Basal forebrain purinergic P2 receptor mechanisms of sleep-wake regulation

 描述(申请人提供)：睡眠问题出现在大多数精神障碍患者(例如精神分裂症、阿尔茨海默氏症和帕金森氏症)中，也常见于许多其他疾病(例如疼痛、癌症、脑损伤、中风)。由神经精神疾病、其他疾病或工作/生活方式引起的嗜睡会对我们的健康和认知表现产生负面影响，有时甚至会导致死亡(例如，在开车时睡着)。许多人自行服用咖啡因等兴奋剂来暂时恢复警觉，而在临床上，患者会被开出莫达非尼等药物来治疗过度嗜睡。然而，目前的治疗策略产生了许多副作用(身体耐受性、依赖性等)。因此，有必要确定可用于防止嗜睡和改善认知能力的新的药理机制。我们实验室和其他实验室以前的工作已经证明了基底前脑(BF)作为促进觉醒的中枢的重要性。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，一种嘌呤能P2受体(P2R)激动剂应用于BF，在体内增加觉醒，并在体外兴奋BF可能的皮质投射神经元。因此，对高炉的研究 P2Rs在睡眠-觉醒调节中的作用可能有助于发现新的治疗靶点，这将使普通公众在消除嗜睡、改善认知表现和防止错误/事故方面受益匪浅。我们将测试我们的主要假设，即BF P2Rs的激活通过兴奋皮质投射的BF神经元来诱导觉醒。这两种特定的AIMS(SAS)都将在小鼠模型中进行测试，瑞士韦伯斯特小鼠的SA1和最先进的转基因小鼠模型(GAD67-GFP敲入小鼠)允许在记录SA2之前识别GABA神经元。SA1将通过反向微透析用局部药物输注特定刺激BF P2R系统，并利用脑电(EEG)/肌电(EMG)记录技术测量睡眠-觉醒状态和功率谱的变化，直接测试P2Rs的作用。我们将使用多种P2R激动剂(对P2XRs类和P2YRs类具有不同的选择性)，并将一种P2XR拮抗剂联合注入BF，以确定BF中的P2XR或P2YR是否参与促进觉醒。我们还将调查急性和慢性部分睡眠剥夺诱导的嗜睡是否可以通过激活BF P2R来逆转，这是进行翻译研究的必要的第一步。SA2将通过重点研究BF皮质投射的胆碱能和GABA能神经元来研究其细胞机制。我们将使用全细胞膜片钳记录单个细胞对P2R激动剂的反应，并确定这些反应是由P2XRs还是P2YRs负责。我们在此提出的研究将促进我们对BF嘌呤能系统在调节皮质活动中的理解，并可能有助于识别新的治疗靶点，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.