Research ArticleNew Research, Neuronal Excitability

Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons

Christina L. Torturo, Zhen-Yu Zhou, Timothy A. Ryan and Hugh C. Hemmings
eNeuro 10 January 2019, 6 (1) ENEURO.0278-18.2018; DOI: https://doi.org/10.1523/ENEURO.0278-18.2018

Abstract

Volatile anesthetics affect neuronal signaling by poorly understood mechanisms. Activation of central dopaminergic pathways has been implicated in emergence from general anesthesia. The volatile anesthetic isoflurane differentially inhibits glutamatergic and GABAergic synaptic vesicle (SV) exocytosis by reducing presynaptic Ca2+ influx without affecting the Ca2+-exocytosis relationship, but its effects on dopaminergic exocytosis are unclear. We tested the hypothesis that isoflurane inhibits exocytosis in dopaminergic neurons. We used electrical stimulation or depolarization by elevated extracellular KCl to evoke exocytosis measured by quantitative live-cell fluorescence imaging in cultured rat ventral tegmental area neurons. Using trains of electrically evoked action potentials (APs), isoflurane inhibited exocytosis in dopaminergic neurons to a greater extent (30 ± 4% inhibition; p < 0.0001) than in non-dopaminergic neurons (15 ± 5% inhibition; p = 0.014). Isoflurane also inhibited exocytosis evoked by elevated KCl in dopaminergic neurons (35 ± 6% inhibition; p = 0.0007), but not in non-dopaminergic neurons (2 ± 4% inhibition). Pharmacological isolation of presynaptic Ca2+ channel subtypes showed that isoflurane inhibited KCl-evoked exocytosis mediated exclusively by either CaV2.1 (P/Q-type Ca2+ channels; 30 ± 5% inhibition; p = 0.0002) or by CaV2.2 (N-type Ca2+ channels; 35 ± 11% inhibition; p = 0.015). Additionally, isoflurane inhibited single AP-evoked Ca2+ influx by 41 ± 3% and single AP-evoked exocytosis by 34 ± 6%. Comparable reductions in exocytosis and Ca2+ influx were produced by lowering extracellular [Ca2+]. Thus, isoflurane inhibits exocytosis from dopaminergic neurons by a mechanism distinct from that in non-dopaminergic neurons involving reduced Ca2+ entry through CaV2.1 and/or CaV2.2.

Significance Statement

Despite their medical importance, the mechanisms of action of general anesthetics have not been fully elucidated. Isoflurane, a widely used volatile anesthetic, inhibits voltage-gated sodium channels and differentially inhibits synaptic vesicle exocytosis depending on neurotransmitter phenotype. Here, we show that in dopaminergic neurons of the ventral tegmental area isoflurane acts via a sodium channel-independent mechanism to inhibit synaptic vesicle exocytosis in proportion to reduced presynaptic Ca2+ flux mediated by CaV2.1 and/or CaV2.2, in contrast to its effects in non-dopaminergic neurons. These findings provide a synaptic mechanism for the observed role of reduced dopamine release in anesthetic-induced unconsciousness and implicate presynaptic Ca2+ channels of dopaminergic neurons as important targets of isoflurane.

Introduction

General anesthetics are essential medicines that induce a reversible state of amnesia, unconsciousness, and immobility in the face of intensely painful stimuli. Despite their widespread use in modern medicine, their mechanisms of action are not well understood (Hemmings et al., 2005b). The amnestic, hypnotic, and immobilizing effects of anesthetics differ in dose dependence, neuroanatomical regions involved, and molecular targets consistent with multiple mechanisms working in parallel to produce the state of anesthetic-induced unresponsiveness (Brown et al., 2011). However, general anesthesia can produce serious adverse side effects, including cardiovascular, respiratory, and cognitive dysfunction. It is therefore critical to identify the anesthetic mechanisms relevant for both their on-target and off-target actions, with the ultimate goals of designing safer and more selective anesthetics and of using currently available anesthetics in a rational mechanism-based manner to maximize therapeutic ratio.

Volatile anesthetics such as isoflurane modulate synaptic and extrasynaptic neurotransmission through multiple postsynaptic targets, primarily by potentiating inhibitory GABAA receptors and depressing excitatory glutamatergic transmission via ionotropic glutamate receptors (Rudolph and Antkowiak, 2004). However, the GABAA receptor antagonist bicuculline does not antagonize isoflurane-induced immobility, indicating a role for other targets in this effect (Zhang et al., 2004). The presynaptic effects of volatile anesthetics are not as well characterized as their postsynaptic effects due to the small sizes of nerve terminals and technical limitations of conventional electrophysiological techniques in recording presynaptically. Nevertheless, considerable neurochemical and neurophysiological evidence indicates that volatile anesthetics directly inhibit neurotransmitter release (Hemmings et al., 2005a,b).

Synaptic vesicle (SV) exocytosis is tightly coupled to the amount of Ca2+ entering the presynaptic bouton (Wu et al., 2004), which is determined primarily by presynaptic voltage-gated ion channels (Na+, Ca2+, and K+ channels) and modulatory receptors. Isoflurane depresses action potential (AP) amplitude in axons and boutons, which results in downstream reductions in Ca2+ influx and neurotransmitter release (Wu et al., 2004; Hemmings et al., 2005a; Ouyang and Hemmings, 2005). Isoflurane also inhibits neurotransmitter release from isolated nerve terminals with greater potency from glutamatergic than from GABAergic terminals (Westphalen and Hemmings, 2003, 2006), consistent with neurotransmitter-specific presynaptic anesthetic mechanisms. The cellular and molecular bases of this synaptic selectivity are unclear.

