Induction of Physical Dependence Upon Ethanol and the Associated Behavioral Changes in Rats

8/12/2019 Induction of Physical Dependence Upon Ethanol and the Associated Behavioral Changes in Rats

1/10

Psychopharm acologia (Berl .) 43, 24 5- 25 4 (t975) - 9 by Springer-Verlag 1975

Induct io n o f Phy s ica l D ep enden ce u po n E t ha no land the Assoc ia ted Behav iora l Changes in Rats

E D W A R D M A J C H R O W I C ZLab orato ry of Alcohol Research, N ational Inst itute on Alcohol Abuse and Alcoholism, A DA MH A,William A. White Building, Saint Elizabeths Hosp ital, Washington, D.C.

Received September 25, 1974; Final Version May 1, 1975A b s t r a c t . This paper reports findings relative to a simple,rapid an d repro ducible technique for the induction o f physicaldependence upon ethanol in the rat. The dependence wasinduced by intragastric intuba tion o f 20 % (w/v) ethanol solu-t ions a t 9 -1 5 g/kg in 3 - 5 fract ional doses daily for 4 days ,maintaining blood ethanol concentra t ions above a thresholdlevel sufficient to sustain observable sedation thro ugh out theentire period of intubation. Two phases were distinguishedduring the withdrawal period: 1. P r o d r o m a l d e t o x i c a t i o ncharacterized by a spe ctrum o f signs and responses of diminish-ing severity, related to the decline in blood ethanol concen-trations (mg/dl): death, > 640; coma, 78 0 - 460; loss of right-

ing reflex, 64 0-4 40 ; a taxia 3 -1 , 57 0-2 50 ; sedation,340-190; neutra l i ty, 220-130; 2. E t h a n o l d e p e n d e n c e charac-terized by a spectrum of withdrawal signs and reactions o fprogressively increasing severity as blood ehtanol concen-tration approached 100mg/dl: hyperactivity, tremors, aki-nesia, spastic rigidity, and induced and spontaneous convul-sions. A rapid succession of two diverse clusters o f signs andreactions represents a reversal of the central nervo us systemfunct ion from the extremes of e thanol intoxicat ion (CNSdepression) to the extremes of ethanol dependence (CNShyperexcitability) during the withdrawal period. Bothextremes may term inate in death.

K e y w o r d s : Ethano l - Ethanol Dependence - Physical Dependence - Induct ion of Ethanol Dependence - Prodro malDetoxicat ion - Alcohol Withdrawal Syndrome - Withdrawal S igns and Responses - Blood Ethanol Levels - Convuls ions -Trem or - CNS Hyperact ivi ty - CNS Depression - Assessment of Doses - Intoxicat ion-Dose Relat ionship - Rat ing ofWithdrawal Syndrom e - Spectrum and Continuum of Ethanol Intoxicat ion and Withdrawal.

T h e a c q u i s i t io n o f t o l e r a n c e a n d p h y s i c a l d e p e n d e n c eu p o n d r u g s s u c h a s e t h a n o l i n v o l v e s p r i m a r i l y s o m ea s y e t u n i d e n t if i e d c h a n g e s i n t h e f u n c t i o n o f t h e c e n -t r a l n e r v o u s s y s te m ( C N S ) a n d p e r h a p s i n o t h e r o r g a n so f t h e b o d y a s w e l l. A l t h o u g h a v a r i e t y o f b i o lo g i c a la n d p s y c h o l o g i c a l d e t e r m i n a n t s o f a d d i c t i o n t o e t h a -n o l c a n b e r e l a t i v e l y e a s i l y i n v e s t i g a t e d i n h u m a n s ,t h e s t u d ie s o f t h e n e u r o b i o l o g i c a l a b e r r a t i o n s p r o -d u c e d b y e t h a n o l i n t h e C N S a t t h e s u b c e l l u l a r a n dm o l e c u l a r l e v e l a r e l i m i t e d i n h u m a n s .b y a n u m b e ro f o b v i o u s c o n s i d e r a t i o n s . T h e r e f o r e , t h e a v a i l a b i li t yo f a n a p p r o p r i a t e a n i m a l m o d e l i s a n e s s e n ti a l p re -r e q u i s it e f o r t h e u n d e r s t a n d i n g o f th e n e u r o c h e m i c a lc o r r e l a t e s o f e i th e r a c u t e i n t o x i c a t i o n o r c h r o n i ca d d i c t i o n t o e t h a n o l .

S e v e r a l a u t h o r s r e p o r t e d i n d u c t i o n o f p h y s i c a ld e p e n d e n c e u p o n e t h a n o l i n m a n ( I s b e l l e t a l . 1955;M e n d e l s o n , 1 9 64 ) , m o n k e y ( E l li s a n d P i c k , 1 9 7 0 a ;P i e p e r a n d S k e e n , 1 9 7 2 b ) ; c h i m p a n z e e ( P i e p e r e t a l .

* This paper was presented in part a t the M eetings of theAm erican Society for Pharmacolog y and Experimental Th era-peutics, East Lansing, Michigan, August 1973, and in M on-treal, C anada , Au gust 1974, and was published as abstractsin Pharm acolog ist (15, abs. 159, 1973, and 16, abs. 646, 1974).

1 9 7 2 a ) , d o g ( E s s i g a n d L a m , 1 9 6 8 ; E l l i s a n d P i c k ,1 9 7 0 b ) , r a t ( L e s t e r , 1 9 61 ; B r a n c h e y e t a l . 1971 ; Ra t -c l i f f e , 1 9 7 2 ; F a l k , 1 9 7 2 ; M a j c h r o w i c z , 1 9 7 3 ; R o a c h e ta l . 1 9 7 3 ; W a l l g r e n e t a l . 1 9 73 ) a n d m o u s e ( M c Q u a r r i ea n d F i n g l e , 1 9 5 8 ; F r e u n d , 1 9 6 9 ; G o l d s t e i n a n d P a l ,1 97 1 ; W a l k e r a n d Z o r n e t z e r , 1 9 7 4 ) u s i n g a v a r i e t y o fo r a l ( R a t c l i f f e , 1 9 72 ) , in t r a g a s t r i c ( E l li s a n d P i c k , 1 9 7 0 ;E s s i g a n d L a i n , 1 9 6 8 ; M a j c h r o w i c z , 1 9 7 3 ) , l i q u i d d i e t( P i e p e r a n d S k e e n , 1 9 7 2 ; B r a n c h e y e t a l . 1971;F r e u n d , 1 9 6 9 ; L i e b e r e t a l . 1 9 73 ) , i n t r a v e n o u s ( W o o da n d W i n g e r , 1 9 7 1 ) a n d i n h a l a t i o n ( G o l d s t e i n a n d P a l ,1 97 1 ; R o a c h e t a l . 1 9 7 3 ) t e c h n i q u e s f o r t h e a d m i n i s -t r a t i o n o f e th a n o l . E x t e n s i v e r e vi e w s a n d c o m p a r a t i v ee v a l u a t i o n o f th e c r i t e ri a f o r a n a n i m a l m o d e l o fa l c o h o l i s m ( L e s t e r a n d F r e e d , 1 9 73 ) a n d f i n d in g s c o n -c e r n i n g t h e t e c h n i q u e s o f in d u c i n g p h y s i c a l d e p e n -d e n c e ( M e l l o , 1 9 7 3 ) u p o n e t h a n o l h a v e b e e n r e c e n t l yp r e s e n t e d .

A c c o r d i n g t o g e n e r a l l y h e l d v i e w s, p h y s i c a l d e p e n -d e n c e u p o n e t h a n o l a n d t h e a s s o c i a t e d a b s t i n e n c es y n d r o m e i n h u m a n s a n d i n e x p e r i m e n t a l a n i m a l sr e q u i r e p r i o r c o n s u m p t i o n o f la r g e v o l u m e s o f a lc o h o l i cb e v e r a g e s f o r e x t e n s i v e p e r i o d s o f t i m e ( I s b e l l e t a l .1 9 5 5 ; V i c t o r a n d A d a m s , 1 9 53 ). H o w e v e r , c l in i c al


2/10

246 Psy cho pha rm aco logi a (Berl .) , Vol . 43 , Fasc . 3 (1975)o b s e r v a t i o n ( M e n d e l s o n , 1 9 6 4; M a j c h r o w i c z a n dM e n d e l s o n , 1 9 71 a ) s u g g e st s t h a t t h i s m a y n o t b e t r u e .A c c o r d i n g l y , t h e p r e s e n t s t u d y t e s te d t h e h y p o t h e s i st h a t a c o n t i n u o u s l y s u s t a i n e d e l e v a t io n o f r e l at i v e lyh i g h 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 b y f o r c e d i n tr a -g a s t r ic a d m i n i s t r a t i o n f o r o n l y a fe w d a y s m a y p r o v ea d e q u a t e a n d i s a n e s s e nt i al c o n d i t i o n f o r r a p i d i n d u c -t i o n o f p h y s i c a l d e p e n d e n c e u p o n e t h a n o l i n t h e r at .

