Clozapine inhibits synaptic transmission at GABAergic synapses established by ventral tegmental area neurones in culture

Abstract

Elucidation of the mechanism of action of the atypical antipsychotic clozapine is complicated by the finding that this molecule interacts with multiple targets including dopaminergic and serotonergic receptors. Binding studies have suggested that clozapine also antagonises GABA_A receptors, but physiological evidence for such a block at functional synapses is lacking. In this study, we explored this antagonism by using electrophysiological techniques on GABAergic neurones of the ventral tegmental area in culture. Inhibitory post-synaptic currents (IPSCs) evoked in isolated GABAergic neurones were found to be dose-dependently inhibited by clozapine. Compatible with a post-synaptic mechanism, we found that membrane currents evoked by exogenous applications of GABA were similarly dose-dependently inhibited by clozapine. An analysis of miniature inhibitory post-synaptic currents (mIPSCs) showed that clozapine reduced the amplitude of quantal events in a way similar to SR-95531, a specific GABA_A receptor antagonist. Both drugs caused a similar leftward shift of the cumulative probability distribution of mIPSC amplitudes. This suggests that clozapine acts on both synaptic and extrasynaptic GABA_A receptors. In conclusion, our work demonstrates that clozapine produces a functional antagonism of GABA_A receptors at synapses. Because this effect occurs at concentrations that could be found in the brain of patients treated with clozapine, a reduction in GABAergic synaptic transmission could be implicated in the therapeutic actions and/or side-effects of clozapine.

Introduction

Although atypical antipsychotic drugs such as clozapine are widely used in the treatment of schizophrenia (Andersson et al., 1998; Brunello et al., 1995), their exact mechanism of action remains ill-defined. The discovery that classical antipsychotics are potent antagonists of dopamine (DA) receptors has been instrumental in the elaboration of a “dopamine hypothesis” for the pathophysiology of schizophrenia (Bacopoulos et al., 1979; Davis et al., 1991; Grace, 1991; Joyce, 1993; Reynolds, 1983; Seeman, 1992). Although a number of variants of this hypothesis have been proposed, most of these suggest that, one way or another, some aspect of the functioning of the central dopaminergic pathways is perturbed in schizophrenia and that most antipsychotics act by interacting with dopamine receptors.

Despite a large number of studies on clozapine, our understanding of its mechanism of action remains fragmentary. This drug interacts not only with all DA receptors [the D4 subtype showing the highest affinity (∼9 nM) (Van Tol et al., 1991)], but also with other metabotropic receptors such as those for serotonin (5HT1 and 5HT2) (Canton et al., 1990), norepinephrine (Coward, 1992), acetylcholine (Snyder et al., 1974) and histamine (Coward, 1992) (for review see Brunello et al., 1995; Coward, 1992). It has also been shown that clozapine can interact with some ionotropic receptors such as the NMDA-subtype glutamate receptor (Arvanov et al., 1997; Ossowska et al., 1999) and the GABA_A receptor (Squires and Saederup, 1991).

The possibility that GABAergic receptors could be a relevant target for clozapine can be considered interesting in part because dopaminergic neurones such as those in the ventral tegmental area (VTA) are known to be regulated by the activity of GABAergic neurones both of local origin and from areas such as the nucleus accumbens (Ikemoto et al., 1997; Johnson and North, 1992; Steffensen et al., 1998). Most evidence in favour of an interaction between clozapine and GABA receptors has originated from binding studies. Squires and Saederup (1991) demonstrated that clozapine in the micromolar range can reverse the inhibitory effect of GABA on the binding of [${}^{35}\text{S}$]TBPS (${}^{35}\text{S}$-t-butylbicyclophosphorothionate), a competitive GABA_A receptor antagonist, to rat brain membranes. A clozapine metabolite, N-desmethylclozapine, seems to have the same effect (Wong et al., 1996). The same authors have suggested recently that in addition to clozapine, several other antipsychotic drugs may also act as antagonists at GABA_A receptors (Squires and Saederup, 1997, Squires and Saederup, 1998). These observations have been confirmed by Korpi et al. (1995) who found that clozapine at 10 μM significantly reduces GABA-mediated Cl⁻ influx in brain vesicles and that at 100 μM it regionally antagonises GABA inhibition of [${}^{35}\text{S}$]TBPS binding in brain sections. Finally, using a system allowing the expression of various combinations of recombinant GABA_A receptor subunits, this same group has demonstrated that clozapine interacts preferentially with GABA_A receptors containing specific combinations of α and β subunits (Korpi et al., 1995). Although physiological evidence is lacking, taken together these experiments support the idea that clozapine can act to reduce GABA_A receptor activation.

In this report we have sought to determine whether the reported antagonism of GABA_A receptors by clozapine could be of physiological importance at the synapse. Using GABAergic neurones from the VTA in primary culture we have tested whether clozapine acts to regulate the synaptic activation of GABA_A receptors.