CHOLINERGIC INFLUENCE ON CIRCADIAN FUNCTION

所有哺乳动物都有一个昼夜节律钟，它支配着生物体的神经、激素和代谢功能，周期近24小时。大脑下丘脑内的视交叉上核（SCN）容纳了这个时钟。SCN与大脑的各个区域有神经连接。这些连接中最突出的是来自负责睡眠和警觉的大脑区域的胆碱能投射，即基底前脑（BF）和脑干。关于SCN内的细胞内信号转导途径，有相当多的工作是可用的。体外药理学研究表明，毒蕈碱乙酰胆碱受体（M1 AChR）的M1亚型是介导胆碱能药物对昼夜节律系统影响的受体。最近，国际药理学联合会受体命名和药物分类委员会对用于在胆碱能信号级联中涉及M1AChR的药物的特异性提出了质疑。代替特定的药理学，必须采用其他方法来确定受体介导胆碱能昼夜信号。在这项提议中，将在缺乏M1AChR（M1 KO）的小鼠中进行电生理，行为和解剖学研究。首先，将在体外和体内检测B6129 PF 1/J小鼠（一种与M1 K 0具有相似遗传背景的小鼠）对胆碱能激动剂卡巴胆碱的敏感性。其次，将测试M1 KO对卡巴胆碱的敏感性降低。最后，双标记荧光共聚焦显微镜将被用来评估是否在SCN内的胆碱能终端并列M1AChR-含有细胞，并映射其分布。

All mammals possess a circadian clock that governs daily oscillations with neural, hormonal, and metabolic functions for the organism with a near 24-h period. The suprachiasmatic nucleus (SCN) within the brain's hypothalamus houses this clock. The SCN has neural connections with various regions of the brain. Prominent among these connections are cholinergic projections from regions of the brain responsible for asleep and alerting, namely the basal forebrain (BF) and brainstem. A considerable body of work is available regarding intracellular signal transduction pathways within SCN. Pharmacological studies in vitro have implicated the M1-subtype of the muscarinic acetylcholine receptor (M1AChR) as the receptor mediating the effects of cholinergics on the circadian system. Recently, the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification has questioned the specificity of the drugs used to implicate the M1AChR in the cholinergic signaling cascade. In lieu of specific pharmacology, other methods must be employed to confidently identify the receptor(s) mediating cholinergic circadian signaling. In this proposal, electrophysiological, behavioral and anatomical studies will be carried out in mice devoid of the M1AChR (M1KO). First, the B6129PF1/J mouse, a mouse of similar genetic background to the M1K0, will be tested for sensitivity to the cholinergic agonist, carbachol, both in vitro and in vivo. Second, the M1KO will be tested for decreased sensitivity to carbachol. Finally, double-label fluorescence confocal microscopy will be used to evaluate whether the cholinergic terminals within the SCN are juxtaposed to M1AChR-containing cells and to map their distribution.