Optogenetic Control of Hypocretin Neurons and Stress

描述（由申请人提供）：下丘脑分泌素神经元和应激的光遗传学控制急性应激反应的神经基础涉及产生促皮质激素释放因子（CRF）的神经元的激活和多个稳态回路之间的相互作用。然而，人们对这些相互作用的细节知之甚少。下丘脑外侧（LH）产生下丘脑分泌素（Hcrt，也称为食欲素）的神经元对于维持觉醒稳定性很重要，因为在小鼠、狗和人类中，Hcrt功能的丧失与嗜睡症有关。下丘脑分泌素神经元被急性应激激活，接受来自CRF末梢的神经支配，并被CRF去极化。相反，注入Hcrt-1激活下丘脑-垂体-肾上腺（HPA）轴，下丘脑分泌素受体拮抗剂可以阻断急性应激诱导的ACTH释放。在这里，我们建议使用一种新开发的光遗传学方法来测试下丘脑分泌素神经元的活性是否是激活下丘脑轴的必要和充分条件。在第一个目标中，我们将通过监测Hcrt缺陷小鼠的急性应激反应来确定Hcrt神经元是否需要激活HPA轴。在具体目标2中，我们将利用光遗传学方法确定Hcrt神经元的活性是否足以诱导应激样反应。我们还将通过使用在下丘脑分泌素细胞中表达光激活氯通道的慢病毒转导小鼠来测试何时需要这种激活。这项技术将使我们能够破译与应激反应相关的下丘脑分泌素网络的神经密码。在第三个目标中，我们将测试Hcrt的功能连通性。我们将测试光刺激对下丘脑轴的影响是由室旁下丘脑核的CRF信号直接介导的，还是通过其他脑结构间接介导的。本修订提案中收集的数据将以前所未有的时间分辨率增强我们对应激反应的神经基础的理解，并可能导致新的应激障碍和相关疾病的治疗方法，以及确定靶向Hcrt系统治疗其他疾病的药物的潜在副作用。

DESCRIPTION (provided by applicant): Optogenetic control of hypocretin neurons and stress The neural underpinnings of the response to acute stress involve activation of neurons producing corticotrophin releasing factor (CRF) and interactions between multiple homeostatic circuits. However, the details of these interactions are poorly understood. Hypocretin (Hcrt, also known as Orexin)-producing neurons in the lateral hypothalamus (LH) are important for maintaining arousal stability since loss of Hcrt function has been linked to narcolepsy in mouse, dog and human subjects. Hypocretin neurons are activated by acute stress, receive innervation from CRF terminals and are depolarized by CRF. Conversely, infusion of Hcrt-1 activates the hypothalamo-pituitary-adrenal (HPA) axis, and hypocretin receptor antagonists can block the release of ACTH induced by acute stress. Here we propose to use a newly developed optogenetic method to test whether the activity of hypocretin neurons is necessary and sufficient to activate the HPA axis. In the first aim, we will determine whether Hcrt neurons are necessary to activate the HPA axis by monitoring the acute stress response in Hcrt-deficient mice. In specific aim 2, we will determine whether the activity of Hcrt neurons is sufficient to induce a stress-like response by using an optogenetic approach. We will also test when is this activation required by using mice transduced with a lentivirus expressing a photoactivatable chloride channel in hypocretin cells. This technology will allow us to decipher the neural code of the hypocretin network that is associated with the stress response. In the third aim, we will test the functional connectivity of Hcrt. We will test whether the effects of photostimulation on the HPA axis are mediated directly by CRF signaling in the paraventricular hypothalamic nucleus, or whether the effect is indirect through other brain structures. The data collected in this revised proposal will enhance our understanding of the neural basis of the stress response with unprecedented temporal resolution and may lead to novel therapeutics for stress disorders and related diseases, as well as identify potential side effects for drugs that target the Hcrt system for other disorders. PUBLIC HEALTH RELEVANCE: The data collected in this research proposal will enhance our understanding of stress response and may lead to novel therapeutics for stress disorders and related diseases in the general public.