- Email: [email protected]
Antagonism of ethanol withdrawal convulsions in withdrawal seizure prone mice by diazepam and abecarnil
Jottrtml of Pltarttiacolugy, 221 (1992) 85-90 0 1992 Elsevicr Science Publishers B.V. All rights resewed 0014-2999/92/$05.00
Ettropw
EJP 52673
Antagonism of ethanol withdrawal convulsions in Withdrawal Seizure Prone mice by diazepam and abecarnil John C ‘Irabbe Research Sewice, VA Medical Cetrter uttd Deparmmetrrs of Mcdicul Psychology and Pharmacology,
Oregon Health Sciences Uttirersity. Portland,
OR 97201, USA
Received 30 June 1992, accepted 7 July 1992
Abccarnil, a in a number clinical
@-carbolinc
application
abecarnil
acting at benzodiazcpinc
of models. It has rcduccd of diazcpam
with diazepam
scvcrc handling-induced
to prcvcnt
cxperimcnt
or diazepam
Withdrawal
clcvation
to diazcpam. genetically
reduced handling-induced
its effects were shorter-lasting.
of handling-induced
Similar
withdrawal;
1. Introduction Benzodiazepine receptors bind a number of compounds with agonist, antagonist, and inverse agonist properties. The p-carboline derivative, abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-~-carboline-3carboxylate: ZK 112119), has been shown to have partial agonist effects including high anxiolytic potency with little to no sedative effects (Stephens et al., 1990) and a distinctive anticonvulsant profile in various animal models (Turski et al., 1990). Benzodiazepines remain the drugs of choice for clinical treatment of alcohol withdrawal where withdrawal seizures might be expected to occur. While their sedative effects may be useful in treating the early stages of alcohol withdrawal, their sedative and motor incoordinating effects may be problematic in later stages of treatment. They also have clinical application as antiepileptics and anxiolytics. There is a continuing
Correspondence IO: 3. Crabbe, Research Career Scientist (ISIW). Department of Vctcrans Affairs Medical Center, 3710 SW US Vcterans Hospital Road, Portland, OR 97201, USA. Tel. 1.503.273 5298. fax I SO3.273 535 I.
in naive WSP mice.
Handling-induced (Genetic
convulsion
results were seen in an injection.
Abecarnil
Single doses of abecamil or
convulsion scores suggcstivc of a withdrawal
cffccts; &Carbolines;
to compare
sclcctcd to develop
were exposed to ethanol vapor for 24 h.
had significantly
convulsion scores seen
properties
Given the wide
model was employed
convulsions were assessed after a single high-dose ethanol
scizurc prone mouse (WSPI; Ethanol Anticonvulsant
animal
(WSP) mice,
from chronic ethanol treatment,
also reduced the smaller handling-induced
did not lcad to a rebound
Seizure Prone
at the peak of withdrawal
was slightly more potent than diazcpam. handling-induced
effects in comparison
seizures, a genetic
cffccts. Withdrawal
convulsions after withdrawal
where withdrawal
and diazepam diazcpam
abccamil
has been shown to have anxiolytic and anticonvulsant
and incoordinating
alcohol withdrawal
for its anti-withdrawal
WSP mice given doses of abccarnil scores. While
rcccptors,
musdc relaxant
convulsions; Abecarnil;
reac!ion.
Diazcpam;
animal model)
search for benzodiazepine ligands with more selective anticonvulsant (or anxiolytic) effects versus sedative/ ataxic effects. The handling-induced convulsion score in mice was originally proposed as a useful index of central nervous system hyperactivity during drug withdrawal (Goldstein and Pal, 1971). Ethanol withdrawal handling-induced convulsions were found to be antagonized by most drugs displaying cross-tolerance and cross-dependence with ethanol (Goldstein, 1972), and heritable differences in the severity of ethanol withdrawal convulsions were reported (Crabbe et al., 1983a). We have developed a genetic animal model of sensitivity to ethanol physical dependence by selectively breeding Withdrawal Seizure Prone (WSP) mice to display severe handling-induced convulsions after cessation of chronic ethanol vapor inhalation (Crabbe et al., 1985). The genetic sensitivity of WSP mice has been shown to generalize to withdrawal from barbiturates (Belknap et al., 1988), diazepam (Belknap et al., 1989), and nitrous oxide (Belknap et al., 1987). After many generations of selection, WSP mice are so sensitive that significant elevations in withdrawal handling-induced convulsion scores can be seen several hours after a single injection of several compounds with sedative effects on the
t.WItd WlYOkJSS~SttSll(ClXbht2et al..
IWlil).
En-
stt~ed central nervous system activity (charactcrizcd by hwered seizure thresholds) following acute ethanol treatment was first reported by McQuarrie and Fingl f 195~1. In the current
study. we evaluated
the efficacy of
ct al.. 19S3b). At O7:OO h, mice were injected 2.2 g/kg
ethanol
wire-mesh
(20%
hanging
v/v
cages
in saline)
i.p. with
and placed in
in a chamber
containing
ethanol vapor. At 2 and 6 h after injection, 30 mice wcrc briefly rcmcived from the chamber and a 20 ~1 tail-blood
sample was drawn. At 24 h after injection, all
abecamil as an anticonvulsant against ethanol withdrdwal in WSP mice and compared it with diazcpam,
mice were removed from the chamber, blcs; samples were taken. and the mice were scored for handling-in-
using the handling-induced convulsion score after acute or chronic ethanol exposure.
duced convulsions each h for 15 h, and again at 24 and 25 h. Handling-induced
convulsion scores were deter-
mined using a previously published scale (Crab& 2. Materials
and methods
(score = II to sevcrc, tonic-clonic Adult.
ct al.,
19Yla): non-zero scores range from minimal tonic seizures which must be elicited by spinning by the tail
female WSP mice were bred in our colonies
in Portland. Oregon. USA. Al! animals were naive at the time of testing. Mice were maintained in groups of five in individually ventilated cages (Thorcns) with ad iibitum access to food and water under a 12 : 12 h light : dark cycle at 2h + 1°C.
seizures elicited sim-
ply by removing the cage lid (score = 7). Vapor concentrations were maintained ethanol/l air by adjusting
at approximately 14.5 mg the flow of ethanol onto a
chromatographic paper wick. Blood samples were prepared (Crabbe et al., 1989) and analyzed for ethanol according to gas chromatographic ~i~iii& (Crabbc ei. al., 1982) which have been previously drscribed. Peak withdrawal handling-induced convulsion scores
Ethanol (Midwest Solvents. Muscatinc. IA, USA) was diluted to 20% v/v in saline. Diazcpam was a gift oi Hoffmann LaRochc, Inc., Nutley. NJ, USA. Allecarnil W;IS a gift of Dr. D.N. Stcphcns, Schcring, Berlin. Drugs were initially dissolved in a drop of Tween 80. and saline was added to a final volume of 5 cc.
arc attained
under !hese conditions approximatc!y
h after mice were injected remova?
~EYXII
of abccarnil
(0.5,
4-6
the chambers. At 5 h 3 min, i.p. with vehicle, one of four doses 1.0, 2.0 or 4.0 mg/kg)
or diazepam
(same doses). As most mice had evidently recovered from drug-induced suppression of withdrawal by 8 h, injections wcrc rcpcated
at 7 h 45 min.
