th e e t h a n o l c h a m b e r a n d t h e n u m b e ro f m i c e s t a y i n g i n th e a i r c h a m b e r w e r e s c o r e d . T h e d i s tr i -b u t i o n o f m i c e w a s a l s o c o u n t e d a t 8 : 0 0 a . m . e a c h d a y , p r i o rt o t h e d a i l y b r e a k . I n a d d i t i o n , t h e m i c e w e r e s c o r e d a t an u m b e r o f n ig h t l y h o u r s o n D a y 10 .Measurement of Withdrawal Convulsions

A t 8 : 0 0 a .m . o n D a y 11 o f E x p e r i m e n t 1 , t h e m i c e w e r et a k e n f r o m t h e a p p a r a t u s . E x c e p t f o r th e m i c e u s e d f o rm e a s u r i n g b l o o d e t h a n o l l e v e l s ( s ee b e l o w ) , a l l a n i m a l s w e r es u b j e c t e d t o c o n v u l s i o n t e s t i n g , s t a r t i n g i m m e d i a t e l y a f t e rw i t h d r a w a l a n d c o n t i n u i n g a t h o u r l y i n t e r v a ls f o r u p t o 6 h ra f t e r w i th d r a w a l . W i t h d r a w a l c o n v u l s io n s w e r e s c o r e d w i t h as l i g h tl y m o d i f i e d v e r s i o n o f G o l d s t e i n ' s m e t h o d [ 4] . C o n v u l -s i o n s w e r e i n d u c e d b y h a n d l i n g o f t h e m i c e . A s c o r e o f I w a sg i v e n t o a n i m a l s d i s p l a y i n g a t o n i c c o n v u l s i o n w h e n l i f t e d b yt h e t a f t a n d t u r n e d g e n t l y 1 8 0 C. A s c o r e o f 2 w a s a s s i g n e d w h e na m o r e i n t e n s e t o n i c - c l o n i c c o n v u l s i o n w a s e l i c it e d b y t h et u r n in g . S c o r e s o f 3 a n d 4 w e r e g i v e n t o t o n i c - c l o n i c c o n v u l -s i o n s n o t r e q u i r i n g a n y t u r n i n g , w i t h a s c o r e o f 4 a s s i g n e d t o


3/7

M O U S E M O D E L O F A L C O H O L I S M 835

FIG . 1 . V iew of the two cham bers and the in te rconnec t ing b r idge . The f igure shows the four segm ents runn ingacross the b r idge .

v i o l e n t c o n v u l s i o n s . W i t h d r a w a l c o n v u l s i o n s w e r e a l s os c o r e d f o r a l l o f th e m i c e f r o m E x p e r i m e n t 2 . T h i s w a s d o n eo n D a y 1 4, i m m e d i a t e l y a f t e r w i t h d r a w a l , a n d 3 - 7 h r l a t e r a th o u r l y i n t e r v a l s .Measurement o f Blood Ethanol Levels