C o n s e q u e n t l y , t h i s p a p e r r e p o r t s f i n d i n g s r e l a t i v et o a si m p l e, r a p i d a n d r e p r o d u c i b l e t e c h n i q u e f o r th ei n d u c t i o n o f p h y s i ca l d e p e n d e n c e u p o n e t h a n o l i n t her a t. T h i s t e c h n i q u e u s e s t h e a d m i n i s t r a t i o n o f e t h a n o lb y s t o m a c h t u b e w i t h o u t a n y o t h e r a n c il l ar y m e a s u r es ,t h u s c l o s e l y r e s e m b l i n g t h e r o u t e o f e t h a n o l i n t a k e i nm a n . T h e s t u d y e x p l o r e d i n d et a i l t h e r e l a t io n s h i pb e t w e e n t h e o n s e t o f t h e w i t h d r a w a l s y n d r o m e a n dt h e a n t e c e d e n t e l e v a t i o n o f b l o o d e t h a n o l c o n c e n t r a -t i on w h i c h a c c o m p a n i e s o v e r t i n t o x ic a t io n . T h e p a p e rd e s c r ib e s s i gn s a n d r e a c t i o n s o f t h e e t h a n o l d e t o x i -c a t io n p h a s e a n d o f t h e e th a n o l d e p e n d e n c e p h a s e o ft h e w i t h d r a w a l p e r i o d .

MethodsAnimals Spr a gue - Da wl e y ma l e r a t s , we i gh i ng be t we e n 200a nd 350 g, we r e ob t a i ne d f i ' om Ca r w or t h , I nc . , a nd we r e u s e di n g r oups o f 30 . Fo l l owi ng de l i ve r y t o t he l a bo r a t o r y , t hea n i ma l s we r e a l l owe d t o a c c l i ma t e t o t he a n i ma l hous e c on -d i t i ons a t l e a s t 2 - 4 da ys . The a n i ma l s we r e hous e d i n l o t so f 6 pe r c a ge a nd ha d un l i mi t e d a c c e s s t o r ode n t pe ll et s ( Pu r i naChow) a nd wa t e r t h r oughou t t he e n t i r e e xpe r i me n t a l pe r i od .E t ha no l ( a 20 % w/ v s o l u t i on o f de hyd r a t e d a l c oho l ; a b s o l u t ee t h y l a l c o h o l ; U S P R e a g e n t G r a d e 9 9 . 5 % b y v o l u m eU. S . P . H . S . S . C . C . ) wa s a dmi n i s t e r e d f o r 4 da ys by i n t r aga s t r i ci n t nba t i on u s i ng a pe d i a t r i c f e e d ing t ube ( Am e r i c a n Hos p i t a lSupp l y , C a t a l og No . 19897 -815, K - B1) . Onc e da i l y t hea n i ma l s a l s o r e c e i ve d a pp r ox i ma t e l y 0 .5 ml o f a mu l t i v i t a mi na nd m i ne r a l s upp l e me n t ( V i D a y l i n -M) . A s er ie s o f e xp l o r a t o r ye xpe r i me n t s i nd i c a te d t ha t t he a dm i n i s t r a ti on o f e t ha no l f o r4 c ons e c u t i ve da ys r e s u l t s i n ma x i ma l r e s pons e s a nd r e p r o -ducible resul ts . Thu s , the 4-day per iod o f e than ol admin is-t ra t ion was se lec ted for the s tudies repor ted in th is paper .Assessment of the Doses I n o r de r t o ma i n t a i n a c on t i nuouse l e va t ion o f b l ood e t ha no l c onc e n t r a t i on i t wa s ne c e s s a ry t oa dmi n i s t e r e t ha no l a t t he r a t e o f 9 - 1 5 g / kg i n t h r e e t o fi vef r a c t i ona l dos e s ove r e a c h 24 - h r pe r iod . Ea c h e x pe r i me n t wa si n i ti a t e d by a dm i n i s te r i ng a p r i mi ng e t ha no l d os e o f 5 g / kgbod y we i gh t. How e ve r , t he a s s e s sme n t o f s ubs e que n t dos e swa s p r e d i c a t e d upo n t he e x i s ti ng b l ood e t ha no l c onc e n t r a t i on .No ne o f t he a va il a b le me t ho ds f o r t he de te r mi na t i on o f b l oodethanol i s suf f ic ient ly rapid to turn out approximate ly th i r tyde t e r mi na t i ons o f e t ha no l i n a bo u t 10 mi n . To bypa s s t h i sp r ob l e m, a n i n t ox i c a t i on - dos e r e l a t i ons h i p wa s e s t a b l i s he d(Table 1). The d oses were assessed individual ly for ea cha n i ma l a t e ve r y i n t uba t i on s e s s i on de pe nd i ng upon t heobs e r ve d de g r e e o f i n t ox i c a t i on a s s how n i n Ta b l e 1. Thec o r r e l a t i on be t we e n the b l ood e t ha no l c onc e n t r a t i ons a nd t her a t i ngs o f s e ve r it y o f i n t ox ic a t i on wa s de t e r mi ne d du r i ng t hep r od r o ma l de t ox i c a t i on pha s e o f t he w i t hd r a wa l pe r i od ( s e ebe l ow). B l oo d s a mp l e s we r e t a ke n a t hou r l y i n te r va ls i mm e -dia te ly af ter the assessment o f the degree of in toxica t ion . Th is

Ta b l e 1 . I n t ox i c a t i on - dos e r e l a t i ons h i p i n r a t sS i gns B l ood e t ha no l Dos e o f e t ha no lo f i n t ox i c a t ion c onc e n t r a t i on ( g / kg )(mg/dl* , me ans S .D. ) Ea r l y La t eC om a (11) 649 148 0 0Los s o f r i gh t ingreflex (17) 507 83 0 0Atax ia 3 (24) 450 /04 0 1Ata xia 2 (25) 384 77 1 2Ata xia 1 (40) 335 67 2 3Sed atio n (59) 283 58 3 4Neu tra l i ty (63) 216 73 4 5The nu mbe r s i n pa re n t he s e s r e f e r t o t he numb e r o f obs e r -va t i ons ma de on t he 63 a n i ma l s u s e d f o r t he a s s e s s me n t o fs i gns a nd r e s pons e s o f e t ha no l i n t ox i c a t ion a nd f o r t hede t e r mi na t i on o f b l ood e t ha n o l c onc e n t r a t i ons . The dos e so f e t ha no l a dm i n i s t er e d a t e a c h i n t uba t i on s e s si on ( 5 da i l y )we r e a s s e s s e d on t he ba s i s o f p r i o r e va l ua t i on o f t he de g r e eo f i n t ox i c a t i on o f e a c h a n i ma l .* mg /dl = mi l l igrams per deci l i te r; 100 mg /dl = 0 .1%.

c o r r e l a t i on be t we e n t he be ha v i o r a l m e a s u r e s o f t he obs e r ve dde g r e e o f i n tox i c a t i on a nd t he e x i s ti ng b l ood e t ha no l c onc e n -t r a t i on pe r mi t s r a p i d a s s e s sme n t o f t he dos e t o be a dmi n -is tered and a lso a fa i r es t imate of the exis t ing b loo d e than olc onc e n t r a t i on i n t he obs e r ve d a n i ma l s .The a m oun t o f e tha no l a dm i n i s te r e d a t a ny pa r t i c u l a rt i me de pe nde d u pon t he obs e r ve d de g r e e o f i n t ox ic a t i on a ndpa r t l y upon t he p r i o r du r a t i on o f t he e xpe r ime n t . Thus , t hedos e s o f e t ha no l a dmi n i s t e r e d f o r t he s a me de g r e e o f i n t ox i -c a t i on w e r e s ome w ha t l owe r du r i ng t he f ir st 2 da ys o f thee xpe r i me n t ( Ta b l e I , e a r l y ) t ha n du r i ng t he l as t 2 da ysaf ter the anim als develop ed a s igni f icant degree of to leranceto e than ol (Table 1 , la te ) .Observations during Withdrawal Period The e n t i r e w i t h -d r a wa l p e r i od wa s de f i ne d a s a n i n t e r va l be t we e n t he a dm i n -i s t r at i on o f the l a s t dos e o f e t ha no l a n d t he d i s a ppe a r a nc eo f s igns a nd r e s pons e s o f t he e t ha no l w i t hd r a wa l s ynd r ome .S t a r t ing 6 - 7 h r s a f t e r the l a s t dos e o f e tha no l a t m i dn i gh t ,the animals were evaluated a t hour ly in tervals for the d is -a ppe a r a nc e o f s igns a nd r e s pons e s o f e tha no l i n t ox i c a t i ona nd a f t e r wa r ds f o r t he ons e t o f the e t ha no l w i t hd r a wa ls ynd r ome . T he o bs e r va t i ons o f be ha v i o r a l me a s u r e s o f in t ox i -c a t i on a n d wi t hd r a wa l we r e c a r r i e d ou t r ou t i ne l y by s e ve ra lope r a t o r s i n a n i n t e r c ha nge a b l e a nd b l i nd f a s h i on . Fo r t hebe ha v i o r a l obs e r va t i ons t he a n i ma l s we r e t r a ns f e r r e d f r omt he i r home c a ge s i n t o a n obs e r va t i on a r e na t a k i ng c a r e t oa vo i d e xc es si ve s t i mu l a ti on . T he obs e r va t i on a r e na wa s m a deo f unpo l i s he d p l yw ood wi t h a n op e n t op ( 55 c m wide , 60 c mlong, 15 cm high).At each observat ion sess ion animals were assessed s imul-t a ne ous l y f o r c ompr e he ns i ve be ha v i o r a l a nd phys i o l og i cc ond i t i ons : voc a l i z a t i on , body pos i t i on a nd body pos t u r e ,l oc omot o r a c t i v i t y , ga i t , p e l v i c a nd a bdomi na l e l e va t i on ,sedat ion , eye c losure , m uscle tone , spas t ic i ty , muscle r ig id i ty ,muscle fasc icula t ions , gr ip s t rength , t remors , res t lessness ,hyperac t iv i ty , i r r i tabi l i ty , convuls ive se izures , b izar re behav-ior , secre t ions and excre t ions (sa l iva t ion , lacr imat ion, d iar -rhea , defecat ion , ur ina t ion , sweat ing , re tching and vomit ing) .The s e pa r a me t e r s a r e e xpone n t s o f be ha v i o r a l, ne u r o l og i ca nd a u t on om i c r e s pons e s e it he r t o e t ha no l i n t ox i c a ti on du r i ng


