Brain Research. 550 11991) 1-6 © 1991 Elsevier Science Publishers B.V. (~106-8993/91/$03.511 A I)ONIS (~1068993911671)3Z BRES 167113

Research Reports

Effects of convulsants on handling-induced convulsions in mice selected for ethanol withdrawal severity J o h n C. Crabbe ~'2, Catherine D. Merrill I and J o h n K. B e i k n a p ~'2 IResearch Service (151W), VA Medical ('enter, Portland, OR 97201 (U.S.A.) and "Departments of Medical Psychology and Pharmacolog);, Oregon Health Sciences University. Portland, OR 97201 (U.S.A.) (Accepted 15 January 1991) Key word~: Pharmacogcnctics: (Tonvulsion; Convulsant drug: 7-Aminobutyric acid: Excitatory amino acid; Withdrawal seizure-prone mouse; Withdrawal seizure-resistant mouse: Alcohol withdrawal

Withdrawal seizure-prone (WSP) mice were genetically selected to express severe handling-induced convulsions (ILK') upon cessation of chronic ethanol vapor inhalation. The ItlC is a sensitive measurc of ('NS excitability, ~,nd the current paper compares the effects of eleven convulsant drugs on the HIC in WSP and WSR (withdrawal seizure-resistant) mice, the latter selected for minimal alcohol withdrawal HIC. If WSP and WSR mice wcrc differentially sensitive to a subset of the tested drugs, a common mechanism of action for that subset would imply that genes influencing that mechanism wcrc important in determining ethanol withdrawal severity. All drugs significantly enhanced HIC in WSP mice. The magnitude of enhancement was small for N-methylq~-aspartate (NMDAI, kainic acid, BAY K 8644, Ro 15-4513, and strychnine; greater enhancement in WSP mice was sccn after nicotine, and the direct and indirect 7-aminobutyric acid (GABA) antagonists bicuculline, 3-mercaptopropionic acid, picrotoxin, t-butylcyclophosphorothionatc (TBPS), and pcntylenetetrazol. Only two drugs, picrotoxin and pcntylcnctctrazol, had a marked effect on WSR mice: maximal effect of these drugs was equivalent in WSP and WSR mice. However, picrotoxin and pcntylcnctctrazol were more potent in WSP than in WSR mice. Three other GABA antagonists, bicuculline, 3mcrcaptopropionic acid. and TBPS, had a ,,'cry small effect in WSR mice: these drugs also seemed to be more potent in WSP than in WSR mice. For all other tested drugs, maximal effect in WSP mice was much greater in WSP than in WSR mice. Given the minimal response of WSR mice to these other drugs, it was not possible to estimate potency, or to compare it with potency in WSP mice. WSP mice were clearly more sensitive to all tested drugs than WSR mice. Since WSP and WSR mice differed least in sensitivity to GABA-related compounds, these results suggest that differences in brain GABA sy~tems may not bc critical for the expression of genetic susceptibility or resistance to ethanol withdrawal severity. However, similar studies in animals withdrawing from ethanol may provide a better method for evaluating potential ncuropharmaeological correlates of withdrawal.

INTRODU('TION

We thought that it might be possible to identify the biological substratc for the differential w i t h d r a w a l seizure

s e i z u r e - p r o n e ( W S P ) mice w e r e geneti-

susceptibility in W S P and W S R mice by f o l l o w i n g the

cally selected to express s e v e r e h a n d l i n g - i n d u c e d convul-

Withdrawal

g e n e r a l strategy of a d m i n i s t e r i n g a n u m b e r of c o n v u l s a n t

sions ( H I C ) after cessation of chronic e t h a n o l ( E t O H )

agents with differing m e c h a n i s m s of action to W S P and

v a p o r inhalation s. E a r l i e r studies c o m p a r e d W S P with

W S R mice. Wc a d m i n i s t e r e d relatively low doses o f such

w i t h d r a w a l seizure-resistant ( W S R ) mice selected for low

drugs, and used the H I C as an index of C N S sensitivity.

