Genetic Analysis of Neuromuscular Synaptic Structure and Function

指导我研究的核心问题是：突触是如何起作用的？它们是如何发展的？我们有动力研究这些问题，以更好地理解神经系统和大脑是如何引起健康和疾病的行为的。为了解决我们的问题，我的研究团队将黑腹果蝇的经典遗传学和分子遗传学与发育分析和突触生理学测量相结合。由于在细胞，遗传和分子水平上的高度保守，我们研究果蝇产生的信息转化为对神经元如何发育和功能的更一般的理解。我们的基本方法是以系统的方式使用扰动基因，然后确定突触如何反应。我们使用的主要技术方法是果蝇遗传学、突触传递电生理学、成像技术，包括共聚焦、活细胞和电子显微镜；我们也按要求做分子生物学和生物化学。NSERC发现基金一直支持我们在这个框架内的探索。在这项资助中，使用最新的遗传工具，我们将研究发育竞争和合作在突触生理学中的作用。在哺乳动物的NMJs中，当多个神经元支配目标肌肉时，突触竞争就会发生，随后神经元之间的相互作用导致一个“赢家”和其他神经末梢的退出。然而，在包括果蝇在内的节肢动物NMJs中，两个或两个以上功能特化的运动神经元支配同一个肌肉细胞是正常的，通常非常接近；它们似乎相互合作，以激活目标细胞。我们现在有能力选择性地驱动这些神经网络中的一种或另一种基因表达，并询问NMJ在生理和发育上的反应。例如，我们将通过驱动凋亡诱导基因杀死其中一个神经元，并确定在缺少伙伴神经元的情况下，剩余的运动神经元如何发育和功能。在第二组实验中，我们将使用K+通道表达式来控制电兴奋性。最后，我们将通过表达众所周知的囊泡周期调节因子，通过减少两个运动神经元中的一个的突触传递来靶向突触活动。在每种情况下，我们将描述未治疗的运动神经元的功能和形态，在没有其伴侣的情况下。基于初步数据，我们将设计一个正向筛选，利用果蝇遗传学的真正优势，揭示决定两个神经支配神经元之间通信的关键分子。由于突触发育和功能的错误与各种各样的神经、认知和行为疾病有关，拟议的实验将产生新的数据，这将有助于我们了解突触的基本发育和功能；所获得的知识将进一步告知我们的神经发育和神经生理障碍的概念。

The central questions that guide my research are: How do synapses function? How do they develop? We are motivated to study these questions to better understand how the nervous system and the brain give rise to behaviors in both health and disease. To address our questions my research team combines classical and molecular genetics in Drosophila melanogaster with developmental analysis and measurements of synaptic physiology. Due to the high degree of conservation at the cellular, genetic and molecular level, the information we generate studying flies translates to a more general understanding of how neurons develop and function. Our basic approach is to use perturb genes in a systematic way and then determine how the synapses respond. The main technical approaches we use are fly genetics electrophysiology of synaptic transmission, imaging technologies, including confocal, live-cell, and electron microscopy; we also do molecular biology and biochemistry as required. NSERC Discovery Grants have continuously supported our exploration within this framework. In this grant, using newly available genetic tools, we will examine the role of developmental competition and co-operation on synaptic physiology. At mammalian NMJs, synaptic competition occurs when more than one neuron innervates a target muscle and subsequent interactions between neurons leads to one `winner' and the withdrawal of the other nerve terminals. However, at arthropod NMJs, including Drosophila, it is normal for two or more functionally specialized motor neurons to innervate the same muscle cell, often in very close proximity; they appear to co-operate to innervate the target cell. We now have the ability to selectively drive gene expression in one or the other of these MNs and ask how the NMJ responds physiologically and developmentally. As one example, we will kill one of the neurons by driving apoptosis-inducing genes and determine how the remaining motor neuron develops and functions in the absence of the partner neuron. In a second set of experiments, we will control electrical excitability using K+ channel expression. Finally, we will target synaptic activity by reducing synaptic transmission in one of the two motor neurons by expressing well known regulators, of the vesicle cycle. In each case, we will characterize the function and morphology of the untreated motor neuron, in the absence of its partner. Based upon preliminary data, we will design a forward screen, to harness the real advantage of Drosophila genetics, to uncover molecules crucial to determining communication between two innervating neurons. Since errors in synaptic development and function are associated with a wide variety of neurological, cognitive and behavioural diseases the proposed experiments will yield new data that will help us understand the basic development and function of synapses; the knowledge gained will further inform our concept of neurodevelopmental and neurophysiological disorders.