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Histaminergic neurons of the tuberomamillary nucleus display pacemaker properties; their firing rate is regulated according to behavioural state by gabaergic inhibition. Whole-cell recordings and single-cell RT-PCR from acutely isolated rat tuberomamillary neurons were used to characterize GABA -evoked currents and to correlate them with the expression pattern of 12 GABAA receptor subunits. We report differences in sensitivity to GABA and zinc as well as in the modulation of IPSC-decay times by zolpidem in histaminergic neurons expressing gamma-subunits at different levels. Immunocytochemistry and pharmacological analysis of whole-cell GABA-currents in these neurons revealed that all carry the gamma2-subunit protein and that all receptors contain at least one gamma-subunit. Neurons with different expression levels of gamma-subunits displayed a difference in cooperativity of GABA and zolpidem binding which we explain by the presence of one vs. two gamma-subunits in one receptor. Thus, we describe here native GABAA receptor function in relation to its stoichiometry.

The modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration-response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was approximately 90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 mM glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds.

The actions of Cu2+ ions on GABAA receptor-mediated currents in acutely isolated Purkinje cells from rat cerebellum were studied using the whole-cell patch-clamp technique and a rapid perfusion system. Bath application of Cu2+ reduced currents induced by 2 microM gamma-aminobutyric acid (GABA) in a concentration-dependent manner with an IC50 of 35 nM. The Cu2+-induced block of GABA responses was not voltage-dependent. Increasing the GABA concentration (from 2 to 50 microM) decreased the blocking effect of Cu2+. Dose-response analysis for activation of GABAA receptors revealed a twofold decrease in apparent affinity for GABA in the presence of 0.1 microM Cu2+. Recovery from the block required several minutes after removal of Cu2+ from the medium. The block was removed by histidine, which preferentially forms complexes with Cu2+, or by other chelating substances. Application of 10 microM histidine immediately before application of 2 microM GABA completely relieved the block of GABA responses produced by 0.1 microM Cu2+. The effect of histidine was concentration-dependent with an EC50 of 0.75 microM. The results demonstrate that Cu2+ is a potent inhibitor of GABA-evoked responses in rat Purkinje cells. Copper may be an endogenous synaptic modulating factor. Cu2+ toxicity, notably in Wilson's disease, could result to some extent from chronic GABAA receptor blockade.

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