Tianeptine modulates synaptic vesicle dynamics and favors synaptic mitochondria processes in socially isolated rats.

Deregulation of synaptic function and neurotransmission has been linked with the development of major depression disorder (MDD). Tianeptine (Tian) has been used as antidepressant with anxiolytic properties and recently as a nootropic to improve cognitive performance, but its mechanism of action is unknown. We conducted a proteomic study on the hippocampal synaptosomal fractions of adult male Wistar rats exposed to chronic social isolation (CSIS, 6 weeks), an animal model of depression and after chronic Tian treatment in controls (nootropic effect) and CSIS-exposed rats (lasting 3 weeks of 6-week CSIS) (therapeutic effect). Increased expression of Syn1 and Camk2-related neurotransmission, vesicle transport and energy processes in Tian-treated controls were found. CSIS led to upregulation of proteins associated with actin cytoskeleton, signaling transduction and glucose metabolism. In CSIS rats, Tian up-regulated proteins involved in mitochondrial energy production, mitochondrial transport and dynamics, antioxidative defense and glutamate clearance, while attenuating the CSIS-increased glycolytic pathway and cytoskeleton organization proteins expression and decreased the expression of proteins involved in V-ATPase and vesicle endocytosis. Our overall findings revealed that synaptic vesicle dynamics, specifically exocytosis, and mitochondria-related energy processes might be key biological pathways modulated by the effective nootropic and antidepressant treatment with Tian and be a potential target for therapeutic efficacy of the stress-related mood disorders.

Phytic acid attenuates upregulation of GSK-3ß and disturbance of synaptic vesicle recycling in MPTP-induced Parkinson's disease models.

Heightened activity of glycogen synthase kinase-3ß (GSK-3ß) is linked to the degeneration of dopaminergic neurons in Parkinson's disease (PD). Phytic acid (PA), a naturally occurring compound with potent antioxidant property, has been shown to confer neuroprotection on dopaminergic neurons in PD. However, the underlying mechanism remains unclear. In the present study, MPTP and MPP+ treatments were used to model PD in mice and SH-SY5Y cells, respectively. We observed reduced tissue dopamine, disrupted synaptic vesicle recycling, and defective neurotransmitter exocytosis. Furthermore, expression of GSK-3ß was upregulated while that of ß-catenin was downregulated, concentration of cytosolic calcium was increased, and expressions of two dopamine carriers, dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) were decreased. PA treatment attenuated the MPTP-induced upregulation of GSK-3ß, increase in cytosolic calcium concentration, decreases in the levels of DAT, VMAT2, tissue dopamine, and synaptic vesicle recycling. Importantly, disturbances in synaptic vesicle recycling are thought to be early events in PD pathology. These findings suggest that PA is a promising therapeutic agent to treat early events in PD.

Impaired synaptic vesicle recycling contributes to presynaptic dysfunction in lipoprotein lipase-deficient mice.

Lipoprotein lipase (LPL) is expressed at high levels in hippocampal neurons, although its function is unclear. We previously reported that LPL-deficient mice have learning and memory impairment and fewer synaptic vesicles in hippocampal neurons, but properties of synaptic activity in LPL-deficient neurons remain unexplored. In this study, we found reduced frequency of miniature excitatory postsynaptic currents (mEPSCs) and readily releasable pool (RRP) size in LPL-deficient neurons, which led to presynaptic dysfunction and plasticity impairment without altering postsynaptic activity. We demonstrated that synaptic vesicle recycling, which is known to play an important role in maintaining the RRP size in active synapses, is impaired in LPL-deficient neurons. Moreover, lipid assay revealed deficient docosahexaenoic acid (DHA) and arachidonic acid (AA) in the hippocampus of LPL-deficient mice; exogenous DHA or AA supplement partially restored synaptic vesicle recycling capability. These results suggest that impaired synaptic vesicle recycling results from deficient DHA and AA and contributes to the presynaptic dysfunction and plasticity impairment in LPL-deficient neurons.

Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

Simvastatin treatment highlights a new role for the isoprenoid/cholesterol biosynthetic pathway in the modulation of emotional reactivity and cognitive performance in rats.

