Modulation Of γ-aminobutyric Acid Neuronal System By Myricetin In Rat Hypothalamic Paraventricular Nucleus

Epidemiological studies have shown that over 50% of the adult population suffers fromsleep disorders, in which the majorities are classified as insomnia. Masses of drugs totreat insomnia in clinics produce sedative effects by facilitating theγ-aminobutyric acid(GABA) type A receptors' function. However, most of the drugs have more or less severeside effects. Traditional herbal medicines have a great potential to treat insomnia.However, the pharmacological action of most of the over-the-counter herbal medicineremains largely unknown. Recent studies in Epithelia cell biology research centre ofChinese university of Hong Kong demonstrated that an herbal extracts from theAmpelopsis family could not only reduce sleep latency but also significantly improveNREM and REM duration during the sleeping period in both rats and humans. The activecomponent was also identified as myricetin. However, the possible mechanismsunderlying myricein induced sedation are still unknown. GABA is the predominantneurotransmitter in the central nervous system. Modulation of GABA-A receptormediated synaptic currents is one of the major targets to treat insomnia. It has beenreported that some flavonoids could interact with benzoldiazepine (BZ) binding site onGABA-A receptor. Several studies also showed that some flavonoids have anxiolytic andanticonvulsant effects. However, the modulation effect of myricetin on GABA-A receptoris still unknown. By a combination of whole cell patch clamp recording, molecularbiology and calcium imaging, we investigated effects of myricetin on GABA-A receptormediated inhibitory postsynaptic currents (IPSCs) and voltage gated calcium channel(VGCC) currents in hypothalamic brain slices; tested the action of myricetin onintracellular calcium concentration in primary cultured hypothalamic neurons; comparedthe changes of mRNA expressions of GABA-A receptor, histamine receptor andhypocretin receptor after myricetin treatment. The results were as follows:1. In the tested 14 hypothalamic paraventricular nucleus (PVN) neurons, the half decay time of GABA-A receptor mediated miniature inhibitory postsynaptic currents(mIPSCs) was significantly prolonged by myricetin (P＜0.01); in which, 5 neuronsshowed an increase of mIPSCs' frequency, 3 showed a decrease and 1 showed anincrease of mlPSCs' amplitude (K-S test, P＜0.05).2. The half decay time of GABA-A receptor mediated spontaneous inhibitorypostsynaptic currents (sIPSCs) was significantly prolonged by myricetin inhypothalamic PVN neurons (P＜0.01). However, the slPSCs' frequency was notchanged after myricetin treatment.3. Myricetin-induced prolongation of mIPSCs' half decay time could not be blocked byflumazenil——the specific BZ binding site antagonist. However, it could be blockedby KN-62——the calcium-calmodulin dependent protein kinaseâ…¡(CaM-Kâ…¡)inhibitor.4. The VGCC currents in hypothalamic PVN neurons increased after myricetin treatment(P＜0.01). The current-voltage (â… -â…¤) relationship curves also shifted to thehyperpolarized direction by myricetin. Further studies showed that both high voltageactivated (HVA) calcium channel currents and low voltage activated (LVA) calciumchannels currents were enhanced by myricetin (P＜0.05).5. The fura-2/AM fluorescence intensity significantly increased by myricetin in primarycultured hypothalamic neurons. This effect could be partly blocked by NiCl₂ and/orCdCl₂. Myricetin-induced increase of fluorescence intensity partly recovered whenperfusion with calcium free solution.6. The hypothalamic PVN neurons could be divided into three types according to theirfiring mode: tonic firing neurons, burst firing neurons and silent neurons. The actionpotential frequency in tonic firing neurons was markedly inhibited, (P＜0.05) and themembrane potential in silent neurons was hyperpolarized by myricetin (P＜0.05).7. The potassium currents increased by myricetin in hypothalamic PVN neurons(P＜0.05); this effect disappeared when perfusion with calcium free solution. 8. The GABA-A receptor a1 subunit mRNA expression down-regulated after myricetinor zolpidem treatment for 8 h (P＜0.05 for myricetin treatment, P＜0.01 for zolpidemtreatment) in hypothalamus. However, no changes could be detected after 2 htreatment (P＞0.05).9. The H1 receptor and Hcrt2 receptor mRNA expressions showed some tendency ofdown-regulation after myricetin or zolpidem treatment for 2 h, however no significantdifference can be detected (P＞0.05). The mRNA expressions returned to the normallevel after 8 h treatment.These results suggested that myricetin somehow facilitate GABA-A receptors' functionby enhancing GABA-A receptor mediated IPSCs. The mechanisms underlyingmyricetin-mediated modulation were different from that of zolpidem——a selectiveGABA-A receptor a1 subunit activator. The Ca²⁺/CaM-Kâ…¡pathway activation rather thanBZ binding site regulation was involved in myricetin-mediated modulation of GABA-Areceptors' function. The intracellular calcium concentration also increased bymyricetin-induced enhancement of VGCC currents, which may be contribute to theactivation of Ca²⁺/CaM-Kâ…¡pathway and Ca²⁺-actwlted potassium channels. Themolecular biology studies showed that several sleep-related genes expressions modulatedby myricetin. However, effects of myricetin on GABA-A receptor a1 subunit, H1 receptorand Hcrt2 receptor genes expressions were similar to that of zolpidem. The modulationeffects of myricetin on these genes expressions may be mediated by an indirect way.These data demonstrated that the mechanisms underlying myricetin-induced sedationwere to some extend different from that of zolpidem. This work may provide a usefulframework to advance our knowledge of myricetin-mediated modulation of GABAergicsystem and may permit the development of a novel alternative approach to treat insomnia.