3/10

E. Majchrowicz: Physical Dep enden ce up on Eth ano l and Associated Behavioral Changes in Rats 247an early prodro ma l detoxification phase or to etha nol depen-dence during the dependence phase o f the ethano l withdrawalperiod. The ent i re procedure of observing 20-30 animalsand taking samples from 1 6 - 20 animals required 30 - 45 min.Blood Ethano l Determination. During the detoxicat ion phaseof the ethanol withdrawal period when ethanol was stillpresent, the capillary bloo d samples 0.05 ml) were taken fro mthe dorsal tail vein at hourly intervals until the ethanol hadbeen completely eliminated Majchrowicz et a l . 1967).Ethanol was determined by an automated adaptat ion Maj-chrowicz, 197 1b) of the gas chromatographic method ofRoac h and Creaven 1968). The blood ethanol concentra tionsare expressed in milligrams per deciliter mg/dl).

Resu l t sBlood Ethanol Concentrat ions during the InductionPeriod. A p r e r e q u i s i t e f o r t h e i n d u c t i o n o f p h y si c a ld e p e n d e n c e u p o n e t h a n o l w a s th e m a i n t e n a n c e o fe l e v a t e d 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 t h r o u g h o u tt h e e n t i r e p e r i o d o f i n d u c t io n . A c c o r d i n g l y , t h e a n i -m a l s w e r e a d m i n i s t e r e d h i g h d o s e s o f e t h a n o l r a n g i n gf r o m 9 - 1 5 g / k g / d a y i n t h r e e t o f iv e f r a c t i o n a l d o s e sf o r 4 da y s , w i th c o n c u r r e n t m o n i t o r i n g o f t h e d e g r e eo f i n to x i c a t i o n . D u r i n g t h e e n t i re i n d u c t i o n p e r i o dt h e a n i m a l s w e r e i n t o x i c a t e d t o v a r y i n g d e g r e e s r a n g -i n g f r o m c o m a t o s e d a t i o n . 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 a n d t h e c o r r e s p o n d i n g r a t i n g s o f t h e s e v e ri t yo f in t o x i c a t i o n a t t h e e n d o f t h e 6 - 7 h r i n t e r v a lf o l l o w i n g t h e m i d n i g h t i n t u b a t i o n s e ss i on a r e s h o w ni n T a b l e I a n d F i g . 1 , w h e r e a s t h e t y p i c a l e l e v a t i o no f b l o o d e t h a n o l b e f o r e t h e m i d m o r n i n g i n t u b a t io ns e s s io n i n t h r e e s e p a r a t e g r o u p s o f r a t s i s s h o w n i nT a b l e 2 .

Withdrawal Per iodT h e l a s t d o s e o f e t h a n o l m a r k e d t h e w i t h d r a w a l o f t h ea n i m a l s f r o m e t h a n o l a n d t h e i n i ti a t i o n o f th e w i t h -d r a w a l p e r i o d . I n i t i a l l y , t h e a n i m a l s w e r e u s u a l l yi n t o x i c a t e d t o a v a r i a b l e d e g r e e d e p e n d i n g u p o n t h eb 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 i n c e t h e a n i m a l s w h i c hw e r e h i g h l y i n t o x i c a t e d a t t h e t i m e o f t h e l a st i n t u b a -t i o n r e c e i v e d o n l y a s m a ll d o s e o r n o e t h a n o l , t h e o n s e to f t h e w i t h d r a w a l s y n d r o m e i n t h e s e a n i m a l s e v o l v e dp r o m p t l y a f t e r t h e i n i t i a ti o n o f t h e o b s e r v a t i o n s . I nc o n t r a s t , t h e a n i m a l s w i t h s l ig h t d e g r e e o f i n t o x i c a t io nr e c e i v e d r e la t i v el y l a rg e d o s e s o f e t h a n o l . T h e yr e m a i n e d i n t o x i c a t e d f o r l o n g e r p e r i o d s o f t i m e a n dt h e w i t h d ra w a l s y n d r o m e d e v e l o p e d a t 1 6 - 2 4 h rsa f t e r t h e i n i ti a t i o n o f t h e w i t h d r a w a l p e r i o d .

D u r i n g t h e e n t i r e w i t h d r a w a l p e r i o d , t w o s u c c e s-s i v e p h a s e s e v o l v e d w i t h i n r e l a t i v e l y w e l l d e f i n e dt e m p o r a l l i m i t s . T h e e a r l y p h a s e , c a l l e d t h e p r o d r o ma lde tox ica t ionphase w a s c h a r a c t e r i z e d b y t h e d i s a p p e a r -a n c e o f a v a r i e t y o f s ig n s a n d r e s p o n s e s o f i n t o x i c a t i o nw i t h a g r a d u a l l y d e c r e a s i n g d e g r e e o f s e v e r i ty F i g . 1 ).

Table 2. Blood ethanol concen tra t ion during the induct ionperiodDay Blood ethanol concentra t ionsof mg/dl)experiment

G r o u p 1 G r o u p 2 G r o u p 31. 394_+153 8) 298 + 97 28) 312+_197 19)2 . 330+116 11) 347_+ 124 22) 276_+ 69 18)3. 398_+ 49 10) 26 2+1 38 13 ) -4. 358_+ 73 8) 305-+128 9) 405 _+ 99 17)Bloo d samples from tail vein were taken at 9.0 0 a.m. eachday of the induction period in three separate groups of ratsbefore the midmorning intubat ion sess ion. Blood ethanollevels were determined by gas chromatography. Figures inparentheses represent the number of animals used for theobservations. Th e results are means _+ stan dar d dev iations.

A s e c o n d p h a s e o r t h e ethanol dependence phasee v o l v e d a s 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 c u r v et a i le d o f f t o w a r d t h e z e r o l e v e l a n d w a s c h a r a c t e r i z e db y t h e o n s e t a n d p r o g r e s s iv e p o t e n t i a t i o n o f t h ee t h a n o l w i t h d r a w a l s y n d r o m e F i g . 1 ).

1. P r o d r o m a l D e t o x i c a t i o n P h a s eT h e t r a n s i t i o n b e t w e e n s u c c e s s i v e s i gn s o f i n t o x i c a -t i o n w a s r e l a t iv e l y r a p i d . T h e a n i m a l s r e m a i n e d i n e a c hi n t o x i c a t i o n f o r r e l a ti v e l y s h o r t p e r i o d s o f t i m e u s u a l l yn o t e x c e e d i n g 1 o r 2 h r s b e f o r e p r o g r e s s i n g t o a l e sss e v e r e s t a g e o f i n t o x i c a t i o n . O c c a s i o n a l l y , t h e r e c o v e r yw a s s o r a p i d t h a t t h e t r a n s i t i o n a m o n g t h e t h r e e s ta g e so f m i n i m a l i n t o x i c a t i o n o c c u r r e d b e t w e e n t w o c o n -s e c u ti v e h o u r l y o b s e r v a t i o n s : F o r e x a m p l e , t h e t r a n -s i t io n f r o m A t a x i a 1 t o t h e N e u t r a l i t y s t ag e w a s o c c a -s i o n a ll y a c h i e v e d w i t h i n 1 h r w i t h o u t o b s e r v i n g t h ei n t e r m e d i a r y S e d a t i o n s t ag e F i g . 1 ). I t is n o t e w o r t h yt h a t a l l t h e d e p r e s s iv e a n d e x c i t a t o r y c h a n g e s f o l l o w e de a c h o t h e r i n a c o n t i n u o u s f a s h i o n w i t h n o c l e a rd e m a r c a t i o n b e t w e e n a d j a c e n t s t a g e s . A s c a n b e s e e ni n Fi g . l , 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 B E C )a s s o c i a te d w i t h a n y p a r t i c u l a r p a r a m e t e r o f in t o x i -c a t i o n o v e r l a p o v e r a r e l a t iv e l y w i d e m a r g i n . A d e s c r ip -t i o n o f t h e s e s t a g e s f o l l o w s .Death . A l t h o u g h t h e l e t h a l 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 v a r i e d c o n s i d e r a b l y a m o n g t h e a n i m a l s , a na v e r a g e e s t i m a t e s u gg e s ts t h a t d e a t h u s u a l l y o c c u r r e dw h e n 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 a t t a i n e d 7 0 0 m g / d lo r h i g h e r . O c c a s i o n a l l y w e o b s e r v e d B E C u p t o8 0 0 m g / d l 6 - 7 h r s a f t e r t h e l as t d o s e o f e th a n o l . T h e s eh i g h B E C i n d i c a t e th e a c q u i s i t i o n o f a h ig h d e g r e e o ft o l e r a n c e t o e t h a n o l . A t t h e b e g i n n i n g t o t h e s es t ud i e s t h e m o r t a l i t y r a t e r e a c h e d 3 0 - 5 0 ~o , p r i m a r i l yd u e t o o v e r d o s a g e o r a c c i d e n t a l d e l i v e r y o f e t h a n o li n t o t h e l u n g s r e s u l t i n g i n d r o w n i n g . A f t e r e s t a b l is h i n g