The aim of the present work was to shed light on the role played by the isoprenoid/cholesterol biosynthetic pathway in the modulation of emotional reactivity and memory consolidation in rodents through the inhibition of the key and rate-limiting enzyme 3-hydroxy 3-methylglutaryl Coenzyme A reductase (HMGR) both in vivo and in vitro with simvastatin. Three-month-old male Wistar rats treated for 21 days with simvastatin or vehicle were tested in the social interaction, elevated plus-maze, and inhibitory avoidance tasks; after behavioral testing, the amygdala, hippocampus, prefrontal cortex, dorsal, and ventral striatum were dissected out for biochemical assays. In order to delve deeper into the molecular mechanisms underlying the observed effects, primary rat hippocampal neurons were used. Our results show that HMGR inhibition by simvastatin induces anxiogenic-like effects in the social interaction but not in the elevated plus-maze test, and improves memory consolidation in the inhibitory avoidance task. These effects are accompanied by imbalances in the activity of specific prenylated proteins, Rab3 and RhoA, involved in neurotransmitter release, and synaptic plasticity, respectively. Taken together, the present findings indicate that the isoprenoid/cholesterol biosynthetic pathway is critically involved in the physiological modulation of both emotional and cognitive processes in rodents.

Regulation of thalamocortical axon branching by BDNF and synaptic vesicle cycling.

During development, axons form branches in response to extracellular molecules. Little is known about the underlying molecular mechanisms. Here, we investigate how neurotrophin-induced axon branching is related to synaptic vesicle cycling for thalamocortical axons. The exogenous application of brain-derived neurotrophic factor (BDNF) markedly increased axon branching in thalamocortical co-cultures, while removal of endogenous BDNF reduced branching. Over-expression of a C-terminal fragment of AP180 that inhibits clathrin-mediated endocytosis affected the laminar distribution and the number of branch points. A dominant-negative synaptotagmin mutant that selectively targets synaptic vesicle cycling, strongly suppressed axon branching. Moreover, axons expressing the mutant synaptotagmin were resistant to the branch-promoting effect of BDNF. These results suggest that synaptic vesicle cycling might regulate BDNF induced branching during the development of the axonal arbor.

Rabconnectin3α promotes stable activity of the H+ pump on synaptic vesicles in hair cells.

Acidification of synaptic vesicles relies on the vacuolar-type ATPase (V-ATPase) and provides the electrochemical driving force for neurotransmitter exchange. The regulatory mechanisms that ensure assembly of the V-ATPase holoenzyme on synaptic vesicles are unknown. Rabconnectin3α (Rbc3α) is a potential candidate for regulation of V-ATPase activity because of its association with synaptic vesicles and its requirement for acidification of intracellular compartments. Here, we provide the first evidence for a role of Rbc3α in synaptic vesicle acidification and neurotransmission. In this study, we characterized mutant alleles of rbc3α isolated from a large-scale screen for zebrafish with auditory/vestibular defects. We show that Rbc3α is localized to basal regions of hair cells in which synaptic vesicles are present. To determine whether Rbc3α regulates V-ATPase activity, we examined the acidification of synaptic vesicles and localization of the V-ATPase in hair cells. In contrast to wild-type hair cells, we observed that synaptic vesicles had elevated pH, and a cytosolic subunit of the V-ATPase was no longer enriched in synaptic regions of mutant hair cells. As a consequence of defective acidification of synaptic vesicles, afferent neurons in rbc3α mutants had reduced firing rates and reduced accuracy of phase-locked action potentials in response to mechanical stimulation of hair cells. Collectively, our data suggest that Rbc3α modulates synaptic transmission in hair cells by promoting V-ATPase activity in synaptic vesicles.

Quantal amplitude at the cone ribbon synapse can be adjusted by changes in cytosolic glutamate.

