This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Article: New Research | Neuronal Excitability Isoflurane inhibits dopaminergic synaptic vesicle exocytosis coupled to CaV2.1 and CaV2.2 in rat midbrain neurons Christina L. Torturo1,2, Zhen-Yu Zhou1, Timothy A. Ryan1,3 and Hugh C. Hemmings1,2 1Departments of Anesthesiology, Weill Cornell Medicine, New York, NY 10065 2Pharmacology, Weill Cornell Medicine, New York, NY 10065 3Biochemistry, Weill Cornell Medicine, New York, NY 10065 https://doi.org/10.1523/ENEURO.0278-18.2018 Received: 16 July 2018 Revised: 18 December 2018 Accepted: 21 December 2018 Published: 10 January 2019 Author Contributions: CLT, ZZ, TAR and HCH designed the research; CLT performed the research, TAR contributed unpublished reagents/analytic tools; CLT and ZZ analyzed the data; CLT, ZZ, TAR, and HCH wrote the paper. Funding: http://doi.org/10.13039/100000002HHS | National Institutes of Health (NIH) GM58055 Conflict of Interest: HCH: Editor-in-Chief of the British Journal of Anaesthesia; consultant for Elsevier. Funding Sources: NIH GM58055 Corresponding author: Hugh C. Hemmings, E-mail: [email protected] Cite as: eNeuro 2019; 10.1523/ENEURO.0278-18.2018 Alerts: Sign up at www.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 Torturo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Title: Isoflurane inhibits dopaminergic synaptic vesicle exocytosis coupled to CaV2.1 and CaV2.2 in rat 2 midbrain neurons 3 Short Title: Isoflurane inhibits exocytosis in DA neurons 4 Author Affiliation: Christina L. Torturo1, 2, Zhen-Yu Zhou1, Timothy A. Ryan,1,3 Hugh C. Hemmings Jr.1, 2 * 5 Departments of 1Anesthesiology, 2Pharmacology, and 3Biochemistry, Weill Cornell Medicine, New York, NY 6 10065 7 Author Contributions: 8 CLT, ZZ, TAR and HCH designed the research; CLT performed the research, TAR contributed unpublished 9 reagents/analytic tools; CLT and ZZ analyzed the data; CLT, ZZ, TAR, and HCH wrote the paper. 10 *Corresponding author: E-mail: [email protected], Weill Cornell Medical College, Department of 11 Anesthesiology, 1300 York Avenue, New York, NY, 10065, USA. 12 Number of figures: 8 figures 13 Number of words: Abstract- 249/250, Significance Statement- 110, Introduction- 677, Discussion- 1493 14 Acknowledgments: 15 This work was supported by National Institutes of Health grant R01GM58055 (HCH). We are grateful to 16 Robert Edwards for generously providing plasmids; and Joel Baumgart, David Sulzer, Yelena Canter, and 17 Ping-Yue Pan for technical advice. We thank members of the Hemmings and Ryan laboratories for 18 constructive comments and critical reading of the manuscript. 19 Conflict of Interest: HCH: Editor-in-Chief of the British Journal of Anaesthesia; consultant for Elsevier. 20 Funding Sources: NIH GM58055 21 1 22 ABSTRACT 23 Volatile anesthetics affect neuronal signaling by poorly understood mechanisms. Activation of central 24 dopaminergic pathways has been implicated in emergence from general anesthesia. The volatile 25 anesthetic isoflurane differentially inhibits glutamatergic and GABAergic synaptic vesicle exocytosis by 26 reducing presynaptic Ca2+ influx without affecting the Ca2+-exocytosis relationship, but its effects on 27 dopaminergic exocytosis are unclear. We tested the hypothesis that isoflurane inhibits exocytosis in 28 dopaminergic neurons. We used electrical stimulation or depolarization by elevated extracellular KCl to 29 evoke exocytosis measured by quantitative live-cell fluorescence imaging in cultured rat ventral 30 tegmental area neurons. Using trains of electrically evoked action potentials (APs), isoflurane inhibited 31 exocytosis in dopaminergic neurons to a greater extent (30 ± 4% inhibition; p < 0.0001) than in non- 32 dopaminergic neurons (15 ± 5% inhibition; p = 0.014). Isoflurane also inhibited exocytosis evoked by 33 elevated KCl in dopaminergic neurons (35 ± 6% inhibition; p = 0.0007), but not in non-dopaminergic 34 neurons (2 ± 4% inhibition). Pharmacological isolation of presynaptic Ca2+ channel subtypes showed that 2+ 35 isoflurane inhibited KCl-evoked exocytosis mediated exclusively by either CaV2.1 (P/Q-type Ca 2+ 36 channels) (30 ± 5% inhibition; p = 0.0002) or by CaV2.2 (N-type Ca channels) (35 ± 11% inhibition; p = 37 0.015). Additionally, isoflurane inhibited single AP-evoked Ca2+ influx by 41 ± 3% and single AP-evoked 38 exocytosis by 34 ± 6%. Comparable reductions in exocytosis and Ca2+ influx were produced by lowering 39 extracellular [Ca2+]. Thus, isoflurane inhibits exocytosis from dopaminergic neurons by a mechanism 2+ 40 distinct from that in non-dopaminergic neurons involving reduced Ca entry through CaV2.1 and/or 41 CaV2.2. 