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Effect of hypnotics on mice genetically selected for sensitivity to ethanol
Pharmacology Biochemistry & Behavior, Vol 4, pp. 679-683 Copyright © 1976 by ANKHO International Inc All rights of reproduction m any form reserved. Printed m the U.S A.
Effect of Hypnotics on Mice Genetically Selected for Sensitivity to Ethanol V. G E N E E R W I N , W. D. W. H E S T O N 1 , G. E. M C C L E A R N
School o f Pharmacy and Institute for Behavioral Genetics UniversiO, o f Colorado, Boulder, CO 80302 AND R A DEITRICH
Department o f Pharmacology, University o f Colorado School o f Medicine 4200 East Ninth Avenue, Denver, CO 80220 (Received 9 J a n u a r y 1976)
ERWIN, V G., W. D W. HESTON, G. MCCLEARN AND R. A. DEITRICH. Effect of hypnotics on mice genetically selected for sensitivity to ethanol. PHARMAC. BIOCHEM. BEHAV. 4(6) 679-683, 1976. - It was previously shown that the rate of disappearance of blood ethanol was tdentical for 2 lines of mice selectwely bred for differences in sleep-time after ethanol admimstration The ED s o values for the loss of nghtung response with ethanol were significantly different at 3 64 g per kg for the SS line and 1.65 g per kg for the LS hne In the present study the mean sleep ume is 367 sec for SS mice and 9342 sec for LS mice The ED s 0 values remain essentially the same as previously reported. Unchanged LD s o values for ethanol, however, are not different at 4 8 g per kg for the SS and 4 5 g per kg for the LS hne of mice The ED s o value for loss for righting response following administration of methanol, butanol and t-butanol Is approximately 2 fold greater for the SS line of mice than for the LS hne The ED s 0 values for sodium pentobarbltal or ether m the 2 hnes of mice for loss of righting response are virtually identical In addition, the sleep-time values obtained after the admimstratlon of pentobarbital, chloral hydrate, tHchloroethanol and paraldehyde are not significantly different. These data indicate that while the SS and LS lines of mice differ in central nervous system sensltlwty to ethanol, methanol, butanol and t-butanol It is Implied that they do not differ in central nervous system sensitivity to other hypnotic agents tested. Proof of this latter suggestion awaits determination of metabohc rates, and brain levels of these other depressants Ethanol
Central Nervous System Sensltwlty
Genetics
R E C E N T studies o n the a c t i o n s o f e t h a n o l , [4,5] have r e p o r t e d o n the utilization o f 2 lines o f mice w h i c h were derived f r o m a genetically h e t e r o g e n e o u s HS s t o c k b y m e a n s of a selective b r e e d i n g p r o g r a m The h e t e r o g e n e o u s s t o c k was e s t a b l i s h e d b y i n t e r c r o s s i n g o f 8 i n b r e d strains o f mice (A, A K R , B A L B / c , C 3 H / 2 , C57BL, D B A / 2 , ls/Bi and R i l l ) and s u b s e q u e n t l y m a i n t a i n e d b y a r a n d o m m a t i n g procedure. Details o f this p r o c e d u r e were given previously [11 ]. A f t e r d e t e r m i n i n g the sleep-time ( f o l l o w i n g 3.4 g e t h a n o l per Kg b o d y weaght) o f a large n u m b e r o f mice f r o m t h e h e t e r o g e n e o u s s t o c k , t h o s e a m m a l s w i t h the s h o r t e s t sleep-time were m a t e d , and their p r o g e n y was t h e first g e n e r a t i o n o f the short-sleep (SS) line o f mice. The first g e n e r a t i o n o f long-sleep (LS) mice was p r o d u c e d in a c o m p a r a b l e m a n n e r b y mating t h o s e animals w i t h the longest sleep-time. In s u b s e q u e n t g e n e r a t i o n s o f the s h o r t sleep hne, selection pressure was m a i n t a i n e d b y o n l y b r e e d i n g animals w i t h the s h o r t e s t sleeping times. Sxmilarly, the long-sleep line was p r o d u c e d b y utilizing as p a r e n t s m
Alcohols
Hypnotics
each successive g e n e r a t i o n o n l y t h o s e a m m a l s w longest sleeping t i m e . Previously, we r e p o r t e d t] actiwtles o f liver a l c o h o l d e h y d r o g e n a s e and the i b l o o d e t h a n o l d i s a p p e a r a n c e were virtually identi samples o f the 2 lines o f mice [5 ]. The ED~ 0 values o f righting r e s p o n s e w i t h e t h a n o l were a p p r o x i m fold greater m the SS a m m a l s t h a n in t h e LS m i c e suggested t h a t the 2 lines o f mice differ m their nervous s y s t e m sensltivlty to e t h a n o l . Prior investigations [14,15] o f t h e e n z y m e s resl: for e t h a n o l m e t a b o l i s m d e m o n s t r a t e d d i f f e r e n c e s activity o f liver e n z y m e s r e s p o n s i b l e for e t h a n o l olism in various i n b r e d strains o f mice. O t h e r inves have s h o w n t h a t mice f r o m various inbred stratus d sleep-time i n d u c e d b y b a r b i t u r a t e s [ 6 , 1 7 ] . Liver somal fractaons xsolated f r o m t h e s e inbred stratus d i f f e r e d in rates o f h e x o b a r b i t a l m e t a b o h s m H studies o f possible d i f f e r e n c e s in brain sensiti' b a r b i t u r a t e s were n o t r e p o r t e d . It has b e e n not~
~Present Address Department of Pharmacology and Experimental Therapeutrcs, The Johns Hopkins School of Medicine, Bz Maryland 679
680
E R W I N , H E S T O N , M C C L E A R N A N D DE!
