Synaptobrevin2-expressing vesicles in rat astrocytes: insights into molecular characterization, dynamics and exocytosis
1 Istituto di Neuroscienze CNR and Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Viale G. Colombo 3, 35121 Padova, Italy
2 International School For Advanced Studied (SISSA-ISAS), Neuroscience Program, Trieste, Italy
3 Dipartimento di Farmacologia Medica and Istituto di Neuroscienze CNR, Università di Milano, Via Vanvitelli 32, 20129 Milano, Italy
Abstract
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The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)L-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2–EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte.
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Introduction
Over the last decade, neurone–astrocyte reciprocal communication has emerged as a novel signalling pathway that plays distinct, multiple roles in brain function. Astrocytes can respond to the release of neurotransmitters as diverse as glutamate (Porter & McCarthy, 1996; Pasti et al. 1997), GABA (Kang et al. 1998), noradrenaline (Duffy & MacVicar, 1995; Kulik et al. 1999) and acetylcholine (Shelton & McCarthy, 2000; Araque et al. 2002) with transient, often repetitive, elevations in the concentration of intracellular Ca2+ ([Ca2+]i) which regulate the release from these cells of various neurone-active molecules, including classical transmitters (Parpura et al. 1994; Pasti et al. 1997; Bezzi et al. 1998; Kang et al. 1998). Among these, glutamate is the most extensively studied. Once released from activated astrocytes in hippocampal slices, glutamate has been shown to act on: (i) ionotropic glutamate receptors of interneurones to modulate their excitability and potentiate inhibitory transmission (Kang et al. 1998; Liu et al. 2004); (ii) presynaptic mGluR receptors to increase the probability of spontaneous glutamate release from axon terminals (Fiacco & McCarthy, 2004); and (iii) extrasynaptic NMDA receptors to promote synchronous activity in CA1 pyramidal neurones (Fellin et al. 2004).
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Various mechanisms have been proposed to explain glutamate release from astrocytes including reverse operation of glutamate transporters (Szatkowski et al. 1990; Attwell et al. 1993) and opening of large conductance channels in the astrocytic membrane, i.e. volume-operated channels (Kimelberg et al. 1990; Kimelberg & Mongin, 1998; Nedergaard et al. 2002), hemichannels (Ye et al. 2003) and purinergic (P2X7) receptors (Duan et al. 2003). While these different mechanisms of release may coexist and be preferentially operative under different physiological and pathological conditions, a number of studies provides evidence that glutamate can be released from astrocytes also through a process that (i) depends on Ca2+ (Parpura et al. 1995a,b; Jeftinija et al. 1997; Bezzi et al. 1998), (ii) depends on the same set of proteins that regulate neuronal exocytosis (Jeftinija et al. 1997; Calegari et al. 1999; Araque et al. 2000; Pasti et al. 2001) although differences may exist (Kreft et al. 2004; Zhang et al. 2004a). (iii) is sensitive to bafilomycin A1, an inhibitor of the vesicle ATPase the activity of which allows glutamate loading onto vesicles (Araque et al. 2000; Pasti et al. 2001; Montana et al. 2004), (iv) evokes quantal-like events in glutamate biosensor cells (Pasti et al. 2001), and (v) results in an increase in membrane capacitance (Kreft et al. 2004; Zhang et al. 2004b). All these findings point to a vesicle-mediated mechanism similar to exocytosis in neuronal cells. Furthermore, by means of total internal reflection fluorescence microscopy, a recent study in cultured astrocytes demonstrated that vesicles tagged with a vesicular glutamate transporter–EGFP (enhanced green fluorescent protein) chimera fuse with the membrane upon stimuli that trigger [Ca2+]i elevations in transfected astrocytes and deliver the transmitter glutamate (Bezzi et al. 2004).
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The aim of this study was to provide further insights into the molecular features and dynamics of astrocytic vesicles. We found that synaptobrevin2/VAMP2 (vesicle-associated membrane protein) (Syb2)-positive vesicles are clear organelles quite heterogeneous in size, containing, beside the v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) Syb2, Syb3, synaptotagmin IV, the vesicular glutamate transporter v-Glut-2, the AMPA receptor subunits GluR2,3 and the microtubule-dependent motor kinesin. By time lapse confocal microscopy we also found that vesicles tagged with a Syb2–EGFP chimera are highly mobile and fuse with the membrane upon stimuli that trigger Ca2+-dependent release of glutamate.
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Methods
Cell culture
All experimental procedures were in strict accordance with the Italian and EU regulation on animal welfare and were authorized by the Italian Ministry of Health. Neonatal Wistar rats were deeply anaesthetized by a cocktail of xilazina, tiletamina and zolazepan (10 mg/kg) and then killed by cervical dislocation.
For the immunocytochemistry and the immunoisolation of vesicles, hippocampal mixed glia cultures were obtained from embryonic rat pups (E18) using previously described methods (Calegari et al. 1999). Briefly, after dissection, the hippocampi were dissociated by treatment with trypsin (0.25% for 15 min at 37°C), followed by fragmentation with a fire-polished Pasteur pipette. Dissociated cells were plated on either glass coverslips or tissue culture dishes at a density of 0.5 x 106 cells ml–1 of glial medium: minimal essential medium (Invitrogen) supplemented with 20% FCS (fetal calf serum) (Euroclone Ltd, UK) and glucose at a final concentration of 5.5 g l–1. To obtain a pure astrocyte monolayer, any microglial cells were harvested by shaking 3-week-old cultures.
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For studies of vesicle dynamics and exocytosis, and for the monitoring of [Ca2+]i changes, primary cultures of cortical or hippocampal astrocytes were prepared from neonatal Wistar rats as previously described (Pasti et al. 1995). For purification of the astrocyte culture, 14 days after plating, cells were subjected to 12 h of continuous shaking, washed to remove floating microglia and dead cells, and then incubated for 5 min with 0.25% trypsin. Detached cells were then collected and replated on 24 mm coverslips. In the experiments performed, 10–14 day primary astrocytes or 7–10 day secondary astrocytes were used, as indicated for each experiment. No significant difference was observed in the response to stimuli between hippocampal and cortical astrocytes, or between primary or secondary cultures.
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Transfection
For the immunocytochemistry and the immunoisolation of vesicles, astrocytes were trypsinized 1 day before transfection. Cells were transfected using Fugene reagent (Roche, Milano, Italy) according to the manufacturer's instructions and monitored 2–3 days after.
For the study of vesicle dynamics and [Ca2+]i changes, astrocytes were transfected with the cDNA encoding Syb2–EGFP (Syb2 cDNA cloned into the multiple cloning site of the pEGFP-N1 expression vector from Clontech) using a modification of the calcium phosphate precipitation technique (Xia et al. 1996). Briefly, conditioned medium was removed from cultured astrocytes on 24 mm coverslips and replaced with 1.5 ml transfection medium (Hepes-buffered MEM medium, with Hanks' salts) at pH 7.85. Then 150 μl of a DNA–Ca2+ phosphate buffer mixture containing 10 μg DNA was added to each coverslip and incubated for 30–45 min at 37°C. After two washes with the transfection medium, this medium was replaced with the original, conditioned medium. Thereafter, astrocytes were maintained at 37°C in 5% CO2 and used for experiments 36–48 h after transfection.
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Subcellular fractionation and immunoblotting
Astrocytes were grown on Petri dishes until near confluence, harvested by scraping, washed and resuspended in homogenization buffer (10 mM Hepes-KOH pH 7.4, 250 mM sucrose, 1 mM magnesium acetate, protease inhibitor cocktail (1:1000 concentration, P8340 Sigma)). The cells were homogenized using a cell cracker (European Molecular Biology Laboratory, Heidelberg, Germany) and centrifuged at 1000 g for 10 min, to prepare the postnuclear supernatant. This supernatant was loaded on a 0.4–1.8 M continuous sucrose gradient and spun in a SW41 rotor (Beckman Instruments, Inc.) at 80.000 g for 18 h. Fractions of 1 ml were collected and analysed by SDS-PAGE followed by Western blotting as previously described (Calegari et al. 1999; Coco et al. 2003). Briefly, after electrophoresis, the proteins were transferred to nitrocellulose filters which, after being incubated in blocking buffer (5% milk, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl) were labelled with primary antibodies followed by appropriate secondary antibodies, conjugated to peroxidase, and diluted in blocking buffer containing 0.1–0.3% Tween 20. After extensive washes, the immunodecoration pattern was revealed using an enhanced chemiluminescence system (SuperSignal from Pierce, Rockford, IL, USA) following the manufacturer's protocol.
