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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Anesthesiology. 2019 Jul;131(1):94–104. doi: 10.1097/ALN.0000000000002753

Isoflurane Modulates Hippocampal CA1 Pyramidal Neuron Excitability by Inhibition of both Transient and Persistent Sodium Currents in Mice

Wenling Zhao 1,2,#, Mingyue Zhang 1,2,#, Jin Liu 1,2, Peng Liang 2, Rurong Wang 2, Hugh C Hemmings 3, Cheng Zhou 1,2,*
PMCID: PMC6586485  NIHMSID: NIHMS1525294  PMID: 31166240

Abstract

Background:

Volatile anesthetics inhibit presynaptic voltage-gated sodium channels to reduce neurotransmitter release, but their effects on excitatory neuron excitability by sodium current inhibition are unclear. We hypothesized that inhibition of transient and persistent neuronal sodium currents by the volatile anesthetic isoflurane contributes to reduced hippocampal pyramidal neuron excitability.

Methods:

Whole-cell patch-clamp recordings of sodium currents of hippocampal CA1 pyramidal neurons were performed in acute mouse brain slices. The actions of isoflurane on both transient and persistent sodium currents were analyzed at clinically relevant concentrations of isoflurane.

Results:

The IC50 (median inhibitory concentration) of isoflurane for inhibition of transient sodium currents (INaT) was 1.0 ± 0.3 mM (~3.7 minimum alveolar concentration, MAC) from a physiological holding potential of −70 mV. Currents from a hyperpolarized holding potential of −120 mV were minimally inhibited (IC50 = 3.6 ± 0.7 mM, ~13.3 MAC). Isoflurane (0.55 mM; ~2 MAC) shifted the voltage-dependence of steady-state inactivation by −6.5 ± 1.0 mV (n = 11, P < 0.0001), but did not affect the voltage-dependence of activation. Isoflurane increased the time constant for sodium channel recovery from 7.5 ± 0.6 to 12.7 ± 1.3 ms (n = 13, P < 0.001). Isoflurane also reduced persistent sodium current (INaP) density (IC50 = 0.4 ± 0.1 mM, ~1.5 MAC) and resurgent currents. Isoflurane (0.55 mM; ~2 MAC) reduced action potential amplitude, and hyperpolarized resting membrane potential from −54.6 ± 2.3 to −58.7 ± 2.1 mV (n = 16, P = 0.001).

Conclusions:

Isoflurane at clinically relevant concentrations inhibits both transient and persistent sodium currents in hippocampal CA1 pyramidal neurons. These mechanisms may contribute to reductions in both hippocampal neuron excitability and synaptic neurotransmission.

Introduction

While general anesthetics have been used clinically for over 170 years, their molecular and cellular mechanisms of action are still not clear. Compared to intravenous general anesthetics like propofol, the molecular mechanisms of volatile anesthetics such as isoflurane and sevoflurane are more complex since they interact with multiple molecular targets.1,2 Volatile anesthetics produce the desirable pharmacological endpoints of amnesia, unconsciousness and immobility, but also unwanted side effects including respiratory and cardiovascular depression and developmental neurotoxicity.3–5 Therefore, understanding the various molecular targets that contribute to specific desired and undesired endpoints is important for a complete pharmacological understanding and for development of novel anesthetics with improved safety profiles.

Volatile anesthetics depress neurotransmitter release with greater potency at excitatory than inhibitory synapses, which contributes to volatile anesthetic depression of central nervous system function.6 Volatile anesthetics at clinically relevant concentrations inhibit sodium currents in transfected cells,7,8 nerve terminals8,9 and dorsal root ganglion (DRG) neurons.10 Voltage-gated sodium channels (Nav) are important targets for the presynaptic effects of volatile anesthetics on excitatory neurotransmitter release.11,12 However, whether volatile anesthetics can directly depress activity of excitatory neurons by inhibiting sodium currents is unclear.

