alpha-Pinene, a Major Constituent of Pine Tree Oils, Enhances Non-Rapid Eye Movement Sleep in Mice through GABA(A)-benzodiazepine Receptors

Abstract

alpha-Pinene is a major monoterpene of the pine tree essential oils. It has been reported that alpha-pinene shows anxiolytic and hypnotic effects upon inhaled administration. However, hypnotic effect by oral supplementation and the molecular mechanism of alpha-pinene have not been determined yet. By combining in vivo sleep behavior, ex vivo electrophysiological recording from brain slices, and in silico molecular modeling, we demonstrate that (2)-alpha-pinene shows sleep enhancing property through a direct binding to GABA(A)-benzodiazepine (BZD) receptors by acting as a partialmodulator at the BZDbinding site. The effect of (2)-alpha-pinene on sleep-wake profiles was evaluated by recording electroencephalogram and electromyogram. The molecular mechanism of (2)-alpha-pinene was investigated by electrophysiology and molecular docking study. (2)-alpha-pinene significantly increased the duration of non-rapid eye movement sleep (NREMS) and reduced the sleep latency by oral administration without affecting duration of rapid eye movement sleep and delta activity. (2)-alpha-pinene potentiated the GABA(A) receptor-mediated synaptic response by increasing the decay time constant of sIPSCs in hippocampal CA1 pyramidal neurons. These effects of (2)-alpha-pinene on sleep and inhibitory synaptic response were mimicked by zolpidem, acting as a modulator for GABA(A)-BZD receptors, and fully antagonized by flumazenil, an antagonist for GABA(A)-BZD receptor. (2)-alpha-pinene was found to bind to aromatic residues of alpha 1- and -gamma 2 subunits of GABA(A)-BZD receptors in the molecular model. We conclude that (2)-alpha-pinene enhances the quantity of NREMS without affecting the intensity of NREMS by prolonging GABAergic synaptic transmission, acting as a partial modulator of GABA(A)-BZD receptors and directly binding to the BZD binding site of GABA(A) receptor.