Evaluation of an integrated care program for schizophrenia: concept and study design.

Concept and design of an independent scientific evaluation of different pathways of care for schizophrenia patients in Germany with respect to effectiveness and efficiency are presented. In this prospective, observational study, schizophrenia patients receiving an integrated care treatment, the intervention group (IG), are compared with patients under routine care conditions treated by the same physician (first control group, CG 1). A second control group (CG 2) of patients treated by office-based psychiatrists not participating in the integrated care program will be recruited and their data compared with the two other groups. The total amount of psychiatric hospital days after 12 months is defined as primary outcome parameter. Secondary outcome parameters comprise the frequency of psychiatric inpatient readmissions, severity of schizophrenia symptoms, remission rates and quality of life. Patients undergo assessments at baseline, month 6 and 12 using standardized and experimental questionnaires. Routine data of a regional German social health insurance fund complement information on included patients. Additionally, a cost-effectiveness and cost-utility analysis will be performed. Until now, 137 psychiatrists included 980 patients in the integrated care project in Lower Saxony, Germany, and 47 psychiatrists (IG and both CGs) are willing to participate in the independent evaluation. For the first time, a prospective observational controlled evaluation study of a countrywide integrated care project planning to recruit 500 schizophrenia patients has started using comprehensive assessments as well as routine data of a social health insurance fund.

[The health-care situation in outpatient psychiatry--results of a survey among members of the Germany Association of Psychotherapists (DPtV)]. / Zur Versorgungslage im Bereich der ambulanten Psychotherapie--Ergebnisse einer Erhebung unter den Mitgliedern der Deutschen Psychotherapeuten Vereinigung (DPtV).

BACKGROUND: Mental-health problems are of increasing importance in the German population. Nonetheless there is a lack of data concerning outpatient psychotherapeutic care, especially with a focus on psychotherapy sessions approved by social health insurances and performed by psychological psychotherapists and paediatric psychotherapists. METHODS: The study presents the results of a survey among all members of the German Association of Psychotherapists (DPtV) and is based on questionnaires filled in by 2,497 psychotherapists (return rate 33.3%). The assessment is based on the therapists' data without an external validation. Main topics of the survey were characteristics of the supply of psychotherapeutic care, therapeutic time contingents per patient and their utilisation and demographic and socio-economic features of patients. Evaluating the survey, the results were analysed overall and by groups of therapists varying in demographic features, professional qualifications and regional criteria, using bivariate as well as multivariate methods. RESULTS: The study provides evidence indicating an underprovision of outpatient psychotherapeutic care. Psychotherapists who use a waiting list reported average waiting times of more than 2.5 months. Additionally there are differences in psychotherapeutic care between various patient groups. In regions with lower population density we found less provision of psychotherapeutic care compared to regions with higher population density. Taking into account epidemiological data, the study indicates that the participation of men, persons of older age and patients with lower levels of education in psychotherapeutic care is below average. CONCLUSION: The results suggest a considerable degree of underprovision and inadequate provision of outpatient psychotherapeutic care. With regard to special population groups, further research is necessary to identify utilisation barriers towards psychotherapy.

[Health-based risk adjustment. Effects and side effects]. / Morbiditätsorientierter Risikostrukturausgleich. Wirkungen und Nebenwirkungen.

Numerous health systems have introduced competition between health plans while banning risk-rated premiums. Risk adjustment for health plans is introduced to reduce incentives for risk selection and to create incentives for health plans to permanently invest in care for the chronically ill. According to the international health economics state of the art, risk adjustment in the German social health insurance system has used information on health status (measured by diagnoses and drug prescriptions) on top of demographic information since 2009. In non-competitive health care systems similar mechanisms are sometimes established, e.g. to achieve an equitable distribution of resources between regions. An evaluation of the first year of health-based risk adjustment demonstrates a superior performance in comparison to the old, demographic risk adjustment. The old risk adjustment formula (without ex post high-cost pooling) showed R(2) of 5.8%, CPM of 10.4% and MAPE of 2,226 €, in contrast to the new health status-based risk adjustment formula (without cash benefit for sick allowance) which reaches R(2) 20.2%, CPM 22.5% and MAPE 1,817 €. However, to make competition between health plans functional for improvement of quality and efficiency of health care, health plans must be granted additional instruments to act as prudent buyers of health care.

Synaptic vesicles studied by dynamic light scattering.