PURPOSE: Vision is encoded at photoreceptor synapses by the number of released vesicles and size of the post-synaptic response. We hypothesized that elevating cytosolic glutamate could enhance quantal size by increasing glutamate in vesicles. METHODS: We introduced glutamate (10-40 mM) into cone terminals through a patch pipette and recorded excitatory post-synaptic currents (EPSCs) from horizontal or OFF bipolar cells in the Ambystoma tigrinum retinal slice preparation. RESULTS: Elevating cytosolic glutamate in cone terminals enhanced EPSCs as well as quantal miniature EPSCs (mEPSCs). Enhancement was prevented by inhibiting vesicular glutamate transport with 1S,3R-1-aminocyclopentane-1,3-dicarboxylate in the patch pipette. A low affinity glutamate receptor antagonist, γD-glutamylglycine (1 mM), less effectively inhibited EPSCs evoked from cones loaded with glutamate than control cones indicating that release from cones with supplemental glutamate produced higher glutamate levels in the synaptic cleft. Raising presynaptic glutamate did not alter exocytotic capacitance responses and exocytosis was observed after inhibiting glutamate loading with the vesicular ATPase inhibitor, concanamycin A, suggesting that release capability is not restricted by low vesicular glutamate levels. Variance-mean analysis of currents evoked by flash photolysis of caged glutamate indicated that horizontal cell AMPA receptors have a single channel conductance of 10.1 pS suggesting that ~8.7 GluRs contribute to each mEPSC. CONCLUSIONS: Quantal amplitude at the cone ribbon synapse is capable of adjustment by changes in cytosolic glutamate levels. The small number of channels contributing to each mEPSC suggests that stochastic variability in channel opening could be an important source of quantal variability.

Dysfunction of the fusion of pre-synaptic plasma membranes and synaptic vesicles caused by oxidative stress, and its prevention by vitamin E.

To define whether hyperoxia induces the dysfunction of membrane fusion between synaptic vesicles with pre-synaptic plasma membranes in the nerve terminals, and whether vitamin E prevents this abnormal event, we investigated the influence of hyperoxia on the fusion ability of isolated synaptic vesicles and the inside-out type pre-synaptic plasma membrane vesicles from rat brain using the fluorescence tracing method. The membrane fusion ability of both membranes from rats subjected to hyperoxia was markedly decreased compared with the membranes from a normal rat. Rats subjected to hyperoxia in the form of oxidative stress showed significant increases in the levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes, and protein carbonyl moieties in both synaptic vesicles and pre-synaptic plasma membranes. When rats were supplemented with vitamin E, these abnormalities were inhibited even when rats were subjected to hyperoxia.

Post-tetanic increase in the fast-releasing synaptic vesicle pool at the expense of the slowly releasing pool.

Post-tetanic potentiation (PTP) at the calyx of Held synapse is caused by increases not only in release probability (P(r)) but also in the readily releasable pool size estimated from a cumulative plot of excitatory post-synaptic current amplitudes (RRP(cum)), which contribute to the augmentation phase and the late phase of PTP, respectively. The vesicle pool dynamics underlying the latter has not been investigated, because PTP is abolished by presynaptic whole-cell patch clamp. We found that supplement of recombinant calmodulin to the presynaptic pipette solution rescued the increase in the RRP(cum) after high-frequency stimulation (100 Hz for 4-s duration, HFS), but not the increase in P(r). Release-competent synaptic vesicles (SVs) are heterogeneous in their releasing kinetics. To investigate post-tetanic changes of fast and slowly releasing SV pool (FRP and SRP) sizes, we estimated quantal release rates before and 40 s after HFS using the deconvolution method. After HFS, the FRP size increased by 19.1% and the SRP size decreased by 25.4%, whereas the sum of FRP and SRP sizes did not increase. Similar changes in the RRP were induced by a single long depolarizing pulse (100 ms). The post-tetanic complementary changes of FRP and SRP sizes were abolished by inhibitors of myosin II or myosin light chain kinase. The post-tetanic increase in the FRP size coupled to a decrease in the SRP size provides the first line of evidence for the idea that a slowly releasing SV can be converted to a fast releasing one.

Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals.

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca(2+)-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca(2+) influx. Consistent with this, omega-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca(2+) channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca(2+) release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca(2+) influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca(2+) channel activation through a signaling cascade involving PKC.

Noradrenaline triggers multivesicular release at glutamatergic synapses in the hypothalamus.

