Goel Pragya, Li Xiling, Dickman Dion
Department of Neurobiology, University of Southern California, Los Angeles, USA.
Bio Protoc. 2019 Jan 5;9(1). doi: 10.21769/BioProtoc.3127.
Presynaptic boutons at nerve terminals are densely packed with synaptic vesicles, specialized organelles for rapid and regulated neurotransmitter secretion. Upon depolarization of the nerve terminal, synaptic vesicles fuse at specializations called active zones that are localized at discrete compartments in the plasma membrane to initiate synaptic transmission. A small proportion of synaptic vesicles are docked and primed for immediate fusion upon synaptic stimulation, which together comprise the readily releasable pool. The size of the readily releasable pool is an important property of synapses, which influences release probability and can dynamically change during various forms of plasticity. Here we describe a detailed protocol for estimating the readily releasable pool at a model glutamatergic synapse, the neuromuscular junction. This synapse is experimentally robust and amenable to sophisticated genetic, imaging, electrophysiological, and pharmacological approaches. We detail the experimental design, electrophysiological recording procedure, and quantitative analysis necessary to determine the readily releasable pool size. This technique requires the use of a two-electrode voltage-clamp recording configuration in elevated external Ca with high frequency stimulation. We have used this assay to measure the readily releasable pool size and reveal that a form of homeostatic plasticity modulates this pool with synapse-specific and compartmentalized precision. This powerful approach can be utilized to illuminate the dynamics of synaptic vesicle trafficking and plasticity and determine how synaptic function adapts and deteriorates during states of altered development, stress and neuromuscular disease.
神经末梢的突触前终扣密集地填充着突触小泡，这是用于快速且受调控的神经递质分泌的特殊细胞器。当神经末梢去极化时，突触小泡在称为活性区的特化部位融合，这些活性区位于质膜的离散区域，以启动突触传递。一小部分突触小泡对接并准备好在突触刺激时立即融合，它们共同构成了易释放池。易释放池的大小是突触的一个重要特性，它影响释放概率，并且在各种形式的可塑性过程中会动态变化。在这里，我们描述了一种在模型谷氨酸能突触——神经肌肉接头处估计易释放池的详细方案。这个突触在实验上很稳定，并且适用于复杂的遗传学、成像、电生理学和药理学方法。我们详细介绍了确定易释放池大小所需的实验设计、电生理记录程序和定量分析。这项技术需要在高细胞外钙浓度下使用双电极电压钳记录配置并进行高频刺激。我们已经使用这个测定法来测量易释放池的大小，并揭示了一种稳态可塑性形式以突触特异性和区室化的精度调节这个池。这种强大的方法可用于阐明突触小泡运输和可塑性的动态过程，并确定在发育改变、应激和神经肌肉疾病状态下突触功能是如何适应和恶化的。