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Immunoisolation of vesicles and electron microscopy
Syb2-positive vesicles were immunoisolated from a postnuclear supernatant of cultured hippocampal astrocytes with Dynabeads M-450 (Dynal, Oslo, Norway) rat anti-mouse IgG1, according to the manufacturer's instructions. Bound vesicles were either further analysed by SDS-PAGE and Western blotting or processed for electron microscopy. For the latter, the beads were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, overnight at 4°C and after several washes in cacodylate buffer were postfixed with 2% OsO4 for 2 h. Samples were dehydrated in a graded ethanol series and embedded in EPON (Fluka) epoxy resin. Sections were cut on a Reichert Ultracut E microtome, counterstained and examined with a Philips CM10 electron microscope. Vesicle diameters were evaluated on images at 52 500 x final magnification (number of analysed vesicles = 300).
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Vesicular (3H)L-glutamate transport assay
Subcellular fractionation of cultured hippocampal astrocytes was performed on a continuous sucrose gradient as described above. Fractions enriched in synaptobrevin (fractions 11–14, ranging from 0.6 to 1.1 M sucrose) or secretogranin (fractions 15–17, ranging from 1.2 to 1.5 M sucrose) and control fractions (collected at the bottom of the gradient, fractions 21–22, ranging from 1.7 to 1.8 M sucrose) were pooled, diluted in homogenization buffer and spun at 165 000 g for 2 h. A crude fraction of synaptic vesicles (LP2) was prepared from rat brain as previously described (Huttner et al. 1983). The pellet was resuspended in 320 mM sucrose, 10 mM Tris-HCl, pH 7.4, to a concentration of 1 mg ml–1. The uptake assay was performed as previously described (Varoqui et al. 2002). Briefly, 100 μl aliquots of LP2, synaptobrevin or dense core granule-enriched membranes containing 100 μg of proteins were mixed with 50 μl of a buffer containing 110 mM potassium tartrate, 20 mM Hepes pH 7.4 (buffer A) and incubated at 32°C for 2 min in the presence or absence of 1 μM Bafilomycin A1. After preincubation, a solution (50 μl) containing 20 mM Mg2+-ATP, 200 μM glutamate, 2 μCi of (3H)L-glutamate (NET 490, 40–80 Ci mmol–1, PerkinElmer Life Science Inc., Boston, MA, USA) and plus or minus Bafilomycin A1 (1 μM) was added to the samples. After 5 min at 32°C, the uptake reaction was stopped by placing the tubes in ice and the samples were rapidly vacuum filtered through fibre filters GF/F (Whatman, Biomap, Milano, Italy) and washed with 15 ml of buffer A containing 10 mM Mg2SO4. Radioactivity bound to the filters was counted using the Filter-Count cocktail (PerkinElmer). Experiments were done in duplicate and data were expressed as pmol of glutamate (mg of proteins)–1. Experimental values (pmol (mg of protein)–1) were obtained upon subtraction of background radioactivity, calculated from control fractions (21–22), either in the presence or in the absence of Bafilomycin A1.
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Confocal microscopy
The 2nd day after transfection, astrocyte coverslips were mounted in an apposite cell chamber, containing 1 ml of extracellular solution maintained at 37°C and consisting of (mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 1 Na3PO4, 5.5 glucose and 20 Hepes, at pH 7.4 with NaOH. Cells were observed in either a Nikon inverted microscope (Eclipse TE200), equipped with a 100 x oil immersion objective and connected with a Perkin Elmer confocal system, or on a Leica confocal microscope (TCS SP2 RS AOBS) equipped with a 63 x immersion objective.
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Vesicle analysis
Cytoplasmic regions of monitored astrocytes were analysed off-line for vesicle number and tracking with the public domain ImageJ program (developed at the United States National Institutes of Health by W. Rasband and available on the internet at http://rsb.info.nih.gov/ij/). A threshold of 85% of maximal fluorescence intensity was applied. The number of vesicles was determined assuming that a vesicle is comprised of between 5 and 50 pixels. Astrocytes showing clear punctuate fluorescence were selected and monitored for a control period of 10–30 min. During this period, to verify the possible variability in vesicle number, Syb2–EGFP vesicles were estimated from images that were acquired in two to six time-lapse series of images of 30 s duration (exposure time, 300–800 ms). To measure mobility, we analysed vesicles composed of 5–20 pixels (which represented 86.3 ± 0.6% of the total number of vesicles in our sample). From the sequence of images, coordinates of individual vesicles at a given time were used to construct a vector tracing of the vesicle path. The pixel with maximal fluorescence intensity was used to determine the coordinates of each vesicle. The vector was also used to measure the maximal displacement for each vesicle (Wacker et al. 1997). The maximal displacement was calculated in all vesicles by measuring the distance between the two most distant points in the vector tracing. In all vesicles the maximal displacement was calculated over a fixed period of 6 s. When vesicles remained in focus for periods as long as 10–20 s (this occurred more frequently for non-directional vesicles), the maximal displacement was calculated as a mean of the values calculated for two or more subsequent 6-s periods. The velocity of each Syb2–EGFP vesicle was calculated by determining the travel distance of individual vesicles per second over an observation period of at least 6 s. To stimulate glutamate release from astrocytes, 3 nM-latrotoxin, 100 μML-quisqualate or 10 μM ionomycin were added to the extracellular solution in the cell chamber, and time-lapse series were acquired at intervals of 2–5 min, beginning at 30 s after stimulus addition. Only astrocytes that displayed stable values in vesicle number during resting conditions were considered. To verify the Ca2+ dependency of this release, 100 μML-quisqualate was applied in astrocytes preincubated for 45 min with 20 μM BAPTA-1 AM (Molecular Probes).
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Statistical analysis was performed using Student's t test. Unless specified, values are expressed as mean ±S.E.M.
To load vesicles with acridine orange (AO), astrocytes were incubated with 5 μM AO (Molecular Probes) for 15 min at room temperature in the dark. After extensive washing astrocytes were monitored with a TCS SP2 RS AOBS Leica confocal microscope. Counting of AO-loaded vesicles was performed by using the same approach described above for counting of Syb2–EGFP vesicles. Confocal planes of AO-positive vesicles were obtained using two channels: one channel collected red-emitting vesicles (at a window of 600–680 nm), while the other channel collected green-emitting ones (at a window of 500–560 nm). These images were pseudo-coloured red and green, respectively, and an overlay generated. A colour-dependent threshold was carried out using the Plugin ‘Threshold Colour’ (by G. Landini and B. Dougherty; available through the same URL as above), to specifically select yellow vesicles.
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Enzymatic assay of glutamate release
The release of glutamate from cultured astrocytes was monitored using an enzymatic assay. A 2 ml cuvette, containing extracellular solution at 37°C under continuous stirring, was lodged inside a Perkin-Elmer LS50B computerized spectrofluorometer. After glutamate dehydrogenase (GDH, 40 U ml–1, Sigma G4387), 1 mM NADP+ and the release-inducing stimulus (3 nM-LTX, 10 μM ionomycin or 100 μML-quisqualate) were added to the solution, this solution was applied to astrocyte cultures maintained at 37°C, for a period depending on the type of stimulus. The solution was then re-collected in the cuvette. Glutamate released from astrocytes was oxidized by GDH to -ketoglutarate, with consequent formation of NADPH, and NADPH fluorescence emission at 430 nm was measured through the spectrofluorometer. Release was quantified referring to standard curves constructed with known amounts of exogenous glutamate and normalized for the protein content of each sample (calculated with Bradford's method).
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Calcium imaging
Digital fluorescence microscopy (Nikon, RCM8000) was used for monitoring the change in indo-1 emission after cell loading with indo-1 AM (Molecular Probes) as previously described (Pasti et al. 1997). After excitation at 351 nm, the emitted light was separated into its two components (405 and 485 nm), and the ratio (405/485) was displayed as a pseudocolour scale. During experiments, cultured cells were continuously perfused (1.5–3 ml min–1) at room temperature with an extracellular solution consisting of (mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 1 Na3PO4, 5.5 glucose and 20 Hepes, pH 7.4, with NaOH. Sampling rate was 2 s, and 16 images were averaged for each frame.
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Chemicals
Antibodies against rat segretograninII were raised in rabbits, purified by affinity chromatography, and characterized as previously described (Calegari et al. 1999). Monoclonal antibodies against Syb2 and polyclonal antibodies directed against vGlut2 were from Synaptic System GbmH (Goettingen, Germany). Antibodies against synaptotagmin IV, kinesin and rab7 were a kind gift of Mitsunori Fukuda (RIKEN, Wako City, Japan), Francesca Navone (Milano, Italy) and Reinhard Jahn (MPI, Goettingen, Germany), respectively. Antibodies against VAMP7/TI-VAMP, SNAP-23 and cellubrevin/Syb3 were a kind gift of Thierry Galli (INSERM, Paris). Polyclonal antibodies against GluR1 and GluR2/3 were purchased from Chemicon (Temecula, CA, USA). Secondary antibodies conjugated to fluorescein isothiocyanate, Texas Red or horseradish peroxidase were obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA).
