Do benzodiazepine receptors mediate the anticonflict action of pentobarbital?

Life Sciences, Vol. 32, pp. 2241-2246 Printed in the U.S.A.

Pergamon Press

DO BENZODIAZEPINE RECEPTORS MEDIATE THE ANTICONFLICT ACTION OF PENTOBARBITAL? Wallace B. Mendelson a, Ted Davis a, Steven M. Paul h, and P h i l S k o l n i c k c

aAdult P s y c h i a t r y Branch and C l i n i c a l Psychobiology Branch, NIMH; c . b e l i n i c a l Neuroscience Branch, NINtf; and L a b o r a t o r y of B l o o r g a n i c Chemistry, NIADDK, N a t i o n a l I n s t i t u t e s of H e a i t h , Bethesda, Maryland 20205 U.S.A. (Received in final form February 10, 1983) Summary The effects of the benzodiazepine antagonist CGS 8216 (2-phenylpyrazolo[4,3-c]quinoline-3(SH)-one) were examined in a thirsty rat conflict test in the presence and absence of pentobarbital. CGS 8216 (2.5-10 mg/kg i.p.) did not affect nonpunished responding, but doses of 5 and I0 mg/kg significantly reduced the rate of punished responding (i.e., the number of 3 second drinking episodes in a "shock" contingency). However, a dose of CGS 8216 which did not significantly alter punished responding (2.5 mg/kg) antagonized the anticonflict actions of pentobarbital. These observations suggest that while high doses of CGS 8216 may elicit an "anxiogenic" response in rodents, lower doses of CGS 8216 antagonize the anticonflict actions of a compound which has been shown to enhance benzodiazepine affinity in vitro. These data imply that the anticonflict actions of pentobarbital may be mediated through benzodiazepine receptors. Although the anxiolytic actions of barbiturates such as pentobarbital have been well described in both experimental animals (i) and man (2), the mechanism(s) by which this drug exerts this action remains unclear. Recently, it has been reported that pentobarbital and related barbiturates can regulate the affinity of CNS benzodiazepine receptors in vitro. A stereospecific, anion dependent enhancement of [3H]benzodiazepine binding by pentobarhital has been described (3,4) at concentrations corresponding to those necessary to achieve anesthesia. A good correlation has been reported between the potencies of a series of barbiturates to enhance benzodiazepine receptor affinity in vitro and the anesthetic potencies of such barbiturates (3). At lower concentrations (i.e. those concentrations achieved in vivo following anxiolytic/sedative doses), ~entobarbital potentiates submaximum concentrations of GABA in facilitating [~H]benzodiazepine binding to these receptors (4,5). Although the pharmacological sequellae of these neurochemical actions are unknown, such observations suggest that some of the pharmacologic actions of pentobarbital could be via an interaction with benzodiazepine receptors. The discovery of compounds which bind to benzodiazepine receptors with high affinities and antagonize some of the pharmacologic actions of henzodiazepines (6) now permits an analysis of the involvment of benzodiazepine receptors in the anxiolytic actions of pentobarbital. Using a modification of the "thirsty rat" conflict procedure (i), we now report that the henzodiazepine antagonist CGS 8216 (7) reverses the increases in punished responding by rats pretreated with pentobarbital. 0024-3205/83/192241-06503.00/0

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This action of CGS 8216 was observed at a dose which did not by itself effect responding in either a punished or nonpunished situation. These data imply that the anticonflict actions of pentobarbital in rats may be mediated through benzodiazepine receptors. Materials