O n D a y 11 o f E x p e r i m e n t 1 , 15 m i c e ( 2 - 5 p e r s e g m e n t )w e r e r a n d o m l y p i c k e d f r o m a m o n g t h o s e a n i m a l s s t a y i n g i nt h e e t h a n o l c h a m b e r a t t h e t i m e o f w i t h d r a w a l . I n a d d i t i o n , 6m i c e ( 3 f r o m o n e s e g m e n t ; 3 f ro m a n o t h e r ) w e r e r a n d o m l yt a k e n f r o m t h e n u m b e r o f a n i m a l s p r e s e n t i n th e a i r c h a m b e r .A l l t h e s e l e c t e d m i c e w e r e i m m e d i a t e l y d e c a p i t a t e d , 0 . 5 m lt r u n k b l o o d w a s c o l l e c t e d in a h e p a r i n i z e d c u p a n d w a s i m -m e d i a t e l y f r o z e n o n d r y i c e . B l o o d s a m p l e s w e r e t r e a t e d a sp r e v i o u s l y d e s c r i b e d [ 2] . D e t e r m i n a t i o n s w e r e m a d e w i t h aP e r k i n - E l m e r F - 3 0 g a s c h r o m a t o g r a p h a n d a P e r k i n - E l m e ra u t o s a m p l e r A S 4 1 w i th a f l a m e i o n i z a t i o n d e t e c t o r a n d n i -t r o g e n a s t h e c a r r i e r g a s a t a f l o w r a t e o f 4 0 m l / m i n . T h et e m p e r a t u r e o f t h e c o l u m n w a s 1 80 0C ; d e t e c t o r a n d i n j e c ti o np o r t t e m p e r a t u r e w a s 2 5 00 C. A 2 m , 4 m m i n s i d e d i a m e t e rg l a s s c o l u m n w a s l a a c k e d w i t h P o r a p a k Q , 8 0 t o I 0 0 m e s h( . W a t e rs ) a n d c o n d i t i o n e d b y h e a t i n g o v e r n i g h t a t 2 00 C w i t hc a r r i e r g a s f l o w . P e a k a r e a s w e r e d e t e r m i n e d u s i n g th e V a r -i a n C h r o m a t o g r a p h y D a t a S y s t e m 2 40 .Statistical nalyses

F o r t h e s e r f - a d m i n i s t r a t i o n d a t a , i t w a s a s s u m e d t h a t i fm i c e n e i t h e r p r e f e r r e d n o r a v o i d e d th e e th a n o l , 5 0 o f t h e

a n i m a l s w o u l d b e i n th e e t h a n o l c h a m b e r a t a n y g i v e n t im e .O n e s c o r e w a s d e r i v e d f o r e a c h t e s t d a y f o r e a c h s e g m e n t b ya v e r a g i n g t h e h o u r l y d i s t r i b u t i o n s f o r t h a t d a y . W h e n 5 0 o rm o r e o f th e m i c e f r o m o n e s e g m e n t ( a v e r a g e d o v e r r e p e a t e dh o u r l y c o u n t s ) s e l e c t e d t h e e t h a n o l c h a m b e r o n a t e s t in gd a y , a + s c o r e w a s a s s i g n e d ; f o r l e s s t h a n 5 0 , a - s c o r ew a s g i v e n . T h u s , f o r 3 t e s t in g d a y s a n d 4 s e g m e n t s a t o t a l o f1 2 + s c o r e s w o u l d s i g n a l f l a w l e s s s e l e c t io n o f t h e e t h a n o lc h a m b e r b y t h e m a j o r i t y o f th e a n i m a l s i n e a c h s e g m e n t .D e v i a t i o n f r o m c h a n c e l e v e l w a s t e s t e d w i t h t h e X 2 -o nes a m p l e t e s t . I t s h o u l d b e n o t e d t h a t s o c i a l b o n d s a r e n o t ac o n f o u n d i n g f a c t o r i n t h i s a n a l y s i s . I f m i c e i n a s e g m e n t w e r et o c h o o s e a s a g r o u p o n e o r th e o t h e r c h a m b e r , a c h a n c ed i s t r i b u t i o n o f + a n d - s c o r e s w o u l d b e e x p e c t e d a c r o s sd a y s i f e t h a n o l w e r e n o t a r e i n f o r c i n g a g e n t . I n f a c t , w e k n o wf r o m u n p u b l i s h e d c o n t r o l s t u d i e s th a t i f b o t h c h a m b e r s c o n -t a i n e i t h e r e t h a n o l o r a i r , m i c e a s a g r o u p d e v e l o p e d a p o s i -t i o n p r e f e r e n c e . T h a t i s , t h e y s e t t l e d d o w n e a c h d a y i n e i t h e rt h e l e f t o r t h e r i g h t c h a m b e r . I f m i c e f r o m t h e p r e s e n t e x p e r -i m e n t s w o u l d h a v e d o n e t h e s a m e , a c h a n c e n u m b e r o f +s c o r e s w o u l d h a v e b e e n r e c o r d e d ( 6 in th e a b o v e e x a m p l e ) .T h e w i t h d r a w a l d a t a w e r e a n a l y z e d a s f o l lo w s . T h e m e a nc o n v u l s i o n s c o r e a n d c o r r e s p o n d i n g s t a n d a r d e r r o r w e r e c a l -c u l a t e d a t e a c h p o i n t a n d p l o t t e d t o s h o w t h e t i m e c o u r s e o ft h e w i t h d r a w a l r e a c t i o n . D i f f e r e n c e s b e t w e e n s u b g r o u p s o fm i c e w e r e e v a l u a t e d f o r e a c h t i m e p o i n t w i t h a m o d i f i e dx 2 - t e s t [ 1 1] . D i f f e r e n c e s i n m e a n l e v e l s o f e t h a n o l i n b l o o db e t w e e n s u b g r o u p s o f an i m a l s w e r e a n a l y z e d b y m e a n s o f at w o - t a i l e d S t u d e n t ' s t - t e s t .