e t h a n o l w i t h d r a w a l H I C . T h e s e studies found that W S P

O u r g e n e r a l hypothesis was that any d r u g e n h a n c i n g H I C

mice also had m o r e s e v e r e H I C than W S R mice after

t h r o u g h the s a m e m e c h a n i s m s as those o p e r a t i n g during

w i t h d r a w a l from chronic p h e n o b a r b i t a l 5. d i a z e p a m ~', or

e t h a n o l w i t h d r a w a l w o u l d show large ( s e v e r a l - f o l d ) W S P

nitrous o x i d e 4. Since W S P and W S R mice w e r e bred

vs W S R sensitivity d i f f e r e n c e s , similar in m a g n i t u d e to

solely on the basis of E t O H w i t h d r a w a l responses, this

those seen during w i t h d r a w a l f r o m e t h a n o l . C o n v e r s e l y ,

suggests strongly that

drugs o p e r a t i n g t h r o u g h c o m p l e t e l y i n d e p e n d e n t m e c h -

genes modulating

drawal also influence withdrawal

EtOH

with-

from phenobarbital,

anisms f r o m those u n d e r l y i n g e t h a n o l w i t h d r a w a l w o u l d

d i a z e p a m and nitrous oxide. W S P mice, t h e r e f o r e , constitute an animal m o d e l for genetic p r o p e n s i t y to

show no d i f f e r e n c e s b e t w e e n the lines. F o u r possible o u t c o m e s w e r e e n v i s i o n e d . First, W S P and W S R mice

d e v e l o p physical d e p e n d e n c e on a n u m b e r of drugs of

might not differ in sensitivity to any c o n v u l s a n t drug. This

abuse which share in c o m m o n a d e p r e s s a n t effect on central n e r v o u s system ( C N S ) function.

o u t c o m e w o u l d suggest that e i t h e r the site of the E t O H w i t h d r a w a l d i f f e r e n c e b e t w e e n the lines is not one w h e r e

Correspondence: J.C. Crabbe. Research Service (151W), VA Medical Center, Portland. OR 97291, U.S.A.

any tested drugs act, or that differences between WSP and WSR mice arise only during E t O H treatment, and are absent or not easily detectable prior to E t O H exposure. Second, WSP mice might be more sensitive to all convuisants than WSR mice. During the course of selective breeding, we could simply have selected WSP mice for seizure susceptibility, and WSR mice for seizure resistance, regardless of the cause of the seizures. Third. WSP and WSR mice might be differentially sensitive to one or more of the tested drugs. This outcome, which reflects our general strategy, might then allow us to identify through a common mechanism or site of action of such convulsants a critical neurochemical pathway for E t O H withdrawal. However, even in a replicated selective breeding study with two independent WSP and two independent WSR lines, a fourth possibility cannot be discounted. WSP and WSR mice might differ in sensitivity to some drugs due to the chance fixation of some genes in the homozygous state due to genetic drift. We could rule out this possibility with some security by finding an identical pattern of differences in both sets of WSP and WSR mice. Furthermore, in the case of the GABAergic drugs, we presumed that convergent validity would lead to detection of multiple drugs differentially effective in WSP and WSR mice. The choice of convulsants was guided by the potential role of particular neurotransmitters as potentially important sites of the differences in E t O H withdrawal-induced convulsions between the WSP and WSR lines of mice. Differences in functional activity at the ~,-aminobutyric acid ( G A B A ) receptor-mediated chloride ionophore have been shown to correlate with genetically determined sensitivity to the hypnotic effects of E t O H in mice and rats 3. Furthermore, increased number of dihydropyridine-sensitive calcium channel binding sites have been shown to accompany chronic E t O H treatment ~7, and to correlate genetically with genetic susceptibility to EtOH withdrawal 7. Finally, the number of excitatory amino acid modulated ion channels bound by MK-801 increases during E t O H dependence 16, and WSP mice have more MK-801 binding sites than WSR mice 22. Given these considerations, the drugs studied for their effects on HIC were picrotoxin, [35S]t-butylcyclophosphorothionate (TBPS), pentylenetetrazol (PTZ), bicuculline, Ro 15-4513, 3mercaptopropionic acid (3-MPA), BAY K 8644, NmethyI-D-aspartate (NMDA), kainic acid, strychnine. and nicotine. MATERIALS AND METHODS

Subjects W S P and W S R mice were bred in our laboratory in Portland, O R . U . S . A . Drug-naive adult mice (age range 59-100 days) of one sex from selected generations 32-34 were used to test each drug. The

WSP/WSR selective breeding experiment is replicated ~, thus there are two independently derived WSP and two W S R lines. For each test drug, mice of both WSP and both W S R lines were tested. Since the replicate lines did not differ, results were combined for data analysis and presentation. Mice were maintained under a light-dark cycle of 06.00-18.00 h light, and testing was performed between 119.00 and 14.(111 h.