and Methods

A modification of the thirsty rat conflict test (1,8) was used in these studies. Male Sprague-Dawley rats (~200 g, Zivic-Miller Co., Allison Park, PA) were water deprived for 48 hours prior to testing. The rats were briefly placed in a i0 3/4" x 8" x 8 1/2" chamber (Lafayette Instrument Co., Lafayette, IN) and allowed to locate a drinking nipple (this procedure usually required less than one minute). The rats were removed from the chamber, injected (intraperitoneally) with drug or vehicle, and returned to the home cage prior to a three minute trial in the experimental chamber. During the trial, 0.55 mA shocks (I sec duration) were delivered through the ~eeding nipple after the animal accumulated three seconds of contact with the tube. Statistical significance was assessed using a one-way analysis of variance for independent groups, and a post-hoc comparison of any two groups was then performed using a Least Significance Difference test. COS 8216 was the gift of Dr. W. Cash, Ciba-Geigy Corp., Ardsley, N.Y. CGS 8216 was suspended in 10% DMSO and administered in a volume of 2 ml/kg. Pentobarbital sodium (V-PENTO; A.J. Buck and Son, Cockeysville, MD)RWaS injected intrappritoneally in a concentration of 65 mg/ml. Diazepam (Valium-; HoffmanLaRoche, Nutley, NJ) was injected in a concentration of 5 mg/ml. Control animals received equivalent volumes of the appropriate vehicle. Results Effects of diazepam in the thirsty rat conflict test: .... In order to validate this modified thirsty rat conflict test, three groups of ten water-deprived rats were injected with diazepam vehicle or diazepam (0.5-2 mg/kg) 15 min. prior to testing. For each animal, the three min. trials of vehicle and diazepam were given in randomized order at a one-week interval. Diazepam elicited a dose dependent increase in punished responding which was significantly greater than vehicle at doses of l and 2 mg/kg (Fig. i). E f f e c t s o f C6S 8216 on p u n i s h e d and n o n p u n i s h e d r e s p o n d i n g : In o r d e r to d e t e r m i n e i t COS 8216 had a n y e f f e c t s on d r i n k i n g b e h a v i o r , w a t e r - d e p r i v e d r a t s w e r e a d m i n i s t e r e d 2 . 5 - 1 0 m g / k g and t h e n u m b e r o f c u m u l a t e d (3 s e c ) d r i n k i n g e p i s o d e s were m e a s u r e d i n a t h r e e rain. "no s h o c k " ( i . e . unpunished) situation. T h e s e d o s e s ot CGS 8216 d i d n o t e f f e c t d r i n k i n g b e h a v i o r ( T a b l e I). In c o n t r a s t , d o s e s o f 5 and 10 C6S 8216 e l i c i t e d significant reductions i n t h e number- o f d r i n k i n g e p i s o d e s i n a " s h o c k " ( i . e . , punished) situation (Table 1). Effects of Pentobarbital and CGS 8216 i n t h e c o n f l i c t Lest: Pentobarbital (5 m g / k g ) e l i c i t e d a significant i n c r e a s e in the number of d r i n k i n g e p i s o d e s d u r i n g t h e t h r e e rain. " s h o c k " s i t u a t i o n compared to vehicle injected rats. H o w e v e r , a c o m b i n a t i o n o f CGS 8216 ( 2 . 5 and 5 m g / k g ) and p e n t o b a r b i t a l (5 m g / k g ) r e d u c e d t h e n u m b e r o f d r i n k i n g e p i s o d e s t o v a l u e s w h i c h w e r e riot significantly different from c o n t r o l .

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34 32 30

28 26 24 o

O

22

~

DIAZEPAM

~

VEHICLE

m 16 14 z

12

=<0.02 P<0.002 (n=10) (n=10)

10

P<0.15 (n=10) l

I

L

0.5

1.0

2

DOSE (mg/kg)

FIG.

1

Effects o f d i a z e p a m on c o n f l i c t responding in rats. Each pair of points represents a separate s t u d y i n w h i c h 10 r a t s r e c e i v e d d i a z e p a m o r v e h i c l e in r a n d o m i z e d s e q u e n c e w i t h a o n e week i n t e r v a l between studies. Diazepam produced a dose-dependent increase in the number of shocks received.

TABLE I Effects of CGS 8216 on Drinking Behavior Dose of CGS 8216 (mg/kg) 0 2.5 5 I0

Nonpunished 23.7 23.3 17.9 25.8

i ++ i

1.5 3.6 2.5 1.6

Punished 14.0 12.0 6.9 2.2

i i i i

2.5 2.6 1.2 0.3

Values represent the X i SEM of 3 second drinking episodes recorded during a three minute trial. The experimental details are described in Methods. An analysis of variance demonstrated no significant effect of COS 8216 on nonpunished responding, and a highly significant effect (p<0.0006) on punished responding. A post hoc analysis of the effects of COS 8216 on punished responding revealed highly significant reductions in punished responding with both 5 and i0 mg/kg of COS 8216 compared to vehicle control (p<.Ol and p< .0002, respectively). Eight to ten rats were used in each group.

