Life Sciences, Vol. 26, pp. 771-776 Pergamon Press Printed in the U.S.A.

BENZODZAZEPINES MODULATE STRIATAL ENKEPHALIN LEVELS VIA A GABAERGIC MECHANISM

Theodora Duka, Michael Wflster, and Albert Herz

Department of Neuropharmacology, Max-Planck-lnstitut fur Psychiatrle,

Kraepelinstrasse 2, D-8000 Mttnchen 40 Federal Republic of Germany

(Received in final form January I0, 1980)

Summary

As measured by a highly specific radloimmunoassay, dlazepam treatment of rats results in a rapid decrease of enkephalin levels in the striatum whilst these are increased in the hypothalamus. This striatal effect is mimicked by the GABA agonlst musclmol and the GABA-trans- aminase inhibitor aminooxyacetlc acid (AOAA}. It is furthermore blocked by the GABA antagonist bicuculline and is thus GABAergic in nature. Further, the diazepam effect upon strlatal enkephalln levels is antagonized by low doses of naloxone (1.O mg/kg, i.p.). In the hypothalamus, diazepam effects were neither mimicked nor modulated by any of a variety of agonlsts and anta- gonists tested, suggesting that benzodiazepine effects on enkephalin levels in this structure are not mediated via a GABAergic mechanism.

Introduction

Evidence has accumulated which suggests that benzodiazepines, extensively used as minor tranquilizers, exert their wide-spread effects via an influence upon transmission within GABA pathways (I,2,3). Other data, however, indicates that at least some 'specl- fic' benzodlazepine actions are mediated independently of the GABA system (4,5}. Certainly, it is clear that both benzodiazepines and GABA interact with CNS-endogenous opiates, as is apparent from biochemical (6,7) and behavioural studies (8,9).

Recently, we have demonstrated that acute treatment of rats with dlazepam induces rapid changes in the met- and leu-enkephalin concentrations of particular areas of the CNS (10). In this study, it was shown that diazepam dose-dependently decreases striatal enkephalln levels whereas those in the hypothalamus are increased. Although this latter effect has been attributed (10) to an anxio- lytic action of benzodlazepines, the mechanism underlying the dl- azepam-induced changes is largely unknown. The present investiga- tion considers the ability of certain representatives of different classes of drugs, to mimlck or abolish dlazepam-effected changes in striatal and hypothalamlc enkephalln levels.

0024-3205/80/100771-06502.00/0 Copyright (c) 1980 Pergamon Press Lid

772 Benzodiazepines Modulate Enkephalin Levels Vol. 26, No. i0, 1980

Methods

Methods used for the estimation of met-enkephalin levels have been described in detail elsewhere (10). Briefly, male Sprague- Dawley rats were decapitated and the brains quickly removed. After dissecting out the corpus strlatum and hypothalamus (11), met-enke- phalin was estimated by a highly selective and sensitive radioimmu- noassay (12). Animals were killed between 9 and 11 a.m., and the injection schedule (Table I) arranged according to the particular times at which the drugs display their peak effectsi. Controls killed alternately with experimental animals, received intravenous injections of diazepam-vehicle 5 min prior to killing. In naloxone, amlnooxyacetic acid and bicuculline animals, the vehicle was also injected, i.v., 5 mln prior to killing.

Drugs used: 'H-met-enkephalln [spec. activity 50 Ci/nmole] (Amersham), met-enkephalln (Peninsula), diazepam, dlazepam-vehicle (Roche), muscimol (Fluka), fentanyl (Janssen), hexobarbital (Bayer), bicuculline (Serva), naloxone (Endo), aminooxyacetic acid hemi- chloride [AOAA] (Sigma).

TABLE I

Injection schedule

time of application dose (min prior (mg/kg) to killing)

route of administration

Diazepam 5 2.5 i.v.

Muscimol 30 1.5 i.v.

AOAA 30 40.0 i.p.

Fentanyl 5 0.005 i.v.

Hexobarbital 5 10.O i.v.

Bicuculline 15 2.5 i.p.

Naloxone 15 1.O and 10.O i.p.

Results

The effects of certain agonists, of a variety of drug types, on met-enkephalin concentrations in corpus strlatum and hypothala- mus are shown in figure I. Diazepam treatment decreased striatal enkephalin levels by 25% and increased those in the hypothalamus by about 33%, as compared to their respective vehicle-treated controls. The GABA agonist muscimol mimicked this action in the striatum but failed to change enkephalin concentrations in the hypothalamus. Both hexobarbltal and the opiate agonist fentanyl were ineffective in modulating concentrations in these two struc- tures. The GABA transamlnase inhibitor AOAA is known to augment GABAerglc function by blocking enzymatic degradation. In this in- vestigation, AOAA effectively decreased the enkephalln levels in the strlatum but did not change those in the hypothalamus.

The GABA receptor antagonist blcuculline and the opiate recep- tor antagonist naloxone were tested for their effects either alone

Vol. 26, No. 10, 1980 Benzodlazeplnes Modulate Enkephalln Levels 773

or in combination with dlazepam. Administration of these compounds did not induce gross behavioural changes, namely the blcuculline dose employed failed to evoke seizures in the animals. Concerning enkephalln levels, the antagonists when administered alone neither displayed an effect in each of the structures investigated (Fig. 2,3), nor did they affect diazepam action in the hypothalamus. However, both bicuculline and naloxone in the doses tested comple- tely reversed the diazepam-induced decrease in strlatal enkephalln content.

i D

m iii ! ! i

FIG. 1 Met-enkaphalin concentrations in hypothalamus and corpus striat~Im of rats following treatment with various agonists. Each bar represents the mean + SEM of 15 animals. ** p < O.OO1, when compared To vehlcle-treated animals.

