Acute complexin knockout abates spontaneous and evoked transmitter release

SNARE-mediated synaptic vesicle (SV) fusion is controlled by multiple regulatory proteins that determine neurotransmitter release efficiency. Complexins are essential SNARE regulators whose mode of action is unclear, as available evidence indicates positive SV fusion facilitation and negative 'fusion clamp'-like activities, with the latter occurring only in certain contexts. Because these contradictory findings likely originate in part from different experimental perturbation strategies, we attempted to resolve them by examining a conditional complexin-knockout mouse line as the most stringent genetic perturbation model available. We found that acute complexin loss after synaptogenesis in autaptic and mass-cultured hippocampal neurons reduces SV fusion probability and thus abates the rates of spontaneous, synchronous, asynchronous, and delayed transmitter release but does not affect SV priming or cause 'unclamping' of spontaneous SV fusion. Thus, complexins act as facilitators of SV fusion but are dispensable for 'fusion clamping' in mammalian forebrain neurons

Phorbol esters modulate spontaneous and Ca2+-evoked transmitter release via acting on both munc13 and protein kinase C

Diacylglycerol (DAG) and phorbol esters strongly potentiate transmitter release at synapses by activating protein kinase C (PKC) and members of the Munc13 family of presynaptic vesicle priming proteins. This PKC/Munc13 pathway has emerged as a crucial regulator of release probability during various forms of activity-dependent enhancement of release. Here, we investigated the relative roles of PKC and Munc13-1 in the phorbol ester potentiation of evoked and spontaneous transmitter release at the calyx of Held synapse. The phorbol ester phorbol 12,13-dibutyrate (1 mu M) potentiated the frequency of miniature EPSCs, and the amplitudes of evoked EPSCs with a similar time course. Preincubating slices with the PKC blocker Ro31-82200 reduced the potentiation, mainly by affecting a late phase of the phorbol ester potentiation. The Ro31-8220-insensitive potentiation was most likely mediated by Munc13-1, because in organotypic slices of Munc13-1H567K knock-in mice, in which DAG binding to Munc13-1 is abolished, the potentiation of spontaneous release by phorbol ester was strongly suppressed. Using direct presynaptic depolarizations in paired recordings, we show that the phorbol ester potentiation does not go along with an increase in the number of readily releasable vesicles, despite an increase in the cumulative EPSC amplitude during 100 Hz stimulation trains. Our data indicate that activation of Munc13 and PKC both contribute to an enhancement of the fusion probability of readily releasable vesicles. Thus, docked and readily releasable vesicles are a substrate for modulation via intracellular second-messenger pathways that act via Munc13 and PKC

Munc13-1 is a Ca2+-phospholipid-dependent vesicle priming hub that shapes synaptic short-term plasticity and enables sustained neurotransmission

During ongoing presynaptic action potential (AP) firing, transmitter release is limited by the availability of release-ready synaptic vesicles (SVs). The rate of SV recruitment (SVR) to release sites is strongly upregulated at high AP frequencies to balance SV consumption. We show that Munc13-1-an essential SV priming protein-regulates SVR via a Ca2+-phospholipid-dependent mechanism. Using knockin mouse lines with point mutations in the Ca2+-phospholipid-binding C2B domain of Munc13-1, we demonstrate that abolishing Ca2+-phospholipid binding increases synaptic depression, slows recovery of synaptic strength after SV pool depletion, and reduces temporal fidelity of synaptic transmission, while increased Ca2+-phospholipid binding has the opposite effects. Thus, Ca2+-phospholipid binding to the Munc13-1-C2B domain accelerates SVR, reduces short-term synaptic depression, and increases the endurance and temporal fidelity of neurotransmission, demonstrating that Munc13-1 is a core vesicle priming hub that adjusts SV re-supply to demand

Structurally and functionally unique complexins at retinal ribbon synapses

Ribbon synapses in retinal sensory neurons maintain large pools of readily releasable synaptic vesicles. This allows them to release several hundreds of vesicles per second at every presynaptic release site. The molecular components that cause this high transmitter release efficiency of ribbon synapses are unknown. In the present study, we identified and characterized two novel vertebrate complexins (CPXs), CPXs III and IV, that are the only CPX isoforms present in retinal ribbon synapses. CPXs III and IV are COOH-terminally farnesylated, and, like CPXs I and II, bind to SNAP receptor complexes. CPXs III and IV can functionally replace CPXs I and II, and their COOH-terminal farnesylation regulates their synaptic targeting and modulatory function in transmitter release. The novel CPXs III and IV may contribute to the unique release efficacy of retinal sensory neurons

