The role of vesicle recycling in synaptic depression

描述（由申请人提供）：高频动作电位放电突触表现出广泛的抑制。这种常见形式的突触可塑性通过增加神经元对突触输入的细微时间变化的敏感性，为突触回路带来了计算优势。这种抑制被认为是突触前终末囊泡循环和囊泡耗竭动力学的直接结果。然而，突触囊泡周期动力学与突触抑制期之间的明确机制联系仍然缺乏。该项目的主要目标是利用海马体突触的电生理学、光学成像、电子显微镜和分子生物学的强大结合来弥合这一差距。我们最近的研究表明，synaptotagmin7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循环速率。这一观察结果为旨在理解突触囊泡循环和突触输出之间确切关系的研究奠定了基础。为实现这一目标，提出了三个具体目标。在第一个特定目标中，将使用囊泡循环和电生理学的荧光测量来研究活性和第二信使（如钙和二酰基甘油）对囊泡循环和突触抑制的调节。在第二个具体目标中，将通过synaptotagmin7剪接变异体的过表达来分子解剖快速和慢循环，以检查它们各自在突触前动力学和神经递质释放调节中的作用。这些功能实验将辅以电子显微镜对突触结构的形态学分析。在第三个具体目标中，将通过对转染突触的结构-功能分析来鉴定synaptotagmin7中控制突触囊泡循环的结构元件。这些协同的研究将使我们能够越来越精确地理解突触囊泡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等几种神经和精神疾病的病理学也至关重要。

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.