Handling-induced convulsion scoring Twenty to 30 min and immediately before i.p. drug administration. and at several appropriate intervals after each drug, mice were scored for H I C severity. The scale we employed was modified from that first described by Goldstein ~s. Each m o u s e was picked up by the tail, and if that failed to elicit a convulsion, the m o u s e was spun gently through a 180 ° arc. H I C scores independently assigned by two trained observers in our laboratory correlate greater than 0 9 0 (unpublished). The rating scale used has been published 9"~°, and ranges from 0 to 7. where 7 represents a severe, tonic-clonic convulsion, with quick onset and tong duration, which is either s p o n t a n e o u s or elicited by mild environmental stimuli, such as lifting the cage t o p For each drug. pilot testing at sc~,cral doses was employed to identify the highest dose which was still s o m e w h a t below that causing more severe, drug-specific convulsions other than H I C (e.g. tonic hindlimb extensor or wild running), since these interfere with HIC scoring. As a result, the dose range used was 'subconvulsive" for these more severe seizures, but was efficacious for eliciting HI(? in at least the WSP mice.

Index of effects on HIC severity and statistical analysis The two pre-drug baseline scores of each subject were averaged (PRE). Since most W S R mice scored zero or one u n d e r all test conditions, their data were not easlily comparable to WSP mice and were separately analyzed. G r o u p s of 6-21 mice were given vehicle or one of the indicated drugs tested and scored for H I C at several times thereafter. Multiple subconvulsant doses were tested. Drug effects on HIC were quantified in three ways. In drug-treated groups, one index was the difference from P R E of the greatest post-drug score for each subject: this index can overestimate drug responsiveness by capitalizing on any chance elevations in HIC. A second, related m e a s u r e was the mean difference from P R E at the time of average greatest drug effect for each dose group. Finally. drug and vehicle groups were directly compared at the time of average greatest drug effect. All 3 m e a s u r e s yielded very similar rcsults, so we generally statistically analyzed drug effects as differences from baseline at the time of average greatest drug effect. Significance of the drug effects was tested with a repeated m e a s u r e s one-way A N O V A for the P R E - and post-drug H I C score versus a null hypothesis of no difference. To explore dose-effect relationships in more detail, we report for P T Z a factorial A N O V A with post-hoe comparisons made by the m e t h o d of N e u m a n - K e u l s -'3.

Drug source.~ and preparation Picrotoxin, pentylenetetrazol (PTZ), bicuculline, 3-MPA, N M D A , kainic acid, and strychnine sulfate were purchased from Sigma Chemical Co. (St. Louis). T B P S was purchased from Research Biochemicals Inc. (Natick. MA). Ro 15-4513 was a gift of Dr. R. Adron Harris, and B A Y K 8644 was a gift of Dr. A a r o n Janowsky. Picrotoxin, P T Z , nicotine, 3-MPA, N M D A , kainic acid. and strychnine were dissolved in saline. T B P S was dissolved in a solution of 2% dimethylsulfoxide ( D M S O ) in saline and sonicated. Bicuculline was dissolved in a few drops of glacial acetic acid. diluted with saline, and pH adjusted using 5 M N a O H to a value of 5.1). Ro 15-4513 was dissolved in a 0.3% Tween 20 in distilled HzO solution (= 1 drop of Tween 20 in 10 ml H 2 0 ). B A Y K 8644 was dissolved by placing 3 drops of Tween 20 on the drug and bringing the final volume to 10 ml with saline. B A Y K 8644 was kept in foil on ice. Nicotine was dissolved in a 1:9 solution of (I.5 M HEPES-Tris buffer:saline. All drugs were administered i.p. at an ambient temperature of 22 _+ 1 ° C

