Clinical Toxicology
ISSN: 0009-9309 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ictx18
Barbiturate Physical Dependence in Mice: Effects of Neuroleptics and Diazepam on the Withdrawal Syndrome J. K. Belknap To cite this article: J. K. Belknap (1978) Barbiturate Physical Dependence in Mice: Effects of Neuroleptics and Diazepam on the Withdrawal Syndrome, Clinical Toxicology, 12:4, 427-434, DOI: 10.3109/15563657809150013 To link to this article: http://dx.doi.org/10.3109/15563657809150013
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CLINICAL TOXICOLOGY 12(4), pp. 427-434 (1978)
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Barbiturate Physical Dependence in Mice: Effects of Neuroleptics and Diazepam on the Withdrawal Syndrome
J. K. BELKXAP
Department of Psychology University of Texas a t Austin Austin, Texas 78712
The pharmacologic treatment of alcohol withdrawal in alcoholic patients has involved primarily sedative-hypnotic agents (especially benzodiazepines) and to a l e s s e r extent neuroleptic agents [l, 21. The most common treatment of barbiturate ( o r the newer sedative-hypnotic) withdrawal syndrome has been the us e of barbiturates, usually pentobarbital, in gradually decreasing doses [3]. In principle, the s a m e drugs that are effective in alcohol withdrawal should be effective in barbiturate withdrawal, s i nce the syndromes a r e virtually identical and cross-dependence exists between alcohol and the barbiturates [3]. Neuroleptics a r e often used because they do not fost er continuing drug dependence, i. e. , they produce no euphoria o r cross-dependence, nor do they produce ataxia o r boisterousness. They are sometimes useful in treating agitation, delirium, hallucinosis, and e m e s i s [ 11. Unfortunately, some neuroleptics may produce unwanted hypotension, lower the convulsive threshold, and (in the ca se of promazine) inc r e a s e mortality. These untoward effects a r e almost always associ ated with aliphatic o r piperidine phenothiazines, suggesting that the piperazines o r butyrophenones, which produce relatively little hypotension o r lowered s ei zur e threshold, may be much safer in these respects [2, 41. 427 Copyright 0 1978 by Marcel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical. including photocopying, microfilming, and recording, or by any information storage and retrieval system. without permission in writing from the publisher
428
BELKNAP
The purpose of the present report is to compare the effects of three neuroleptics, chlorpromazine (an aliphatic phenothiazine), haloperidol ( a butyrophenone), and reserpine with a benzodiazepine (diazepam) on the barbiturate (phenobarbital) withdrawal syndrome in mice. An inhibitor of catecholamine biosynthesis (alpha-methyl-ptyrosine) was also tested.
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METHOD Physical dependence on phenobarbital ( P B ) was produced by the method of Belknap et al. [5, 61. At 15 to 20 weeks of age, 77 male C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.) were housed singly and introduced to a milled diet (Puri na Lab Chow) dispensed from a glass feeder jar. After 2 days, phenobarbital (Sigma Chemical Co. ) was thoroughly mixed with the milled diet (3.2 gm P B p er kg d iet) and administered ad lib f or 7 days. Following this, the unadulterated diet was substituted f or the PB diet a t the time of light onset (12L: 12D light cycle) and the animals monitored for withdrawal symptoms at 4-hr intervals beginning a t 14 h r aft er withdrawal and ending a t 38 h r a f t er withdrawal. This interval encompassed almost a l l of the withdrawal symptom incidence and all of the sei zure incidence. A 1%body weight loss occurred during the intoxication phase; an additional 9% loss occurred 24 h r af t er withdrawal. Mean consumption of phenobarbital was 382 mg/kg/day (Experiment 1) and 361 mg/ kg/day (Experiment 2). The animals were divided into eight groups (Experiment 1) with each group receiving either saline, haloperidol (Haldol injection, McNeil Laboratories), chlorpromazine (Thorazine injection, Smith, Kline, and French Laboratories), reserpine (Sigma Chemical Co. ), a-methyl-p-tyrosine (AMT, Sigma Chemical Co. ), o r diazepam (Valium, kindly provided by Hoffman-LaRoche, Inc.). The groups and their respective dosages are shown in Table 1. The saline, haloperidol, and chlorpromazine injections were given i.p. at 9 h r aft er withdrawal and again a t 2 1 h r af t er withdrawal. AMT and diazepam were given only once (i.p.) at 9 h r after withdrawal, followed by saline at 21 hr. Reserpine was given once at 0 hr. The injection volume was 5 ml/ k g in a saline vehicle. A l l doses are in t er ms of the free drug (not the salt). The exact s a m e procedure was followed in Experiment 2 with an additional 45 animals, except that s i x observations were made ( r a t h e r than seven) since the withdrawal symptoms ceased somewhat e a r l i e r than in Experiment 1. The doses and groups employed are shown in Table 2. Withdrawal severity was as s es s ed by suspending each animal by the tail f o r 10 s e c , returning it to i t s home cage, and recording the
