Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle-or fusion-related mechanism.During the past decade there has been increasing evidence for both integrative and dynamic roles for astrocytes in the central nervous system. Following activation of G protein-coupled receptors, astrocytes exhibit calcium oscillations, leading to the release of the chemical transmitters glutamate and ATP (1-3). Studies in vitro and in brain slices have led to the hypothesis of tripartite synaptic transmission (4); neuronal activity causes elevations of synaptically associated calcium in astrocytes, which in turn leads to the release of chemical transmitters from these glial cells to locally modulate synaptic transmission (2, 5-7).The mechanisms mediating the release of these transmitters from astrocytes are, however, ill-defined and are still the subject of intense debate. At least three distinct release pathways have been proposed as mediating the calcium-dependent release of glutamate from astrocytes: the reversal of plasma membrane glutamate transporters, anion transporter mediate release mechanisms, and calcium-dependent exocytosis (8 -10). Because the release of glutamate is stimulated by calcium elevations and is not affected by glutamate transport inhibitors and because changes in cell volume have not been detected coincident with release, it has been proposed that this transmitter is released through a vesicle-mediated exocytotic pathway.Several observations made using cultured astrocytes support such a vesicle-mediated exocytotic mechanism of glutamate release, including the calcium depend...
Kiss-and-run exocytosis, consisting of reversible fusion between the vesicle membrane and the plasma membrane, is considered to lead to full fusion after stimulation of vesicles containing classical transmitters. However, whether this is also the case in the fusion of peptidergic vesicles is unknown.Previously, we have observed that spontaneous neuropeptide discharge from a single vesicle is slower than stimulated release, because of the kinetic constraints of fusion pore opening. To explore whether slow spontaneous release also reflects a relatively narrow fusion pore, we analyzed the permeation of FM 4-64 dye and HEPES molecules through spontaneously forming fusion pores in lactotroph vesicles expressing synaptopHluorin, a pH-dependent fluorescent fusion marker. Confocal imaging showed that half of the spontaneous exocytotic events exhibited fusion pore openings associated with a change in synaptopHluorin fluorescence but were impermeable to FM 4-64 and HEPES. Together with membrane capacitance measurements, these findings indicate an open fusion pore diameter Ͻ0.5 nm, much smaller than the neuropeptides. In stimulated cells, Ͼ70% of exocytotic events exhibited a larger, FM 4-64-permeable pore (Ͼ1 nm). Interestingly, capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. Stimulation increased the frequency of transient events and the fusion pore dwell time but decreased the fraction of events with lowest measurable fusion pore.Kiss-and-run is the predominant mode of exocytosis in resting and in stimulated peptidergic vesicles. Stimulation prolongs the effective opening of the fusion pore and expands its primary subnanometer diameter to enable hormone secretion without full fusion.
Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca 2؉ ] i also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca 2؉ ] i attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca 2؉ -dependent. This was confirmed by patchclamp studies on HEK-293T cells transfected with P2X 3 receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca 2؉ . Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.Many recent studies demonstrate that astrocytes play a significant modulatory role in synaptic physiology (1-4). Astrocytes respond to neurotransmitters, integrate different inputs, and signal back to neurons or forward information to neighboring or more distant astrocytes (2). In response to stimulation they release several chemical substances (5-7), termed gliotransmitters, which can interfere with the neuronal communicating pathways (8 -10).One major extracellular messenger important for coordinating the function of astrocytes, as well as for the cross-talk between them and other cell types, is ATP (11). Whereas several lines of evidence support the idea of ATP release from astrocytes (12-14), the release mechanisms are not completely understood. Some studies have described a connexin hemichannel-mediated release (12, 15, 16) in both resting and activated conditions. Other possible mechanisms, like volume-regulated anion channels (17, 18) and ATP-binding cassette transporters (multidrug resistance P-glycoprotein (19), or cystic fibrosis transmembrane conductance regulator (20) have also been reported. On the contrary, only few studies have focused on the possibility of exocytotic, vesicular ATP release mechanism operating in astrocytes (13, 14), even though it has been shown that astrocytes express the element...
Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.
PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.
Astrocytes, a subtype of glial cells, have numerous characteristics that were previously considered exclusive for neurons. One of these characteristics is a cytosolic [Ca2+] oscillation that controls the release of the chemical transmitter glutamate and atrial natriuretic peptide. These chemical messengers appear to be released from astrocytes via Ca(2+)-dependent exocytosis. In the present study, patch-clamp membrane capacitance measurements were used to monitor changes in the membrane area of a single astrocyte, while the photolysis of caged calcium compounds by a UV flash was used to elicit steps in [Ca2+]i to determine the exocytotic properties of astrocytes. Experiments show that astrocytes exhibit Ca(2+)-dependent increases in membrane capacitance, with an apparent Kd value of approximately 20 microM [Ca2+]i. The delay between the flash delivery and the peak rate in membrane capacitance increase is in the range of tens to hundreds of milliseconds. The pretreatment of astrocytes by the tetanus neurotoxin, which specifically cleaves the neuronal/neuroendocrine type of SNARE protein synaptobrevin, abolished flash-induced membrane capacitance increases, suggesting that Ca(2+)-dependent membrane capacitance changes involve tetanus neurotoxin-sensitive SNARE-mediated vesicular exocytosis. Immunocytochemical experiments show distinct populations of vesicles containing glutamate and atrial natriuretic peptide in astrocytes. We conclude that the recorded Ca(2+)-dependent changes in membrane capacitance represent regulated exocytosis from multiple types of vesicles, about 100 times slower than the exocytotic response in neurons.
SummarySynaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.
Hormones are released from cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. In stimulated exocytosis vesicle content is discharged swiftly. Although rapid vesicle discharge has also been proposed to mediate basal secretion, this has not been studied directly. We investigated basal hormone release by preloading fluorescent peptides into single vesicles. The hormone discharge, monitored with confocal microscopy, was compared with the simultaneous loading of vesicle by FM styryl dye. In stimulated vesicles FM 4-64 (4 microM), loading and hormone discharge occurs within seconds. In contrast, in approximately 50% of spontaneously releasing vesicles, the vesicle content discharge and the FM 4-64 loading were slow (approximately 3 min). These results show that in peptide secreting neuroendocrine cells the elementary vesicle content discharge differs in basal and in stimulated exocytosis. It is proposed that the view dating back for some decades, which is that, at rest, the vesicle discharge of hormones and neurotransmitters is similar to that occurring after stimulation, needs to be extended. In addition to the classical paradigm that secretory capacity of a cell is determined by controlling the probability of occurrence of elementary exocytotic events, one will have to consider activity modulation of elementary exocytotic events as well.
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