Melatonin suppresses sympathetic vasomotor tone through enhancing GABAA receptor activity in the hypothalamus

Abstract

Introduction: Melatonin (5-methoxy-N-acetyl-tryptamine) is a circadian hormone synthesized and secreted by the pineal gland. In addition to regulating circadian rhythms of many physiological functions, melatonin is involved in regulating autonomic nervous function and blood pressure. Hypothalamus paraventricular nucleus (PVN), receiving melatonin projections from the superchiasmatic nucleus, is a critical brain region to regulate neuroendocrine and cardiovascular function. Here, we determined the synaptic mechanisms involved in the effect of melatonin on the sympathetic outflow and blood pressure. Methods and Results: Microinjection of melatonin into the PVN produced a depressor effect and decreased renal sympathetic nerve activity (RSNA). While microinjection of luzindole, a non-selective melatonin receptor antagonist, into the PVN did not change melatonin-induced sympathoinhibition, GABA_A receptor antagonist bicuculline eliminated melatonin-induced sympathoinhibition. Furthermore, melatonin decreased firing rate of retrogradely labeled PVN neurons which project to the rostral ventrolateral medulla (RVLM), an effect was not altered by luzindole but eliminated by bicuculline. Melatonin significantly increased the amplitude of spontaneous and evoked GABAergic inhibitory synaptic currents, as well as GABA-induced currents. Conclusion: These data suggest that melatonin in the PVN suppresses sympathetic vasomotor tone through enhancing GABA_A receptor activity. This study provides novel information for understanding the cellular mechanisms involved in the effect of melatonin on regulating blood pressure and sympathetic output.

Keywords: GABAA receptor; autonomic nervous system; circadian rhythms; hypothalamus; melatonin; sympathetic nervous system.

FIGURE 1

Microinjection of melatonin into the PVN reduced ABP and sympathetic outflow. (A), Representative recordings show that the effect of bilateral microinjection of melatonin (0.1 mM, 100 nL) into the PVN on mean ABP, RSNA, HR, and integrated RSNA (Int-RSNA). (B), Representative recordings show microinjection of non-selective antagonist luzindole (10 mM, 100 nL) failed to alter the melatonin-induced reduction of ABP and RSNA. (C–E), Summary data show change of mean ABP, RSNA, and HR in response to microinjection of melatonin and luzindole, an antagonist for MT1 and MT2 receptors, into the PVN. Data are mean ± SEM. One-way ANOVA with the Bonferroni’s post hoc test was used to determine differences between groups (n = 7 rats in each group). *p < 0.05, compared with the respective baseline each group. MLT, melatonin; LZD, luzindole.

FIGURE 2

Melatonin reduced the firing activity of RVLM projecting PVN neurons. (A), microphotography (upper panel) and FluoSphere-labeled PVN neuron (*) in brain slice viewed with and infrared differential interference contrast optics (middle panel) and fluorescence illumination (lower panel). The recording electrode was marked with an arrow. (B), upper panel: Representative raw tracings show that bath application of melatonin (100 nM) decreased the firing activity of RVLM projecting PVN neurons. Lower panel: MT receptor antagonist luzindole (100 μM) did not alter the baseline firing activity and melatonin-induced decrease in the firing activity of RVLM projecting PVN neurons. (C) and (D), Summary data of firing rats (C) and membrane potential (D) show melatonin decreased the spontaneous firing rats and hyperpolarized the labeled RVLM projecting PVN neurons in both vehicle and MT receptor antagonist luzindole (100 μM) (n = 7 neurons from 3 rats in each group). Data are mean ± SEM. One-way ANOVA with the Bonferroni’s post hoc test was used to determine differences between groups *p < 0.05 compared with the basal values in each group. MLT, melatonin; LZD, luzindole.

