Integrated research for the mechanism of sleep/wakefulness regulation

睡眠/觉醒是一种生理现象，只在整个身体中观察到。因此，用整个动物进行研究是必要的。光遗传学是最近发展起来的一项技术，通过表达光激活蛋白，如通道视紫红质2（ChR2）或halorhodopsin（Halo）来控制神经元的活性。这项技术使我们能够在体内和体外利用光来控制产生食欲素的神经元（食欲素神经元）的活动。首先，我们利用薄片膜片钳确定了CHR2和Halo在食欲素神经元中的功能，并确定了光照条件。接下来，我们用有意识的小鼠进行体内实验。双侧下丘脑植入光纤。通过光纤照射到下丘脑的橙色光抑制了食欲素神经元的活动。同时，细胞外记录中缝核5 -羟色胺能神经元的活动。食欲素神经元的急性抑制诱导慢波睡眠，血清素能神经元的活性随慢波睡眠的开始而下降。结果表明，抑制食欲素神经元可诱导小鼠慢波睡眠。

Sleep/wakefulness is a physiological phenomenon which observed only in the whole body. Thus, it is essential to study using whole animal. Optogenetics is a recently developed technique to control the activity of neurons by expressing light activated proteins, such as channelrhodopsin2(ChR2) or halorhodopsin(Halo). This technique allows us to control the activity of orexin-producing neurons(orexin neurons) using light in vivo and in vitro. First, we confirmed the function of CHR2 and Halo in the orexin neurons using slice patch clamp and decided a condition of light illumination. Next, we proceeded in vivo experiment using conscious mouse. Optical fibers were bilaterally implanted into the hypothalamus. The activity of orexin neurons was inhibited by illuminating orange light into the hypothalamus through the optical fibers. At the same time, the activity of serotonergic neurons in the raphe nucleus is recorded by the extracellular recordings. Acute inhibition of orexin neurons induced slow wave sleep and the activity of serotonergic neurons was decreased in accordance with initiation of slow wave sleep. This result showed that inhibition of orexin neurons induced slow wave sleep in mice.