Local control of the action potential in axons

描述（由申请人提供）：在轴突中，动作电位（AP）波形决定神经传递的时间和强度。虽然AP被经典地认为是一个刻板的“全部或没有”信号，但最近的研究表明，轴突AP波形比曾经认为的更具可塑性。一种解释是电压敏感性钾（Kv）通道的非均匀分布存在于不同的轴突隔室中。电生理学对轴突的相对不可及性阻碍了对这一主题的探索。这 对于中间神经元的小轴突尤其如此，其中尚未直接观察到轴突AP波形。为了研究中间神经元轴突中的AP，我开发了一种双光子（2 P）电压成像技术，以高空间和时间分辨率准确地报告快速电压变化。选择紧凑的小脑星状细胞，因为这些中间神经元提供小脑内抑制的唯一来源，并在小脑输出的时间整合中发挥重要作用。初步结果表明，AP的形状在个别的终扣由本地表示的千伏信道，允许AP波形的变化发生在一个终扣，而不干扰AP在附近的终扣。此外，AP的活动依赖性增宽发生在终扣处，而不是在连接轴突轴中，这表明在终扣处表达的Kv通道失活是造成这种效应的原因。本提案将更详细地探讨本地AP控制。第一个目标将确定哪些Kv亚型在终扣处局部表达以及局部控制如何影响轴突内的突触强度，利用膜片钳记录和2 P电压和Ca 2+成像/释放Kv抑制剂。第二个目标将揭示哪些Kv亚型允许快速活动依赖性增宽以及这种现象对突触传递的影响。这些目标的结果将提供关于中间神经元轴突如何以特定位点的方式执行复杂计算的新数据，从而更全面地了解神经元处理。

DESCRIPTION (provided by applicant): In axons, the action potential (AP) waveform determines the timing and strength of neurotransmission. Although the AP is classically thought of as a stereotyped "all or none" signal, recent studies have shown that the axonal AP waveform is more malleable then once thought. One explanation is that a non-uniform distribution of voltage-sensitive potassium (Kv) channels exists within different axonal compartments. The relative inaccessibility of axons to electrophysiology has hampered exploration of this topic. This is especially true with respect to the small axons of interneurons, in which the axonal AP waveform has not been directly observed. To study the AP in interneuron axons, I developed a 2-photon (2P) voltage imaging technique to accurately report fast voltage changes with high spatial and temporal resolution. The compact cerebellar stellate cell was chosen, as these interneurons provide the sole source of inhibition within the cerebellum and play a vital role in the temporal integration of cerebellar output. Preliminary results show APs are shaped at individual boutons by locally expressed Kv channels, allowing AP waveform changes to occur at one bouton without perturbing the AP at nearby boutons. In addition, activity-dependent broadening of the AP occurred at boutons but not in connecting axon shafts, suggesting that inactivating Kv channels expressed at boutons were responsible for this effect. This proposal will explore local AP control in more detail. The 1st aim will determine which Kv subtypes are locally expressed at boutons as well as how local control influences synaptic strength within axons, utilizing patch-clamp recordings and 2P voltage and Ca2+ imaging/uncaging Kv inhibitor. The 2nd aim will uncover which Kv subtypes allow for rapid activity-dependent broadening and the impact of this phenomenon on synaptic transmission. Results from these aims will present new data on how interneuron axons perform complex computations in a site-specific manner, leading to a more complete understanding of neuronal processing.