Initiation and propagation of action potentials (APs) are important for synaptic transmission and neuronal plasticity.13 Even a small change in AP properties can lead to significant modulation of synaptic transmission.14 Transient sodium currents (INaT) are crucial for membrane excitability, including initiation and propagation of APs.13 Nav can also produce persistent (INaP) and resurgent (INaR) sodium currents to modulate excitability of neuronal networks.15,16 Slowly inactivating or non-inactivating sodium currents (INaP) are activated at sub-threshold voltages and enhance repetitive firing.17 Resurgent sodium currents (INaR) occur with repolarization following a prior period of depolarization; neurons with significant INaR share the capacity for rapid spontaneous firing or burst firing.16 Changes in INaP or INaR are implicated in several diseases including epilepsy, paramyotonia congenita, and extreme painsyndromes.17,18 However, whether volatile anesthetics directly modulate INaP and/or INaR is unknown.

The hippocampus is critical to many brain functions including learning and memory,19 which are sensitive to the actions of general anesthetics.20–22 Pyramidal neurons are the principal excitatory neurons in the hippocampus and express multiple subtypes of Nav.23 We designed the present study to test the hypothesis that the volatile anesthetic isoflurane directly modulates excitability of hippocampal pyramidal neurons by inhibiting both transient and persistent sodium currents.

Materials and Methods

Materials

Working solutions of isoflurane were prepared from saturated solutions (12–15 mM isoflurane, as measured by gas chromatography24) in extracellular solution consisting of (in mM) 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 15 glucose, pH = 7.3 ± 0.5. The saturated solution was prepared by rotation in a gas-tight vial for at least 24 h. The saturated solution was diluted to experimental concentrations immediately before perfusion, and final concentrations of isoflurane were confirmed by gas chromatography.24 Isoflurane 0.27 mM was used as the predicted minimum alveolar concentration (MAC, equivalent to the EC50 for immobilization) for mouse adjusted to room temperature (~25°C).25–27 Isoflurane was purchased from Abbott Pharmaceutical Co. Ltd. (Shanghai, China), tetrodotoxin (TTX) was purchased from Alomone Labs (Jerusalem, Israel), and other compounds were obtained from Sigma-Aldrich (Shanghai, China).

Preparation of mouse hippocampal slices

Procedures were approved by the Animal Ethics Committee of Sichuan University (Chengdu, China). Randomization and blinding methods were not used in these electrophysiological recordings. C57BL/6 mice (28 male and 25 female, 53 in total) at 7–10 postnatal days were anesthetized with ketamine/xylazine (60/10 mg/kg) and decapitated. The brain was rapidly removed and put into ice-cold oxygenated (95% O2/5% CO2) sucrose-substituted dissecting solution containing (in mM): 87 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 7.5 MgCl2, 75 sucrose and 25 glucose. Horizontal hippocampal slices (thickness of 270 μm) were cut using a vibratome (Leica VT1000 A, Buffalo Grove, IL, USA), incubated for 30 min at 37°C and then at room temperature (23–25°C) in extracellular solution aerated with 95% O2/5% CO2. After incubation, hippocampal slices were mounted in the recording chamber for electrophysiological recordings at room temperature.8