b a r b i t u r a t e s a n d general a n e s t h e t i c s as well as a l i p h a t i c a l c o h o l s depress t h e r e t i c u l a r a c t i v a t i n g s y s t e m w h i c h subserves w a k e f u l n e s s [ 8 , 1 3 ] . C o n s e q u e n t l y it a p p e a r e d t h a t t h e LS a n d SS lines o f mice w h i c h d i f f e r in b r a i n sensitivity to e t h a n o l m i g h t be o f value m c o m p a r i n g t h e m e c h a n i s m s o f a c t i o n o f various c e n t r a l n e r v o u s s y s t e m d e p r e s s a n t s . In t h e p r e s e n t s t u d y we c o m p a r e d t h e h y p n o t i c p o t e n c i e s a n d d u r a t i o n o f r e s p o n s e o f various c e n t r a l n e r v o u s s y s t e m d e p r e s s a n t s in t h e LS a n d SS lines o f mice. Some o f t h e s e d a t a have a p p e a r e d in a b s t r a c t f o r m [ 4 ] . MATERIALS AND METHOD Mice o f t h e long-sleep (LS) a n d t h e short-sleep (SS) line of the 1 4 t h g e n e r a t i o n were o b t a i n e d f r o m t h e I n s t i t u t e f o r B e h a v i o r a l G e n e t i c s ( I B G ) a n d a f t e r at least a week o f a d a p t a t i o n to t h e t e s t i n g r o o m in a n a d j a c e n t l a b o r a t o r y , were t e s t e d b e t w e e n 9 0 - 2 0 0 days o f age HS a n i m a l s were also t e s t e d m 1 e x p e r i m e n t . E q u a l n u m b e r s o f male a n d female mice were t e s t e d at e a c h dose. Doses o f e t h a n o l o f 0 . 0 0 8 - 0 . 0 1 2 ml/g were i n j e c t e d IP (25% v/v in sahne). T h e mice weighed b e t w e e n 1 8 - 3 4 g. R e s p o n s e to t h e a l c o h o l s a n d t h e various s e d a t w e h y p n o t i c a g e n t s was m e a s u r e d b y d e t e r m i n a t i o n o f sleeping time. Because a w a k e n i n g f r o m a l c o h o l is n o t as clear cut as a w a k e n i n g f r o m o t h e r sedative h y p n o t i c s , t h e a m m a l was not c o n s i d e r e d a w a k e u n t d it h a d r i g h t e d itself 3 t i m e s w i t h i n 30 sec. F o r t h e ED~o studies, t h e a m m a l was c o n s i d e r e d to have lost its r i g h t i n g r e s p o n s e ff it r e m a i n e d o n its b a c k for 1 m i n or longer. F o r t h e LD~o a c u t e t o x i c i t y s t u d y , t h e a n i m a l s were e x a m i n e d a f t e r 24 hr h a d elapsed a n d t h e n u m b e r o f d e a t h s at t h a t dose r e c o r d e d [ 2 ] . T h e ED 5 o doses were d e t e r m i n e d b y t h e m e t h o d o f Litchfleld and Wilcoxon [9]. T o o b t a i n ED~ o values for e t h e r , a m m a l s were placed in 4 1 jars w i t h air t i g h t lids a n d e t h e r was i n j e c t e d t h r o u g h a rubber septum. Animals tested without ether adminis-
t r a t i o n s h o w e d no signs o f necessary for testing.
hypoxia
during
RESULTS T a b l e 1 illustrates t h e sleep-time scores for t h e sample o f a n i m a l s f r o m the 1 4 t h g e n e r a t i o n sleep-time selection lines. A m e a n sleep-time score sec for t h e SS line a n d 9 3 4 2 sec for t h e LS line is o following a dose of 3.75 g/kg o f e t h a n o l IP. These can b e c o m p a r e d w i t h t i m e s a f t e r a d m i n i s t r a t i o n of h y p n o t i c drugs. B o t h m e t h a n o l a n d b u t a n o l cause of t h e LS h n e to sleep significantly longer t h a n thos, SS line. H o w e v e r , t h e r e is n o statistically sig d i f f e r e n c e a m o n g the lines in p e n t o b a r b i t a l , chlc drate, p a r a l d e h y d e or t r i c h l o r o e t h a n o l i n d u c e d sle~ T h e ED s o for loss of r i g h t i n g r e s p o n s e s h o u l d be definitive measure of CNS sensitivity for these 2 1 animals, since a n y m e t a b o l i c changes wall b e less lm m t h e few m i n r e q u i r e d to p r o d u c e loss o f r i g h t i n g rq m t h e a n i m a l s t h a n d u r i n g the course o f sle d e t e r m i n a t i o n . S u c h e x p e r i m e n t s were carried o u t results p r e s e n t e d in Fig. 1. T h e dose r e s p o n s e h essentially parallel. In T a b l e 2 are listed the ED s 0 w i t h the 95% c o n f i d e n c e limits for t h e s e alcohols s o d i u m p e n t o b a r b l t a l a n d e t h e r It can be seen fr d a t a t h a t t h e r e is a p p r o x i m a t e l y a 2 fold differ, EDs0 for t h e various a l c o h o l s b e t w e e n LS and SS However, t h e r e is n o d i f f e r e n c e b e t w e e n HS, LS lines in ED 50 for Na p e n t o b a r b i t a l or b e t w e e n the LS mice m ED 5 0 for e t h e r . Previously we h a d f o u n d n o d i f f e r e n c e m t h e m e t a b o l i s m o f e t h a n o l in t h e 2 lines, yet t h e LS lin m u c h longer. O b v i o u s l y t h e n , t h e SS animals m u s t at h i g h e r b l o o d a l c o h o l levels t h a n do t h e LS amm~ p r e d i c t i o n is fulfilled as i l l u s t r a t e d in Table 3. T a b l e 4 lists t h e LD~ 0 doses for e t h a n o l . T h e LS
TABLE
1
LONG SLEEP ILS). SHORT SLEEP (SS) AND HETEROGENEOUS STOCK (HS~ SLEEP TIMES WITH VARIOUS HYPNOTIC AGENTS* Animal Line
Drug
(n)
SS LS SS LS SS LS SS LS HS SS LS SS LS SS LS
Ethanol Ethanol Methanol Methanol n-Butanol n-Butanol Pentobarbltal Pentobarbltal Pentobarbltal Chloral Hydrate Chloral Hydrate Paraldehyde Paraldehyde Tnchloroethanol Trlchloroethanol
l0 3.75 l0 3 75 10 4 5 10 4 5 10 0 567 10 0 567 70 0 060 56 0 060 I0 0 060 19 0 45 20 0 45 20 1 0 19 I 0 10 0 225 10 0 225
th
Doset
Sleep Time (sec) 367 9,342 52 18,456 280 2,169 2,107 1,414 2,500 5,502 7,203 2,766 3,720 1,502 2,542
(+_547)~: (+--5,969) (_+77) (-10,750) (_+384) (_+890) ( ~- 1,678) (_+873) (_+ 1,035) (4-1,842) ~-+ 1,662) (+- 1,172) (_+846) (_+ 1,263) (+-844)
p Value <0 01 <0 01 <0 01 ns ns ns ns ns
*Sleep times were taken as the time from the loss of nghtmg reflex to the time the animal was able to nght itself three t~mes within 30 sec. t i P dose m g/kg ~:Represents +- the standard devmtton
GENETICS
OF CNS SENSITIVITY
TO ETHANOL
Bu
Bu t-Bu SS LS
t-Bu S,S
Et
Me LS
/
z
80
n 09
70-
Me S,S
j]/ ! ! I
z
IE)
Et SS
! I I
I 1
I
I '
,I
/
,I
/ I--"
z Iii 0
40
ILl
20.
30
10
/
/i .
I
i
I !
I !