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Results
Electron microscopy characterization of Syb2-expressing vesicles
Syb2-positive vesicles are known to be present in astrocytes, though their dynamics and secretory content remains largely unknown (Parpura et al. 1995a; Jeftinija et al. 1997; Maienschein et al. 1999). To further characterize this vesicle population we performed subcellular fractionation of cultured hippocampal astrocytes on a continuous sucrose gradient. As previously reported (Coco et al. 2003), fractions 9–16 are enriched in Syb2 (Fig. 1A). These fractions, which appear to be partly distinct from fractions 10–22 containing the marker for large dense core vesicles secretogranin II (SgII) peaking in fractions 11–19 (Coco et al. 2003), are also enriched in the ubiquitous SNARE (cellubrevin/Syb3) (Fig. 1B). A very small amount of SgII is also detected at the top of the gradient (fractions 1–3, corresponding to the loading region) and most probably represent SgII released from granules damaged during the homogenization procedures. To test whether Syb2-positive vesicles contain and release glutamate following appropriate stimuli, fractions enriched in Syb2 (fractions 11–14; 0.6–1.1 M sucrose) were pooled and vesicles were pelleted by centrifugation. The obtained vesicle fraction was assayed for (3H)L-glutamate transport. Although less efficiently than synaptic vesicles, astrocytic vesicles were able to actively uptake glutamate (Fig. 1C). The neurotransmitter was specifically transported into this vesicle fraction since there was no glutamate uptake above background in a fraction corresponding to dense core granules (Fig. 1C, fractions 15–17; 1.2–1.5 M sucrose). Furthermore the uptake of glutamate could be specifically reduced by the inhibitor of the vesicular H+-ATPase bafilomycin A1 (pM (mg of protein)–1: 49.3 ± 2.18, control; 5.97 ± 0.35, bafilomycin A1; P < 0.01). Residual glutamate uptake, detected in the presence of bafilomycin A1, was comparable to that obtained in synaptic vesicles upon bafilomycin treatment (Fig. 1C).
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A and B, sucrose equilibrium gradient analysis of relative densities of the vesicles containing Syb2, Syb3 and SgII in astrocytes. The post-nuclear supernatant from astrocyte cultures was centrifuged through a 0.4–1.8 M sucrose gradient. Twenty two fractions were collected and pelleted in 80% acetone at –20°C. Aliquots of each fraction were analysed by immunoblotting using antibodies against Syb2, cellubrevin/Syb3 and SgII. C, quantitative analysis of (3H)L-glutamate uptake in Syb2-enriched fractions (11–14) of astrocytic vesicles (n= 3) and crude synaptic vesicles (n= 3) (white and grey bars, respectively). In the presence of 1 μM bafilomycin A1 applied 2 min before measurements, the uptake of glutamate was significantly reduced to levels comparable in astrocytic (n= 2) and synaptic (n= 2) vesicles. P < 0.01. (3H)L-glutamate uptake was undetectable in a fraction corresponding to dense core granules (fractions 15–17).
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To perform a detailed characterization of this vesicle population, Syb2-positive vesicles were immunoisolated from a low speed supernatant (LSS) prepared from cultured hippocampal astrocytes (Fig. 2A). Electron microscopy of magnetic beads, carrying astrocytic vesicles bound to anti-Syb2 antibodies, revealed that these vesicles were of variable size ranging from 30 nm to above 110 nm (total number of vesicles examined: 300; Fig. 2B). The majority of these vesicles had diameters of 30–80 nm. Astrocytic, Syb2-positive vesicles appear thus more heterogeneous in size compared with small, clear synaptic vesicles (40–60 nm diameter) (De Camilli & Navone, 1987). Only sporadically, anti-Syb2-coated beads carried, in addition to clear vesicles, dense core vesicles that corresponded to 2.34% of the total number of vesicles examined (n= 513).
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A, electron microscopy images showing anti-Syb2 antibody-coated beads with bound astrocytic vesicles. Clear vesicles, heterogeneous in size represent the large majority of Syb2-positive organelles. Dense core vesicles were also sporadically observed. The highest extent of membrane contamination in Syb2 antibody-coated beads is shown in the bottom panel. Calibration bars, 140 nm. B, histogram showing the size distribution (percentage of total) of clear vesicles bound to Syb2 antibody-coated beads (n= 300). The actual number of vesicles analysed is reported within the bars. C, Western blot analysis of low speed supernatant (LSS) and immunoisolated organelles (IP vesicles) for Syb2 (VAMP2), cellubrevin/Syb3 (VAMP3), TI-VAMP/VAMP7 (VAMP7), v-Glut-2, rab7, kinesin (u-kin), the AMPA receptor subunits GluR1,2,3, Syt IV and the t-SNARE SNAP-23. D, Western blot analysis of brain synaptosomes, astrocyte homogenates, low speed supernatant (LSS) and immunoisolated organelles (IP vesicles) for Syt I. Note that Syt I immunoreactivity is detectable in synaptosomes but not astrocytes.
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To characterize their molecular composition, immunoisolated, Syb2-positive astrocytic vesicles were analysed by SDS PAGE and Western blotting. Syb2-positive vesicles were found to lack the vSNARE TI-VAMP/VAMP7 (Galli et al. 1998), but contained the ubiquitous v-SNARE cellubrevin/Syb3. Rab7, a marker for late endosomes/lysosomes, was totally absent from the immunoisolated vesicles, further confirming the specificity of the procedure. The ubiquitous form of kinesin was associated, although not enriched, with immunoisolated vesicles, thus suggesting that the isolated particles possess molecular motors specific for microtubule-based movement. In accordance with the ability of the vesicles to internalize glutamate, Syb2-positive vesicles were immunoreactive for the vesicular glutamate transporter v-Glut-2, which co-enriched together with Syb2 from the LSS. Furthermore, Syb2-positive vesicles were immunopositive for the AMPA receptor subunits GluR2,3 and, although to a lesser extent, for GluR1 (Fig. 2C). We did not detect synaptophysin on isolated vesicles (not shown). Furthermore, although clearly detectable in brain synaptosomes, synaptotagmin I immunoreactivity was absent from astrocyte homogenates and immunoisolated vesicles (Fig. 2D). Syb2-positive vesicles were instead immunoreactive for synaptotagmin IV. Finally, differently from neuronal synaptic vesicles which contain major pools of the t-SNARE SNAP-25 recycling from the plasma membrane (Walch-Solimena et al. 1995), they did not contain detectable amount of the t-SNARE SNAP-23 (Fig. 2C).
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Astrocytic Syb2–EGFP vesicles are highly mobile
confocal microscopy imaging of astrocytes transfected with the Syb2–EGFP construct a punctate pattern of fluorescence is evident throughout the cytoplasm of soma and processes (Fig. 3A).
A, confocal image of a Syb2–EGFP transfected astrocyte displaying a punctate pattern of fluorescence. Due to scattering of the fluorescent signal the size of the vesicles seem larger than that of Syb2-immunoisolated vesicles as measured by electron microscopy. Scale bar, 5 μm. B, the bar histogram shows the distribution of the velocities of non-directional (white bars) and directional (grey bars) Syb2–EGFP vesicles. C, vector tracings report the path of several vesicles. Examples of the non-directional (left) and directional (right) types of motion are illustrated. The value of maximal displacement for each vesicle is reported (±S.E.M., in the case of multiple measurements). The number on the path of the vesicle corresponds to the number that marks the same vesicle path in the Movie 1b in Supplemental material. Time frame, 1 s. D, sequence of imaging illustrating the movement of a vesicle along an astrocyte process towards the growth cone. The arrows in each frame indicate the position of the mobile vesicle. Time frame, 1 s; scale bar, 3 μm.
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Time-lapse confocal microscopy imaging revealed that, while a majority of vesicles are immobile, a subpopulation of Syb2–EGFP vesicles, corresponding to about 25% of the total number of vesicles, shows different types of motion (see Supplementary material, Movie 1a). Vesicles move at a mean velocity of 0.65 ± 0.04 μm s–1 (n= 81). The bar histogram that reports the velocity of each vesicle motion shows, however, a clear bimodal distribution suggesting the presence of two distinct populations of vesicles that move at different velocities (Fig. 3B). By measuring the maximal displacement for individual vesicles, two types of motion were also distinguishable. Vesicles with maximal displacement below and above 1 μm were classified as short-range, non-directional and long-range, directional vesicles, respectively (Potokar et al. 2005) (Supplementary material, Movie 1a). The motion of these latter vesicles involves occasionally rapid changes, or even reversals in direction as well as pauses. The path travelled by some vesicles through the two types of motion is reported in Fig. 3C (the path of some of these vesicles, labelled by various colours, is presented in Supplementary material, Movie 1b). The mean values of the maximal displacement of non-directional (0.6 ± 0.1 μm, n= 33) and directional (3.5 ± 0.4 μm, n= 48) vesicles was significantly different (P < 0.001). It appears that non-directional vesicles move preferentially at slow velocity (below 0.4 μm s–1) while directional vesicles move preferentially at a velocity above 0.6 μm s–1 (Fig. 3B).
