An approach for quantitative mapping of synaptic periactive zone architecture and organization

Abstract

Following exocytosis at active zones, synaptic vesicle membranes and membrane-bound proteins must be recycled. The endocytic machinery that drives this recycling accumulates in the periactive zone (PAZ), a region of the synapse adjacent to active zones, but the organization of this machinery within the PAZ, and how PAZ composition relates to active zone release properties remains unknown. The PAZ is also enriched for cell adhesion proteins, but their function at these sites is poorly understood. Here, using Airyscan and STED imaging of Drosophila synapses, we develop a quantitative framework describing the organization and ultrastructure of the PAZ. Different endocytic proteins localize to distinct regions of the PAZ, suggesting that sub-domains are specialized for distinct biochemical activities, stages of membrane remodeling, or synaptic functions. We find that the accumulation and distribution of endocytic but not adhesion PAZ proteins correlate with the abundance of the scaffolding protein Bruchpilot at active zones - a structural correlate of release probability. These data suggest that endocytic and exocytic activities are spatially correlated. Taken together, our results identify novel relationships between the exocytic and endocytic apparatus at the synapse and provide a new conceptual framework to quantify synaptic architecture.

Specific Extracellular Vesicles, Generated and Operating at Synapses, Contribute to Neuronal Effects and Signaling

“…The processes of various PAZ regions have been found to correspond to distinct functions, suggesting a correlation between various PAZ subdomains and sites of ongoing processes. These data suggest the neuronal exocytic and endocytic recycling activities to be spatially correlated [30]. Similar conclusions were reached by an analysis of a specific protein, the activity-related cytoskeletal protein (Arc), associated with EVs in pre-synaptic terminals.…”

“…The present, comprehensive short review illustrates the neuronal EVs generated and active at pre-synaptic terminals. These neuronal EVs, identified about 15 years ago [21,22], have been investigated from 2021 [18][19][20][23][24][25] up to now [26][27][28][29][30][31][32]. The EVs generated at synapses appear different from those generated in other neuronal areas.…”

“…In functional terms, the role of synaptic EVs appears highly relevant. Their activities emerge from a communication platform, active in signaling organization [30]. Their observed operation could be a modulation of canonical neurotransmission signaling.…”

“…The various functions of EVs could be relevant for synapses due to their possible involvement in wireless communications, protein trans-synaptic transmission, structural organization and other processes [28][29][30][31]. In the future, studies in these fields might be expanded.…”

High-resolution imaging of presynaptic ER networks inAtlastinmutants

“…To overcome this limitation, we developed a reusable imaging slide to acquire live super-resolution images of neuronal ER in Drosophila larval fillets, using high numerical aperture oil objectives and Airyscan microscopy ( Figure 1A ). Our system provides axial resolution of <170 nm after processing (Del Signore et al, 2022). We imaged larval fillets using this device on both inverted and upright microscopes within 40-50 min from dissection, a time frame in which this preparation maintains muscle potential and the capacity for evoked neurotransmitter release, even once the axon is severed from the cell body (Feng et al, 2004).…”