The role of vesicle recycling in synaptic depression

描述（由申请人提供）：响应于高频动作电位放电，突触表现出广泛的抑制。突触可塑性的这种常见形式通过增加神经元对突触输入中细微的时间变化的敏感性，为突触电路带来了计算优势。这种抑制被认为是突触前末梢中囊泡回收和囊泡耗尽的动力学的直接结果。然而，突触囊泡周期的动态和突触抑制的阶段之间的明确的机制联系仍然缺乏。该项目的主要目标是在海马突触中使用电生理学，光学成像，电子显微镜和分子生物学的强大组合来弥合这一差距。我们最近的研究表明，synaptotagmin 7形成一个分子开关，通过其可变剪接变体以双向方式控制囊泡回收的速率。这一观察结果为旨在了解突触囊泡再循环和突触输出之间的确切关系的研究奠定了基础。为了实现这一目标，提出了三个具体目标。在第一个具体的目标，通过活动和第二信使，如钙和甘油二酯，将研究使用荧光测量囊泡回收和电生理的调节囊泡回收和突触抑制。在第二个具体的目标，快速和缓慢的回收将通过过表达的synaptotagmin 7剪接变体的分子解剖，以检查其各自的作用，在调节突触前动力学和神经递质释放。这些功能实验将通过使用电子显微镜的突触结构的形态学分析来补充。在第三个具体目标中，将通过转染突触中的结构-功能分析来鉴定控制突触囊泡再循环的突触结合蛋白7内的结构元件。这些协调一致的调查将使我们能够更精确地了解突触囊泡的再循环和短期突触可塑性之间的机制联系。这些信息对于更好地理解从癫痫到精神分裂症的几种神经和精神疾病的病理基础也至关重要。

DESCRIPTION (provided by applicant): In response to high frequency action potential firing synapses exhibit extensive depression. This common form of synaptic plasticity brings computational advantage to synaptic circuits by increasing the sensitivity of neurons to subtle temporal changes in synaptic inputs. This depression is thought to be a direct outcome of the dynamics of vesicle recycling and vesicle depletion in presynaptic terminals. However, a clear mechanistic link between the dynamics of synaptic vesicle cycle and phases of synaptic depression is still lacking. The main goal of this project is to bridge this gap using a powerful combination of electrophysiology, optical imaging, electron microscopy and molecular biology in hippocampal synapses. Our studies have recently shown that synaptotagmin7 forms a molecular switch controlling the rate of vesicle recycling in a bi-directional manner through its alternative splice variants. This observation sets the stage for studies aimed at understanding the exact relationship between synaptic vesicle recycling and synaptic output. To fulfill this goal three specific aims are proposed. In the first specific aim, the regulation of vesicle recycling and synaptic depression by activity and second messengers, such as calcium and diacylglycerol, will be studied using fluorescent measurement of vesicle recycling and electrophysiology. In the second specific aim, the fast and slow recycling will be molecularly dissected through overexpression of synaptotagmin7 splice variants to examine their respective roles in regulation of presynaptic dynamics and neurotransmitter release. These functional experiments will be complemented by morphological analysis of synapse structure using electron microscopy. In the third specific aim, structural elements within synaptotagmin7 that control synaptic vesicle recycling will be identified through structure-function analysis in transfected synapses. These concerted investigations will enable us to understand with increasing precision the mechanistic link between recycling of synaptic vesicles and short-term synaptic plasticity. This information will also be critical for a better understanding of the pathologies underlying several neurological and psychiatric illnesses ranging from epilepsy to schizophrenia.