Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

突触囊泡循环 突触小泡 神经科学 去极化 神经传递 胞吐 生物 突触 肌萎缩侧索硬化 细胞生物学 化学 生物物理学 小泡 生物化学 医学 病理 受体 疾病
作者
Karthik Krishnamurthy,Davide Trotti,Piera Pasinelli,Brigid K. Jensen
出处
期刊:Journal of Visualized Experiments [MyJOVE]
卷期号: (173) 被引量:3
标识
DOI:10.3791/62813
摘要

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.

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