clozapine and gabazine

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.

Topics: Animals; Animals, Newborn; Cells, Cultured; Clozapine; Dose-Response Relationship, Drug; GABA Antagonists; Membrane Potentials; Neurons; Pyridazines; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Synapses; Synaptic Transmission; Ventral Tegmental Area

1. The loop diuretic furosemide is known to antagonize the function of gamma-aminobutyric acid type A (GABAA) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABAA receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits. 2. Autoradiography with [35S]-t-butylbicyclophosphorothionate ([35S]-TBPS) as a ligand indicated that furosemide (0.1-1 mM) reversed the 5 microM GABA-induced inhibition of binding only in the cerebellar granule cell layer of rat brain sections. In all other regions studied, notably also in the hippocampal and thalamic areas, furosemide failed to antagonize GABA. Furosemide 1 mM decreased [35S]-TBPS binding only in a limited number of brain regions, but facilitation of the GABA-inhibition of the binding was much more widespread. 3. In well-washed rat cerebellar, but not cerebrocortical, membranes, furosemide enhanced the [35S]-TBPS binding over basal level in the absence of added GABA. The GABAA antagonist, SR 95531, and the convulsant, Ro 5-4864, blocked this furosemide-induced increase. Both interactions with the furosemide enhancement are likely to be allosteric, since furosemide affected the binding of [3H]-SR 95531 and [3H]-Ro 5-4864 identically in the cerebellar and cerebrocortical membranes. Maximal GABA-antagonism induced by furosemide in cerebellar membranes was further increased by SR 95531 but not by Ro 5-4864, indicating additive antagonism only for SR 95531. In human cerebellar receptors, only GABA antagonism by furosemide, but not the enhancement without added GABA, was observed. 4. In recombinant GABAA receptors, furosemide antagonism of GABA-inhibition of [35S]-TBPS binding depended only on the presence of alpha 6 and beta 2/3 subunits, irrespective of the presence or absence of gamma 2 or delta subunits. 5. In alpha 6 beta 3 gamma 2 receptors, clozapine reversed the enhancement of [35S]-TBPS binding by furosemide in the absence of GABA. However, it failed to affect the GABA-antagonism of furosemide, suggesting that the enhancement of basal binding and the GABA antagonism might represent two different allosteric actions of furosemide. 6. In conclusion, the present results indicate that furosemide is a subtype-selective GABAA antagonist with a mode of action not shared by several other antagonists, which makes furosemide a unique compound for development of potential GABAA rece

Topics: Animals; Autoradiography; Brain; Cell Line; Clozapine; Diuretics; Furosemide; GABA Antagonists; Humans; Male; Pyridazines; Rats; Rats, Wistar; Receptors, GABA-A; Recombinant Proteins

Clozapine, an atypical neuroleptic, functionally antagonizes the gamma-aminobutyric acid-induced chloride uptake via the main central inhibitory receptor, gamma-aminobutyric acid type A (GABAA) receptor, in brain vesicles. GABAA antagonism by micromolar concentrations of clozapine is more efficient in rat cerebrocortical and hippocampal membranes than in cerebellar membranes, as evidenced by clozapine reversal GABA-inhibition of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding. A typical neuroleptic, haloperidol, failed to antagonize GABA in any of these brain regions, while the specific GABAA antagonist 2'-(3'-carboxy-2',3'-propyl)-3-amino-6-p -methoxyphenylpyrazinium bromide (SR 95531) was efficient in all three brain regions. Clozapine action on [35S]TBPS binding was unaffected by the benzodiazepine receptor antagonist flumazenil. Clozapine inhibited the binding of [3H]muscimol and [3H]SR 95531 to the GABA recognition site, but this effect only partially correlated with the regional differences in and the potency of clozapine antagonism of GABA-inhibition of [35S]TBPS binding, suggesting that also other than GABA sites may mediate clozapine actions. Autoradiography of [35S]TBPS binding revealed GABA antagonism by clozapine in most brain regions. Main exceptions were cerebellar granule cell and molecular layers, olfactory bulb external plexiform and glomerular layers and primary olfactory cortex, where clozapine antagonized GABA inhibition less than average, and lateral hypothalamic and preoptic areas where its antagonism was greater than average. Recombinant alpha 6 beta 2 gamma 2 receptors, the predominant alpha 6 subunit-containing receptor subtype in cerebellar granule cells, failed to show GABA antagonism by clozapine up to 100 microM. In contrast, recombinant alpha 1 beta 2 gamma 2 receptors, forming the predominant receptor subtype in the brain, were clozapine sensitive. Recombinant alpha 6 beta 2 gamma 2 and alpha 6 beta 3 gamma 2 receptors resulted in clozapine-insensitive receptors, whereas alpha 6 beta 1 gamma 2 receptors were clozapine sensitive. The efficacy of clozapine to antagonize GABA in alpha 1 beta x gamma 2 receptors decreased in the order of alpha 1 beta 1 gamma 2 > alpha 1 beta 2 gamma 2> alpha 1 beta 3 gamma 2. The results indicate that clozapine antagonizes the function of most GABAA receptor subtypes, and that the interaction is determined by the interaction of the alpha and beta subunit variants. GABA antagonism is a u

Topics: Animals; Antipsychotic Agents; Brain; Bridged Bicyclo Compounds, Heterocyclic; Clozapine; GABA Antagonists; GABA-A Receptor Antagonists; Male; Muscimol; Pyridazines; Rats; Rats, Wistar; Receptors, GABA-A