Effects of isoflurane on dopaminergic neurone synaptic vesicle exocytosis

Abstract

Despite widespread clinical use of anaesthetics, their pharmacological mechanisms of action are poorly understood. Volatile anaesthetics such as isoflurane induce a reversible state of amnesia, unconsciousness, and immobility in response to painful stimuli.1 At the cellular level, volatile anaesthetics exert prominent effects on synaptic transmission. While these synaptic effects involve predominately postsynaptic actions, data from our laboratory support presynaptic mechanisms as well.2 At presynaptic sites, anaesthetics differentially inhibit synaptic vesicle (SV) exocytosis depending on neurone phenotype, with greater inhibition of excitatory glutamatergic SV release than of inhibitory GABAergic SV release from hippocampal neurones.3,4 However, anaesthetic effects on dopamine release from dopaminergic (DA) neurones have not been studied, even though dopaminergic mechanisms have been implicated in emergence from anaesthesia.5 We investigated the effects of isoflurane on SV release in DA neurones (identified by post hoc tyrosine hydroxylase immunoreactivity) from rat ventral tegmental area (VTA) neurones using live-cell imaging to measure SV exocytosis and Ca2+ influx. We employed a pH-sensitive variant of eGFP (pHluorin) fused to the luminal domain of the vesicular monoamine transporter (vMAT) to measure exocytosis and the cell permeant fluorescent Ca2+ indicator Fluo-5F to measure intracellular Ca2+ concentration. Isoflurane differentially inhibited SV exocytosis and Ca2+ influx induced by electrically stimulated action potentials from both DA (by 29 ± 4%, n = 13, p < 0.0001 and 42 ± 3%, n = 6, p < 0.0001, respectively) and non-DA neurones (by 16 ± 5%, n = 10, p < 0.0055 and 58 ± 5%, n = 8, p < 0.0001, respectively). In contrast to other neurotransmitter phenotypes, isoflurane also inhibited SV exocytosis evoked by elevated K+ in DA neurones, suggesting a voltage-gated Na+ channel (Nav) independent mechanism of anaesthetic action in this neuronal population. This contrasts with SV exocytosis from glutamate or GABA releasing neurones, which requires Nav. SV release is known to be tightly coupled to Ca2+ entry6. The degree of inhibition of SV exocytosis by isoflurane in DA neurons was proportional to the reduction in Ca2+ influx, and could be mimicked by reducing extracellular Ca2+ concentration. Use of subtype-specific voltage-gated Ca2+ channel toxins revealed that SV exocytosis in DA neurones was solely mediated by P/Q-type and N-type Ca2+ channels. Isoflurane inhibited SV exocytosis mediated by both P/Q-type and N-type Ca2+ channels by a Nav-independent mechanism, supporting a role for inhibition of these Ca2+ channel subtypes in the presynaptic effects of isoflurane in DA neurones. These findings shed light on the presynaptic targets of isoflurane in DA neurones, and provide a molecular target for isoflurane induced unconsciousness and emergence from isoflurane anaesthesia. Funding: US NIH grant GM58055. 1.Hemmings HC Jr, Akabas MH, Goldstein PA, et al. Trends Pharmacol Sci. 2005; 26: 503–10.2.Hemmings HC Jr, Yan W, Westphalen RI, et al. Mol Pharmacol. 2005; 67: 1591–9.3.Westphalen RI, Hemmings HC Jr. J Pharmacol Exp Ther. 2006; 316: 216–23.4.Baumgart JP, Zhou ZY, Hara M, et al. Proc Natl Acad Sci USA. 2015; 112: 11959–64.5.Solt K, Van Dort CJ, Chemali JJ, et al. Anesthesiology. 2014; 121: 311–9.6.Dodge FA Jr, Rahamimoff R. J Physiol. 1967; 193: 419–32.