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In astrocytic processes, the long-range, directional motion is largely dominant while the non-directional type of motion is only occasionally observed. An example of a vesicle that moves rapidly towards the growth cone of an astrocytic process is reported in Fig. 3D (arrows) and dynamically illustrated in the Movie 2 (in Supplementary material). In the Movie 1b, an additional example of the movement of a vesicle (the path of this vesicle is shown also in Fig. 3C, track 3) along a process is marked as a coloured path. Once the growth cone is reached, vesicles stabilize their motion. Indeed, average velocity of vesicles in the growth cone is 0.096 ± 0.006 μm s–1 (n= 16).
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Ca2+-dependent membrane fusion of Syb2–EGFP vesicles
To investigate the Ca2+-dependent exocytosis of Syb2–EGFP vesicles, we first used the potent secretory stimulus ionomycin that triggers in cultured astrocytes a large increase in the concentration of intracellular Ca2+ ([Ca2+]i). By means of a fluorescence enzymatic assay we confirmed previous observations and demonstrated the ability of ionomycin to trigger a significant release of glutamate from astrocytes. Specifically, we found that 10 μM ionomycin induced the release of 49.5 ± 2.1 nmol glutamate (mg of protein)–1 (n= 3). After binarization of the image, the number of Syb2–EGFP vesicles in the cytoplasm of astrocytes transfected with our Syb2–EGFP construct was counted at a single focal plane from successive confocal microscope images taken at 1 Hz. In these transfected astrocytes ionomycin resulted in a significant reduction of fluorescent puncta (Fig. 4A and D). The decrease in vesicle number after ionomycin was not due to a movement of vesicles out of focus. In two astrocytes five images were taken at different focal planes, before and 5 min after ionomycin stimulation, and the number of vesicles in each plane was counted. The values of the relative reduction in vesicle number in the different images taken at the same focal plane after ionomycin were similar. Specifically, in the two astrocytes analysed, after ionomycin vesicles counted from the five images taken at distinct focal planes were reduced to 62.3 ± 6.1 and 72.9 ± 6.0% (mean ±S.D.) with respect to vesicles counted from images taken at the same focal planes before ionomycin. To ensure that the parameters under study maintained stable values, astrocytes were monitored over a period of at least 15 min before ionomycin application. During this period, the number of vesicles measured at a single focal plane in transfected astrocytes (n= 8) did not change significantly. In additional control experiments, we confirmed the stability of this value over a period of 30 min by monitoring astrocytes for 30 s every 5 min. Furthermore, in control astrocytes the number of vesicles measured in the stacked images over a period of 10 min (n= 1) and 15 min (n= 2) did not change significantly (relative change, mean ±S.D., astrocyte 1, –4.5 ± 2.9%; astrocyte 2, 2.2 ± 9.9%; astrocyte 3, –3.8 ± 5.2%). In the example illustrated in Fig. 4, we counted a mean number of 42 ± 1 vesicles in 30 images (taken at 1 frame s–1) before ionomycin and 17 ± 0.6 vesicles in the 60 images taken immediately after ionomycin application (Fig. 4A and B; see also Movie 3a and b). Upon ionomycin stimulation, several vesicles (arrowheads in Fig. 4A) are seen to be associated with the membrane. The movement of these vesicles in the region immediately beneath the astrocytic membrane and their association with the membrane is also dynamically illustrated in the Movie 3b. As a consequence of this event, the fluorescence associated with the membrane increases in parallel to a decrease in the number of vesicles in the cytoplasm (Fig. 4C). Probably due to the association of a large number of vesicles with the membrane, we could observe that after ionomycin stimulation, processes from some astrocytes, as in the example illustrated in Fig. 4, underwent a change in morphology. Results obtained from a total of eight experiments are summarized in Fig. 4D. The time course of ionomycin effect shows a certain degree of variability. In 6 of the 8 astrocytes examined it acted rapidly resulting in a significant decrease in vesicle number within 2 min, while in the two remaining astrocytes its effect was delayed, being observed between 3 and 5 min after its application. The average values of the reduction in vesicle number at different times after ionomycin application are reported in Fig. 4D.
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A, the two images show a transfected astrocyte at basal conditions (left panel) and 30 s after 10 μM ionomycin application (right panel). Stimulation causes a prompt reduction in the number of visible vesicles in the cytoplasm and a parallel increase in the fluorescence associated with the plasma membrane (arrowheads). B, time course of the vesicle number in the astrocyte shown in A, before (35 images; 1 s–1) and after ionomycin (60 images, starting 30 s after stimulation). C, bar graph showing the mean reduction in vesicle number in the cytoplasm (white bars) and the mean increase in the fluorescence of the plasma membrane (grey bars), as expressed in arbitrary units at basal conditions and after ionomycin stimulation. In this and in the following figures, P < 0.001. D, bar graph showing the average reduction in vesicle number with respect to control values occurring within 2 and 5 min of ionomycin application. Each bar reports the number of performed experiments. In this and in the following figures, P < 0.05.
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As an additional, powerful secretory stimulus for vesicle fusion we used -latrotoxin (-LTX). This toxin causes the release of neurotransmitters in neurones via both Ca2+-dependent and Ca2+-independent mechanisms (Davletov et al. 1998). As previously observed (Parpura et al. 1995b), the application of 3 nM-LTX triggered the release of glutamate from cultured astrocytes (34.8 nmol (mg of protein)–1). In transfected cells (n= 4), the number of Syb2–EGFP vesicles, following application of -LTX, was significantly reduced (average reduction, 52.2 ± 19.4% with respect to basal conditions, P < 0.05). All these observations are consistent with the view that astrocytes possess a regulated exocytosis of glutamate-containing vesicles.
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Additional experiments were then performed to investigate whether a more physiological activation of the [Ca2+]i response in astrocytes, such as the [Ca2+]i increase mediated by activation of mGluRs, can also trigger membrane fusion of Syb2–EGFP vesicles. We found that [Ca2+]i elevations in astrocytes mediated by the mGlu and AMPA receptor agonist L-quisqualate (100 μM), induced a glutamate release of 31 ± 2.4 nmol (mg of protein)–1 (n= 6) as well as a significant decrease in vesicle number. Figure 5 shows a Syb2–EGFP transfected astrocyte at basal conditions, and at 1 and 3 min after the application of L-quisqualate (Fig. 5A). In this astrocyte, at 1 and 3 min after stimulation the number of vesicles was reduced to 78.0 ± 2.5 and 58.0 ± 3.2%, respectively (Fig. 5B). From all the experiments, the average number of vesicles were significantly reduced at 3 min but not at 1 min after L-quisqualate stimulation (Fig. 5C). The change in morphology that we occasionally observed in the astrocyte upon ionomycin stimulation was never observed upon L-quisqualate stimulation. Taken together, these results demonstrate that stimuli that in astrocytes trigger both [Ca2+]i elevations and glutamate release also trigger the exocytosis of Syb2–EGFP vesicles.
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A, the three images show a transfected astrocyte at basal conditions (left panel), at 1 min (central panel) and at 3 min (right panel) after 100 μML-quisqualate application. The small decrease in vesicle number that can be observed at 1 min is significant at 3 min. Scale bar, 5 μm. B, time course of vesicle number as measured from each image (taken at 1 s intervals) of the astrocyte shown in A, before (19 images), 1 (20 images) and 3 min (19 images) after L-quisqualate. C, bar graph showing the average reduction in vesicle number with respect to control values occurring at 2 and 5 min after L-quisqualate application. Each bar reports the number of astrocytes analysed. D, bar graph showing the reduction in vesicle number at the level of cell body and processes following stimulation of astrocytes with either ionomycin (n= 4) or L-quisqualate (n= 3). The distinct values of vesicle number reduction at the cell body and processes are 63 ± 12% and 18 ± 14%, for ionomycin experiments, and 56 ± 9% and 39 ± 13%, for L-quisqualate experiments.
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In view of the possibility that glutamate release may preferentially occur at the level of astrocyte processes, we compared the reduction in the number of vesicles evoked by astrocyte stimulation at the processes and the soma. For this analysis we considered only cells (n= 7) in which the processes could be clearly visualized. The rate of vesicle fusion was found to be higher in the processes than in the cell body for each astrocyte analysed except one. As a mean, the reduction in the number of vesicles at the processes was significantly higher than that at the soma (Fig. 5D).
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After 45 min incubation of astrocytes with the calcium chelator BAPTA-1 AM (20 μM), L-quisqualate, which in control astrocytes caused a prompt [Ca2+]i increase, failed to trigger [Ca2+]i elevations (Fig. 6A and B). Under these conditions, L-quisqualate triggered a low glutamate release (7.8 ± 1.3 nmol (ng of protein)–1, n= 4; Fig. 6D). Accordingly, in all the transfected astrocytes examined (n= 10), the number of Syb2–EGFP vesicles was not significantly changed after L-quisqualate stimulation (98.5 ± 6.7%, Fig. 6E).
