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Objective Sleep deprivation (SD) is considered a risk factor for various disorders involving behavior, emotion, attention, learning ability, and immunological functions.It has been reported that SD-induced physiological functional disturbances are associated with reduction of melatonin.Animal experiments unequivocally show that SD can lead to melatonin reduction in the pineal gland of rats.However, the underlying mechanism remains unknown.The habenular nucleus (Hb) is an important structure that regulates the function of the pineal gland, which may affect melatonin content in the pineal gland after SD.In the present study, we want to confirm that the reduction of melatonin in pineal gland after SD is related to the changed activity of the Hdb, revealing a central mechanism of sleeping regulation.Methods The content of melatonin in the pineal gland and the content of glutamate (Glu) and γ-aminobutyric acid (GABA) in the Hb were measured by high performance liquid chromatography.The number of Fos-positive neurons in the Hb was measured by immunohistochemistry.Results In the present study, the melatonin content in the pineal gland was significantly reduced, and GABA content in the Hb was significantly increased after SD.Furthermore, the melatonin content in the pineal gland was markedly reduced after Hb lesion under normal sleep and SD conditions.The number of Fos-positive neurons was significantly decreased in the lateral and medial Hb after SD.Conclusion The results demonstrate that the reduction of melatonin in the pineal gland after SD is related to decreased activity of Hb neurons, and that the Hb can regulate sleep-wake rhythm by influencing melatonin secretion in the pineal gland.