, ;
P
/
,
:
, l
:
l
I
I
I
52
I
0.,'
I
,
0.3 0.4
I
I
I
t
I
I
0.6 0.8 1.0
I
2.0
I
I
t
3.0 4.0 5.0
(gin per Kg BodyWe,ght)
DOSE
FIG 1. Dose response curve for alcohol m SS and LS mice Methanol; (Me) LS mace 1" . . . . . . - ) , SS rmce (A. . . . . . ~) Ethanol (Et) LS mice (e e); SS mice (o c) Butanol (Bu) LS rmce(, . . . . =), SS mice (c~. . . . a) t-Butanol (tBu) LS mice (~. . . . . *), SS mice
(~ . . . .
~)
TABLE TABLE
2
EDs0 V A L U E S FOR LOSS O F R I G H T I N G R E F L E X WITH V A R I O U S H Y P N O T I C AGENTS*
Ammal Line Drug
EDso (g/kg, IP)
SS LS SS LS SS
Ethanol Ethanol Methanol Methanol Butanol
3 7 I 65 50 2 75 0 535
LS SS LS SS LS HS SS LS
Butanol t-Butanol t-Butanol Na Pentobarb~tal Na Pentobarbnal NaPentobarb~tal Ether Ether
0 245 (_+0 011) 1 39 ( ± 0 065)§ 0 84 ( ± 0 0785§ 0.039 (_+0.0028) 0 038 (-+-+00026) 0040(±0.0027)§ 1 04 (_+0 26)$§ 104 (±0.3255§
(±0.115¢ ( ± 0 09) ( _ 0 24) (_+0 11) (_+0.026)
EDs0 Ratio SS/LS 2 24
3
B L O O D A L C O H O L C O N C E N T R A T I O N S IN SS AND LS TIME OF A W A K E N I N G
Ammal Line SS LS
Sleep Time sec _+ SD (n)
Blood Alcoh, m g ~ ± SD (
3937 ± 2502 (10) 16069 _+ 5443 (125 p < < 0 001
380 2 -'- 52 6 ( 230 0 ± 43.5 ( p < < 0 001
1 82 2 18 1 65
*EDso values determined by the m e t h o d of Litchfield and Wilcoxon ( n = 10 animals each at four different doses) f R e p r e s e n t s 95% confidence hm~ts ~mM/l by inhalation §Tested m the 18th generation or c o n t e m p o r a n e o u s HS stock
M~ce of each hne were given 4 1 g/kg ethanol as outhn text This dose was n e c e s s a r y to insure loss of righting re! all SS mace At the time of regaining the righting reflex, b taken from the retro-orbltal sinus and blood alcohol deter: gas c h r o m a t o g r a p h y as prevaously described [5].
are o n l y s l i g h t l y m o r e s e n s i t i v e to t h e a c u t e e t h a n o l . It s h o u l d b e n o t e d t h a t at t h e methanol and t-butanol ammals were often following day, even though they had not lost the righting response.
t o x i c el higher ( found C always
DISCUSSION In c e r t a i n g e n e r a t i o n s , a s a m p l e o f H S m~ce ~s te ethanol sleep time contemporaneously w i t h t h e LS
682
ERWIN, H E S T O N , M C C L E A R N A N D DEI TABLE 4
LDs0 VALUES FOR ETIqANOL IN SS AND LS SELECTED LINES OF MICE*
Animal Line
LDs0~
SS LS
g/kg 4 8 ~±0 2)~ 4.5 I±0 2)
*Lethahty was determined by the number of deaths occurring within 24 hr followmg IP injections of various doses of ethanol (n=8 at each of 5 doses) tAccordlng to the method of Ltchfield and Wdcoxon [9] ~:Represents 95% confidence hmns mice These samples s h o w t h e e x p e c t e d gaussian dist r i b u t i o n o f sleep-times t h a t was p r e s e n t m t h e f o u n d a t i o n stock HS animals. T h e SS a n d LS mice are m a t e d m s u c h a way t h a t m a t i n g parrs n e v e r have c o m m o n g r a n d p a r e n t s , T h u s , in d i s t i n c t i o n to i n b r e d m o u s e stratus t h a t s h o w d i f f e r e n c e s m sleep t i m e s similar to t h o s e o b s e r v e d m ttus r e p o r t [ 7 , 1 2 ] , t h e g e n e t i c v a r i a b i l i t y p r e s e n t initially m the HS s t o c k w i t h respect to p h e n o t y p e s u n r e l a t e d to e t h a n o l sleep t i m e is preserved as m u c h as possible. This preservation o f h e t e r o g e m t y is s h o w n b y the fact t h a t the r e s p o n s e s of HS, LS a n d SS mice to p e n t o b a r b l t a l do n o t differ. T h e r e is a significant d i f f e r e n c e in ED~ 0 b e t w e e n lines for loss o f r i g h t i n g r e s p o n s e for m e t h a n o l , e t h a n o l , butanol and t-butanol, P r e v i o u s l y we h a d d e m o n s t r a t e d t h a t t h e r e was n o change m t h e rate of a l c o h o l m e t a b o l i s m b e t w e e n t h e 2 lines [ 5 ] . We have n o t m a d e similar d e t e r m i n a t i o n s for t h e o t h e r a l c o h o l s t e s t e d here H o w e v e r , e a c h a l c o h o l t e s t e d is m e t a b o h z e d via a d i f f e r e n t e n z y m a t i c system. T h e m e t a b olic r o u t e for m e t h a n o l m t h e r o d e n t is p r i m a r i l y via the catalase s y s t e m [ 1 0 ] . E t h a n o l is m e t a b o h z e d p n m a r d y via the a l c o h o l d e h y d r o g e n a s e s y s t e m , t - B u t a n o l is o n l y slowly m e t a b o l i z e d if at all via c o n j u g a t i o n w i t h g l u c o r o n l d e [ 1 8 ] , a l t h o u g h o t h e r possible r o u t e s o f m e t a b o h s m have n o t b e e n studied. It w o u l d seem u n h k e l y t h a t we have selected for similar changes m several diverse m e t a b o h c p a t h w a y s , a n d we c o n c l u d e , t h e r e f o r e , t h a t the selective b r e e d i n g p r o g r a m has d e v e l o p e d lines t h a t differ in b r a i n sensitivity to alcohols m general, n o t j u s t to e t h a n o l It a p p e a r s t h a t we have n o t selected for a general CNS s e n s l t w i t y to d e p r e s s a n t s b e c a u s e n o n e o f t h e o t h e r CNS d e p r e s s a n t s s h o w d i f f e r e n t i a l effects m t h e two lines. This o b s e r v a t i o n is s o m e w h a t s u r p r i s m g since all t h e t e s t e d c o m p o u n d s ( p e n t o b a r b l t a l , e t h e r , chloral h y d r a t e a n d p a r a l d e h y d e ) are at least additive w i t h e t h a n o l m t h e i r effects o n t h e CNS. In p a r t i c u l a r e t h e r , chloral h y d r a t e a n d p a r a l d e h y d e have long b e e n t h o u g h t to possess a similar ff n o t i d e n t i c a l m e c h a n t s m to t h a t o f e t h a n o l [ 1 3 ] . T h e s e results p e r m i t a c o n c l u s i o n t h a t t h e m e c h a n i s m o f these c o m p o u n d s all differ in s o m e r e s p e c t f r o m t h a t o f e t h a n o l even t h o u g h t h e final c o m m o n result ~s the same. F i r m c o n c l u s i o n s c o n c e r n i n g t h e n o n - a l c o h o l drugs m u s t await analysis of t h e i r m e t a b o l i s m in t h e 2 lines of mice, b u t again it is u n h k e l y s e l e c t i o n has b e e n change s i m u l t a n e o u s l y in a large, diverse g r o u p o f d i s p o s i t i o n p a t h w a y s .
