Social factors influence solo and rat dyads exploration of an unfamiliar open field.

Exploring new and unfamiliar environments is critical for survival, providing information on food, shelter, mates, and sources of danger. The open field paradigm is commonly used to study exploration and anxiety-like behaviors in the lab. Many social animals, like humans and rats, may explore their environments in social groups; however, relatively few studies have investigated the influence of conspecifics on open field activity. Here, we provide a comparison of individual (solo) or pairs of male rats (dyads) exploring and interacting across repeated exposures to an unfamiliar (Day 1) or more familiar (Day 2) open field. Both solo rats and dyads explored a larger area, traveled further, and spent less time near the maze walls on the second maze exposure. Solo rats explored a larger area and spent less time near the maze walls than dyads on both days because dyads spent more time socializing rather than exploring the environment. Furthermore, we compared familiar dyads that were co-housed for seven days versus stranger dyads that met for the first time in the open field. While familiar and stranger dyads did not differ in maze exploration, strangers spent more time interacting nose to nose than nose to anogenital. These results indicate that the degree of familiarity with the environment does not interact with the tendency of dyads to socialize rather than explore the environment.

Proximity ligation assay reveals both pre- and postsynaptic localization of the APP-processing enzymes ADAM10 and BACE1 in rat and human adult brain.

BACKGROUND: Synaptic degeneration and accumulation of amyloid ß-peptides (Aß) are hallmarks of the Alzheimer diseased brain. Aß is synaptotoxic and produced by sequential cleavage of the amyloid precursor protein (APP) by the ß-secretase BACE1 and by γ-secretase. If APP is instead cleaved by the α-secretase ADAM10, Aß will not be generated. Although BACE1 is considered to be a presynaptic protein and ADAM10 has been reported to mainly localize to the postsynaptic density, we have previously shown that both ADAM10 and BACE1 are highly enriched in synaptic vesicles of rat brain and mouse primary hippocampal neurons. RESULTS: Here, using brightfield proximity ligation assay, we expanded our previous result in primary neurons and investigated the in situ synaptic localization of ADAM10 and BACE1 in rat and human adult brain using both pre- and postsynaptic markers. We found that ADAM10 and BACE1 were in close proximity with both the presynaptic marker synaptophysin and the postsynaptic marker PSD-95. The substrate APP was also detected both pre- and postsynaptically. Subcellular fractionation confirmed that ADAM10 and BACE1 are enriched to a similar degree in synaptic vesicles and as well as in the postsynaptic density. CONCLUSIONS: We show that the α-secretase ADAM10 and the ß-secretase BACE1 are located in both the pre- and postsynaptic compartments in intact brain sections. These findings increase our understanding of the regulation of APP processing, thereby facilitating development of more specific treatment strategies.

Real-time MRI for dynamic assessment of gastroesophageal reflux disease: Comparison to pH-metry and impedance.

PURPOSE: To evaluate the diagnostic potential of real-time MRI for dynamic assessment of gastroesophageal reflux in patients with GERD (gastroesophageal reflux disease)-like symptoms compared to pH-metry and impedance. METHODS: Patients who underwent real-time MRI and pH-metry between 2015-2018 were included in this retrospective study. Real-time MRI at 3 T was achieved by undersampled radial FLASH acquisitions with iterative image reconstruction by NLINV. Real-time MRI visualized transit of pineapple juice through the gastroesophageal junction and during Valsalva maneuver. MRI results were compared to 24 h pH-metry to assess acidic reflux (following Lyon Consensus guidelines) and to impedance to assess non-acidic reflux. A standard 2 × 2 table was chosen to calculate diagnostic performance. RESULTS: 91/93 eligible patients fulfilled inclusion criteria (male n = 49; female n = 42; median age 55 y). All MRI studies were successfully completed without adverse events at a mean examination time of 15 min. On real-time MRI, reflux was evident in 60 patients (66 %). pH-metry revealed reflux in 41 patients (45 %), and impedance in 54 patients (59 %). Compared to pH-metry and impedance, real-time MRI sensitivity was 0.78 (95 % CI: 0.66-0.87), specificity 0.67 (95 % CI: 0.45-0.84) and PPV 0.87 (95 % CI: 0.75-0.94). CONCLUSION: Real-time MRI is an imaging method for assessment of gastroesophageal reflux in patients with GERD-like symptoms. Considering its high positive predictive value, real-time MRI can accurately identify patients in which further invasive testing with pH-metry and impedance might be considered.

