Imaging synapse formation using novel microfluidic platforms

描述（由申请人提供）：突触是高度特化的细胞-细胞连接，介导神经元之间的通信。这些结构由突触前和突触后终端组成，是大脑中复杂电路的基础。大多数兴奋性突触的突触后终末采用树突棘的形式，树突棘是从树突轴发出的富含肌动蛋白的突起。毫不奇怪，树突棘和突触的形成和可塑性在认知功能中起着核心作用，这些结构的异常与许多神经系统疾病有关。尽管脊髓和突触在中枢神经系统中很重要，但调控这些结构形成的分子机制尚不清楚。在确定调节脊椎和突触形成的关键分子方面的一个限制是，观察突触形成的过程非常困难。我们正在开发新的微流体装置，使我们能够动态观察突触的形成（具体目标1）。这些设备设计上的一些创新将显著提高我们以高空间和时间分辨率成像突触形成早期步骤的能力。在Specific Aim II中，我们将应用这项技术来检查突触组装过程中肌动蛋白的时空动态。此外，我们将验证我们的假设，即Rho家族gtpase的活性是肌动蛋白的关键调节因子，在突触的初始组装和成熟中至关重要。对于这些实验，我们将使用尖端的显微镜技术，包括FRAP，光激活和FRET来检查突触形成过程中的肌动蛋白动力学和调节。这些微流控平台的发展将为神经生物学家提供一个识别调节突触组装的关键分子信号的平台，从而引起极大的兴趣和利益。

DESCRIPTION (provided by applicant): Synapses are highly specialized cell-cell junctions that mediate communication between neurons. These structures are composed of pre- and post-synaptic terminals and are the basis for the complex circuitry found in the brain. Most postsynaptic terminals of excitatory synapses take the form of dendritic spines, which are actin-rich protrusions that emanate from the dendrite shaft. Not surprisingly, the formation and plasticity of dendritic spines and synapses play a central role in cognitive function and abnormalities in these structures are associated with a number of neurological disorders. Despite the importance of spines and synapses in the central nervous system, the molecular mechanisms that regulate the formation of these structures are not well understood. A limitation toward identifying key molecules that regulate spine and synapse formation has been the great difficulty in observing synapses as they form. We are developing novel microfluidic devices that will allow us to dynamically observe forming synapses (Specific Aim I). Several innovations in the design of these devices will significantly enhance our ability to image the early steps of synapse formation with high spatial and temporal resolution. In Specific Aim II, we will apply this technology to examining the spatiotemporal dynamics of actin during synaptic assembly. In addition, we will test our hypothesis that the activity of Rho family GTPases, which are key regulators of actin, is critical in the initial assembly and maturation of synapses. For these experiments, we will use cutting-edge microscopy technologies, including FRAP, photoactivation, and FRET to examine actin dynamics and regulation during synapse formation. The development of these microfluidic platforms will be of great interest and benefit to neurobiologist by providing a platform for identifying the key molecular signals that regulate the assembly of synapses. PUBLIC HEALTH RELEVANCE: Project Narrative Abnormalities in the number, size, and morphology of dendritic spines and synapses are associated with many neurological and psychiatric disorders, including mental retardation, schizophrenia, autism, epilepsy, and Alzheimer's disease. We are developing novel microfluidic devices to dynamically image the molecular assembly of these structures. A better understanding of the key molecules that regulate spine and synapse formation could lead to novel therapeutic approaches for treating these disorders.