Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must ‘dock’ to refill vacated release sites. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. After stimulation, the total number of docked vesicles across all synapses decreases by ~40%. Within 14 ms, new vesicles are recruited and fully replenish the docked pool, but this docking is transient and they either undock or fuse within 100 ms. These results demonstrate that recruitment of synaptic vesicles to release sites is rapid and reversible.

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must refill vacated release sites. How many vesicles can fuse at a single active zone, where they fuse within the active zone, and how quickly they are replaced with new vesicles is not well-established. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone; this multivesicular release is augmented by increasing the extracellular calcium concentration. Synchronous fusions are distributed throughout the active zone, whereas asynchronous fusions are biased toward the center of the active zone. Immediately after stimulation a large fraction of vesicles become undocked. Between 8 and 14 ms, new vesicles are recruited to the plasma membrane and fully replenish the docked pool, but docking of these vesicles is transient and they either undock or fuse within 100 ms. These results demonstrate that recruitment of synaptic vesicles to release sites is rapid and reversible.3 When an action potential invades a synaptic bouton, voltage-gated calcium channels open and calcium influx triggers vesicle fusion to release neurotransmitter. Synaptic vesicle fusion takes place at a specialized membrane domain: the active zone 1 . It is believed that the active zone is organized into one or more release sites, which are individual units at which a single synaptic vesicle can fuse 2 . Ultrastructural studies demonstrate that some synaptic vesicles are in contact with the plasma membrane and define the 'docked' pool 3,4 . Since both docking and physiological readiness require engaged SNARE proteins 4-6 , docked vesicles are thought to represent fusion-competent vesicles. There are several features of fusion that could be addressed by ultrastructural studies if methods existed to capture such rapid events as fusion and docking, specifically: how many vesicles fuse in response to an action potential, when and where they fuse, and how vesicle docking refills fusion sites.How many vesicles fuse in response to an action potential has been intensely debated. A large body of work argues that, even when release probability is high, small central synapses act as binary operators: no more than one vesicle can fuse per action potential 7-10 . One possible mechanism is that the fusion of a vesicle blocks further fusions by 'lateral inhibition' 11,12 . By contrast, evidence from electrophysiology [13][14][15][16] , optical imaging 17-21 and electron microscopy 22,23 suggests that multiple vesicles can fuse at a synapse 24 .Evoked neurotransmitter release takes place in two phases, with a synchronous component that begins within a millisecond of an action potential followed by an asynchronous component that can last for hundreds of milliseconds 25 . As ...

Highlights d Casein kinase 1 d (CK1d) stabilizes nervous system architecture after axon outgrowth d CK1d phosphorylates and inhibits SSUP-72, an RNA polymerase II CTD phosphatase d CK1d inhibits transcription termination to promote giant Ankyrin expression d Expression of giant Ankyrin in CK1d mutants rescues axon maturation defects

Nano-resolution fl uorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are fi rst imaged using a super-resolution fl uorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present fi ve technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fl uorescence, the use of ground-state depletion instead of photoactivation for temporal control of fl uorescence, the use of organic fl uorophores instead of fl uorescent proteins to obtain brighter fl uorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.

Pituitary tumors are frequently associated with mutations in the AIP gene and are sometimes associated with hypersecretion of growth hormone. It is unclear whether other factors besides an enlarged pituitary contribute to the hypersecretion. In a genetic screen for suppressors of reduced neurotransmitter release, we identified a mutation in Caenorhabditis elegans AIPR-1 (AIP-related-1), which causes profound increases in evoked and spontaneous neurotransmitter release, a high frequency of spontaneous calcium transients in motor neurons and an enlarged readily releasable pool of vesicles. Calcium bursts and hypersecretion are reversed by mutations in the ryanodine receptor but not in the voltage-gated calcium channel, indicating that these phenotypes are caused by a leaky ryanodine receptor. AIPR-1 is physically associated with the ryanodine receptor at synapses. Finally, the phenotypes in aipr-1 mutants can be rescued by presynaptic expression of mouse AIP, demonstrating that a conserved function of AIP proteins is to inhibit calcium release from ryanodine receptors.

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