Future liver remnant growth after various portal vein embolization regimens: a quantitative comparison.

Purpose: To compare the efficacy of right portal vein embolization using ethylene vinyl alcohol (EVOH-PVE) compared to other embolic agents and surgical right portal vein ligation (PVL).Material and methods: Patients with right sided liver malignancies scheduled for extensive surgery and receiving induction of liver hypertrophy via right portal vein embolization/ligature between 2010-2016 were retrospectively evaluated. Treatments included were ethylene vinyl alcohol copolymer (Onyx®, EVOH-PVE), ethiodized oil (Lipiodol®, Lipiodol/PVA-PVE), polyvinyl alcohol (PVA-PVE) or surgical ligature (PVL). Liver segments S2/3 were used to assess hypertrophy. Primary outcome was future liver remnant growth in ml/day.Results: Forty-one patients were included (EVOH-PVE n = 11; Lipiodol/PVA-PVE n = 10; PVA-PVE n = 8; PVL n = 12), the majority presenting with cholangiocarcinoma and colorectal metastases (n = 11; n = 27). Pre-interventional liver volumes were comparable (p = .095). Liver hypertrophy was successfully induced in all but one patient receiving Lipiodol/PVA-PVE. Liver segment S2/3 growth was largest for EVOH-PVE (5.38 ml/d) followed by PVA-PVE (2.5 ml/d), with significantly higher growth rates than PVL (1.24 ml/d; p < .001; p = .007). No significant difference was evident for Lipiodol/PVA-PVE (1.43 ml/d, p = .809).Conclusions: Portal vein embolization using EVOH demonstrates fastest S2/3 growth rates compared to other embolic agents and PVL, potentially due to its permanent portal vein embolization and induction of hepatic inflammation.

Splenic torsion with involvement of pancreas and descending colon in a 9-year-old boy.

Splenic torsion is an uncommon condition becoming clinically apparent when the spleen twists or rotates around the organ's vascular pedicle. In the case of a wandering spleen the organ is only attached to an elongated vascular pedicle while the peritoneal attachments are absent. However, splenic torsion could also occur in patients with abnormal laxity of the splenic peritoneal attachments. We report a case of a splenic torsion due to absence of splenic ligaments with pancreatic volvulus and partial involvement of descending colon in a 9-year-old boy.

Synaptotagmin-3 drives AMPA receptor endocytosis, depression of synapse strength, and forgetting.

Forgetting is important. Without it, the relative importance of acquired memories in a changing environment is lost. We discovered that synaptotagmin-3 (Syt3) localizes to postsynaptic endocytic zones and removes AMPA receptors from synaptic plasma membranes in response to stimulation. AMPA receptor internalization, long-term depression (LTD), and decay of long-term potentiation (LTP) of synaptic strength required calcium-sensing by Syt3 and were abolished through Syt3 knockout. In spatial memory tasks, mice in which Syt3 was knocked out learned normally but exhibited a lack of forgetting. Disrupting Syt3:GluA2 binding in a wild-type background mimicked the lack of LTP decay and lack of forgetting, and these effects were occluded in the Syt3 knockout background. Our findings provide evidence for a molecular mechanism in which Syt3 internalizes AMPA receptors to depress synaptic strength and promote forgetting.

Regulation of Dendritic Spine Morphology in Hippocampal Neurons by Copine-6.

