A Preferentially Segregated Recycling Vesicle Pool of Limited Size Supports Neurotransmission in Native Central Synapses

Abstract

SummaryAt small central synapses, efficient turnover of vesicles is crucial for stimulus-driven transmission, but how the structure of this recycling pool relates to its functional role remains unclear. Here we characterize the organizational principles of functional vesicles at native hippocampal synapses with nanoscale resolution using fluorescent dye labeling and electron microscopy. We show that the recycling pool broadly scales with the magnitude of the total vesicle pool, but its average size is small (∼45 vesicles), highly variable, and regulated by CDK5/calcineurin activity. Spatial analysis demonstrates that recycling vesicles are preferentially arranged near the active zone and this segregation is abolished by actin stabilization, slowing the rate of activity-driven exocytosis. Our approach reveals a similarly biased recycling pool distribution at synapses in visual cortex activated by sensory stimulation in vivo. We suggest that in small native central synapses, efficient release of a limited pool of vesicles relies on their favored spatial positioning within the terminal.

Myosin II Regulates Activity Dependent Compensatory Endocytosis at Central Synapses

“…We attribute most of the decrease in evoked synaptic strength, however, to a presynaptic defect that reduces the release of neurotransmitter in response to an action potential without affecting spontaneous release. This finding is surprising given the uncertainty about whether or not there is a presynaptic requirement for actin (Sankaranarayanan et al, 2003;Bourne et al, 2006;Marra et al, 2012). Thus, our results provide new evidence indicating that actin and MII work together presynaptically to support synaptic vesicle recycling.…”

“…There is some evidence suggesting that a recycling pool of vesicles recently retrieved by CME is preferentially recruited for exocytosis and that directed trafficking spatially segregates recycling vesicles from the resting (reluctant) pool (Cheung et al, 2010;Alabi and Tsien, 2012;Marra et al, 2012;Peng et al, 2012). Therefore we measured the distance of HRP-positive vesicles from the active zone in the different samples.…”

“…Since HRP-positive vesicles in cultures fixed immediately after the initial depolarization represent the recently recycled population and may have a higher probability of fusing with the plasma membrane in response to subsequent stimulation (Marra et al, 2012), an important parameter for predicting release probability may be the percentage of HRP-positive vesicles that are physically docked. Therefore we calculated the percentage of HRP-positive docked vesicles per synapse.…”

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

“…This approach has been used extensively and highly successfully in cultured neurons 7,10,24,28,30,[32][33][34] , a number of large, peripheral terminals [26][27][28][29]31,[35][36][37] , and large central release sites such as calyx of Held 25 revealing important information about organizational principles of vesicle pools. However, only recently 38 has this method been successfully applied to small central synapses in native brain tissue, where neurons are retained in relevant circuits with defined cytoarchitecture. This reflects the fact that the thick tissue of a brain slice presents a number of technical challenges compared to, for example, cultured neurons.…”

“…1a-f). The principal dye-labelling step is based on an established approach adopted in a number of previous studies 7,13,33,[38][39][40][41][42] and we outline key strategies to validate its success. Subsequently, detailed methodology for rapid microwave-enhanced fixation 43 and calibrated photoconversion of the target region are provided.…”