Voltage-gated Ca2+ channels play an essential role in neurotransmission by mediating Ca2+ influx that is closely coupled to exocytosis. Presynaptic Ca2+ channels are possible targets for inhibition of neurotransmitter release by volatile anesthetics, and are also involved in producing myocardial depression and vasodilation leading to significant cardiovascular side effects (Lynch et al., 1981; Bosnjak et al., 1991). Synaptic transmission at most central nervous system synapses is mediated by multiple Ca2+ channel subtypes that are closely coupled to SV exocytosis, most prominently CaV2.1 (P/Q-type Ca2+ channels) and CaV2.2 (N-type Ca2+ channels; Wheeler et al., 1994; Wu et al., 1999). The degree of CaV2.1 and CaV2.2 involvement differ not only between different neuron classes (Murakami et al., 2002; Evans and Zamponi, 2006) but also between nerve terminals of the same afferent axon (Reid et al., 1997; Ariel et al., 2013). Reports of the effects of volatile anesthetics on specific Ca2+ channel subtypes are inconsistent (Hall et al., 1994; Study, 1994; White et al., 2005; Joksovic et al., 2009) such that the extent to which inhibition of Ca2+ channels contributes to inhibition of SV exocytosis remains unclear.

Recent work suggests that presynaptic Ca2+ channels are not the principal targets involved in the inhibition of glutamate and GABA release by volatile anesthetics (Westphalen et al., 2013). Despite their central roles in wakefulness and arousal (Monti and Monti, 2007), few studies have investigated anesthetic effects on aminergic neurons, which have distinct mechanisms of transmitter release (Liu et al., 2018). Electrical stimulation of dopaminergic neurons in the rat ventral tegmental area (VTA), one of the principal midbrain dopaminergic nuclei (Barrot, 2014), induces emergence from isoflurane anesthesia in rats (Solt et al., 2014). We sought to clarify the neurotransmitter selectivity and presynaptic targets of volatile anesthetics by investigating the effects of isoflurane, a representative halogenated ether anesthetic, on SV exocytosis from central dopaminergic neurons to test the hypothesis that isoflurane inhibits dopamine release by a mechanism distinct from that involved in non-dopaminergic neurons.

Materials and Methods

Reagents and solutions

Isoflurane was obtained from Abbott, ω-conotoxin GVIA and ω-agatoxin IVA from Alomone Labs, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (2R)-amino-5-phosphonovaleric acid (AP5) from Tocris. All other reagents were from Sigma-Aldrich. Rat vMAT2-pHluorin was kindly provided by Robert Edwards (University of California, San Francisco, CA), and mouse VAMP-mCherry was from [Timothy Ryan]. Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM, MgCl2, 25 mM HEPES, and 30 mM glucose; pH 7.4) was used as the standard buffer in all experiments. The glutamate receptor antagonists CNQX (10 μM) and AP5 (50 μM) were added to Tyrode’s solution to block postsynaptic excitatory synaptic transmission. For single AP studies, Tyrode’s solution contained 4 mM CaCl2. Saturated stock solutions of isoflurane were prepared and diluted to 0.7 mM [two times the minimum alveolar concentration (2 MAC)] in Tyrode’s solution and into gas-tight glass syringes for focal perfusion onto imaged neurons through a 150-μm diameter polytetrafluoroethylene tube in the imaging chamber. Accounting for 10–20% loss, the predicted final concentration of isoflurane was 0.64 mM, which corresponds to 2 MAC in rats, a clinically relevant concentration equivalent to two time the ED50, corrected to the experimental temperature of 30°C (Franks and Lieb, 1993). Isoflurane was applied for 5 min before imaging to allow uptake and equilibration. At the conclusion of each experiment, a sample was taken from the chamber for analysis of delivered isoflurane concentration using a Shimadzu GC-2010 Plus gas chromatography with external standard calibration (Ratnakumari and Hemmings, 1998).

Cell culture

Experiments were conducted according to protocols approved by the [Weill Cornell Medicine] Institutional Animal Care and Use Committee and conformed to National Institutes of Health Guidelines for the Care and Use of Animals. Glial monolayers were prepared from cerebral cortex as feeder layers for primary VTA neuron cultures from Sprague Dawley postnatal day 1 male and female rats (Charles River Laboratories) as described previously (Mena et al., 1997). After 7 d in vitro (DIV), neurons were transfected with vMAT2-pHluorin or VAMP-mCherry using a DNA-calcium phosphate coprecipitation protocol (Goetze et al., 2004; Jiang and Chen, 2006) modified to ensure low density transfection so that images could be obtained from a single neuron. Data were acquired from only one neuron per coverslip to avoid the contaminating and potentially irreversible effects of each drug treatment. Each experimental group contained coverslips from two to four different batches of primary neuron cultures to minimize artifacts due to differing culture conditions.

Imaging SV exocytosis

Live-cell epifluorescence imaging employed a Zeiss Axio Observer microscope with images acquired using an Andor iXon+ CCD camera (model DU-897E-BV) and APs were evoked with 1-ms current pulses delivered via platinum-iridium electrodes. Depolarization with elevated K+ Tyrode’s solution (50 mM KCl substituted for 50 mM NaCl and buffered to pH 7.4) was used to evoke SV exocytosis independent of Nav involvement (57). Elevated K+ Tyrode’s solution was applied onto imaged neurons using a pressurized injector (PDES System, ALA) for 4 s at 29 μl/s as the chamber was continuously perfused with Tyrode’s solution with or without added drugs. Fluorescence data were acquired as described, and total pool (TP) of SVs was identified by perfusion with Tyrode’s solution containing 50 mM NH4Cl (substituted for 50 mM NaCl and buffered to pH 7.4).

Imaging calcium influx

VAMP-mCherry, a red fluorescent protein fused to VAMP (vesicle associated membrane protein), was used to identify synaptic boutons for Ca2+ imaging experiments. Transfected neurons were loaded with 7 μM Fluo-5F AM, incubated for 10 min at 30°C, and washed by superfusion with Tyrode’s solution for 15 min. Neurons were stimulated with a single AP 5 times at 2-min intervals during superfusion with Tyrode’s solution containing 2 mM Ca2+ with or without 2 MAC isoflurane.

