SYNAPTIC BASIS OF SLEEP CYCLE CONTROL

这一系列研究的主要目的是利用动物实验， 了解其生理和药理机制 控制睡眠，从而为睡眠提供声音基础。 人类睡眠障碍的理解和治疗， 其次是医疗和物理条件。 以前的工作 极大地推进了我们对脑干机制的认识 控制睡眠的快速眼动（REM）阶段， 脑干神经元使用乙酰胆碱作为神经递质 （胆碱能神经元）促进这一阶段的睡眠。 本申请 建立并扩展了这项工作。 要使用的关键技术是 微透析和细胞外单位记录的新组合 在自由活动的猫中，自然睡眠的猫的细胞内记录 和大鼠离体切片制备。 待研究的假设包括腺苷增加是否 在长时间的觉醒（以及代谢活动增加）之后， 作为减少觉醒（W）和增加慢波的因素 睡眠（SWS或非REM）阶段。 我们假设腺苷 增加对基底节胆碱能神经元的作用最强 前脑和中桥脑区，促进W和激活 脑电图（EEG）。 新的数据表明， 中缝背核（DRN）的状态改变的影响。 细胞外腺苷的微透析测定和腺苷的递送 腺苷转运抑制剂和伴随单位记录将 用于体内试验，而体外研究将检查机制 的行动。 含有胡萝卜素的DRN神经元解除抑制的假说 胆碱能神经元和允许REM睡眠发生时，这些DRN 还将评估SWS和REM期间的神经元慢放电; 在体内和体外技术将检查的程度， 四种因素可能控制DRN放电的减慢：GABA， 5-HT侧支反馈、腺苷和突触前障碍 肾上腺素能输入 我们将使用细胞内体内记录， 双标记以确定REM-on神经元（=放电）是否 活动选择性REM睡眠，并可能控制这种状态） 以及觉醒和REM神经元（W/R-on，可能控制 在中脑脑桥中记录的W和REM中的EEG激活 胆碱能区可被确定为胆碱能区。 使用 微透析和单位记录，我们将测试的假设， 该胆碱能区的REM-on神经元不同于W/R-on神经元 通过DRN输入作用于5 HT 1A受体的可重复性。 最后，我们将检查的腹外侧视前区（VLPOA） 下丘脑，早期对cFos蛋白的研究表明， SWS期间神经元群体的选择性激活。 使用 微透析（MD），我们将评估是否自发细胞外 GABA水平在SWS期间降低，表明去抑制， 是否MD灌注荷包牡丹碱促进SWS，因为它在 初步数据。 我们还将评估胆碱能的程度 控制（来自基底前脑）和组胺能控制（来自 结节乳头核）。 体外研究将检查后-和 这些和其他神经递质对VLPOA的突触前作用 用生物胞素和GAD免疫组织化学鉴定神经元 标签。

The broad purpose of this series of studies is to use work in animals to understand the physiological and pharmacological mechanism controlling sleep, and thereby provide a sound baiss for the understanding and treatment of human sleep disorders, both primary and secondard to medical and physchiatric conditions. Previous work has greatly advanced our knowledge of brainstem mechanisms controlling the rapid eye movement (REM) phase of sleep, suggesting that brainstem neurons using acetylcholine as a neurotransmitter (cholinergic neurons) promote this phase of sleep. This application builds on, and extends this work. The key techniques to be used are a novel combination of microdialysis and extramcellular unit recording in freely moving cats, intracellular recordings in naturally sleeping cats and the rat in vitro slice preparation. Hypotheses to be investigated include whether increases in adenosine following prolonged wakefulness (and increased etabolic activity) act as a factor reducing wakefulness (W) and increasing the Slow Wave Sleep (SWS or nonREM) phase of sleep. We hypothesize adenosine increases act most strongly on cholinergic neurons in the basal forebrain and the mesopontine area that promote W and an activated electroencephalogram (EEG). New data suggest strong adenosine state-altering effects on the dorsal raphe nucleus (DRN) also. Microdialysis measurements of extracellular adenosine and delivery of adenosine transport inhibitors and concomitant unit recordings will be used for in vivo tests, while in vitro studies will examine mechanisms of action. The hypothesis that serotonin-containing DRN neurons disinhibit cholinergic neurons and allow REM sleep to occur when these DRN neurons slow discharge during SWS and REM will also be evaluated; in vivo and in vitro techniques will examine the degree to which each of four factors may control the slowing of DRN discharge: GABA, 5HT collateral feedback, adenosine, and presynaptic disfacilitation of adrenergic input. We will use intracellular in vivo recording and double labeling to determine if the REM-on neurons (=discharge activity selective for REM sleep, and possibly controlling this state) and the Waking- and REM-on neurons (W/R-on, possibly controlling EEG activation in both W and REM) recorded in the mesopontine cholinergic zone can be positively identified as cholinergic. Using microdialysis and unit recording we will test the hypothesis that the REM-on neurons of this cholinergic zone differ from W/R-on neurons by their inhibtability by DRN input acting on 5HT1A receptors. Finally we will examine the Ventrolateral preoptic Area (VLPOA) of hypothalamus, where earlier work with cFos protein indicated a selective activation of a population of neurons during SWS. Using microdialysis (MD) we will evaluate whether spontaneous extracellular GABA levels decrease during SWS, suggesting disinhibition, and whether MD-perfused bicuculline promotes SWS, as it did in preliminary data. We will also evaluate the extent of cholinergic control (from basal forebrain) and of histaminergic control (from the tuberomammillary nucleus). In vitro work will examine the post- and pre-synaptic effects of these and other neurotransmitters on VLPOA neurons identified with biocytin and GAD immunohistochemical labeling.