The Action Potential as a Modulator of Synaptic Transmission

项目摘要 动作电位(AP)是一种控制电压门控钙离子活性的指令信号 经络(CAV)和神经传递。AP波形传统上被认为是统一的， 然而，最近的研究表明，存在一种令人惊讶的 由电压门控Na+的不均匀分布引起的波形宽度的变化量 和突触上的K+通道。在海马区，神经元在经历了 波形展宽虽然还不清楚这种展宽的后果是什么。存在根本性的鸿沟 在了解AP波形的变化如何机械地影响神经传递的实验中 需要具有能够在微秒时间尺度上积分的亚细胞分辨率的方法。这个 光遗传学的发展为在小范围内操纵和成像活动提供了机会 中枢神经系统的突触，如在海马体中。这些实验将提供 首次对单个海马神经元突触前AP、Ca~(2+)内流和胞吐进行光学测量。 AP波形的调制是通过抑制一系列电压门控K+通道在药理学上实现的 这导致了AP的可预测的加宽。在这种治疗中出现了意想不到的表型 兴奋性神经元：胞吐功能的急剧增加与钙离子的轻微增加相对应，这表明 钙离子与囊泡融合关系的解偶联和突触的增强 功效。这一提议的中心假设是，加宽的AP波形改变了钙离子的贡献 改变钙离子微域的半径和/或以不同方式激活囊泡融合 机械设备。这项建议的总体目标是研究转导An的机制。 电动作电位信号向神经递质的化学释放。长期目标是 确定AP波形的异质性如何影响突触的强度和可塑性。 具体目标1将确定波形展宽如何调制突触前Cav异构体贡献 选择性毒素，用于分离具有和不具有扩大AP的特定异构体。具体目标2将决定如何 AP形状改变了钙微区和囊泡胞吐作用。利用光遗传学、基因沉默和 药理学这些实验将证明AP的加宽是否影响微域的半径或 差异化地激活介导胞吐作用的蛋白质钙感受器。考虑到这个系统的复杂性， 以及可兴奋细胞中电信号的本质，毫不奇怪，电压的突变- 控制AP形状的门控通道与多种疾病有关，包括癫痫、共济失调 还有心律失常。由于AP作为信息载体的作用，它通过调节细胞内钙和 因此，迫切需要更好地了解这一水平的神经传递 监管。

Project Summary The action potential (AP) is a command signal that sharply controls the activity of voltage-gated Ca2+ channels (Cavs) and neurotransmission. The AP waveform has traditionally been considered to be a uniform, binary signal as it propagates across an axonal arbor, however recent work has suggested there is a surprising amount of variability in the width of the waveform arising from a heterogeneous distribution of voltage-gated Na+ and K+ channels across synapses. In the hippocampus, neurons burst in high frequency trains that undergo waveform broadening though it is unclear what the ramifications of this broadening are. A fundamental gap exists in understanding how variations in the AP waveform mechanistically affect neurotransmission as an experimental approach is required with subcellular resolution that can integrate at the microsecond time scale. The development of optogenetics has provided opportunities for manipulating and imaging activity within the small en passant synapses of the central nervous system such as in the hippocampus. These experiments will provide the first optical measurements of presynaptic APs, Ca2+ influx and exocytosis in single hippocampal neurons. Modulation of the AP waveform is achieved pharmacologically by inhibiting a family of voltage-gated K+ channels which results in a predictable broadening of the AP. An unexpected phenotype occurs with this treatment in excitatory neurons: a dramatic increase in exocytosis corresponds with a minimal increase in Ca2+, suggesting an uncoupling of the clearly defined Ca2+ and vesicle fusion relationship and an enhancement of synaptic efficacy. The central hypotheses of this proposal are that a broadened AP waveform alters the Ca2+ contribution of a specific Cav isoform, changes the radius of Ca2+ microdomains and/or differentially activates vesicle fusion machinery. The overall objective of this proposal is to investigate the mechanisms behind the transduction of an electrical action potential signal to the chemical release of neurotransmitter. The long-term goals are to determine how heterogeneity of the AP waveform informs synaptic strength and plasticity. Specific Aim 1 will determine how waveform broadening modulates presynaptic Cav isoform contribution using selective toxins to isolate specific isoforms with and without a broadened AP. Specific Aim 2 will determine how AP shape alters Ca2+ microdomains and vesicle exocytosis. Using optogenetics, gene silencing and pharmacology these experiments will demonstrate if AP broadening influences the radius of microdomains or differentially activates the protein Ca2+ sensors that mediate exocytosis. Given the complexity of this system as well as the essential nature of electrical signaling in excitable cells it is unsurprising that mutations in the voltage- gated channels which control the shape of the AP are implicated in several diseases including epilepsy, ataxia and arrhythmias. Due to the role the AP plays as an information carrier by modulating intracellular Ca2+ and subsequently neurotransmission, there is a critical need for a better understanding of this level of neural regulation.