Immunocytochemistry

Post hoc immunolabelling with mouse anti-tyrosine hydroxylase (TH) monoclonal antibody (MAB318, Millipore) was used to identify dopaminergic neurons following live cell imaging. Fixed neurons were immunolabelled with either a 1:1000 dilution of Alexa Fluor 594 goat anti-mouse (for SV exocytosis experiments using vMAT2-pHluorin) or Alexa Fluor 488 goat anti-mouse (for Ca2+ imaging experiments). Imaged neurons were identified by coordinates on the coverslips and photographed.

Image and statistical analysis

Fluorescence data were analyzed in ImageJ (http://rsb.info.nih.gov/ij) with a custom plug-in (http://rsb.info.nih.gov/ij/plugins/time-series.html). Transfected boutons were selected as regions of interest (ROIs) based on their response to 50 mM NH4Cl for SV exocytosis experiments or labeling with VAMP-mCherry for Ca2+ measurements. Each bouton was subjected to a signal-to-noise ratio (SNR) calculation based on its response to the first control electrical stimulation, and ΔF was calculated as the difference of the average intensities between Fpeak and Fbaseline. Fluorescence intensity changes for Ca2+ measurements were normalized to baseline as ΔF/F: (Fpeak – Fbaseline)/Fbaseline. Boutons with SNR > 5 were used in the analysis. Data are expressed as mean ± SD. To allow expression of inhibition or potentiation, drug effects are shown as a percentage of either TP or control response. Statistical significance was determined by paired or unpaired two-tailed or one-tailed Student’s t tests and by paired or unpaired one-way ANOVA with Tukey’s post hoc test, with p < 0.05 considered significant. Normality was assayed using the Shapiro–Wilk normality test. All statistical data are displayed in Table 1. Statistical analysis and graph preparation used GraphPad Prism v7.05 (GraphPad Software, Inc.).

Results

We used high resolution microscopy to quantify exocytosis at dopaminergic nerve terminals by the fluorescence change of pH-sensitive pHluorin fused to the luminal domain of the vesicular mononamine transporter vMAT2 (Anantharam et al., 2010; Pan and Ryan, 2012). Cultured rat midbrain neurons transfected with vMAT2-pHluorin were stimulated with trains of 100 APs at 10 Hz to elicit SV exocytosis (Fig. 1A). Increases in fluorescence (ΔF) following stimulation indicate alkalization of intravesicular pHluorin due to SV exocytosis. The difference between baseline and stimulus-evoked peak fluorescence reflects the amount of SV exocytosis; quenching of fluorescence in the post-stimulus period indicates SV endocytosis and re-acidification (Sankaranarayanan et al., 2000; Atluri and Ryan, 2006; Fig. 1B). The biosensor vMAT2-pHluorin reliably measured SV exocytosis over time with minimal decay in signal over the course of three control stimulations (stimulation 1 = 6.7 ± 0.9% of TP; stimulation 2 = 6.9 ± 0.9% of TP; stimulation 3 = 6.8 ± 0.9% of TP; n = 8; p = 0.93). Fluorescence data from each cell were normalized to the total SV pool defined by perfusion with 50 mM NH4Cl, which alkalizes the acidic SV interior and unquenches pHlourin fluorescence of the entire SV pool (Fig. 1A,B). Both dopaminergic and non-dopaminergic neurons can be transfected by vMAT2-pHluorin; dopaminergic neurons were positively identified by post hoc immunolabeling with mouse anti-TH (Fig. 1C).

Figure 1.
Figure 1.

Measurement of SV exocytosis in dopaminergic neurons. A, Representative fluorescence images of a cultured VTA neuron transfected with vMAT2-pHluorin at rest (left), after stimulation with a train of 100 APs delivered at 10 Hz (middle), and after perfusion with 50 mM NH4Cl in Tyrode’s solution (with an equivalent reduction in NaCl) to alkalinize the SV interior (right). Scale bar, 10 μm. B, Representative traces of fluorescence responses to 100 APs at 10 Hz after 5 s of baseline fluorescence values (top), and to perfusion with 50 mM NH4Cl (bottom). Vertical arrow represents the change in fluorescence (ΔF). Blue bars indicate electrical stimulation (top) or NH4Cl perfusion that defines the TP (bottom). C, Fluorescence images of a neuron transfected with vMAT2-pHluorin (green, left) and stained post hoc with anti-TH (red, middle). Composite image shows overlap of vMAT2-pHluorin and TH indicating this neuron is dopaminergic (right). Scale bar, 10 μm.

Isoflurane inhibits SV exocytosis in dopaminergic neurons

Isoflurane at an immobilizing concentration (0.7 mM) inhibited SV exocytosis evoked by trains of 100 APs at 10 Hz in both dopaminergic (TH+) neurons and non-dopaminergic (TH–) neurons (Fig. 2). In dopaminergic neurons, control exocytosis was 9.4 ± 0.8% of TP, which was reduced to 6.7 ± 0.7% of TP by 0.7 mM (∼2× ED50) isoflurane (30 ± 4% inhibition; p < 0.0001; n = 12)a. In non-dopaminergic neurons, control exocytosis was 10.4 ± 0.9% of TP, which was reduced to 8.6 ± 0.7% of TP by isoflurane (15 ± 5% inhibition; p = 0.014; n = 9)b. The degree of inhibition of exocytosis was greater in dopaminergic neurons (p = 0.017; Fig. 2D)c. The time constant of pHluorin recovery was not significantly affected by isoflurane (DA Ctrl = 53 ± 15 s vs Iso = 45 ± 17 s; n = 12; non-DA Ctrl = 58 ± 11 s vs Iso = 46 ± 13 s; n = 9).

Figure 2.
Figure 2.