It is also of i n t e r e s t t h a t t h e LD s 0 values for eth not differ in t h e t w o lines i n d i c a t i n g t h a t t h e mecl r e s p o n s i b l e for sleep are n o t t h e same as t h o s e resI for e v e n t u a l d e a t h f r o m overdosage Sleep t i m e d e t e r m i n a t i o n s w i t h these animals are to a n u m b e r of v a r i a t i o n s f r o m e x p e r i m e n t to e x p as e v i d e n t in T a b l e 3 a n d o u r p r e v i o u s results [: values of 3 9 3 7 sec vs 1250 sec for t h e SS line a n d 1 6 2 5 0 sec for t h e LS line shows t h e c o n s i s t e n c y m d i f f e r e n c e s b u t also illustrate variability m t h e a n u m b e r s . T h e reasons for t h e variability are u n k n o certainly d i f f e r e n t i n j e c t i o n t e c h n i q u e s a n d dIffer~ o r a t o r y e n v t r o n m e n t s c o n t r i b u t e . In a n y given exp~ the age o f all o f t h e a n i m a l s was a p p r o x i m a t e l y t h F r o m e x p e r i m e n t to e x p e r i m e n t t h e r e were age diff and this m a y a c c o u n t for some of the v a r i a t i o n s is also critical to p o i n t o u t t h a t t h e a b s o l u t e differ sleep t i m e s b e t w e e n SS a n d LS mice is d e p e n d e n t ul b l o o d level o f a l c o h o l achieved. This is best illustr~ c o n s i d e r i n g the case w h e r e the level is so low t h a t mice lose the r i g h t i n g r e s p o n s e b u t the LS mice s u b s t a n t i a l time. F r o m Table 3 it is seen t h a t such would b e a p p r o x i m a t e l y 327 rag% G i v e n t h a t t h e I m u s t r e a c h 2 3 0 mg% for half o f t h e mice to a w a k e a the rate o f m e t a b o l i s m is a b o u t 19 mg% per 1000 the LS mice w o u l d sleep a b o u t 5 0 0 0 sec o n t h e ~ These c a l c u l a t i o n s are possible because o f t h e zer, rate o f e t h a n o l e h m m a t l o n S i e m e n s a n d C h a n [16] r e p o r t e d t h a t pentol p r o d u c e d a significantly longer loss o f r i g h t i n g retie mice t h a n m LS mice b u t t h a t this was d u e to a d a p p a r e n t volume of d i s t r i b u t i o n o f t h e drug m t hnes. In t h e present s t u d y we did n o t observe a stati significant d i f f e r e n c e m the sleep t i m e s b u t did slightly longer loss of r i g h t i n g r e s p o n s e in t h e SS mic G o l d s t e l n and K a k l h a n a [3] tested the SS and 1 for severity of w i t h d r a w a l a f t e r 3 days of m t o x i T h e y find t h a t t h e SS mice have m o r e severe wltt r e a c t i o n s t h a n d o t h e LS mice and t h e s e d i f f e r e n c e s casually related to t h e line d t f f e r e n c e s m acute effects. R e c e n t l y Collins ( p e r s o n a l c o m m u n i c a t i o n ) fore the i n f l u e n c e of e t h a n o l o n t h e t u r n o v e r o f do differed b y a b o u t 2 fold m t h e SS a n d LS mice althc d i f f e r e n c e s in n o r e p m e p h r m e or s e r o t o n m t u r n o w o b s e r v e d Analysis o f t h e m a j o r s y n t h e t i c and degr e n z y m e s o f t h e d o p a m m e p a t h w a y s revealed n o dlff~ Fuller t p e r s o n a l c o m m u n i c a t i o n ) has o b s e r v e d a dlf in t h e sleep time i n d u c e d b y salsolinol ( t h e c o n d e product of dopamme and acetaldehyde) when i i n t r a c r a m a l l y . F r o m these data t h e n it would appc s t u d y of the d o p a m i n e r g i c s y s t e m m i g h t prove fru the s e a r c h for a b i o c h e m i c a l e x p l a n a t i o n o f results r~ in this paper. ACKNOWLEDGEMENTS Supported by grants No. AA00263, MH18948, NIMH Grant No. MH 11167, and a Career Development Award No GM-10475. The authors wish to express their apprec Mrs Candace Owen tor her excellent techmcal assistance.
G E N E T I C S O F CNS S E N S I T I V I T Y TO E T H A N O L
REFERENCES 1. Collins, A. C., T N. Yeager, M E. Lebsack and S. Panter. Varlahons m Alcohol Metabohsm Influence of Sex and Age. Pharmac. Blochem Behav. 3: 973-978, 1975. 2. Foster, R. H K. Standardization of safety margin J. Pharmac. exp. Ther. 65: 1-17, 1939. 3. Goldstem, D B. and R Kaklhana. Alcohol Withdrawal reactions m "Long-Sleep" and "Short-Sleep-Mice" Pharmacologtst 17: 197, 1975 4. Heston, W. D. W., S. M Anderson. V. G. Erwm and G . E . McClearn. A comparison of the actions of various hypnotics on rmce selectwely bred for sensthvtty to ethanol Behav Genet 3: 402-403, 1973 5. Heston, W D W., V G. Erwm, S. M. Anderson and H. Robbms. A comparison of the effects of alcohol on mice selectwely bred for differences m ethanol sleep-time Life SCL 14: 365-370, 1974. 6. Jay, G. E , Jr Varmtlons m response of various mouse stratus to hexobarbltal (Evlpal) Proc exp. Btol Med 90: 378-380, 1955. 7. Kaklhana, R , D. R. Brown, G E McClearn and I R. Tabershaw. Brain sens~tlvlty to alcohol m ~nbred mouse stratus Sctence 154: 1574-1575, 1966. 8. Kalant, H. Effects of ethanol on the nervous system. In Alcohols and Derwatwes, Vol. 1, Int. Encycl Pharmac Ther. Sec 20, edited by J Tr~moheies. London Pergamon Press, 1970, p. 189. 9 Lltchfleld, J. T., Jr. and F. W Wdcoxon. A slmphfied method of evaluating dose-effect experiments. J Pharmac exp Ther. 96: 99-113, 1949.
