Purinergic Mechanisms in Homeostatic Sleep Control

这个建议的主要目的是了解控制嗜睡的细胞机制。睡眠 约40%的军事人员在部署后立即经历剥夺（SD）和失眠， SD是一个众所周知的风险因素，对退伍军人的心理健康产生不利影响。基底前脑（BF） 这是一个重要的大脑区域，用于控制SD后增加的睡眠倾向，即睡眠稳态。 以前的研究周期研究了睡眠稳态是如何通过抑制BF促醒作用介导的， 神经元通过增加细胞外浓度的腺苷（ADex），并研究的作用， 细胞外三磷酸腺苷（ATPex）作为ADex的来源。然而，电路和神经递质的基础 ATPex和ADex的这些增加仍然没有得到解决。 BF内至少有三种类型的皮质投射唤醒神经元共存：胆碱能（ChAT+）， 表达GABA能（PV+）和谷氨酸能（vGluT 2+）的小清蛋白。BF ChAT+的选择性病变研究 神经元表明，它们是SD诱导的ADex增加和睡眠稳态所必需的。然而，在这方面， 不可逆的损伤可引起超越ChAT+神经元损失的退行性变化。照经上所 重要的是使用可逆刺激或失活实验来测试ChAT+神经元的作用。此外，委员会认为， 对完整ChAT+神经元的需求可能反映了ChAT+神经元自身的ATPex/ADex释放 和/或ChAT+调节邻近的非胆碱能神经元。因此，在本申请中，我们将使用我们的新颖的 视透析的方法学（Zant等人，2016）结合联合收割机可逆的，神经元亚型特异性光遗传学 同时在体内微透析的操作，以测量神经化学变化的存在和 缺乏特定神经递质受体的选择性拮抗剂来描述功能性相互作用 神经元亚型之间的区别最近的报道表明，光遗传学刺激vGluT 2+神经元兴奋 PV+神经元对ChAT+神经元的影响较弱。此外，BF 谷氨酸受体激活促进皮层乙酰胆碱（ACh）释放，并增加BF中ADex水平。 这些发现为我们的假设提供了基础，即BF ChAT+和vGluT 2+之间的局部正反馈 而PV+神经元不导致ATPex/ADex增加，这是稳态睡眠反应的基础。三 具体的目标（SA）提出了实现这一目标：我们将研究胆碱能，小清蛋白的作用， 使用光遗传学刺激在睡眠稳态中表达GABA能和多巴胺能神经元， 抑制（SA 1）和BF增加的ATPex和ADex使用光透析（SA 2）。利用体外电生理学 和在体内视透析，SA 3将检查的假设，胆碱能和 SD期间，多巴胺能神经元导致BF ATPex/ADex增加。成功完成这些研究将 提供了深入了解细胞和神经化学机制下的稳态睡眠反应。 因此，促进了治疗措施的发展，以减少睡眠不足对睡眠质量的有害影响。 军人、退伍军人和睡眠障碍者。

The broad objective of this proposal is to understand the cellular mechanisms which control sleepiness. Sleep deprivation (SD) and insomnia are experienced by ~40% of military personnel immediately after deployment and SD is a well-known risk factor that adversely affects the mental health of veterans. The basal forebrain (BF) is an important brain region for controlling the increased propensity for sleep after SD i.e. sleep homeostasis. Previous grant cycles investigated how sleep homeostasis is mediated by inhibition of BF wake-promoting neurons via increases in the extracellular concentration of adenosine (ADex), and investigated the role of extracellular adenosine triphosphate (ATPex) as a source of ADex. However, the circuit and neurotransmitter basis for these increases in ATPex and ADex has remained unresolved. At least three types of cortically-projecting, wake-active neurons coexist within BF: cholinergic (ChAT+), parvalbumin expressing GABAergic (PV+) and glutamatergic (vGluT2+). Selective lesion studies of BF ChAT+ neurons suggested that they are required for SD-induced increases in ADex and for sleep homeostasis. However, irreversible lesions may cause degenerative changes which go beyond loss of ChAT+ neurons. Thus, it is important to test the role of ChAT+ neurons using reversible stimulation or inactivation experiments. Furthermore, the need for intact ChAT+ neurons could reflect either ATPex/ADex release from ChAT+ neurons themselves and/or ChAT+ modulation of neighboring, non-cholinergic neurons. Thus, in this application we will use our novel methodology of optodialysis (Zant et al., 2016) to combine reversible, neuronal-subtype-specific optogenetic manipulations with simultaneous in vivo microdialysis to measure neurochemical changes in the presence and absence of selective antagonists for particular neurotransmitter receptors to delineate functional interactions between neuronal subtypes. Recent reports showed that optogenetic stimulation of vGluT2+ neurons excited ChAT+ neurons whereas PV+ neurons had only a weak influence on ChAT+ neurons. Furthermore, BF glutamate receptor activation enhanced acetylcholine (ACh) release in cortex and increased ADex levels in BF. These findings provide the basis for our hypothesis that local positive feedback between BF ChAT+ and vGluT2+ but not PV+ neurons leads to the ATPex/ADex increases which underlie the homeostatic sleep response. Three specific aims (SA) are proposed towards this goal: We will examine the role of cholinergic, parvalbumin expressing GABAergic, and glutamatergic neurons in sleep homeostasis using optogenetic stimulation and inhibition (SA1) and BF increases of ATPex and ADex using optodialysis (SA2). Using in vitro electrophysiology and in vivo optodialysis, SA3 will examine the hypothesis that a local positive feedback between cholinergic and glutamatergic neurons leads to BF ATPex/ADex increases during SD. Successful completion of these studies will provide insight into the cellular and neurochemical mechanisms underlying the homeostatic sleep response. Thereby, facilitating the development of therapeutic measures to reduce the deleterious effects of sleep loss in military personnel, veterans and people with sleep disorders.