Jefferson Weinberg ALS Center, Thomas Jefferson University;
Jefferson Weinberg ALS Center, Thomas Jefferson University.
J Vis Exp. 2021 Jul 16(173). doi: 10.3791/62813.
Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons. Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.
在神经元变性之前，肌萎缩侧索硬化症（ALS）和/或额颞叶痴呆（FTLD）患者的运动和认知缺陷的原因是神经元和运动神经元与肌肉之间的通讯功能障碍。突触传递的潜在过程涉及膜去极化依赖性突触囊泡融合和神经递质释放到突触中。这个过程通过局部钙流入突触前末梢（突触囊泡所在的地方）来发生。这里，该方案描述了荧光为基础的活细胞成像方法，该方法可可靠地报告培养神经元中去极化介导的突触囊泡胞吐作用和突触前末梢钙流入动力学。使用整合到突触囊泡膜中的苯乙烯基染料，阐明了突触囊泡的释放。另一方面，为了研究钙进入，使用了 Gcamp6m，这是一种遗传编码的荧光报告蛋白。我们采用高钾氯化物介导的去极化来模拟神经元活动。为了明确无误地量化突触囊泡胞吐作用，我们测量归一化苯乙烯基染料荧光随时间的损失。在相似的刺激条件下，在钙流入的情况下，Gcamp6m 荧光增加。以与苯乙烯基染料方案类似的方式对这种荧光变化进行归一化和量化。这些方法可以与基于转染的荧光标记突变蛋白的过表达进行多路复用。这些方案已被广泛用于研究 FUS-ALS 和 C9ORF72-ALS 的模型中的突触功能障碍，利用原代啮齿动物皮质和运动神经元。这些方案允许快速筛选可能改善神经元通讯的化合物，因此，这些方法不仅对 ALS 的研究有价值，而且对神经退行性和发育神经科学研究的所有领域都有价值。