4/10

248 Psych opha rmac ologia (Berl.), Vol. 43, Fasc. 3 (1975)CNS DEPRES SION J(INTOXICATION) ~. 'CNS HYPERACTIVITY~' (WITHDRAWAL)J

DEATHCOMAL RT, RFXATAXIA-3ATAXIA-2ATAX IA- ISEDATION

NEUTRALITY

.JL_ i

7 _ i800 600 400

BLOOD ETHANOL(mg/dl)

~ -- - . . . . . . . . . ~/ // // // // // /A~ -- -~ . . . . . . . 4 // // // // // // // A

~ - - ..t . . . . . . t l // / // / // / // / // / /A

~ - - - t- . . . . ~ / / // / / // / / // / / // / / /A

~-- ---- . I / / / / / / / / / / / / / / / / / / / / / /A~---P--4U/ / / / / / / / / / / / / / / / / / /7~

~--- --4 / / / / / / / / / / / / / / / / / / / / / / / / / / /A~ P ~ / / / / / / / / / / / / / / / / / / / / / / / / / / / ~ J~----~/ / / / / / / / / / / / / / / / / / / / / / / / / / / / / /A

- ~ / / @ / / / / / // / / / / // / / / // / / / / // / d~/ / / / / / / / / / / / / / / / / / / / /17/ / / / / / / / / / / / /A

200 I00 0dr ithdrawalSigns Signs

I0 t2 ~, 14-16 hrs 9hrsTIME AFTER LAST INTUBATION

DEATHSPONZ CONVULSIONSINDUCED CONVULSIONSCHATTERING OF TEETHWET SHAKESINDUCED RUNNING EPISODESHEAD TREMORSGENERAL TREMORSGENERAL SPASTICITYTREMORS OF CAUDAL REGIONTAIL SPASTIClTYTAiL TREMORSGENERAL HYPERACTIVITY

PRODROMAL DETOXICATION ETHANOL DEPENDENCEPHASE PHASEALCOHOL WITHDRAWAL PERIOD

Fig. 1. Spec trum and co ntinu um o f ethanol intoxicatio n and w ithdrawal in rats. Th e entire withdrawal p eriod is dividedinto 2 phases : pro drom al detoxicat ion phase and ethanol dependence phase. The extreme ethanol intoxicat ion may terminatein death due to central nervo us system depression whereas the ethanol withdrawal phase m ay terminate in death due to centralnervous sys tem hyperact ivi ty as shown by the wide arrow o n the top of the diagram. The s igns and react ions of both theprodro mal de toxieat ion phase and the e thanol dependence phase increase in severity from the bot to m tow ard the top as indicatedby the arrow s on each s ide of the f igure . The s igns of the detoxicat ion phase are direct ly re la ted to blood ethanol concentra t ion(heavy vertical lines represe nt the means whereas the standa rd deviations are indicated by dott ed vertical lines). Sign s andreact ions of the e thanol dependence phase are arrang ed according to subjective evaluat ion rela tive to order o f appearance,specificity, frequency of oceurance, and the degree o f severity. Thus, the earliest, th e least specific, the m ost freq uentlyobserved, and th e least severe signs and reactions are at the bo ttom of the diagram. In parenthesis are shown the num bers ofanimals used f or assessment of the s igns of the detox ication phase: com a (11); loss of righting reflex (17); ataxia 3 (24);ataxia 2 (25); ataxia 1 (40); sedation (59); neutrality (63). The signs and reactions of the withdrawal p hase were based on theevalua tion of 119 animals an d w ere classified into 3 categories acco rding to the degree of severity (Table 3)

t h e i n t o x i c a t i o n - d o s e r e la t i o n sh i p ( T a b l e 1 ) a n d a f t erp e r f e c t i n g t h e i n t u b a t i o n t e c h n i q u e t h e m o r t a l i t y r a ted r o p p e d t o a p p r o x i m a t e l y 2 0 o r l es s. D e a t h u s u a l l ye n s u e d a s a r e s u l t o f r e s p i r a t o r y d e p r e s s i o n f o l l o w i n ga n o c c a s i o n a l o v e r d o s a g e w i t h e t h a n o l .C o m a . T h i s w a s d e f i n e d a s a s t a t e o f d e e p i n t o x i c a t i o nw h e n r a t s d e m o n s t r a t e d n o s ig n s o f a n y m o v e m e n t ,w e r e t o t a l l y u n r e s p o n s i v e d u r i n g t r a n s f e r t o t h e o b s e r -v a t i o n a r e n a , a n d d e m o n s t r a t e d t o t a l p a l p e b r a l c l o s u r ea n d t o t a l a b s e n c e o f th e e y e b l i n k r e fl ex . N o a t t e m p t sw e r e m a d e t o d i f f e r e n t i a te a m o n g d r o w s i n e s s , s t u p o r ,u n c o n s c i o u s n e s s a n d s l ee p . H o w e v e r , d e e p l y c o m a t o s ea n i m a l s w e r e d e f i n e d a s t h o s e t h a t h a d b a r e l y p e r -c e p t i b l e r e s p i r a t i o n , a w e a k h e a r t b e a t a n d n o s w a l -l o w i n g r e f l e x . S u c h a n i m a l s h a d a p o o r p r o g n o s i s f o rs u r v iv a l . T h e i r r e c o v e r y w a s o c c a s i o n a l l y a i d e d b ys t i m u l a t i n g r e s p i r a t i o n w i t h b r i e f i n h a l a t i o n o f c a r b o nd i o x i d e . T h e s w a l l o w i n g r e f le x w a s o c c a s i o n a l l y s t im u -

l a t e d b y p l a c i n g a f e w d r o p s o f d i l u te s o l u t i o n o f s u g a re.g., f r u c t o s e ) o n t h e t o n g u e o f t h e a n i m a l .A s c a n b e s e e n in T a b l e 1 a n d F i g . 1, m o s t a n i m a l s

w e r e c o m a t o s e w h e n t h e B E C w a s a p p r o x i m a t e l y6 4 0 m g / d l . O c c a s i o n a l l y a n i m a l s w e r e c o m a t o s e w i t ht h e B E C a s h i g h a s 7 30 m g / d l o r a s l o w a s 46 0 m g / d l .L o s s o r R i g h t in g R e f l e x L R R ) . T h i s w a s d e f i n e d a sa n i n a b i li t y o f t h e a n i m a l t o r i g h t h i m s e l f w i t h i na p p r o x i m a t e l y 3 s e c a f t e r b e i n g p l a c e d o n h i s b a c k .R a t s w h i c h h a d o n l y a m o d e r a t e l y s u p p r e s s e d r i g h t in gr e f le x u s u a l l y m a d e c o n s i d e r a b l e e f f o r t t o r i g h t t h e m -s el v es . U s u a l l y t h e s e a n i m a l s h a d a n o r m a l s w a l l o w i n gr e f le x a n d t h e i r b r e a t h i n g a n d h e a r t r a t e a p p e a r e du n i m p a i r e d . T h e y h a d a f a i rl y sa t i s f a c to r y e y e b l i n kr e f le x a n d t h e i r p a l p e b r a w e r e o n l y p a r t l y c l o se d .W h e n p l a c e d i n a s u p i n e p o s i t i o n , a n i m a l s a t t e m p t e dt o m o v e a f t e r s l i g h t p r o d d i n g . H o w e v e r , i n g e n e r a l ,t h e a n i m a l s w e r e h i g h l y i n t o x i c a t e d , s t u p o r o u s , a n d