In a second study, naive WSP mice were scored for handling-induced convulsions and injected with 4 g/kg All procedures adhere to United States Public Health Service-National Institutes of Health Guidelines for the care and use of laboratory animals and were approved by the two local institutional Animal Care XX! Use Committees. The apparatus and method for inducing ethanol dependence by exposure to ethanol vapor have been dcscribcd previously (Crabbc
O
1
2
3
4
5
6
t
7
8
9101112131415
t TIME (HOURS)
2425
ethanol in saline (20%
v/v).
Handling-induced
convul-
sions were scored at 2, 4, 5 and 6 h, when peak acute withdrawal scores were attained (Crabbe et al., 199la). At
6 h 30 min
after
ethanol
injection,
mice
were
injected i.p. with vehicle, abecarnil (0.1, 0.2 or 0.5 mg/kgl, or diazepam (0.2 or 0.5 mg/kg). Handling-induccd convulsions wire again scored, at 10 min intcr-
0
1
2
3
4
5
6 t
7
B
9 101112131415
2425
t TIME (HOURS)
Fig.. I. hleai; !aar:Ging-indocd ca)bdsion scores in groups of WSP rnicc withdrawing irum 24 h chronic rnhalatbi~ of ethimcil vapor. lnjcctions ot vehicle or ihe mdicated doses of ahecarnil (A) or diazepam (B) were given at the timczsindicated by arrow%. Vehicle group (N = IX) is shown in both panels. N = ‘#./drug dose. S.E.s arc omitted for clarity. and range from 0.19 (smaller than symhol size) to 0.07. Open circles, vehicle. Filled circles. 0.5 mg/kg. Filled triangles. 1.0 mg/kg. Filled squnrcs . . 2.0mg/kg. Filled dlamcrilds. 4.0 mg/kg.
x7
vals starting to II h.
10 min after injection,
and hourly !-cm 7
In a third study, the effect of abecarnil on handlinginduced convulsions was tested in naive WSP mice. After
scoring basclinc
handling-induced
convulsions,
placebo, 0,02,0.2 or 2.0 mg/kg doses of abecarnil were administered, and handling-induced convulsions were scored at 10 min intervals for I h, and again 2, 3 and 4 h after injectlon. In a fourth
study, the possibility that single doses of
abccarnil or diazepam might themselves provoke acute withdrawal responses was evaluated. Handling-induced convulsions were scored, and vehicle, doses of each
drug
were
z
4 PRE : : 2: : 4
I
1.0 or 2.0 mg/kg
administered
to scparatc
groups of mice. Handling-induced convulsions were scored hourly for 8 h as described, beginning 1 h after injection.
Fig. 2. Mean
TIM&OURS)
handling-induced
mice aculely withdrawing arrow).
Injections
ahecarr,il
(indicated
or diazepam
Handling-induced
convulsion scores in groups of WSP
from 4 g/kg
ethanol (given at hour 0: large
by smail arrow) of vehicle or doses of
were
given at h h 30 min after
thus, the 30 min score occurred 7 h post-ethanol. (0.1 mg/kg
For most analysts, was employed, Newman-Keuls,
single- or two-factor
ANOVA
and post-hoc tests used the method of employing
the
studentized
range
mg/kg
abecarnil
upward
triangles.
It.9 mg/kg
Results of the first experiment vapor exposure induced
are shown in fig. 1. stable blood alcohol
levels. At 6 h after exposure. mean ) S.E. blood alcohol concentrations were 1.97 + 0.1 I mg/ml, and trcatmcnt groups did not differ significantly (F(8. 21) = 0.5, P > 0.10). Mice had blood ethanol concentrations averaging 2.09 + 0.11 mg/ml at the time of withdrawal, and no significant diffcrcnccs among groups wcrc seen (F(8, 81) = 1.3 P > 0. IO). Handling-induced convulsion scores, which typically average 2 in naive WSP mice (Crabbc ct al., lY9lbL wcrc rcduccd by ethanol during the first hour of withdrawal. Between 1 and 3 h of withdrawal, as the animals eliminated blood ethanol, they
began
0.2 mg/kg
ahecarnil.
Filled
ahecarnil. squares,
diamonds. ti.S mg/kg
Results
Ethanol
for
to display
characteristically
cxaccrbatcd
handling-induced convulsions, which pcakcd at 4-S h after removal. Abecarnil (fig. IA) and diazepam (fig. IB), administered IS min bcforc the 6 h scoring, inhibitcd withdrawal handling-induced convulsions dose dcpcndently. The change in handling-induced convulsion scores bctwccn 5 and 6 h diffcrcd significantly among groups (F(X, 81) = 5.7. P < 0.0001). Post-hoc tests versus the vehicle-treated group revealed that abecarnil was signilicantiy cffcctivc at the 2 and 4 mg/kg doses, while diazcpam had significant effects only at the 4 mg/kg dose (P < 0.01). This suggests that abecarnil
N = IX (vehicle).
IO
groups) or 16 (both 0.2
ciarity, and range from I) to
0.61. Open circles. vehicle. Filled circles. 0.1 mg/kg
statistic. qF.
3.
group. hoth 0.5 mg/kg
S.E.s iire <:did
gmiips).
ethanol.
convulsion scores were taken 10 and 20 min later:
Fi!led
ahccarnil.
downward
0.2 mg/kp
Filled
triangles.
diazepam
Filled
diazepam.
prc-drug) and 6 h (first post-drug) scores was calculated for each animal. These data for the eight drug groups were reanalyzed in a two-factor ANOVA (drug X dose). The effect of drug was sl:..nificant (F(1, 64) = 6.2, P = 0.02): abecarnil-treated mice averaged handling-induced convulsion score reductions of 1.8 units, while diazcpam-treated mice had handling-induced convulsion score reductions of 0.8 units. The effect of dose approached significance (F(3, 64) = 2.5, P = 0.061, and the interaction was non-significant (P > 0.10). It seems likely that the dose-related effects apparent in fig. 1 (A,B) did not reach significance due to the rclativcly small group sizes and corresponding magnitude of within-group error. Analyses of responses to the second injection revcalcd a similar pattern of outcomes. The suppression of h;lliC!!ing-i!K!tJCCC! convulsions by di:tzepam appeared to have been of longer duration, at least at the highest dose, for ahccarnil-trcatcd mice had rccovcrcd to control levels of handling-induced convulsion scores by 3 h after the second injection. This is not surprising, since abccarnil has a shorter half-life
than diazcpam
in mice
(Dr. D.N. Stcphcns, personal communication). The withdrawal response following a single acute ethanol dose was also supprcsscd by diazcpam and abecarnil; fig. 2 shows the results of the second study. Ethanol
initially
reduced handling-induced
convulsion
this possibility. we reanJyzed the data in a way that would facilitate direct comparison. The vehicle group
>,corcs in ali groups (compare scores at 2 and 4 h with those (PRE) just before injection). By 6 h. handling-induced convulsion scores had rebounded to levels abo;e
was cxcludcd,
prc-ethanol
may have been rnorc potent tharr diazcpam.