Dendritic spines compartmentalize information in the brain, and their morphological characteristics are thought to underly synaptic plasticity. Here we identify copine-6 as a novel modulator of dendritic spine morphology. We found that brain-derived neurotrophic factor (BDNF) - a molecule essential for long-term potentiation of synaptic strength - upregulated and recruited copine-6 to dendritic spines in hippocampal neurons. Overexpression of copine-6 increased mushroom spine number and decreased filopodia number, while copine-6 knockdown had the opposite effect and dramatically increased the number of filopodia, which lacked PSD95. Functionally, manipulation of post-synaptic copine-6 levels affected miniature excitatory post-synaptic current (mEPSC) kinetics and evoked synaptic vesicle recycling in contacting boutons, and post-synaptic knockdown of copine-6 reduced hippocampal LTP and increased LTD. Mechanistically, copine-6 promotes BDNF-TrkB signaling and recycling of activated TrkB receptors back to the plasma membrane surface, and is necessary for BDNF-induced increases in mushroom spines in hippocampal neurons. Thus copine-6 regulates BDNF-dependent changes in dendritic spine morphology to promote synaptic plasticity.

Capture of Dense Core Vesicles at Synapses by JNK-Dependent Phosphorylation of Synaptotagmin-4.

Delivery of neurotrophins and neuropeptides via long-range trafficking of dense core vesicles (DCVs) from the cell soma to nerve terminals is essential for synapse modulation and circuit function. But the mechanism by which transiting DCVs are captured at specific sites is unknown. Here, we discovered that Synaptotagmin-4 (Syt4) regulates the capture and spatial distribution of DCVs in hippocampal neurons. We found that DCVs are highly mobile and undergo long-range translocation but switch directions only at the distal ends of axons, revealing a circular trafficking pattern. Phosphorylation of serine 135 of Syt4 by JNK steers DCV trafficking by destabilizing Syt4-Kif1A interaction, leading to a transition from microtubule-dependent DCV trafficking to capture at en passant presynaptic boutons by actin. Furthermore, neuronal activity increased DCV capture via JNK-dependent phosphorylation of the S135 site of Syt4. Our data reveal a mechanism that ensures rapid, site-specific delivery of DCVs to synapses.

TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus.

TRPV1 is an ion channel activated by heat and pungent agents including capsaicin, and has been extensively studied in nociception of sensory neurons. However, the location and function of TRPV1 in the hippocampus is debated. We found that TRPV1 is expressed in oriens-lacunosum-moleculare (OLM) interneurons in the hippocampus, and promotes excitatory innervation. TRPV1 knockout mice have reduced glutamatergic innervation of OLM neurons. When activated by capsaicin, TRPV1 recruits more glutamatergic, but not GABAergic, terminals to OLM neurons in vitro. When TRPV1 is blocked, glutamatergic input to OLM neurons is dramatically reduced. Heterologous expression of TRPV1 also increases excitatory innervation. Moreover, TRPV1 knockouts have reduced Schaffer collateral LTP, which is rescued by activating OLM neurons with nicotine-via α2ß2-containing nicotinic receptors-to bypass innervation defects. Our results reveal a synaptogenic function of TRPV1 in a specific interneuron population in the hippocampus, where it is important for gating hippocampal plasticity.

A novel method for culturing stellate astrocytes reveals spatially distinct Ca2+ signaling and vesicle recycling in astrocytic processes.

Interactions between astrocytes and neurons rely on the release and uptake of glial and neuronal molecules. But whether astrocytic vesicles exist and exocytose in a regulated or constitutive fashion is under debate. The majority of studies have relied on indirect methods or on astrocyte cultures that do not resemble stellate astrocytes found in vivo. Here, to investigate vesicle-associated proteins and exocytosis in stellate astrocytes specifically, we developed a simple, fast, and economical method for growing stellate astrocyte monocultures. This method is superior to other monocultures in terms of astrocyte morphology, mRNA expression profile, protein expression of cell maturity markers, and Ca2+ fluctuations: In astrocytes transduced with GFAP promoter-driven Lck-GCaMP3, spontaneous Ca2+ events in distinct domains (somata, branchlets, and microdomains) are similar to those in astrocytes co-cultured with other glia and neurons but unlike Ca2+ events in astrocytes prepared using the McCarthy and de Vellis (MD) method and immunopanned (IP) astrocytes. We identify two distinct populations of constitutively recycling vesicles (harboring either VAMP2 or SYT7) specifically in branchlets of cultured stellate astrocytes. SYT7 is developmentally regulated in these astrocytes, and we observe significantly fewer synapses in wild-type mouse neurons grown on Syt7-/- astrocytes. SYT7 may thus be involved in trafficking or releasing synaptogenic factors. In summary, our novel method yields stellate astrocyte monocultures that can be used to study Ca2+ signaling and vesicle recycling and dynamics in astrocytic processes.