4/7

836 R1JK, CRABBE AND RIGTERM e d i a n p e r c e n t a g eo f m i c e p r e f e r r i n ga l c o h o l

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FIG. 2. Median percentage of mice preferring the ethanol chamberin Experiment 1 and 2. The data points are the median percentagesof ethanol-selecting mice across all segments and all times of obser-vation for a particular day. The bars represent semi-interquartileranges.

M e d i a n p e r c e n t a g eo f m i c e p r e f e r r i n ga l c o h o l1 0 0 - D ay 7 ~ . . ' . . ~ . t . . j / ~ . ' ~ .

7 5 ///5 0

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1'1 12a . m .

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FIG. 3. Median percentage of mice preferring the ethanol chamberin Experiment 1 for each time of observation during daytime:

R E S U L T S

Ethanol Self-AdministrationWhen mice are housed in an ethanol chamb er from whichno escape is possible and thus forcedly exposed to concen-trations of ethanol va pour similar to those used in the presen texperiments, they will develop tolerance and, upon cessa-tion of ethanol treatment, withdrawal convulsions [10]. Themethod described here offered the mice an escape route, butduring the first six days of the experiments the dark-lightdifference between the ethanol and the air chambersprompted the animals to select the former as their principalterritory. This primin g procedure was highly successful.

At virtually all times of observation during these first sixdays, more than 85-90% of the mice from each segment werepresent in the (dark) ethanol chamber. When placed in the airchamber at the beginning of each day, mice would exploreboth chambers for about 30-60 min and then settle down tosleep in the ethanol chamber. During daytime hours, amouse would occasionally wake up and move from thecrowded ethanol chamber to the quiet air chamber to eat o rdrink. These short excursions accounted for the fact that atmany times of observation a few mice were seen in eachsegment of the air chamber. At night, these short excursionswere more frequent. Nevertheless at any one time more than85% of the mice were in the ethanol chamber.The self-sought exposure to ethanol during the first sixdays resulted in the death of a few animals, 1 or 2 mice fromthree out of four segments (a total of 4 animals in bothstudies). During the testing period, no further deaths oc-curred.The actual test of self-administration, the unpromptedselection of ethanol chamber, was begun on Day 7. In Exper-iment 1, preference testing was continued through Day 10,and in Experiment 2 through Day 13. In both studies, themedian percentage of mice continuing to prefer the ethanolchamber was 90% or more during the first three testing days(Days 7-9; Fig. 2). When the data from Days 7-9 were sub-mitted to statistical analysis, mice were shown to select theethanol chamber significantly above chance level (both ex-periments: X2=8.3, dr= l p


5/7


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F I G . 4 . M e d i a n p e rc e n t a g e o f m i c e p r e f e rr i n g t h e e t h a n o l c h a m b e r o n D a y 1 0 o fExper im ent 1 and 2 fo r each t ime o f obse rva t ion dur ing day t ime and n igh t . Thebars represen t semi- in te rquar t i l e r anges .