BARBITURATE DEPENDENCE IN MICE
42 9
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TABLE 1. Relative Frequencies of the Withdrawal Symptoms Expressed a s the Per cent of the Total Number of Observations in Experiment la TR
CH
ST
SZ
N
Nobs
ws
28
10
17
12
20
140
4.4
i 2.5
22
11
18
10
9
63
4.2
i 2.1
31
2Eib
23
11
8
56
5.8
i 3.1
52b
11
16
13
8
56
5.7
* 2.0
5gb
3gb
20
21b
8
56
8.8
i 3.6'
44
20
52b
3gb
8
48
9. 5 * 3. 7b
7 gb
11
14
23'
8
56
7.7
i 2.6'
gb
oc
8
56
1.0
f
oc
2c
b 1.1
aThere were a total of seven observations made p e r mouse a t 4-hr intervals. The number of mice p e r group ( N ) , the total number of observations (Nabs), and the withdrawal s c o r e s (WS) a r e al so given (mean i S. D. ). bp < .01, two-tailed. Cp < .05, two-tailed. presence o r absence of four symptom categories during the next one minute. These symptom categories were convulsions on handling (CH) -clonic s p a s ms and tightening of the muscles of the face, usually extending to the r e s t of the body; t r e m o r (TR)-marked tremulousness of the en tir e body; straub t ai l (ST)-arching of the tail over the back, a sign of muscular rigidity; s ei zur es (SZ)-tonic-clonic (nonlethal) o r tonic (lethal) s ei zur es (LSZ). A l l scoring was done on a blind basis. Convulsions on handling was scored while the animal was suspended by the tail; the other t hr ee symptom categories were scored while the animal was in its home cage. Convulsions on handling ( a mild tonicclonic s e i z u r e ) appeared to be virtually identical to the convulsions on handling described by Goldstein [7, 81 with respect to alcohol with-
430
BELKNAP
TABLE 2. Relative Frequencies of the Withdrawal Symptoms Expressed as the Percent of the Total Number of Observations in Experiment 2a CH
ST
SZ
N
Nobs
ws
13
9
11
7
9
54
2.4
* 1.4
Haloperidol ( 1 m g / k g x 2)
8
11
14
8
6
36
2.4
* 2.0
Haloperidol ( 5 m g / k g x 2)
19
21
18
6
12
72
3.6
* 1.9
Chlorpromazine ( 1 mg/kgX 2)
19
14
8
6
6
36
2.8
i 1.7
C hlorpromazine ( 5 m g / k g x 2)
31b
17
14
18b
12
72
5.0
* 2.6 b
Saline
Downloaded by [University of California, San Diego] at 02:28 06 November 2015
TR
a
T h e r e were a total of s i x observations made p e r mouse at 4-hr intervals. The number of mice p e r group (N), the total number of observations (Nabs), and the withdrawal s c o r e s (WS) a r e a l s o given (mean i S. D. ). bp < .05, two-tailed. drawal in mice, and h e r terminology is adopted h e r e f o r this reason. These symptom categories w e r e chosen because they can be quickly and reliably s c o r e d and are distinct from any behavior seen in m i c e not exposed to phenobarbital. In addition to recording the occurrence of each symptom, a withdrawal s c o r e (four-point rating scale) was a l s o assigned as follows: 0, no appreciable symptoms compared to nondrugged animals; 1, the presence of one o r m o r e of the following: CH, TR, ST; 2, SZ; and 3, LSZ. The assigned withdrawal score (WS) was based only on the most s e v e r e symptom observed. Thus, an animal exhibiting an SZ was given a s c o r e of 2 r e g a r d l e s s of the p r e s e n c e of CH, TR, o r ST. This was done s i n c e a n SZ o r LSZ occurring e a r l y in the observation period greatly diminished the opportunity f o r the m i l d e r symptoms to manifest themselves. The withdrawal s c o r e s w e r e summed f o r each mouse a c r o s s all seven ( o r s i x ) observations, and this s u m was used in subsequent analyses. The peak in withdrawal s e v e r i t y f o r C57 m a l e s o c c u r s a t 18 to 24 h r a f t e r withdrawal [5]. Statistical analyses w e r e performed using two-tailed chi s q u a r e t e s t s based on the r a w frequencies f o r each symptom category. Withdrawal s c o r e s w e r e subjected to two-tailed Mann-Whitney U t e s t s based on ranked s c o r e s f o r individual mice.
