Secretory vesicles dock at their target in preparation for fusion. Using single-vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18-1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking-deficient munc18-1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t-SNARE and Munc18-1 binding partner syntaxin, but not the v-SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18-1/syntaxin-dependent, strongly tethered, fusioncompetent state.

Ca²+-evoked transmitter release shows a high dynamic range over spontaneous release. We investigated the role of the Ca²+ sensor protein, Synaptotagmin2 (Syt2), in both spontaneous and Ca²+-evoked release under direct control of presynaptic [Ca²+](i), using an in vivo rescue approach at the calyx of Held. Re-expression of Syt2 rescued the highly Ca²+ cooperative release and suppressed the elevated spontaneous release seen in Syt2 KO synapses. This latter release clamping function was partially mediated by the poly-lysine motif of the C₂B domain. Using an aspartate mutation in the C₂B domain (D364N) in which Ca²+ triggering was abolished but release clamping remained intact, we show that Syt2 strongly suppresses the action of another, near-linear Ca²+ sensor that mediates release over a wide range of [Ca²+](i). Thus, Syt2 increases the dynamic range of synapses by driving release with a high Ca²+ cooperativity, as well as by suppressing a remaining, near-linear Ca²+ sensor.

Exocytosis of secretory or synaptic vesicles is executed by a mechanism including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Munc18-1 is a part of this fusion machinery, but its role is controversial because it is indispensable for fusion but also inhibits the assembly of purified SNAREs in vitro. This inhibition reflects the binding of Munc18-1 to a closed conformation of the target-SNARE syntaxin1. The controversy would be solved if binding to closed syntaxin1 were shown to be stimulatory for vesicle fusion and/or additional essential interactions were identified between Munc18-1 and the fusion machinery. Here, we provide evidence for both notions by dissecting sequential steps of the exocytotic cascade while expressing Munc18 variants in the Munc18-1 null background. In Munc18-1 null chromaffin cells, vesicle docking is abolished and syntaxin levels are reduced. A mutation that diminished Munc18 binding to syntaxin1 in vitro attenuated the vesicle-docking step but rescued vesicle priming in excess of docking. Conversely, expressing the Munc18-2 isoform, which also displays binding to closed syntaxin1, rescued vesicle docking identical with Munc18-1 but impaired more downstream vesicle priming steps. All Munc18 variants restored syntaxin1 levels at least to wild-type levels, showing that the docking phenotype is not caused by syntaxin1 reduction. None of the Munc18 variants affected vesicle fusion kinetics or fusion pore duration. In conclusion, binding of Munc18-1 to closed syntaxin1 stimulates vesicle docking and a distinct interaction mode regulates the consecutive priming step.

The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is the most common genetic cause of Parkinson's disease (PD), accounting for a significant proportion of both autosomal dominant familial and sporadic PD cases. Our aim in the present study is to generate a mammalian model of mutant G2019S LRRK2 pathogenesis, which reproduces the robust nigral neurodegeneration characteristic of PD. We developed adenoviral vectors to drive neuron-specific expression of full-length wild-type or mutant G2019S human LRRK2 in the nigrostriatal system of adult rats. Wild-type LRRK2 did not induce any significant neuronal loss. In contrast, under the same conditions and levels of expression, G2019S mutant LRRK2 causes a progressive degeneration of nigral dopaminergic neurons. Our data provide a novel rat model of PD, based on a prevalent genetic cause, that reproduces a cardinal feature of the disease within a rapid time frame suitable for testing of neuroprotective strategies.

Developmental refinement of synaptic transmission can occur via changes in several pre-and postsynaptic factors, but it has been unknown whether the intrinsic Ca 2+ sensitivity of vesicle fusion in the nerve terminal can be regulated during development. Using the calyx of Held, a giant synapse in the auditory pathway, we studied the presynaptic mechanisms underlying the developmental regulation of Ca 2+ -secretion coupling, comparing a time period before, and shortly after the onset of hearing in rats. We found an ∼2-fold leftward shift in the relationship between EPSC amplitude and presynaptic

Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a ‘conventional’ PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP.DOI: http://dx.doi.org/10.7554/eLife.01715.001

The amygdala is a brain area critical for the formation of fear memories. However, the nature of the teaching signal(s) that drive plasticity in the amygdala are still under debate. Here, we use optogenetic methods to investigate the contribution of ventral tegmental area (VTA) dopamine neurons to auditory-cued fear learning in male mice. Using anterograde and retrograde labeling, we found that a sparse and relatively evenly distributed population of VTA neurons projects to the basal amygdala (BA). In vivo optrode recordings in behaving mice showed that many VTA neurons, among them putative dopamine neurons, are excited by footshocks, and acquire a response to auditory stimuli during fear learning. Combined cfos imaging and retrograde labeling in dopamine transporter (DAT) Cre mice revealed that a large majority of BA projectors (.95%) are dopamine neurons, and that BA projectors become activated by the tone-footshock pairing of fear learning protocols. Finally, silencing VTA dopamine neurons, or their axon terminals in the BA during the footshock, reduced the strength of fear memory as tested 1 d later, whereas silencing the VTA-central amygdala (CeA) projection had no effect. Thus, VTA dopamine neurons projecting to the BA contribute to fear memory formation, by coding for the saliency of the footshock event and by signaling such events to the basal amygdala.

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