Isoflurane differentially inhibits SV exocytosis in dopaminergic and non-dopaminergic neurons. A, Schematic of protocol used to assess the effect of isoflurane on SV exocytosis. 100 APs were delivered at 10 Hz (blue arrows) under control conditions, followed by perfusion with 0.7 mM (2 MAC) isoflurane for 5 min (red bar), and a second stimulation of 100 APs at 10 Hz in the presence of isoflurane. Lastly, NH4Cl was perfused to determine the TP (blue bar). B, C, Mean values of vMAT2-pHluorin response amplitudes in control and isoflurane-treated neurons stimulated with 100 APs at 10 Hz; ****p < 0.0001; *p < 0.05 by two-tailed paired t test. Representative raw traces from a dopaminergic (DA) and a non-dopaminergic (non-DA) neuron are shown. D, The effect 0.7 mM isoflurane on 100 AP-evoked exocytosis was greater in dopaminergic than in non-dopaminergic neurons; *p < 0.05 by one-tailed t test.

Isoflurane effect is Na+ channel independent in dopaminergic neurons

Ca2+-dependent SV exocytosis evoked by elevated extracellular KCl occurs by sustained depolarization that is independent of NaV activation: it is insensitive to the specific Nav blocker tetrodotoxin (TTX), in contrast to phasic AP-evoked SV exocytosis, which is completely blocked by TTX (Westphalen and Hemmings, 2003). Superfusion of TTX abolished SV exocytosis evoked electrically to –0.5 ± 0.2% of TP, yet had no effect on the response to elevated KCl (control with KCl = 12.9 ± 1.8% of TP, KCl with TTX = 13.3 ± 1.7% of TP; p = 0.92; n = 6; Fig. 3)d. We compared the effects of isoflurane on SV exocytosis evoked by elevated KCl, which directly activates Ca2+ channels, to SV exocytosis evoked electrically to determine whether isoflurane acts upstream or downstream of Ca2+ entry (Fig. 4A). We used a KCl concentration that evoked similar peak SV exocytosis compared to that obtained with the 100 AP stimulus train. Isoflurane inhibited elevated KCl-evoked exocytosis in dopaminergic neurons from 11.4 ± 1.3% of TP in control to 7.8 ± 1.5% of TP with isoflurane (35 ± 6% inhibition; p = 0.0007; n = 8; Fig. 4B,D)e. In contrast, isoflurane did not inhibit KCl-evoked SV exocytosis in non-dopaminergic neurons: 10.4 ± 0.9% of TP in control and 10.3 ± 1.1% of TP with isoflurane (2 ± 4% inhibition; p = 0.72; n = 6; Fig. 4C)f. Thus, isoflurane inhibited SV exocytosis in dopaminergic neurons by an NaV-independent pathway, in contrast to its Nav-dependent inhibition of SV exocytosis in non-dopaminergic neurons (Wu et al., 2004; Westphalen et al., 2013; Baumgart et al., 2015).

Figure 3.
Figure 3.

Elevated KCl depolarization-evoked SV exocytosis is NaV independent. A, Schematic of the protocol used to determine the effect of TTX on exocytosis evoked by elevated KCl or electrical stimulation. B, Representative traces of vMAT2-pHluorin response to elevated KCl depolarization or electrical stimulation in the absence or presence of 250 nM TTX (left). Mean values of vMAT2-pHluorin response amplitudes (right). ns, not significant by paired one-way ANOVA with Tukey’s post hoc test.

Figure 4.
Figure 4.

Isoflurane differentially inhibits SV exocytosis in dopaminergic neurons via a Nav-independent mechanism. A, Schematic of protocol using a pulse of 50 mM KCl (with an equivalent reduction in NaCl, green bars) to induce exocytosis in the absence or presence of isoflurane. B, C, Mean values of vMAT2-pHluorin response amplitudes in control and 0.7 mM isoflurane-treated neurons stimulated with elevated KCl. Traces show representative raw traces from a dopaminergic (DA) and non-dopaminergic (non-DA) neuron; ***p < 0.001; ns, not significant by two-tailed paired t test. D, Comparison of the effect of isoflurane on KCl-evoked exocytosis; ***p < 0.001 by one-tailed t test.

Ca2+ channel subtypes mediating exocytosis in dopaminergic neurons

The role of specific Ca2+ channel subtypes in isoflurane inhibition of SV exocytosis was studied using the subtype-specific neurotoxin ω-conotoxin GVIA to block CaV2.2 or ω-agatoxin IVA to block CaV2.1 (Fig. 5A). Conotoxin alone inhibited SV exocytosis by 43 ± 3% (n = 9) in dopaminergic neurons and by 68 ± 3% (n = 5) in non-dopaminergic neurons, consistent with a greater contribution of CaV2.2 in non-dopaminergic neurons than in dopaminergic neurons (p = 0.015; Fig. 5B)g. The CaV2.1 blocker agatoxin alone inhibited SV exocytosis by 83 ± 5% (n = 7) in dopaminergic neurons and by 63 ± 11% (n = 6) in non-dopaminergic neurons, confirming a greater contribution of CaV2.1 to SV exocytosis in dopaminergic neurons (conotoxin inhibition = 43 ± 3%; agatoxin inhibition = 83 ± 5%; p < 0.0001)h. In non-dopaminergic neurons, there was no significant difference in inhibition by conotoxin or agatoxin (p = 0.99)i, indicating similar contributions by both CaV2.1 and CaV2.2. There was no effect of the L-type Ca2+ channel inhibitor nimodipine (10 µM, Nimo) on SV exocytosis from dopaminergic or non-dopaminergic neurons (DA Ctrl = 7.8 ± 0.4% vs DA + Nimo = 7.2 ± 0.5% of TP, n = 7, p = 0.139; non-DA Ctrl = 10.1 ± 1.8% vs non-DA + Nimo = 9.8 ± 1.9% of TP, n = 5, p = 0.122). SV exocytosis in both dopaminergic and non-dopaminergic neurons was mediated exclusively by CaV2.1 and CaV2.2 since conotoxin and agatoxin together completely blocked SV exocytosis (Fig. 5B). A bouton by bouton analysis from all recorded neurons examined the effect of conotoxin (Fig. 5C) and agatoxin (Fig. 5D) in dopaminergic neurons and showed that the effects of the toxins correlated to the averaged effects (Fig. 5B).

