Basal forebrain purinergic P2 receptor mechanisms of sleep-wake regulation

 描述（由申请人提供）：睡眠问题发生在大多数精神障碍患者（例如精神分裂症、阿尔茨海默病和帕金森病）中，在许多其他疾病（例如疼痛、癌症、脑损伤、中风）中也很常见。由神经精神疾病、其他疾病或工作/生活方式引起的困倦对我们的健康和认知能力有负面影响，有时甚至会导致死亡（例如在驾驶过程中睡着）。许多人用咖啡因等兴奋剂自我催眠，以暂时恢复警觉性，而临床上，患者被处方药物，如莫达非尼，以治疗过度嗜睡。然而，目前的治疗策略产生许多副作用（身体耐受性，依赖性等）。因此，非常需要鉴定可用于预防困倦和改善认知表现的新的药理学机制。我们实验室和其他人的先前工作已经证明了基底前脑（BF）作为觉醒促进中心的重要性。它的皮质投射系统在调节皮质活动、可塑性和认知方面起着重要作用。我们的新的初步数据表明，嘌呤能P2受体（P2 R）激动剂应用于BF增加清醒在体内和兴奋BF假定的皮质投射神经元在体外。因此， P2 Rs在睡眠-觉醒调节中的作用可能有助于发现新的治疗靶点，这可以极大地有利于公众消除困倦，改善认知能力和预防错误/事故。 我们将测试我们的总体假设，即激活BF P2 Rs通过兴奋皮质投射BF神经元诱导觉醒。将在小鼠模型中测试这两种特定目标（SA），使用Swiss-Webster小鼠进行SA 1测试，使用最先进的转基因小鼠模型（GAD 67-GFP敲入小鼠）进行SA 2测试，该模型允许在记录之前识别GABA神经元。SA 1将通过使用反向微透析局部药物输注特异性刺激BF P2 R系统，并使用脑电图（EEG）/肌电图（EMG）记录技术测量睡眠-觉醒状态和功率谱的变化来直接测试P2 Rs的作用。我们将使用多种P2 R激动剂（对P2 XR类和P2 YR类具有不同的选择性），并将P2 XR拮抗剂共输注到BF中，以确定BF中的P2 XR或P2 YR是否参与促进觉醒。我们还将研究急性和慢性部分睡眠剥夺诱导的嗜睡是否可以通过BF P2 R激活来逆转，这是转化研究的重要第一步。SA 2将通过关注BF皮质投射胆碱能和GABA能神经元来研究细胞机制。我们将使用全细胞膜片钳记录对P2 R激动剂的个体细胞反应，并确定P2 XRs或P2 YRs是否负责这些反应。我们的研究将促进我们对BF嘌呤能系统在操纵皮层活动中的理解，并有助于确定新的治疗靶点，以消除嗜睡和改善认知。

 DESCRIPTION (provided by applicant): Sleep problems occur in most people with mental disorders (e.g. Schizophrenia, Alzheimer's and Parkinson's diseases) and are common in many other disorders (e.g. pain, cancer, brain injury, stroke). Drowsiness caused by neuropsychiatric conditions, other disorders or work/life-style has a negative impact on our health & cognitive performance and sometimes can even lead to death (e.g. falling asleep during driving). Many people self-medicate with stimulants such as caffeine to temporarily restore alertness, while clinically, patients are prescribed medications such as modafinil to treat excessive sleepiness. However, current treatment strategies produce many side effects (physical tolerance, dependence, etc.). Thus, there is a substantial need to identify new pharmacological mechanisms that can be utilized to prevent drowsiness, and improve cognitive performance. Previous work from our laboratory and others has demonstrated the significance of basal forebrain (BF) as a wakefulness-promoting center. Its cortically-projecting systems play an important role in regulating cortical activity, plasticity and cognition. Our novel preliminary dat suggest that a purinergic P2 receptor (P2R) agonist applied into BF increases wakefulness in vivo and excites BF putative cortically-projecting neurons in vitro. Therefore, investigation of BF P2Rs' role in sleep-wake regulation may contribute to the discovery of novel therapeutic targets, which can greatly benefit the general public in removing drowsiness, improving cognitive performance and preventing errors/accidents. We will test our overarching hypothesis that activation of BF P2Rs induces wakefulness by exciting cortically-projecting BF neurons. Both specific aims (SAs) will be tested in mouse models, with Swiss-Webster mice for SA1 and a state-of-the-art transgenic mouse model (GAD67-GFP knock-in mice) which allows the identification of GABA neurons prior to recording for SA2. SA1 will directly test the role of P2Rs by specifically stimulating the BF P2R system with local drug infusion using reverse microdialysis and measuring the changes of sleep-wake states and power spectra with electroencephalogram (EEG)/electromyogram (EMG) recording techniques. We will use multiple P2R agonists (with different selectivity to class P2XRs vs class P2YRs) and co-infusion of a P2XR antagonist into BF to determine whether P2XRs or P2YRs in BF are involved in promoting wakefulness. We will also investigate if acute and chronic partial sleep-deprivation-induced drowsiness can be reversed with BF P2R activation, an essential first step towards translational studies. SA2 will investigate the cellular mechanisms by focusing on BF cortically-projecting cholinergic and GABAergic neurons. We will use whole-cell patch clamp to record the individual cellular responses to the P2R agonists and determine if P2XRs or P2YRs are responsible for these responses. Our study proposed here will advance our understanding of the BF purinergic system in manipulating cortical activity and could contribute to the identification of novel therapeutic targets for removing drowsiness and improving cognition.