An optogenetic tool for acute modulation of inhibitory synaptic function

大脑通过修改单个突触来对世界上的经验做出反应。更改 突触结构是学习和记忆的细胞基础，突触功能障碍导致 一系列神经发育和精神疾病，包括癫痫、自闭症和精神分裂症。的 突触连接的强度由突触后的潜在分子结构决定。 密度;神经递质受体，粘附蛋白，信号分子和细胞骨架元素都相互作用 以短暂和高度调节的方式塑造神经传递。模块化支架蛋白质在 在这个组织中的作用。最近对突触兴奋性突触的研究表明， 突触后支架蛋白不是均匀分布的，而是聚集在突触前膜附近。 活动区进入突触后膜内的突触下“纳米结构域”。该组织被认为是 便于快速有效的传输。抑制性突触的突触下组织仍然很差 虽然类似的原则可能适用。这种纳米尺度的功能意义 兴奋性或抑制性突触的组织仍然不清楚，因为缺乏诱导的工具。 可逆地破坏分子结构，同时测量突触功能。 在这项提案中，我将使用我们已经开发的一种新的光遗传学方法来解决这个问题， 秒）和可逆地（在几分钟内）扰乱主要抑制性的纳米级结构。 突触后支架蛋白Gephyrin我将利用光学二聚化蛋白质p22 olig，它可以自我- 在暴露于488 nm光12的数秒内寡聚化，附着于针对桥蛋白的胞内抗体（IGF 2寡聚体， GephIB）。这种新的光遗传学工具提供了一种急性干扰内源性桥蛋白组织的方法 在真实的时间里。在初步的实验中，我们发现抑制性突触强度的持续下降 在光诱导的交联的60-120秒内在表达p53 2 olig-GephIB的细胞中。我会用这个 光遗传学工具结合活细胞成像、电生理学和超分辨率显微镜， 直接测试操纵突触下支架结构域对突触传递的影响。这 这种方法不仅为突触功能提供了新的见解，而且还将填补光遗传学领域的一个主要空白。 通过快速和直接操纵突触强度来研究电路动力学的新方法工具包。

The brain responds to experiences in the world through the modification of individual synapses. Changes to synaptic architecture underlie the cellular basis for learning and memory and synaptic dysfunction results in a range of neurodevelopmental and psychiatric disorders including epilepsy, autisms, and schizophrenia. The strength of synaptic connections is governed by the underlying molecular architecture at the post-synaptic density; neurotransmitter receptors, adhesion proteins, signaling molecules and cytoskeletal elements all interact in transient and highly regulated ways to shape neurotransmission. Modular scaffolding proteins play a decisive role in this organization. Recent studies at glutamatergic excitatory synapses have demonstrated that postsynaptic scaffolding proteins are not homogeneously distributed but instead are clustered near pre-synaptic active zones into subsynaptic “nanodomains” within the postsynaptic membrane. This organization is thought to facilitate fast, efficient transmission. The subsynaptic organization of inhibitory synapses remains poorly characterized, although analogous principles likely apply. The functional significance of this nano-scale organization at either excitatory or inhibitory synapses remains unclear due to a lack of tools for inducibly and reversibly disrupting molecular architecture while simultaneously measuring synaptic function. In this proposal I will address this using a novel optogenetic approach we have developed for rapidly (within seconds) and reversibly (within minutes) perturbing the nanoscale architecture of the major inhibitory postsynaptic scaffolding protein Gephyrin. I will utilize the optical dimerization protein CRY2olig, which self- oligomerizes within seconds of exposure to 488 nm light12, attached to an intrabody against gephyrin (CRY2olig- GephIB). This novel optogenetic tool provides an approach to acutely perturb endogenous gephyrin organization in real time. In preliminary experiments we find a robust and persistent decrease to inhibitory synaptic strength in cells expressing CRY2olig-GephIB within 60-120 seconds of photo induced cross-linking. I will use this optogenetic tool in combination with live cell imaging, electrophysiology and super-resolution microscopy to directly test the effect of manipulating subsynaptic scaffolding domains on synaptic transmission. This approach will not only provide novel insight into synaptic function but will also fill a major gap in the optogenetic toolkit for new approaches studying circuit dynamics through rapid and direct manipulation of synaptic strength.