The size polydispersity distribution of synaptic vesicles (SVs) is characterized under quasi-physiological conditions by dynamic light scattering (DLS). Highly purified fractions of SVs obtained from rat brain still contain a small amount of larger contaminant structures, which can be quantified by DLS and further reduced by asymmetric-flow field-flow (AFFF) fractionation. The intensity autocorrelation functions g (2)(τ) recorded from these samples are analyzed by a constrained regularization method as well as by an alternative direct modeling approach. The results are in quantitative agreement with the polydispersity obtained from cryogenic electron microscopy of vitrified SVs. Next, different vesicle fusion assays based on samples composed of SVs and small unilamellar proteoliposomes with the fusion proteins syntaxin 1 and SNAP-25A are characterized by DLS. The size increase of the proteoliposomes due to SNARE-dependent fusion with SVs is quantified by DLS under quasi-physiological conditions.

Effects of media stories featuring coping with suicidal crises on psychiatric patients: Randomized controlled trial.

BACKGROUND: Accumulating evidence suggests beneficial effects of media stories featuring individuals mastering their suicidal crises, but effects have not been assessed for psychiatric patients. METHODS: We randomized n = 172 adult psychiatric patients (n = 172, 97.1% inpatients) to read an educative article featuring a person mastering a suicidal crisis (n = 92) or an unrelated article (n = 80) in a single-blind randomized controlled trial. Questionnaire data were collected before (T1) and after exposure (T2) as well as 1 week later (study end-point, T3). The primary outcome was suicidal ideation as assessed with the Reasons for Living Inventory; secondary outcomes were help-seeking intentions, mood, hopelessness, and stigmatization. Differences between patients with affective versus other diagnoses were explored based on interaction tests. RESULTS: We found that patients with affective disorders (n = 99) experienced a small-sized reduction of suicidal ideation at 1-week follow up (mean difference to control group [MD] at T3 = -0.17 [95% CI -0.33, -0.03], d = -0.15), whereas patients with nonaffective diagnoses (n = 73) experienced a small-sized increase (T2: MD = 0.24 [95% CI 0.06, 0.42], d = 0.19). Intervention group participants further experienced a nonsustained increase of help-seeking intentions (T2: MD = 0.53 [95% CI 0.11, 0.95], d = 0.19) and a nonsustained deterioration of mood (T2: MD = -0.14 [95% CI -0.27, -0.02], d = -0.17). CONCLUSIONS: This study suggests that patients with affective disorders appear to benefit from media materials featuring mastery of suicidal crises. More research is needed to better understand which patient groups are at possible risk of unintended effects.

Controlled assembly of silane-based polymers: chemically robust thin-films.

We describe the controlled assembly of silane-based copolymers on various interfaces that have surface silanol groups. This assembly occurs as a result of the formation of very robust siloxane bonds (Si-O-Si) due to a condensation reaction between the alkoxysilane groups of the polymers and surface hydroxyl groups of the substrates. Deposition of these copolymers is not self-limiting; therefore, they could not be assembled into discrete monolayers. However, UV-visible data collected as a function of deposition cycle reveals a linear relationship, confirming the deposition of a constant amount of polymer in each deposition cycle. A linear variation of layer thickness with deposition cycles is also observed. The assembled polymer layers are found to be very robust and resistant even when exposed to piranha solution for several hours.

Clostridial neurotoxins: new tools for dissecting exocytosis.

Tetanus toxin and botulinal toxins are potent inhibitors of neuronal exocytosis. Within the past five years the protein sequences of all eight neurotoxins have been determined, their mode of action as metalloproteases has been established, and their intraneuronal targets have been identified. The toxins act by selectively proteolysing the synaptic vesicle protein synaptobrevin (VAMP) or the presynaptic membrane proteins syntaxin (HPC-1) and SNAP-25. These three proteins form the core of a complex that mediates fusion of carrier vesicles to target membranes. Tetanus and botulinal neurotoxins could serve in the future as tools to study membrane trafficking events, or even higher brain functions such as behaviour and learning.

Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins.

Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.

Synaptophysin (p38) at the frog neuromuscular junction: its incorporation into the axolemma and recycling after intense quantal secretion.

Recycling of synaptophysin (p38), a synaptic vesicle integral membrane protein, was studied by the use of antisera raised against the protein purified from frog brain. When frog cutaneous pectoris muscles were fixed at rest, a bright, specific immunofluorescent signal was observed in nerve-terminal regions only if their plasma membranes had been previously permeabilized. When muscles were fixed after they had been treated for 1 h with a low dose of alpha-latrotoxin in Ca2+-free medium, an equally intense fluorescence could be observed without previous permeabilization. Under this condition, alpha-latrotoxin depletes nerve terminals of their quantal store of acetylcholine and of synaptic vesicles. These results indicate that fusion of synaptic vesicles leads to the exposure of intravesicular antigenic determinants of synaptophysin on the outer surface of the axolemma, and provide direct support for the vesicle hypothesis of neurotransmitter release. After 1 h treatment with the same dose of alpha-latrotoxin in the presence of 1.8 mM extracellular Ca2+, immunofluorescent images were obtained only after permeabilization with detergents. Under this condition, the vesicle population was maintained by an active process of recycling and more than two times the initial store of quanta were secreted. Thus, despite the active turnover of synaptic vesicles and of quanta of neurotransmitter, no extensive intermixing occurs between components of the vesicle and presynaptic plasma membrane.