10. Mannerlng, G J , A. Makar, D. R. Van-Harken, ar Tephly. A species difference m the oxidation of rr Toxic. appl Pharmac. 7: 490, 1965. 11. McClearn, G. E , J R Wdson and W Meredith trtbutions to Behavtor - Genetw Analysts The Mc Prototype, edited by G Llndzey and G G Lmdsey a Thzessen New York Appleton-Century Crofts, 1~. 3-22 12. Randall, C L., J. A. Carpenter, D. Lester and it J. F Locomotor activity differences between C57BL an mtce after ethanol Fedn. Proc Abs 34: 720, 1975. 13. Rltchle, J. M. The ahphatlc alcohols In The Pharma, Basts ofTherapeuttcs, 4 t h e d , edited by L. S. Goodma Gilman. New York MacMlllan Co., 1970, p. 135. 14 Rodgers, D A , G. E. McClearn, E. L. Bennett and M Alcohol Preference as a function of its caloric utdtty tv comp. phystol. Psychol. 56: 666-672, 1963. 15. Sheppard, J R., P. Albershelm and G E McClearn actw~t~es and ethanol preference m mice Btochem. ( 205-212, 1968 16. Siemens, A. J , and A. W. K. Chan Effects of Pentob., Mice Selectwely bred for different sensitivities to Pharmacologist 17: 197, 1975 17. Vessell, E. S. Genetic and envtronmental factors hexobarbltal metabohsm in mice. Ann N. Y. Acad. ' 900-912, 1968. 18, Wilhams, R. T. Detoxtcatton Mechanisms, 2nd ed., Ne Wdey, 1959, p. 68.
Effect of Hypnotics on Mice Genetically Selected for Sensitivity to Ethanol V. G E N E E R W I N , W. D. W. H E S T O N 1 , G. E. M C C L E A R N
School o f Pharmacy and Institute for Behavioral Genetics UniversiO, o f Colorado, Boulder, CO 80302 AND R A DEITRICH
Department o f Pharmacology, University o f Colorado School o f Medicine 4200 East Ninth Avenue, Denver, CO 80220 (Received 9 J a n u a r y 1976)
ERWIN, V G., W. D W. HESTON, G. MCCLEARN AND R. A. DEITRICH. Effect of hypnotics on mice genetically selected for sensitivity to ethanol. PHARMAC. BIOCHEM. BEHAV. 4(6) 679-683, 1976. - It was previously shown that the rate of disappearance of blood ethanol was tdentical for 2 lines of mice selectwely bred for differences in sleep-time after ethanol admimstration The ED s o values for the loss of nghtung response with ethanol were significantly different at 3 64 g per kg for the SS line and 1.65 g per kg for the LS hne In the present study the mean sleep ume is 367 sec for SS mice and 9342 sec for LS mice The ED s 0 values remain essentially the same as previously reported. Unchanged LD s o values for ethanol, however, are not different at 4 8 g per kg for the SS and 4 5 g per kg for the LS hne of mice The ED s o value for loss for righting response following administration of methanol, butanol and t-butanol Is approximately 2 fold greater for the SS line of mice than for the LS hne The ED s 0 values for sodium pentobarbltal or ether m the 2 hnes of mice for loss of righting response are virtually identical In addition, the sleep-time values obtained after the admimstratlon of pentobarbital, chloral hydrate, tHchloroethanol and paraldehyde are not significantly different. These data indicate that while the SS and LS lines of mice differ in central nervous system sensltlwty to ethanol, methanol, butanol and t-butanol It is Implied that they do not differ in central nervous system sensitivity to other hypnotic agents tested. Proof of this latter suggestion awaits determination of metabohc rates, and brain levels of these other depressants Ethanol
Central Nervous System Sensltwlty
Genetics
R E C E N T studies o n the a c t i o n s o f e t h a n o l , [4,5] have r e p o r t e d o n the utilization o f 2 lines o f mice w h i c h were derived f r o m a genetically h e t e r o g e n e o u s HS s t o c k b y m e a n s of a selective b r e e d i n g p r o g r a m The h e t e r o g e n e o u s s t o c k was e s t a b l i s h e d b y i n t e r c r o s s i n g o f 8 i n b r e d strains o f mice (A, A K R , B A L B / c , C 3 H / 2 , C57BL, D B A / 2 , ls/Bi and R i l l ) and s u b s e q u e n t l y m a i n t a i n e d b y a r a n d o m m a t i n g procedure. Details o f this p r o c e d u r e were given previously [11 ]. A f t e r d e t e r m i n i n g the sleep-time ( f o l l o w i n g 3.4 g e t h a n o l per Kg b o d y weaght) o f a large n u m b e r o f mice f r o m t h e h e t e r o g e n e o u s s t o c k , t h o s e a m m a l s w i t h the s h o r t e s t sleep-time were m a t e d , and their p r o g e n y was t h e first g e n e r a t i o n o f the short-sleep (SS) line o f mice. The first g e n e r a t i o n o f long-sleep (LS) mice was p r o d u c e d in a c o m p a r a b l e m a n n e r b y mating t h o s e animals w i t h the longest sleep-time. In s u b s e q u e n t g e n e r a t i o n s o f the s h o r t sleep hne, selection pressure was m a i n t a i n e d b y o n l y b r e e d i n g animals w i t h the s h o r t e s t sleeping times. Sxmilarly, the long-sleep line was p r o d u c e d b y utilizing as p a r e n t s m
Alcohols
Hypnotics
each successive g e n e r a t i o n o n l y t h o s e a m m a l s w longest sleeping t i m e . Previously, we r e p o r t e d t] actiwtles o f liver a l c o h o l d e h y d r o g e n a s e and the i b l o o d e t h a n o l d i s a p p e a r a n c e were virtually identi samples o f the 2 lines o f mice [5 ]. The ED~ 0 values o f righting r e s p o n s e w i t h e t h a n o l were a p p r o x i m fold greater m the SS a m m a l s t h a n in t h e LS m i c e suggested t h a t the 2 lines o f mice differ m their nervous s y s t e m sensltivlty to e t h a n o l . Prior investigations [14,15] o f t h e e n z y m e s resl: for e t h a n o l m e t a b o l i s m d e m o n s t r a t e d d i f f e r e n c e s activity o f liver e n z y m e s r e s p o n s i b l e for e t h a n o l olism in various i n b r e d strains o f mice. O t h e r inves have s h o w n t h a t mice f r o m various inbred stratus d sleep-time i n d u c e d b y b a r b i t u r a t e s [ 6 , 1 7 ] . Liver somal fractaons xsolated f r o m t h e s e inbred stratus d i f f e r e d in rates o f h e x o b a r b i t a l m e t a b o h s m H studies o f possible d i f f e r e n c e s in brain sensiti' b a r b i t u r a t e s were n o t r e p o r t e d . It has b e e n not~
~Present Address Department of Pharmacology and Experimental Therapeutrcs, The Johns Hopkins School of Medicine, Bz Maryland 679
680
E R W I N , H E S T O N , M C C L E A R N A N D DE!