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i i i , )I 3 5

T i m e a f t e r w i t h d r a w a l I h o u r s )

FIG . 5 . The t ime c ourse o f wi thdrawal reac t iv i ty fo r mice f romExper iment 1 . Da ta po in t s a re mean convu ls ion scores ; ba r s a res t a n d a r d e r r o r s o f th e m e a n . T h e u p p e r c u r v e s a r e f o r m i c e f r o m t h eexper im enta l g roup . Th ere a re separa te cu rves fo r mice tha t were int h e e t h a n o l c h a m b e r ( n = 5 2 ) a n d t h e a i r c h a m b e r ( n = l l ) , r e s p e c -t ive ly , a t the t im e o f w i thdrawal ( l e f t pane l ) . A d i f fe ren t g roup inginvo lved the m ice f rom the th ree segments wi th e thano l p re fe rencec o n t i n u e d t h r o u g h t h e l a s t d a y o f t es t i n g ( h i g h - p r e f e r e n c e , n = 4 4 ) ,and mice f rom the four th segm ent whose p re fe rence dec l ined on thelas t day o f t e s t ing ( low -pre fe rence , n= 19) ( r igh t pane l ) . In bo thpane l s the resu l t s a re shown fo r con t ro l mice tha t d id no t haveaccess to e thano l bu t had been g iven da l ly in jec t ions wi th e i the rsa l ine (c rosses ) o r py razo le ( t r i ang les ) (n=20 each) . Fo r the sake o fc la r i ty , the s tandard e r ro r s o f the mean a re no t shown fo r the da tapo in t s o f the con t ro l g roups ; these va lues ranged be tween 0 and0.13.

hamber Ethanol oncentrationsE t h a n o l c o n c e n t r a t i o n s e a c h d a y w e r e k e p t w i t h i n t h e s e tr a n g e , 7 - 9 m g e th a n o l / l ai r . E t h a n o l c o n c e n t r a t i o n s w e r ef a i r l y c o n s t a n t d u r i n g d a y t i m e , a n d u p t o m i d n i g h t . H o w -e v e r , i n b o t h s t u d i e s , t h e a v e r a g e c o n c e n t r a t i o n m e a s u r e d a t8 : 00 a . m . w a s l o w e r th a n t h e a v e r a g e c o n c e n t r a t i o n m e a s -u r e d b e t w e e n 1 1 :0 0 a . m . - 1 6 : 0 0 p . m . ( E x p e r i m e n t 1 , 8 . 0- + 0. 1v s . 7 . 3 - + 0 .2 ra g/ 1 a i r , t ( 4 9 ) = 3 . 5 0 , p < 0 . 0 1 ; E x p e r i m e n t 2 ,8 .8 _+ 0 .2 v s . 7 . 2_ + 0.5 m g / 1 a i r , t ( 3 0 ) = 3 .0 2 , p < 0 ,0 1 ) . E t h a n o lc o n c e n t r a t i o n i n t h e a i r c h a m b e r w a s l o w e r t h a n 0 .1 m g /1 a ir

w h e n m i c e w e r e a b s e n t f r o m t h e c h a m b e r , b u t o c c a s i o n a ll yr o s e t o v a l t ie s o f 1 -2 m g q a i r , w h e n t h e a n i m a l s m a d e e x c u r -s i o n s t o t h e a i r c h a m b e r .Withdrawal