Whole-cell patch-clamp recording

Hippocampal slices were placed in a recording chamber and continuously perfused with extracellular solution at 2 ml/min. Pyramidal neurons in the hippocampal CA1 region were directly visualized and identified by their shape and size with infrared differential interference contrast imaging microscopy. Whole-cell voltage-clamp recordings were applied to record sodium currents. Electrophysiological recordings were conducted using an Axopatch 200B amplifier, 1440 Digidata and coupled with pClamp 10.2 software (Molecular Devices, Sunnyvale, CA, USA). Currents were sampled at 20 kHz and filtered at 5 kHz. The external solution was the same as the incubation solution but supplemented with 25 mM TEA-Cl to block potassium currents. The resistance of glass pipettes was 3–4 MΩ and the internal pipette solution contained (in mM): 110 CsF, 9 NaCl, 1.8 MgCl2, 4 Mg-ATP, 0.3 Na-GTP, 0.09 EGTA, 0.018 CaCl2, 9 HEPES, 10 TEA-Cl. CsOH was used to adjust to pH = 7.38 (290–310 mOsm). Tetrodotoxin (TTX)-sensitive currents were confirmed by subtraction after application of 500 nM TTX. Series resistance was compensated by ~70–75%, and cells were rejected when series resistance exceeded 15 MΩ. APs were recorded under current-clamp mode with an extracellular solution containing (in mM): 125 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, 15 glucose, and 10 μM bicuculline or 100 μM picrotoxin to block possible effects from GABAergic inputs. The internal pipette solution for current-clamp mode contained (in mM): 122 K-methanesulfonate, 9 NaCl, 1.8 MgCl2, 4 Mg-ATP, 0.3 Na-GTP, 0.09 EGTA, 0.018 CaCl2 and 9 HEPES (pH adjusted to 7.35 with KOH, 290–310 mOsm).

Statistical analysis

No power calculations were conducted prior to the study. Sample sizes were based on our experience with similar experimental designs. No data were lost, but data were not collected when cell series resistance exceeded 15 MΩ. Electrophysiological data were analyzed using Clampfit 10.0 software (Molecular Devices), Graph-Pad Prism 6 (Graph-Pad Software Inc., San Diego, CA, USA) and Origin 10.0 (OriginLab, Northampton, MA, USA). Half-maximal inhibitory concentration (IC50) values were obtained by least squares fitting to the Hill equation: Y = 1/(1 + 10((logIC50 − X)*h), where Y is the effect, X is the measured concentration of isoflurane and h is the Hill slope. For INaT, voltage-dependence of half-maximal activation (V½act) and half-maximal inactivation (V½inact) were fitted to the Boltzmann equation: G/Gmax = 1/[1 + exp (V½ − V/k)]. Sodium current recovery traces and time constants (τ) were determined by fitting to the mono-exponential function: Y = exp (−τ*n) + AP, where τ is the time constant, AP is the plateau and n is stimulus number based on complete recovery time. Inhibition of peak INaT was normalized as %inhibition vs. control and compared by chi-square test. For INaP, currents were measured as the preserved currents from 450 to 475 ms after stimulus. INaR refers to the peak current during the first 50 ms following step back. Both INaP and INaR were corrected for each individual trace by subtracting current recorded in the presence of TTX. Normal distribution of data was tested by the Shapiro-Wilk test. All data are presented as mean ± SD. Repeated measures data were analyzed by two-way ANOVA with Bonferroni post hoc testing. Two-tailed independent-samples or paired Student’s t-test were used for comparison between control and isoflurane conditions. Statistical significance was set as P < 0.05.

Results

Isoflurane inhibits transient sodium channel current (INaT)

Transient sodium channel current (INaT) was activated by depolarization to 0 mV from holding potentials of −70 or −120 mV. At a clinically relevant concentration (0.55 mM, ~2 MAC), isoflurane inhibited INaT in a voltage-dependent manner (fig. 1a). Inhibition was significantly greater at the physiological holding potential of −70 mV (37.8 ± 4.6% inhibition, n = 9) than at a hyperpolarized holding potential of −120 mV (3.1 ± 7.6% inhibition, n = 9, P = 0.012 vs. −70 mV) (fig. 1b). The IC50 (median inhibitory concentration) of isoflurane for inhibition of INaT was 1.0 ± 0.3 mM (~3.7 MAC) from a holding potential of −70 mV, while the currents from a holding potential of −120 mV were minimally inhibited (IC50 = 3.6 ± 0.7 mM, ~13.3 MAC) (fig. 1c). With repeated 5-ms pulses depolarizations at 50 Hz (fig. 1d), normalizing INa of each pulse to that of the first pulse (Pulsen/Pulse1) removed the effect of resting block by isoflurane.24 Thus the reduced INa at the 10th pulse reflected activity-dependent inhibition as a result of repeated membrane depolarizations. From a holding potential of −70 mV, isoflurane reduced Pulse10/Pulse1 ratio from 0.49 ± 0.02 to 0.38 ± 0.03 (fig. 1f, n = 7, P = 0.010). This suggests that isoflurane leads to progressive inhibition of INa during trains of APs due to delayed recovery from inactivation produced by isoflurane.