b a r b i t u r a t e s a n d general a n e s t h e t i c s as well as a l i p h a t i c a l c o h o l s depress t h e r e t i c u l a r a c t i v a t i n g s y s t e m w h i c h subserves w a k e f u l n e s s [ 8 , 1 3 ] . C o n s e q u e n t l y it a p p e a r e d t h a t t h e LS a n d SS lines o f mice w h i c h d i f f e r in b r a i n sensitivity to e t h a n o l m i g h t be o f value m c o m p a r i n g t h e m e c h a n i s m s o f a c t i o n o f various c e n t r a l n e r v o u s s y s t e m d e p r e s s a n t s . In t h e p r e s e n t s t u d y we c o m p a r e d t h e h y p n o t i c p o t e n c i e s a n d d u r a t i o n o f r e s p o n s e o f various c e n t r a l n e r v o u s s y s t e m d e p r e s s a n t s in t h e LS a n d SS lines o f mice. Some o f t h e s e d a t a have a p p e a r e d in a b s t r a c t f o r m [ 4 ] . MATERIALS AND METHOD Mice o f t h e long-sleep (LS) a n d t h e short-sleep (SS) line of the 1 4 t h g e n e r a t i o n were o b t a i n e d f r o m t h e I n s t i t u t e f o r B e h a v i o r a l G e n e t i c s ( I B G ) a n d a f t e r at least a week o f a d a p t a t i o n to t h e t e s t i n g r o o m in a n a d j a c e n t l a b o r a t o r y , were t e s t e d b e t w e e n 9 0 - 2 0 0 days o f age HS a n i m a l s were also t e s t e d m 1 e x p e r i m e n t . E q u a l n u m b e r s o f male a n d female mice were t e s t e d at e a c h dose. Doses o f e t h a n o l o f 0 . 0 0 8 - 0 . 0 1 2 ml/g were i n j e c t e d IP (25% v/v in sahne). T h e mice weighed b e t w e e n 1 8 - 3 4 g. R e s p o n s e to t h e a l c o h o l s a n d t h e various s e d a t w e h y p n o t i c a g e n t s was m e a s u r e d b y d e t e r m i n a t i o n o f sleeping time. Because a w a k e n i n g f r o m a l c o h o l is n o t as clear cut as a w a k e n i n g f r o m o t h e r sedative h y p n o t i c s , t h e a m m a l was not c o n s i d e r e d a w a k e u n t d it h a d r i g h t e d itself 3 t i m e s w i t h i n 30 sec. F o r t h e ED~o studies, t h e a m m a l was c o n s i d e r e d to have lost its r i g h t i n g r e s p o n s e ff it r e m a i n e d o n its b a c k for 1 m i n or longer. F o r t h e LD~o a c u t e t o x i c i t y s t u d y , t h e a n i m a l s were e x a m i n e d a f t e r 24 hr h a d elapsed a n d t h e n u m b e r o f d e a t h s at t h a t dose r e c o r d e d [ 2 ] . T h e ED 5 o doses were d e t e r m i n e d b y t h e m e t h o d o f Litchfleld and Wilcoxon [9]. T o o b t a i n ED~ o values for e t h e r , a m m a l s were placed in 4 1 jars w i t h air t i g h t lids a n d e t h e r was i n j e c t e d t h r o u g h a rubber septum. Animals tested without ether adminis-
t r a t i o n s h o w e d no signs o f necessary for testing.
hypoxia
during
RESULTS T a b l e 1 illustrates t h e sleep-time scores for t h e sample o f a n i m a l s f r o m the 1 4 t h g e n e r a t i o n sleep-time selection lines. A m e a n sleep-time score sec for t h e SS line a n d 9 3 4 2 sec for t h e LS line is o following a dose of 3.75 g/kg o f e t h a n o l IP. These can b e c o m p a r e d w i t h t i m e s a f t e r a d m i n i s t r a t i o n of h y p n o t i c drugs. B o t h m e t h a n o l a n d b u t a n o l cause of t h e LS h n e to sleep significantly longer t h a n thos, SS line. H o w e v e r , t h e r e is n o statistically sig d i f f e r e n c e a m o n g the lines in p e n t o b a r b i t a l , chlc drate, p a r a l d e h y d e or t r i c h l o r o e t h a n o l i n d u c e d sle~ T h e ED s o for loss of r i g h t i n g r e s p o n s e s h o u l d be definitive measure of CNS sensitivity for these 2 1 animals, since a n y m e t a b o l i c changes wall b e less lm m t h e few m i n r e q u i r e d to p r o d u c e loss o f r i g h t i n g rq m t h e a n i m a l s t h a n d u r i n g the course o f sle d e t e r m i n a t i o n . S u c h e x p e r i m e n t s were carried o u t results p r e s e n t e d in Fig. 1. T h e dose r e s p o n s e h essentially parallel. In T a b l e 2 are listed the ED s 0 w i t h the 95% c o n f i d e n c e limits for t h e s e alcohols s o d i u m p e n t o b a r b l t a l a n d e t h e r It can be seen fr d a t a t h a t t h e r e is a p p r o x i m a t e l y a 2 fold differ, EDs0 for t h e various a l c o h o l s b e t w e e n LS and SS However, t h e r e is n o d i f f e r e n c e b e t w e e n HS, LS lines in ED 50 for Na p e n t o b a r b i t a l or b e t w e e n the LS mice m ED 5 0 for e t h e r . Previously we h a d f o u n d n o d i f f e r e n c e m t h e m e t a b o l i s m o f e t h a n o l in t h e 2 lines, yet t h e LS lin m u c h longer. O b v i o u s l y t h e n , t h e SS animals m u s t at h i g h e r b l o o d a l c o h o l levels t h a n do t h e LS amm~ p r e d i c t i o n is fulfilled as i l l u s t r a t e d in Table 3. T a b l e 4 lists t h e LD~ 0 doses for e t h a n o l . T h e LS
TABLE
1
LONG SLEEP ILS). SHORT SLEEP (SS) AND HETEROGENEOUS STOCK (HS~ SLEEP TIMES WITH VARIOUS HYPNOTIC AGENTS* Animal Line
Drug
(n)
SS LS SS LS SS LS SS LS HS SS LS SS LS SS LS
Ethanol Ethanol Methanol Methanol n-Butanol n-Butanol Pentobarbltal Pentobarbltal Pentobarbltal Chloral Hydrate Chloral Hydrate Paraldehyde Paraldehyde Tnchloroethanol Trlchloroethanol
l0 3.75 l0 3 75 10 4 5 10 4 5 10 0 567 10 0 567 70 0 060 56 0 060 I0 0 060 19 0 45 20 0 45 20 1 0 19 I 0 10 0 225 10 0 225
th
Doset
Sleep Time (sec) 367 9,342 52 18,456 280 2,169 2,107 1,414 2,500 5,502 7,203 2,766 3,720 1,502 2,542
(+_547)~: (+--5,969) (_+77) (-10,750) (_+384) (_+890) ( ~- 1,678) (_+873) (_+ 1,035) (4-1,842) ~-+ 1,662) (+- 1,172) (_+846) (_+ 1,263) (+-844)
p Value <0 01 <0 01 <0 01 ns ns ns ns ns
*Sleep times were taken as the time from the loss of nghtmg reflex to the time the animal was able to nght itself three t~mes within 30 sec. t i P dose m g/kg ~:Represents +- the standard devmtton
GENETICS
OF CNS SENSITIVITY
TO ETHANOL
Bu
Bu t-Bu SS LS
t-Bu S,S
Et
Me LS
/
z
80
n 09
70-
Me S,S
j]/ ! ! I
z
IE)
Et SS
! I I
I 1
I
I '
,I
/
,I
/ I--"
z Iii 0
40
ILl
20.