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25

2O

o u~ 0

15

10

TREATMENT

FIG. 2 Effects of CGS 8216 and pentobarbital on conflict responding. Values are the ± SEM of six independent groups with i0 rats each. Symbols: *, p<.01 compared to vehicle injected rats; CGS, CGS 8216; PB, pentobarbital; VEH, vehicle. Other significant contrasts include PB + CGS VEH vs PB + CGS 2.5 mg/kg (p<0.01); PB VEH + COS 2.5 mg/kg vs PB + CGS 2.5 mg/kg (p<0.04); and PB VEH + CGS 5 mg/kg vs PB + C6S 5 mg/kg (p<0.01).

Discuss,on The discovery of compounds such as CGS 8216, Ro 15-1788, and 3-carboethoxy-~-carboline (~-CCE) which bind to benzodiazepine receptors with high affinities and antagonize some of the pharmacologic properties of benzodiazepines (6) permits an examination of the putative relationship of benzodiazepine receptors to the pharmacologic actions of pentobarbital (3-5). Although the initial reports on the pharmacology of benzodiazepine antagonists did not reveal any intrinsic actions (9-12), subsequent reports have detailed a number of effects of these compounds when administered alone. For example, ~-CCE elicits behaviors in both rodents (13-15) and mammals (16) suggestive of an anxiogenic action. ~-CCE will also produce electroencephalographic seizures in rats (Skolnick et al., submitted) and motor seizures in primates (J. Barrett, personal communication). Ro 15-1788 has been reported to reduce social interaction in rats, which File (16) has suggested represents an anxiogenic action, while higher doses of Ro 15-1788 may have both anticonflict (17) and anticonvulsant actions (17,18) in rodents. These observations necessitated examining the intrinsic actions of a benzodiazepine

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antagonist in a paradigm with sufficient sensitivity to detect reductions as well as increases in.punished responding (13) prior to an examination of the effects of this compound on the anticonflict actions of pentoharbital. In accord with previous studies (i), both diazepam and pentobarbital elicited significant increases in punished responding (Fig. 1,2). Despite the lack of effect of CGS 8216 on nonpunished responding, this compound elicited a significant reduction in punished behavior at 5 and 10 mg/kg (Table I). These results suggest that CGS 8216 at doses of 5 and i0 mg/kg may possess an anxiogenic action. Similar reductions in punished responding have been reported with ~-CCE (13), which elicits a syndrome reminiscent of clinical anxiety in primates (16). Nonetheless, a dose of CGS 8216 (2.5 mg/kg) which did not affect punished responding abolished the anticonflict actions of pentobarbital. These observations suggest that pentobarbital could produce an anticonflict effect through an interaction with benzodiazepine receptors. However, Bernard et al. (i0) have reported that CGS 8216 antagonizes the anticonflict actions of meprobamate, phenobarbital and diazepam, while Petersen et al. (15) demonstrated that ~-CCE, but not Ro 15-1788, antagonized the anticonflict actions of phenobarbital. These observations imply that Ro 15-1788 may have a somewhat different spectrum of effects than does CGS 8216, although both appear to have relatively specific binding properties to the benzodiazepine recognition site. However, it is not known if the doses of CGS 8216 used to antagonize the anticonflict actions of meprobamate and phenobarbital (i0) in a Geller-Seifter test would affect punished responding when administered alone, or if there is a difference in the potency of CGS 8216 in antagonizing the anticonflict actions of barbituates, benzodiazepines and propanediol carbamates. The present results provide evidence for a functional link between the anticonflict actions of pentobarbital and the benzodiazepine receptor complex. This interaction could be due to pentobarbital binding to the chloride ionophore ("the barbiturate receptor"), with CGS disrupting functional coupling between these two sites. ACKNOWLEDGEMENT The authors thank Ms. Cindy Owen for excellent technical assistance and Colleen LePore for typing the manuscript. REFERENCES i. 2. 3. 4. 5.

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