ADAM10 and BACE1 are localized to synaptic vesicles.

Synaptic degeneration and accumulation of the neurotoxic amyloid ß-peptide (Aß) in the brain are hallmarks of Alzheimer disease. Aß is produced by sequential cleavage of the amyloid precursor protein (APP), by the ß-secretase ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. However, Aß generation is precluded if APP is cleaved by the α-secretase ADAM10 instead of BACE1. We have previously shown that Aß can be produced locally at the synapse. To study the synaptic localization of the APP processing enzymes we used western blotting to demonstrate that, compared to total brain homogenate, ADAM10 and BACE1 were greatly enriched in synaptic vesicles isolated from rat brain using controlled-pore glass chromatography, whereas Presenilin1 was the only enriched component of the γ-secretase complex. Moreover, we detected ADAM10 activity in synaptic vesicles and enrichment of the intermediate APP-C-terminal fragments (APP-CTFs). We confirmed the western blotting findings using in situ proximity ligation assay to demonstrate close proximity of ADAM10 and BACE1 with the synaptic vesicle marker synaptophysin in intact mouse primary hippocampal neurons. In contrast, only sparse co-localization of active γ-secretase and synaptophysin was detected. These results indicate that the first step of APP processing occurs in synaptic vesicles whereas the final step is more likely to take place elsewhere.

Modulation of Presynaptic Release Probability by the Vertebrate-Specific Protein Mover.

Mover, a member of the exquisitely small group of vertebrate-specific presynaptic proteins, has been discovered as an interaction partner of the scaffolding protein Bassoon, yet its function has not been elucidated. We used adeno-associated virus (AAV)-mediated shRNA expression to knock down Mover in the calyx of Held in vivo. Although spontaneous synaptic transmission remained unaffected, we found a strong increase of the evoked EPSC amplitude. The size of the readily releasable pool was unaltered, but short-term depression was accelerated and enhanced, consistent with an increase in release probability after Mover knockdown. This increase in release probability was not caused by alterations in Ca(2+) influx but rather by a higher Ca(2+) sensitivity of the release machinery, as demonstrated by presynaptic Ca(2+) uncaging. We therefore conclude that Mover expression in certain subsets of synapses negatively regulates synaptic release probability, constituting a novel mechanism to tune synaptic transmission.

Long-term depression is differentially expressed in distinct lamina of hippocampal CA1 dendrites.

Information storage in CA1 hippocampal pyramidal neurons is compartmentalized in proximal vs. distal apical dendrites, cell bodies, and basal dendrites. This compartmentalization is thought to be essential for synaptic integration. Differences in the expression of long-term potentiation (LTP) in each of these compartments have been described, but less is known regarding potential differences in long-term depression (LTD). Here, to directly compare LTD expression in each compartment and to bypass possible differences in input-specificity and stimulation of presynaptic inputs, we used global application of NMDA to induce LTD. We then examined LTD expression in each dendritic sub-region-proximal and distal apical, and basal dendrites-and in cell bodies. Interestingly, we found that distal apical dendrites exhibited the greatest magnitude of LTD of all areas tested and this LTD was maintained, whereas LTD in proximal apical dendrites was not maintained. In basal dendrites, LTD was also maintained, but the magnitude of LTD was less than in distal apical dendrites. Blockade of inhibition blocked LTD maintenance in both distal apical and basal dendrites. Population spikes recorded from the cell body layer correlated with apical dendrite field EPSP (fEPSP), where LTD was maintained in distal dendrites and decayed in proximal dendrites. On the other hand, LTD of basal dendrite fEPSPs was maintained but population spike responses were not. Thus E-S coupling was distinct in basal and apical dendrites. Our data demonstrate cell autonomous differential information processing in somas and dendritic sub-regions of CA1 pyramidal neurons in the hippocampus, where LTD expression is intrinsic to distinct dendritic regions, and does not depend on the nature of stimulation and input specificity.