The origin of large-amplitude miniature EPSCs (mEPSCs) at central synapses remains to be firmly established. Here, we show that at excitatory synapses onto magnocellular neurosecretory cells in the hypothalamus, noradrenaline induces a rapid and robust increase in mEPSC amplitude that requires alpha1-adrenoceptor activation but is impervious to postsynaptic manipulations that block the putative insertion of AMPA receptors. In response to noradrenaline, mEPSCs exhibit a putative multimodal amplitude histogram distribution that is not attributable to random temporal summation, the unveiling of a quiescent synapse, or the release of large vesicles. Large-amplitude mEPSCs are sensitive to a high dose of ryanodine and are associated with an enhanced glutamate cleft concentration. Together, these data are consistent with the hypothesis that large-amplitude mEPSCs result from the synchronous release of multiple vesicles via rapid presynaptic calcium expulsion from intracellular stores.

Nicotine enhancement of dopamine release by a calcium-dependent increase in the size of the readily releasable pool of synaptic vesicles.

A major factor underlying compulsive tobacco use is nicotine-induced modulation of dopamine release in the mesolimbic reward pathway (Wise and Rompre, 1989). An established biochemical mechanism for nicotine-enhanced dopamine release is by activating presynaptic nicotinic acetylcholine receptors (nAChRs) (Wonnacott, 1997). Prolonged application of 10(-7) to 10(-5) m nicotine to striatal synaptosomes promoted a sustained efflux of [3H]dopamine. This nicotine effect was mediated by non-alpha7 nAChRs, because it was blocked by 5 mum mecamylamine but was resistant to 100 nm alpha-bungarotoxin (alphaBgTx). Dopamine release was diminished by omitting Na+ or by applying peptide calcium channel blockers, indicating that nAChRs trigger release by depolarizing the nerve terminals. However, because alpha7 receptors rapidly desensitize in the continuous presence of agonists, a repetitive stimulation protocol was used to evaluate the possible significance of desensitization. This protocol produced a transient increase in [3H]dopamine released by depolarization and a significant increase in the response to hypertonic solutions that measure the size of the readily releasable pool (RRP) of synaptic vesicles. The nicotine-induced increase in the size of the readily releasable pool was blocked by alphaBgTx and by the calmodulin antagonist calmidazolium, suggesting that Ca2+ entry through alpha7 nAChRs specifically enhances synaptic vesicle mobilization at dopamine terminals. Thus, nicotine enhances dopamine release by two complementary actions mediated by discrete nAChR subtypes and suggest that the alpha7 nAChR-mediated pathway is tightly and specifically coupled to refilling of the RRP of vesicles in dopamine terminals.

Toxic effect of chloromycetin on the ultrastructures of the motor neurons of the Chinese tree shrew (Tupaia belangeri).

This paper describes the toxic effects of chloromycetin on the motor neurons of the Chinese tree shrew (Tupaia belangeri chinensis) with horse radish peroxidase (HRP) as the labeling enzyme. When chloromycetin was administered orally at 2.5 mg/kg (body weight)/day for 3 days, Chinese tree shrews showed evidence of neurotoxicity. This included damage in cortical motor neuron synapses ending on neurons of the red nucleus and the ultrastructural changes in the mitochondria such as swelling of these organelles and blurring of their cristae. There was an increase of the mitochondrial matrix density and of the thickness of the synaptic membranes. These observations indicate that chloromycetin can lead to ultrastructural change of terminals of the cortical motor axons, and that Chinese tree shrews are sensitive animal model for chloromycetin neurotoxicity.

PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells.

To determine the possible role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the development of the sympathoadrenal cell lineage, we have examined the effects of this neurotrophic peptide, in comparison to nerve growth factor (NGF), on the morphology, electrophysiological properties, expression of neuronal and neuroendocrine marker genes, and activity of transcription factors during differentiation of sympathoadrenal-derived cells, using the rat pheochromocytoma PC12 cell model. Both PACAP and NGF elicited rapid neurite outgrowth, which was accompanied by induction of cell excitability and the development of both sodium and calcium currents. Concurrently, PACAP and NGF increased the expression of a marker of synaptic vesicles. By contrast, PACAP, but not NGF, regulated the expression of different constituents of neuroendocrine large dense core vesicles in PC12 cells. Furthermore, PACAP and NGF differentially regulated the expression of mammalian achaete-scute homologue and paired homeobox 2b genes, transcription factors instrumental for sympathoadrenal development. To compare downstream effectors activated by PACAP and NGF, we studied the effects of these factors on the binding activity of consensus 12-O-tetradecanoylphorbol-13-acetate- and cAMP-responsive elements to nuclear extracts of differentiating PC12 cells. We found that both PACAP and NGF markedly increase the binding activity of these cis-regulatory sequences and that PACAP preferentially recruits activator protein-1-like transcription factors to these elements. Taken together, these results show that PACAP and NGF exert common as well as different effects on neuronal and neuroendocrine traits in differentiating PC12 cells, strongly suggesting that these two trophic factors could play complementary roles in the development of the sympathoadrenal cell lineage.