BARBITURATE DEPENDENCE IN MICE
43 1
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RESULTS The r e s u l t s f o r Experiment 1 are shown in Table 1. Only diazepam (8 mg/kg) produced a significant reduction in frequency f o r any of the symptom categories, and this reduction was significant in all four symptom categories (chi square). This dose produced v e r y little ataxia o r o t h e r signs of g r o s s intoxication in withdrawn animals. None of the remaining groups exhibited a significant reduction f o r any of the symptom categories (relative to controls). Only the low dose of haloperidol ( 2 mg/kg X 2) did not worsen withdrawal severity; the o t h e r groups exhibited a t l e a s t one symptom that was significantly exacerbated by the drug treatments ( p < .05, chi square). S e i z u r e s (90% of which were tonic-clonic) w e r e significantly worsened by the 8 mg/kg X 2 dose of chlorpromazine and by r e s e r p i n e and AMT, but no effect was seen f o r e i t h e r dose of haloperidol (chi square). The drug effects on g r o s s t r e m o r w e r e v e r y s i m i l a r to that s e e n with s e i z u r e s . Convulsions on handling w e r e significantly worsened by the high doses of haloperidol and chlorpromazine but not by any of the other treatments. Straub tail was only worsened by r e s e r pine. T h r e e lethal s e i z u r e s w e r e observed; all of t h e s e o c c u r r e d in the r e s e r p i n e group. In Experiment 1, withdrawal severity based on withdrawal s c o r e s (WS) was not significantly affected by e i t h e r dose of haloperidol; however, the high dose ( 8 mg/kg X 2) of chlorpromazine did elevate withdrawal s c o r e s vs the controls ( p < .05, U test) as did r e s e r p i n e ( p < .01) and AMT ( p < .05). Only diazepam significantly reduced withdrawal s c o r e s compared with the controls ( p < .01, U test). The results f o r Experiment 2 are shown in Table 2. Neither dose of haloperidol significantly increased the incidence of any symptom category; however, the 5 mg/kg dose of chlorpromazine significantly elevated the incidence of t r e m o r and s e i z u r e s ( p-s < .05, chi square). No lethal s e i z u r e s were observed. In Experiment 2, withdrawal severity based on withdrawal s c o r e s was not significantly affected by e i t h e r dose of haloperidol; however, the 5 mg/kg dose of chlorpromazine did i n c r e a s e withdrawal s c o r e s ( p < .05, U t e s t ) relative to the controls. DISCUSSION Diazepam presumably owes its therapeutic effectiveness in P B withdrawal to cross-dependence with phenobarbital and o t h e r sedativehypnotic agents. This finding is consistent with studies employing benzodiazepines with withdrawn alcoholics [ 1, 21, withdrawn ethanoldependent m i c e [8, 91, and withdrawn barbital-dependent r a t s [ l o ] . The neuroleptic drug treatments (and AMT) e i t h e r had no effect
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432
BELKNAP
o r they worsened the P B withdrawal syndrome. All of these drugs, in doses s i m i l a r to those used here, a r e known to reduce catecholamine function [ll-131, increase audiogenic sei zure susceptibility in mice [13, 141 and r a t s [15], and exacerbate convulsions on handling in ethanol-dependent mice [8, 9, 161. Intracerebral injections of norepinephrine o r dopamine significantly reduced ethanol withdrawal in mice; norepinephrine was 10 to 20 times more potent than dopamine [17]. Changes in norepinephrine levels had a great er effect on audiogenic s eiz u r e s than did changes in dopamine levels [ 151. These results are consistent with the hypothesis [13, 141 that decreases in catecholamine function lead to increases in seizure susceptibility, with norepinephrine perhaps playing a m or e important role than dopamine. It was a t f ir s t s ur pr i s i ng to us that haloperidol and chlorpromazine were approximately equally potent in their overall effects on barbiturate withdrawal. Haloperidol is over 40 times m ore potent than chlorpromazine in the suppression of schizophrenic symptoms [18], in the inhibition of dopamine release in rat corpus striatum [19], and in i t s antidopaminergic action [ll, 121. In contrast, haloperidol is much l e s s potent than chlorpromazine in i t s antimuscarinic effects [20], and when used clinically, haloperidol produces much less a-adrenergic blocking effect than chlorpromazine [21]. Overall, these findings suggest that the antidopaminergic effects of these two drugs were apparently not the p r imar y cause of the exacerbation of withdrawal symptoms, since the expected g r e at er potency of haloperidol did not materialize. Of course, the antidopaminergic effects may be the important mechanism of action in reducing hallucinosis, agitation, delirium, and e m e s i s in withdrawn alcoholics, but none of these possibilities are readily testable with animal models. Overall, diazepam would appear to be the drug treatment of choice in barbiturate withdrawal in comparison with the neuroleptics. A l l of the monitored symptoms of withdrawal ( s ei zures, t rem or, st raub tail, and convulsions on handling) responded promptly and effectively to diazepam administration. SUMMARY Diazepam ( 8 mg/kg) quickly and effectively reduced the incidence of withdrawal symptoms in phenobarbital dependent C 57BL/6J male mice. In contrast, the neuroleptic agents employed (chlorpromazine, haloperidol, o r reserpine) o r a-methyl-p-tyrosine tended to exacerbate the withdrawal syndrome. Chlorpromazine and haloperidol were approximately equally potent in their effects; this would suggest that their antidopaminergic effects w er e not the pri m ary mechanism of action f o r the increased incidence of withdrawal symptoms.