and the diffcrcncc
To pursue:
bctwccn the 5 h (last
basclinc, indicating a state of ~CLI~Cethanol
witbdr~wal similar to that previously reported (Crabhe et ai.. I99is). Since groups did not differ significantly
interaction (F(32.440) = 2.X. P < 0.0001). A separate one-way ANOVA of the scores I h post-injection was
(P > tl. it)) at 6 h. a similar degree of acute dependence cm ethanol in alI groups may be inferred. injections of abevarnii or diazcpam :,I h ,h 30 min after ethanol
yses showed that the 2 mg/kg
reduced
inje&,‘tion
handling-induced
convulsion
scores
significant (F(4, 55) = 4.7, P < 0.01) and post-hoc analdose groups of either
drug, and the I mg/kg dose group treated with diazepam, had significantly reduced handling-induced
dose dependently. ANOVA for the three initial postdrug scores revealed only a significant effect of treat-
convulsion scores versus the placebo group (P < 0.01).
ment group. Post-hoc tests revealed that the groups treated with 05 mg/kg doses of abecarnil or diazepam
lowered handling-induced
had significantly attcnuatcd
withdrawal
scores (P < 0.01
and t.OS. respectively). Although groups treated iower doses appeared to have somewhat lower
By 2 h, only the diazcpam-trcatcd This suggests. once longer-lasting effect.
mice still showed
convulsion scores (P < O.OI).
again,
that
diazepam
had
the
with han-
dling-induced coavuision scores than vehicle-trcatcd mice (see fig. 2). these differences were not significant. Abecamii also effectively reduced handling-induced convulsion scores in naive mice. Mean ( + S.E.) baseline handling-induced convulsion scores for the 29 mice tested in Esperiment 3 were 3.2 + 0. I. and handling-induced conkruision scores 40 min after ir?jcction were 1-S + 0.1. 0.4 + 0 3. :ir.d !I i il. f01 the groups given 0_(12, 0.2. and 2 mg/kg ahccarnii. respectively. The latter two groups were significantly rcduccd from bascline means (P < O.Oi). By I80 min after injection, the respective handling-induced convulsion scores were 2.3 f 0.2. 2.4 -+ 0.2. and 2.7 + 0.4. These did not differ significantly from each other. or from baseline scores. Figure 3 again shows that abecarnii or diazepam effectively reduced handling-induced convulsion scores in naive mice. No treatments employed showed signs of provoking an acute withdrawal response. since no groups significantly excccdcd baseline scores during the S h test. Analysrb indicated a significant effect of treatment group (F(4. 55) = 35. P < O.Oi ) and time (RX 440) =4-S. P < O.oUOi), as wcli as a significant
4. Discussion The finding withdrawal
that diazepam
handling-induced
could suppress ethanol convulsions
in mice was
;lot surprising. Goldstein (1972) reported such antagonism in genetically heterogeneous mice, but she used a lonpcr ncriod of dure
that
c!had
included
r;xyosurc (72 h), and a proccadjunct
treatment
with
pyrazole
hydrochloride. an alcohol dehydrogenase inhibitor. Howcvcr. she found a SO mg/kg dose of diazepam (but not 5 or 20 mg/kg) that 20-100 mg/kg
to be significantly effective, and chloridazepoxide was also effec-
tive. In the current studies, a 4 mg/kg dose of diazepam inhihited withdrawal. Greater sensitivity to diazepam was seen in the current experiments:
this could
bc due to our USC of WSP mice. rather than the generic Swiss-Webster used by Qr. Goldstein, or to the greater degree of dependence instilled by use of 72 h inhalation coupled tion.
with
pyrazole-induced
metabolic
inhibi-
Abecarnil clearly inhibited handling-induced convulsions seen after withdrawal from 24 h chronic ethanol intoxication. it appeared to have been more potent than diazepam. although the difference was not marked. it seems likely that its cffcct was of somewhat shorter duration. Both drugs also an!agnnized wi!hdrawal in the single-injection acute withdrawal model, at cvcn lower doses (0.5 mg/kg). In the abscncc of information
about brain or plasma levels for the two
drugs. firm conclusions regarding relative potency cannot bc drawn from thcsc data. ::nd await further studies. Abecarnil
is a potent anticonvulsant
in several ani-
mal models of cxpcrimcntal seizures (Turski et al., IYYO; Fischer et al., 1991). We have previously re-
4
;
4
:
Ii
7
I
ported enhancement of handling-induced convulsions in naive WSP mice with the bcnzodiazcpinc inverse
8
nME(HOLJRS) Fig. 3. Mran + S.E. handling-inducedccmvulsion WSP (open Open
agonist, Ro IS-45 1.3, at doses from 0.1 to 10.0 mg/kg SCW~S in gmqx
of
mice
(each N = I?). Injections (indic:!rcd hy arrow) of vchiclc rquart?i) llr cimr% +.Tfabcrarnil or di;lzcplm wcrc given i.n. circles.
arepam.
I
mg/kg
dia7epam.
Filled lrianplrs.
I mg/kg
Open
triangles,
ahecarnil.Filled
ahecarnil.
2 mg/kg
Ji-
circles. 2 mg/kg
(Crabbe et al., 199lb). In addition, many other drugs including psychostimulants, direct and indirect GABA antagonists. the calcium channel agonist BAY K 8644, corticosteronc, and excitatory amino acid receptor agonists, clcvatc
handling-induced
convulsions
in WSP
XY
mice (Crabbe et a!., 1991b; Roberts et a!., 1991; Crabbe and Kosobud, 1990; Phillips and Crabbe, 1991). Abecarni! clearly antagonized handling-induced convulsions, both in ethanol-withdrawing and naive mice, thus resembling drugs with central nervous system depressant effects (Crabbe et a!., 199la; Kosobud and Crabbe, 19861, and several other anticonvulsants (Crabbe and Kosobud, 1990; Phillips and Crabbe, 1991). In Experiment 4, we saw no signs that withdrawal from a single dose of abecarni! or diazepam led to a rebound exacerbation of handling-induced convulsions. This agrees with unpublished data with diazepam in our laboratory. WSP mice are extremely sensitive to such acute withdrawal effects, which are easily seen after ethanol (Crabbe et a!., 1991a; Kosobud and Crabbe, 1986), t-butano!, pentobarbita!, and acetaldehydc (Kosobud and Crabbe, 1986). Earlier studies had reported that cessation of thrice daily administration of 5 mg/kg abecarni! to rats led to no marked signs of spontaneous withdrawal during several days after trcatment Uscher et a!., 1991). and 6 weeks of daily administration of 4 mg/kg to dogs produced no spontaneous withdrawal reaction even when f!umazeni!-prccipitated Escher et a!., 1990). Thus, existing data suggest that the risk of physical dependence on this compound is relatively low. Comparison of the results seen in Experiments l-3 with published literature suggests that the effects of abecarnil against alcohol withdrawal may be dissociable from its genera! anticonvulsant effects. while the same would apparently not be true of diazepam. The cffectivc dose for producing sedative effects in mice clearly depends upon the specific test employed, and probably on the strain of mice. Nonetheless, when the hanging wire and rotarod tests of ataxia were employed in NMRI strain mice (Stephens et a!., 19901, the ED+ for abecarni! were 38 and > 100 mg/kg, respectively; for diazepam, the comparable values were 0.99 and 14 mg/kg, respectively. Our studies showed efficacy of both of these compounds against alcohol withdrawal at doses no higher than 4 mg/kg. In conclusion, abecarni! or diazepam, given to genetically WSP mice at the peak zf chronic ethanol withdrawal, significantly reduced handling-induced convuision scores. While abecarni! was somewhat more potent than diazepam, its effects were shorter-lasting. Similar results were seen in an cxpcrimcnt where withdrawal was assessed after a single high-dose ethanol injection. Abecarni! and diazepam also inhibited the lower handling-induced convulsion scores seen in naive mice, indicating that this convulsive sign in mice is sensitive to anticonvulsants. Single administrations of diazcpam or a!?ccarni! did not lcad to spontaneous withdrawal-induced increases of handling-inducetl CcJI1vu!sion severity. Doses of abecarni! effective in reducing withdrawal handling-induced convulsion scores
were lower than published values for tests of ataxia, suggesting that it might be possible to dissociate the antiwithdrawal and ataxic effects of abecarni!.
