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  • Cy3 maleimide (non-sulfonated) Methods br Results br Discuss

    2022-09-24

    Methods
    Results
    Discussion SV exocytosis from excitatory boutons is more sensitive to inhibition by isoflurane than exocytosis from inhibitory boutons,4, 31 but the mechanism(s) of this synaptic selectivity is unknown. Although P/Q-type VGCCs contributed quantitatively more to SV exocytosis than did N-type VGCCs in rat hippocampal neurones, there was no significant difference in their functional sensitivities to isoflurane. These results indicate that differential expression, or coupling of presynaptic VGCC subtypes to SV exocytosis, or both are not the basis for the synaptic selectivity of isoflurane in hippocampus, which likely involves targets upstream of Ca2+ entry. The greater contribution of P/Q-type VGCCs to SV exocytosis in hippocampal excitatory neurones, evident in the greater degree of inhibition by the specific toxin agatoxin than conotoxin, has been reported by others, although we observed a greater contribution of P/Q-type VGCCs. Electrophysiological studies also show a dominant contribution of P/Q-type VGCCs to neurotransmission in hippocampus.27, 29 In contrast, greater inhibition of SV exocytosis by conotoxin has been reported in cultured neurones from the rat hippocampal CA3–CA1 subregion,15, 32 which might be attributable to subregion-specific differences or days in culture. In immature hippocampal neurones (e.g., before 15 DIV corresponding to postnatal day 10), conotoxin inhibits synaptic transmission more than agatoxin because of a relative predominance of N-type VGCCs. In contrast, later in development (e.g., after 21 DIV corresponding to postnatal day 16), agatoxin leads to greater inhibition of exocytosis as P/Q-type Cy3 maleimide (non-sulfonated) predominate.33, 34 Our results are consistent with these findings, although the age, in days, of the rats used to produce the cultures is different. Taken together, these data indicate that expression of VGCC subtypes at synaptic boutons varies depending on factors including neuronal phenotype and culture age. The basis of the greater sensitivity to inhibition by isoflurane of glutamate release compared with GABA release is unclear.4, 35, 36 One hypothesis is that differential expression of critical ion channels with greater anaesthetic sensitivity leads to such differences between synapses. However, our results indicate that differential presynaptic VGCC subtype expression does not contribute to differences in sensitivity of SV exocytosis to isoflurane in hippocampal neurones. These results are consistent with previous electrophysiological studies showing that isoflurane inhibits P/Q- and N-type Ca2+ currents to a similar extent in rat hippocampal pyramidal neurones, cultured cerebellar granule neurones, and Xenopus oocytes heterologously expressing mammalian N- or P/Q-type channels. In contrast, isoflurane was reported to inhibit N-type currents to a greater extent than P/Q-type currents in rat dorsal root ganglion neurones. The reason for this difference in sensitivity to isoflurane is not clear, although it may reflect differences in channel subunit compositions. The auxiliary subunits of VGCCs are differentially distributed amongst brain regions,38, 39 such that pharmacological properties might vary between brain regions. Several limitations must be considered in interpreting our results. Experiments were performed at 29–30°C to facilitate electrophysiological recording stability and volatile anaesthetic use. The kinetics of SV exocytosis and endocytosis are faster at higher physiological temperatures, but exhibit similar fundamental properties at 30°C. Use of SV-targeted pHluorins such as vGlut1-pHlourin is an established and validated method for measuring vesicular exocytosis and endocytosis in live cells.4, 15, 25, 32 Although this approach does not directly see membrane fusion, as in the classic fast freeze electron micrographs of Heuser and Reese,41, 42 membrane fusion is necessary to neutralise the vesicle lumen to induce the fluorescence change. Live-cell imaging also has the advantage of providing dynamic rather than static imaging of vesicle fusion and re-endocytosis. To determine isoflurane effects, we analysed boutons with SNR ≥ 5 in response to 100 APs in the presence of VGCC blockers, which biases data to boutons with larger fluorescent signals. We used a primary hippocampal neurone culture system in which functional synapses are formed.16, 17, 18 Given that expression of VGCC subtypes in individual boutons is known to be determined by factors including the type of postsynaptic cell,28, 29, 30 differential contributions of P/Q- and N-type VGCCs to SV exocytosis between different boutons of the same transfected neurone as observed in this study may result from differences in their postsynaptic targets. Cultured hippocampal neurones consist of various phenotypes,28, 29, 43 so the isoflurane effects observed represent a heterogeneous neuronal population. The effects of isoflurane on SV exocytosis in other brain regions with different properties may differ from those observed in hippocampus. Our studies focus on the hippocampus as a model given the large body of data available on its fundamental neurophysiology, sensitivity to anaesthetics, and critical role as a target for anaesthetics.44, 45 Finally, we used a relatively high, but clinically relevant, concentration of isoflurane for these mechanistic studies to maximise detection of possible presynaptic inhibitory effects. It is possible that the lack of inhibition reported by Hall and colleagues was because of the lower concentration they used, but also to the fact that they measured electrophysiological effects on somatic, rather than presynaptic, channels.