Modifications in brain CaM kinase II after long-term treatment with desmethylimipramine.

The present study investigated the effect of long-term (15 mg/kg for 15 days) and acute (15 mg/kg, single administration) treatment with desmethylimipramine, a tricyclic antidepressant drug, on calcium/calmodulin-dependent protein kinase II (CaMKII), a kinase implicated in the mechanism of antidepressant drug action. Similar to selective and non-selective serotonin reuptake inhibitors, long-term, but not acute, treatment with desmethylimipramine markedly increased the activity of CaMKII in the hippocampal synaptic vesicle fraction (+51.9%). The kinase activity was also increased in the same fraction of frontal cortex (+24.2%) and in the striatum (+45.9%), although in this last area the mechanism appeared to be different because the protein level of the kinase was also markedly increased (+43.7%). However, the effect of treatment was not restricted to the presynaptic kinase, because CaMKII activity was also increased in the total cellular cytosol in cortical areas. The autonomous (calcium-independent) activity of CaMKII was assayed for the first time after antidepressant treatment, and found to be increased in synaptic vesicles of all three areas. These results confirmed the involvement of CaMKII in antidepressant drug action and suggested that modulation of transmitter release is a primary component in the action of psychotropic drugs.

The hypocretins/orexins: novel hypothalamic neuropeptides involved in different physiological systems.

The hypothalamus regulates diverse physiological functions, including the control of energy metabolism, circadian rhythms, stress and anxiety, sexual and reproductive behaviors. An overview of the most prevalent hypothalamus-enriched mRNAs revealed that this area of the brain specializes in producing intercellular signaling molecules. Two new secreted peptides derived from a single neuropeptide precursor, named hypocretins and orexins by two different groups, are synthesized in a small set of neurons in the perifornical area of the hypothalamus. Intracerebroventricular injection of the hypocretins/orexins increases food consumption in rats. Here we review recent progress in identifying the role of the hypocretins/orexins in the control of energy balance and in other physiological systems.

Modifications in brain cAMP- and calcium/calmodulin-dependent protein kinases induced by treatment with S-adenosylmethionine.

Several lines of evidence suggest that the mechanism of action of antidepressant drugs (AD) involves adaptive changes occurring in intraneuronal post-receptor signal transduction cascades. Protein phosphorylation has a key role in signal transduction and was previously found to be a target in the action of AD (5-HT and/or NA reuptake blockers). Several studies showed that cAMP- and type II Ca2+/calmodulin-dependent protein kinases (PKA and CaMKII) are markedly affected by typical AD in two different and complementary cellular districts, respectively microtubules (a somatodendritic compartment) and synaptic vesicles (a presynaptic terminal compartment). In order to investigate whether the effect on protein kinases may be involved in the therapeutic action of drugs it is interesting to compare the effect of atypical AD with that of typical drugs. In this study the effect of the atypical AD S-adenosylmethionine (SAMe) was tested. Repeated (12 days) SAMe treatment induced in cerebrocortical microtubules an increase in the binding of cAMP to the RII PKA regulatory subunit and an increase in the endogenous phosphorylation of microtubule-associated protein 2, an effect resembling that of typical AD. In synaptic terminals the treatment induced an increase in the activity of CaMKII and in the endogenous phosphorylation of vesicular substrates. However, this modification was found in the cerebral cortex rather than in the hippocampus, where typical AD affect CaMKII. In addition the synapsin I level was decreased in the hippocampus and increased in the cerebral cortex, an effect not detected with typical AD.

Cysteine string protein is required for calcium secretion coupling of evoked neurotransmission in drosophila but not for vesicle recycling.

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Drosophila. CSP has been found to interact in vitro with the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons of csp mutant neuromuscular junctions fail to destain at 32 degrees C after K+ depolarization, and that FM1-43 dye uptake cannot be evoked by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that the csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.