Acknowledgments These studies were supported ment of Veterans AAOXb2I.
Affairs.
DAtii22X).
I thank Catherine
by a grant from the US Depart-
by grants from
and USPHS-NIDA
Merrill
the USP!IS
Contract
(AAO624.3.
No. 271-90-7405.
for her expert help with these experiments.
and Drs. David N. Stephens and Graham Jones (Schering-Berlin)
for
their comments on the manuscript.
References Belknap.
J.K..
S.E.
Laursen
and J.C. Crabbe.
Belknap.
J “... P.W.
Ethanol
Danielson.
and barbiturate
codetermined
Belknap.J.K.,
and
and J.C. Crabbe.
19x8.
convulsions are extensively
in mice. Alcohol 5. 167. J-C. Crabbe
diazepam withdrawal WSP and WSR Crabbe.
M. Lam6
withdrawal
19x7, Ethanol
convulsions by similar
nitrous oxide produce withdrawal-induced mechanisms in mice. Life Sci. 41. 2033.
and S.E.
Laursen,
1989, Ethanol
and
convulsions are extensively codete:mined
in
mice. Life Sci. 44, 2075.
J.C. and A.
Kosobud,
1990. Alcohol
withdrawal
seizures:
Genetic animal models. in: Alcohol and Seizures. eds. R.J. Porter. R F!. Mattcon, Philadelphia)
.I.A.
Cramer
and
1. Diamond
(FA
Davis Co.,
p. 126.
Crabbe. J.C.. J. Janowsky, E.R. Young, A. Kosobud. J. Stack and H. Rigter.
19X2. Tolerance
Genotypic
to ethanol
correlations
with
hypothermia
behavioral
in inbred mice:
responses.
Alcoholism:
Clin. Exp. Res. 6, 44f1. Crahbe. J.C.. E.R.
Young
tions with ethanol hav. IX (SuppI. Crahbe.
J.C..
and A. Kosobud,
withdrawal
Kosohud.
E.R.
Polygenic and single-gene
J.C..
A.
withdrawal J.C..
tolerance
Kosobud.
inbred
D.J.
E.R.
Feller
Young.
C.D.
hypothermia
and J.K. Bclknap.
drugs is determined
Neurohehav.
Tam
and J.D.
D.B.,
1989,
!5. 521.
Sensitivity
and
in gcnctically selected IO9la.
Acute dcpcn-
hy common genes in
and J.K.
Belknap,
IYYlh,
Effecis
of
convulsions in mice sclccted for
severity, Brain Rcs. 550. I.
1972, An animal model for testing effects of drugs
on alcohol withdrawal
reactions, J. Pharmacol.
Exp. Ther.
D.B. and N. Pal. 1971. Alcohol depcndcnce
mice by inhalation
of ethanol: Grading
jcience 172. 2:X Kosobud, A. and J.C. Cr,ibbe. to be ~rnetically withdrawal
Mc-
Exp. Ther. 257. 663.
Merrill
ethanol withdrawal
14. Goldstein.
B.R.
J.S. Dorow,
convulsants on handling-induced Goldsrem,
strains,
Exp. Ther. 249. 456.
mice, J. Pharmacol. J.C..
and
Merrill
dcncc on depressant Crahbe.
mouse
selection for susceptibility to elbano
to ethanol-induced
J.C., C.D.
19S3h.
of response to ethanol
seizures in M1r.srnrrscrdus, Behav. Genet.
mice, J. Pharmacol. Crabhc.
and J. Janowsky.
5, 1X1.
Swigan. 1985. Bidirectional Crahhe.
Young
determination
recombi!lant
Toxicol. Teratol. Crahhe.
correla-
Biochem. Be-
I). 541.
A.
in BXD/Ty
I9X3a. Genetic
severity. Pharmacol.
rcizures,
or resistant
J. Pharmacol.
L&her. W.D.. D. Hiinack. l99[), Pharmacokinclics. fects of the /$carboline
produced in
the withdrawal
1986. Ethanol withdrawal
prone t WSP)
(WSR)
reaction.
in mice hrcd to ethanol
Exp. Ther. 238. 170.
R. Scherkl. A. Hashcm and ti:H. anticonvulsant efficacy and ;IdWSc abccarnil,
1X.3.
a novel lig~d
Frey. cf-
for brnzodi-
azepine receptors. ::tter acute and chronic administration in dogs. J. Phamrrac~& Exp. Ther. 255. 541. Liischer. W.. C. Rundfeldt and D. Hiinack. 1991. Tolerance to anticonvulsant effects of the partial henzodiazepine receptor agonist ahzarnil in kindled T.IISinvolves learning. Eur. J. Pharmacol. 2tc 303. M&uarrie. D.G. and E. Fingl. 19%. Effects of single doses and chronic administration of ethanol on experimental seizures in mice. J. Pharnmcol. Exp. Ther. 124, 264. Phillips. T.J. and J.C. Cmhhe. IWI. Behavioral studies of genetic differences in al~xrhol action. in: The Genetic Basis of Alcohol and Drug Actions. eds. J.C. Crahhe and R.A. ffarris (Plenum Publishing Corp.. New York) p. 25. R&ens. A.J.. H.-P Chu. J.C. Crathbe and L.D. Keith. 1991. Differ-
ential modulation by the stress axis of ethanol withdrawal seizure expression in WSP and WSR mice, Alcoholism: Clin. Exp. Res. IS. 412. Stephens, D.N., H.H. Schneider, W. Kehr, J.S. Andrews, K.-J. Rettig. L. Turski. R. Schmiechen, J.D. Turner, L.H. Jensen, E.N. Petersen, T. Honore and J. Bondo Hansen, 1990. Ahecarnil. a stable. anxioselective fl-carboline acting at benzodiazepine receptors. J. Pharmacol. Exp. Ther. 253, 334. Turski, L.. D.N. Stephens. L.H. Jensen, E.N. Petersen, B.S. Meldrum. S. Patel, J. Bondo Hansen. W. Liischer, H.H. Schneider and R. Schmiechen, 1990, Anticonvulsant action of the P-carboline ahecarnil: Studies in rodents and baboon, f~pio papio, J. Fharmacol. Exp. Ther. 253, 344.