5/10

E. Majchrowicz: Physical Dependence upon Ethanol and Associated Behavioral Changes in Rats 249demonstr ated little or no spontaneous motor activity.Usually the BEC in the animals who lost their rightingreflex ranged between 640 and 440 mg/dl with themean value of 507 mg/dl.A t a x i a 3 . The main criterion for this category was therecovery of the righting reflex. However, their motorcoordination was heavily impaired as indicated bytheir broad staggering gait, absence of pelvic andabdominal elevation, heavily depressed reflexes, flaccidmuscles, heavy general sedation and lethargic appear-ance. Only occasionally did animals make a spon-taneous effort to move or to walk. Ataxia 3 was usuallyobserved when BEC ranged between 580 and 370 mg/dlwith the mean value of 450 mg/dl.A t a x i a 2 . This marked a stage of intoxication inter-media ry between ataxia I and ataxia 3. All criteriaof behavior observed were less accentuated than inataxia 3 but more advanced than in ataxia 1. A typicalsign was an accentuated staggering gait. However,during the fo rward movement there was a considerableelevation of abdomen and pelvis. The BEC rangedbetween 490 and 290 mg/dl with a mean value o f384 mg/dl.A t a x i a 1 . This stage of intoxication marked the lowestdegree o f gait impairment in addition to heavy seda-tion, pronounced muscle incoordination and sluggishmovements. During the forward movement, there wasa markedly sustained elevation of the abdomen andpelvis despite a staggering gait. Ataxia 1 usuallyoccurred when BEC declined to approximately 400 to250 mg/dl with the mean value of 335 mg/dl.Seda t i on . The disappearance of the overt signs ofmotor incoordination and gait impairment was usedto distinguish between Ataxia 1 and Sedation. Thissign of intoxication was marked by the presence ofreduced muscle tone, general sedation, dulled andrelaxed appearance and slow locomotor activity.The muscle tone was assessed by palpation whileholding the animal in the palm of the hand. Thesedation signs emerged at the mean blood ethanolconcentration of 283 mg/dl with a range of 360 to190 mg/dl.Neu t ra l i t y . During further decrease of BEC theassociated detoxication was followed by the onset ofan intermediate stage of neutrality which constitutesa transition from depression observed during theprodromal ethanol detoxication phase to hyperexcit-ability associated with the ethanol dependence phase.This stage is marked by the absence of the observablesigns of either overt ethanol intoxication or ethanolwithdrawal. The neutrality stage is characterized bythe emergence of transient normal body and muscle

tone which remarkably contrasts with the flabby andrelaxed muscles during the preceding sedation andthe hyperactivity to evolve immediately afterwardsin animals experiencing the ethanol withdrawal syn-drome. Furthermore, it is marked by the emergenceof the normal reflexes like fur-grooming, face cleaning,exploratory activity and the onset of the startlereflex. However, these short-lasting signs of neut ralitywere rapidly superseded by the gradually evolvingsigns of the ethanol withdrawal syndrome. It isremarkable tha t the observed neutrality stage usuallyemerged at relatively high blood ethanol concentra-tions ranging between 300 and 130 mg/dl with themean value of 216 mg/dl.

2. Ethanol Dependence PhaseThe observed signs and reactions of the ethanolwithdrawal syndrome are arranged in Fig. 1 accordingto subjective evaluations relative to order o f appear-ance, specificity, frequency of occurrence and degreeof severity. The assessment of the withdrawal syn-drome was carried out interchangeably by 2 - 4 observ-ers using 119 animals. The onset of ethanol depen-dence was marked by the progressive appearance ofsigns and reactions of an ethanol withdrawal syndromeeven before complete clearance of ethanol from theblood. Usually the maximum intensity of the charac-teristic withdrawal signs developed within 2-6 hrsafter blood ethanol clearance. However, fully devel-oped signs o f withdrawal including convulsive seizureswere occasionally observed when BEC was 100 mg/dlor close to the zero level which is indicated in thediagram by the dotted arrows Fig. l). The mostsalient characteristics of these signs and reactions aredescribed below in the order of their appearanceduring the development o f the withdrawal syndrome.Genera l Hyperac t i v i t y . The onset of general hyper-activity was usually observed after blood ethanolconcentrations descended to the zero level. However,the maximal accentuation of hyperactivity occurredwithin 0-6 hrs following clearance of alcohol fromthe blood. Hyperactivity was expressed by a varietyof responses which ensued in an unpredictable fashionin different animals, e.g . frantic frenzied) explorationof the cage, sniffing other animals, excessive locomotoractivity, enhanced startle reflex, agi tation and restless-ness. These signs of hyperact ivity were observed withvarying degrees of severity in most animals whichdeveloped the syndrome of ethanol withdrawal.Further exacerbation of these signs and reactions wasprobably reflected in various behavioral parametersclassified as bizarre behavior.


6/10

250 Psychopharmacologia (Berl.), Vol. 43, Fasc. 3 (1975)Tai l Tremors and Tai l S t i f fness The earliest typicalsigns of ethanol withdrawal consisted of subtletremors and slight stiffness of the tail that wereobserved when blood ethanol concentrations declinedto approximately 100 mg/dl. The maximum intensityof tail tremors was manifested up to 6 hrs followingcomplete clearance of ethanol from the blood. Con-trary to earlier observations of tail coiling or tailelevation in mice (Goldstein and Pal, 1971), duringthe entire dependence phase the tails of our animalswere usually rigidly extended in a horizontal plane,pressing downward against the bottom of the cage.The stiffness of the tail was similar to an inflexible rodin contrast to pliable and elastic tail found in untreatedanima ls.The tail stiffnesswas most pronounced after theanimals were tested for grip strength (while suspendedby his forelimbs on a thin metal rod) and during theperiod of maximal withdrawal.General Tremors and Spastic i ty These were observedin several body regions as the BEC further decreased.Tremors spread rostrally and evolved to the body andhead, either simultaneously or within 2 or 3 hrs later.Usually when the fully developed withdrawal syn-drome was severe, all tremors developed over a shortperiod of time or nearly simultaneously. The generalspasticity was characterized by rigid posture of theentire body including the limbs and was associatedwith little flexibility and resiliance to handling.Tremors o f the head usually appeared after eliminationof ethanol and only in animals tha t developed a severewithdrawal syndrome.We t Shakes and Chatter ing Tee th were observed onlyin rats undergoing a very severe withdrawal syndrome.Often these responses preceded or followed clonic-tonic convulsions. Chattering teeth could be detectedonly by continuous observations in a room free fromexcessive background noise.Induced Convulsions and Running Episodes Theseresponses were induced by jingling a bunch of keys assuggested by Fa lk (1972) and were observed inanimals with relatively high blood ethanol concen-trations and before the development of standard signsof withdrawal which regularly appeared in a spon-taneous fashion.Spontaneous Convulsions Convulsive seizures wereobserved in 35 of 119 animals used for the observationof the withdrawal syndrome (Table 3). The mostsevere convulsions were usually observed after com-plete clearance of ethanol from the blood. However,57 ~o of the rats which experienced convulsions andwere rated Severe did so with a mean BEC of93 + 58 mg/dl, while the rest (43 ~) had no ethanolin the blood. In contrast, only 20 ~ of the Severe

withdrawing rats with no convulsions, developedmaximal signs and responses with BEC of 47 + 28 rag/dl, while the others (80 ~o) experienced maximum sever-ity after complete clearance of ethanol (Table 3). Noindicative behavior or prodromal signs were observedprior to the onset of convulsive seizures. The convul-sions emerged suddenly in animals that either alreadyhad the well developed withdrawal syndrome or lessfrequently in animals that h ad no overt signs of with-drawal prior to complete clearance of ethanol fromthe blood. After an animal experienced convulsionslittle or no overt signs of withdrawal were manifestedand these animals appeared sedated and tired.

Occasionally the convulsions terminated in eitherimmediate or delayed death. Multiple seizures wereusually fatal. Recurrent seizures were observed inanimals that were treated with ethanol for at least4 - 6 days or longer. The generalized convulsions wereof short dura tion (approximately 5 - 10 sec), with theclonic component most prevalent. The number ofanimals undergoing convulsions increased with theduration of ethanol treatment periods. Thus, after1 day of treatment only an occasional animal experi-enced convulsions, whereas after 4 - 6 days of treat-ment, the convulsions were observed in approximately20-40 ~o of those developing the withdrawal syn-drome. It should be noted that the number of animalsundergoing convulsions is only approximate, becauseit was impractical to observe them continuously for2 4 - 48 hrs or longer. Most of the recorded convulsionscoincided with the time of routine observations andtaking o f blood samples. Thus, a considerable numberof convulsions probably remained unobserved.Bizarre Behavior In additi on to the typical withdrawalsigns and reactions already described, a considerablenumber of animals developed a variety of atypicalresponses which are reflections of increased hyper-excitability of the central nervous system. Theseresponses were usually observed in animals treatedwith ethanol for at least 4 - 5 days. This cluster ofresponses includes retropulsion, apparently aimlesslocomo tor activity, stereotyped body movement, head-search activity, aggressiveness, biting other animals,cannabalism, uncontrolled running episodes andothers. The stereotyped body movement involvedrepetitive shifting of the upper thorax and both fore-limbs from side to side or restless turning of the headfrom side to side. Unusual locomotor activity such asmoving in circles was slow at first but occasionallybecame accelerated into uncontrolled running epi-sodes, giving an impression of an excape attempt.Duration and Sever i ty o f the Wi thdrawal SyndromeIn the studies reported here the onset of the with-drawal syndrome was manifested by the emergence


7/10

E. Majchrowicz: Physical Dependence upon Ethanol and Associated Behavioral Changes in Rats 251Table 3

Score Se ve ri ty Num be r ~o B.E.C.of atanimals maximumseveritymg/dl)

3 Severe Total) 86 72.3with con- 35)vulsions)no con- 51)vulsions)2 Mode ra te 20 16.81 Mild 13 10.9

29.4 93 + 58 20)0 15)42.9 47 _+ 28 10)0 41)

Total 119 100Rating of the severity of the withdrawal syndromewas basedon visual evaluation of the intensity of the withdrawal signsand reactions. These were rated on scale 3 to i and classifiedinto Severe including convulsions), Moderate and Mild.B.E.C. = mean blood ethanol concentrations + S.D. Figuresin parentheses denote number of animals.

of subtle signs and reactions at any time between 6and 22 hrs after the administration of the last doseof ethanol. However, in most cases the first with-drawal signs appeared at 10-12hrs when BECdecreased to 200 and 100 mg/dl Fig. 1). The meanBEC -4-_ S.D.) at the onset of the signs and react ionsof withdrawal was 132 +_ 64 mg/dl for 97 animals.The emergence of the maximum intensity of the with-drawal syndrome was observed after the BECdescended to zero level and 2 - 6 hrs later Fig. 1).The ratings of the relative severity of the with-drawal syndrome shown in Table 3 are classifiedinto 3 groups according to the degree of the severity:Severe including convulsions), Moderate , and Mild.The basic criterion used for the identification of thesyndrome was the presence of at least three of thefollowing signs and reactions: Tail tremors, tailrigidity, tremors of the caudal region, general rigidity,general tremors, general hyperactivity and convul-sions. Except for the convulsive seizures, the presenceof all of these signs at the maximum intensity wasalways observed in animals with the severe form ofthe withdrawal syndrome. Animals with the mildform o f the syndrome developed at least three of thesesigns in just observable form. The severity of the syn-drome in animals rated moderate was intermediatebetween those of the severe and mild. The mostremarkable observation was the finding that allanimals that survived the induc tion period developedclearly defined withdrawal signs and reactions ofvarying severity. Furthermore, most of the animalsdeveloped the severe form of the syndrome Table 3).

