Integrated research for the mechanism of sleep/wakefulness regulation

睡眠/觉醒是一种只在全身观察到的生理现象。因此，利用整个动物进行研究是必要的。光遗传学是最近开发的通过表达光激活蛋白如通道视紫红质2（ChR 2）或盐视紫红质（Halo）来控制神经元活性的技术。这项技术使我们能够在体内和体外使用光来控制产生食欲素的神经元（食欲素神经元）的活性。首先，我们用膜片钳技术证实了CHR 2和Halo在食欲素神经元中的功能，并确定了光照条件。接下来，我们使用清醒小鼠进行体内实验。将光纤双侧植入下丘脑。橙子光通过光导纤维照射下丘脑可抑制食欲素神经元的活性。同时，细胞外记录也记录到中缝核内多巴胺能神经元的活动。急性抑制食欲素神经元可诱导慢波睡眠，而食欲素能神经元的活性随着慢波睡眠的开始而降低。这一结果表明，抑制食欲素神经元诱导小鼠慢波睡眠。

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.