Structure of synaptogyrin (p29) defines novel synaptic vesicle protein.

Synaptogyrin (p29) is a synaptic vesicle protein that is uniformly distributed in the nervous system (Baumert et al., 1990). We have cloned and sequenced the cDNA encoding synaptogyrin, and the sequence predicts a protein with a molecular mass of 25,900 D with four membrane-spanning domains. The topology of the protein was confirmed by limited proteolysis using domain-specific antibodies. Database searches revealed several cDNA sequences coding polypeptides with sequence identities ranging from 32 to 46%, suggesting that synaptogyrin is a member of a multigene family. When the synaptogyrin cDNA is expressed in COS cells, the generated protein is indistinguishable from native synaptogyrin. To study intracellular sorting, synaptogyrin was expressed in CHO cells that revealed a punctate staining that was very similar to that of synaptophysin and endogenously expressed cellubrevin. Significant overlap with transferrin staining was also observed, suggesting that synaptogyrin is targeted to a recycling compartment involved in membrane traffic to and from the plasma membrane.

Microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of presynaptic nerve terminals.

Nerve endings of the posterior pituitary are densely populated by dense-core neurosecretory granules which are the storage sites for peptide neurohormones. In addition, they contain numerous clear microvesicles which are the same size as small synaptic vesicles of typical presynaptic nerve terminals. Several of the major proteins of small synaptic vesicles of presynaptic nerve terminals are present at high concentration in the posterior pituitary. We have now investigated the subcellular localization of such proteins. By immunogold electron microscopy carried out on bovine neurohypophysis we have found that three of these proteins, synapsin I, Protein III, and synaptophysin (protein p38) were concentrated on microvesicles but were not detectable in the membranes of neurosecretory granules. In addition, we have studied the distribution of the same proteins and of the synaptic vesicle protein p65 in subcellular fractions of bovine posterior pituitaries obtained by sucrose density centrifugation. We have found that the intrinsic membrane proteins synaptophysin and p65 had an identical distribution and were restricted to low density fractions of the gradient which contained numerous clear microvesicles with a size range the same as that of small synaptic vesicles. The peripheral membrane proteins synapsin I and Protein III exhibited a broader distribution extending into the denser part of the gradient. However, the amount of these proteins clearly declined in the fractions preceding the peak of neurosecretory granules. Our results suggest that microvesicles of the neurohypophysis are biochemically related to small synaptic vesicles of all other nerve terminals and argue against the hypothesis that such vesicles represent an endocytic byproduct of exocytosis of neurosecretory granules.

Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells.

An intrinsic membrane protein of brain synaptic vesicles with Mr 38,000 (p38, synaptophysin) has recently been partially characterized (Jahn, R., W. Schiebler, C. Ouimet, and P. Greengard, 1985, Proc. Natl. Acad. Sci. USA, 83:4137-4141; Wiedenmann, B., and W. W. Franke, 1985, Cell, 41:1017-1028). We have now studied the presence of p38 in a variety of tissues by light and electron microscopy immunocytochemistry and by immunochemistry. Our results indicate that, within the nervous system, p38, like the neuron-specific phosphoprotein synapsin I, is present in virtually all nerve terminals and is selectively associated with small synaptic vesicles (SSVs). No p38 was detectable on large dense-core vesicles (LDCVs). p38 and synapsin I were found to be present in similar concentrations throughout the brain. Outside the nervous system, p38 was found in a variety of neuroendocrine cells, but not in any other cell type. In neuroendocrine cells p38 was localized on a pleiomorphic population of small, smooth-surfaced vesicles, which were interspersed among secretory granules and concentrated in the Golgi area, but not on the secretory granules themselves. Immunoblot analysis of endocrine tissues and cell lines revealed a band with a mobility slightly different from that of neuronal p38. This difference was attributable to a difference in glycosylation. The finding that p38, like synapsin I, is a component of SSVs of virtually all neurons, but not of LDCVs, supports the idea that SSVs and LDCVs are organelles of two distinct pathways for regulated neuronal secretion. In addition, our results indicate the presence in a variety of neuroendocrine cells of an endomembrane system, which is related to SSVs of neurons but is distinct from secretory granules.

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling.

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.

Export of cellubrevin from the endoplasmic reticulum is controlled by BAP31.

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.

A cell-free system for regulated exocytosis in PC12 cells.

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.

Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis.

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites.

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal-dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow-labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.

Association of Rab3A with synaptic vesicles at late stages of the secretory pathway.

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t-SNAREs participate in SV recycling in what may be functionally distinct forms.