Fig. 1.

Fig. 1.

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Isoflurane inhibits Na+ currents in a voltage- and activity-dependent manner. (a, b) Representative current traces recorded from the same pyramidal neuron before and after isoflurane at a clinically relevant concentration (0.55 mM, ~2 MAC) application from holding potentials of −70 mV and −120 mV (a), and the normalized inhibition (b) showing voltage-dependent inhibition of Na+ currents with greater inhibition by isoflurane from a holding potential of −70 mV than of −120 mV (n = 9, P = 0.012). (c) Concentration-effect curves of isoflurane on Na+ currents from holding potentials of −70 mV or −120 mV, respectively. The shadow indicates clinically relevant concentrations of isoflurane (0.5–2 MAC). The IC50 (median inhibitory concentration) from a holding potential of −70 mV was 1.0 ± 0.3 mM (~3.7 MAC), while 3.6 ± 0.7 mM (~13.3 MAC) at a holding potential of −120 mV (n = 9–12,P < 0.0001). (d-f) Activity-dependent inhibition by isoflurane. (d) Representative currents from the same neuron before (top) and after (bottom) isoflurane. (e, f) Normalized Na+ currents (e) and the last pulse (f) from individual normalized currents (Pulse10/Pulse1) in the absence or presence of isoflurane (n = 7, P = 0.010). Data are mean ± SD. *P < 0.05, **P < 0.01. Analyzed by two-tailed unpaired t-test (b), two-tailed paired t-test (f). CTL: control, ISO: isoflurane.

Isoflurane modulates transient sodium channel gating

To test the effects of isoflurane on voltage-gated sodium channel activation, sodium current was activated by a series of voltage steps from −70 to +70 mV preceded by a −110 mV prepulse to relieve channel inactivation (fig. 2a). Isoflurane at 0.42 mM (~1.6 MAC) did not shift the current-voltage (I−V) relationship, and maximum INa for both control and isoflurane conditions occurred at −20 mV (fig. 2b). Voltage-dependent activation was unchanged by isoflurane: V½act was −23.9 ± 0.2 mV for control compared to −24.2 ± 0.2 mV with isoflurane (fig. 2c, n = 8, P = 0.144). The effect of isoflurane on steady-state inactivation was determined by eliciting currents at 0 mV after a 20-ms prepulse to voltages from −120 to +10 mV (fig. 2d). Normalized INa/INamax values reflected the fraction of channels inactivated during the prepulse.9 Isoflurane (0.42 mM, ~1.6 MAC) shifted the steady-state inactivation curve in the hyperpolarizing direction; with a V½inact of −46.7 ± 0.9 mV for control compared to −53.2 ± 0.8 mV with isoflurane (fig. 2e, n = 11, P < 0.0001). These data suggest that isoflurane inhibits peak INa by increasing the fraction of inactivated channels.

Fig. 2.

Fig. 2.

