Nitric Oxide and the Regulation of Behavioral State

虽然我们已经对调节行为状态的递质系统有了很多了解，但我们对导致大脑睡眠和醒来的实际分子机制知之甚少。本项目旨在界定这些机制。投射到基底前脑（BF）的背侧被盖/脚桥被盖核的胆碱能神经元以及BF的内源性胆碱能神经元都含有一氧化氮合酶（NOS），并且在清醒时活跃。我们发现：1）NO引起腺苷的释放， 2）透析入BF的NOS抑制剂抑制大鼠睡眠剥夺后的NREM睡眠，而透析入BF的一氧化氮供体增加NREM睡眠。这些观察结果是激发该项目的两个主要假设之一的基础，即在清醒期间由胆碱能神经元在BF中释放的NO是清醒期间腺苷释放的主要刺激物，因此是调节这种重要的睡眠素的细胞外水平的关键因素。此外，我们还发现：1）cGMP降解酶环核苷酸磷酸二酯酶具有高亲和力， (PDE)9A在BF的大神经元中表达; 2）透析到BF中的cGMP降解PDE的抑制剂增加NREM睡眠。基于这些观察结果，我们假设NO也具有与行为状态调节相关的重要作用，这些作用由NO/cGMP信号通路介导，并且独立于NO的腺苷释放作用。 1)使用微透析与行为和脑电图监测， 使用NOS抑制剂和NO供体测试NO在大鼠稳态睡眠调节中的作用。 2)确定NO/cGMP信号转导系统（cGMP水解环核苷酸磷酸二酯酶、可溶性鸟苷酸环化酶、NO合酶、蛋白激酶G）组分在与睡眠/觉醒调节相关的大脑区域中的解剖学和细胞定位。 3)采用基底前脑/视前区脑片制备，表征NO对BF和腹外侧视前核（VLPO）神经元的电生理效应。

Although much has been learned about the transmitter systems regulating behavioral state, we know little about the actual molecular mechanisms causing the brain to sleep and to wake up. This project is directed at defining these mechanisms. Cholinergic neurons of the laterodorsal tegrnental/pedunculopontine tegmental nucleus, which project to the basal forebrain (BF), as well as intrinsic cholinergic neurons of the BF, both contain nitric oxide synthase (NOS), and are active during waking. We found that: 1) NO evokes the release of adenosine from forebrain neurons in culture by a cGMP-independent mechanism; 2) A NOS inhibitor dialyzed into the BF suppressed NREM sleep following sleep deprivation in rats, and a nitric oxide donor dialyzed into the BF increased NREM sleep. These observations are the basis for one of two major hypothesis motivating this project, which is that NO, released during wakefulness in the BF by cholinergic neurons, is a major stimulus to the release of adenosine during waking, and thus is a key factor regulating the extracellular level of this important somnogen. In addition, we found that: 1) The high affinity cGMP degrading enzyme cyclic nucleotide phosphodiesterase (PDE) 9A is expressed in large neurons of the BF; 2) An inhibitor of cGMP degrading PDEs dialyzed into the BF increased NREM sleep. On the basis of these observations, we hypothesize that NO also has important effects relevant to the regulation of behavioral state that are mediated by the NO/cGMP signaling pathway, and are independent of the adenosine-releasing effects of NO. The specific aims of this project are to: 1) Use mierodialysis with behavioral and electroeneephalographie monitoring to test for the role of NO in homeostatic sleep regulation in the rat using NOS inhibitors and NO donors. 2) Determine the anatomic and cellular localization of the components of the NO/cGMP signal transduction system (cGMP hydrolyzing cyclic nucleotide phosphodiesterases, soluble guanylyl cyclase, NO synthase, protein kinase G) in regions of the brain relevant for sleep/wake regulation. 3) Characterize the electrophysiological effects of NO on neurons of the BF and ventrolateral preoptic nucleus (VLPO) using a basal forebrain/preoptic area BF/POA) slice preparation.