Synaptic Vesicle Exocytosis

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Our goal is to advance the understanding of how exocytosis is mediated at the nanoscale single-molecule level with high temporal resolution. The progress of the last year is described below. 1. Despite decades of pharmacological studies, how the ubiquitous cytoskeletal actin regulates synaptic transmission remains poorly understood. We addressed this issue with a tissue-specific knockout of actin β-isoform or γ-isoform, combined with recordings of postsynaptic EPSCs, presynaptic capacitance jumps or fluorescent synaptophysin-pHluorin changes, and electron microscopy in large calyx-type and small conventional hippocampal synapses. We found that actin restrains basal synaptic transmission during single action potential firings by lowering the readily releasable vesicle’s release probability. Such an inhibition of basal synaptic transmission is turned into facilitation during repetitive firings by slowing down depletion of the readily releasable vesicle pool and, thus, short-term synaptic depression, leading to more effective synaptic transmission for a longer time. These mechanisms, together with the previous finding that actin promotes vesicle replenishment to the readily releasable pool, may control synaptic transmission and short-term synaptic plasticity at many synapses, contributing to neurological disorders caused by actin cytoskeleton impairment. 2. Dense-core vesicles (DCVs) are found in various types of cells, such as neurons, pancreatic β- cells, and chromaffin cells. These vesicles release transmitters, peptides, and hormones to regulate diverse functions, such as the stress response, immune response, behavior, and blood glucose levels. In traditional electron microscopy after chemical fixation, it is often reported that the dense cores occupy a portion of the vesicle toward the center and are surrounded by a clear halo. With electron microscopy after cryofixation in adrenal chromaffin cells, we report here that we did not observe halos, but dense cores filling up the entire vesicles suggesting that halos are likely the product of chemical fixation. More importantly, we observed that a fraction of DCVs contained 36–168 nm clear-core vesicles. A similar fraction of DCVs labeled with fluorescent false neurotransmitter FFN 511 or the dense-core matrix protein chromogranin A (CGA) were colocalized with fluorescently labeled or endogenous CD63 or ALIX, the membrane or lumen marker of ∼40–160 nm exosomes. These results suggest that DCVs contain exosomes. Since exosomes are generally thought to reside within multivesicular bodies in the cytosol and are released to the extracellular space to mediate diverse cell-to-cell communications, our findings suggest that DCV fusion from many cell types is a new source for releasing exosomes to mediate intercellular communications. Given that DCV fusion mediates many physiological functions, such as stress responses, immune responses, behavior regulation, and blood glucose regulation, exosome release from DCV fusion might contribute to mediating these important functions. 3. Exocytosis, which mediates important functions like synaptic transmission and stress responses, has been postulated to release all transmitter molecules in the vesicle in the “all-or-none” quantal hypothesis. Challenging this hypothesis, amperometric current recordings of catecholamine release propose that sub-quantal or partial transmitter release is dominant in various cell types, particularly chromaffin cells. The sub-quantal hypothesis predicts that fusion pore closure (kiss-and-run fusion), the cause of sub-quantal release, is dominant, and blocking pore closure increases quantal size. We tested these predictions by imaging fusion pore closure and amperometric recording of catecholamine release in chromaffin cells during high potassium application, the most-used stimulation protocol for sub-quantal release study. We found that fusion pore closure is not predominant, and inhibition of the fusion pore closure does not increase the quantal size calculated from the amperometric current charge when a sufficiently long integration time is used. These results suggest that sub-quantal release is not prevalent during high potassium application in adrenal chromaffin cells. 4. We provided a review of a series of studies that examine the membrane’s role as a signaling platform, the proteins that drive fusion and budding, and the physical principles underlying these events.