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（A1 R）可抑制脑干和基底前脑胆碱能唤醒中心的神经元，并促进慢波睡眠，如外源性AD局部应用于这些中心的致眠作用所证明。这表明了这样的假设：与神经元活动相关的AD局部增加足以促进慢波睡眠（SWS）。该假设可以通过使用具有重组酶Cre的选择性促进的转基因和表达重组酶Cre的腺相关病毒载体诱导时间和解剖学上限制的A1 R基因缺失来测试。Cre重组酶将催化转基因小鼠中的基因缺失，其中loxP序列侧接靶标，功能上必需的A1 R外显子。据预测，这种删除会增加基线条件下的清醒，并抑制睡眠剥夺后的睡眠反弹反应。增加A1 R抑制的生理学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平衡，因此，对细胞外AD介导的抑制和睡眠/觉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和蛋白质水平的表达水平和定位方面进行表征。将使用分子生物学、蛋白质生物化学和神经药理学方法的组合来研究AK的蛋白质磷酸化/去磷酸化对细胞内腺苷的潜在调节。将在整个动物（大鼠和小鼠）和脑切片中评估腺苷激酶跨行为状态以及响应睡眠债和反弹睡眠的磷酸化状态。这可能为NMDA受体和细胞外AD的其他潜在调节剂提供必要的靶细胞机制。

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.