Ethanol inhibits long-term potentiation in hippocampal CA1 neurons, irrespective of lamina and stimulus strength, through neurosteroidogenesis.

Ethanol inhibits memory encoding and the induction of long-term potentiation (LTP) in CA1 neurons of the hippocampus. Hippocampal LTP at Schaffer collateral synapses onto CA1 pyramidal neurons has been widely studied as a cellular model of learning and memory, but there is striking heterogeneity in the underlying molecular mechanisms in distinct regions and in response to distinct stimuli. Basal and apical dendrites differ in terms of innervation, input specificity, and molecular mechanisms of LTP induction and maintenance, and different stimuli determine distinct molecular pathways of potentiation. However, lamina or stimulus-dependent effects of ethanol on LTP have not been investigated. Here, we tested the effect of acute application of 60 mM ethanol on LTP induction in distinct dendritic compartments (apical versus basal) of CA1 neurons, and in response to distinct stimulation paradigms (single versus repeated, spaced high frequency stimulation). We found that ethanol completely blocks LTP in apical dendrites, whereas it reduces the magnitude of LTP in basal dendrites. Acute ethanol treatment for just 15 min altered pre- and post-synaptic protein expression. Interestingly, ethanol increases the neurosteroid allopregnanolone, which causes ethanol-dependent inhibition of LTP, more prominently in apical dendrites, where ethanol has greater effects on LTP. This suggests that ethanol has general effects on fundamental properties of synaptic plasticity, but the magnitude of its effect on LTP differs depending on hippocampal sub-region and stimulus strength.

BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) is widely reported to enhance synaptic vesicle (SV) exocytosis and neurotransmitter release. But it is still unclear whether BDNF enhances SV recycling at excitatory terminals only, or at both excitatory and inhibitory terminals. In the present study, in a direct comparison using cultured rat hippocampal neurons, we demonstrate that BDNF enhances both spontaneous and activity-dependent neurotransmitter release from excitatory terminals, but not from inhibitory terminals. BDNF treatment for 5 min or 48 h increased both spontaneous and activity-induced anti-synaptotagmin1 (SYT1) antibody uptake at excitatory terminals marked with vGluT1. Conversely, BDNF treatment did not enhance spontaneous or activity-induced uptake of anti-SYT1 antibodies in inhibitory terminals marked with vGAT. Time-lapse imaging of FM1-43 dye destaining in excitatory and inhibitory terminals visualized by post-hoc immunostaining of vGluT1 and vGAT also showed the same result: The rate of spontaneous and activity-induced destaining was increased by BDNF at excitatory synapses, but not at inhibitory synapses. These data demonstrate that BDNF enhances SV exocytosis in excitatory but not inhibitory terminals. Moreover, BDNF enhanced evoked SV exocytosis, even if vesicles were loaded under spontaneous vesicle recycling conditions. Thus, BDNF enhances both spontaneous and activity-dependent neurotransmitter release on both short and long time-scales, by the same mechanism.

Activity-independent release of the amyloid ß-peptide from rat brain nerve terminals.

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease. The molecular mechanism underlying this degeneration is not fully elucidated but one key player appears to be the synaptotoxic amyloid ß-peptide (Aß). The exact localization of the production of Aß and the mechanisms whereby Aß is released remain elusive. We have earlier shown that Aß can be produced in crude synaptic vesicle fractions and it has been reported that increased synaptic activity results in increased secreted but decreased intracellular Aß levels. Therefore, we considered whether Aß could be produced in synaptic vesicles and/or released through the same mechanisms as neurotransmitters in synaptic vesicle exocytosis. Small amounts of Aß were found to be produced in pure synaptic vesicle preparations. We also studied the release of glutamate and Aß from rat cortical nerve terminals (synaptosomes). We found that large amounts of Aß were secreted from non-stimulated synaptosomes, from which glutamate was not released. On the contrary, we could not detect any differences in Aß release between non-stimulated synaptosomes and synaptosomes stimulated with KCl or 4-aminopyridine, whereas glutamate release was readily inducible in this system. To conclude, our results indicate that the major release mechanism of Aß from isolated nerve terminals differs from the synaptic release of glutamate and that the activity-dependent increase of secreted Aß, reported by several groups using intact cells, is likely dependent on post-synaptic events, trafficking and/or protein synthesis mechanisms.

