The Role of Adenosine in Wake/Sleep Transition

说明(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，并促进慢波睡眠，这一点从外源性AD局部应用于这些中心的催眠效应中得到证明。这表明，AD局部增加与神经元活动相关的假设足以促进慢波睡眠(SWS)。这一假说可以通过使用选择性促进重组酶Cre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受限的A1R基因缺失来检验。Cre重组酶将催化带有loxP序列的转基因小鼠的基因缺失，靶基因是功能上必不可少的A1R外显子。据预测，该缺失会增加基线条件下的觉醒，并抑制睡眠剥夺后的睡眠反弹反应。增强A1R抑制作用的一个生理途径是激活NMDA受体。这将在使用体外切片和全细胞电压钳记录技术的LDT中得到表征。AD胞浆水平与细胞外水平是平衡的，因此，对细胞外、AD介导的抑制和睡眠/觉醒状态的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)的神经元形式将从RNA和蛋白质水平的表达水平和定位方面进行表征。将结合分子生物学、蛋白质生化和神经药理学方法研究AK蛋白磷酸化/去磷酸化对细胞内腺苷的潜在调节作用。在整个动物(包括大鼠和小鼠)和脑片中，将评估在整个动物(包括大鼠和小鼠)和脑片中对行为状态和对睡眠欠债和恢复睡眠的反应的腺苷激酶的磷酸化状态。这可能为N-甲基-D-天冬氨酸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(S)。

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.