Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus

Abstract

Zolpidem is a widely prescribed sleep aid with relative selectivity for GABA(A) receptors containing alpha1-3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABA(A) receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABA(A) receptor-mediated tonic current (I(tonic)) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that alpha1-3 subunits are expressed in synaptic GABA(A) receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABA(A) receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABA(A) receptors containing alpha1-3 subunits contribute to the I(tonic).

Fig. 1

Effects of bath application of zolpidem on tetrodotoxin (TTX)-resistant mIPSCs recorded in DMV neurons. A. Recordings of mIPSCs before and during the application of zolpidem (1 μM). B. Averaged mIPSCs in control ACSF and in the presence of zolpidem (1 μM; arrow). C. Averaged mIPSCs in flumazenil (10 μM) and with addition of zolpidem (1 μM). D. Effects of zolpidem (0.01–30 μM) on mIPSC amplitude (D1), frequency (D2), rise time (D3) and weighted decay time (D4). The values obtained in different concentrations of zolpidem are plotted as ratios over control values measured within each recording; each point represents the mean ± SEM of 5–6 neurons. Asterisks indicate significant change in zolpidem versus measurements in control ACSF (p < 0.05). Kynurenic acid (1 mM) and TTX (1 μM) were present for all recordings.

Fig. 2

Effect of zolpidem on muscimol-evoked currents. A. Muscimol microapplication evoked whole-cell currents at various membrane potentials. Each trace represents the average of three to five responses to muscimol. Traces shown at holding potentials of –60, –30, 0, 15 mV, and 30 mV; KCl intracellular solution. Bottom, I–V curve of the peak currents evoked at each holding potential. B. The current evoked by muscimol (100 μM) was blocked by bath-applied bicuculline (50 μM). C. The muscimol-evoked currents were examined before and after bath application of zolpidem (1 μM). Bottom, summary graph (n = 6) of the effects of zolpidem on the peak current and decay time constant (τ) of currents evoked by muscimol. Asterisk indicates significance versus currents evoked by muscimol in control ACSF (p < 0.05). Cell was voltage-clamped at –60 mV for experiments in B and C.

Fig. 3

Effect of zolpidem on tonic GABA current (I_tonic) and action potential firing activity in DMV neurons. A. Bath application of zolpidem (1 μM) did not significantly change the baseline holding current. Addition of bicuculline (30 μM) revealed the GABA_A receptor-mediated I_tonic. B. Graph showing the I_tonic amplitude in control ACSF and after addition of zolpidem (1 μM) to ACSF. Number of replicates in parentheses. C. Current-clamp recording at resting membrane potential showing that zolpidem (1 μM) decreased the action potential frequency and bicuculline (30 μM) reversed the effects caused by zolpidem. D, E. Histograms showing the effects of zolpidem and bicuculline on the resting membrane potential (D) and action potential firing frequency (E) in DMV neurons. Asterisks indicate significant change between groups (p < 0.05). Number of replicates in parentheses.

Fig. 4

Zolpidem decreased the input resistance in whole-cell current-clamp recordings. A. Current-clamp recording from a DMV neuron showing a decreased voltage response to current-steps injection after zolpidem (1 μM) application; bicuculline (30 μM) reversed the effect of zolpidem. B. Bar graph showing a decrease in input resistance caused by zolpidem in 9 DMV neurons, and a reversal of the effect with bicuculline. Asterisks indicate significant differences between groups indicated (p < 0.05). Number of replicates in parentheses. C. Current versus voltage (I–V) plot from the same DMV neuron in A illustrating the decrease in slope input resistance after zolpidem perfusion. Bic, bicuculline.

Fig. 5

Zolpidem increased the I_tonic in DMV neurons in the presence of nipecotic acid. A. In the presence of nipecotic acid (1 mM), application of zolpidem (1 μM) significantly enhanced the amplitude of I_tonic. Right, graph showing the I_tonic amplitude after adding zolpidem to the nipecotic acid-containing ACSF. Asterisk indicates significant change in current versus nipecotic acid alone (p < 0.05). Number of replicates in parentheses. B. The increase in I_tonic amplitude induced by zolpidem in the presence of nipecotic acid was largely suppressed by application of flumazenil (10 μM). Right, summary graph shows the I_tonic amplitude changes induced by zolpidem in the presence and absence of flumazenil. Both groups obtained from nipecotic acid-containing ACSF. Asterisk indicates significant difference between the two groups (p < 0.05). Nip, nipecotic acid; Bic, bicuculline.

Fig. 6

Two different responses to zolpidem in gabazine-containing ACSF. A. In the presence of nipecotic acid (1 mM) and gabazine (0.5 μM), the effect of zolpidem (1 μM) on holding current was either partially maintained (upper trace) or blocked (lower trace). B. Comparison of the zolpidem effect on I_tonic in the gabazine-attenuated and the gabazine-blocked groups. Asterisk indicates significant difference between gabazine treatment groups (p < 0.05). Number of replicates in parentheses. Bic, bicuculline; GBZ, gabazine; Nip, nipecotic acid.