Antagonism of diazepam against central anticholinergic drug-induced hyperactivity in mice: Involvement of a GABA mechanism

ANTAGONISM OF DIAZEPAM AGAINST CENTRAL ANTICHOLIN~RGIC DRUG-INDUCED HYPERACTIVITY IN MICE: INVOLVEMENT OF A GABA MECHANISM P. SOUBRIB P. SIMON and J. R. BCIISSIER Unite de Neuropsychopharmacolgie de l’lnserm, 2, rue d’Alesia, F75014 Paris. France (Accepteci s IMLI!.1976) mice tested for 30 min in photometer activity boxes , an increased activity (hyperactivity) was observed after intraperitoneal administration of benztropine (8 mg/kg), scopolamine (2 mg/kg). atropine (24 mg/kg) and trihexyphenidyl (8 mg/kg). Such hyperactrvity was reduced or suppressed by diazepam (0.5. I or 2 mg/kg, Lp.). At these doses no reduction of spontaneous activity was found with diazepam. Picrotoxin (1 or 2 mg/kg, s.c.), but neither thiosemi~arbazid~ (444 mgikg, s.c.) nor strychnine (0.2s and 0.5 mg/kg, SC) antagonized the inhibitory effects of diazepam on each anticholinergic drug-induced hyperactivity. Picrotoxin (0.5, 1 or 2 mg/kg) did not modify the decreased spontaneous activity elicited by diazepam (4 mg/kg). These results suggest that diazepam may exert its antagonistic effect on the hyperactivity induced by anticholinergic drugs through a link with the y-aminobutryic acid (GABA) system and more precisely through direct stimulation of GABA receptors. However, since picrotoxin did not modify the reduction of the spontaneous activity induced by diazepam, one can assume that diazepam-elicited decreased activity does not involve a GABA-ergic component and furthermore does not fully determine the effects of this benzodiazepine against the hyperactivity induced by antichoiinergic drugs.

Summary-h

Various data suggest that some of the biochemical (Fuxe, Hiikfelt, Agnati, Ljungdahl, Perez de la Mora and Johansson, 1975; Perez de la Mora, Fuxe, Agnati, HSkfeit and Ljungdahl, 1975) and eiectrophysiotogical (Barnes and Moolenaar, 1971; Stratten and Barnes, 1971; Banna, Jabbur and Saade, 1974) effects of benzodiazepines could be attributed to their actions on y-aminobutyric acid (GABA) mechanisms. The protective effect of benzodi~epines against the epileptic seizures induced by a wide range of agents known to lower the levels of GABA at its receptor sites (Costa, Guidotti, Mao and Suria, 1975) gives pharmacological support for this view. The purpose of this work was to determine if another pharmacological effect of benzodiazepines, the antagonism against the increased locomotor activity induced in mice by anticholinergic drugs [Simon, Soubrie and Boissier, 1974), could be related to the action of the benzodiazepines on GABA systems. With that aim, the effects of picrotoxin, a GABA receptor blocking agent, and of thiosemicarbazide, a GABA synthesis blocking agent, were studied on the antagonism exerted by diazepam on the hyperactivity induced by four anticholinergic drugs: benztropine, scopolamine, atropine and trihexyphenidyl. However, recent results obtained in receptor binding experiments do not support a direct interaction between benzodi~epines and GABA mechanisms, but suggest that benzodiazepines interact with glycine receptors (Synder and Enna, 1975). Accordingly, the effects of strychnine, a glycine receptor blocking

agent, were also studied on the antagonism exerted by diazepam against the hyperactivity induced by the four anticholinergic drugs.

MATERIAL

AND

METHODS

The experiments were carried out on male Swiss NMRI mice (18-22 g). The animals were housed eight to a cage with free access to food and water, and maintained on a 12 hr light-dark cycle. Mice were individually tested for motor activity in translucent actophotometer boxes. The boxes (26 x 21 x 10 cm) were crossed at a height of 1 cm above the floor by two perpendicular light beams centred on photoelectric cells connected with meters. The number of light beams crossed by each animal (referred to as activity or hyperactivity) was recorded during the 30 min immediately after placing the mouse into the test-box. To obtain equivalent increases of activity with the four antichoIinergic drugs, the doses used ~determined by previous experiments) were as follows: benztropine mesyIate 8 mgfig; scopoiamine hydrobromide, 2 mg/ kg; atropine sulphate, 24 mg!kg and trihexyphenidyl chlorhydrate, 8 mgikg. These drugs and diazepam were injected intraperitoneally. Previous experiments have shown that the effects of diazepam injected either through the same route as anticholinergic drugs or through a different route (subcutaneous) were equivalent. Picrotoxin, thiosemicarbazide or strychnine were injected subcutaneously. All drugs were administered 30 min before testing. Drugs were preKey words: Anticholinergic drugs, diazepam, picrotoxin, strychnine, GABA. mice. pared as solution in distilled water or as suspension 773

