Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca ²⁺ influx, not Ca ²⁺ -exocytosis coupling

Abstract

Identifying presynaptic mechanisms of general anesthetics is critical to understanding their effects on synaptic transmission. We show that the volatile anesthetic isoflurane inhibits synaptic vesicle (SV) exocytosis at nerve terminals in dissociated rat hippocampal neurons through inhibition of presynaptic Ca 2+ influx without significantly altering the Ca 2+ sensitivity of SV exocytosis. A clinically relevant concentration of isoflurane (0.7 mM) inhibited changes in [Ca 2+ ] i driven by single action potentials (APs) by 25 ± 3%, which in turn led to 62 ± 3% inhibition of single AP-triggered exocytosis at 4 mM extracellular Ca 2+ ([Ca 2+ ] e ). Lowering external Ca 2+ to match the isoflurane-induced reduction in Ca 2+ entry led to an equivalent reduction in exocytosis. These data thus indicate that anesthetic inhibition of neurotransmitter release from small SVs occurs primarily through reduced axon terminal Ca 2+ entry without significant direct effects on Ca 2+ -exocytosis coupling or on the SV fusion machinery. Isoflurane inhibition of exocytosis and Ca 2+ influx was greater in glutamatergic compared with GABAergic nerve terminals, consistent with selective inhibition of excitatory synaptic transmission. Such alteration in the balance of excitatory to inhibitory transmission could mediate reduced neuronal interactions and network-selective effects observed in the anesthetized central nervous system. GCaMP3 | pHlourin | mechanisms of anesthesia | live cell imaging | presynaptic T he molecular and cellular mechanisms of anesthetic-induced amnesia, unconsciousness and immobilization are incompletely understood, particularly for the modern halogenated ether derivatives like isoflurane. General anesthetics, which are essential to both medical practice and experimental neuroscience, have potent and selective effects on neurotransmission (1), including both presynaptic actions (reduced neurotransmitter release) and postsynaptic actions (modulation of receptor function). These effects contribute to anesthetic-induced reductions in neuronal interactions, which are critical to information processing and consciousness (2-4). Knowledge of the fundamental synaptic effects of anesthetics is therefore essential to a molecular and physiological understanding of anesthetic mechanisms, and to development of more selective and safer anesthetics.Although postsynaptic electrophysiological effects of anesthetics can be assessed directly using whole cell recordings and heterologous expression of putative molecular targets, their presynaptic actions have been difficult to resolve by conventional approaches that do not clearly discriminate between presynaptic and postsynaptic contributions. Direct evidence for presynaptic effects of volatile anesthetics includes selective inhibition of glutamate release from isolated nerve terminals (5, 6) and of synaptic vesicle (SV) exocytosis in intact hippocampal neurons (7). However, it remains controversial whether these effects involve direct inhibition of SV exocytosis itself or of upstrea...

α2-Adrenergic Receptor and Isoflurane Modulation of Presynaptic Ca2+ Influx and Exocytosis in Hippocampal Neurons

“…Silent boutons, defined as those where the response to 100 APs was smaller than the standard deviation of the baseline before stimulation (Δ F 100 – σ ≤ 0), were excluded from analysis; < 10% of boutons did not respond to stimulation and were excluded from analysis. Based on previous studies, 13,14,22,23 sample sizes of ≥ 5 were used. Data are shown as mean ± s.e.m.…”

“…11–14 Since α 2 -AR agonists reduce requirements for general anesthetics, 15 we hypothesized that they also affect non-adrenergic synaptic transmission through presynaptic effects on evoked neurotransmitter release. Reduced excitatory transmission resulting in alteration of the balance between neuronal excitation and inhibition has been implicated in the effects volatile anesthetics, 1,16 and provides a plausible mechanism for the well known pharmacological interaction underlying the anesthetic-sparing effects of α 2 -AR agonists.…”

Anti-Allergic Drugs Tranilast and Ketotifen Dose-Dependently Exert Mast Cell-Stabilizing Properties

“…Inhaled anesthetics, such as sevoflurane or isoflurane, were demonstrated to depress the activity of mast cells or inhibit the process of exocytosis in synaptic vesicles by reducing the Ca 2+ influx into the cells [25,26]. In the present study, however, despite the use of isoflurane during the initial isolation of rat peritoneal mast cells, compound 48/80 (10 µg/ml) induced degranulation in 79.8 ± 3.4 % of the mast cells observed (n=6; Fig.…”

What is unconsciousness in a fly or a worm? A review of general anesthesia in different animal models

“…Thus, small changes in neuronal excitability can dramatically decrease neurotransmitter release, contributing to the clinical effects of volatile anesthetics. Other evidence points to presynaptic sodium and calcium channels as a potential mechanism whereby diverse general anesthetics impair neurotransmission (Baumgart et al, 2015;Lingamaneni et al, 2001).…”