The duration of the peak severity of the withdrawalsyndrome extended for varying periods of the timeranging from 2 - 6 hrs Fig. 1), after which time therewas a gradual subsidence of the signs and reactionsover the next 2- 3 days. The sequence of disappear-ance of particular signs was in reverse order to thatobserved during their emergence. The most severesigns, which emerged relatively late after the onset ofthe withdrawal syndrome, were the first to subside.The tremors of the caudal region and slight tail stiffnesswere still manifested at 2 or 3 .days after init iation ofthe withdrawal period.Recovery and Surv iva l Ra tes . The survival rates wererelatively high in rats which developed severe reactionsof the withdrawal syndrome, both with and withoutthe concurrent convulsions. Forty-eight rats 95~ )without convulsions and 31 rats 90~o) with convul-sions survived the entire withdrawal episode. Allsurviving animals regained their original body weightwithin 10-14 days after the initiation of the with-drawal period, and demonstrated no further signs ofdisease.Food In take a nd Body Weigh t Losses . Since theinduction of physical dependence upon ethanol waspredicated upon sustained elevation of blood ethanolconcentrations during the entire period of induction,the animals manifested prolonged episodes of severeintoxication. Consequently, the animals were toostuporous to consume solid food even though it wasavailable. This lack of apetite resulted in mean lossesof 17 ~ in body weight for 68 animals.Excre t ions and Secre t ions . Throughout the entireexperimental period, i.e. during the induction andwithdrawal periods, urination and defecation werenot significantly different from those observed inuntreated and acutely treated animals. It should benoted th at in o ther species and particularly in man thegastrointestinal disorders are standard symptoms ofethanol withdrawal Victor and Adams, 1953; Men-delson, 1964). A few animals in each experiment hadconstipation, which was detected by occasional pal-pation of the abdominal cavity. A number of responseswhich are regularly observed in man and nonhumanprimates Victor and Adams, 1953; Mendelson, 1964;Victor, 1966; Ellis, 1970a; Pieper et al. 1972a; Pieperand Skeen, 1972b) were absent during the ethano lwithdrawal period in rats. Thus, we did not observevomiting, retching, diarrhea, salivation, lacrimationand muscle fasciculation.Vocalization. Squealing was recorded occasionallywhen the BEC declined to approximately 300 to200 mg/dl. The majority of animals that had a fullydeveloped withdrawal syndrome including convulsions


8/10

252 Psychopharmacologia (Berl.), Vol. 43, Fasc. 3 (1975)exhibited no signs of vocalization. Accordingly, thisvocal response in the rat does not appea r to be a typicalsign associated with ethanol withdrawal.Ventro Medio Distal Flexion (VMF) is a responsethat to the knowledge of the author has not beenreported in the literature. VMF of either forelimb wasinducible after BEC declined to below 300 to 200 mg/dl.This response was elicited by holding the animal atthe fold of the skin at the back of the neck andraising it vertically. Then either one or bo th forelimbswere drawn downward pressing against the body.In untr eated rats the limbs were stretched horizontallyat right angle to the body s main axis. Abou t 9 0- 95of the animals tested during the withdrawal perioddeveloped the VMF response. This response can alsobe seen in contro l rats treated with subconvulsive dosesof pentylenetetrazole (unpublished observation byW. A. Hunt). This suggests that VMF is a nonspecificsign of hyperactivity, due to a neurological defectresulting from ethanol intoxication.Keratosis and Dryness of the Eyes were nonspecificside effects that developed during the induction ofphysical dependence upon ethanol. These lesionsdeveloped as a consequence of sustained ethanolintoxication which resulted in the abolition of theeyeblink reflex for prolonged periods of time. Mostof these lesions were mild opacities of the cornea,which developed after a day or two of ethanol intoxi-cation and usually disappeared a week or two followingthe ethanol withdrawal period. The most severelesions, were observed after 4- 6 days of ethanolintoxication, occasionally resulted in severe and irre-versible scarring of the cornea.

DiscussionThe results of the studies reported in this paperdemonstrate that a continuously sustained elevationof blood ethanol concen trations for 4 days is sufficientfor the inducti on of physical dependence upon ethanoland for the manifestation of fully developed with-drawal signs and reactions reminiscent of tremulous,spastic and convulsive stages observed in man andin nonh uman primates.Furthermore, these correlative studies establishfor the first time an accurate temporal relationshipbetween the blood ethanol disappearance and thebehavioral, neurologic and autonomic responsesobserved during the prodromal detoxication phaseof the withdrawal period in the rat and the onset ofthe ethanol withdrawal signs and reactions.The results of these studies demonstrate that thediverse signs and reactions observed during bothphases of the withdrawal period are not discrete

responses. The successive onset and disappearanceof the observed signs and reactions during the pro-dromal detoxication phase and later dependencephase constitute a continuum of effects and actions.This continu um is further accentuated by the absenceof clear lines of demarcation between both phasesof the withdrawal period and between the individualsigns within each phase. This continuum between thetwo phases of the withdrawal period is linked throughan intermediate neutrality stage which is characterizedby the definitive recession of the signs of overt intoxi-cation and a subtle onset of progressively severe signsand reactions of the ethanol withdrawal syndrome.The findings of these studies indicate that a rapidsuccession of two clusters of signs and reactionsrepresent a subtle but definitive reversal in centralnervous system function from the extremes of ethanolintoxication to the extremes of ethanol withdrawalwithin a relatively short period of time. Both o f theseextremes may result in death due to the severity ofresponses associated with either excessive depressionor with the extremes of excessive hyperexcitabilityof the central nervous system (CNS).The rapidity of the reversal in CNS function mayexplain why a number of confounding reports werea source of confusion in the diagnosis and treatmentof alcoholic intoxication and the withdrawal syn-drome. As po inted out by Victor (1966) the literaturehas been characterized by outstanding confusion,inexactness and contradictions, the major shortcom-ings of which were that no clear distinction has beenmade between the problems of acute alcoholic intoxi-cation, alcohol addiction, mild withdrawal signs andsymptoms, and delirium tremens.In view of the findings reported in this paper, it isnot surprising that such confusion existed for a longtime, since within a period of 1 or 2 hrs there mayoccur a total reversal in the clinical responses fromsevere depression to severe hyperactivity. The mostconfounding might have beeri the finding that occa-sionally the sudden emergence of a fully developedwithdrawal syndrome including convulsive seizuresoccurred at BEC as high as 100 mg/dl.

The observation of the reversal in CNS functionis in agreement with the studies on seizure thresholdto either electroconvulsive shock and to pentylene-tetrazole-induced seizures (McQuarrie and Fingle,1958; Hunt, 1973). It has been reported that duringthe period of ethanol intoxication, the thresholdto pentylenetetrazole-induced seizures is elevated asa result of the depressive action of ethanol. On theother hand, during the dependence phase of the with-drawal period, when there was no ethanol in the body,the threshold was lowered as a consequence of theoverall increase in the CNS excitability (Hunt, 1973).


