The Role of Adenosine in Wake/Sleep Transition

描述（由申请人提供）：腺苷（AD）激活A1腺苷受体（A1R）可以抑制脑干和基底前脑胆碱能觉醒中心的神经元，促进慢波睡眠，外源性AD局部应用于这些中心的致睡作用证明了这一点。这表明AD的局部增加与神经元活动相关的假设足以促进慢波睡眠（SWS）。这一假设可以通过转基因诱导暂时和解剖上受限的A1R基因缺失，选择性地促进重组酶Cre和表达重组酶Cre的腺相关病毒载体来验证。Cre重组酶将催化带有loxP序列的转基因小鼠的基因缺失，这些loxP序列位于功能必需的A1R外显子的侧翼。预计这种缺失会增加基线条件下的清醒时间，并抑制睡眠剥夺后的反弹睡眠反应。增加A1R抑制的生理相关手段是通过激活NMDA受体。这将在LDT中使用体外切片和全细胞电压钳记录技术进行表征。AD细胞质水平与细胞外水平处于平衡状态，因此对细胞外、AD介导的抑制和睡眠/清醒状态调节具有潜在的重要性。信号转导过程在调节AD细胞质水平中的作用将被研究。主要的腺苷代谢酶腺苷激酶（AK）的神经元形式将在RNA和蛋白质水平上的表达水平和定位方面具有特征。AK蛋白磷酸化/去磷酸化对细胞内腺苷的潜在调控将通过分子生物学、蛋白生化和神经药理学方法的结合进行研究。在整个动物（大鼠和小鼠）和大脑切片中，将评估腺苷激酶磷酸化在行为状态、睡眠债务和反弹睡眠反应中的状态。这可能为NMDA受体和其他潜在的细胞外AD调节剂提供了必要的靶细胞机制。

DESCRIPTION (provided by applicant): Adenosine (AD) activation of A1 adenosine receptors (A1R) can inhibit neurons of the brainstem and basal forebrain cholinergic arousal centers and facilitate slow wave sleep as demonstrated by the somnogenic effect of local application of exogenous AD to these centers. This suggests the hypothesis that local increases in AD, correlated with neuronal activity, are sufficient to facilitate slow wave sleep (SWS). This hypothesis may be tested by induction of temporally and anatomically restricted A1R gene deletion using transgenes with selective promotion of the recombinase, Cre, and with an adeno-associated viral vector that expresses the recombinase, Cre. The Cre recombinase will catalyze the gene deletion in transgenic mice with loxP sequences flanking the target, the functionally essential A1R exon. The deletion is predicted to increase waking under baseline conditions and to dampen the rebound sleep response to sleep deprivation. A physiologically relevant means of increasing A1R inhibition is by activation of NMDA receptors. This will be characterized in LDT using in vitro slice and whole cell voltage clamp recording techniques. AD cytoplasmic levels are in equilibrium with extracellular levels, and are, accordingly, of potential importance to extracellular, AD mediated inhibition and sleep/wake state modulation. The role of signal transduction processes in regulating the AD cytoplasmic levels will be investigated. The neuronal form of the major adenosine metabolizing enzyme, adenosine kinase (AK), will be characterized with regard to expression levels and localization at the RNA and protein level. The potential regulation of intracellular adenosine by protein phosphorylation/dephosphorylation of AK will be studied using a combination of molecular biological, protein biochemical, and neuropharmacological approaches. The state of phosphorylation of adenosine kinase across behavioral states and in response to sleep debt and rebound sleep will be assessed in whole animals (both rats and mice) and in brain slices. This may provide the requisite target cellular mechanism(s) for NMDA receptors and other potential modulators of extracellular AD.