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A, sequence of pseudocolour images illustrating the [Ca2+]i change in Indo-1-loaded astrocytes upon 100 μM quisqualate stimulation. Astrocytes display a prompt [Ca2+]i elevation upon a first L-quisqualate stimulation (a and b), and failed to respond upon a second L-quisqualate challenge that was performed after BAPTA-1 AM incubation (c and d). Scale bar, 10 μm. B, time course of the [Ca2+]i changes in the astrocytes labelled 1 and 2. R corresponds to the ratio of the intensity of the light emitted by Indo-1 at the two wavelengths (405 and 485 nm). C, average [Ca2+]i changes following L-quisqualate application that was performed either before and after BAPTA-1 AM incubation. Each bar reports the number of astrocytes analysed in four different experiments. D, glutamate released from astrocytes as measured by a fluorimetric assay. After astrocyte incubation with BAPTA-1 AM, the release of glutamate triggered by L-quisqualate is drastically reduced. Each bar reports the number of experiments. E, bar graph showing the average number of vesicles in the cytoplasm of transfected astrocytes after L-quisqualate stimulation (n= 10) in control slices and in slices preincubated with BAPTA-1 AM (n= 10).
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These results indicate that Ca2+-dependent release of glutamate is accompanied by a disappearance of Syb2–EGFP vesicles from astrocyte processes, further supporting the idea that Syb2-positive vesicles are indeed the source of glutamate in astrocytes.
Staining with acridine orange (AO) has been used recently to tag vesicles in cultured astrocytes (Bezzi et al. 2004). Following accumulation into acidic compartments, such as vesicles, this dye changes its spectral properties and shifts its emission from green to red light. By combining this approach with the total internal reflection fluorescence microscope technique (TIRF), following astrocyte stimulation a subpopulation of AO-loaded vesicles have been observed to undergo rapid membrane fusion (Bezzi et al. 2004). We thus investigated whether and to what extent our Syb2–EGFP vesicles could be labelled by AO and whether their number in the cytoplasm is reduced rapidly upon L-quisqualate stimulation.
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We first found that the number of Syb2 vesicles in transfected astrocytes was significantly higher than that of vesicles that were loaded with AO in the same astrocytes (n= 14) and only part of these AO vesicles were Syb2-positive (36%, Fig. 7A). Figure 7B–D shows an example of these different populations of vesicles in a transfected astrocyte. What is noteworthy is that the mean number of AO vesicles in transfected cells was not significantly different from that of AO vesicles in non-transfected cells (87 ± 12, n= 14, versus 70 ± 5, n= 21).
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A, bar graph showing the mean number of the different subpopulations of vesicles. P < 0.0001. B, AO-labelled vesicles in a Syb2–EGFP transfected astrocyte. From this image 61 AO-loaded vesicles were counted Scale bar, 5.5 μm. C and D, higher magnifications of the boxed region in B showing the green (C, Syb2–EGFP-positive vesicles) image and the merge (D) of the green and red signals. Arrows indicate yellow vesicles double fluorescent for AO and Syb2–EGFP. The total number of double fluorescent vesicles in this astrocyte was 50, while that of Syb2–EGFP vesicles, measured before loading with AO, was 151. Scale bar, 1.5 μm.
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AO-labelled vesicles were then monitored in astrocytes after astrocyte stimulation with L-quisqualate. In these experiments, the number of AO-labelled vesicles was not significantly reduced 30 s after the onset of stimulation (93.4 ± 6%, n= 22).
Discussion
Over the last decade the ability of astrocytes to release glutamate through a Ca2+-dependent, vesicle-mediated mechanism has been the subject of intense debate. Only recent studies were able to provide direct evidence for the presence in astrocytes of a regulated exocytosis of glutamate-containing vesicles (Bezzi et al. 2004; Zhang et al. 2004b).
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In our study we demonstrated that subcellular fractions enriched in the SNARE protein Syb2 are able to specifically take up glutamate via a bafilomycin-sensitive mechanism. The expression of a functional glutamate transporter of the v-Glut family that we report here follows the immunocytochemical demonstration that v-Gluts are expressed in astrocytes in situ (Bezzi et al. 2004; Montana et al. 2004; Zhang et al. 2004b) and colocalized with glutamate in vesicles from cultured astrocytes (Anlauf & Derouiche, 2005). These results represent the first direct evidence that astrocytes possess a functional machinery devoted to glutamate loading into vesicular compartments containing Syb2.
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Immunoisolation of vesicles, endogenously expressing Syb2, using magnetic beads bound to specific antibodies allowed the molecular characterization of these organelles. The Western blot analysis of the Syb2 vesicles revealed the presence of the vesicular glutamate transporter v-Glut-2, thus indicating that Syb2 vesicles are endowed with the molecular machinery necessary for glutamate loading.
In line with the recent evidence that synaptotagmin IV, and not the neuronal isoform synaptotagmin I, regulates glial glutamate release (Zhang et al. 2004b), synaptotagmin IV was enriched in the Syb2-positive vesicle population immunoisolated from astrocytes. This result further supports its possible role as a Ca2+ sensor for gliotransmission. As an additional difference from neuronal vesicles, which contain SNAP-25 (Walch-Solimena et al. 1995), astrocytic vesicles do not contain detectable amounts of the non-neuronal homologue SNAP-23, nor do they contain synaptophysin. These subtle differences may reflect differences in the molecular mechanism at the basis of vesicle fusion in neurones and astrocytes.
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Interestingly, Syb2-positive vesicles appeared to be also positive for the ubiquitous SNARE cellubrevin/Syb3. Our data open up the possibility that a redundancy of v-SNAREs might occur in astrocytic vesicles, as previously described in other experimental models (Chilcote et al. 1995). Whereas the possibility that cellubrevin/Syb3 may support ‘per se’ regulated secretion could also be taken into consideration (Chilcote et al. 1995; Regazzi et al. 1996; Bezzi et al. 2004), the presence of Syb2 in v-Glut1-2-positive astrocytic vesicles provides a clear molecular basis for regulated exocytosis (Randhawa et al. 2000; Chen & Scheller, 2001; Jahn et al. 2003).
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Finally, we show here that Syb2-positive vesicles contain the AMPA receptor subunits GluR2,3 and, to a lesser extent, GluR1. This observation is in line with the recent demonstration that a major pool of presynaptic GluR2,3 and GluR1 subunits is associated with synaptic vesicle membranes and is recruited to the presynaptic membrane in response to depolarization (Schenk et al. 2003). The predominant presence of GluR2,3 subunits of AMPA receptors on Syb2-positive vesicles suggests the occurrence in astrocytes of a subunit-specific regulation of AMPA receptor delivery to the plasma membrane, with receptors cycling between intracellular pools and the membrane surface, as previously described in neurones (Passafaro et al. 2001; Shi et al. 2001; Schenk et al. 2003). Astrocytic glutamate receptors are involved, upon activation, in the regulation of proliferation and differentiation, as well as in the modulation of synaptic efficacy (reviewed in Gallo & Ghiani, 2000). Syb2-positive vesicles fusing with the astrocytic plasma membrane as a consequence of intracellular Ca2+ elevations may provide the pathway responsible for a regulated delivery of AMPA receptor subunits to the surface of astrocytic cells.
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Electron microscopy analysis of Syb2-positive vesicles revealed a heterogeneous size of these organelles. This feature adds to the demonstration that, whereas synaptic vesicles are organized in tight clusters inside the nerve terminal, astrocytic vesicles show a more diffuse distribution inside processes (Bezzi et al. 2004). The size heterogeneity of astrocytic vesicles that we report here appears to be in contrast with the recent demonstration that vesicles detected by electron microscopy in astrocytes, both in situ and in culture, are homogeneous in size (around 30 nm) (Bezzi et al. 2004). The use of different experimental approaches (electron microscopy of vesicles in astrocyte processes in situversus isolation of the whole population of vesicles endogenously expressing Syb2) may at least partially explain this discrepancy. Interestingly, the lack, in the Syb2-positive vesicles, of endosomal markers such as rab7, and of other v-SNAREs, such as TI-VAMP/VAMP7, indicate that vesicle heterogeneity is not due to a contamination of this fraction by other organelles.
