REST/NRSF governs the expression of dense-core vesicle gliosecretion in astrocytes.

Astrocytes are the brain nonnerve cells that are competent for gliosecretion, i.e., for expression and regulated exocytosis of clear and dense-core vesicles (DCVs). We investigated whether expression of astrocyte DCVs is governed by RE-1-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), the transcription repressor that orchestrates nerve cell differentiation. Rat astrocyte cultures exhibited high levels of REST and expressed neither DCVs nor their markers (granins, peptides, and membrane proteins). Transfection of a dominant-negative construct of REST induced the appearance of DCVs filled with secretogranin 2 and neuropeptide Y (NPY) and distinct from other organelles. Total internal reflection fluorescence analysis revealed NPY-monomeric red fluorescent protein-labeled DCVs to undergo Ca(2+)-dependent exocytosis, which was largely prevented by botulinum toxin B. In the I-II layers of the human temporal brain cortex, all neurons and microglia exhibited the expected inappreciable and high levels of REST, respectively. In contrast, astrocyte REST was variable, going from inappreciable to high, and accompanied by a variable expression of DCVs. In conclusion, astrocyte DCV expression and gliosecretion are governed by REST. The variable in situ REST levels may contribute to the well-known structural/functional heterogeneity of astrocytes.

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.

SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: Imaging analysis at the single-vesicle level with TIRF microscopy

Chemokines are small chemotactic molecules widely expressed throughout the central nervous system. A number of papers, during the past few years, have suggested that they have physiological functions in addition to their roles in neuroinflammatory diseases. In this context, the best evidence concerns the CXC-chemokine stromal cell-derived factor (SDF-1alpha or CXCL12) and its receptor CXCR4, whose signalling cascade is also implicated in the glutamate release process from astrocytes. Recently, astrocytic synaptic like microvesicles (SLMVs) that express vesicular glutamate transporters (VGLUTs) and are able to release glutamate by Ca(2+)-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Here, in order to elucidate whether SDF-1alpha/CXCR4 system can participate to the brain fast communication systems, we investigated whether the activation of CXCR4 receptor triggers glutamate exocytosis in astrocytes. By using total internal reflection (TIRF) microscopy and the membrane-fluorescent styryl dye FM4-64, we adapted an imaging methodology recently developed to measure exocytosis and recycling in synaptic terminals, and monitored the CXCR4-mediated exocytosis of SLMVs in astrocytes. We analyzed the co-localization of VGLUT with the FM dye at single-vesicle level, and observed the kinetics of the FM dye release during single fusion events. We found that the activation of CXCR4 receptors triggered a burst of exocytosis on a millisecond time scale that involved the release of Ca(2+) from internal stores. These results support the idea that astrocytes can respond to external stimuli and communicate with the neighboring cells via fast release of glutamate.

Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

Role of Rab5 in synaptic vesicle recycling

During synaptic transmission, NT-filled synaptic vesicles are released by Ca2+-triggered exocytosis at the active zone. Following exocytosis, SV membrane is immediately re-internalized and synaptic vesicles (SVs) are regenerated by a local recycling mechanism within the presynaptic terminal. It is debated whether an endosomal compartment is involved in this recycling process. In contrast, it is well known from cultured mammalian cells, that endocytic vesicles fuse to the early sorting endosome. The early endosome is a major sorting station of the cell where cargo is send into the degradative pathway to late endosome and lysosome or towards recycling. Each trafficking step is mediated by a certain protein of the Rab family. Rab proteins are small GTPases belonging to the Ras superfamily. They accumulate at their target compartments and have thereby been used as markers for the different endocytic organelles in cultured mammalian cells. Rab5 controls trafficking from the PM to the early endosome and has thereby been used as marker for this compartment. A second marker is based on the specific binding of the FYVE zinc finger protein domain to the lipid PI(3)P that is specifically generated at the early endosomal membrane. This study used the Drosophila NMJ as a model system to investigate the SV recycling process. In particular, three questions were addressed: First, is an endosomal compartment present at the synapse? Second, do SVs recycle through an endosome? Third, is Rab5 involved in SV recycling? We used GFP fusions of Rab5 and 2xFYVE to visualize endosomal compartments at the presynaptic terminal of Drosophila third instar larval NMJs. Furthermore, the endosomes are located within the pool of recycling SVs, labeled with the styryl-dye FM5-95. Using the temperature-sensitive mutation in Dynamin, shibirets, we showed that SV recycling involves trafficking through an intermediate endosomal compartment. In cultured mammalian cells, interfering with Rab5 function by expressing the dominant negative version, Rab5SN causes the fragmentation of the endosome and the accumulation of endocytic vesicles. In contrast, when Rab5 is overexpressed enlarged endosomal compartments were observed. In Drosophila, the endosomal compartment was disrupted when loss of function and dominant negative mutants of Rab5 were expressed. In addition, at the ultrastructural we observed an accumulation of endocytic vesicles in Rab5S43N expressing terminals and enlarged endosomes when Rab5 was overexpressed. Furthermore, interfering with Rab5 function using the dominant negative Rab5S43N caused a decrease in the SV recycling kinetics as shown by FM1-43 experiments. In contrast, overexpression of Rab5 or GFP-Rab5 caused an increase in the FM1-43 internalization rate. Finally, standard electrophysiological techniques were used to measure synaptic function. We found that the Rab5-mediated endosomal SV recycling pathway generates vesicles with a higher fusion efficacy during Ca2+-triggered release, compared to SVs recycled when Rab5 function was impaired. We therefore suggest a model in which the endosome serves as organelle to control the SV fusion efficacy and thereby the synaptic strength. Since changes in the synaptic strength are occuring during learning and memory processes, controlling endosomal SV recycling might be a new molecular mechanism involved in learning and memory.