9/10

E. Majchrowicz: Physical Dependence upon Ethanol and Associated Behavioral Changes in Rats 253O u r e a r l i e r s t u d i e s (M a j c h ro w i c z e t a l . 1968;

M e e k e t a I . 1967) demons t ra ted tha t ra t s wh ich wereg iven so lu t ions o f e than o l (5, 10 and 20 %) as the i r so lesource o f f lu id fo r up to 11 mo n ths d i sp layed noo b s e rv a b l e s i g ns o f p h y s i c a l d e p e n d e n c e u p o n e t h a n o l.Th e r a t e g ro w t h o f t h e s e r a t s w a s r e t a rd e d a n d t h emo rta l i ty ra tes w ere l a rges t in the g ro up d r ink ing 20 ~oe t h a n o l s o l u t i o n, U n d o u b t e d l Y t h e i n d u c t io n o f v o l i-t iona l in take o f e thano l in amo un ts su f f ic ien t to sus ta inp ro l o n g e d p e r i o d s o f o b s e rv a b l e in t o x i c a ti o n w o u l dbe an idea l so lu t ion . However , desp i te the fac t tha ta n i m a l s m a y b e i n d u c e d i n t o e t h a n o l - s e e k i n g b e h a v i o r ,th i s i s ach ieved on ly by app ly ing s t reso rs o f vary ingdegrees resu l t ing in severe changes in bas ic phys io -l o g ic a l p ro c e s s e s w h i c h c a n n o t b e e q u a t e d w i t h v o li -t i o n a l i n ta k e o f e t h a n o l i n m a n . F o r e x a m p l e , th e p o l y -d i ps i a p r o t o c o l r e q u ir e s a 2 0 ~ r e d u c ti o n o f b o d yweigh t p r io r to admin is t ra t ion o f e thano l (Fa lk , 1972).F u r t h e rm o re , d u r i n g t h e a d m i n i s tr a t i o n o f e t h a n o l ,the an imals d r ink f lu ids up to 40 ~0 o f the i r bo dyweigh ts . Transpos ing th i s s i tua t ion to man , th i s i se q u i v a l e n t t o t h e i n t a k e o f 2 8 1 o f f l u id s p e r d a y p e r70 kg individual .

Go lds te in (1971) has repor ted tha t e thano l depen-d e n c e c a n b e i n d u c e d b y i n h a l a t io n o f e t h a n o l v a p o r sw i t h c o n c o m i t a n t tr e a t m e n t w i t h p yr a z o le , a n d A D Hinh ib i to r . Th e pur pose o f the py razo te i s to s t ab i l izet h e B EC t h e re b y i m p ro v i n g t h e c h a n c e s o f i n d u c ti o na n d d e c re a si n g t h e m o r t a l i t y c a u s e d b y w i d e v a r i a t i o n so f B EC fo l lo w i n g o r a l a d m i n i s t r a t i o n o f e t ha n o l .I n c o n t r a s t t o t h e w e l l k n o w n p a t t e rn o f a c q u is i t io n o fp h y s i c a l d e p e n d e n c e u p o n e t h a n o l i n m a n fo l l o w i n gora l inges t ion o f la rge quan t i t i es o f a lcoho l ic bever-a g e s , th e i n d u c t i o n o f p h y s ic a l d e p e n d e n c e b y e t h a n o li n h a l a t io n w h i l e b l o c k in g t h e m e t a b o l i s m o f e t h a n o lwi th py razo le (Go lds te in , 1971) in exper imen ta l an i -m a l s i s a s s o c i a te d w i t h t h e m e t a b o l i s m o f c o n s i d e ra b l ys m a l le r q u a n t i t ie s o f e t h a n o l t h ro u g h o u t t h e e n t ir einduct ion per iod . Th is p robab ly wi l l resu l t in thefo rm a t i o n o f s m a l l e r q u a n t i ti e s o f a v a r i e t y o f m e t -abo l ic in te rmed iar ies and a co rrespond ing def ic i t o ft h e r e d u c e d c o fa c t o r s a n d t h e e n d p ro d u c t s . F u r t h e r -m o re , p y ra z o l e a l o n e a n d i n c o m b i n a t i o n w i t h e t h a n o le x e r ts a n u m b e r o f h e p a t o x i c a n d b e h a v i o ra l e f f ec t s(Wi l son and Bo t t ig l i e r i , 1962 ; Le tbach , 1971 ; Liebere t a l . 1 9 7 0 ; M a g n u s s o n e t a l . 1972) wh ich may a l socom pl ica te the in te rp re ta t ion o f the resu l ts .S ince a l l an imals in th i s s tudy invar iab ly deve l -o p e d a c o m p l e t e s p e c t ru m o f s ig n s a nd r e a c t io n s o fe t h a n o l w i t h d ra w a I , a m o s t c r it ic a l p a r a m e t e r f o r t h ei n d u c t i o n o f p h y s i c a l d e p e n d e n c e a p p e a r s t o b e t h em a i n t e n a n c e o f u n i n t e r ru p t e d e l e v a t i o n o f b l o o de t h a n o l c o n c e n t r a t i o n s a b o v e a c e r t a i n t h r e s h o l dva lue th ro ugh ou t 4 c lays o f in t ragas t r ic adm in is t ra t iono f e t ha n o l . T h i s t h r e s h o l d c o r r e s p o n d s t o t h e c o n c e n -

t r a t i o n o f b l o o d e t h a n o l w h i c h i s s uf f ic i e n t t o i n d u c ea t l eas t the min im al deg ree o f observab le in tox ica t ion ,i . e . sed at io n (283 _+ 58 mg/dt) . Th eref ore, the roleo f o ther var iab les such as ind iv idua l suscep t ib i l i ty ,gene t ic p red i spos i t ion , p r io r t ra in ing and p rev iousi n d u c t i o n o f t o l e r a n c e t o e t h a n o l u n d e r t h e s e c o n d i-t ions had l i tt l e e f fec t on the de ve lopm en t o f depen-dence .

I t has been~also observe d tha t i f the an imals werea l lowed to rega in the s tage o f neu t ra l i ty and conse-quen t ly the BEC a l lowed to dec l ine to the zero l eve l ,these an imals e i ther deve loped in te rmi t t en t w i th -d ra w a l s y n d ro m e o f r e l a ti v e ly m o d e ra t e s e v e r it ya n d / o r t h e i r c a p a c i t y fo r d e v e l o p i n g w i t h d ra w a l s y n -d ro m e w a s c o n s i d e ra b l y d e l a y e d . Th i s i s s i m i l a r t otha t found recen t ly by Golds te in (1974) in mice .

In d u c t i o n o f p h y s ic a l d e p e n d e n c e u p o n e t h a n o ldescr ibed in th i s paper fa l l s sho r t o f Les te r ' s (1973)i d e a l m o d e l r e f e r r e d t o e a r l i e r . H o w e v e r , F r e u n d(1975) p ropo sed a no the r se t o f c r i t e r i a fo r induct iono f p h y s ic a l d e p e n d e n c e u p o n e t h a n o l w h i c h i n c lu d e :1 . rep roducib i l i ty , 2 . in the shor tes t poss ib le per iodof t ime, 3 . by s imple p roce dures , 4 . in t roduct ion o ft h e f e w e s t v a r ia b l e s b e s i d e s th e a d m i n i s tr a t i o n o fe thano l , 5 . resu l t s in spon taneous majo r wi thd rawals igns and 6 . in a l l t rea ted an im als . In conc lus ion i ts e e m s th a t t h e m e t h o d fo r i n d u c ti o n o f p h y si c a ld e p e n d e n c e u p o n e t h a n o l d e s c r i b e d i n t h i s p a p e rfulfi lls these c ondit io ns.

A c k n o w l e d g e m e n t s . The author thanks Mr. Richard D.Taylor and Mrs. Narcissus Wh itley for their excellent echnicalassistance. I am indebted to D r. W alter A. Hu nt for stimu-lating discussions throughout the course o f this study and toDr. Nancy K. M ello for her interest in the e arly stages ofthis investigation. I also thank Dr. Darwin Che ney for hisvaluable suggestions concerning the hand ling of the anim als.

R e f e r e n c e sBranchey, M., Rausche r, G., Kissin, B. : Modifications in theresponse to alcohol following establishment of physical

dependence. Psychopharmacologia (Berl.) 22, 314-322(1971)Etlis, F. W ., Pick, J. R .: Experimentally ind uce d ethanoldependence in rhesus monkeys. J. Pharmacol. e xp. Ther.175, 88--93 (1970a)Ellis, F. W ., Pick, J. R. : Evide nce of ethanol depende nce indogs. Fed. P roc. 29, 649 abs (1970b)Essig, C. F., La in, R. C. : Convulsions and hallucinatorybehavior following alcohol withdrawal in the dog. A rch.Neurol. (Chic.) 18, 626- 632 (1968)Falk, J. L. : Behavioral maintenance of high blood ethanol andphysical dependence in the rat. Science 177, 811-813(1972)Freund, G. : Alcohol w ithdrawal syndro me in mice. Arch.Neurol. (Chic.) 21, 315- 320 (1969)


10/10

254 Psy cho pha rm aco logia (Berl .) , Vol . 43 , Fasc . 3 (1975)F r e und , G . : I nduc t i on o f phys ic a l de pe nde nc e on a l c oho l i nr ode n t s . I n : B i oc he m i c a l pha r m a c o l o gy o f e t ha no l . E .Ma j c h r ow i c z , e d. , Adva nc . e xp . Me d . B i ol . 56, 31 1 - 326.N e w Y o r k - L o n d o n : P l e n u m P r e ss 1 9 75Gol ds t e i n , D . B . : Ra t e s o f ons e t a nd de c a y o f a l c oho l phys i c a ldepen dence in mice . J . Pha rma col . exp. Ther . 190, 37 7- 383