BARBITURATE DEPENDENCE IN MICE
433
ACKNOWLEDGMENT T h i s work was supported by NIDA G r a n t DA-01215 and DA-01800 awarded to J. K. Belknap, Dept. of Psychology.
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REFERENCES J. 0. Cole and C. Ryback, "Pharmacological T r e a t m e n t , " in, Alcoholism (R. E. T a r t e r and A. A. Sugerman, eds.), AddisonWesley, Reading, Mass., 1976. E. M. S e l l e r s and H. Kalant, Alcohol intoxication and withdrawal, New Engl. J. Med., 294, 757 (1976). J. H. Jaffe, "Drug A a c t i o n and D r u g Abuse," in The P h a r m a cological B a s i s of T h e r a p e u t i c s , (L. Goodman and A. Gilman, eds.), Macmillan, New York, 1975. M. L. Palestine, Drug t r e a t m e n t of the alcohol withdrawal synd r o m e and delirium t r e m e n s , Quart. J. Stud. Alc., 34, 185 (1973). J. K. Belknap, S. Waddingham, and G. Ondrusek, B a r b i t u r a t e dependence in m i c e induced by a s i m p l e s h o r t - t e r m o r a l p r o c e d u r e , Physiol. Psych., 1, 394 (1973). J. K. Belknap, G. Ondrusek, J. Berg, and S. Waddingham, B a r biturate dependence in mice: Effects of continuous vs. discontinuous d r u g administration, Psychopharmacology, 51, 195 (1977). D. B. Goldstein, Convulsions elicited by handling: A s e n s i t i v e method of m e a s u r i n g CNS excitation in mice t r e a t e d with r e s e r pine o r convulsant d r u g s , Psychopharmacologia, 32, 27 (1973). D. B. Goldstein, Physical dependence on alcohol i n m i c e , F e d e r ation Proc., 34, 1953 (1975). K. Blum, J. D. Eubanks, J. E. Wallace, and H. Hamilton, Enhancement of alcohol withdrawal convulsions i n m i c e by haloperidol, Clin. Toxicol., 9, 427 (1976). P. R. E. Norton, The e f g c t s of d r u g s on b a r b i t u r a t e withdrawal convulsions in the r a t , J. P h a r m . P h a r m a c o l . , 22, 763 (1970). I. C r e e s e , D. R. Burt, and S. H. Snyder, DopamKe r e c e p t o r binding predicts clinical and pharmacological potencies of antischizophrenic drugs, ~ Science, 192, _ 481 (1976). _ S. H. Snyder, The dopamine hypothesis of schizophrenia: Focus on the dopamine r e c e p t o r , Am. J. Psychiat., 133, 197 (1976). K. Schlesinger and S. S h a r p l e s s , Audiogenic s e i z u r e s and acoustic priming, in Psychopharmacogenetics (B. Eleftheriou, ed. ), Plenum, New York, 1975. A. Lehman, Audiogenic s e i z u r e data in m i c e supporting new
434
[15] [16]
BELKNAP theories of biogenic amine mechanisms in the central nervous system, Life Sci., 2,1423 (1967). P. Jobe, A. Picchioni, and L. Chin, Role of brain norepinephrine in audiogenic s ei zur es in the rat, J. Pharmacol. Exptl. Therap., 184, 1 (1973). T J . Griffiths, J. M. Littleton, and A. Ortiz, Changes in monoamine concentrations in mouse brain associated with ethanol dependence and withdrawal, Brit. J. Pharmacol., 489 (1974). H. 0.J. Collier, M. D. Hammond, and C. Schneider, Effects of drugs affecting endogenous amines o r cyclic nucleotides on ethanol withdrawal head twitches in mice, Brit. J. Pharmacol., 58, 9 (1976). T M . Davis, Comparative doses and costs of antipsychotic medication, Arch. Gen. Psychiat., 33, 858 (1976). P. Seeman and T. Lee. AntiusvFhotic drugs: Direct correlation between clinical potency and presynaptic actions on dopamine neurons, _ Science, _ _ -188, 1217 (1975). S. H. Snyder, D. Greenberg, and H. I. Yamamura, Antischizophrenic drugs and brain cholinergic receptors, Arch. Gen. Psychiat., 31, 58 ( 1974). R. Byck, "Drugs in the Treatment of Psychiatric Disorders," in The Pharmacological Basis of Therapeutics (L. Goodman and A. Gilman, eds.), Macmillan, New York, 1975).
so,
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[17]
-f l8 -1 1191 L
J
[20] [21]
ISSN: 0009-9309 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ictx18
Barbiturate Physical Dependence in Mice: Effects of Neuroleptics and Diazepam on the Withdrawal Syndrome J. K. Belknap To cite this article: J. K. Belknap (1978) Barbiturate Physical Dependence in Mice: Effects of Neuroleptics and Diazepam on the Withdrawal Syndrome, Clinical Toxicology, 12:4, 427-434, DOI: 10.3109/15563657809150013 To link to this article: http://dx.doi.org/10.3109/15563657809150013
Published online: 25 Sep 2008.