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Isoflurane modulates Nav gating. (a, b) Isoflurane at a clinically relevant concentration (0.42 mM, ~1.6 MAC) does not alter the voltage-dependence of Nav activation. (a) Representative current traces recorded from the same pyramidal neuron before (left) and after (right) isoflurane application. (c) Voltage of half-maximal activation (V½act) was not altered by isoflurane (n = 8; P = 0.144). (d, e) Isoflurane shifts the voltage-dependence of Nav inactivation in the hyperpolarized direction. (d) Steady-state current curves recorded from the same neuron in the absence (left) or presence (right) of isoflurane. (e) Voltage of half-maximal inactivation (V½inact) is shifted in the hyperpolarized direction by isoflurane (n = 8, P < 0.0001). (f, g) Isoflurane significantly delays recovery from inactivation of Nav. (f) Traces recorded in the absence (top) or presence (bottom) of isoflurane using a paired-pulse protocol. (g) Normalized peak current was plotted against duration of the inter-pulse interval, which varied from 1–400 ms (inset), for CTL and ISO, and time constants were determined from mono-exponential fits for individual neuron data (left panel; n = 13, P = 0.0002). Data are mean ± SD. *P < 0.05, **P < 0.01 by two-tailed paired t-test. CTL: control, ISO: isoflurane.

As neuronal firing frequency depends in part on how fast Nav can cycle through its various activation states, we measured the time-course of recovery from inactivation. Peak INa was recorded in response to two 10-ms pulses to 0 mV from a holding potential of −70 mV, where the duration between the two pulses was varied from 1 to 400 ms (fig. 2f). Recovery time-courses were fit to a mono-exponential function for both control and isoflurane conditions, indicating that channels predominantly entered a single fast-inactivated state.24 Isoflurane increased the time required for channel recovery, with the recovery time constant increasing from 7.5 ± 0.6 to 12.7 ± 1.3 ms (fig. 2g, n = 13, P < 0.0001).

Isoflurane inhibits persistent and resurgent sodium currents

Persistent currents (INaP) after fast-inactivated transient currents were recorded with 500-ms voltage steps from −70 to +20 mV (fig. 3a). The mean residual currents during 450 to 475 ms were calculated as INaP.18 The largest persistent currents were found at a voltage step to −40 mV (fig. 3b). Isoflurane inhibited INaP at nearly all voltage steps from −60 to 0 mV (fig. 3c). The IC50 of isoflurane for INaP was 0.4 ± 0.1 mM (~1.5 MAC) from a holding potential of −70 mV with depolarization to −40 mV (the voltage for maximal persistent currents) (fig. 3d). INaP were corrected for each individual trace by subtracting current recorded in the presence of TTX (500 nM). When INaP was evoked by a ramp depolarization stimulus (from −80 to 0 mV at 30 mV/s),28 isoflurane decreased INaP density (fig. 3e–f, n = 11). INaP was TTX-sensitive as it was inhibited completely by 500 nM TTX (fig. 3e). INaR is the residual fraction of the slow sodium current that can be activated by the next depolarization.16 Isoflurane inhibited the resurgent current evoked by voltage steps from −70 to 0 mV with a 20-ms prepulse to +30 mV (fig. 3g–h, n = 7, all P < 0.05 for voltages from −70 to −10 mV).

Fig. 3.

Fig. 3.

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Isoflurane inhibits persistent (INaP) and resurgent (INaR) sodium channel currents. (a-f) Isoflurane at a clinically relevant concentration (0.49 mM, ~1.8 MAC) inhibits INaP in pyramidal neurons. (a) Representative traces recorded from the same neuron using a prolonged step protocol shown in the inset (INaP was determined as the mean persistent currents from 450–475 ms). (b) The largest currents were elicited by depolarization to −40 mV; recordings before and after isoflurane perfusion are shown. (c) Density of INaP evoked by depolarization from −70 mV to 0 mV (n = 7). (d) Concentration-effect curve of isoflurane for INaP from a holding potential of −70 mV. The shading indicates clinically relevant concentrations of isoflurane (0.5–2 MAC). (e) Mean INaP curves obtained from seven neurons, evoked with a ramp protocol from −80 mV to 0 mV (30 mV/s). (f) Isoflurane reduced the density of INaP. (g, h) Representative resurgent sodium currents (g) and density (h) indicates that isoflurane depressed INaR in pyramidal neurons (n = 17). INaR refers to the peak current during the first 50 ms following step back. Data are mean ± SD. *P < 0.05 by two-way ANOVA. CTL: control, ISO: isoflurane.