Discussion

The present report confirms earlier studies, demonstrating that acute dlazepam treatment rapidly changes met-enkephalln levels in corpus striatum and hypothalamus (10}. Thus, diazepam decreases strlatal enkephalln content, whereas concentrations are increased in the hypothalamus. The fact that dlazepam does not change enke- phalin levels in other brain areas such as medulla oblongata/pons and midbrain (IO) hints at the specificity of these effects. More- over, the failure of other drugs, such as morphine or haloperldol, to acutely modulate brain enkephalin levels has been demonstrated (13,14). In order to further characterize the specificity of the dlazepam effect, musclmol, fentanyl and hexobarbital, representa- tive agonists of different classes of drugs and AOAA, a potent GABA transaminase inhibitor, were included in the present study. Apparently, the dlazepam effects on striatal enkephalin are selec- tively mimicked by the GABA agonlst musclmol and AOAA (Fig. I).

These findings are in agreement with the ability of the GABA antagonist bicuculline to block diazepam-induced changes in the striatum, and strongly suggests an involvement of GABAerglc neu- tones in the observed effects of dlazepa,L. The observation that benzodiazepines and oploids exhibit opposite effects on the GABA

774 8enzodlazeplnes Modulate Enkephalln Levels Vol. 26, No. I0, 1980

HYPOTHALAMUS

"

FIG. 2 Met-enkephalin concentrations in hypothalamus follow- ing treatment with antagonists, either alone or in combination with diazepam. Each bar represents the mean + SEM of at least 10 animals. * p < 0.005, • * p < O.OO1 when compared to vehicle-treated animals.

turnover in certain brain areas (6,15) has led us to consider the reduced enkephalin levels upon dlazepam treatment in terms of a stimulated release (and subsequently increased function). The pre- sent experiments employing muscimol and AOAA are supportative of this hypothesis (IO), and indicate that diazepam may not be acting directly at a strlatal locus, but is affecting GABAergic transmis- sion in nigro-striatal pathways.

The above suggestion that an increase in GABAergic activity is associated with a presumably increased release of striatal enkepha- fin is not in llne with the previous in vitro demonstration of an inhibitory impact of GABA and muscimol on K -stimulated release of met-enkephalin from striatal slices (7). However, as non-striatal brain structures are conceivably involved in the phenomena observed in vivo, in vitro results from isolated striata may not be expected to coincide precisely with those obtained in vivo.

Diazepam treatment apparently increases met-enkephalin levels in the hypothalamus, but there is evidence that this effect may, to be more exact, reflect the prevention of a decrease in levels (10). Thus, the observed changes have been regarded as an indirect effect, as the anxlolytic activity of diazepam may result in a reduced release of enkephalins during stress, leading to an appar- ent increase in peptide levels as compared to controls. The inabi- lity to mlmick this effect by manipulating GABAergic transmission by musclmol or AOAA supports the idea that the benzodiazepine re- ceptors concerned are not linked to the GABA system (16). It has, in fact, been suggested that the anxiolytic action of benzodiaze- pines is GABA-independent, probably acting via hypothalamic ACTH (4,5).

The reversal by the opiate antagonist naloxone of the effect of diazepam on striatal enkephalin levels appears of particular interest. Although it has been postulated that naloxone exerts

Vol. 26, No. 10, 1980 Benzodlazeplnes Modulate Enkephalln Levels 775

I

l

!

,Ji jJ! Ji FIG. 3

Met-enkephalin concentrations in corpus strlatum. For further explanation refer to figure 2.

GABA-antagonistic properties (17,18), these effects occur only at excessive doses (60-240 mg/kg), whereas the low doses employed here (I.O and 10.O mg/kg) may be regarded as opiate receptor spe- sific. Thus, the naloxone antagonism seen appears indicative of the involvement of an opiate receptor bearing neurone in the con- trol of striatal met-enkephalin release. The opposing observation that the opiate agonist fentanyl fails to modulate enkephalln levels, may be assimilable into this framework, if one invokes the concept of multiple opiate receptors, with each preferentially binding particular ligands.

The conclusion that the contrasting effects of dlazepam on enkephali~ levels in strlatum as compared to hypothalamus are mediated by different mechanisms (10) gains further support from the present investigation. The enkephalin pool in the striatum is apparently controlled by a GABAergic pathway, whereas that of hypothalamlc enkephalln is not thus modulated. Further studies are in progress in an attempt to elucidate the significance of these biochemical changes with respect to the various known effects of benzodiazeplnes.

Acknowled@ements

We are grateful to Dr. L. Maitre, Ciba-Gelgy, for a gift of musclmol. The stylistic revision of the English text by M.J. Millan is qratefully acknowledged.

Supported by Deutsche Forschungsgemeinschaft, Bonn.

776 Benzodlazepines Modulate Enkephalln Levels Vol. 26, No. I0, 1980

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