(1974)GoI ds t e i n , D . B . , Pa l , N . : A l c oho l de pe nde nc e p r odu c e d i nmi c e by i nha l a t ion o f e t ha no l : g r a d i ng t he w i t hd r a wa lreac t ion . Science 172, 28 8 - 290 (~ 971)Hu n t , W. A . : Cha ng e s i n t he ne u r o - e xc i t a b i l it y o f a l c oho l -d e p e n d e n t r a t s u n d e r g o i n g w i t h d r a w a l a s m e a s u r e d b yt he pe n t y l e ne t e t r a z o l e s e i z u r e t h r e s ho l d . Ne u r opha r ma -c o l o g y 12 , 1 0 9 7 - 1 t 0 2 ( 19 73 )Isbell , H. , Fraser, H. F. , Wikler, A. , Belleville, R. E. , Eisen-m a n , A . J . : A n e x p e ri m e n ta l s t u d y o f th e e t io l o g y o f r u mfi t s and del i r ium t remens . Quar t . J . S tud. Alcoh ol 16 ,1 - 33 (1955)Le l ba e h , W. K . : Expe r i me n t a l he pa t oc e l l u l a r ne c r os i s induc e dby e t ha no l a f t e r pa r t i a l i nh i b i t i on o f l ive r a l c oho l de hyd r o -ge na s e . I n : Me t a bo l i c c ha nge s i nduc e d by a l c oho l , G . A .Ma r t i n i a nd Ch . Bode , e d s ., pp . 62 - 6 9 . Be r l in - He i de l -b e r g - N e w Y o r k : S p r i ng e r 1 9 71Le s t e r , D . : Se l f - ma i n t e na nc e o f i n t ox i c a t i on i n t he r a t .Qua r t . J . S t ud . A l c oho l 22 , 22 3 - 23 1 ( 1961)Le s t e r , D . , F r e e d , E . X . : C r i t e r i a f o r a n a n i ma l mo de l o fa l c oho l i s m. Pha r ma c o l . B i oc he m. Be ha v . 1 , 103 - 107(1973)L i e be r , C . S ., De Ca r l i , L . M . : E t ha no l de pe nde n c e a nd t o l e r -a nc e . A nu t r i t i ona l l y c on t r o l l e d e xpe r i me n t a l mode l i nt h e r a t . R e p . C o m m . C h e m . P a t h . P h a r m . 6 , 9 8 3 - 9 9 I(1973)Lieber , C. S . , Rubin , E . , DeCar l i , L . M. , Misra , P . , Gang, H. :E f f e c ts o f py r a z o l e on he pa t i c f unc t i on a nd s t r uc t u r e . La b .I nve s t . 22 , 615 - 62 I ( 1970 )Ma gnus s on , G . , Nyb e r g , J . A . , Bod i n , N . O . , Ha ns s o n , E . :Tox ic it y o f py r a z o l e a nd 4 - me t hy l py r a z o l e i n mi c e a ndra ts . Exper ient ia (Basel ) 28 , 1198-1200 (1972)M a j c h r o w i c z , E . : D e t e r m i n a t i o n o f e t h a n o l , m e t h a n o l a n da c e t one i n b i o log i c a l f l u id s by a u t om a t e d ga s c h r oma t o g -raph y. Am er . chem . Soc . Div . Biol . Che m. 298 abs (1971b)Ma j c h r ow i c z , E . : I nduc t i on o f phys i c al de pe nde nc e on a l c oho la nd t he a s s oc i a t e d me t a bo l i c a nd be ha v i o r a l c ha nge s i nra ts . Pharmacologis t 15 , 159 abs (1973)Ma j c h r ow i c z , E . , B e r c a w, B . L . , Co l e , W. M. , Gr e g o r y , D .H . : N i c o t i na m i de a de n i ne d i nuc l e o t i de a nd t he m e t a b -o l i sm o f e t ha no l a nd a c e t a l de hyde . Qua r t . J . S t ud . A l c oh o l2 8 , 2 1 3 - 2 2 4 ( 19 67 )Ma j c h r ow i c z , E . , L i p t on , M. A . , Me e k , J , L . , Ha l l , L . :E f fe c ts o f c h r o n i c e t h a n o l c o n s u m p t i o n o n c l e a ra n c e o fa c u t e l y a dmi n i s t e r e d e t ha no l a nd a c e t a l de hyde f r om b l oodin ra ts . Quar t . J . S tud. Alco ho l 29 , 55 3 - 557 (1968)

Ma j c h r owi c z , E . , Me nde l s on , J . H . : B l ood me t h a no l c o nc e n -t r a t i ons du r i ng e xpe r i me n t a l l y i nduc e d e t ha no l i n t ox i -ca t ion in a lcohol ics . J. Pharm acol . exp. Ther . 179, 29 3- 300(1971a)M cQu arr ie , D. G. , F ingle , E . : Ef fec ts of s ingle doses andc h r on i c a dm i n i s t r a t ion o f e t ha no l on e xpe r i me n t a l s ei zu r esin mice . J . Phar ma col . exp. Ther . 124, 26 4- 27 1 (1958)Me e k , J . L . , Ma j c h r owi c z , E . , L i p t on , M . L . : Ra t e s o f C14Ozpr oduc t i on f r om l a be l e d e t ha no l , a c e t a t e a nd g l uc os e i na l c oho l d r i nk i ng a nd non - d r i nk i ng r a t s . P r oc . Soc . e xp .Biol . (N.Y. ) 126, 379-383 (1967)Me l l o , N . K . : A r e v i e w o f me t hod s t o i nduc e a l c oho l a dd i c t i oni n an i ma l s . Pha r ma c o l . B i oc he m. Be ha v . 1 , 89 - 1 01 ( 1973 )Me nde l s on , J . H . ( e d . ): Expe r i me n t a l l y i nduc e d c h r on i cin toxica t ion and wi thdrawal in a lcohol ics . Quar t . J . S tud,Alcohol , Suppl . No. 2 (1964)Pieper , W. A. , Skeen, M. J . , McClure , H. M. , Bourne , P . G. :The c h i mpa nz e e a s a n a n i ma l mode l f o r i nve s t i ga t i ngalcohol ism . Science 176, 71 - 73 (1972a)P i e pe r , W . A . , Ske e n, M . J . : I nduc t i on o f phys i c a l de pe nde nc eon e t ha no l i n r he s us monke ys u s i ng o r a l a c c e p t a nc et e c hn i que . L i f e Sc i. 11 , 98 9 - 99 7 ( t 972b )Ra t c l if f e , F . : E t ha no l de pe nde nc e i n t he r a t : I t s p r oduc t i o na nd c ha r a c t e ri s ti c s . Ar c h . i n t. Pha r m a c ody n . 196 , 146 - 15 6(1972)Roa c h , M . K . , C r e a ve n , P . J . : A m i c r om e t hod f o r t he de t e r -mi na t i on o f a c e t a lde hyde a nd e t ha no l i n b l ood . C l i n .c h i m. Ac t a 21 , 275 - 278 ( 1968 )Roa c h , M. K . , Kha n , M. M. , Co f f ma n , R . , Pe nn i ng t on , W. ,Da v i s , D . L . : B r a i n ( Na + K + ) - a c t i va te d a de nos i ne t r i phos -pha t a s e a c t i v i t y a nd ne u r o t r a ns mi t t e r up t a ke i n a l c oho l -de pe nde n t r a t s . B r a i n Re s . 63 , 323 - 3 29 ( 1973)Vi c t o r , M . : T r e a t m e n t o f a l c oho l i c in t ox i c a t ion a nd t he w i t h -d r a wa l s yn d r ome . A c r i ti c a l a na ly s i s o f the u s e o f d r ugsa n d o t h e r f o r m s o f th e r a p y . P s y c h o so m . M e d . 3 3 , 6 3 6 - 6 5 0(1966)Vi c t o r , M. , Ad a m s , R . D . : The e f f ec t o f a l c oho l on t he ne r -vou s sys tem. Res . PuN . Ass . nerv . mer i t. Dis . 32 , 5 2 6 - 573(1953)Wa l ke r , D . W. , Zo r ne t z e r , S . F . : A l c oh o l w i t hd r a wa l i n mi c e :E l e c t r oe nc e pha l og r a ph i c a nd be ha v i o r a l c o r r e l a te s . E l e e -t r oe nc e ph , c l in . Ne u r op hys i o l . 36 , 233 - 2 44 ( 1974)Wa l l g r e n , H . , Ko s une n , A . L . , Ah t e e , L . : Te c hn i que f o r p r o -duc i ng a n a l c oho l w i t hd r a wa l s ynd r ome i n r a t s . I s r a e lJ . reed . Sci . , Suppl . 9 , 63-71 (1973)W ilson, W. L. , Bot t ig l ie ri , N. G . : Phas e I s tudies wi th p yra-z o l e . Ca nc e r Che mot he r . Re p . 21 , 137 - 141 ( 1%2)W ood s , J. H . , I ko mi , F . , W i nge r , G . : T i l e r e i n f o r c i ng p r op -e r ti e s o f e t ha no l . I n : B i o l og ic a l a s pe c ts o f a l c oho l is m,M . K . R o a c h , W . M . M c I s a a c a n d P . J . C r e a v e n , e ds .,pp . 371 - 388. Au st in : Un iv . of Texas Press , 1971

D r . E . M a j c h r o w i c z , L a b o r a t o r y o f A l c o h o l R e se a r ch , N a t i o n a l I n s t i tu t e o n A l c o h o l A b u s e a n d A l c o h o li s m , A D A M H A ,Wi l l i a m A . W hi t e Bu i l d ing , S t . E l iz a be t h s Hos p i t a l , W a s h i ng t on , D . C . 20032 , U . S . A .

Documents

Induction of Physical Dependence Upon Ethanol and the Associated Behavioral Changes in Rats