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Fig. I. Effects of picrotoxin on the antagonism exerted by diarepam against the hyperactivity induced in mice by four anticholinergic drugs. Vertical bars represent standard error of the means. In animals injected with diazepam, asterisks indicate that picrotoxin-treated mice differ significantly from picro***P < 0.01. Diazepam (2 mg/kg) induced a non toxin non treated mice at: *P < 0.05; **P < 0.02; significant decrease (20”<,) of the spontaneous activity. Picrotoxin (2 mg/kg) induced a slight but significant decrease (about 35”,,) of the spontaneous activity and of the hyperactivity.

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gum and injected in a volume of 0.1 ml/ 20g of body weight. The data presented are the means t_ S.E.M. of the results obtained in groups of 8-12 mice. Statistical comparisons were made using Student t-test, or analysis of variance after controlling the homogeneity of the variances. RESULTS

Diazepam at the doses studied (0.5 and 1 mg/kg with benztropine, 1 and 2 mg/kg with scopolamine, atropine or trihexyphenidyl) induced a reduction or a suppression of the hyperactivity produced by each anticholinergic drug (Figs, 1 and 2). At these doses, diazepam did not statistically modify the spontaneous activity of mice. At 2 mg/kg diazepam decreased the spontaneous activity by only approx. 20Yb. Picrotoxin by itself decreased the hyperactivities and the spontaneous activity to the same extent: about 20-X:,, at 1 mg/kg and about 30-355,) at 2 mg/kg. Picrotoxin induced a dose-related antagonism against the effect of diazepam on the four druginduced hyperactivities (Fig. 1). In all instances, the linear regressions calculated for the antagonism of picrotoxin against the highest doses of diazepam (1 mg/kg with benztropine; 2 mg/kg with scopolamine, atropine or trihexyphenidyl) were statistically significant: benztropine (F,:,, = 5.42 P < 0.02); scopolamine (F,,,, = 5.12 P < 0.05); atropine (F,,,, = 7.03 P < 0.02); trihexyphenidyl (Ft.,, = 6.38 P = 0.02). Concerning the lowest doses of diazepam (0.5 mg/kg with benztropine; 1 mg/kg with scopolamine, atropine or trihexphenidyl) the linear regressions were statisti-

significant for benztropine (F,,,, = 12.42 cally P < 0.01) and atropine (F ,,5‘$ = 4.08 P < 0.05). To verify that the effect of picrotoxin was not only specific to diazepam, a study was carried out with another benzodiazcpine. Chlordiazepoxide (8 mg/kg). a dose which did not modify spontaneous activity, suppressed the hyperactivity induced by benztropine (number of light beams crossed: benztropine. 765 i 15 ; benztropine + chlordiazepoxide, 200 + 17). The antagonistic effect of chlordiazepoxide was significantly reduced by 1 mg/kg picrotoxin (benztropine + chlordiazepoxide + picrotoxin, 378 + 45). Picrotoxin (0.5, 1. 2 mg/kg) did not antagonize the decrease induced by 4 mg/kg diazepam on the spontaneous motor activity of mice (number of light beams crossed: controls, 246 1 15; diazepam. 172 _+ 13; diazepam + picrotoxin 0.5, 1 or 2 mg/kg. 135 i: 20, 130 * 12. 130 + 15. respectively). Thiosemicarbazide administered at doses ranging from 4 to 64 mg/kg did not antagonize the effect of diazepam (0.5 or 1 mg/kg with benztropine: 1 or 2 mgjkg with scopolamine, atropine or trihexyphenidyl) on the hyperactivity induced by each anticholinergic drug. In no case does the decrease induced by thiosemicarbazide on the activity of mice explain the inefficacity of this agent in counteracting the effect of diazepam against the hyperactivities. Strychnine (Fig. 2) at 0.25 or 0.5 mg/kg did not reliably modify the effect of diazepam (0.5 or 1 mg/kg with benztropine; 1 or 2 mg/kg with scopolamine, atropine or trihexyphenidyl) on the hyperactivity induced by each anticholinergic drug. With strychnine neither the linear regressions nor the group to group comparisons reached a statistically significant level.

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By itself, strychnine at 0.5 mg/kg, induced a slight decrease (2&251<) of the activity (spontaneous or induced by anticholinergic drugs) of mice.