Ettropw
EJP 52673
Antagonism of ethanol withdrawal convulsions in Withdrawal Seizure Prone mice by diazepam and abecarnil John C ‘Irabbe Research Sewice, VA Medical Cetrter uttd Deparmmetrrs of Mcdicul Psychology and Pharmacology,
Oregon Health Sciences Uttirersity. Portland,
OR 97201, USA
Received 30 June 1992, accepted 7 July 1992
Abccarnil, a in a number clinical
@-carbolinc
application
abecarnil
acting at benzodiazcpinc
of models. It has rcduccd of diazcpam
with diazepam
scvcrc handling-induced
to prcvcnt
cxperimcnt
or diazepam
Withdrawal
clcvation
to diazcpam. genetically
reduced handling-induced
its effects were shorter-lasting.
of handling-induced
Similar
withdrawal;
1. Introduction Benzodiazepine receptors bind a number of compounds with agonist, antagonist, and inverse agonist properties. The p-carboline derivative, abecarnil (isopropyl-6-benzyloxy-4-methoxymethyl-~-carboline-3carboxylate: ZK 112119), has been shown to have partial agonist effects including high anxiolytic potency with little to no sedative effects (Stephens et al., 1990) and a distinctive anticonvulsant profile in various animal models (Turski et al., 1990). Benzodiazepines remain the drugs of choice for clinical treatment of alcohol withdrawal where withdrawal seizures might be expected to occur. While their sedative effects may be useful in treating the early stages of alcohol withdrawal, their sedative and motor incoordinating effects may be problematic in later stages of treatment. They also have clinical application as antiepileptics and anxiolytics. There is a continuing
Correspondence IO: 3. Crabbe, Research Career Scientist (ISIW). Department of Vctcrans Affairs Medical Center, 3710 SW US Vcterans Hospital Road, Portland, OR 97201, USA. Tel. 1.503.273 5298. fax I SO3.273 535 I.
in naive WSP mice.
Handling-induced (Genetic
convulsion
results were seen in an injection.
Abecarnil
Single doses of abecamil or
convulsion scores suggcstivc of a withdrawal
cffccts; &Carbolines;
to compare
sclcctcd to develop
were exposed to ethanol vapor for 24 h.
had significantly
convulsion scores seen
properties
Given the wide
model was employed
convulsions were assessed after a single high-dose ethanol
scizurc prone mouse (WSPI; Ethanol Anticonvulsant
animal
(WSP) mice,
from chronic ethanol treatment,
also reduced the smaller handling-induced
did not lcad to a rebound
Seizure Prone
at the peak of withdrawal
was slightly more potent than diazcpam. handling-induced
effects in comparison
seizures, a genetic
cffccts. Withdrawal
convulsions after withdrawal
where withdrawal
and diazepam diazcpam
abccamil
has been shown to have anxiolytic and anticonvulsant
and incoordinating
alcohol withdrawal
for its anti-withdrawal
WSP mice given doses of abccarnil scores. While
rcccptors,
musdc relaxant
convulsions; Abecarnil;
reac!ion.
Diazcpam;
animal model)
search for benzodiazepine ligands with more selective anticonvulsant (or anxiolytic) effects versus sedative/ ataxic effects. The handling-induced convulsion score in mice was originally proposed as a useful index of central nervous system hyperactivity during drug withdrawal (Goldstein and Pal, 1971). Ethanol withdrawal handling-induced convulsions were found to be antagonized by most drugs displaying cross-tolerance and cross-dependence with ethanol (Goldstein, 1972), and heritable differences in the severity of ethanol withdrawal convulsions were reported (Crabbe et al., 1983a). We have developed a genetic animal model of sensitivity to ethanol physical dependence by selectively breeding Withdrawal Seizure Prone (WSP) mice to display severe handling-induced convulsions after cessation of chronic ethanol vapor inhalation (Crabbe et al., 1985). The genetic sensitivity of WSP mice has been shown to generalize to withdrawal from barbiturates (Belknap et al., 1988), diazepam (Belknap et al., 1989), and nitrous oxide (Belknap et al., 1987). After many generations of selection, WSP mice are so sensitive that significant elevations in withdrawal handling-induced convulsion scores can be seen several hours after a single injection of several compounds with sedative effects on the
t.WItd WlYOkJSS~SttSll(ClXbht2et al..
IWlil).
En-
stt~ed central nervous system activity (charactcrizcd by hwered seizure thresholds) following acute ethanol treatment was first reported by McQuarrie and Fingl f 195~1. In the current
study. we evaluated
the efficacy of
ct al.. 19S3b). At O7:OO h, mice were injected 2.2 g/kg
ethanol
wire-mesh
(20%
hanging
v/v
cages
in saline)
i.p. with
and placed in
in a chamber
containing
ethanol vapor. At 2 and 6 h after injection, 30 mice wcrc briefly rcmcived from the chamber and a 20 ~1 tail-blood
sample was drawn. At 24 h after injection, all
abecamil as an anticonvulsant against ethanol withdrdwal in WSP mice and compared it with diazcpam,
mice were removed from the chamber, blcs; samples were taken. and the mice were scored for handling-in-
using the handling-induced convulsion score after acute or chronic ethanol exposure.
duced convulsions each h for 15 h, and again at 24 and 25 h. Handling-induced
convulsion scores were deter-
mined using a previously published scale (Crab& 2. Materials
and methods
(score = II to sevcrc, tonic-clonic Adult.
ct al.,
19Yla): non-zero scores range from minimal tonic seizures which must be elicited by spinning by the tail
female WSP mice were bred in our colonies
in Portland. Oregon. USA. Al! animals were naive at the time of testing. Mice were maintained in groups of five in individually ventilated cages (Thorcns) with ad iibitum access to food and water under a 12 : 12 h light : dark cycle at 2h + 1°C.
seizures elicited sim-
ply by removing the cage lid (score = 7). Vapor concentrations were maintained ethanol/l air by adjusting
at approximately 14.5 mg the flow of ethanol onto a
chromatographic paper wick. Blood samples were prepared (Crabbe et al., 1989) and analyzed for ethanol according to gas chromatographic ~i~iii& (Crabbc ei. al., 1982) which have been previously drscribed. Peak withdrawal handling-induced convulsion scores
Ethanol (Midwest Solvents. Muscatinc. IA, USA) was diluted to 20% v/v in saline. Diazcpam was a gift oi Hoffmann LaRochc, Inc., Nutley. NJ, USA. Allecarnil W;IS a gift of Dr. D.N. Stcphcns, Schcring, Berlin. Drugs were initially dissolved in a drop of Tween 80. and saline was added to a final volume of 5 cc.
arc attained
under !hese conditions approximatc!y
h after mice were injected remova?
~EYXII
of abccarnil
(0.5,
4-6
the chambers. At 5 h 3 min, i.p. with vehicle, one of four doses 1.0, 2.0 or 4.0 mg/kg)
or diazepam
(same doses). As most mice had evidently recovered from drug-induced suppression of withdrawal by 8 h, injections wcrc rcpcated
at 7 h 45 min.