FIGURE 3

Effect of melatonin on GABAA receptor activity in RVLM projecting PVN neurons. (A,B), Representative raw tracings show that melatonin at concentration of 100 nM increased the amplitude of evoked GABAergic IPSCs (A) and the currents induced by puff application of 100 μM GABA (B) onto the labeled RVLM projecting PVN neurons recorded. Please note that application of 20 μM bicuculline abolished evoked IPSCs and puff GABA-induced currents. (C,D), Summary data show that 100 nM melatonin significantly increased the amplitude of evoked IPSCs (n = 6 neurons from 3 rats) and puff GABA-induced currents (n = 6 neurons from 3 rats). Data are mean ± SEM. One-way ANOVA with the Bonferroni’s post hoc test was used to determine differences between groups *p < 0.05 compared with the basal values in each group. MLT, melatonin.

FIGURE 4

Effect of melatonin on GABAergic miniature IPSCs in RVLM projecting PVN neurons (A) Representative tracings from a labeled RVLM projecting PVN neuron show that mIPSCs recorded during control, application of 100 nM melatonin, washout, and application of 20 μM bicuculline. Note that bicuculline completely eliminated mIPSCs. (B,C), Cumulative probability plot analysis of mIPSCs of the same neuron showing the distribution of the interevent interval (B) and peak amplitude (C) during control, melatonin application, and washout. Melatonin shifted the distribution curve of amplitude of mIPSCs to the right (p < 0.05; Kolmgorov—Smirnov test) without changing the distribution of the interevent-interval. (D,E), Summary data show the effect of 100 nM melatonin on the frequency (D) and amplitude (E) of mIPSCs of 8 labeled PVN neurons. Data are presented as means ± SEM (*p < 0.05 compared with the control; Kruskal–Wallis ANOVA, followed by Dunn’s post hoc test). MLT, melatonin.

FIGURE 5

Effect of melatonin on glutamatergic mEPSCs and evoked EPSCs in RVLM projecting PVN neurons. (A) Representative raw tracings from a labeled RVLM projecting PVN neuron show that mEPSCs recorded during control, application of 100 nM melatonin, and application of 20 μM CNQX. (B,C), Summary data show the effect of 100 nM melatonin on the frequency (B) and amplitude (C) of mEPSCs of 6 labeled PVN neurons. (D,E), Representative raw tracings (D) and summary data of amplitude of evoked EPSCs (E) show that 100 nM melatonin did not change the amplitude of evoked EPSCs. Data are presented as means ± SEM (*p < 0.05 compared with the control; ANOVA, followed by Dunn’s post hoc test). MLT, melatonin.

FIGURE 6

GABAA receptor antagonist eliminated melatonin-induced decrease in firing activity of RVLM projecting PVN neurons. (A), Representative raw tracings showing the spontaneous firing activity during application of 100 nM melatonin, 20 μM bicuculline, bicuculline plus melatonin. Please note that application of bicuculline significantly increased the firing activity of RVLM projecting PVN neurons. Furthermore, bicuculline eliminated the effect of melatonin on the firing activity and membrane potentials. (B,C), summary data of firing rate (B) and membrane potentials (C) showing that GABA_A receptor antagonist bicuculline eliminated melatonin-induced decrease in firing activity and hyperonization of RVLM projecting PVN neurons. Please note that bicuculline increased the firing activity and depolarized the membrane potentials of RVLM projecting PVN neurons. Data are presented as means ± SEM (*p < 0.05 compared with the control; ANOVA, followed by Dunn’s post hoc test). MLT, melatonin.

FIGURE 7

Blocking GABAA receptors in the PVN eliminated melatonin-induced decrease in blood pressure and sympathetic outflow. (A), Representative raw traces show the effect of microinjection of bicuculline into the PVN increased ABP, RSNA, and HR. Subsequent microinjection of melatonin into the PVN did not significantly alter the ABP, RSNA, and HR. (B–D), Summary data show changes in mean ABP (B), RSNA (C), and HR (D) in response to microinjection of bicuculine and melatonin into the PVN (n = 6). Data are presented as means ± SEM (*p < 0.05 compared with the control; ANOVA, followed by Dunn’s post hoc test). MLT, melatonin.