30
10
/
/i .
I
i
I !
I !
, ;
P
/
,
:
, l
:
l
I
I
I
52
I
0.,'
I
,
0.3 0.4
I
I
I
t
I
I
0.6 0.8 1.0
I
2.0
I
I
t
3.0 4.0 5.0
(gin per Kg BodyWe,ght)
DOSE
FIG 1. Dose response curve for alcohol m SS and LS mice Methanol; (Me) LS mace 1" . . . . . . - ) , SS rmce (A. . . . . . ~) Ethanol (Et) LS mice (e e); SS mice (o c) Butanol (Bu) LS rmce(, . . . . =), SS mice (c~. . . . a) t-Butanol (tBu) LS mice (~. . . . . *), SS mice
(~ . . . .
~)
TABLE TABLE
2
EDs0 V A L U E S FOR LOSS O F R I G H T I N G R E F L E X WITH V A R I O U S H Y P N O T I C AGENTS*
Ammal Line Drug
EDso (g/kg, IP)
SS LS SS LS SS
Ethanol Ethanol Methanol Methanol Butanol
3 7 I 65 50 2 75 0 535
LS SS LS SS LS HS SS LS
Butanol t-Butanol t-Butanol Na Pentobarb~tal Na Pentobarbnal NaPentobarb~tal Ether Ether
0 245 (_+0 011) 1 39 ( ± 0 065)§ 0 84 ( ± 0 0785§ 0.039 (_+0.0028) 0 038 (-+-+00026) 0040(±0.0027)§ 1 04 (_+0 26)$§ 104 (±0.3255§
(±0.115¢ ( ± 0 09) ( _ 0 24) (_+0 11) (_+0.026)
EDs0 Ratio SS/LS 2 24
3
B L O O D A L C O H O L C O N C E N T R A T I O N S IN SS AND LS TIME OF A W A K E N I N G
Ammal Line SS LS
Sleep Time sec _+ SD (n)
Blood Alcoh, m g ~ ± SD (
3937 ± 2502 (10) 16069 _+ 5443 (125 p < < 0 001
380 2 -'- 52 6 ( 230 0 ± 43.5 ( p < < 0 001
1 82 2 18 1 65
*EDso values determined by the m e t h o d of Litchfield and Wilcoxon ( n = 10 animals each at four different doses) f R e p r e s e n t s 95% confidence hm~ts ~mM/l by inhalation §Tested m the 18th generation or c o n t e m p o r a n e o u s HS stock
M~ce of each hne were given 4 1 g/kg ethanol as outhn text This dose was n e c e s s a r y to insure loss of righting re! all SS mace At the time of regaining the righting reflex, b taken from the retro-orbltal sinus and blood alcohol deter: gas c h r o m a t o g r a p h y as prevaously described [5].
are o n l y s l i g h t l y m o r e s e n s i t i v e to t h e a c u t e e t h a n o l . It s h o u l d b e n o t e d t h a t at t h e methanol and t-butanol ammals were often following day, even though they had not lost the righting response.
t o x i c el higher ( found C always
DISCUSSION In c e r t a i n g e n e r a t i o n s , a s a m p l e o f H S m~ce ~s te ethanol sleep time contemporaneously w i t h t h e LS
682
ERWIN, H E S T O N , M C C L E A R N A N D DEI TABLE 4
LDs0 VALUES FOR ETIqANOL IN SS AND LS SELECTED LINES OF MICE*
Animal Line
LDs0~
SS LS
g/kg 4 8 ~±0 2)~ 4.5 I±0 2)
*Lethahty was determined by the number of deaths occurring within 24 hr followmg IP injections of various doses of ethanol (n=8 at each of 5 doses) tAccordlng to the method of Ltchfield and Wdcoxon [9] ~:Represents 95% confidence hmns mice These samples s h o w t h e e x p e c t e d gaussian dist r i b u t i o n o f sleep-times t h a t was p r e s e n t m t h e f o u n d a t i o n stock HS animals. T h e SS a n d LS mice are m a t e d m s u c h a way t h a t m a t i n g parrs n e v e r have c o m m o n g r a n d p a r e n t s , T h u s , in d i s t i n c t i o n to i n b r e d m o u s e stratus t h a t s h o w d i f f e r e n c e s m sleep t i m e s similar to t h o s e o b s e r v e d m ttus r e p o r t [ 7 , 1 2 ] , t h e g e n e t i c v a r i a b i l i t y p r e s e n t initially m the HS s t o c k w i t h respect to p h e n o t y p e s u n r e l a t e d to e t h a n o l sleep t i m e is preserved as m u c h as possible. This preservation o f h e t e r o g e m t y is s h o w n b y the fact t h a t the r e s p o n s e s of HS, LS a n d SS mice to p e n t o b a r b l t a l do n o t differ. T h e r e is a significant d i f f e r e n c e in ED~ 0 b e t w e e n lines for loss o f r i g h t i n g r e s p o n s e for m e t h a n o l , e t h a n o l , butanol and t-butanol, P r e v i o u s l y we h a d d e m o n s t r a t e d t h a t t h e r e was n o change m t h e rate of a l c o h o l m e t a b o l i s m b e t w e e n t h e 2 lines [ 5 ] . We have n o t m a d e similar d e t e r m i n a t i o n s for t h e o t h e r a l c o h o l s t e s t e d here H o w e v e r , e a c h a l c o h o l t e s t e d is m e t a b o h z e d via a d i f f e r e n t e n z y m a t i c system. T h e m e t a b olic r o u t e for m e t h a n o l m t h e r o d e n t is p r i m a r i l y via the catalase s y s t e m [ 1 0 ] . E t h a n o l is m e t a b o h z e d p n m a r d y via the a l c o h o l d e h y d r o g e n a s e s y s t e m , t - B u t a n o l is o n l y slowly m e t a b o l i z e d if at all via c o n j u g a t i o n w i t h g l u c o r o n l d e [ 1 8 ] , a l t h o u g h o t h e r possible r o u t e s o f m e t a b o h s m have n o t b e e n studied. It w o u l d seem u n h k e l y t h a t we have selected for similar changes m several diverse m e t a b o h c p a t h w a y s , a n d we c o n c l u d e , t h e r e f o r e , t h a t the selective b r e e d i n g p r o g r a m has d e v e l o p e d lines t h a t differ in b r a i n sensitivity to alcohols m general, n o t j u s t to e t h a n o l It a p p e a r s t h a t we have n o t selected for a general CNS s e n s l t w i t y to d e p r e s s a n t s b e c a u s e n o n e o f t h e o t