Isoflurane inhibits sub-threshold currents

Sodium currents at sub-threshold voltages determine the threshold of neuronal activation and are critical to synaptic transmission.29 To test the effects of isoflurane on sub-threshold sodium currents, we applied successive 5-mV step depolarizations at the same rate as the ramp depolarization29 (fig. 4a). Both transient and steady-state INa were evoked at these potentials. The largest transient and steady-state INa densities were found at a voltage step to −60 mV, with current densities of −24.0 ± 7.9 and −2.0 ± 0.3 pA/pF, respectively (fig. 4b). Isoflurane (0.42 mM, ~1.6 MAC) inhibited the transient sodium current at voltage steps to −50 and −45 mV, and steady-state sodium currents at voltage steps to −60 mV (fig. 4c and 4d, n = 9).

Fig. 4.

Fig. 4.

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Isoflurane modulates sub-threshold transient and persistent currents. (a) Representative currents recorded by a stair ramp protocol. Transient (INaT) and state-steady persistent (INaP) currents at each potential were calculated. (b) Sample of initial part of recording. INaT was inhibited by isoflurane at a clinically relevant concentration (0.42 mM, ~1.6 MAC, n = 8, P = 0.047, and P = 0.011) at potentials of −50 and −45 mV (c), while INaP was inhibited at a potential of −60 mV (d, n = 9, P = 0.044). Data are mean ± SD. *P < 0.05 by two-way ANOVA. CTL: control, ISO: isoflurane.

Isoflurane depresses action potentials

Single AP were evoked by injection of 60 pA current for 100 ms in current-clamp mode (fig. 5a). Isoflurane reduced AP amplitude from 104.5 ± 5.1 to 90.0 ± 3.0 mV (fig. 5b left, n = 13, P = 0.001) and increased AP width from 2.3 ± 0.2 to 2.6 ± 0.2 ms (fig. 5b right, n = 13, P = 0.001). By phase-plane plot dV/dt analysis, isoflurane inhibited the whole time course of somatic spikes recorded from hippocampal CA1 pyramidal neurons (fig. 5c). Firing of APs was activated by 1-sec series of current injection from120 to 60 pA (fig. 5d). Isoflurane reduced firing frequency from 10.2 ± 1.6 to 3.3 ± 1.0 Hz for 30 pA injections (fig. 5e, n = 20, P = 0.0002), from 13.2 ± 1.5 to 7.2 ± 1.0 Hz for 60 pA injections (fig. 5e, n = 20, P < 0.0001), and from 12.5 ± 1.5 to 7.6 ± 1.0 Hz for 90 pA injections (fig. 5e, n = 20, P < 0.0001). Isoflurane hyperpolarized the resting membrane potential from −54.6 ± 2.3 to −58.7 ± 2.1 mV (fig. 5f, n = 16, P = 0.001). Rheobase was increased by isoflurane from 21.4 ± 5.7 to 42.9 ± 11.5 pA (fig. 5g, n = 14, P = 0.019) and input resistant was increased from 220 ± 17 to 241 ± 17 MΩ (fig. 5h, n = 12, P < 0.0001). The effects of isoflurane on APs firing frequency, rheobase, neuronal input resistance and resting membrane potential represent the combined results with recordings under bicuculline or picrotoxin.

Fig. 5.

Fig. 5.

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Effects of isoflurane on hippocampal action potentials. (a) The effects of isoflurane at a clinically relevant concentration (0.48 mM, ~1.8 MAC) on single APs. (b) AP amplitude was reduced and width increased by isoflurane (n = 13, P = 0.001), as illustrated by the AP dynamics of dV/dt (c). (d-e) Isoflurane significantly reduced AP frequency (n = 20). (f) Isoflurane significantly hyperpolarized pyramidal neuron resting membrane potential (n = 16, P = 0.001). (g) Isoflurane significantly increased the rheobase that evoked APs (n = 14, P = 0.019). (h) Isoflurane significantly increased the input resistant of neurons (n = 12, P < 0.0001). Data are mean ± SD. *P < 0.05, **P < 0.01 by two-tailed paired t-test (b, g, h) or two-way ANOVA (e) CTL: control, ISO: isoflurane.