In a second study, naive WSP mice were scored for handling-induced convulsions and injected with 4 g/kg All procedures adhere to United States Public Health Service-National Institutes of Health Guidelines for the care and use of laboratory animals and were approved by the two local institutional Animal Care XX! Use Committees. The apparatus and method for inducing ethanol dependence by exposure to ethanol vapor have been dcscribcd previously (Crabbc
O
1
2
3
4
5
6
t
7
8
9101112131415
t TIME (HOURS)
2425
ethanol in saline (20%
v/v).
Handling-induced
convul-
sions were scored at 2, 4, 5 and 6 h, when peak acute withdrawal scores were attained (Crabbe et al., 199la). At
6 h 30 min
after
ethanol
injection,
mice
were
injected i.p. with vehicle, abecarnil (0.1, 0.2 or 0.5 mg/kgl, or diazepam (0.2 or 0.5 mg/kg). Handling-induccd convulsions wire again scored, at 10 min intcr-
0
1
2
3
4
5
6 t
7
B
9 101112131415
2425
t TIME (HOURS)
Fig.. I. hleai; !aar:Ging-indocd ca)bdsion scores in groups of WSP rnicc withdrawing irum 24 h chronic rnhalatbi~ of ethimcil vapor. lnjcctions ot vehicle or ihe mdicated doses of ahecarnil (A) or diazepam (B) were given at the timczsindicated by arrow%. Vehicle group (N = IX) is shown in both panels. N = ‘#./drug dose. S.E.s arc omitted for clarity. and range from 0.19 (smaller than symhol size) to 0.07. Open circles, vehicle. Filled circles. 0.5 mg/kg. Filled triangles. 1.0 mg/kg. Filled squnrcs . . 2.0mg/kg. Filled dlamcrilds. 4.0 mg/kg.
x7
vals starting to II h.
10 min after injection,
and hourly !-cm 7
In a third study, the effect of abecarnil on handlinginduced convulsions was tested in naive WSP mice. After
scoring basclinc
handling-induced
convulsions,
placebo, 0,02,0.2 or 2.0 mg/kg doses of abecarnil were administered, and handling-induced convulsions were scored at 10 min intervals for I h, and again 2, 3 and 4 h after injectlon. In a fourth
study, the possibility that single doses of
abccarnil or diazepam might themselves provoke acute withdrawal responses was evaluated. Handling-induced convulsions were scored, and vehicle, doses of each
drug
were
z
4 PRE : : 2: : 4
I
1.0 or 2.0 mg/kg
administered
to scparatc
groups of mice. Handling-induced convulsions were scored hourly for 8 h as described, beginning 1 h after injection.
Fig. 2. Mean
TIM&OURS)
handling-induced
mice aculely withdrawing arrow).
Injections
ahecarr,il
(indicated
or diazepam
Handling-induced
convulsion scores in groups of WSP
from 4 g/kg
ethanol (given at hour 0: large
by smail arrow) of vehicle or doses of
were
given at h h 30 min after
thus, the 30 min score occurred 7 h post-ethanol. (0.1 mg/kg
For most analysts, was employed, Newman-Keuls,
single- or two-factor
ANOVA
and post-hoc tests used the method of employing
the
studentized
range
mg/kg
abecarnil
upward
triangles.
It.9 mg/kg
Results of the first experiment vapor exposure induced
are shown in fig. 1. stable blood alcohol
levels. At 6 h after exposure. mean ) S.E. blood alcohol concentrations were 1.97 + 0.1 I mg/ml, and trcatmcnt groups did not differ significantly (F(8. 21) = 0.5, P > 0.10). Mice had blood ethanol concentrations averaging 2.09 + 0.11 mg/ml at the time of withdrawal, and no significant diffcrcnccs among groups wcrc seen (F(8, 81) = 1.3 P > 0. IO). Handling-induced convulsion scores, which typically average 2 in naive WSP mice (Crabbc ct al., lY9lbL wcrc rcduccd by ethanol during the first hour of withdrawal. Between 1 and 3 h of withdrawal, as the animals eliminated blood ethanol, they
began
0.2 mg/kg
ahecarnil.
Filled
ahecarnil. squares,
diamonds. ti.S mg/kg
Results
Ethanol
for
to display
characteristically
cxaccrbatcd
handling-induced convulsions, which pcakcd at 4-S h after removal. Abecarnil (fig. IA) and diazepam (fig. IB), administered IS min bcforc the 6 h scoring, inhibitcd withdrawal handling-induced convulsions dose dcpcndently. The change in handling-induced convulsion scores bctwccn 5 and 6 h diffcrcd significantly among groups (F(X, 81) = 5.7. P < 0.0001). Post-hoc tests versus the vehicle-treated group revealed that abecarnil was signilicantiy cffcctivc at the 2 and 4 mg/kg doses, while diazcpam had significant effects only at the 4 mg/kg dose (P < 0.01). This suggests that abecarnil
N = IX (vehicle).
IO
groups) or 16 (both 0.2
ciarity, and range from I) to
0.61. Open circles. vehicle. Filled circles. 0.1 mg/kg
statistic. qF.
3.
group. hoth 0.5 mg/kg
S.E.s iire <:did
gmiips).
ethanol.
convulsion scores were taken 10 and 20 min later:
Fi!led
ahccarnil.
downward
0.2 mg/kp
Filled
triangles.
diazepam
Filled
diazepam.
prc-drug) and 6 h (first post-drug) scores was calculated for each animal. These data for the eight drug groups were reanalyzed in a two-factor ANOVA (drug X dose). The effect of drug was sl:..nificant (F(1, 64) = 6.2, P = 0.02): abecarnil-treated mice averaged handling-induced convulsion score reductions of 1.8 units, while diazcpam-treated mice had handling-induced convulsion score reductions of 0.8 units. The effect of dose approached significance (F(3, 64) = 2.5, P = 0.061, and the interaction was non-significant (P > 0.10). It seems likely that the dose-related effects apparent in fig. 1 (A,B) did not reach significance due to the rclativcly small group sizes and corresponding magnitude of within-group error. Analyses of responses to the second injection revcalcd a similar pattern of outcomes. The suppression of h;lliC!!ing-i!K!tJCCC! convulsions by di:tzepam appeared to have been of longer duration, at least at the highest dose, for ahccarnil-trcatcd mice had rccovcrcd to control levels of handling-induced convulsion scores by 3 h after the second injection. This is not surprising, since abccarnil has a shorter half-life
than diazcpam
in mice
(Dr. D.N. Stcphcns, personal communication). The withdrawal response following a single acute ethanol dose was also supprcsscd by diazcpam and abecarnil; fig. 2 shows the results of the second study. Ethanol
initially
reduced handling-induced
convulsion
this possibility. we reanJyzed the data in a way that would facilitate direct comparison. The vehicle group
>,corcs in ali groups (compare scores at 2 and 4 h with those (PRE) just before injection). By 6 h. handling-induced convulsion scores had rebounded to levels abo;e
was cxcludcd,
prc-ethanol
may have been rnorc potent tharr diazcpam.
