Cheung Giselle, Cousin Michael A
Membrane Biology Group, Centre for integrative Physiology, University of Edinburgh.
J Vis Exp. 2011 Nov 11(57):3143. doi: 10.3791/3143.
After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis(1,2). The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation(1,2). It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli. FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals(3-8). They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane(9). Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal(10), the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified. Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable(11). Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)(12,13).
在中枢神经末梢释放神经递质后，突触小泡（SVs）通过内吞作用迅速回收。回收的突触小泡随后重新填充神经递质，并重新加入循环池，循环池定义为可用于胞吐作用的突触小泡(1,2)。循环池通常可细分为两个不同的池——即时释放池（RRP）和储备池（RP）。顾名思义，即时释放池由可立即用于融合的突触小泡组成，而储备池突触小泡仅在强烈刺激时释放(1,2)。拥有一种可靠的检测方法来报告这些突触小泡池的差异补充情况很重要，以便了解1）突触小泡在不同内吞模式（如网格蛋白依赖性内吞和活性依赖性批量内吞）后如何运输，以及2）控制即时释放池和储备池响应不同刺激而动员的机制。FM染料通常用于定量报告中枢神经末梢的突触小泡周转情况(3-8)。它们有一个疏水的烃尾，可在脂质双层中进行可逆分配，还有一个亲水的头部基团，可阻止其穿过膜。这些染料在水溶液中荧光较弱，但当分配到膜中时，其量子产率会显著增加(9)。因此，FM染料是追踪活跃循环的突触小泡的理想荧光探针。使用FM染料的标准方案如下。首先，将它们应用于神经元，并在内吞过程中被摄取（图1）。从质膜上洗去未内化的染料后，回收的突触小泡在循环池内重新分布。然后使用卸载刺激耗尽这些突触小泡（图1）。由于突触小泡的FM染料标记是量子化的(10)，因此产生的荧光下降与释放的囊泡数量成正比。因此，可以可靠地量化上一轮内吞作用产生的突触小泡的循环和融合情况。在这里，我们提出了一种经过修改的方案，以获得另外两个信息要素。首先，使用顺序卸载刺激来分别卸载即时释放池和储备池，以便对特定突触小泡池的补充情况进行量化。其次，每个神经末梢都要进行两次该方案。因此，可以将同一神经末梢在S1阶段的反应与S2阶段测试物质的存在情况进行比较（图2），提供一个内部对照。这很重要，因为不同神经末梢之间突触小泡循环的程度差异很大(11)。任何贴壁的原代神经元培养物都可用于该方案，不过接种密度、溶液和刺激条件已针对小脑颗粒神经元（CGNs）进行了优化(12,13)。
