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

Abstract

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 mM u-conotoxin GVIA reduced SV exocytosis to 81±5% of control (n¼10). Residual exocytosis mediated by P/Q-type channels was further inhibited by isoflurane to 42±4% of control (n¼10). The P/ Q-type channel inhibitor u-agatoxin IVA at 0.4 mM inhibited SV exocytosis to 29±3% of control (n¼10). Residual exocytosis mediated by N-type channels was further inhibited by isoflurane to 17±3% of control (n¼10). Analysis of isoflurane effects at the level of individual boutons revealed no difference in sensitivity to isoflurane between P/Q-or N-type channelmediated SV exocytosis (P¼0.35). There was no correlation between the effect of agatoxin (P¼0.91) or conotoxin (P¼0.15) 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.

Versatile Surface Electrodes for Combined Electrophysiology and Two-Photon Imaging of the Mouse Central Nervous System

“…It reduces Na + currents while inducing K + currents, lowers resting membrane potential, depresses NMDA receptor-dependent excitatory transmission, and increases GABA A receptor-mediated inhibitory transmission (Franks and Lieb, 1988 ; Rudolph and Antkowiak, 2004 ; Westphalen and Hemmings, 2006 ; Lissek et al, 2016 ; Zhao et al, 2019 ). Thus, neuronal excitability is considerably reduced, leading to the decreased opening probability of voltage-gated Ca 2+ channels for example (Study, 1994 ; Koyanagi et al, 2019 ; Timic Stamenic et al, 2019 ). Hence, higher peak amplitudes are observed at lower isoflurane levels.…”

Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol atDrosophilaMotor Nerve Terminals

“…There is increasing evidence in animal models as well as cell culture preparations that general anesthetics also act presynaptically (van Swinderen et al, 1999;Metz et al, 2007;Herring et al, 2009Herring et al, , 2011Xie et al, 2013;Baumgart et al, 2015;Zalucki et al, 2015;Troup et al, 2019). Exactly how general anesthetics impair neurotransmission remains unclear, with isoflurane affecting presynaptic sodium and calcium channels (Koyanagi et al, 2019;Zhou et al, 2019) as well as SNARE formation (Xie et al, 2013;Troup et al, 2019); however, it is unknown whether intravenous and volatile agents might have common presynaptic effects. Work in rat neurosecretory PC12 cells showed that clinical concentrations of both propofol and isoflurane inhibit neurotransmission from these cells, and that coexpression of a mutant form of the presynaptic protein syntaxin1A preserves neurotransmission in these cells under anesthetic exposure (Herring et al, 2009(Herring et al, , 2011.…”

Advances in precision anaesthesia may be found by testing our resistance to change

“…It remains to be seen what these target sites might be, or even how many sites might be involved in producing the differential actions for each anaesthetic. 4 Growing evidence suggests that multiple sites of action contribute to anaesthesia, including some types of sodium, 8 calcium, 9 and potassium 10 channels, many ligand-gated channels, 11 including channels gated by acetylcholine, 12 catecholamines, glutamate, 13,14 and GABA, 6,15e18 and presynaptic neurotransmitter release proteins. 19e21 Many additional protein targets remain to be explored, and thus there are potentially hundreds of anaesthetic sites of action contributing to the observed biological variability.…”