T h e t i m e c o u r s e o f w i t h d r a w a l r e a c t i v i t y f o r m i c e f r o mE x p e r i m e n t 1 i s i l l u s t ra t e d in F i g . 5 . S e p a r a t e c u r v e s a r es h o w n f o r m i c e t h a t w e r e i n th e e t h a n o l c h a m b e r a n d t h e a i rc h a m b e r , r e s p e c t i v e l y , a t t h e t i m e o f w i t h d r a w a l ( l e f t p a n e l ) .A d i f f e r e n t w a y t o g r o u p t h e d a t a w a s t o c o m p a r e t h e m i c ef r o m t h e t h r e e s e g m e n t s w h e r e e t h a n o l p r e f e r e n c e c o n t i n u e dt h r o u g h t h e l a s t d a y o f t e s ti n g ( i r r e s p e c t i v e o f w h e r e t h e yw e r e a t t h e t i m e o f w i t h d r a w a l ) , a n d m i c e f ro m t h e f o u r t hs e g m e n t , w h o s e p r e f e r e n c e f o r e t h a n o l d e c l i n e d o n t h e l a s td a y o f te s t i n g (r i gh t p an e l ) . M o s t m i c e h a d w i t h d r a w a l c o n -v u l s i o n s , b u t t h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s b e t w e e ns u b g r o u p s a t a n y t i m e o r a c r o s s a l l t i m e s o f m e a s u r e m e n t ,e x c e p t f o r 6 h r a f t e r w i t h d r a w a l ( t ( 61 ) = 2 . 4 1 , l e f t p a n e l ,p < 0 . 0 5 ; t ( 6 1 ) = 4 . 2 0 , r i g h t p a n e l , p < 0 . 0 0 1 ) . T h i s w a s d u e t ol a r g e v a r i a ti o n s i n s c o r e s w i t h i n g r o u p s a n d t h e s m a l l n u m b e ro f m i c e i n t h e a i r a n d t h e l o w - p r e f e r e n c C ' g ro u p s . A l s o ,i t is p o s s i b l e t h a t t h e t i m e p e r i o d d u r i n g w h i c h w i t h d r a w a lc o n v u l s i o n s w e r e f o l l o w e d w a s t o o s h o r t ; t h e c u r v e s i n F i g . 5o n l y s t a r t t o p a r t a t t h e e n d o f t h e t e s t i n g p e r i o d .T h e a v e r a g e c o n v u l s i o n s c o r e f o r c o n t r o l m i c e t h a t h a dn o t r e c e i v e d e t h a n o l b u t o n l y d a i l y i n j e c ti o n s w i t h e i t h e rs a l in e o r p y r a z o l e n e v e r r o s e a b o v e 0 . 3 0 ( s h o w n i n b o t hp a n e l s ) . T h e s e c o n t r o l g r o u p s w e r e c l e a r l y d i ff e r e n t f r o m t h ee x p e r i m e n t a l ( s u b - ) g r o u p s a t a l l t i m e s o f t e s t i n g ( t s > 2 . 3 6 ,p < 0 . 0 5 ) , e x c e p t fo r s o m e o f th e c o m p a r i s o n s a t t h e 6 h r t im ep o i n t .M i c e f r o m E x p e r i m e n t 2 a l s o d i s p l a y e d w i t h d r a w a l c o n -v u l s i o n s ( o v e r a l l a v e r a g e p e a k s c o r e : 1 .9 5_ +0 .1 3) .T h e m i ce f r om t h e l o w - p r e f e r e n c e s e g m e n t o f E x p e r i -m e n t 1 h a d l o w b l o o d e t h a n o l l e v e l s , w h e t h e r o r n o t t h e yw e r e p r e s e n t i n th e e t h a n o l c h a m b e r a t t h e t i m e o f w i t h -d r a w a l (0 .0 3_ +0 m g / m l , n = 2 ; a n d 0 . 2 5 -+ 0 . 1 2 , n = 3 , r a n g e :0 . 0 2 - 0 .3 8 , r e s p e c t i v e l y ) . T h e m i c e f r o m t h e th r e e h i g h -p r e f e r e n c e s e g m e n t s h a d h i g h e r b l o o d e t h a n o l l e v e l s ,w h e t h e r o r n o t t h e y w e r e i n th e e t h a n o l c h a m b e r a t t h e ti m eo f w i t h d r a w a l ( m e a n - + s . e . m . : 0 . 6 0 -+ 0 .1 1 m g e t h a n o l / m lb l o o d , n = 1 3 , r a n g e : 0 . 0 3 - 1 . 3 ; a n d 0 . 5 6 -+ 0 . 0 6 , n = 3 , r a n g e :0 . 4 6 - 0 .6 8 , r e s p e c t i v e l y ) . T h e d i f f e r e n c e b e t w e e n t h e p o o l e d