Submit your article to this journal
Article views: 6
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ictx18 Download by: [University of California, San Diego]
Date: 06 November 2015, At: 02:28
CLINICAL TOXICOLOGY 12(4), pp. 427-434 (1978)
Downloaded by [University of California, San Diego] at 02:28 06 November 2015
Barbiturate Physical Dependence in Mice: Effects of Neuroleptics and Diazepam on the Withdrawal Syndrome
J. K. BELKXAP
Department of Psychology University of Texas a t Austin Austin, Texas 78712
The pharmacologic treatment of alcohol withdrawal in alcoholic patients has involved primarily sedative-hypnotic agents (especially benzodiazepines) and to a l e s s e r extent neuroleptic agents [l, 21. The most common treatment of barbiturate ( o r the newer sedative-hypnotic) withdrawal syndrome has been the us e of barbiturates, usually pentobarbital, in gradually decreasing doses [3]. In principle, the s a m e drugs that are effective in alcohol withdrawal should be effective in barbiturate withdrawal, s i nce the syndromes a r e virtually identical and cross-dependence exists between alcohol and the barbiturates [3]. Neuroleptics a r e often used because they do not fost er continuing drug dependence, i. e. , they produce no euphoria o r cross-dependence, nor do they produce ataxia o r boisterousness. They are sometimes useful in treating agitation, delirium, hallucinosis, and e m e s i s [ 11. Unfortunately, some neuroleptics may produce unwanted hypotension, lower the convulsive threshold, and (in the ca se of promazine) inc r e a s e mortality. These untoward effects a r e almost always associ ated with aliphatic o r piperidine phenothiazines, suggesting that the piperazines o r butyrophenones, which produce relatively little hypotension o r lowered s ei zur e threshold, may be much safer in these respects [2, 41. 427 Copyright 0 1978 by Marcel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical. including photocopying, microfilming, and recording, or by any information storage and retrieval system. without permission in writing from the publisher
428
BELKNAP
The purpose of the present report is to compare the effects of three neuroleptics, chlorpromazine (an aliphatic phenothiazine), haloperidol ( a butyrophenone), and reserpine with a benzodiazepine (diazepam) on the barbiturate (phenobarbital) withdrawal syndrome in mice. An inhibitor of catecholamine biosynthesis (alpha-methyl-ptyrosine) was also tested.
Downloaded by [University of California, San Diego] at 02:28 06 November 2015
METHOD Physical dependence on phenobarbital ( P B ) was produced by the method of Belknap et al. [5, 61. At 15 to 20 weeks of age, 77 male C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.) were housed singly and introduced to a milled diet (Puri na Lab Chow) dispensed from a glass feeder jar. After 2 days, phenobarbital (Sigma Chemical Co. ) was thoroughly mixed with the milled diet (3.2 gm P B p er kg d iet) and administered ad lib f or 7 days. Following this, the unadulterated diet was substituted f or the PB diet a t the time of light onset (12L: 12D light cycle) and the animals monitored for withdrawal symptoms at 4-hr intervals beginning a t 14 h r aft er withdrawal and ending a t 38 h r a f t er withdrawal. This interval encompassed almost a l l of the withdrawal symptom incidence and all of the sei zure incidence. A 1%body weight loss occurred during the intoxication phase; an additional 9% loss occurred 24 h r af t er withdrawal. Mean consumption of phenobarbital was 382 mg/kg/day (Experiment 1) and 361 mg/ kg/day (Experiment 2). The animals were divided into eight groups (Experiment 1) with each group receiving either saline, haloperidol (Haldol injection, McNeil Laboratories), chlorpromazine (Thorazine injection, Smith, Kline, and French Laboratories), reserpine (Sigma Chemical Co. ), a-methyl-p-tyrosine (AMT, Sigma Chemical Co. ), o r diazepam (Valium, kindly provided by Hoffman-LaRoche, Inc.). The groups and their respective dosages are shown in Table 1. The saline, haloperidol, and chlorpromazine injections were given i.p. at 9 h r aft er withdrawal and again a t 2 1 h r af t er withdrawal. AMT and diazepam were given only once (i.p.) at 9 h r after withdrawal, followed by saline at 21 hr. Reserpine was given once at 0 hr. The injection volume was 5 ml/ k g in a saline vehicle. A l l doses are in t er ms of the free drug (not the salt). The exact s a m e procedure was followed in Experiment 2 with an additional 45 animals, except that s i x observations were made ( r a t h e r than seven) since the withdrawal symptoms ceased somewhat e a r l i e r than in Experiment 1. The doses and groups employed are shown in Table 2. Withdrawal severity was as s es s ed by suspending each animal by the tail f o r 10 s e c , returning it to i t s home cage, and recording the
