Figure 1.
The inhibitory effect of menthol on synaptically driven spiking in hippocampal cultures.
(A) Typical traces of spontaneous spiking from one cell in the presence or absence of menthol. (B) Typical traces of zero Mg2+-induced spiking from the same cell in the presence or absence of menthol. (C) The distribution of interspike interval obtained from A and B showing the effect of menthol on spontaneous spiking and elevated spiking. (D) Concentration-dependent inhibition of menthol on spontaneous and zero Mg2+-induced firing rate. (n = 6).
Figure 2.
Synergistic activation of GABAARs by menthol and GABA.
(A) Typical traces showing the currents evoked by 1 µM GABA, 100 µM menthol, 1 mM menthol, 1 µM GABA plus 100 µM menthol and 1 µM GABA plus 1 mM menthol. (B) Histograms showing relative IGABA, Iment, IGABA+ment. (C) The concentration-response relationship of synergic action of menthol with 1 µM GABA. (D) Typical traces showing the current evoked by different concentrations of GABA in the absence or presence of 100 µM menthol. (E) Statistic data showing the relative currents induced by 100 µM menthol plus various concentrations of GABA. * P<0.05 and *** P<0.001, compared with IGABA in the absence of menthol.
Figure 3.
Pharmacological and electrophysiological properties of menthol-activated currents (Iment) in cultured hippocampal neurons.
(A) Typical traces showing the currents induced by various concentration of menthol. (B) Concentration-response relationship of Iment in cultured hippocampal neurons. All peak currents are normalized to the peak amplitude evoked by 1 mM menthol (*). Symbols represent the average response for 6–13 neurons. (C) Typical traces of Iment evoked by 1 mM menthol in the absence or presence of 10 µM BMI (n = 15), 100 µM PTX (n = 5) and 1 µM STR (n = 15). (D) Summary results from all experiments similar to that shown in C showing the relative Iment in the presence of BMI, PTX and STR. Dashed line indicates the control values without antagonist treatment. (E) Typical traces showing Iment evoked by 1 mM menthol at various holding potentials (VH) with [Cl−]i of 153 mM (upper traces) and 33 mM (lower traces). (F) The current-voltage relationships of Iment in the condition of [Cl−]i of 153 mM and 33 mM, respectively. The reversal potential of Iment moved toward hyperpolarizing direction by lowering [Cl−]i. (G) Summary results showing the reversal potentials for Iment in 153 mM [Cl−]i and 33 mM [Cl−]i, respectively.
Figure 4.
Selective enhancement of GABAAR-mediated tonic currents by menthol.
(A) Voltage-clamp recordings from CA1 pyramidal neurons showing GABAergic tonic currents in the absence and presence of 300 µM menthol. The tonic current plotted at 500-ms intervals was revealed by 30 µM BMI. (B) Summary data showing the change of tonic currents by 300 µM menthol (n = 6, **P<0.01, compared with the control group without menthol treatment, paired t-test). (C) Representative traces showing GABAergic mIPSCs in the absence or presence of 300 µM menthol. Averaged mIPSCs in the absence or presence of 300 µM menthol are shown in the pane (D) Summary data showing normalized amplitude, frequency and kinetics of GABAergic mIPSCs in the presence of menthol (n = 14). Dashed line indicates the control values without menthol treatment. (E) Normalized cumulative curves showing the effect of menthol on the amplitude of GABAergic mIPSCs from the sample neuron. (F) Normalized cumulative curves showing the effect of menthol on the frequency of GABAergic mIPSCs from the sample neuron.
Figure 5.
Effects of menthol on seizures of PTZ treated mice and hippocampal kindled rats.
(A) Effect of menthol on the latency from PTZ injection to the clonic (**P<0.01 compared with Ctrl, One-Way ANOVA) and tonic (*P<0.05 compared with Ctrl) convulsion. (B) Effect of menthol on mortality in mice after PTZ injection. (C) Typical traces showing the effect of menthol and PB on the afterdischarge in fully kindled rats. (D) The afterdischarge (AD) threshold induced by first stimulus in rats treated with vehicle, menthol and PB (**P<0.01 compared with Ctrl, n = 10). (E) The seizure stage in fully kindled rats treated with vehicle, menthol and PB (**P<0.01 compared with Ctrl, n = 6). (F) Summary data showing afterdischarge duration in Ctrl, menthol and PB group (**P<0.01 compared with Ctrl, n = 6).
Figure 6.
Effects of menthol isomers, (−)-isopulegol, JE207 and (−)-menthyl chloride on GABAAR.
(A) Chemical structures of menthol isomers, (−)-isopulegol, JE207 and (−)-menthyl chloride. (B) Summary of data showing the relative currents induced by 1 µM GABA in the presence of 300 µM various menthol isomers, (−)-isopulegol, JE207 or (−)-menthyl chloride.