Novel Split GFP Based Intersynaptic Markers to Study Synaptic Specificity in vivo

描述(申请人提供)：神经系统由控制思想和行为的高度复杂的回路组成。为了确保正确的电路形成，神经元必须在形成突触连接之前从它们接触的许多突触中识别合适的突触目标。突触发生的改变被认为在精神分裂症和自闭症等疾病中发挥了作用。尽管突触在回路形成中起着核心作用，但突触的特异性却知之甚少。我已经开发了一种新的方法来可视化体内特定神经元之间的突触。我建议使用这种方法，并利用线虫简单的、特征良好的神经系统来阐明这一基本过程的分子机制。然后，我建议将我的发现扩展到哺乳动物神经元。这项建议与NINDS的任务相关，NINDS的任务是支持与神经疾病原因相关的领域的基础研究，NIMH的任务是支持精神疾病的研究以及大脑和行为的基础基础科学。我的具体目标是：1)可视化线虫特定突触前和突触后神经元之间突触连接的变化，2)发现这个系统中突触特异性的分子决定因素，3)使这种新的标记方法适用于哺乳动物神经元。为了可视化突触连接的变化，我开发了一个突触间标记。我已经将分裂的GFP的互补片段与突触前和突触后的定位蛋白融合在一起，在细胞特异性启动子下表达。我使用已知的突触特异性突变体，成功地在线虫的两个特征良好的回路中可视化了连接性的变化。我感兴趣的是，在复杂的环境中，突触的特异性是如何受到调节的，比如神经束，在这种环境中，平行的神经突起必须区分多个潜在的目标，才能形成适当的连接。因此，我现在正在修改突触间标记，以标记后神经束中特定神经元对之间的突触连接。我将采取一种公正的遗传学方法来发现这个系统中指导突触专一性的分子机制，利用这个标记来检测连接中的缺陷。最后，我正在开发用于培养哺乳动物皮质神经元的突触间标记。从这项研究中获得的见解将有助于理解脊椎动物神经系统复杂环境中的突触特异性。了解调节回路形成的机制将使我们更接近理解和治疗神经系统疾病。

DESCRIPTION (provided by applicant): The nervous system is composed of highly complex circuits that govern thought and behavior. To ensure correct circuit formation, neurons must identify appropriate synaptic targets among the many neurites they contact before forming synaptic connections. Altered synaptogenesis is thought to play a role in disorders such as schizophrenia and autism. Despite its central role in circuit formation, synaptic specificity is poorly understood. I have developed a novel way to visualize synapses between specific neurons in vivo. I propose to use this method and take advantage of the simple, well-characterized nervous system of C. elegans to elucidate molecular mechanisms that underlie this fundamental process. I then propose to extend this my findings to mammalian neurons. This proposal is relevant to the NINDS mission to support basic research in fields related to the causes of neurological disorders, and the NIMH mission to support research on mental disorders and the underlying basic science of brain and behavior. My specific aims are: 1) to visualize changes in synaptic connectivity between a specific pair of pre- and postsynaptic neurons in C. elegans, 2) to discover the molecular determinants of synaptic specificity in this system, and 3) to adapt this new labeling method to mammalian neurons. To visualize changes in synaptic connectivity, I have developed an intersynaptic marker. I have fused complementary fragments of a split GFP to pre- and postsynaptically localized proteins, expressed under cell-specific promoters. I have successfully visualized changes in connectivity in two well-characterized circuits in C. elegans using known synaptic specificity mutants. I am interested in how synaptic specificity is regulated in complex environments, such as nerve bundles, where parallel neurites must distinguish among multiple potential targets to form appropriate connections. Therefore I am now adapting the intersynaptic marker to label synaptic connections between a specific neuron pair in a posterior nerve bundle. I will take an unbiased genetic approach to discover molecular mechanisms guiding synaptic specificity in this system, utilizing this marker to detect defects in connectivity. Finally, I am developing the intersynaptic marker for use in cultured mammalian cortical neurons. Insights gained from this study will aid in understanding synaptic specificity in the complex environments of the vertebrate nervous system. Understanding the mechanisms that regulate circuit formation will bring us closer to understanding and treating neurological diseases.