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Our approach to target vesicles with the Syb2–EGFP chimera allows vesicle movement to be monitored readily with confocal microscopy. Using different stimuli, we provide indirect, though compelling, evidence that, upon [Ca2+]i elevations in transfected astrocytes, Syb2–EGFP vesicles fuse to the membrane and release glutamate. By time-lapse imaging we could also determine the route taken, the distance travelled and the velocity of individual Syb2–EGFP vesicles in living astrocytes. Results obtained revealed that the behaviour of Syb2–EGFP vesicles is similar to that of synaptic vesicles in neurones and secretory granules in neuroendocrine cells (Steyer et al. 1997; Pouli et al. 1998). We also found that Syb2–EGFP vesicles moved in a chaotic fashion while others moved back and forth along linear routes. These two distinct patterns of mobility in a similar percentage of vesicles have been previously observed in astrocytic vesicles tagged with a DNA construct encoding the proatrial natriuretic peptide fused with GFP (Potokar et al. 2005). Interestingly, vesicles in astrocytic processes moved almost exclusively along linear pathways and upon reaching the distal end become immobile. This linear motion and the expression in astrocytes of kinesin are consistent with a model of motor-assisted diffusion in which vesicles are transported along microtubules to distal portions of astrocytic processes until they reach a position that may be adjacent to release sites. The existence of potential release sites in the astrocytic membrane is also supported by a recent high-resolution immunofluorescence study (Anlauf & Derouiche, 2005). This study reveals that the vesicles immunopositive for either glutamate and/or v-Gluts are concentrated in processes and at distinct sections of the cell boundary. The higher reduction in vesicle number that we detected upon stimulation at the level of processes than at the cell body provides further support for the existence of specific sites where exocytosis may preferentially occur.
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After BAPTA-1 AM incubation, astrocyte stimulation with L-quisqualate failed to trigger in astrocytes the release of glutamate as well as the reduction in the number of vesicles otherwise observed in Syb2–EGFP transfected astrocytes, confirming the dependency of both glutamate release and vesicle fusion on [Ca2+]i elevations.
By using total internal reflection fluorescence (TIRF) microscopy and AO staining, the exocytosis of glutamate-containing vesicles has been recently demonstrated in cultured astrocytes (Bezzi et al. 2004). The great majority of vesicle fusion events was observed to occur within 150 ms after the local application of the mGluR agonist (S)-3,5-dihydroxyphenylglcyine (DHPG), whereas rare events were observed thereafter. Our results confirm the ability of astrocyte vesicles to fuse with the membrane upon stimuli that trigger [Ca2+]i elevations, although they fail to reveal a significant decrease in cytoplasmic Syb2–EGFP vesicles shortly after stimulation. This latter result does not necessarily mean that Syb2–EGFP vesicles cannot fuse rapidly with the membrane upon astrocyte stimulation. In previous experiments in cultured astrocytes we have already provided evidence that glutamate can be released rapidly from astrocytes through an exocytotic mechanism, and trigger fast NMDA receptor-mediated inward currents in glutamate sniffer cells (Pasti et al. 2001). By estimating fusion events indirectly from the counting of cytoplasmic vesicles, our experimental approach cannot allow us to detect the initial, rapid phase of exocytosis described by Bezzi et al. (2004). After fusion, Syb2–EGFP vesicles can rapidly re-enter into the pool of releasable vesicles in the cytoplasm. A reduction in the number of cytoplasmic vesicles can thus be detected only after a relatively long-lasting stimulation of the exocytosis that may change the equilibrium between exocytotic and endocytotic events or the kinetics of one or both events.
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On the other hand, in experiments based on AO-labelling of vesicles, a long-lasting exocytotic process may not be revealed. In these experiments, fusion events are detected as green fluorescent light flashes accompanied by the disappearance of red-labelled vesicles. As such, this experimental approach may not allow the following of repetitive episodes of fusion from the same vesicle and thus a prolonged process of continuous exocytosis that may ultimately lead to the reduction of cytoplasmic vesicles that we detected.
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The ability of astrocytes to support a long-lasting exocytosis of glutamate-containing vesicles was previously demonstrated. Indeed, repetitive episodes of fast activation of the NMDA receptor in glutamate sniffer cells, reflecting the exocytosis of glutamate-containing vesicles, were observed for several minutes after stimulation of astrocyte [Ca2+]i oscillations with L-quisqualate (Pasti et al. 2001).
Data reported here also demonstrate that subpopulations of Syb2–EGFP vesicles load AO differently. Indeed, the number of AO vesicles is consistently lower than that of Syb2–EGFP vesicles and only part of the AO vesicles are also Syb2–EGFP positive. This could be related to different stages of vesicle functional maturation. The low accumulation of AO in some Syb2–EGFP vesicles may also be due to a different pH in these vesicles as well as to other factors that are recognized to play important roles in AO accumulation, such as the total internal volume and the nature of the anions that follow the movements of the protons into the vesicle (Palmgren, 1991; Clerc & Barenholz, 1998).
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Given that Syb2–EGFP and AO vesicles represent different populations that overlap only partially, we investigated whether a decrease in the number of AO vesicles could be detected rapidly after stimulation. When L-quisqualate was applied to trigger exocytosis, the number of AO vesicles in the cytoplasm did not change significantly within 30 s of the onset of stimulation. These data are not necessarily in disagreement with those obtained in TIRF experiments that detected a rapid fusion of AO vesicles after astrocyte stimulation. In these experiments, only a subpopulation of total AO vesicles – those that are just beneath the plasma membrane – could be monitored, and only part (34%) of these vesicles undergo a rapid fusion after maximal stimulation of astrocytes (Bezzi et al. 2004). Thus, our results indirectly support the view that vesicles ready to fuse with the membrane represent a small subpopulation of vesicles and their disappearance from the cytoplasm could not result in a significant reduction in the total number of AO vesicles.
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The observations that vesicles in cultured astrocytes are highly mobile and fuse with the membrane upon stimuli that trigger [Ca2+]i elevations give the opportunity to explore in future experiments many aspects in the secretory behaviour of these cells at the cellular and subcellular level. Glutamate release from astrocytes activated by stimuli that evoke Ca2+ elevations in these cells is emerging as a functionally relevant signalling pathway that modulates neuronal transmission in the hippocampus. In this brain region astrocytic glutamate has been reported to act on (i) interneurones to potentiate inhibitory transmission (Kang et al. 1998; Liu et al. 2004), (ii) extrasynaptic mGluR receptors to increase the probability of spontaneous glutamate release from axon terminals (Fiacco & McCarthy, 2004), and (iii) extrasynaptic NMDA receptors to promote synchronized activity in CA1 pyramidal neurones (Fellin et al. 2004). The results that we report here, i.e. the molecular characterization of astrocyte vesicles, the evidence for the functional expression of a glutamate transporter of the v-Glut family and for the ability of these vesicles to fuse with the membrane, provide significant support for the hypothesis that a regulated, sustained release of glutamate-containing vesicles represents a functionally relevant signalling pathway at the basis of astrocyte-to-neurone communication.
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Supplemental material
The online version of this paper can be accessed at: 10.1113/jphysiol.2005.094052
http://jp.physoc.org/cgi/content/full/jphysiol.2005.094052/DC1
and contains supplemental material entitled: Vesicle Movement.
This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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Footnotes
D. Crippa and U. Schenk contributed equally to this work.
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2 International School For Advanced Studied (SISSA-ISAS), Neuroscience Program, Trieste, Italy
3 Dipartimento di Farmacologia Medica and Istituto di Neuroscienze CNR, Università di Milano, Via Vanvitelli 32, 20129 Milano, Italy
Abstract
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The SNARE-dependent exocytosis of glutamate-containing vesicles in astrocytes is increasingly viewed as an important signal at the basis of the astrocyte-to-neurone communication system in the brain. Here we provide further insights into the molecular features and dynamics of vesicles in cultured astrocytes. We found that immunoisolated synaptobrevin2 vesicles are clear vesicles quite heterogenous in size and contain the vesicular glutamate transporter v-Glut-2. Moreover, they are immunopositive for synaptotagmin IV, for AMPA receptor subunits GluR2,3 and, to a lesser extent, for GluR1. We also provide direct evidence for the functional expression of v-Glut-2 in astrocytes and demonstrate that synaptobrevin2-positive vesicles can specifically take up (3H)L-glutamate via a bafilomycin-sensitive mechanism. Finally, by time lapse confocal microscopy, we show that a subpopulation of vesicles (tagged with a synaptobrevin2–EGFP chimera) is highly mobile and can fuse with the plasma membrane, preferentially at the level of the astrocyte processes, in a Ca2+-dependent manner. These latter observations, together with the evidence reported here for the expression of functional v-Glut-2 in synaptobrevin2-positive vesicles, provide a molecular basis for regulated exocytosis in astrocyte.
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Introduction
Over the last decade, neurone–astrocyte reciprocal communication has emerged as a novel signalling pathway that plays distinct, multiple roles in brain function. Astrocytes can respond to the release of neurotransmitters as diverse as glutamate (Porter & McCarthy, 1996; Pasti et al. 1997), GABA (Kang et al. 1998), noradrenaline (Duffy & MacVicar, 1995; Kulik et al. 1999) and acetylcholine (Shelton & McCarthy, 2000; Araque et al. 2002) with transient, often repetitive, elevations in the concentration of intracellular Ca2+ ([Ca2+]i) which regulate the release from these cells of various neurone-active molecules, including classical transmitters (Parpura et al. 1994; Pasti et al. 1997; Bezzi et al. 1998; Kang et al. 1998). Among these, glutamate is the most extensively studied. Once released from activated astrocytes in hippocampal slices, glutamate has been shown to act on: (i) ionotropic glutamate receptors of interneurones to modulate their excitability and potentiate inhibitory transmission (Kang et al. 1998; Liu et al. 2004); (ii) presynaptic mGluR receptors to increase the probability of spontaneous glutamate release from axon terminals (Fiacco & McCarthy, 2004); and (iii) extrasynaptic NMDA receptors to promote synchronous activity in CA1 pyramidal neurones (Fellin et al. 2004).