6/7

838 RIJK, CRABBE AND RIGTER

animals from th e high prefe rence segments and the pooledanimals from the low- prefer ence segment was significant(t(19)=3.50, p


7/7


o b v i o u s i m p o r t a n c e . W e f o u n d a s o m e w h a t l o w e r e d c o n c e n -t r a t i o n o f e t h a n o l i n t h e e t h a n o l c h a m b e r e a r l y i n th e m o r n -i n g ( 8 : 00 a . m . ) , c o m p a r e d w i t h s a m p l e s t a k e n d u r i n g t h em i d d l e o f t h e d a y . T h i s c o u l d b e d u e t o t h e i n c r e a s e d m o t i l i t yo f t h e m i c e d u r i n g t h e n i g h t . T h e a n i m a l s m i g h t h a v e c a r d e de t h a n o l i n t h e i r fu r , o r e x h a l a t e , t o t h e a i r c h a m b e r , t h u sr e d u c i n g t h e c o n c e n t r a t i o n i n t h e e t h a n o l c h a m b e r . A t a n yr a t e , e t h a n o l c o n c e n t r a t i o n s w e r e a l w a y s c l e a r l y h i g h e r i nt h e e t h a n o l c h a m b e r ( 7 - 9 m g/ 1 a i r) t h a n i n t h e a i r c h a m b e r( 1 - 2 m g / l a i r a t m o s t ) . W e h a v e l o o k e d f o r w a y s t o f u r t h e rr e d u c e t h e e t h a n o l c o n c e n t r a t i o n i n t h e a i r c h a m b e r . T h i sc o u l d b e d o n e b y e n h a n c e d v e n t il a ti o n o f t h e a i r c h a m b e r b u tw e h a v e f o u n d t h a t t h e r e s u l t i n g d r a u g h t c a u s e s m i c e t oa v o i d t h e ai r c h a m b e r . W e d e c i d e d t o e n h a n c e v e n t i l a t i o n o ft h e a i r a n d t h e e t h a n o l c h a m b e r t o t h e s a m e e x t e n t , a n d i ns u c h a w a y t h a t e t h a n o l c o n c e n t r a t i o n s i n t h e e t h a n o lc h a m b e r a r e n o t a f f e c t e d w h i l e e t h a n o l c o n c e n t r a t i o n s i n t h ea i r c h a m b e r a r e r e d u c e d t o v i r t u a ll y z e r o l e v el s . U n d e r t h e s ec o n d i t i o n s , m i c e s ti ll s e l e c t c o n s i s t e n t l y t h e e t h a n o l c h a m b e r(R ig te r , u n p u b l i s h e d f in d in g s ) .

T h e m o d e l w e r e p o r t l a r g e l y m e e t s C i c e r o s [ 1 ] f i r s t t h r e ec r i t e r ia f o r a n a n i m a l m o d e l o f a l c o h o l i s m . R e g a r d i n g h i sf o u r t h c r i t e r i o n , w e d o n o t k n o w i f c h r o n i c s e l f -a d m i n i s tr a t io n o f e t h an o l v a p o u r p r o d u c e s b i o m e d i c a l c o m -p l i c a t i o n s, s u c h a s li v e r a n d b r a i n d a m a g e , s i n c e w e h a v e n o ty e t t e s t e d t h is . P r e s u m a b l y , f o r s u c h c o m p l i c a t i o n s t o o c c u r ,s e l f - a d m i n i s tr a t i o n w o u l d n e e d t o b e c o n t i n u e d f o r l o n g e rp e r i o d s o f ti m e t h a n i n t h e p r e s e n t e x p e r i m e n t s . W e u s e d af i x e d r a n g e o f e t h a n o l c o n c e n t r a t i o n s a c r o s s p r i m i n g a n dt e s t i n g d a y s . I t m a y b e t h a t p r e f e r e n c e d i m i n i s h e s b e c a u s e o ft h e d e v e l o p m e n t o f m e t a b o l i c to l e r a n c e , a n d t h a t p r e f e r e n c ec a n b e e x t e n d e d w h e n e t h a n o l c o n c e n t r a t io n s a r e g r a d u a l lyr a i s e d . W e a r e p r e s e n t l y t e s t i n g t h i s p o s s i b il i t y . H o w e v e r , i ts h o u l d b e n o t e d t h a t t h e r e i s n o r e a s o n w h y m i c e s h o u l dm a i n t a i n p r e f e r e n c e p e r m a n e n t l y . I t b e c o m e s in c r e a s i n g l yc l e a r t h a t m a n y h u m a n a l c o h o l i c s o s c i l l a t e b e t w e e n p e r i o d so f e x c e s s i v e s e l f - a d m i n i s t r a t io n a n d p e r i o d s o f a b s t i n e n c e[3,9].C i c e r o [ 1 ] p r o p o s e d t h a t a n i m a l s , i n a r e l e v a n t m o d e l o f