Mover is a homomeric phospho-protein present on synaptic vesicles.

With remarkably few exceptions, the molecules mediating synaptic vesicle exocytosis at active zones are structurally and functionally conserved between vertebrates and invertebrates. Mover was found in a yeast-2-hybrid assay using the vertebrate-specific active zone scaffolding protein bassoon as a bait. Peptides of Mover have been reported in proteomics screens for self-interacting proteins, phosphorylated proteins, and synaptic vesicle proteins, respectively. Here, we tested the predictions arising from these screens. Using flotation assays, carbonate stripping of peripheral membrane proteins, mass spectrometry, immunogold labelling of purified synaptic vesicles, and immuno-organelle isolation, we found that Mover is indeed a peripheral synaptic vesicle membrane protein. In addition, by generating an antibody against phosphorylated Mover and Western blot analysis of fractionated rat brain, we found that Mover is a bona fide phospho-protein. The localization of Mover to synaptic vesicles is phosphorylation dependent; treatment with a phosphatase caused Mover to dissociate from synaptic vesicles. A yeast-2-hybrid screen, co-immunoprecipitation and cell-based optical assays of homomerization revealed that Mover undergoes homophilic interaction, and regions within both the N- and C- terminus of the protein are required for this interaction. Deleting a region required for homomeric interaction abolished presynaptic targeting of recombinant Mover in cultured neurons. Together, these data prove that Mover is associated with synaptic vesicles, and implicate phosphorylation and multimerization in targeting of Mover to synaptic vesicles and presynaptic sites.

Rapid fusion of synaptic vesicles with reconstituted target SNARE membranes.

Neurotransmitter release at neuronal synapses occurs on a timescale of 1 ms or less. Reconstitution of vesicle fusion from purified synaptic proteins and lipids has played a major role in elucidating the synaptic exocytotic fusion machinery with ever increasing detail. However, one limitation of most reconstitution approaches has been the relatively slow rate of fusion that can be produced in these systems. In a related study, a notable exception is an approach measuring fusion of single reconstituted vesicles bearing the vesicle fusion protein synaptobrevin with supported planar membranes harboring the presynaptic plasma membrane proteins syntaxin and SNAP-25. Fusion times of ∼20 ms were achieved in this system. Despite this advance, an important question with reconstituted systems is how well they mimic physiological systems they are supposed to reproduce. In this work, we demonstrate that purified synaptic vesicles from rat brain fuse with acceptor-SNARE containing planar bilayers equally fast as equivalent reconstituted vesicles and that their fusion efficiency is increased by divalent cations. Calcium boosts fusion through a combined general electrostatic and synaptotagmin-specific mechanism.

Small-scale isolation of synaptic vesicles from mammalian brain.

Synaptic vesicles (SVs) are essential organelles that participate in the release of neurotransmitters from a neuron. Biochemical analysis of purified SVs was instrumental in the identification of proteins involved in exocytotic membrane fusion and neurotransmitter uptake. Although numerous protocols have been published detailing the isolation of SVs from the brain, those that give the highest-purity vesicles often have low yields. Here we describe a protocol for the small-scale isolation of SVs from mouse and rat brain. The procedure relies on standard fractionation techniques, including differential centrifugation, rate-zonal centrifugation and size-exclusion chromatography, but it has been optimized for minimal vesicle loss while maintaining a high degree of purity. The protocol can be completed in less than 1 d and allows the recovery of ∼150 µg of vesicle protein from a single mouse brain, thus allowing vesicle isolation from transgenic mice.

Controlling synaptotagmin activity by electrostatic screening.

Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca(2+) sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca(2+), but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca(2+)-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca(2+)-dependent exocytosis.