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Various mechanisms have been proposed to explain glutamate release from astrocytes including reverse operation of glutamate transporters (Szatkowski et al. 1990; Attwell et al. 1993) and opening of large conductance channels in the astrocytic membrane, i.e. volume-operated channels (Kimelberg et al. 1990; Kimelberg & Mongin, 1998; Nedergaard et al. 2002), hemichannels (Ye et al. 2003) and purinergic (P2X7) receptors (Duan et al. 2003). While these different mechanisms of release may coexist and be preferentially operative under different physiological and pathological conditions, a number of studies provides evidence that glutamate can be released from astrocytes also through a process that (i) depends on Ca2+ (Parpura et al. 1995a,b; Jeftinija et al. 1997; Bezzi et al. 1998), (ii) depends on the same set of proteins that regulate neuronal exocytosis (Jeftinija et al. 1997; Calegari et al. 1999; Araque et al. 2000; Pasti et al. 2001) although differences may exist (Kreft et al. 2004; Zhang et al. 2004a). (iii) is sensitive to bafilomycin A1, an inhibitor of the vesicle ATPase the activity of which allows glutamate loading onto vesicles (Araque et al. 2000; Pasti et al. 2001; Montana et al. 2004), (iv) evokes quantal-like events in glutamate biosensor cells (Pasti et al. 2001), and (v) results in an increase in membrane capacitance (Kreft et al. 2004; Zhang et al. 2004b). All these findings point to a vesicle-mediated mechanism similar to exocytosis in neuronal cells. Furthermore, by means of total internal reflection fluorescence microscopy, a recent study in cultured astrocytes demonstrated that vesicles tagged with a vesicular glutamate transporter–EGFP (enhanced green fluorescent protein) chimera fuse with the membrane upon stimuli that trigger [Ca2+]i elevations in transfected astrocytes and deliver the transmitter glutamate (Bezzi et al. 2004).
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The aim of this study was to provide further insights into the molecular features and dynamics of astrocytic vesicles. We found that synaptobrevin2/VAMP2 (vesicle-associated membrane protein) (Syb2)-positive vesicles are clear organelles quite heterogeneous in size, containing, beside the v-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) Syb2, Syb3, synaptotagmin IV, the vesicular glutamate transporter v-Glut-2, the AMPA receptor subunits GluR2,3 and the microtubule-dependent motor kinesin. By time lapse confocal microscopy we also found that vesicles tagged with a Syb2–EGFP chimera are highly mobile and fuse with the membrane upon stimuli that trigger Ca2+-dependent release of glutamate.
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Methods
Cell culture
All experimental procedures were in strict accordance with the Italian and EU regulation on animal welfare and were authorized by the Italian Ministry of Health. Neonatal Wistar rats were deeply anaesthetized by a cocktail of xilazina, tiletamina and zolazepan (10 mg/kg) and then killed by cervical dislocation.
For the immunocytochemistry and the immunoisolation of vesicles, hippocampal mixed glia cultures were obtained from embryonic rat pups (E18) using previously described methods (Calegari et al. 1999). Briefly, after dissection, the hippocampi were dissociated by treatment with trypsin (0.25% for 15 min at 37°C), followed by fragmentation with a fire-polished Pasteur pipette. Dissociated cells were plated on either glass coverslips or tissue culture dishes at a density of 0.5 x 106 cells ml–1 of glial medium: minimal essential medium (Invitrogen) supplemented with 20% FCS (fetal calf serum) (Euroclone Ltd, UK) and glucose at a final concentration of 5.5 g l–1. To obtain a pure astrocyte monolayer, any microglial cells were harvested by shaking 3-week-old cultures.
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For studies of vesicle dynamics and exocytosis, and for the monitoring of [Ca2+]i changes, primary cultures of cortical or hippocampal astrocytes were prepared from neonatal Wistar rats as previously described (Pasti et al. 1995). For purification of the astrocyte culture, 14 days after plating, cells were subjected to 12 h of continuous shaking, washed to remove floating microglia and dead cells, and then incubated for 5 min with 0.25% trypsin. Detached cells were then collected and replated on 24 mm coverslips. In the experiments performed, 10–14 day primary astrocytes or 7–10 day secondary astrocytes were used, as indicated for each experiment. No significant difference was observed in the response to stimuli between hippocampal and cortical astrocytes, or between primary or secondary cultures.
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Transfection
For the immunocytochemistry and the immunoisolation of vesicles, astrocytes were trypsinized 1 day before transfection. Cells were transfected using Fugene reagent (Roche, Milano, Italy) according to the manufacturer's instructions and monitored 2–3 days after.
For the study of vesicle dynamics and [Ca2+]i changes, astrocytes were transfected with the cDNA encoding Syb2–EGFP (Syb2 cDNA cloned into the multiple cloning site of the pEGFP-N1 expression vector from Clontech) using a modification of the calcium phosphate precipitation technique (Xia et al. 1996). Briefly, conditioned medium was removed from cultured astrocytes on 24 mm coverslips and replaced with 1.5 ml transfection medium (Hepes-buffered MEM medium, with Hanks' salts) at pH 7.85. Then 150 μl of a DNA–Ca2+ phosphate buffer mixture containing 10 μg DNA was added to each coverslip and incubated for 30–45 min at 37°C. After two washes with the transfection medium, this medium was replaced with the original, conditioned medium. Thereafter, astrocytes were maintained at 37°C in 5% CO2 and used for experiments 36–48 h after transfection.
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Subcellular fractionation and immunoblotting
Astrocytes were grown on Petri dishes until near confluence, harvested by scraping, washed and resuspended in homogenization buffer (10 mM Hepes-KOH pH 7.4, 250 mM sucrose, 1 mM magnesium acetate, protease inhibitor cocktail (1:1000 concentration, P8340 Sigma)). The cells were homogenized using a cell cracker (European Molecular Biology Laboratory, Heidelberg, Germany) and centrifuged at 1000 g for 10 min, to prepare the postnuclear supernatant. This supernatant was loaded on a 0.4–1.8 M continuous sucrose gradient and spun in a SW41 rotor (Beckman Instruments, Inc.) at 80.000 g for 18 h. Fractions of 1 ml were collected and analysed by SDS-PAGE followed by Western blotting as previously described (Calegari et al. 1999; Coco et al. 2003). Briefly, after electrophoresis, the proteins were transferred to nitrocellulose filters which, after being incubated in blocking buffer (5% milk, 25 mM Tris-HCl, pH 7.5, 150 mM NaCl) were labelled with primary antibodies followed by appropriate secondary antibodies, conjugated to peroxidase, and diluted in blocking buffer containing 0.1–0.3% Tween 20. After extensive washes, the immunodecoration pattern was revealed using an enhanced chemiluminescence system (SuperSignal from Pierce, Rockford, IL, USA) following the manufacturer's protocol.
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Immunoisolation of vesicles and electron microscopy
Syb2-positive vesicles were immunoisolated from a postnuclear supernatant of cultured hippocampal astrocytes with Dynabeads M-450 (Dynal, Oslo, Norway) rat anti-mouse IgG1, according to the manufacturer's instructions. Bound vesicles were either further analysed by SDS-PAGE and Western blotting or processed for electron microscopy. For the latter, the beads were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, overnight at 4°C and after several washes in cacodylate buffer were postfixed with 2% OsO4 for 2 h. Samples were dehydrated in a graded ethanol series and embedded in EPON (Fluka) epoxy resin. Sections were cut on a Reichert Ultracut E microtome, counterstained and examined with a Philips CM10 electron microscope. Vesicle diameters were evaluated on images at 52 500 x final magnification (number of analysed vesicles = 300).