BARBITURATE DEPENDENCE IN MICE
42 9
Downloaded by [University of California, San Diego] at 02:28 06 November 2015
TABLE 1. Relative Frequencies of the Withdrawal Symptoms Expressed a s the Per cent of the Total Number of Observations in Experiment la TR
CH
ST
SZ
N
Nobs
ws
28
10
17
12
20
140
4.4
i 2.5
22
11
18
10
9
63
4.2
i 2.1
31
2Eib
23
11
8
56
5.8
i 3.1
52b
11
16
13
8
56
5.7
* 2.0
5gb
3gb
20
21b
8
56
8.8
i 3.6'
44
20
52b
3gb
8
48
9. 5 * 3. 7b
7 gb
11
14
23'
8
56
7.7
i 2.6'
gb
oc
8
56
1.0
f
oc
2c
b 1.1
aThere were a total of seven observations made p e r mouse a t 4-hr intervals. The number of mice p e r group ( N ) , the total number of observations (Nabs), and the withdrawal s c o r e s (WS) a r e al so given (mean i S. D. ). bp < .01, two-tailed. Cp < .05, two-tailed. presence o r absence of four symptom categories during the next one minute. These symptom categories were convulsions on handling (CH) -clonic s p a s ms and tightening of the muscles of the face, usually extending to the r e s t of the body; t r e m o r (TR)-marked tremulousness of the en tir e body; straub t ai l (ST)-arching of the tail over the back, a sign of muscular rigidity; s ei zur es (SZ)-tonic-clonic (nonlethal) o r tonic (lethal) s ei zur es (LSZ). A l l scoring was done on a blind basis. Convulsions on handling was scored while the animal was suspended by the tail; the other t hr ee symptom categories were scored while the animal was in its home cage. Convulsions on handling ( a mild tonicclonic s e i z u r e ) appeared to be virtually identical to the convulsions on handling described by Goldstein [7, 81 with respect to alcohol with-
430
BELKNAP
TABLE 2. Relative Frequencies of the Withdrawal Symptoms Expressed as the Percent of the Total Number of Observations in Experiment 2a CH
ST
SZ
N
Nobs
ws
13
9
11
7
9
54
2.4
* 1.4
Haloperidol ( 1 m g / k g x 2)
8
11
14
8
6
36
2.4
* 2.0
Haloperidol ( 5 m g / k g x 2)
19
21
18
6
12
72
3.6
* 1.9
Chlorpromazine ( 1 mg/kgX 2)
19
14
8
6
6
36
2.8
i 1.7
C hlorpromazine ( 5 m g / k g x 2)
31b
17
14
18b
12
72
5.0
* 2.6 b
Saline
Downloaded by [University of California, San Diego] at 02:28 06 November 2015
TR
a
T h e r e were a total of s i x observations made p e r mouse at 4-hr intervals. The number of mice p e r group (N), the total number of observations (Nabs), and the withdrawal s c o r e s (WS) a r e a l s o given (mean i S. D. ). bp < .05, two-tailed. drawal in mice, and h e r terminology is adopted h e r e f o r this reason. These symptom categories w e r e chosen because they can be quickly and reliably s c o r e d and are distinct from any behavior seen in m i c e not exposed to phenobarbital. In addition to recording the occurrence of each symptom, a withdrawal s c o r e (four-point rating scale) was a l s o assigned as follows: 0, no appreciable symptoms compared to nondrugged animals; 1, the presence of one o r m o r e of the following: CH, TR, ST; 2, SZ; and 3, LSZ. The assigned withdrawal score (WS) was based only on the most s e v e r e symptom observed. Thus, an animal exhibiting an SZ was given a s c o r e of 2 r e g a r d l e s s of the p r e s e n c e of CH, TR, o r ST. This was done s i n c e a n SZ o r LSZ occurring e a r l y in the observation period greatly diminished the opportunity f o r the m i l d e r symptoms to manifest themselves. The withdrawal s c o r e s w e r e summed f o r each mouse a c r o s s all seven ( o r s i x ) observations, and this s u m was used in subsequent analyses. The peak in withdrawal s e v e r i t y f o r C57 m a l e s o c c u r s a t 18 to 24 h r a f t e r withdrawal [5]. Statistical analyses w e r e performed using two-tailed chi s q u a r e t e s t s based on the r a w frequencies f o r each symptom category. Withdrawal s c o r e s w e r e subjected to two-tailed Mann-Whitney U t e s t s based on ranked s c o r e s f o r individual mice.
