Molecular Neurobiology of Behavioral Rhythms

描述(申请人提供)：建议进行实验，深入研究果蝇节律系统的细胞神经生物学--构成动物休息-活动周期的细胞、分子和系统。一个焦点将涉及某些成年脑神经元，在这些神经元中，时钟基因产物的作用和相互作用被假设为形成昼夜行为节奏的分子和细胞底物：这些蛋白质在体内是如何物理相互作用的？为此，将进行基于FRET的分析，以评估这种具有生物学意义的相互作用。这将补充，甚至可能修改传统的发条蛋白质-蛋白质相互作用的观点，因为后者很大程度上源于对组织提取液或培养的非神经元细胞中蛋白质相互作用的表征。在将在更多的系统层面上进行的实验中，相关脑神经元的功能将通过操纵来自手持时钟基因的转基因来剖析，并通过其假定的起搏器输出因子的指定来识别其他基因。这些神经元的哪些子集执行本身输出为节律性行为的功能，以及哪些参与起搏系统内细胞之间的通信？假定的神经化学输出(上面提到的)是否仅以这种方式起作用，或者它们是否也将反馈功能传递回起搏器(从细胞和分子角度考虑)？关于已知的某些时钟基因产物和假定的时钟输出分子的节律性振荡--这些系统波动中的哪一个对于能够在恒定环境条件下维持的行为节律性是重要的，这种分子维持在一定程度上是系统属性(涉及某些神经元之间的相互作用、细胞反馈现象或两者兼而有之)？果蝇的日常行为波动代表了昼夜节律的一个特征，这种节律在后生动物中是最突出和最普遍的。此外，基因产物的种类和它们形成发条的方式在广泛的物种中都是相似的。因此，在这个模型系统中对时间生物学的脑行为研究可能会为人类某些节律相关疾病的本质提供洞察力，其中一些与果蝇时钟基因同源基因的遗传变异有关

DESCRIPTION (provided by applicant): Experiments are proposed to delve into the cellular neurobiology of Drosophila's rhythm system--the cells, molecules, and systems that underlie the animal's rest-activity cycles. One focus will involve certain adult-brain neurons, within which actions of and interactions among clock-gene products are hypothesized to form the molecular and cellular substrates of circadian behavioral rhythms: How do these proteins physically interact in vivo? To this end, FRET-based analyses will be performed to assess such biologically meaningful interactions. This will complement and may even modify the conventional view of clockwork protein-protein interactions, because the latter stems largely from characterizations of the protein interactions in tissue extracts or cultured non-neuronal cells. In experiments to be carried out at more of a systems level, THE FUNCTIONS [sic] of the brain neurons in question will be dissected by manipulations of transgenes derived from in-hand clock genes, and from the identification of others by virtue of their presumed specification of pacemaker-output factors. Which subsets of these neurons carry out functions that are output into rhythmic behavior per se, and which are involved in communication among cells within the pacemaking system? Do the presumed neurochemical outputs (alluded to above) function in that manner only, or do they also mediate feedback functions back into the pacemakers (considered both cellularly and molecularly)? With regard to the known rhythmic oscillations of certain clock-gene products and putative clock-output molecules--which of these systematic fluctuations are important for the behavioral rhythmicity that can be sustained in constant environmental conditions, and is such molecular maintenance in part a systems property (involving interactions among certain neurons, cellular feedback phenomena, or both)? Daily oscillations of behavior in Drosophila represent a feature of circadian rhythmicity that is among the most prominent and widespread among metazoan organisms. Moreover, the kinds of gene products and the ways they form the clockworks are analogous across a broad array of species. Therefore, brain-behavior studies of chronobiology in this model system may provide insights into the nature of certain rhythm-related disorders of humans, some of which are associated with genetic variations of Drosophila clock gene orthologs