Neuropeptides are essential signaling molecules transported and secreted by dense‐core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo. Super‐resolution and electron microscopy reveal a total pool of 1,400–18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo‐cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1–6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.

Synaptic transmission requires a stable pool of release-ready (primed) vesicles. Here we show that two molecules involved in SNARE-complex assembly, Munc13-1 and Munc18-1, together stabilize release-ready vesicles by preventing de-priming. Replacing neuronal Munc18-1 by a non-neuronal isoform Munc18-2 (Munc18-1/2SWAP) supports activity-dependent priming, but primed vesicles fall back into a non-releasable state (de-prime) within seconds. Munc13-1 deficiency produces a similar defect. Inhibitors of N-ethylmaleimide sensitive factor (NSF), N-ethylmaleimide (NEM) or interfering peptides, prevent de-priming in munc18-1/2SWAP or munc13-1 null synapses, but not in CAPS-1/2 null, another priming-deficient mutant. NEM rescues synaptic transmission in munc13-1 null and munc18-1/2SWAP synapses, in acute munc13-1 null slices and even partially in munc13-1/2 double null synapses. Together these data indicate that Munc13-1 and Munc18-1, but not CAPS-1/2, stabilize primed synaptic vesicles by preventing NSF-dependent de-priming.

The loss of presynaptic proteins Munc18-1, syntaxin-1, or SNAP-25 is known to produce cell death, but the underlying features have not been compared experimentally. Here, we investigated these features in cultured mouse CNS and DRG neurons. Side-by-side comparisons confirmed massive cell death, before synaptogenesis, within 1-4 DIV upon loss of t-SNAREs (syntaxin-1, SNAP-25) or Munc18-1, but not v-SNAREs (synaptobrevins/VAMP1/2/3 using tetanus neurotoxin (TeNT), also in TI-VAMP/VAMP7 knock-out (KO) neurons). A condensed Golgi was the first abnormality observed upon Munc18-1 or SNAP-25 loss within 3 DIV. This phenotype was distinct from the Golgi fragmentation observed in apoptosis. Cell death was too rapid after syntaxin-1 loss to study Golgi abnormalities. Syntaxin-1 and Munc18-1 depend on each other for normal cellular levels. We observed that endogenous syntaxin-1 accumulates at the Golgi of Munc18-1 KO neurons. However, expression of a non-neuronal Munc18 isoform that does not bind syntaxin-1, Munc18-3, in Munc18-1 KO neurons prevented cell death and restored normalGolgi morphology, but not synaptic transmission or syntaxin-1 targeting. Finally, we observed that DRG neurons are the only Munc18-1 KO neurons that do not degenerate or In these neurons, Golgi abnormalities were less severe, with no changes in Golgi shape. Together, these data demonstrate that cell death upon Munc18-1, syntaxin-1, or SNAP-25 loss occurs via a degenerative pathway unrelated to the known synapse function of these proteins and involving earlyGolgi abnormalities, distinct from apoptosis. This study provides new insights in a neurodegeneration pathway triggered by the absence of specific proteins involved in synaptic transmission (syntaxin-1, Munc18-1, SNAP-25), whereas other proteins involved in the same molecular process (synaptobrevins, Munc13-1/2) do not cause degeneration. Massive cell death occurs in cultured neurons upon depleting syntaxin-1, Munc18-1, and/or SNAP-25, well before synapse formation. This study characterizes several relevant cellular phenotypes, especially early Golgi abnormalities, distinct from abnormalities observed during apoptosis, and rules out several other phenotypes as causal (defects in syntaxin-1 targeting and synaptic transmission). As proteins, such as syntaxin-1, Munc18-1, or SNAP-25, modulate α-synuclein neuropathy and/or are dysregulated in Alzheimer's disease, understanding this type of neurodegeneration may provide new links between synaptic defects and neurodegeneration in humans.

Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role.DOI: http://dx.doi.org/10.7554/eLife.05438.001

PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.