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RESULTS The r e s u l t s f o r Experiment 1 are shown in Table 1. Only diazepam (8 mg/kg) produced a significant reduction in frequency f o r any of the symptom categories, and this reduction was significant in all four symptom categories (chi square). This dose produced v e r y little ataxia o r o t h e r signs of g r o s s intoxication in withdrawn animals. None of the remaining groups exhibited a significant reduction f o r any of the symptom categories (relative to controls). Only the low dose of haloperidol ( 2 mg/kg X 2) did not worsen withdrawal severity; the o t h e r groups exhibited a t l e a s t one symptom that was significantly exacerbated by the drug treatments ( p < .05, chi square). S e i z u r e s (90% of which were tonic-clonic) w e r e significantly worsened by the 8 mg/kg X 2 dose of chlorpromazine and by r e s e r p i n e and AMT, but no effect was seen f o r e i t h e r dose of haloperidol (chi square). The drug effects on g r o s s t r e m o r w e r e v e r y s i m i l a r to that s e e n with s e i z u r e s . Convulsions on handling w e r e significantly worsened by the high doses of haloperidol and chlorpromazine but not by any of the other treatments. Straub tail was only worsened by r e s e r pine. T h r e e lethal s e i z u r e s w e r e observed; all of t h e s e o c c u r r e d in the r e s e r p i n e group. In Experiment 1, withdrawal severity based on withdrawal s c o r e s (WS) was not significantly affected by e i t h e r dose of haloperidol; however, the high dose ( 8 mg/kg X 2) of chlorpromazine did elevate withdrawal s c o r e s vs the controls ( p < .05, U test) as did r e s e r p i n e ( p < .01) and AMT ( p < .05). Only diazepam significantly reduced withdrawal s c o r e s compared with the controls ( p < .01, U test). The results f o r Experiment 2 are shown in Table 2. Neither dose of haloperidol significantly increased the incidence of any symptom category; however, the 5 mg/kg dose of chlorpromazine significantly elevated the incidence of t r e m o r and s e i z u r e s ( p-s < .05, chi square). No lethal s e i z u r e s were observed. In Experiment 2, withdrawal severity based on withdrawal s c o r e s was not significantly affected by e i t h e r dose of haloperidol; however, the 5 mg/kg dose of chlorpromazine did i n c r e a s e withdrawal s c o r e s ( p < .05, U t e s t ) relative to the controls. DISCUSSION Diazepam presumably owes its therapeutic effectiveness in P B withdrawal to cross-dependence with phenobarbital and o t h e r sedativehypnotic agents. This finding is consistent with studies employing benzodiazepines with withdrawn alcoholics [ 1, 21, withdrawn ethanoldependent m i c e [8, 91, and withdrawn barbital-dependent r a t s [ l o ] . The neuroleptic drug treatments (and AMT) e i t h e r had no effect
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o r they worsened the P B withdrawal syndrome. All of these drugs, in doses s i m i l a r to those used here, a r e known to reduce catecholamine function [ll-131, increase audiogenic sei zure susceptibility in mice [13, 141 and r a t s [15], and exacerbate convulsions on handling in ethanol-dependent mice [8, 9, 161. Intracerebral injections of norepinephrine o r dopamine significantly reduced ethanol withdrawal in mice; norepinephrine was 10 to 20 times more potent than dopamine [17]. Changes in norepinephrine levels had a great er effect on audiogenic s eiz u r e s than did changes in dopamine levels [ 151. These results are consistent with the hypothesis [13, 141 that decreases in catecholamine function lead to increases in seizure susceptibility, with norepinephrine perhaps playing a m or e important role than dopamine. It was a t f ir s t s ur pr i s i ng to us that haloperidol and chlorpromazine were approximately equally potent in their overall effects on barbiturate withdrawal. Haloperidol is over 40 times m ore potent than chlorpromazine in the suppression of schizophrenic symptoms [18], in the inhibition of dopamine release in rat corpus striatum [19], and in i t s antidopaminergic action [ll, 121. In contrast, haloperidol is much l e s s potent than chlorpromazine in i t s antimuscarinic effects [20], and when used clinically, haloperidol produces much less a-adrenergic blocking effect than chlorpromazine [21]. Overall, these findings suggest that the antidopaminergic effects of these two drugs were apparently not the p r imar y cause of the exacerbation of withdrawal symptoms, since the expected g r e at er potency of haloperidol did not materialize. Of course, the antidopaminergic effects may be the important mechanism of action in reducing hallucinosis, agitation, delirium, and e m e s i s in withdrawn alcoholics, but none of these possibilities are readily testable with animal models. Overall, diazepam would appear to be the drug treatment of choice in barbiturate withdrawal in comparison with the neuroleptics. A l l of the monitored symptoms of withdrawal ( s ei zures, t rem or, st raub tail, and convulsions on handling) responded promptly and effectively to diazepam administration. SUMMARY Diazepam ( 8 mg/kg) quickly and effectively reduced the incidence of withdrawal symptoms in phenobarbital dependent C 57BL/6J male mice. In contrast, the neuroleptic agents employed (chlorpromazine, haloperidol, o r reserpine) o r a-methyl-p-tyrosine tended to exacerbate the withdrawal syndrome. Chlorpromazine and haloperidol were approximately equally potent in their effects; this would suggest that their antidopaminergic effects w er e not the pri m ary mechanism of action f o r the increased incidence of withdrawal symptoms.
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ACKNOWLEDGMENT T h i s work was supported by NIDA G r a n t DA-01215 and DA-01800 awarded to J. K. Belknap, Dept. of Psychology.
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REFERENCES J. 0. Cole and C. Ryback, "Pharmacological T r e a t m e n t , " in, Alcoholism (R. E. T a r t e r and A. A. Sugerman, eds.), AddisonWesley, Reading, Mass., 1976. E. M. S e l l e r s and H. Kalant, Alcohol intoxication and withdrawal, New Engl. J. Med., 294, 757 (1976). J. H. Jaffe, "Drug A a c t i o n and D r u g Abuse," in The P h a r m a cological B a s i s of T h e r a p e u t i c s , (L. Goodman and A. Gilman, eds.), Macmillan, New York, 1975. M. L. Palestine, Drug t r e a t m e n t of the alcohol withdrawal synd r o m e and delirium t r e m e n s , Quart. J. Stud. Alc., 34, 185 (1973). J. K. Belknap, S. Waddingham, and G. Ondrusek, B a r b i t u r a t e dependence in m i c e induced by a s i m p l e s h o r t - t e r m o r a l p r o c e d u r e , Physiol. Psych., 1, 394 (1973). J. K. Belknap, G. Ondrusek, J. Berg, and S. Waddingham, B a r biturate dependence in mice: Effects of continuous vs. discontinuous d r u g administration, Psychopharmacology, 51, 195 (1977). D. B. Goldstein, Convulsions elicited by handling: A s e n s i t i v e method of m e a s u r i n g CNS excitation in mice t r e a t e d with r e s e r pine o r convulsant d r u g s , Psychopharmacologia, 32, 27 (1973). D. B. Goldstein, Physical dependence on alcohol i n m i c e , F e d e r ation Proc., 34, 1953 (1975). K. Blum, J. D. Eubanks, J. E. Wallace, and H. Hamilton, Enhancement of alcohol withdrawal convulsions i n m i c e by haloperidol, Clin. Toxicol., 9, 427 (1976). P. R. E. Norton, The e f g c t s of d r u g s on b a r b i t u r a t e withdrawal convulsions in the r a t , J. P h a r m . P h a r m a c o l . , 22, 763 (1970). I. C r e e s e , D. R. Burt, and S. H. Snyder, DopamKe r e c e p t o r binding predicts clinical and pharmacological potencies of antischizophrenic drugs, ~ Science, 192, _ 481 (1976). _ S. H. Snyder, The dopamine hypothesis of schizophrenia: Focus on the dopamine r e c e p t o r , Am. J. Psychiat., 133, 197 (1976). K. Schlesinger and S. S h a r p l e s s , Audiogenic s e i z u r e s and acoustic priming, in Psychopharmacogenetics (B. Eleftheriou, ed. ), Plenum, New York, 1975. A. Lehman, Audiogenic s e i z u r e data in m i c e supporting new
434
[15] [16]
BELKNAP theories of biogenic amine mechanisms in the central nervous system, Life Sci., 2,1423 (1967). P. Jobe, A. Picchioni, and L. Chin, Role of brain norepinephrine in audiogenic s ei zur es in the rat, J. Pharmacol. Exptl. Therap., 184, 1 (1973). T J . Griffiths, J. M. Littleton, and A. Ortiz, Changes in monoamine concentrations in mouse brain associated with ethanol dependence and withdrawal, Brit. J. Pharmacol., 489 (1974). H. 0.J. Collier, M. D. Hammond, and C. Schneider, Effects of drugs affecting endogenous amines o r cyclic nucleotides on ethanol withdrawal head twitches in mice, Brit. J. Pharmacol., 58, 9 (1976). T M . Davis, Comparative doses and costs of antipsychotic medication, Arch. Gen. Psychiat., 33, 858 (1976). P. Seeman and T. Lee. AntiusvFhotic drugs: Direct correlation between clinical potency and presynaptic actions on dopamine neurons, _ Science, _ _ -188, 1217 (1975). S. H. Snyder, D. Greenberg, and H. I. Yamamura, Antischizophrenic drugs and brain cholinergic receptors, Arch. Gen. Psychiat., 31, 58 ( 1974). R. Byck, "Drugs in the Treatment of Psychiatric Disorders," in The Pharmacological Basis of Therapeutics (L. Goodman and A. Gilman, eds.), Macmillan, New York, 1975).
so,
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