Abstract
Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17–0.19 μm2/s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.
Highlights
Synaptic vesicles are exocytosed at a presynaptic active zone immediately after arrival of an action potential, and their exocytosis can be repeated at high frequencies
In neurons on the neuroligin-coated glass, cytomatrix at the active zone-associated structural protein (CAST)-RFP positive puncta were found on axons under conventional epi-fluorescence illumination, and some of them were observed with total internal reflection fluorescence (TIRF) illumination, indicating that they were located very close to the glass surface
Neurons cultured on the non-coated glass showed CAST-RFP positive puncta under epifluorescence, most of which were undetectable under TIRF illumination (Figure 1B)
Summary
Synaptic vesicles are exocytosed at a presynaptic active zone immediately after arrival of an action potential, and their exocytosis can be repeated at high frequencies. Previous studies have shown characteristics of exocytosis, endocytosis and synaptic vesicle retrieval using electrophysiological membrane capacitance measurement (von Gersdorff and Matthews, 1994; Yamashita et al, 2005; He et al, 2006) and fluorescence imaging (Miesenböck et al, 1998; Zenisek et al, 2000; Wienisch and Klingauf, 2006; Kavalali and Jorgensen, 2014). The dynamics of synaptic vesicle proteins after a single exocytosis event have remained enigmatic, partly because of the limited spatiotemporal resolution of imaging data. Clarification of synaptic vesicle protein movement on the plasma membrane after exocytosis would provide critical information to reveal these dynamics
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