Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons (original) (raw)
Related papers
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 hippo-campal 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 neu-ronal 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 neu-ronal 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 pre-synaptic 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 glu-tamate 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 upstream targets (8, 9). Moreover, the mechanism for the greater sensitivity of glutamate release relative to that of other transmitters is unclear. Neurotransmitter release is supralinearly dependent on pre-synaptic Ca 2+ influx due to the highly cooperative binding of Ca 2+ to synaptotagmin 1, the principal neuronal Ca 2+ sensor for triggering vesicular fusion through increases in the probability of exocytosis (Pv) (10, 11). We used sensitive quantitative fluores-cence imaging approaches to characterize the effects of the widely used volatile anesthetic isoflurane on the Ca 2+ sensitivity of SV exocytosis in cultured rat hippocampal neurons. Although glutamate and GABA have distinct postsynaptic actions, differences in the mechanisms and regulation of SV exocytosis from glutamatergic and GABAergic neurons are not well characterized. Because previous studies indicate that glutamate release is more sensitive to inhibition by volatile anesthetics than GABA release (12), we compared the effects of isoflurane on the coupling of Ca 2+ influx to exocytosis in glutamatergic and GABAergic boutons. Our results indicate that the inhibition of SV exocytosis by isoflurane is driven by a reduction in Ca 2+ influx. This reduction occurs without affecting the apparent sensitivity of exocytosis to Ca 2+. Sensitivity of SV exocytosis to isoflurane is therefore determined by presynaptic targets upstream of exocytosis that determine the magnitude of action potential-evoked Ca 2+ influx. Identification of neurotransmitter-selective effects of anesthetics on SV exocytosis is critical for understanding their pathway specific effects on neuronal interactions (13, 14). Results The effects of isoflurane on SV exocytosis were examined using the chimeric reporters of exocytosis vGlut-pHluorin (vG-pH) or synapto-pHlourin (syn-pH) expressed in dissociated rat hippo-campal neurons in the presence of 2 or 4 mM extracellular Ca 2+ ([Ca 2+ ] e). In resting terminals the pHluorin moiety residing in the acidic SV lumen is quenched, but fluoresces upon exocytosis Significance Clarification of the presynaptic actions of general anesthetics is critical to understanding the molecular and cellular mechanisms of their prominent effects on synaptic transmission. We show that the ether anesthetic isoflurane inhibits synaptic vesicle exocytosis through inhibition of presynaptic Ca 2+ influx in the absence of significant alteration of the Ca 2+ sensitivity of exocytosis. The greater inhibition of glutamate release compared with GABA release is explained by the relative anesthetic resistance of Ca 2+ influx in GABAergic boutons, consistent with overall reduction in excitatory synaptic tone.
The General Anesthetic Isoflurane Bilaterally Modulates Neuronal Excitability
SSRN Electronic Journal, 2019
Volatile anesthetics induce hyperactivity during induction while producing anesthesia at higher concentrations. They also bidirectionally modulate many neuronal functions. However, the neuronal mechanism is unclear. The effects of isoflurane on sodium channel currents were analyzed in acute mouse brain slices, including sodium leak (NALCN) currents and voltage-gated sodium channels (Na v) currents. Isoflurane at sub-anesthetic concentrations increased the spontaneous firing rate of CA3 pyramidal neurons, whereas anesthetic concentrations of isoflurane decreased the firing rate. Isoflurane at sub-anesthetic concentrations enhanced NALCN conductance but minimally inhibited Na v currents. Isoflurane at anesthetic concentrations depressed Na v currents and action potential amplitudes. Isoflurane at sub-anesthetic concentrations depolarized resting membrane potential (RMP) of neurons, whereas hyperpolarized the RMP at anesthetic concentrations. Isoflurane at low concentrations induced hyperactivity in vivo, which was diminished in NALCN knockdown mice. In conclusion, enhancement of NALCN by isoflurane contributes to its bidirectional modulation of neuronal excitability and the hyperactivity during induction.
Anesthesiology, 2008
Background: Isoflurane anesthesia produces cardiovascular and respiratory depression, although the specific mechanisms are not fully understood. Cranial visceral afferents, which innervate the heart and lungs, synapse centrally onto neurons within the medial portion of the nucleus tractus solitarius (NTS). Isoflurane modulation of afferent to NTS synaptic communication may underlie compromised cardiorespiratory reflex function. Methods: Adult rat hindbrain slice preparations containing the solitary tract (ST) and NTS were used. Shocks to ST afferents evoked excitatory postsynaptic currents with low-variability (SEM <200 s) latencies identifying neurons as second order. ST-evoked and miniature excitatory postsynaptic currents as well as miniature inhibitory postsynaptic currents were measured during isoflurane exposure. Perfusion bath samples were taken in each experiment to measure isoflurane concentrations by gas chromatography-mass spectrometry. Results: Isoflurane dose-dependently increased the decaytime constant of miniature inhibitory postsynaptic currents. At greater than 300 M isoflurane, the amplitude of miniature inhibitory postsynaptic currents was decreased, but the frequency of events remained unaffected, whereas at equivalent isoflurane concentrations, the frequency of miniature excitatory postsynaptic currents was decreased. ST-evoked excitatory postsynaptic current amplitudes decreased without altering event kinetics. Isoflurane at greater than 300 M increased the latency to onset and rate of synaptic failures of ST-evoked excitatory postsynaptic currents. Conclusions: In second-order NTS neurons, isoflurane enhances phasic inhibitory transmission via postsynaptic ␥-aminobutyric acid type A receptors while suppressing excitatory transmission through presynaptic mechanisms. These results suggest that isoflurane acts through multiple distinct mechanisms to inhibit neurotransmission within the NTS, which would underlie suppression of homeostatic reflexes.
European Journal of Anaesthesiology, 1998
reduced by 56, 43 and 36% in response to isoflurane 0.5, 1.5 and 3.0%, respectively (for all: P<0.05). Mem-The molecular mechanism of volatile anaesthetic acbrane depolarization evoked a rise in cytosolic free tion on presynaptic glutamate release is not clear. An calcium of ≈34%. Addition of isoflurane (0.5, 1.5 and inhibitory effect on voltage-gated calcium channels 3.0%) produced no significant change in cytosolic free has been proposed. The present study examines the calcium. These results indicate that the isofluraneeffect of isoflurane on cytosolic free calcium and syninduced reduction in presynaptic glutamate release is aptic glutamate release from isolated nerve terminals.
BACKGROUND: Evidence indicates that the anesthetic-sparing effects of α2-adrenergic receptor (AR) agonists involve α2A-AR heteroreceptors on nonadrenergic neurons. Since volatile anesthetics inhibit neurotransmitter release by reducing synaptic vesicle (SV) exocytosis, the authors hypothesized that α2-AR agonists inhibit nonadrenergic SV exocytosis and thereby potentiate presynaptic inhibition of exocytosis by isoflurane. METHODS: Quantitative imaging of fluorescent biosensors of action potential-evoked SV exocytosis (synaptophysin-pHluorin) and Ca influx (GCaMP6) were used to characterize presynaptic actions of the clinically used α2-AR agonists dexmedetomidine and clonidine, and their interaction with isoflurane, in cultured rat hippocampal neurons. RESULTS: Dexmedetomidine (0.1 μM, n = 10) or clonidine (0.5 μM, n = 8) inhibited action potential-evoked exocytosis (54 ± 5% and 59 ± 8% of control, respectively; P < 0.001). Effects on exocytosis were blocked by the subtype-nonselective α2-AR antagonist atipamezole or the α2A-AR-selective antagonist BRL 44408 but not by the α2C-AR-selective antagonist JP 1302. Dexmedetomidine inhibited exocytosis and presynaptic Ca influx without affecting Ca coupling to exocytosis, consistent with an effect upstream of Ca-exocytosis coupling. Exocytosis coupled to both N-type and P/Q-type Ca channels was inhibited by dexmedetomidine or clonidine. Dexmedetomidine potentiated inhibition of exocytosis by 0.7 mM isoflurane (to 42 ± 5%, compared to 63 ± 8% for isoflurane alone; P < 0.05). CONCLUSIONS: Hippocampal SV exocytosis is inhibited by α2A-AR activation in proportion to reduced Ca entry. These effects are additive with those of isoflurane, consistent with a role for α2A-AR presynaptic heteroreceptor inhibition of nonadrenergic synaptic transmission in the anesthetic-sparing effects of α2A-AR agonists.
Isoflurane depresses hippocampal CA1 glutamate nerve terminals without inhibiting fiber volleys
Background: Anesthetic-induced CNS depression is thought to involve reduction of glutamate release from nerve terminals. Recent studies suggest that isoflurane reduces glutamate release by block of Na channels. To further investigate this question we examined the actions of isoflurane, TTX, extracellular Ca 2+ , CNQX and stimulus voltage (stim) on glutamate-mediated transmission at hippocampal excitatory synapses. EPSPs were recorded from CA1 neurons in rat hippocampal brain slices in response to Schaffer-collateral fiber stimulation.
Anesthesiology, 2000
Background Effects of volatile anesthetic agents on N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic transmission have not been well characterized. The authors compared effects produced by halothane and isoflurane on electrophysiologic properties of NMDA and non-NMDA receptor-mediated synaptic responses in slices from the rat hippocampus. Methods Field excitatory postsynaptic potentials (fEPSPs) in the CA1 area were recorded with extracellular electrodes after electrical stimulation of Schaffer-collateral-commissural fiber inputs. NMDA or non-NMDA receptor-mediated fEPSPs were pharmacologically isolated using selective antagonists. Clinically relevant concentrations of halothane or isoflurane were applied to slices in an artificial cerebrospinal fluid perfusate. Paired pulse facilitation was used as a measure of presynaptic effects of the anesthetic agents. Results Clinically relevant concentrations of halothane (1.2 vol% approximately 0.35 mM) depressed fEPSP ampli...
Isoflurane Blocks Synaptic Plasticity in the Mouse Hippocampus
Anesthesiology, 2001
The volatile anesthetic isoflurane depresses glutamatergic transmission. In this study, the authors investigated the effects of isoflurane on the induction of long-term potentiation (LTP) and long-term depression (LTD) in slices from the juvenile and adult mouse hippocampus. Both forms of synaptic plasticity involve the activation of glutamate receptors.
Muscle & Nerve, 2015
Introduction: Sevoflurane and isoflurane are anesthetics that cause muscle relaxation and potentiate the effects of neuromuscular blocking agents. Their presynaptic mechanisms of action are not understood completely, especially at the motor nerve terminal level. Methods: We compared the presynaptic effects of these anesthetics on the exocytosis of synaptic vesicles labeled with the dye FM1-43 at the mouse neuromuscular junction. Results: Neither anesthetic evoked spontaneous exocytosis of synaptic vesicles, but both significantly inhibited the depolarization evoked by 4-aminopyridine and veratridine, suggesting a putative action on sodium channels. Exocytosis evoked by veratridine was inhibited by tetrodotoxin alone or in conjunction with sevoflurane or isoflurane, indicating that both agents may target voltage-gated sodium channels. Conclusion: We suggest that sevoflurane and isoflurane inhibit exocytosis evoked by sodium-dependent depolarization and might act on tetrodotoxin-sensitive sodium channels. These findings contribute to a better understanding of some clinical neuromuscular effects induced by these anesthetics.