Figure 5.
Figure 5.

CaV2.1 and CaV2.2 contribute to SV exocytosis in VTA neurons. A, Schematic of protocol using trains of 100 APs in the absence (black) or presence of ω-conotoxin GVIA (cono, 1 μM, purple bar) alone, ω-agatoxin IVA (aga, 400 nM, orange bar) alone, or both toxins together. B, Comparison of the effect of conotoxin and agatoxin on dopaminergic and non-dopaminergic neurons. The combination of conotoxin and agatoxin abolished exocytosis in both dopaminergic (DA) and non-dopaminergic (non-DA) neurons; ****p < 0.0001; *p < 0.05; ns, not significant by unpaired one-way ANOVA, Tukey’s post hoc test. C, Histogram displaying the bouton-by-bouton effect of conotoxin alone on exocytosis in dopaminergic neurons for all boutons (n = 9 neurons). Data have been constrained from 0% to 100% of control exocytosis. D, Histogram displaying the bouton-by-bouton effect of agatoxin alone on exocytosis in dopaminergic neurons for all of boutons (n = 7 neurons). Data have been constrained from 0% to 100% of control exocytosis.

Isoflurane inhibits exocytosis mediated by CaV2.1 and CaV2.2

We investigated the isoflurane sensitivity of SV exocytosis mediated by either CaV2.1 or CaV2.2 using pharmacological isolation (Fig. 6A). To examine the effect of isoflurane on Ca2+ channels without contributions by inhibition of upstream Na+ channels, we evoked exocytosis with elevated KCl depolarization. Conotoxin reduced KCl-evoked exocytosis to 81 ± 5% of control, and conotoxin plus isoflurane reduced exocytosis to 57 ± 7% of control (30 ± 5% inhibition; p = 0.0002, n = 5; Fig. 6B)j. A similar degree of isoflurane inhibition was obtained using 100 AP electrical stimulation with conotoxin and isoflurane (Cono = 8.3 ± 1.0% vs Cono + Iso = 5.4 ± 0.7% of TP, 35 ± 5% inhibition, n = 8, p < 0.001). Agatoxin reduced KCl-evoked exocytosis to 74 ± 10% of control, and agatoxin plus isoflurane reduced exocytosis to 51 ± 13% of control (35 ± 11% inhibition; p = 0.015; n = 6; Fig. 6C)k. There was no significant difference in the degree of isoflurane inhibition of CaV2.1 versus CaV2.2-mediated KCl-evoked exocytosis in dopaminergic neurons (p = 0.37; Fig. 6D)l.

Figure 6.
Figure 6.

Isoflurane inhibits elevated KCl-evoked SV exocytosis mediated by CaV2.1 or CaV2.2 in dopaminergic neurons. A, Schematic of protocol using depolarizing pulses of 50 mM KCl with 1 μM conotoxin and 0.7 mM isoflurane or with 400 nM agatoxin and 0.7 mM isoflurane in dopaminergic neurons. B, Mean values of vMAT2-pHluorin response amplitudes in conotoxin and conotoxin + isoflurane treated dopaminergic neurons stimulated with elevated KCl; ***p < 0.001 by two-tailed paired t test. C, Mean values of vMAT2-pHluorin response amplitudes in agatoxin and agatoxin + isoflurane treated dopaminergic neurons stimulated with elevated KCl; *p < 0.05 by two-tailed paired t test. D, Comparison of the effect of isoflurane on CaV2.1 and CaV2.2 mediated elevated KCl-evoked exocytosis. ns, not significant by one-tailed t test.

Isoflurane inhibits exocytosis by reducing Ca2+ entry

Isoflurane inhibits SV exocytosis in glutamatergic and GABAergic hippocampal neurons by reducing Ca2+ influx without affecting Ca2+ sensitivity indicated by the relationship between intracellular Ca2+ concentration and SV exocytosis (Baumgart et al., 2015). We examined this in dopaminergic neurons by comparing the effects of reduced extracellular Ca2+ concentration ([Ca2+]e) on SV exocytosis and Ca2+ influx using single AP stimuli to determine Ca2+ sensitivity (Fig. 7A,C). Isoflurane inhibited single AP-evoked SV exocytosis by 34 ± 6% (0.89 ± 0.19% of TP for 4 mM [Ca2+]e control vs 0.58 ± 0.12% of TP with isoflurane; p = 0.005)m. This reduction in exocytosis was mimicked by reducing [Ca2+]e from 4 mM to 2 mM in the absence of isoflurane. Exocytosis in 2 mM [Ca2+]e was 0.51 ± 0.13% of TP (44 ± 5% reduction vs 4 mM [Ca2+]e), which was not significantly different from the inhibition by isoflurane in 4 mM [Ca2+]e (p = 0.63; n = 6; Fig. 7B)n. A comparable effect was observed using the Ca2+ indicator Fluo-5F to measure changes in intracellular [Ca2+] (Fig. 7D–G), which is proportional to presynaptic Ca2+ influx (Hoppa et al., 2012). Isoflurane inhibited Ca2+ influx by 41 ± 3% in 4 mM Ca2+ (0.25 ± 0.02 ΔF/Fo for 4 mM [Ca2+]e control vs 0.15 ± 0.02 ΔF/Fo with isoflurane; p = 0.0003)o. Presynaptic Ca2+ influx was inhibited to the same degree by reducing [Ca2+]e from 4 mM to 2 mM: Ca2+ influx in 2 mM [Ca2+]e was 0.15 ± 0.01 ΔF/Fo (36 ± 8% reduction vs 4 mM [Ca2+]e). Additionally, isoflurane inhibited Ca2+ influx to a similar degree in 2 mM Ca2+ (42 ± 3%; p = 0.0004)p, indicating the noncompetitive nature of isoflurane with respect to the ability of Ca2+ ions to flow through Ca2+ channels. There was no significant difference between Ca2+ influx with 2 mM [Ca2+]e compared with 4 mM [Ca2+]e plus isoflurane (p = 0.99; n = 5; Fig. 7G)q.