and the diffcrcncc
To pursue:
bctwccn the 5 h (last
basclinc, indicating a state of ~CLI~Cethanol
witbdr~wal similar to that previously reported (Crabhe et ai.. I99is). Since groups did not differ significantly
interaction (F(32.440) = 2.X. P < 0.0001). A separate one-way ANOVA of the scores I h post-injection was
(P > tl. it)) at 6 h. a similar degree of acute dependence cm ethanol in alI groups may be inferred. injections of abevarnii or diazcpam :,I h ,h 30 min after ethanol
yses showed that the 2 mg/kg
reduced
inje&,‘tion
handling-induced
convulsion
scores
significant (F(4, 55) = 4.7, P < 0.01) and post-hoc analdose groups of either
drug, and the I mg/kg dose group treated with diazepam, had significantly reduced handling-induced
dose dependently. ANOVA for the three initial postdrug scores revealed only a significant effect of treat-
convulsion scores versus the placebo group (P < 0.01).
ment group. Post-hoc tests revealed that the groups treated with 05 mg/kg doses of abecarnil or diazepam
lowered handling-induced
had significantly attcnuatcd
withdrawal
scores (P < 0.01
and t.OS. respectively). Although groups treated iower doses appeared to have somewhat lower
By 2 h, only the diazcpam-trcatcd This suggests. once longer-lasting effect.
mice still showed
convulsion scores (P < O.OI).
again,
that
diazepam
had
the
with han-
dling-induced coavuision scores than vehicle-trcatcd mice (see fig. 2). these differences were not significant. Abecamii also effectively reduced handling-induced convulsion scores in naive mice. Mean ( + S.E.) baseline handling-induced convulsion scores for the 29 mice tested in Esperiment 3 were 3.2 + 0. I. and handling-induced conkruision scores 40 min after ir?jcction were 1-S + 0.1. 0.4 + 0 3. :ir.d !I i il. f01 the groups given 0_(12, 0.2. and 2 mg/kg ahccarnii. respectively. The latter two groups were significantly rcduccd from bascline means (P < O.Oi). By I80 min after injection, the respective handling-induced convulsion scores were 2.3 f 0.2. 2.4 -+ 0.2. and 2.7 + 0.4. These did not differ significantly from each other. or from baseline scores. Figure 3 again shows that abecarnii or diazepam effectively reduced handling-induced convulsion scores in naive mice. No treatments employed showed signs of provoking an acute withdrawal response. since no groups significantly excccdcd baseline scores during the S h test. Analysrb indicated a significant effect of treatment group (F(4. 55) = 35. P < O.Oi ) and time (RX 440) =4-S. P < O.oUOi), as wcli as a significant
4. Discussion The finding withdrawal
that diazepam
handling-induced
could suppress ethanol convulsions
in mice was
;lot surprising. Goldstein (1972) reported such antagonism in genetically heterogeneous mice, but she used a lonpcr ncriod of dure
that
c!had
included
r;xyosurc (72 h), and a proccadjunct
treatment
with
pyrazole
hydrochloride. an alcohol dehydrogenase inhibitor. Howcvcr. she found a SO mg/kg dose of diazepam (but not 5 or 20 mg/kg) that 20-100 mg/kg
to be significantly effective, and chloridazepoxide was also effec-
tive. In the current studies, a 4 mg/kg dose of diazepam inhihited withdrawal. Greater sensitivity to diazepam was seen in the current experiments:
this could
bc due to our USC of WSP mice. rather than the generic Swiss-Webster used by Qr. Goldstein, or to the greater degree of dependence instilled by use of 72 h inhalation coupled tion.
with
pyrazole-induced
metabolic
inhibi-
Abecarnil clearly inhibited handling-induced convulsions seen after withdrawal from 24 h chronic ethanol intoxication. it appeared to have been more potent than diazepam. although the difference was not marked. it seems likely that its cffcct was of somewhat shorter duration. Both drugs also an!agnnized wi!hdrawal in the single-injection acute withdrawal model, at cvcn lower doses (0.5 mg/kg). In the abscncc of information
about brain or plasma levels for the two
drugs. firm conclusions regarding relative potency cannot bc drawn from thcsc data. ::nd await further studies. Abecarnil
is a potent anticonvulsant
in several ani-
mal models of cxpcrimcntal seizures (Turski et al., IYYO; Fischer et al., 1991). We have previously re-
4
;
4
:
Ii
7
I
ported enhancement of handling-induced convulsions in naive WSP mice with the bcnzodiazcpinc inverse
8
nME(HOLJRS) Fig. 3. Mran + S.E. handling-inducedccmvulsion WSP (open Open
agonist, Ro IS-45 1.3, at doses from 0.1 to 10.0 mg/kg SCW~S in gmqx
of
mice
(each N = I?). Injections (indic:!rcd hy arrow) of vchiclc rquart?i) llr cimr% +.Tfabcrarnil or di;lzcplm wcrc given i.n. circles.
arepam.
I
mg/kg
dia7epam.
Filled lrianplrs.
I mg/kg
Open
triangles,
ahecarnil.Filled
ahecarnil.
2 mg/kg
Ji-
circles. 2 mg/kg
(Crabbe et al., 199lb). In addition, many other drugs including psychostimulants, direct and indirect GABA antagonists. the calcium channel agonist BAY K 8644, corticosteronc, and excitatory amino acid receptor agonists, clcvatc
handling-induced
convulsions
in WSP
XY
mice (Crabbe et a!., 1991b; Roberts et a!., 1991; Crabbe and Kosobud, 1990; Phillips and Crabbe, 1991). Abecarni! clearly antagonized handling-induced convulsions, both in ethanol-withdrawing and naive mice, thus resembling drugs with central nervous system depressant effects (Crabbe et a!., 199la; Kosobud and Crabbe, 19861, and several other anticonvulsants (Crabbe and Kosobud, 1990; Phillips and Crabbe, 1991). In Experiment 4, we saw no signs that withdrawal from a single dose of abecarni! or diazepam led to a rebound exacerbation of handling-induced convulsions. This agrees with unpublished data with diazepam in our laboratory. WSP mice are extremely sensitive to such acute withdrawal effects, which are easily seen after ethanol (Crabbe et a!., 1991a; Kosobud and Crabbe, 1986), t-butano!, pentobarbita!, and acetaldehydc (Kosobud and Crabbe, 1986). Earlier studies had reported that cessation of thrice daily administration of 5 mg/kg abecarni! to rats led to no marked signs of spontaneous withdrawal during several days after trcatment Uscher et a!., 1991). and 6 weeks of daily administration of 4 mg/kg to dogs produced no spontaneous withdrawal reaction even when f!umazeni!-prccipitated Escher et a!., 1990). Thus, existing data suggest that the risk of physical dependence on this compound is relatively low. Comparison of the results seen in Experiments l-3 with published literature suggests that the effects of abecarnil against alcohol withdrawal may be dissociable from its genera! anticonvulsant effects. while the same would apparently not be true of diazepam. The cffectivc dose for producing sedative effects in mice clearly depends upon the specific test employed, and probably on the strain of mice. Nonetheless, when the hanging wire and rotarod tests of ataxia were employed in NMRI strain mice (Stephens et a!., 19901, the ED+ for abecarni! were 38 and > 100 mg/kg, respectively; for diazepam, the comparable values were 0.99 and 14 mg/kg, respectively. Our studies showed efficacy of both of these compounds against alcohol withdrawal at doses no higher than 4 mg/kg. In conclusion, abecarni! or diazepam, given to genetically WSP mice at the peak zf chronic ethanol withdrawal, significantly reduced handling-induced convuision scores. While abecarni! was somewhat more potent than diazepam, its effects were shorter-lasting. Similar results were seen in an cxpcrimcnt where withdrawal was assessed after a single high-dose ethanol injection. Abecarni! and diazepam also inhibited the lower handling-induced convulsion scores seen in naive mice, indicating that this convulsive sign in mice is sensitive to anticonvulsants. Single administrations of diazcpam or a!?ccarni! did not lcad to spontaneous withdrawal-induced increases of handling-inducetl CcJI1vu!sion severity. Doses of abecarni! effective in reducing withdrawal handling-induced convulsion scores
were lower than published values for tests of ataxia, suggesting that it might be possible to dissociate the antiwithdrawal and ataxic effects of abecarni!.
