Kopke Danielle L, Broadie Kendal
Department of Biological Sciences, Vanderbilt University.
Departments of Biological Sciences, Pharmacology, Cell and Developmental Biology, Kennedy Center for Research on Human Development, Vanderbilt University and Medical Center;
J Vis Exp. 2018 May 24(135):57765. doi: 10.3791/57765.
FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a >40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.
FM染料用于研究突触小泡（SV）循环。这些两亲性探针具有亲水头部和疏水尾部，使其具有水溶性，并能够可逆地进出膜脂双层。这些苯乙烯基染料在水性介质中相对无荧光，但插入质膜外小叶会导致荧光增加>40倍。在神经元突触中，FM染料在SV内吞过程中被内化，在SV池内和之间运输，并随SV胞吐作用释放，为可视化神经传递的突触前阶段提供了一个强大的工具。谷氨酸能突触发育和功能的一个主要遗传模型是果蝇神经肌肉接头（NMJ），在那里FM染料成像已被广泛用于在各种突变条件下量化SV动力学。NMJ突触末端易于接近，有一系列漂亮的大突触小体，非常适合成像应用。在这里，我们比较和对比了三种刺激果蝇NMJ以驱动活性依赖的FM1-43染料摄取/释放的方法：1）浴加高[K]使神经肌肉组织去极化，2）吸电极运动神经刺激使突触前神经末梢去极化，3）通道视紫红质变体的靶向转基因表达用于光刺激、空间控制去极化。这些方法中的每一种在研究果蝇NMJ处基因突变对SV循环的影响时都有优点和缺点。我们将讨论这些优点和缺点，以协助选择刺激方法，以及每种策略特有的方法。除了荧光成像外，FM染料还可以被光转化为电子致密信号，使用透射电子显微镜（TEM）进行可视化，以在超微结构水平上研究SV循环机制。我们提供了来自果蝇NMJ不同刺激方法的共聚焦和电子显微镜成像的比较，以帮助指导未来实验范式的选择。