42 2 43 SIGNIFICANCE STATEMENT 44 Despite their medical importance, the mechanisms of action of general anesthetics have not been fully 45 elucidated. Isoflurane, a widely used volatile anesthetic, inhibits voltage-gated sodium channels and 46 differentially inhibits synaptic vesicle exocytosis depending on neurotransmitter phenotype. Here we 47 show that in dopaminergic neurons of the ventral tegmental area isoflurane acts via a sodium channel- 2+ 48 independent mechanism to inhibit synaptic vesicle exocytosis in proportion to reduced presynaptic Ca 49 flux mediated by CaV2.1 and/or CaV2.2, in contrast to its effects in non-dopaminergic neurons. These 50 findings provide a synaptic mechanism for the observed role of reduced dopamine release in anesthetic- 51 induced unconsciousness, and implicate presynaptic Ca2+ channels of dopaminergic neurons as 52 important targets of isoflurane. 3 53 INTRODUCTION 54 General anesthetics are essential medicines that induce a reversible state of amnesia, unconsciousness, 55 and immobility in the face of intensely painful stimuli. Despite their widespread use in modern 56 medicine, their mechanisms of action are not well understood (Hemmings et al., 2005). The amnestic, 57 hypnotic, and immobilizing effects of anesthetics differ in dose-dependence, neuroanatomical regions 58 involved, and molecular targets consistent with multiple mechanisms working in parallel to produce the 59 state of anesthetic-induced unresponsiveness (Brown et al., 2011). However general anesthesia can 60 produce serious adverse side effects including cardiovascular, respiratory, and cognitive dysfunction. It 61 is therefore critical to identify the anesthetic mechanisms relevant for both their on-target and off- 62 target actions, with the ultimate goals of designing safer and more selective anesthetics and of using 63 currently available anesthetics in a rational mechanism-based manner to maximize therapeutic ratio. 64 Volatile anesthetics such as isoflurane modulate synaptic and extrasynaptic neurotransmission 65 through multiple postsynaptic targets, primarily by potentiating inhibitory GABAA receptors and 66 depressing excitatory glutamatergic transmission via ionotropic glutamate receptors (Rudolph and 67 Antkowiak, 2004). However, the GABAA receptor antagonist bicuculline does not antagonize isoflurane- 68 induced immobility, indicating a role for other targets in this effect (Zhang et al., 2004). The presynaptic 69 effects of volatile anesthetics are not as well characterized as their postsynaptic effects due to the small 70 sizes of nerve terminals and technical limitations of conventional electrophysiological techniques in 71 recording presynaptically. Nevertheless considerable neurochemical and neurophysiological evidence 72 indicates that volatile anesthetics directly inhibit neurotransmitter release (Hemmings et al., 2005). 73 Synaptic vesicle (SV) exocytosis is tightly coupled to the amount of Ca2+ entering the presynaptic 74 bouton (Wu et al., 2004), which is determined primarily by presynaptic voltage-gated ion channels (Na+, 75 Ca2+ and K+ channels) and modulatory receptors. Isoflurane depresses action potential (AP) amplitude in 76 axons and boutons, which results in downstream reductions in Ca2+ influx and neurotransmitter release 4 77 (Wu et al., 2004; Hemmings et al., 2005; Ouyang and Hemmings, 2005). Isoflurane also inhibits 78 neurotransmitter release from isolated nerve terminals with greater potency from glutamatergic than 79 from GABAergic terminals (Westphalen and Hemmings, 2003, 2006), consistent with neurotransmitter- 80 specific presynaptic anesthetic mechanisms. The cellular and molecular bases of this synaptic selectivity 81 are unclear. 82 Voltage-gated Ca2+ channels play an essential role in neurotransmission by mediating Ca2+ influx 83 that is closely coupled to exocytosis. Presynaptic Ca2+ channels are possible targets for inhibition of 84 neurotransmitter release by volatile anesthetics, and are also involved in producing myocardial 85 depression and vasodilation leading to significant cardiovascular side effects (Lynch et al., 1981, Bosnjak 86 et al., 1991). Synaptic transmission at most central nervous system synapses is mediated by multiple 2+ 2+ 87 Ca channel subtypes that are closely coupled to SV exocytosis, most prominently CaV2.1 (P/Q-type Ca 2+ 88 channels) and CaV2.2 (N-type Ca channels)