DISCUSSION

Many reports suggest that anticholinergic drugs may exert some of their pharmacological effects by activating catecholamine systems. Anticholinergic drugs potentiate the effects of various agents known to enhance catecholamine receptors activity (Fibiger. Lytle and Campbell, 1970; Scheel-Kruger, 1970; Kuschinsky and Hornykiewickz, 1974). A catecholamine synthesis inhibitor, r-methyltyrosine, reduced the hyperactivities induced in mice by benztropine, scopolamine and atropine (Thornburg and Moore, 1973) and by trihexyphenidyl (Simon et al., 1974). Experiments carried out to obtain biochemical information concerning the effects of anticholinergic drugs on catecholamines give contradictory results. Benztropine inhibits the reuptake of dopamine, but other anticholinergics do not (Farnebo, Fuxe. Hamberger and Ljungdahl, 1970). Thus, this biochemical effect cannot be invoked to explain the observed pharmacological activities of anticholinergic drugs. A decreased turnover of dopamine (Bartholini and Pletscher, 1971; AndCn and Bedard, 1971) and an antagonism of neuroleptic-induced accelerated dopamine turnover (Anden and Bedard, 1971) have been found after anticholinergic drugs. On the other hand. anticholinergic drugs have been shown to enhance norepinephrine turnover (Anden and Bedard, 1971). Injected into the ventricle or into the substantia nigra, anticholinergic

drugs increase striatal dopamine turnover (Bartholini and Pletscher, 1971; Javoy, Agid, Bouvet and Glowinski. 1974). In mice these drugs decrease the accumulation of catecholamines; an effect possibly related to enhanced release (Hitzemann, Loh and Domino, 1972). Therefore, in mice some biochemical and pharmacological data may support the hypothesis that the hyperactivity induced by anticholinergic drugs is partly related to enhanced catecholamine activity induced indirectly by these compounds. If so, it is possible to explain the antagonistic effect of diazepam on hyperactivity in terms of its action on catecholaminergic neurones. In normal or in stressed animals benzodiazepines reduce catecholamine turnover (Lidbrink, Corrodi, Fuxe and Olson, 1973) and counteract the accelerated dopamine turnover induced by neuroleptics (Perez de la Mora et al., 1975). Some recent data suggest that these effects on catecholamines metabolism could be mediated by a link with the y-aminobutyric acid (GABA) system. y-Aminobutyric acid-like drugs decrease the turnover of dopamine, as do benzodiazepines (Fuxe et al., 1975; Noach. Van ZwietenBoot and Van Valkenburg, 1975); and more significantly, the effect of benzodiazepines on catecholamine metabolism can be counteracted by bicuculline, a GABA receptor antagonist, but not by strychnine. a glycine receptor blocking agent (Perez de la Mora et cd., 1975). Since the effects of diazepam on the hyperactivity induced by the -four anticholinergic drugs were reduced by picrotoxin but not by strychnine, one can assume that these effects of diazepam are correlated, not with its actions on glycinergic neurones. but with its stimulating activity on GABAergic receptor sites. Such stimulation leads to de-

P. SOIJBR~~., P. SIMON and

776

creased catecholamines metabolism, which is responsible for the reduction of the hyperactivities. Picrotoxin and bicuculline have been shown to elevate acetylcho~jne content in mouse brain (Svenneby and Roberts. 1974). Although the mechanism of such an effect is not clear, it may be due to decreased acetylcholine release. But since picrotoxin by itself did not potentiate but decreased the hyperactivities induced by the anticholinergic drugs, it seems difficult to attribute its diazepam collnteracting effect to its action on acetylcholine metabolism. Unlike picrotoxin, thiosemicarbazide failed to modify the action of diazepam. This suggests that in this experimental paradigm, the elects of diazepam are not a result of an indirect action through a GABA releasing action, but rather of a direct stimulation of GABA receptors. This hypothesis agrees with biochemical results suggesting a direct action of diazepam on the central GABA receptors (Perez de la

Mora et ul., 1975). However, it seems that GABA synthesis blocking agents have only weak effects on GABA levels (Maynert and Kaji, 1962). and so it is dificult to rule out the possibility that the decrease in GABA content elicited by thiosemicarbazide was not sufficient to impede the effects of diazepam. However, Banna rt al. (1974) have reported that GABA synthesis inhibition can suppress some spinal activities of diazepam, which suggests that diazepam has

diRerent actions at various levels in the nervous system. In conclusion, picrotoxin blocked the antagonism exerted by diazepam on the hyperactivities induced by anticholinergic drugs, but had no effect on the reduction of spontaneous activity elicited by this benzodiazepine. These results provide supplementary arguments to suggest that these two effects of diazepam are not related. These same data seem to indicate that some of the pharmacological effects of benzodiazepines appear to be mediated by GABA, but that others are possibly not. REFERENCES And&n, N. E. and Bedard, P. (1971). Influences of choliner-

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J. R. BOISSIER

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