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。更改为 突触结构是学习和记忆的细胞基础，突触功能障碍会导致 一系列神经发育和精神障碍，包括癫痫、自闭症和精神分裂症。这个 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、黏附蛋白、信号分子和细胞骨架元素都相互作用 以短暂和高度调控的方式塑造神经传递。模块化支架蛋白起决定性作用 在此组织中扮演的角色。最近对谷氨酸能兴奋性突触的研究表明， 突触后支架蛋白不是均匀分布的，而是聚集在突触前附近 活动区进入突触后膜内的突触下“纳域”。这个组织被认为是 促进快速、高效的传输。抑制性突触的亚突触组织仍然很差。 具有特点的，尽管类似的原则可能适用。这种纳米尺度的功能意义 由于缺乏诱导的工具，兴奋性或抑制性突触的组织结构仍不清楚 可逆地扰乱分子结构，同时测量突触功能。 在这项提议中，我将使用我们为快速(在 秒)并且可逆地(在几分钟内)扰乱主要抑制物的纳米尺度架构 突触后支架蛋白。我将利用光学二聚蛋白CRY2olig，它可以自我- 在488 nm光12照射下几秒钟内寡聚，连接到体内抗吉普林(CRY2olig- GephIB)。这一新的光遗传工具提供了一种强烈干扰内源性吉卜林组织的方法 实时的。在初步实验中，我们发现抑制性突触强度显著持续下降 在表达CRY2olig-GephIB的细胞中，在60-120秒内光诱导交联。我要用这个 结合活细胞成像、电生理学和超分辨率显微镜的光遗传工具 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的一个重大空白 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的工具包。

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.