TABLE I

RESUI;FS

EJJ~'cts o f convulsants on HI(" in WSI' and W S R mice

We have argued elsewhere that the handling-induced c o n v u l s i o n is a s e n s i t i v e i n d e x o f C N S e x c i t a b i l i t y ~ " t T h e d o s e s n e e d e d to elicit H I C are g e n e r a l l y well b e l o w t h o s e n e e d e d to elicit o t h e r f o r m s of c o n v u l s i v e activity. W S P m i c e , g e n e t i c a l l y s e l e c t e d o n l y for e n h a n c e d H I C d u r i n g ethanol withdrawal, now have measurable baseline HIC (PRE approximately zero

1 . 5 - 2 ) , w h i l e W S R mice all score

in t h e a b s e n c e

experiments

Mean + S E . M . is shown for the HIC scorc at the time after injection yielding maximal response at the most effective dose for each drug. PRE. average pre-injection baselinc scores for all animals tested; N.]'., not tested at that dose. For PTZ. maximal response for WSP mice was at 10 rain post-injection (5.8 + 0.1 ), but WSR mice showed lesser response at 10 rain (2.3 ± 0.4). so thc 3 rain time point is shown for both lines.

of drug

treatment.

The

current

Drug

Dose (mg:kg)

Time (rain)

r e v e a l e d t h a t in W S P mice t h e H I C was

s e n s i t i v e to low d o s e s o f c o n v u l s a n t d r u g s with a v a r i e t y

20 20 PRE

Picrotoxin

o f m e c h a n i s m s o f a c t i o n . T a b l e l s h o w s t h e r e s p o n s e s of W S P a n d W S R mice to all d o s e s of all drugs. To facilitate presentation,

Table

representative

II

dose,

shows

and

responses

statistical

to

a single

comparisons

g i v e n . Since W S P b a s e l i n e s v a r y s o m e w h a t

PTZ

arc

(e.g. as a

f u n c t i o n o f d r u g v e h i c l e ) , v a l u e s in T a b l e I1 are e x p r e s s e d

0 15 3O 5O

Selected line .... WSP

WSR

5.2 ± 0.2 5.2_+ 0.2 1.6 k 1).3

3.2 -+ 0.4 3.8 + 0.3 0 _+ 0

3

2.3 +_ O.4

0 4- 0

3 3 3 PRE

4.5 + o.3 5.2 ± 0.1 5.5 ± 0.2

0.7 _+ 1).2 1.4 +_ 0.2 3.3 + 0.1

2.0 ± 0.3

0 _+ 0

15 15 15

2.3 _" 0.2 4.0+_0.3 4.9+0.1

0.2_+0.1 0.8+0.1

PRE

1.8 i 0.1

0 4- 0

5 5 5 5 PRE

2.8 3.5 5.0 4.7

_+0.3 _+0.3 _+0.2 _+0.2 1.6 _* 0.I

0. I _+ 0. I 0. I _+0. I 0.2 _+0. I

IO I0

2.4 ± 0.4 4.3+0.3 5.1 4- 0.2 5.4 + 0.2 1.4 ± o.I

0+0 0.1 + 0 . I 0.4 + O.2 0.7 _+0.2

0 + 0 N.T.

PRE

1.7 t 0.4 3.0 ± 0.3 2.9±0.2 2.7 _+0.3 N.T 1.6 ± 0.3

3 3 3

O.7 ± 0.3 4.3.t 0.6 4.2 t 0.5

0 +_ 0 0+0 0.2 ± 0.2

3

PRE

4.7 ± 0.3 1.1 +_0.2

0.5 _+ 0.2 0 ± 0

I0 I0 I0 PRE

3.7 4.4 4.3 2.1

4- 0.4 +_0.2 ± 0.2 ±0.2

0±0 0. I + 0. I 0.2 + 0. I 0±0

as d i f f e r e n c e s f r o m P R E . O u r results s u p p o r t e d to s o m e d e g r e e b o t h t h e s e c o n d a n d t h e t h i r d a l t e r n a t i v e s de-

'IBPS

s c r i b e d a b o v e . In g e n e r a l , W S P m i c e s h o w e d statistically

o o.o125 o.o25

s i g n i f i c a n t e x a c e r b a t i o n of H I ( " a f t e r all drugs, but t h e magnitude NMDA,

o f t h e effects v a r i e d g r e a t l y . k a i n i c acid, a n d B A Y

Ro

15-4513,

K 8644 h a d m a r g i n a l

Bicuculline

0.5 1

e x a c e r b a t i n g effects o n H I C in W S P mice. N i c o t i n e . a n d five i n d i r e c t G A B A