Acknowledgments These studies were supported ment of Veterans AAOXb2I.
Affairs.
DAtii22X).
I thank Catherine
by a grant from the US Depart-
by grants from
and USPHS-NIDA
Merrill
the USP!IS
Contract
(AAO624.3.
No. 271-90-7405.
for her expert help with these experiments.
and Drs. David N. Stephens and Graham Jones (Schering-Berlin)
for
their comments on the manuscript.
References Belknap.
J.K..
S.E.
Laursen
and J.C. Crabbe.
Belknap.
J “... P.W.
Ethanol
Danielson.
and barbiturate
codetermined
Belknap.J.K.,
and
and J.C. Crabbe.
19x8.
convulsions are extensively
in mice. Alcohol 5. 167. J-C. Crabbe
diazepam withdrawal WSP and WSR Crabbe.
M. Lam6
withdrawal
19x7, Ethanol
convulsions by similar
nitrous oxide produce withdrawal-induced mechanisms in mice. Life Sci. 41. 2033.
and S.E.
Laursen,
1989, Ethanol
and
convulsions are extensively codete:mined
in
mice. Life Sci. 44, 2075.
J.C. and A.
Kosobud,
1990. Alcohol
withdrawal
seizures:
Genetic animal models. in: Alcohol and Seizures. eds. R.J. Porter. R F!. Mattcon, Philadelphia)
.I.A.
Cramer
and
1. Diamond
(FA
Davis Co.,
p. 126.
Crabbe. J.C.. J. Janowsky, E.R. Young, A. Kosobud. J. Stack and H. Rigter.
19X2. Tolerance
Genotypic
to ethanol
correlations
with
hypothermia
behavioral
in inbred mice:
responses.
Alcoholism:
Clin. Exp. Res. 6, 44f1. Crahbe. J.C.. E.R.
Young
tions with ethanol hav. IX (SuppI. Crahbe.
J.C..
and A. Kosobud,
withdrawal
Kosohud.
E.R.
Polygenic and single-gene
J.C..
A.
withdrawal J.C..
tolerance
Kosobud.
inbred
D.J.
E.R.
Feller
Young.
C.D.
hypothermia
and J.K. Bclknap.
drugs is determined
Neurohehav.
Tam
and J.D.
D.B.,
1989,
!5. 521.
Sensitivity
and
in gcnctically selected IO9la.
Acute dcpcn-
hy common genes in
and J.K.
Belknap,
IYYlh,
Effecis
of
convulsions in mice sclccted for
severity, Brain Rcs. 550. I.
1972, An animal model for testing effects of drugs
on alcohol withdrawal
reactions, J. Pharmacol.
Exp. Ther.
D.B. and N. Pal. 1971. Alcohol depcndcnce
mice by inhalation
of ethanol: Grading
jcience 172. 2:X Kosobud, A. and J.C. Cr,ibbe. to be ~rnetically withdrawal
Mc-
Exp. Ther. 257. 663.
Merrill
ethanol withdrawal
14. Goldstein.
B.R.
J.S. Dorow,
convulsants on handling-induced Goldsrem,
strains,
Exp. Ther. 249. 456.
mice, J. Pharmacol. J.C..
and
Merrill
dcncc on depressant Crahbe.
mouse
selection for susceptibility to elbano
to ethanol-induced
J.C., C.D.
19S3h.
of response to ethanol
seizures in M1r.srnrrscrdus, Behav. Genet.
mice, J. Pharmacol. Crabhc.
and J. Janowsky.
5, 1X1.
Swigan. 1985. Bidirectional Crahhe.
Young
determination
recombi!lant
Toxicol. Teratol. Crahhe.
correla-
Biochem. Be-
I). 541.
A.
in BXD/Ty
I9X3a. Genetic
severity. Pharmacol.
rcizures,
or resistant
J. Pharmacol.
L&her. W.D.. D. Hiinack. l99[), Pharmacokinclics. fects of the /$carboline
produced in
the withdrawal
1986. Ethanol withdrawal
prone t WSP)
(WSR)
reaction.
in mice hrcd to ethanol
Exp. Ther. 238. 170.
R. Scherkl. A. Hashcm and ti:H. anticonvulsant efficacy and ;IdWSc abccarnil,
1X.3.
a novel lig~d
Frey. cf-
for brnzodi-
azepine receptors. ::tter acute and chronic administration in dogs. J. Phamrrac~& Exp. Ther. 255. 541. Liischer. W.. C. Rundfeldt and D. Hiinack. 1991. Tolerance to anticonvulsant effects of the partial henzodiazepine receptor agonist ahzarnil in kindled T.IISinvolves learning. Eur. J. Pharmacol. 2tc 303. M&uarrie. D.G. and E. Fingl. 19%. Effects of single doses and chronic administration of ethanol on experimental seizures in mice. J. Pharnmcol. Exp. Ther. 124, 264. Phillips. T.J. and J.C. Cmhhe. IWI. Behavioral studies of genetic differences in al~xrhol action. in: The Genetic Basis of Alcohol and Drug Actions. eds. J.C. Crahhe and R.A. ffarris (Plenum Publishing Corp.. New York) p. 25. R&ens. A.J.. H.-P Chu. J.C. Crathbe and L.D. Keith. 1991. Differ-
ential modulation by the stress axis of ethanol withdrawal seizure expression in WSP and WSR mice, Alcoholism: Clin. Exp. Res. IS. 412. Stephens, D.N., H.H. Schneider, W. Kehr, J.S. Andrews, K.-J. Rettig. L. Turski. R. Schmiechen, J.D. Turner, L.H. Jensen, E.N. Petersen, T. Honore and J. Bondo Hansen, 1990. Ahecarnil. a stable. anxioselective fl-carboline acting at benzodiazepine receptors. J. Pharmacol. Exp. Ther. 253, 334. Turski, L.. D.N. Stephens. L.H. Jensen, E.N. Petersen, B.S. Meldrum. S. Patel, J. Bondo Hansen. W. Liischer, H.H. Schneider and R. Schmiechen, 1990, Anticonvulsant action of the P-carboline ahecarnil: Studies in rodents and baboon, f~pio papio, J. Fharmacol. Exp. Ther. 253, 344.