Figure 7.
Figure 7.

Isoflurane inhibits SV exocytosis in dopaminergic neurons by reducing Ca2+ influx. A, Schematic of single AP (1 AP) evoked exocytosis protocol with 2 mM [Ca2+]e (black bar), 4 mM [Ca2+]e (gray bar), and 2 MAC isoflurane in 4 mM [Ca2+]e (red bar). B, Ensemble average traces and mean values of 1 AP-stimulated exocytosis reported by vMAT2-pHluorin; **p < 0.01; ns, not significant by paired one-way ANOVA, Tukey’s post hoc test. C, Schematic of the Ca2+ influx protocol with Fluo-5F (yellow bar) followed by washout and 1 AP stimulation with 4 mM [Ca2+]e (gray bar), 0.7 mM isoflurane in 4 mM [Ca2+]e (red bar) or 2 mM [Ca2+]e (black bar). D, Fluorescence microscopy image of VAMP-mCherry transfected neuron from which Ca2+ signals were recorded. Scale bar, 5 μm. E, Fluorescence traces from individual boutons of the neuron in panel D with and without isoflurane in 4 mM [Ca2+]e. Mean responses are shown in bold. F, Ensemble average traces. G, Mean values of 1 AP-stimulated Ca2+ influx reported by Fluo-5F in 4 mM [Ca2+]e, isoflurane in 4 mM [Ca2+]e, 2 mM [Ca2+]e, or isoflurane in 2 mM [Ca2+]e(right); ***p < 0.001; ns, not significant by paired one-way ANOVA with Tukey’s post hoc test.

Discussion

Isoflurane inhibited SV exocytosis from cultured dopaminergic neurons by reducing Ca2+ entry though both CaV2.1 and CaV2.2 by a mechanism that is independent of Na+ channel activation (Fig. 8). This is in contrast to the predominant Na+ channel-dependent mechanism observed for release of glutamate or GABA in non-dopaminergic cortical and hippocampal neurons (Westphalen et al., 2010; Baumgart et al., 2015). These findings reveal important neurotransmitter-selective differences in the presynaptic mechanisms of isoflurane, a clinically essential volatile anesthetic. These differences provide a pharmacological rationale for developing novel anesthetics targeting specific anesthetic endpoints mediated by a single neurotransmitter system, for example dopaminergic control of emergence from unconsciousness.

Figure 8.
Figure 8.

Presynaptic mechanisms of isoflurane. In non-dopaminergic (non-DA) neurons, isoflurane inhibits SV exocytosis by inhibiting Na+ channels to reduce nerve terminal excitability. In dopaminergic (DA) neurons, isoflurane can also reduce SV exocytosis by a Na+ channel-independent mechanism mediated by inhibition of CaV2.1 and CaV2.2. The fluorescence of vMAT2-pHluorin is quenched by the acidic (∼pH 5.5) SV interior. On exocytosis and fusion with the plasma membrane, vMAT2-pHluorin fluoresces on exposure to the pH neutral (∼pH 7.4) synaptic cleft.

Relationship between Ca2+ channel subtypes and exocytosis

The Ca2+ channel subtypes present in dopaminergic neuron somata identified using whole-cell voltage-clamp recordings do not necessarily reflect the presynaptic Ca2+ channels involved in SV exocytosis. In rat dopaminergic midbrain neurons, somatic Ca2+ currents are inhibited by the L-type channel blocker nimodipine (by 28%), the CaV2.2 blocker conotoxin (by 22%), and the CaV2.1 blocker agatoxin (by 37%; Cardozo and Bean, 1995). However, synaptic boutons in rat dopaminergic neurons are too small for such direct electrophysiological recording of presynaptic Ca2+ currents. We used high-resolution live-cell imaging to measure SV exocytosis and Ca2+ influx, employing specific Ca2+ channel toxins to determine contributions of the major presynaptic Ca2+ channel subtypes. SV exocytosis in rat dopaminergic VTA neurons was mediated exclusively by CaV2.1 and CaV2.2, with CaV2.1 predominating. Using KCl-induced depolarization to evoke SV exocytosis independent of Na+ channel involvement, isoflurane inhibited exocytosis mediated by either CaV2.1 or CaV2.2 to a similar degree, suggesting a lack of subtype selectivity. Alternatively, the effects of isoflurane are mediated via an unknown mechanism distinct to dopaminergic VTA neurons. One possibility is that K+ channel activity alters the resting membrane potential and therefore modifies open probability of CaV2.1 and CaV2.2.

Isoflurane inhibited SV exocytosis in dopaminergic neurons evoked by electrical stimulation of APs or elevated KCl depolarization, in contrast to non-dopaminergic neurons in which isoflurane inhibited AP-evoked but not KCl-evoked exocytosis. AP-evoked exocytosis requires activation of Na+ channels to sufficiently depolarize boutons and activate the presynaptic Ca2+ channels linked to exocytosis. Clamped depolarization by elevated KCl is less physiologic than repetitive electrical stimulation by causing sustained depolarization (Tibbs et al., 1989), which could alter Ca2+ channel relationship to exocytosis. This is suggested by the finding that with electrical stimulation conotoxin inhibited exocytosis in dopaminergic neurons by ∼40% and agatoxin inhibited by ∼80%, while with KCl-evoked depolarization both conotoxin and agatoxin inhibited exocytosis by 20–30%. The linkage between SV exocytosis to critical presynaptic Ca2+ channels in dopaminergic neurons is selectively sensitive to isoflurane, since isoflurane inhibition of KCl-evoked exocytosis was observed in dopaminergic, but not in non-dopaminergic, neurons. This fundamental neurotransmitter-specific difference in the relationship between Ca2+ channels and exocytosis results in profound neurotransmitter-specific differences in anesthetic sensitivity with potential neuropharmacological implications. This neurochemical difference is preserved in nigrostriatal dopaminergic nerve terminals prepared from rat striatum in which isoflurane also inhibits dopamine release via a Na+ channel-independent mechanism, an action that might contribute to the motor effects of anesthetics (Westphalen et al., 2013). The cellular and molecular attributes underlying this selective anesthetic pharmacology in dopaminergic neurons are unknown and await further characterization of the neurobiology of dopaminergic compared to non-dopaminergic neurons.