2 3

a n t a g o n i s t s , p r o d u c e d m a r k e d ele-

v a t i o n s in I t I C in W S P mice. O n l y t w o d r u g s , p i c r o t o x i n a n d P T Z . h a d a m a r k e d effect o n W S R mice. F o r this

3-MPA

r e a s o n , t h e W S P vs W S R d i f f e r e n c e s w e r e t h e s m a l l e s t for

these

two

convulsants.

For

three

0 4- 0

other

GABA

o 20 3o 4o

a n t a g o n i s t s , b i c u c u l l i n e , T B P S , a n d 3 - M P A , W S R mice

I0

I0 PRE

0.7 + 0.2 o + o

o + o

s h o w s i g n i f i c a n t , b u t v e r y s m a l l i n c r e a s e s in H I C (See Ro15-4513

T a b l e 11). D o s e - e f f e c t c u r v e s for P T Z for W S P a n d W S R mice arc

given

in

Fig.

1. T h e s e

lwo-factor ANOVA, line ( F =

data

were

analyzed

0 0. I

2

I

2 2 2

I0 30

by

w h i c h r e v e a l e d s i g n i f i c a n t effects of

2

0. I +0. I 0.2 4- 0. I 0. I + 0. I 0 + 0

50.4, df = 1,89, P < 0.0001) a n d d o s e ( F =

70.5, df = 3,89, P < 0.0001). T h e i n t e r a c t i o n o f line a n d

Nicotine

0

1.5 1.8 2.9

d o s e was also s i g n i f i c a n t , b u t to a lesser d e g r e e ( F = 6.2, df = 3,89, P < 11.001), P o s t - h o c a n a l y s e s r e v e a l e d t h a t W S P mice r e s p o n d e d m o r e t h a n W S R to P T Z at the 15 a n d 30 m g / k g d o s e s ( P < 0.01)I), but not at the 0 o r 511

B A Y K 8644

mg/kg doses (n.s.). T h e s e d i f f e r e n c e s m a y be c h a r a c t e r i z e d in t w o ways.

0.4 (I.8 1.6

First, t h e y s h o w t h a t t h e m a x i m a l effect in W S P mice was g r e a t e r t h a n t h a t in W S R mice for all but two d r u g s ,

Kainic acid

25 50

I0 I0 PRE

3.0 ± 0.2 3.1 ±0.2 13) ± o.2

0.3 4 0.2 0. I + 0 . l 0+0

NMDA

3O 6(1

3 3

2.0+_0.2 2.4 ± 0.5

0. I +0. I 0±0

PRE

2.1 z 0 . 2

0_+0

5 5 5 PRE

2.5 ± O.3 3.2 £1).2 4.3±0.3 2.0 + o.2

0 + (} o_+o 0. I ±0.1 0±0

picrotoxin and PTZ. However, picrotoxin and pentylenet e t r a z o l w e r e m o r e p o t e n t in W S P t h a n in W S R mice. Three other GABA

antagonists, bicuculline, 3-MPA and

T B P S , also s e e m e d to b c m o r e p o t e n t in W S P t h a n in W S R mice. G i v e n t h e m i n i m a l r e s p o n s e o f W S R mice to all d r u g s o t h e r

than

p o s s i b l e to e s t i m a t e

GABA

antagonists,

it was not

p o t e n c i e s for t h o s e drugs,

o r to

Strychnine

0.25 0.5 1

T A B L E II

M a x i m u m H I C increase over baseline

Values are the mean _+ S.E.M. increase over PRE at the indicated dose, assessed at the time indicated in Table I. Difference scores are not identical to mean HIC minus PRE from Table I because PRE for each dose x line group varied. N.S. = P > 0. l(J. Drug

Dose (mg/kg)

Selected line . . . WSP

.

.

.

3.8 _+ 0.3 3.3 + 0.2 0.8 _+_0. I 11.7 + 0.2 0.5 +_ 0.2

171.0 357.[I 36.[) 17.0 10.8