Discussion

Specific molecular targets mediate the neurophysiological actions of general anesthetics on critical properties and processes such as neuronal excitability, axonal conduction and synaptic transmission.30,31 Voltage-gated sodium channels are essential ion channels in mediating the rising phase of action potentials13,32 and are implicated as presynaptic targets for general anesthetics.3 Although early studies on the squid giant axon found that APs were relatively insensitive to clinical concentrations of general anesthetics,33 more recent studies report significant sensitivity of sodium channels to volatile anesthetics at clinically relevant concentrations.9,34 Supporting in vivo animal studies show that intravenous administration of the Nav blocker lidocaine reduces MAC for several volatile anesthetics both in animals and humans.35–37 Moreover, intrathecal injection of TTX reduces MAC for isoflurane in rats,35 while intrathecal veratridine, a toxin that promotes the Nav open state, increases MAC and antagonizes the effect of intrathecal TTX.38

Volatile anesthetics including isoflurane and sevoflurane inhibit heterologously expressed recombinant brain Nav at clinical concentrations by selectively interacting with the inactivated state of the channel.31 Our findings support and extend these findings by demonstrating effects of isoflurane on INa and APs in native hippocampal neurons in situ. Voltage- and activity-dependent inhibition of Nav by volatile anesthetics can be explained by preferential interaction with the inactivated state to impede transition from the inactivated state back to the resting state.24,31 The IC50 of isoflurane for inhibition of INaT from a holding potential of −70 mV is relevant to the clinically used concentrations, while isoflurane did not inhibit INaT at clinically relevant concentrations from a holding potential of −120 mV. Even perfusion with saturated isoflurane stock solution (12–14 mM, ~48 MAC) only depressed INaT by ~60% from a holding potential of −120 mV. The partial inactivation state sodium channels existing at a holding potential of −70 mV can explain this result. Volatile anesthetics including isoflurane also display activity-dependent inhibition of neuronal sodium channels in isolated rat neurophysical nerve terminals,9 neuroblastoma cells24 and dorsal root ganglion neurons,10 and in the bacterial Nav homologue NaChBac,39,40 but there was no prior evidence that volatile anesthetics directly depress activity of excitatory neurons by inhibiting neuronal sodium channels.

The major anion in our pipette solution was F−, which has been reported to affect inactivation of Nav in squid giant axons.41 We used F− instead of Cl− in the pipette solution for Nav recordings7,42 to avoid changes in intracellular Cl− that might affect neuronal excitability, signaling and plasticity of hippocampal pyramidal neurons.43,44 Since isoflurane may also potentiate Cl− permeable GABAARs, the level of intracellular Cl− has important consequences for its actions in a manner similar to that recently shown for propofol.45,46

We provide evidence that isoflurane at clinically relevant concentrations inhibits INaT, INaP and INaR at physiological holding potentials in mouse hippocampal CA1 pyramidal neurons. For INaT, isoflurane inhibits peak sodium current and modulates channel gating by shifting the voltage-dependence of Nav inactivation, and by delaying recovery from inactivation. Isoflurane also inhibits INaP and INaR at clinically relevant concentrations.6,47 From a holding potential of −70 mV, the IC50 of isoflurane for INaP inhibition was lower than for INaT (0.4 ± 0.1 vs. 1.0 ± 0.3 mM), indicating that isoflurane selectively inhibits the persistent currents of sodium channels. This inhibition by isoflurane of INaP at clinically relevant concentrations might reduce neuronal excitability such as lower APs firing frequency and increased rheobase.