CAPS facilitates filling of the rapidly releasable pool of large dense-core vesicles

Calcium-activator protein for secretion (CAPS) is a cytosolic protein that associates with large dense-core vesicles and is involved in their secretion. Mammals express two CAPS isoforms, which share a similar domain structure including a Munc13 homology domain that is believed to be involved in the priming of secretory vesicles. A variety of studies designed to perturb CAPS function indicate that CAPS is involved in the secretion of large dense-core vesicles, but where in the secretory pathway CAPS acts is still under debate. Mice in which one allele of the CAPS-1 gene is deleted exhibit a deficit in catecholamine secretion from chromaffin cells. We have examined catecholamine secretion from chromaffin cells in which both CAPS genes were deleted and show that the deletion of both CAPS isoforms causes a strong reduction in the pool of rapidly releasable chromaffin granules and of sustained release during ongoing stimulation. We conclude that CAPS is required for the adequate refilling and/or maintenance of a rapidly releasable granule pool

Structural plasticity of spines at giant mossy fiber synapses

The granule cells of the dentate gyrus give rise to thin unmyelinated axons, the mossy fibers. They form giant presynaptic boutons impinging on large complex spines on the proximal dendritic portions of hilar mossy cells and CA3 pyramidal neurons. While these anatomical characteristics have been known for some time, it remained unclear whether functional changes at mossy fiber synapses such as long-term potentiation (LTP) are associated with structural changes. Since subtle structural changes may escape a fine-structural analysis when the tissue is fixed by using aldehydes and is dehydrated in ethanol, rapid high-pressure freezing (HPF) of the tissue was applied. Slice cultures of hippocampus were prepared and incubated in vitro for 2 weeks. Then, chemical LTP (cLTP) was induced by the application of 25 mM tetraethylammonium (TEA) for 10 min. Whole-cell patch-clamp recordings from CA3 pyramidal neurons revealed a highly significant potentiation of mossy fiber synapses when compared to control conditions before the application of TEA. Next, the slice cultures were subjected to HPF, cryosubstitution, and embedding in Epon for a fine-structural analysis. When compared to control tissue, we noticed a significant decrease of synaptic vesicles in mossy fiber boutons and a concomitant increase in the length of the presynaptic membrane. On the postsynaptic side, we observed the formation of small, finger-like protrusions, emanating from the large complex spines. These short protrusions gave rise to active zones that were shorter than those normally found on the thorny excrescences. However, the total number of active zones was significantly increased. Of note, none of these cLTP-induced structural changes was observed in slice cultures from Munc13-1 deficient mouse mutants showing severely impaired vesicle priming and docking. In conclusion, application of HPF allowed us to monitor cLTP-induced structural reorganization of mossy fiber synapses

1H, 13C and 15N resonance assignments of the Calmodulin-Munc13-1 peptide complex

Ca2+-Calmodulin binding to the variable N-terminal region of the diacylglycerol/phorbol ester-binding UNC13/Munc13 family of proteins modulates the short-term synaptic plasticity characteristics in neurons. Here, we report the sequential backbone and side chain resonance assignment of the Ca2+-Calmodulin/Munc13-1458–492 peptide complex at pH 6.8 and 35°C (BMRB No. 15470)

The RacGAP β2-Chimaerin Selectively Mediates Axonal Pruning in the Hippocampus

SummaryAxon pruning and synapse elimination promote neural connectivity and synaptic plasticity. Stereotyped pruning of axons that originate in the hippocampal dentate gyrus (DG) and extend along the infrapyramidal tract (IPT) occurs during postnatal murine development by neurite retraction and resembles axon repulsion. The chemorepellent Sema3F is required for IPT axon pruning, dendritic spine remodeling, and repulsion of DG axons. The signaling events that regulate IPT axon pruning are not known. We find that inhibition of the small G protein Rac1 by the Rac GTPase-activating protein (GAP) β2-Chimaerin (β2Chn) mediates Sema3F-dependent pruning. The Sema3F receptor neuropilin-2 selectively binds β2Chn, and ligand engagement activates this GAP to ultimately restrain Rac1-dependent effects on cytoskeletal reorganization. β2Chn is necessary for axon pruning both in vitro and in vivo, but it is dispensable for axon repulsion and spine remodeling. Therefore, a Npn2/β2Chn/Rac1 signaling axis distinguishes DG axon pruning from the effects of Sema3F on repulsion and dendritic spine remodeling

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling

CAPS1 Regulates Catecholamine Loading of Large Dense-Core Vesicles

SummaryCAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs