The role of gephyrin translation in inhibitory synaptic plasticity

项目摘要 突触可塑性，即神经元连接强度的活动依赖性改变，形成了分子 学习记忆和认知的基础兴奋性和抑制性连接都经历双向的 突触可塑性调节神经元的兴奋性，塑造神经回路，并协调兴奋的平衡 抑制（E/I平衡）。GABA能突触可塑性对维持E/I平衡至关重要， 功能障碍与诸如癫痫、精神分裂症和自闭症谱系障碍的病理学有关。 然而，抑制性突触可塑性及其分子基础的研究严重不足。一个关键 抑制性突触可塑性的调节剂是桥蛋白，其是GABAA受体必需的突触支架蛋白 聚集和抑制性突触传递。在抑制性突触可塑性的一种形式中， 增强（iLTP），桥蛋白簇在树突抑制性突触处迅速增加，以加强 刺激后20分钟，GABA能突触仅在树突中。iLTP的早期机制是 独立于翻译，但关于iLTP持续性以及关键蛋白质的翻译方式知之甚少 维持iLTP。回答这些问题将有助于深入了解突触可塑性的机制，以及如何 GABA能突触强度的变化长期塑造神经回路。在本提案中，我将解决这一问题， 通过关注桥蛋白翻译在维持iLTP中的作用，更具体地说， 它发生在哪里，以及如何调节它以产生抑制性突触的精确和持久的增强作用。 连接.我假设，局部桥蛋白翻译支持iLTP只在树突， 桥蛋白的翻译调节影响桥蛋白突触聚集和抑制性突触， 实力我将利用荧光原位标记的mRNA和可视化的积极翻译蛋白质， 确定iLTP后桥蛋白转录物在树突中的定位和翻译（目的1）。miR 153是一种 短的非编码转录本被鉴定为桥蛋白翻译的关键调节因子，并与学习有关， 记忆和认知。通过调节其在神经元中的表达水平， 通过免疫细胞化学和电生理学，我将确定miR 153功能对桥蛋白的影响 iLTP期间的聚集和抑制性突触强度（Aim 2）。总之，这种方法将阐明 桥蛋白翻译的定位和调节影响抑制性突触的特异性和持久性变化 并最终揭示了驱动抑制性突触可塑性以维持E/I的机制 平衡神经功能

PROJECT SUMMARY Synaptic plasticity, the activity-dependent alteration in the strength of neuronal connections, forms the molecular basis of learning, memory, and cognition. Both excitatory and inhibitory connections undergo bidirectional synaptic plasticity to tune neuronal excitability, sculpt neural circuits, and coordinate the balance of excitation and inhibition (E/I balance). GABAergic synaptic plasticity is crucial for maintaining E/I balance, and its dysfunction is implicated in pathologies such as epilepsy, schizophrenia, and autism spectrum disorders. However, inhibitory synaptic plasticity and its molecular underpinnings are critically understudied. A crucial regulator of inhibitory synaptic plasticity is gephyrin, a synaptic scaffolding protein essential for GABAA receptor clustering and inhibitory synaptic transmission. In one form of inhibitory synaptic plasticity, inhibitory long-term potentiation (iLTP), gephyrin clustering increases rapidly at dendritic inhibitory synapses to strengthen GABAergic synapses exclusively in dendrites by 20 min post-stimulation. Early mechanisms of iLTP are independent of translation, but much less is known about iLTP persistence and how translation of key proteins maintains iLTP. Answering these questions will provide insights into mechanisms of synaptic plasticity and how changes in GABAergic synaptic strength shape neural circuits long-term. In this proposal, I will address this knowledge gap by focusing on the role of gephyrin translation for maintaining iLTP and more specifically where it occurs and how it is regulated to enact precise and persistent potentiation of inhibitory synaptic connections. I hypothesize that local gephyrin translation supports iLTP exclusively in the dendrites and that translational regulation of gephyrin influences gephyrin synaptic clustering and inhibitory synaptic strength. I will utilize fluorescent in situ labeling of mRNA and visualization of actively translating proteins to determine localization and translation of gephyrin transcripts in dendrites following iLTP (Aim 1). miR153 is a short, non-coding transcript identified as a key regulator of gephyrin translation and implicated in learning, memory, and cognition. By modulating its expression levels in neurons and employing a combination of immunocytochemistry and electrophysiology, I will determine the impact of miR153 function on gephyrin clustering and inhibitory synaptic strength during iLTP (Aim 2). Together, this approach will elucidate how the localization and regulation of gephyrin translation impact specific and lasting changes to inhibitory synaptic strength during iLTP and ultimately reveal the mechanisms driving inhibitory synaptic plasticity to maintain E/I balance for proper neural function.