a l c o h o l i s m , s h o u l d d r i n k t h e a l c o h o l . C l e a r l y , o u r m o d e ld o e s n o t m e e t t h is p o s s ib l e r e q u i r e m e n t . H o w e v e r , i t isq u e s t i o n a b l e w h e t h e r t h i s i s p r o h i b i t o r y . A t t e m p t s t o d e -v e l o p r o d e n t d r i n k i n g m o d e l s o f a l co h o l i s m g e n e r a l l y h a v en o t b e e n c o m p l e t e l y s u c c e s s f u l [ 1 ] . I f t h e o r a l r o u t e o fe t h a n o l c o n s u m p t i o n w o u l d b e a n a b s o lu t e r e q u i r em e n t , w ee x p e c t t h a t a n i m a l m o d e l s , i f f e a s i b le a t a l l , w i ll b e e i t h e rt i m e - c o n s u m i n g o r e x p e n s i v e ( e . g . , u s i n g p r i m a t e s ) .A n o t h e r p o s s i b l e l i m i t a ti o n i s t h e u s e o f p y r a z o l e s i n c et h i s i n h ib i t o r o f t h e m e t a b o l i s m o f e th a n o l h a s a n u m b e r o fs i d e e ff e c t s [ 6] . H o w e v e r , p y r a z o l e i s i m p o r t a n t f o r t h e m a i n -t e n a n c e o f s t a b l e b l o o d a l c o h o l le v e l s. T h e r e s u l t s f r o m u n -p u b l i s h e d c o n t r o l e x p e r i m e n t s i n d i c a t e d t h a t p y r a z o l e -t r e a t e d m i c e d i d n o t d i f f e r s u b s t a n t i a l l y f r o m s a l i n e - t r e a t e dm i c e i n s hu t tl i ng b e t w e e n c h a m b e r s w h e n n o e t h a n o l v a p o u rw a s i n f u s e d . A l s o , p y r a z o l e i t s e f f c o u l d n o t e x p l a i n t h e s e -v e r e c o n v u l s i o n s s e e n i n m a n y e t h a n o l - w i t h d r a w n m i c e .T h u s , i t i s u n l i k e l y t h a t p y r a z o l e i s a n i n s u r m o u n t a b l y c o n -f o u n d i n g f a c t o r i n t h e p r e s e n t m o d e l . N e v e r t h e l e s s , w e a r el o o k i n g f o r w a y s t o o m i t p y r a z o l e t r e a t m e n t . A s i n s tu d i e s o fe t h a n o l w i t h d r a w a l [ 6 ] , i t m a y b e p o s s i b l e t h a t s e r f -a d m i n i s t r a t i o n w i l l d e v e l o p i n m i c e t h a t a r e n o t i n j e c t e d w i t h