Mechanisms of dopamine SV exocytosis

Monoamine neurotransmitters such as dopamine are packaged into both small SVs and large dense core vesicles (LDCVs) for release. The biosensor vMAT2-pHluorin labels both small SVs and LDCVs (Fei et al., 2008); however, only small SVs localize to presynaptic active zones of synaptic boutons for exocytosis, while LDCVs engage primarily in extrasynaptic exocytosis (Thureson-Klein, 1983; Südhof and Rizo, 2012). Moreover, the kinetics of SV exocytosis from small SVs and LDCVs are distinct: small SVs fuse within 1 ms of Ca2+ channel opening (Cohen et al., 1991), while LDCV fusion is 100-fold slower and therefore less tightly regulated to AP stimulation (Almers, 1990; Martin, 1994). Based on these characteristics, the SV exocytosis measured by the vMAT2-pHluorin method is primarily from small SVs (Pothos et al., 2000; Leenders et al., 2002). Anesthetic effects on asynchronous neuronal LDCV exocytosis might involve distinct mechanisms.

Neuroendocrine cells such as adrenal chromaffin cells or PC12 cells are frequently used to study catecholaminergic SV exocytosis but exhibit release mostly from LDCVs (Voets et al., 2001). This is an important distinction as the subcellular and molecular organization of the secretory machinery differ between dopaminergic neurons and neuroendocrine cells, which makes the latter poor models for midbrain neurons. For example, neuroendocrine cells do not have active release zones co-localized with Ca2+ microdomains, and the functional linkage of Ca2+ channels to release sites is not as tight as in neurons (Wu et al., 2009). Moreover, the Ca2+ channel subtypes linked to SV exocytosis differ between small SVs and LDCVs, with L-type channels closely linked to LDCV exocytosis (Park and Kim, 2009). In contrast, we found dopaminergic SV exocytosis to be independent of L-type Ca2+ channels.

Differences between dopaminergic and non-dopaminergic neurons

When comparing the effects of isoflurane on dopaminergic and non-dopaminergic neurons it is important to consider that vMAT2 is not endogenously expressed in non-dopaminergic neurons (Yoo et al., 2016). However, vMAT2-pHluorin is still effective in measuring SV exocytosis in non-dopaminergic neurons due to ectopic expression. Transfection of vMAT2-pHluorin involves overexpression of vMAT2, which can increase quantal size, but this does not interfere with its use as an indicator of SV fusion (Pothos et al., 2000; Erickson et al., 2006).

In contrast to dopaminergic neurons, elevated KCl-evoked SV exocytosis from non-dopaminergic neurons was insensitive to isoflurane. This is consistent with previous observations of a NaV-dependent/Cav-independent mechanism for rat glutamatergic and GABAergic hippocampal neurons (Hemmings et al., 2005a; Westphalen and Hemmings, 2006), despite their expression of both CaV2.1 and CaV2.2 (Qian and Noebels, 2001; Kamp et al., 2012). There are other important differences between dopaminergic and non-dopaminergic neurons that could explain their differential sensitivities to anesthetics. Non-dopaminergic neurons from the VTA are primarily GABAergic, some of which are capable of co-releasing glutamate (Carr and Sesack, 2000; Creed et al., 2014; Barker et al., 2016). In cultured rat hippocampal neurons, isoflurane inhibits SV exocytosis from glutamatergic boutons more potently than from GABAergic boutons due to a greater reduction in presynaptic Ca2+ influx (Baumgart et al., 2015), indicating that GABAergic neurons are less sensitive to isoflurane. This neuronal phenotypic difference in anesthetic sensitivity is consistent with the data presented here, as isoflurane more potently inhibited electrically-evoked SV exocytosis in dopaminergic neurons than in non-dopaminergic VTA neurons. It is likely that the non-dopaminergic VTA neurons were primarily GABAergic given their abundance in this nucleus. These neurotransmitter-specific differences in presynaptic sensitivity to isoflurane are likely due to differential presynaptic expression of specific ion channel subtypes (Johnson et al., 2017) with different anesthetic sensitivities that contribute to SV exocytosis.

Considering their numerous possible subunit compositions and splice variants, different ion channel subtypes and variants could contribute to presynaptic Ca2+ entry and SV release in different boutons (Meir et al., 1999). Differences in ion channel expression and degree of functional linkage (tight or loose) between Ca2+ entry and SV exocytosis also exist between various neuronal phenotypes (Eggermann et al., 2011). Differences in expression of various presynaptic Ca2+ binding proteins (e.g., calmodulin, present in all neurons, or calbindin, selectively expressed in some dopaminergic neurons) might also determine observed nerve terminal-specific differences in anesthetic sensitivity (Pan and Ryan, 2012). Further studies are necessary to determine the molecular specializations that underlie these presynaptic differences in anesthetic sensitivity.

