Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface

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.

Tracking Newly Released Synaptic Vesicle Proteins at Ribbon Active Zones

“…Release site clearance is likely a complex process and may be influenced by multiple factors (Haucke et al., 2011, Neher, 2010). Previous measurements of diffusion coefficients of membrane proteins have ranged from 1–300 nm 2 /ms (Bannai et al., 2009, Dahan et al., 2003, Funahashi et al., 2018, Gomez-Varela et al., 2010, Groc et al., 2008, Mercer et al., 2011, Mikasova et al., 2008), placing our values of ∼42 nm 2 /ms during the burst phase and ∼10 nm 2 /ms during the sustained phase at the low end of membrane protein measurements and much lower than the previous report on synaptophysin in hippocampal synapses (180 nm 2 /ms) (Funahashi et al., 2018). We propose the following three hypotheses for why our results may differ: (1) the ribbon synapse may be more densely packed with protein and other diffusion barriers than a conventional synapse or perisynaptic regions; (2) our cells exhibit minimally expressing synaptophysin-pHluorin molecules, whereas previous measurements were maximized for visualizing release and hence exhibited a much higher concentration of released synaptophysin; if fixed synaptophysin-binding proteins reside within the active zone, binding sites may be saturated in their model allowing for free diffusion; and (3) synaptophysin may have different mobility in the membrane of fish bipolar cells than in mouse hippocampal neurons.…”

Amyloid‐β oligomers suppress subunit‐specific glutamate receptor increase during LTP

“…Taking all these facts together, we suggest that PSLM retains critical properties related to synaptic plasticity. We have recently developed methods to visualize individual endocytosis events, and also to form active‐zone like membrane on an NLG‐coated glass surface [49,50]. These methods could also be useful for analyzing the effects of AβOs on endocytosis of AMPARs and presynaptic function in future studies.

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The Decade of Super-Resolution Microscopy of the Presynapse

“…Indeed, it was shown using live cell STED microscopy that Synaptotagmin1 remains clustered after SV exocytosis (Willig et al, 2006). In contrast, other reports claim rapid dispersion of SV proteins by diffusion upon exocytosis (Wienisch and Klingauf, 2006;Funahashi et al, 2018) and re-sorting and clustering into patches at the peri-AZ (Hua et al, 2011). In this context the exact role of adaptor proteins like, e.g., AP2, Stonin2 and AP180 in productive cargo clustering at endocytic sites is still not fully understood since knockdown or knockout in neurons often resulted in only minor inhibition of SV retrieval (for review see Gauthier-Kemper et al, 2015).…”