Role of specific presynaptic calcium channel subtypes in isoflurane inhibition of synaptic vesicle exocytosis in rat hippocampal neurones.

BACKGROUND: P/Q- and N-type voltage-gated calcium channels (VGCC) are the principal subtypes mediating synaptic vesicle (SV) exocytosis. Both the degree of isoflurane inhibition of SV exocytosis and VGCC subtype expression vary between brain regions and neurotransmitter phenotype. We hypothesised that differences in VGCC subtype expression contribute to synapse-selective presynaptic effects of isoflurane. METHODS: We used quantitative live-cell imaging to measure exocytosis in cultured rat hippocampal neurones after transfection of the fluorescent biosensor vGlut1-pHluorin. Selective inhibitors of P/Q- and N-type VGCCs were used to isolate subtype-specific effects of isoflurane. RESULTS: Inhibition of N-type channels by 1 µM ω-conotoxin GVIA reduced SV exocytosis to 81±5% of control (n=10). Residual exocytosis mediated by P/Q-type channels was further inhibited by isoflurane to 42±4% of control (n=10). The P/Q-type channel inhibitor ω-agatoxin IVA at 0.4 µM inhibited SV exocytosis to 29±3% of control (n=10). Residual exocytosis mediated by N-type channels was further inhibited by isoflurane to 17±3% of control (n=10). Analysis of isoflurane effects at the level of individual boutons revealed no difference in sensitivity to isoflurane between P/Q- or N-type channel-mediated SV exocytosis (P=0.35). There was no correlation between the effect of agatoxin (P=0.91) or conotoxin (P=0.15) and the effect of isoflurane on exocytosis. CONCLUSIONS: Sensitivity of SV exocytosis to isoflurane in rat hippocampal neurones is independent of the specific VGCC subtype coupled to exocytosis. The differential sensitivity of VGCC subtypes to isoflurane does not explain the observed neurotransmitter-selective effects of isoflurane in hippocampus.

Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons.

Volatile anesthetics affect neuronal signaling by poorly understood mechanisms. Activation of central dopaminergic pathways has been implicated in emergence from general anesthesia. The volatile anesthetic isoflurane differentially inhibits glutamatergic and GABAergic synaptic vesicle (SV) exocytosis by reducing presynaptic Ca2+ influx without affecting the Ca2+-exocytosis relationship, but its effects on dopaminergic exocytosis are unclear. We tested the hypothesis that isoflurane inhibits exocytosis in dopaminergic neurons. We used electrical stimulation or depolarization by elevated extracellular KCl to evoke exocytosis measured by quantitative live-cell fluorescence imaging in cultured rat ventral tegmental area neurons. Using trains of electrically evoked action potentials (APs), isoflurane inhibited exocytosis in dopaminergic neurons to a greater extent (30 ± 4% inhibition; p < 0.0001) than in non-dopaminergic neurons (15 ± 5% inhibition; p = 0.014). Isoflurane also inhibited exocytosis evoked by elevated KCl in dopaminergic neurons (35 ± 6% inhibition; p = 0.0007), but not in non-dopaminergic neurons (2 ± 4% inhibition). Pharmacological isolation of presynaptic Ca2+ channel subtypes showed that isoflurane inhibited KCl-evoked exocytosis mediated exclusively by either CaV2.1 (P/Q-type Ca2+ channels; 30 ± 5% inhibition; p = 0.0002) or by CaV2.2 (N-type Ca2+ channels; 35 ± 11% inhibition; p = 0.015). Additionally, isoflurane inhibited single AP-evoked Ca2+ influx by 41 ± 3% and single AP-evoked exocytosis by 34 ± 6%. Comparable reductions in exocytosis and Ca2+ influx were produced by lowering extracellular [Ca2+]. Thus, isoflurane inhibits exocytosis from dopaminergic neurons by a mechanism distinct from that in non-dopaminergic neurons involving reduced Ca2+ entry through CaV2.1 and/or CaV2.2.