Vesicle motion during sustained exocytosis in chromaffin cells

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles’ arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that including the fusion-time statistics in our model does not produce any significant changes on the results. These findings indicate that the motion of the whole ensemble of vesicles towards the membrane is directed and reflected in the amperometric signals. Our results confirm the conclusions of previous imaging studies performed on single vesicles that vesicles’ motion underneath plasma membranes is not purely random, but biased towards the membrane.

Self-assembly of a peptide amphiphile containing l-carnosine and its mixtures with a multilamellar vesicle forming lipid

The self-assembly of the peptide amphiphile (PA) hexadecyl-(β-alaninehistidine) is examined in aqueous solution, along with its mixtures with multilamellar vesicles formed by DPPC (dipalmitoyl phosphatidylcholine). This PA, denoted C16-βAH, contains a dipeptide headgroup corresponding to the bioactive molecule L-carnosine. It is found to selfassemble into nanotapes based on stacked layers of molecules. Bilayers are found to coexist with monolayers in which the PA molecules pack with alternating up−down arrangement so that the headgroups decorate both surfaces. The bilayers become dehydrated as PA concentration increases and the number of layers in the stack decreases to produce ultrathin nanotapes comprised of 2−3 bilayers. Addition of the PA to DPPC multilamellar vesicles leads to a transition to well-defined unilamellar vesicles. The unique ability to modulate the stacking of this PA as a function of concentration, combined with its ability to induce a multilamellar to unilamellar thinning of DPPC vesicles, may be useful in biomaterials applications where the presentation of the peptide function at the surface of self-assembled nanostructures is crucial.

Synaptic vesicle glycoprotein 2A modulates vesicular release and calcium channel function at peripheral sympathetic synapses

Synaptic vesicle glycoprotein (SV)2A is a transmembrane protein found in secretory vesicles and is critical for Ca2+-dependent exocytosis in central neurons, although its mechanism of action remains uncertain. Previous studies have proposed, variously, a role of SV2 in the maintenance and formation of the readily releasable pool (RRP) or in the regulation of Ca2+ responsiveness of primed vesicles. Such previous studies have typically used genetic approaches to ablate SV2 levels; here, we used a strategy involving small interference RNA (siRNA) injection to knockdown solely presynaptic SV2A levels in rat superior cervical ganglion (SCG) neuron synapses. Moreover, we investigated the effects of SV2A knockdown on voltage-dependent Ca2+ channel (VDCC) function in SCG neurons. Thus, we extended the studies of SV2A mechanisms by investigating the effects on vesicular transmitter release and VDCC function in peripheral sympathetic neurons. We first demonstrated an siRNA-mediated SV2A knockdown. We showed that this SV2A knockdown markedly affected presynaptic function, causing an attenuated RRP size, increased paired-pulse depression and delayed RRP recovery after stimulus-dependent depletion. We further demonstrated that the SV2A–siRNA-mediated effects on vesicular release were accompanied by a reduction in VDCC current density in isolated SCG neurons. Together, our data showed that SV2A is required for correct transmitter release at sympathetic neurons. Mechanistically, we demonstrated that presynaptic SV2A: (i) acted to direct normal synaptic transmission by maintaining RRP size, (ii) had a facilitatory role in recovery from synaptic depression, and that (iii) SV2A deficits were associated with aberrant Ca2+ current density, which may contribute to the secretory phenotype in sympathetic peripheral neurons.

Ionization and Structural Changes of the DMPG Vesicle along Its Anomalous Gel-Fluid Phase Transition: A Study with Different Lipid Concentrations

