Mechanism Study For The Arousal Effect Of Morphine

Objective:Morphine exerting its effect primarily via opioid receptors is the most efficacious and widely prescribed treatment for pain. Unfortunately, morphine has numerous side effects including sleep disruption. Clinic doses of morphine decrease deep sleep (stage4) and rapid eye movement sleep. In animals, acute administration of morphine at low doses results in arousal effect. There are three opioid receptor subtypes, including mu, kappa and delta receptor subtype. The ventrolateral preoptic nucleus (VLPO) has been reported to play an important role in sleep promotion. It has been reported that the VLPO region represses mu and kappa receptor mRNA. Thereby, we hypothesized that the VLPO may therefore function as the neuroanatomic substrate through which morphine promotes wakefulness. Employing the electrophysiological approach, the effect of morphine on the VLPO neurons and the mechanism were investigated in the cellular level. In addition, by the sleep bioassay system, the role of opioid receptor in the VLPO in the regulation of sleep-wake cycle by morphine was examined in systemic levels.Methods:The VLPO mainly consists of two types of the neurons, sleep-promoting neurons and interneurons. The sleep-promoting neurons showing the characteristic triangular or multipolar shape, are characterized by a potent low-threshold spike (LTS), and are inhibited by the transmitter related with wakefulness, such as noradrenaline (NA). The interneurons with typical bipolar fusiform aspect, lack a LTS, and are depolarized by NA. In in vitro brain slice, according to the response of the neurons to NA, VLPO neurons were classified and identified, and we observed the effects of morphine on the two types of neurons and receptors mechanism through which morphine affected these neurons. In addition, by cutting-edge automatic sleep bioassay systems, combined with the implantation of guide canal into the VLPO, the role of the VLPO in morphine-induced arousal was evaluated. Different doses of morphine (0.3,1and3mg/kg) were injected subcutaneously in order to obtain optimal dose for further research. The mu receptor antagonist CTOP (0.05,0.15and0.5nmol/side) or kappa receptor antagonist nor-BIN (0.5and1.5pmol/side) was locally bilaterally injected into the VLPO15minutes before the subcutaneous injection of morphine. The mu receptor antagonist CTOP (0.5nmol/side) or kappa receptor antagonist nor-BIN (1.5pmol/side) alone was bilaterally injected into the VLPO to measure the function of opioid receptor in the regulation of physiological sleep-wake cycle.Results:In in vitro brain slice, morphine at a concentration of1,10, or100μM completely inhibited the action potential of sleep-promoting neurons but0.1μM morphine did not affect the frequency of action potential of sleep-promoting neurons However, the largest dose of morphine (100μM) showed no effects on the action potential of the interneurons. In the presence of TTX,10μM morphine induced the hyperpolarization of sleep-promoting neurons, which could be antagonized by mu receptor antagonist, CTOP. In the presence of CTOP,10μM morphine still inhibited the firing of sleep-promoting, and this inhibitory effects could be reversed by kappa receptor antagonist, nor-BIN. Moreover, kappa receptor agonist U50488H, also inhibited the firing of sleep-promoting neurons. Kappa receptor agonist U50488H inhibited the frequency of miniature excitatory postsynaptic current but not its amplitude. These findings indicated that kappa receptor presynaptically mediated the glutamatergic synaptic transmission to restrain the sleep-promoting neurons indirectly.In behavioral studies, morphine injected at0.3,1,3mg/kg subcutaneously dose-dependently promoted wakefulness. Morphine at1and3mg/kg increased the total amount of wakefulness in3hour after morphine injection by63.5%and71.5%, respectively. Morphine at0.3mg/kg had no essential statistical change on sleep-wakefulness, compared with vehicle control. Bilateral microinjection of CTOP (0.5,0.15or0.5nmol/side) decreased morphine-induced wakefulness and increased the NREM and REM sleep in dose-dependent manner, while pretreatment of nor-BIN0.5,1.5pmol/side didn’t antagonize morphine-induced arousal. In addition, bilateral microinjection of CTOP or nor-BIN into the VLPO did not affect sleep-wakefulness behavior under baseline conditions.Summary:In in vitro brain slice, morphine exerted inhibitory tone on sleep-promoting neurons in the VLPO, which was mediated through mu and kappa receptors. Systemically, morphine induced arousal mainly via mu receptors in the VLPO.