At least three activation states have been identified for neuronal sodium channels depending on membrane potential: resting (closed), activated (open), and inactivated.16 INaT are evoked by step depolarization and rapidly activate, while the subsequent residual sodium currents that last throughout the step are referred to as INaP.17 Upon repolarization, closed Nav reopen to produce resurgent currents, known as INaR.16 Although these three components of sodium currents all flow through the same channels, they have different kinetics and physiological roles. INaT are pivotal for initiation and propagation of APs,32 while INaP and INaR amplify responses of neurons to synaptic input and enhance repetitive firing.18 The rapid kinetics and all-or-none properties of INaT mediate the upstroke of fast neuronal APs.13 INaP and INaR are activated by small synaptic depolarization and are slower than INaT. Despite their small amplitudes compared to INaT, INaP and INaR can profoundly alter neuronal firing behavior, especially at sub-threshold voltages.16,17 In dendrites, INaP can boost distal synaptic potentials to propagate to the neuronal soma.15 In proximal axons and peripheral axons, INaP can affect initiation of APs.48,49 Both INaP and INaR also play critical roles in the regulation of neuronal firing behavior due to their high densities near the axon initial segment.50 INaR is enhanced by specific pathogenic mutations in sodium channels, thereby increasing AP firing rate and duration, causing neuronal hyperexcitability.51 Inhibition by isoflurane of these neuronal sodium channel currents provides neurophysiological mechanisms to support previous reports that isoflurane reduces excitability of CA1 pyramidal neurons by depressing APs.

Neuronal information coding occurs by varying frequencies and patterns of APs between somata and axons, where they can rapidly propagate information over distance. APs are fundamentally important for synaptic transmission and neuroplasticity.52,53 Neurotransmitter release is largely determined by the shape, frequency and pattern of presynaptic APs during the process of information transfer.14 Release of many neurotransmitters is inhibited by volatile anesthetics in a sodium channel-dependent manner.54,55 Thus Nav is a plausible presynaptic molecular target for volatile anesthetics.

Isoflurane decreased excitability of mouse CA1 pyramidal neurons by shaping the APs and hyperpolarizing the resting membrane potential. The generation of APs depends on multiple ion channels including Nav for rapid depolarization, Cav for slow depolarization and K+ channels for repolarization.56 Analysis of AP kinetics (dV/dt) showed that isoflurane slowed AP kinetics throughout, consistent with net inhibition. Isoflurane decreased AP amplitude mainly from inhibition of transient Nav currents, as seen in rat neurohypophysial terminals.9 Prolonged AP duration impairs neuronal responses to high frequency stimuli, enhancing the depression of synaptic transmission. The increased AP half-width by isoflurane is likely due to inhibition of potassium channels.57 Isoflurane at clinical concentrations can enhance and stabilize the inactivation state of Nav, resulting in activity-dependent reduction of INa with high frequency stimuli which could partly contribute to slow the rapid depolarization of AP by isoflurane. Inhibition by isoflurane of persistent INa may also contribute to its effect on AP amplitude and duration. Isoflurane slightly hyperpolarized resting membrane potential possibly due to enhancement of volatile anesthetic-sensitive background K+ channels.58 The effects of isoflurane on resting membrane potential have been reported to be varied47,59 and voltage-dependent;60 expression of different K+ channel subtypes may contribute to these differential effects.58

In conclusion, isoflurane can inhibit three components of INa, which contribute to its depression of neuronal excitability and APs in hippocampal CA1 pyramidal neurons. Voltage-dependent and activity-dependent inhibition by isoflurane, and possibly other volatile anesthetics, depresses neuronal excitability and may contribute to anesthetic effects on neurotransmitter release and synaptic plasticity.

Funding

Supported by grants No. 81771486 (to C.Z.) and No. 81600918 (to P.L.) from the National Natural Science Foundation of China, and R01 GM-58055 from the National Institutes of Health, Bethesda MD, USA (to H.C.H.).

Footnotes

Prior Presentation

This study was presented at the 2018 annual meeting of the American Society of Anesthesiologists, San Francisco, CA, USA.

Conflicts of Interest

The authors declare no competing interests.

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