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

描述（由申请人提供）：神经系统由管理思想和行为的高度复杂的电路组成。为了确保正确的回路形成，神经元必须在形成突触连接之前在它们接触的许多神经突中识别适当的突触目标。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作用。尽管它在回路形成中起着中心作用，但突触特异性却知之甚少。我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，并利用简单的，良好的特点，神经系统的C。elegans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。该建议与NINDS的使命有关，以支持与神经系统疾病的原因有关的领域的基础研究，以及NIMH的使命，以支持对精神疾病和大脑和行为的基础科学的研究。我的具体目标是：1）可视化C中一对特定的突触前和突触后神经元之间突触连接的变化。elegans，2）发现该系统中突触特异性的分子决定因素，以及3）使这种新的标记方法适用于哺乳动物神经元。为了观察突触连接的变化，我开发了一种突触间标记。我已经融合了一个分裂的绿色荧光蛋白的互补片段，前和突触后本地化的蛋白质，在细胞特异性启动子下表达。我已经成功地在C语言中可视化了两个具有良好特征的电路中的连通性变化。使用已知的突触特异性突变体。我感兴趣的是在复杂的环境中，突触特异性是如何调节的，例如神经束，平行的神经突必须区分多个潜在的目标，以形成适当的连接。因此，我现在正在调整突触间标记，以标记后神经束中特定神经元对之间的突触连接。我将采取无偏见的遗传方法来发现在这个系统中引导突触特异性的分子机制，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层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.