Role of dopaminergic neurons in general anesthesia

Mammalian dopaminergic neurons are located primarily in the substantia nigra pars compacta and the VTA. They project widely to forebrain regions including the dorsal striatum and nucleus accumbens, where dopamine release is essential to motor function and motivated behaviors, respectively (Wise, 2004). Electrical stimulation of the VTA, but not of the substantia nigra, facilitates emergence from isoflurane anesthesia in adult rats (Solt et al., 2014; Taylor et al., 2016). This effect is mediated by D1 dopamine receptors since selective pharmacological activation of D1, but not D2, receptors induces emergence from isoflurane anesthesia (Taylor et al., 2013). Moreover, the dopamine transporter (DAT) inhibitors methylphenidate and dextro-amphetamine restore conscious behaviors in rats anesthetized with isoflurane, propofol, or sevoflurane (Solt et al., 2011; Chemali et al., 2012; Kenny et al., 2015). Our findings that isoflurane inhibits SV exocytosis from midbrain dopamine neurons provides mechanistic support for the hypothesis that reduced dopamine signaling is associated with isoflurane-induced unconsciousness through reduced D1 receptor activation due to reduced dopamine exocytosis.

Conclusions

Isoflurane inhibits SV exocytosis from rat dopaminergic neurons through direct inhibition of Ca2+ influx mediated by CaV2.1 and/or CaV2.2 independent of Na+ channel involvement. This neurotransmitter-selective presynaptic mechanism provides a molecular substrate for the role for dopaminergic VTA neurons in modulating isoflurane-induced unconsciousness. Improved understanding of these mechanisms is critical to elucidating how general anesthetics work to optimize their safe use and develop more specific drugs with fewer adverse effects.

View this table:
Table 1

Statistical Data

Acknowledgments

Acknowledgments: We thank Robert Edwards for generously providing plasmids; and Joel Baumgart, David Sulzer, Yelena Canter, and Ping-Yue Pan for technical advice. We thank members of the Hemmings and Ryan laboratories for constructive comments and critical reading of this manuscript.

Footnotes

  • H.C.H. is Editor-in-Chief of the British Journal of Anaesthesia and consultant for Elsevier. All other authors declare no competing financial interests.

  • This work was supported by the National Institutes of Health Grant GM58055 (to H.C.H.).

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.

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Synthesis

Reviewing Editor: Katalin Toth, Universite Laval

Decisions are customarily a result of the Reviewing Editor and the peer reviewers coming together and discussing their recommendations until a consensus is reached. When revisions are invited, a fact-based synthesis statement explaining their decision and outlining what is needed to prepare a revision will be listed below. The following reviewer(s) agreed to reveal their identity: NONE.

The current manuscript addresses how the volatile anesthetic isoflurane affects synaptic transmission in rat VTA dopaminergic neurons. The authors note significant differences between dopaminergic and non-dopaminergic neurons. The study is technically solid and the results support the authors' conclusions. The potential impact of the manuscripts should be increased by providing information on the following 3 main questions:

1. The authors should carry out a dose response curve to measure the calcium sensitivity of release and determine how the isoflurane impacts the Ca2+ sensitivity of release in dopaminergic neurons.

2. Anesthetic concentration: A dose-response relationship would be useful to determine the magnitude of the effect at lower doses of isoflurane that are sufficient to produce unconsciousness.

3. Please, elaborate in the text about the potential cell-types of the non-dopaminergic neurons.

Reviewers also identified few additional points that should be addressed by the authors:

Rev.#1

In this study the authors found that isoflurane significantly reduces presynaptic dopamine release from cultured rat VTA neurons, through direct inhibition of calcium influx mediated by P/Q-type or N-type calcium channels. Dopamine release from the VTA was previously shown to reverse the state of general anesthesia in rats and mice, and this study suggests that inhibition of dopamine release by VTA neurons may be one mechanism by which isoflurane produces unconsciousness. The manuscript is well written and easy to follow. Volatile anesthetics can be difficult to work with, but the authors took the appropriate steps to ensure that evaporative loss of isoflurane was minimized, and made the necessary calculations to account for the difference between experimental and physiological temperature. My main concern is that 2 MAC was used as the anesthetic concentration. This is higher than the dose required for unconsciousness, which is less than 1 MAC. Considering that the dose of anesthetic was quite high, the observed reduction in dopamine release was rather modest. A dose-response relationship would be useful to determine the magnitude of the effect at lower doses of isoflurane that are sufficient to produce unconsciousness.

Rev. #2.

1) The authors should provide images of the calcium imaging experiments and demonstrate that the Ca2+ signals were not contaminated by dendritic Ca2+ signals.

2) The results are based on averaged values and do not contain any single bouton analysis. It would be helpful to see some single bouton analysis for Figure 5 and Figure 6.

3) it needs to be specified how many different coverslips were used for each experiment. It is well known that primary cultured neurons can have artifacts due to culturing conditions. Therefore, it is important for the reader to know how many different coverslips and if they were from different batches of primary cultured neurons.

4) Data listed as data not shown in the manuscript. Should be shown.

5) N Values are not reported for Figure 5.

6) Values in all graphs should be shown as a bar graph with scatter plot, this will give a better approximation of the data. In addition, normalization graphs of Aga and Conotoxin sensitivity of the individual neurons in Figure 5 and 6 should be plotted.

7) The term coupling should be avoided in the manuscript. Coupling implies physical distance between channels and vesicles. This was not measured in this manuscript. It is better to say exocytosis being P/Q or N type dependent.

8) Please replace P/Q and N type labeling with current voltage gated calcium channel nomenclature Cav2.1 and Cav2.1 etc etc....

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eneuro: 6 (1)
eNeuro
Vol. 6, Issue 1
January/February 2019
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Isoflurane Inhibits Dopaminergic Synaptic Vesicle Exocytosis Coupled to CaV2.1 and CaV2.2 in Rat Midbrain Neurons
Christina L. Torturo, Zhen-Yu Zhou, Timothy A. Ryan, Hugh C. Hemmings
eNeuro 10 January 2019, 6 (1) ENEURO.0278-18.2018; DOI: 10.1523/ENEURO.0278-18.2018
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