h e r CNS d e p r e s s a n t s s h o w d i f f e r e n t i a l effects m t h e two lines. This o b s e r v a t i o n is s o m e w h a t s u r p r i s m g since all t h e t e s t e d c o m p o u n d s ( p e n t o b a r b l t a l , e t h e r , chloral h y d r a t e a n d p a r a l d e h y d e ) are at least additive w i t h e t h a n o l m t h e i r effects o n t h e CNS. In p a r t i c u l a r e t h e r , chloral h y d r a t e a n d p a r a l d e h y d e have long b e e n t h o u g h t to possess a similar ff n o t i d e n t i c a l m e c h a n t s m to t h a t o f e t h a n o l [ 1 3 ] . T h e s e results p e r m i t a c o n c l u s i o n t h a t t h e m e c h a n i s m o f these c o m p o u n d s all differ in s o m e r e s p e c t f r o m t h a t o f e t h a n o l even t h o u g h t h e final c o m m o n result ~s the same. F i r m c o n c l u s i o n s c o n c e r n i n g t h e n o n - a l c o h o l drugs m u s t await analysis of t h e i r m e t a b o l i s m in t h e 2 lines of mice, b u t again it is u n h k e l y s e l e c t i o n has b e e n change s i m u l t a n e o u s l y in a large, diverse g r o u p o f d i s p o s i t i o n p a t h w a y s .
It is also of i n t e r e s t t h a t t h e LD s 0 values for eth not differ in t h e t w o lines i n d i c a t i n g t h a t t h e mecl r e s p o n s i b l e for sleep are n o t t h e same as t h o s e resI for e v e n t u a l d e a t h f r o m overdosage Sleep t i m e d e t e r m i n a t i o n s w i t h these animals are to a n u m b e r of v a r i a t i o n s f r o m e x p e r i m e n t to e x p as e v i d e n t in T a b l e 3 a n d o u r p r e v i o u s results [: values of 3 9 3 7 sec vs 1250 sec for t h e SS line a n d 1 6 2 5 0 sec for t h e LS line shows t h e c o n s i s t e n c y m d i f f e r e n c e s b u t also illustrate variability m t h e a n u m b e r s . T h e reasons for t h e variability are u n k n o certainly d i f f e r e n t i n j e c t i o n t e c h n i q u e s a n d dIffer~ o r a t o r y e n v t r o n m e n t s c o n t r i b u t e . In a n y given exp~ the age o f all o f t h e a n i m a l s was a p p r o x i m a t e l y t h F r o m e x p e r i m e n t to e x p e r i m e n t t h e r e were age diff and this m a y a c c o u n t for some of the v a r i a t i o n s is also critical to p o i n t o u t t h a t t h e a b s o l u t e differ sleep t i m e s b e t w e e n SS a n d LS mice is d e p e n d e n t ul b l o o d level o f a l c o h o l achieved. This is best illustr~ c o n s i d e r i n g the case w h e r e the level is so low t h a t mice lose the r i g h t i n g r e s p o n s e b u t the LS mice s u b s t a n t i a l time. F r o m Table 3 it is seen t h a t such would b e a p p r o x i m a t e l y 327 rag% G i v e n t h a t t h e I m u s t r e a c h 2 3 0 mg% for half o f t h e mice to a w a k e a the rate o f m e t a b o l i s m is a b o u t 19 mg% per 1000 the LS mice w o u l d sleep a b o u t 5 0 0 0 sec o n t h e ~ These c a l c u l a t i o n s are possible because o f t h e zer, rate o f e t h a n o l e h m m a t l o n S i e m e n s a n d C h a n [16] r e p o r t e d t h a t pentol p r o d u c e d a significantly longer loss o f r i g h t i n g retie mice t h a n m LS mice b u t t h a t this was d u e to a d a p p a r e n t volume of d i s t r i b u t i o n o f t h e drug m t hnes. In t h e present s t u d y we did n o t observe a stati significant d i f f e r e n c e m the sleep t i m e s b u t did slightly longer loss of r i g h t i n g r e s p o n s e in t h e SS mic G o l d s t e l n and K a k l h a n a [3] tested the SS and 1 for severity of w i t h d r a w a l a f t e r 3 days of m t o x i T h e y find t h a t t h e SS mice have m o r e severe wltt r e a c t i o n s t h a n d o t h e LS mice and t h e s e d i f f e r e n c e s casually related to t h e line d t f f e r e n c e s m acute effects. R e c e n t l y Collins ( p e r s o n a l c o m m u n i c a t i o n ) fore the i n f l u e n c e of e t h a n o l o n t h e t u r n o v e r o f do differed b y a b o u t 2 fold m t h e SS a n d LS mice althc d i f f e r e n c e s in n o r e p m e p h r m e or s e r o t o n m t u r n o w o b s e r v e d Analysis o f t h e m a j o r s y n t h e t i c and degr e n z y m e s o f t h e d o p a m m e p a t h w a y s revealed n o dlff~ Fuller t p e r s o n a l c o m m u n i c a t i o n ) has o b s e r v e d a dlf in t h e sleep time i n d u c e d b y salsolinol ( t h e c o n d e product of dopamme and acetaldehyde) when i i n t r a c r a m a l l y . F r o m these data t h e n it would appc s t u d y of the d o p a m i n e r g i c s y s t e m m i g h t prove fru the s e a r c h for a b i o c h e m i c a l e x p l a n a t i o n o f results r~ in this paper. ACKNOWLEDGEMENTS Supported by grants No. AA00263, MH18948, NIMH Grant No. MH 11167, and a Career Development Award No GM-10475. The authors wish to express their apprec Mrs Candace Owen tor her excellent techmcal assistance.
G E N E T I C S O F CNS S E N S I T I V I T Y TO E T H A N O L
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