Dispersions of saturated anionic phospholipid dimyristoyl phosphatidylglycerol (DMPG) have been extensively studied regarding their peculiar thermostructural behavior. At low ionic strength, the gel-fluid transition is spread along nearly 17 degrees C, displaying several thermal events in the calorimetric profile that is quite different from the single sharp peak around 23 degrees C found for higher ionic strength DMPG dispersions. To investigate the role of charge in the bilayer transition, we carefully examine the temperature dependence of the electrical conductivity of DMPG dispersions at different concentrations, correlating the data with the corresponding differential scanning calorimetry (DSC) traces. Electrical conductivity together with electrophoretic mobility measurements allowed the calculation of the dependence of the degree of ionization of DMPG vesicles on lipid concentration and temperature. It was shown that there is a decrease in vesicle charge as the lipid concentration increases, which is probably correlated with the increase in the concentration of bulk Na(+). Apart from the known increase in the electrical conductivity along the DMPG temperature transition region, a sharp rise was observed at the bilayer pretransition for all lipid concentrations studied, possibly indicating that the beginning of the chain melting process is associated with an increase in bilayer ionization. It is confirmed here that the gel-fluid transition of DMPG at low ionic strength is accompanied by a huge increase in the dispersion viscosity. However, it is shown that this measured macroviscosity is distinct from the local viscosity felt by either charged ions or DMPG charged aggregates in measurements of electrical conductivity or electrophoretic mobility, Data presented here give support to the idea that DMPG vesicles, at low ionic strength, get more ionized along the temperature transition region and could be perforated and/or deformed vesicle structures.

Synthesis, characterization and biological analysis of the complex [VO(Hdhp)(2)] (H(2)dhp=2,3-dihydroxypyridine)

A new vanadium (IV) complex with the monoanion of 2,3-dihydroxypyridine (H(2)dhp), or 3-hydroxy-2(1H)-pyridone, was synthesized, characterized by physicochemical techniques and tested biologically. The EPR data for the [VO(Hdhp)(2)] complex in DMF are: g(x) = 1.9768, g(y) = 1.9768 and g(z) = 1.9390; A values (10(-4) cm(-1)): A(x), 59.4; A(y//), 59.4; A(z), 171.0. The vV=O band in the IR spectrum of the complex is at 986 cm(-1). The complex is paramagnetic, with mu(eff) = 1.65 BM (d(1), spin-only) at 25 degrees C. The irreversible oxidation process [V(V)/V(IV)] of the [VO(Hdhp)(2)] complex, as revealed in a cyclic voltammogram, occurs at 876 mV. The calculated molecular structure of [VO(Hdhp)(2)] shows the vanadium(IV) center in a distorted square pyramidal environment, with the oxo ligand in the apical position and the oxygen donor atoms of the Hdhp ligands in the basal positions. The ability of [VO(Hdhp)(2)] to mimic insulin, and its toxicity to hepato-biliary functions, were investigated in streptozotocin-induced diabetic rats and it was concluded that the length of treatment and the amount of [VO(Hdhp)(2)] administered were effective in reducing experimental diabetes.

Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synthesis, characterization and biological analysis of the complex [VO(Hdhp)(2)] (H(2)dhp=2,3-dihydroxypyridine)

A new vanadium (IV) complex with the monoanion of 2,3-dihydroxypyridine (H(2)dhp), or 3-hydroxy-2(1H)-pyridone, was synthesized, characterized by physicochemical techniques and tested biologically. The EPR data for the [VO(Hdhp)(2)] complex in DMF are: g(x) = 1.9768, g(y) = 1.9768 and g(z) = 1.9390; A values (10(-4) cm(-1)): A(x), 59.4; A(y//), 59.4; A(z), 171.0. The vV=O band in the IR spectrum of the complex is at 986 cm(-1). The complex is paramagnetic, with mu(eff) = 1.65 BM (d(1), spin-only) at 25 degrees C. The irreversible oxidation process [V(V)/V(IV)] of the [VO(Hdhp)(2)] complex, as revealed in a cyclic voltammogram, occurs at 876 mV. The calculated molecular structure of [VO(Hdhp)(2)] shows the vanadium(IV) center in a distorted square pyramidal environment, with the oxo ligand in the apical position and the oxygen donor atoms of the Hdhp ligands in the basal positions. The ability of [VO(Hdhp)(2)] to mimic insulin, and its toxicity to hepato-biliary functions, were investigated in streptozotocin-induced diabetic rats and it was concluded that the length of treatment and the amount of [VO(Hdhp)(2)] administered were effective in reducing experimental diabetes.

A histological study of the seminal vesicle of the armoured catfish Corydoras aeneus

Most species of Corydoras exhibited a reproductive behaviour called 'T-position', and exhibited an accessory gland in the male genital tract, called the seminal vesicle. It appeared that both the structure and the composition of the fluid varied considerably between the species investigated. Consequently, different opinions were proposed regarding the possible role of seminal vesicle on this particular reproductive behaviour. Male adults of Corydoras aeneus were collected, anaesthetized, and samples of seminal vesicle were fixed in Bouin's solution. The sections were stained with haematoxylin-eosin and periodic acid Schiff. The seminal vesicle showed a system of anastomosed secretory tubules, forming a vesicular collective network, which gave rise to the vesicular ducts. The latter fused with the testicular efferent ducts and formed the spermatic ducts. Considering this fusion, when the sperm cells reached the spermatic ducts, the fluid produced at the seminal vesicle covered them. Histochemical studies evidenced the presence of neutral and acid glycosaminoglycans in the seminal fluid. Considering the reproductive behaviour of C. aeneus, it is believed that the protection associated with the immobilization of the sperm cells assures the sperm integrity during the passage through female's intestine until fertilization.

Evaluation of the ethanol intake on the Calomys callosus seminal vesicle structure

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)