Imaging Synapse Assembly in Hippocampal Circuit Development

海马回路开发中的突触组装成像

• 批准号: 9109956
• 负责人: ZACHARY P WILLS
• 金额: $ 22.78万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-08 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9109956
• 关键词: Address; Alzheimer' s Disease; Animals; Autistic Disorder; Automobile Driving; Axon; Behavior; Calcium; Calcium Signaling; Characteristics; Coupling; Data; Dendrites; Development; Disease; Employee Strikes; Event; Excitatory Synapse; Family; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Future; Glutamates; Goals; Growth; Growth Cones; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Image; Image Analysis; Injury; Knowledge; Label; Learning; Life; Link; Mediating; Mediator of activation protein; Mental Retardation; Molecular; Monomeric GTP-Binding Proteins; Mus; Natural regeneration; Nervous System Trauma; Neurons; Play; Positioning Attribute; Process; Proteins; RNA Interference; Receptor Signaling; Research; Role; Schizophrenia; Signal Transduction; Signaling Molecule; Site; Specificity; Staging; Synapses; Testing; Time; Work; calcium indicator; insight; microscopic imaging; nervous system disorder; neural circuit; novel; postsynaptic; presynaptic; programs; public health relevance; receptor; relating to nervous system; research study; sensor; synaptogenesis; tool

 DESCRIPTION (provided by applicant): How mammalian neural circuits are assembled during development remains a largely unanswered question. We recently identified a mechanism restricting neural circuit assembly, a process we determined is mediated by Nogo receptor signaling via the GTPase RhoA1. We propose to define the principal characteristics of this synapse restriction mechanism in this study. A major obstacle limiting our understanding of the neural circuit assembly process has been the inability to visualize the synaptic wiring process live while simultaneously recording the activity of proteins regulating synaptogenesis. Here, we demonstrate we have overcome these obstacles. Combining state-of-the-art live imaging microscopy with multiple labeling approaches in concert with the introduction of fluorescent sensors in axons and dendrites undergoing synapse development, we can covisualize signaling molecules that we propose restrict (RhoA) or promote synapse assembly (calcium). We hypothesize that NgRs block axo-filopodial contact formation by RhoA-mediated inhibition of postsynaptic calcium signaling. Importantly, our preliminary studies reveal we can detect distinct RhoA and calcium signals in dendrites and axons, observe changes in our sensors during axo-dendritic contact and track the fidelity and strength of these contacts over time. These findings demonstrate we are positioned to address our central hypothesis. We aim to Define RhoA's Role in Restricting Synapse Assembly (Aim 1), determining whether focal activation or global inhibition of RhoA alters synaptic contact stability and then examine the effects of NgR family loss on synaptic contact fidelity (Aim 3). Our preliminary work also reveals that RhoA activity peaks at short-lived but not stable synaptic contacts, suggesting RhoA may inhibit contact stabilization. Further, NgR loss profoundly calcium and RhoA signaling at synaptic contact sites. These findings strongly support our proposed hypothesis. The experiments in this proposal will give us an unprecedented view of the signals underlying synapse assembly and the role the NgR family plays in controlling them. These studies will provide a roadmap for defining the events and signals driving synapse assembly. A roadmap we plan to build on in the future using other sensors and assembly molecules to define how numerous circuits are assembled during development and disrupted in disease.

 描述（由适用提供）：在开发过程中如何组装哺乳动物的神经元电路仍然是一个未解决的问题。我们最近确定了一种限制神经元电路组件的机制，我们确定的过程是由NoGo接收器信号通过GTPase RhoA1介导的。我们建议在本研究中定义该突触限制机制的主要特征。限制我们对神经元电路组装过程的理解的主要障碍是无法可视化突触接线过程，同时记录了蛋白质的蛋白质恢复突触的活性。在这里，我们证明我们已经克服了这些障碍。将最先进的实时成像显微镜与多种标记方法结合在一起，与正在进行突触发育的轴突和树突中引入荧光传感器一起，我们可以将我们提出的限制（RhoA）的信号分子（RHOA）或促进突触组装（钙）（钙）进行共辩驳。我们假设NGRS通过RhoA介导的突触后钙信号传导的抑制作用来阻断Axo轴突媒体接触形成。重要的是，我们的初步研究表明，我们可以在树突和轴突中检测到不同的RhoA和钙信号，观察轴向树突接触期间传感器的变化，并随着时间的推移跟踪这些接触的忠诚度和强度。这些发现表明，我们可以定位解决我们的中心假设。我们的目的是定义Rhoa在限制突触组装中的作用（AIM 1），确定局灶性激活或全球抑制RhoA会改变突触接触稳定性，然后检查NGR家族损失对突触接触忠诚度的影响（AIM 3）。我们的初步工作还表明，RhoA活性在短暂而不是稳定的合成接触处达到峰值，这表明RHOA可能会抑制接触稳定。此外，NGR损失在突触接触部位深刻钙和RhoA信号传导。这些发现强烈支持我们提出的假设。该提案中的实验将使我们对突触组件的信号以及NGR家族在控制它们中所扮演的作用前所未有。这些研究将为定义驱动突触组件的事件和信号提供路线图。我们计划将来使用其他传感器和组装分子来确定未来的路线图，以定义在发育过程中组装多少电路并破坏疾病。