p y r a z o l e b u t a r e e x p o s e d t o h i g h e r c o n c e n t r a ti o n s o f e t h an o lf o r l o n g e r p e r i o d s o f t i m e t h a n u s e d s o f a r .I n c o n c l u s i o n , s e r f - a d m i n i s t r at i o n o f e t h a n o l v a p o u r , w i t hr e s u l t i n g t o l e r a n c e a n d w i t h d r a w a l r e a c t i v i t y , m a y b e u s e da s a m o d e l o f a l c o h o l i s m . T h e m e t h o d s d e s c r i b e d h e r e a r en o t y e t p e r f e c t . P o s s i b l e a m e n d m e n t s a r e : d e l e t i o n o fp y r a z o l e t r e a t m e n t , a u t o m a t i c c o n t r o l o f e t h a n o l c o n c e n t r a -t i o n s i n a i r d u r i n g d a y a n d n i g h t , m o n i t o r i n g o f b e h a v i o u r o fi n d i v i du a l m i c e , a n d c o n s t r u c t i o n o f m o r e s e g m e n t s t o i n -c r e a s e p o w e r o f s t a t is t i ca l a n a l y s i s . I n a d d i t i o n , a s t r i n g o fm o r e t h a n t w o c h a m b e r s c o u n d b e m a d e , w i t h o n e a i rc h a m b e r a n d a n u m b e r o f c h a m b e r s w i t h d i ff e re n t c o n c e n -t r a t i o n s o f e th a n o l . T h u s , t h e a n i m a l s w o u l d n o t b e f o r c e d t om a k e a n a l l - o r - n o n e c h o i c e b e t w e e n e t h a n o l a n d a i r , b u tw o u l d b e a b l e t o t i t r a t e t h e i r e t h a n o l s a m p l i n g b e h a v i o u ra c c o r d i n g t o t h e i r l e v e l o f p r e f e r e n c e .

R E F E R E N E S1. Cicero, T. J. Alc oho l self-administration, tolera nce and with-drawal in humans and animals: theoretical and methodologicalissues. In: A l c o h o l T o l e r a n c e a n d D e p e n d e n c e edited by H.Rigter and J . C. Crabbe. Amsterdam: Elsevier/North-HollandBiomedical Press, 1980, pp. 1-51.2. Crabbe, J. C., H. Rigter, J. Uijlen and C. Strijbos. Rapid devel-opment o f to le rance to the hypothermic e ffec t o f e thano l inmice. J . P h a r m a c . e x p . T h e r . 208: 128-133, 1979.3. Ed ward s, G. The alcohol depend ence syndrom e: usefulness ofan idea. In: A l c o h ol is m . N e w K n o w l e d g e a n d N e w R e s p o n s e s

edited by G. Edw ards and M. Grant. Lon don : Croon and Helm,1977, pp. 136-156.4. Goldstein, D. B. Relationship of alcohol dose to intensity ofwithdrawal signs in mice. J . P h a r m a c . e x p . T h e r . 180: 203-215,1972.5. Goldstein, D. B . Rate of onset and de cay of alcohol physicaldependen ce in mice. J . P h a r m a c . e x p . T h e r . 190: 377-383, 1974.

6. Goldstein, D. B. Inhalation of ethanol vapor. In: A l c o h o lT o l e r a n c e a n d D e p e n d e n c e edited by H. Rigter and J . C.Crabbe. Amst erdam : Elsevier/North-Holland Biomedical Press,1980, pp. 81-92.7. Goldstein, D . B. and N. Pal. Alco hol dependen ce produce d inmic e by inhalation o f ethanol: grading the withdraw al reaction.S c i e n c e 172: 288-290, 1971.8. Grieve, S. J . and J . M. Littleton. The rapid developme nt ofcellular tolerance to ethanol in mice. J . P h a r m . P h a r m a c . 31:605-610, 1979.9. Pattison, E. M., M. B. Sobell and L. C. Sobell. E m e r g i n g C o n -c e p t s o f A l c o h o l D e p e n d e n c e . New York: Springer, 1977.10. Rigter, H . , H . Rijk and J . C. Crabbe. Tolerance to ethanol andseverity of withdrawal in mice are enhanced by a vasopressinfragment. E u r . J . P h a r m a c . 64: 53-68, 1980.11. Yates , F. T he analysis of contingenc y tables with groupingsbased on quantita tive characters . B i o m e t r i k a 35: 1978, 1948.

a mouse model of alcoholism

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