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Vesicular (3H)L-glutamate transport assay
Subcellular fractionation of cultured hippocampal astrocytes was performed on a continuous sucrose gradient as described above. Fractions enriched in synaptobrevin (fractions 11–14, ranging from 0.6 to 1.1 M sucrose) or secretogranin (fractions 15–17, ranging from 1.2 to 1.5 M sucrose) and control fractions (collected at the bottom of the gradient, fractions 21–22, ranging from 1.7 to 1.8 M sucrose) were pooled, diluted in homogenization buffer and spun at 165 000 g for 2 h. A crude fraction of synaptic vesicles (LP2) was prepared from rat brain as previously described (Huttner et al. 1983). The pellet was resuspended in 320 mM sucrose, 10 mM Tris-HCl, pH 7.4, to a concentration of 1 mg ml–1. The uptake assay was performed as previously described (Varoqui et al. 2002). Briefly, 100 μl aliquots of LP2, synaptobrevin or dense core granule-enriched membranes containing 100 μg of proteins were mixed with 50 μl of a buffer containing 110 mM potassium tartrate, 20 mM Hepes pH 7.4 (buffer A) and incubated at 32°C for 2 min in the presence or absence of 1 μM Bafilomycin A1. After preincubation, a solution (50 μl) containing 20 mM Mg2+-ATP, 200 μM glutamate, 2 μCi of (3H)L-glutamate (NET 490, 40–80 Ci mmol–1, PerkinElmer Life Science Inc., Boston, MA, USA) and plus or minus Bafilomycin A1 (1 μM) was added to the samples. After 5 min at 32°C, the uptake reaction was stopped by placing the tubes in ice and the samples were rapidly vacuum filtered through fibre filters GF/F (Whatman, Biomap, Milano, Italy) and washed with 15 ml of buffer A containing 10 mM Mg2SO4. Radioactivity bound to the filters was counted using the Filter-Count cocktail (PerkinElmer). Experiments were done in duplicate and data were expressed as pmol of glutamate (mg of proteins)–1. Experimental values (pmol (mg of protein)–1) were obtained upon subtraction of background radioactivity, calculated from control fractions (21–22), either in the presence or in the absence of Bafilomycin A1.
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Confocal microscopy
The 2nd day after transfection, astrocyte coverslips were mounted in an apposite cell chamber, containing 1 ml of extracellular solution maintained at 37°C and consisting of (mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 1 Na3PO4, 5.5 glucose and 20 Hepes, at pH 7.4 with NaOH. Cells were observed in either a Nikon inverted microscope (Eclipse TE200), equipped with a 100 x oil immersion objective and connected with a Perkin Elmer confocal system, or on a Leica confocal microscope (TCS SP2 RS AOBS) equipped with a 63 x immersion objective.
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Vesicle analysis
Cytoplasmic regions of monitored astrocytes were analysed off-line for vesicle number and tracking with the public domain ImageJ program (developed at the United States National Institutes of Health by W. Rasband and available on the internet at http://rsb.info.nih.gov/ij/). A threshold of 85% of maximal fluorescence intensity was applied. The number of vesicles was determined assuming that a vesicle is comprised of between 5 and 50 pixels. Astrocytes showing clear punctuate fluorescence were selected and monitored for a control period of 10–30 min. During this period, to verify the possible variability in vesicle number, Syb2–EGFP vesicles were estimated from images that were acquired in two to six time-lapse series of images of 30 s duration (exposure time, 300–800 ms). To measure mobility, we analysed vesicles composed of 5–20 pixels (which represented 86.3 ± 0.6% of the total number of vesicles in our sample). From the sequence of images, coordinates of individual vesicles at a given time were used to construct a vector tracing of the vesicle path. The pixel with maximal fluorescence intensity was used to determine the coordinates of each vesicle. The vector was also used to measure the maximal displacement for each vesicle (Wacker et al. 1997). The maximal displacement was calculated in all vesicles by measuring the distance between the two most distant points in the vector tracing. In all vesicles the maximal displacement was calculated over a fixed period of 6 s. When vesicles remained in focus for periods as long as 10–20 s (this occurred more frequently for non-directional vesicles), the maximal displacement was calculated as a mean of the values calculated for two or more subsequent 6-s periods. The velocity of each Syb2–EGFP vesicle was calculated by determining the travel distance of individual vesicles per second over an observation period of at least 6 s. To stimulate glutamate release from astrocytes, 3 nM-latrotoxin, 100 μML-quisqualate or 10 μM ionomycin were added to the extracellular solution in the cell chamber, and time-lapse series were acquired at intervals of 2–5 min, beginning at 30 s after stimulus addition. Only astrocytes that displayed stable values in vesicle number during resting conditions were considered. To verify the Ca2+ dependency of this release, 100 μML-quisqualate was applied in astrocytes preincubated for 45 min with 20 μM BAPTA-1 AM (Molecular Probes).
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Statistical analysis was performed using Student's t test. Unless specified, values are expressed as mean ±S.E.M.
To load vesicles with acridine orange (AO), astrocytes were incubated with 5 μM AO (Molecular Probes) for 15 min at room temperature in the dark. After extensive washing astrocytes were monitored with a TCS SP2 RS AOBS Leica confocal microscope. Counting of AO-loaded vesicles was performed by using the same approach described above for counting of Syb2–EGFP vesicles. Confocal planes of AO-positive vesicles were obtained using two channels: one channel collected red-emitting vesicles (at a window of 600–680 nm), while the other channel collected green-emitting ones (at a window of 500–560 nm). These images were pseudo-coloured red and green, respectively, and an overlay generated. A colour-dependent threshold was carried out using the Plugin ‘Threshold Colour’ (by G. Landini and B. Dougherty; available through the same URL as above), to specifically select yellow vesicles.
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Enzymatic assay of glutamate release
The release of glutamate from cultured astrocytes was monitored using an enzymatic assay. A 2 ml cuvette, containing extracellular solution at 37°C under continuous stirring, was lodged inside a Perkin-Elmer LS50B computerized spectrofluorometer. After glutamate dehydrogenase (GDH, 40 U ml–1, Sigma G4387), 1 mM NADP+ and the release-inducing stimulus (3 nM-LTX, 10 μM ionomycin or 100 μML-quisqualate) were added to the solution, this solution was applied to astrocyte cultures maintained at 37°C, for a period depending on the type of stimulus. The solution was then re-collected in the cuvette. Glutamate released from astrocytes was oxidized by GDH to -ketoglutarate, with consequent formation of NADPH, and NADPH fluorescence emission at 430 nm was measured through the spectrofluorometer. Release was quantified referring to standard curves constructed with known amounts of exogenous glutamate and normalized for the protein content of each sample (calculated with Bradford's method).
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Calcium imaging
Digital fluorescence microscopy (Nikon, RCM8000) was used for monitoring the change in indo-1 emission after cell loading with indo-1 AM (Molecular Probes) as previously described (Pasti et al. 1997). After excitation at 351 nm, the emitted light was separated into its two components (405 and 485 nm), and the ratio (405/485) was displayed as a pseudocolour scale. During experiments, cultured cells were continuously perfused (1.5–3 ml min–1) at room temperature with an extracellular solution consisting of (mM): 125 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 1 Na3PO4, 5.5 glucose and 20 Hepes, pH 7.4, with NaOH. Sampling rate was 2 s, and 16 images were averaged for each frame.
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Chemicals
Antibodies against rat segretograninII were raised in rabbits, purified by affinity chromatography, and characterized as previously described (Calegari et al. 1999). Monoclonal antibodies against Syb2 and polyclonal antibodies directed against vGlut2 were from Synaptic System GbmH (Goettingen, Germany). Antibodies against synaptotagmin IV, kinesin and rab7 were a kind gift of Mitsunori Fukuda (RIKEN, Wako City, Japan), Francesca Navone (Milano, Italy) and Reinhard Jahn (MPI, Goettingen, Germany), respectively. Antibodies against VAMP7/TI-VAMP, SNAP-23 and cellubrevin/Syb3 were a kind gift of Thierry Galli (INSERM, Paris). Polyclonal antibodies against GluR1 and GluR2/3 were purchased from Chemicon (Temecula, CA, USA). Secondary antibodies conjugated to fluorescein isothiocyanate, Texas Red or horseradish peroxidase were obtained from Jackson Immunoresearch Laboratories (West Grove, PA, USA).
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Results
Electron microscopy characterization of Syb2-expressing vesicles
Syb2-positive vesicles are known to be present in astrocytes, though their dynamics and secretory content remains largely unknown (Parpura et al. 1995a; Jeftinija et al. 1997; Maienschein et al. 1999). To further characterize this vesicle population we performed subcellular fractionation of cultured hippocampal astrocytes on a continuous sucrose gradient. As previously reported (Coco et al. 2003), fractions 9–16 are enriched in Syb2 (Fig. 1A). These fractions, which appear to be partly distinct from fractions 10–22 containing the marker for large dense core vesicles secretogranin II (SgII) peaking in fractions 11–19 (Coco et al. 2003), are also enriched in the ubiquitous SNARE (cellubrevin/Syb3) (Fig. 1B). A very small amount of SgII is also detected at the top of the gradient (fractions 1–3, corresponding to the loading region) and most probably represent SgII released from granules damaged during the homogenization procedures. To test whether Syb2-positive vesicles contain and release glutamate following appropriate stimuli, fractions enriched in Syb2 (fractions 11–14; 0.6–1.1 M sucrose) were pooled and vesicles were pelleted by centrifugation. The obtained vesicle fraction was assayed for (3H)L-glutamate transport. Although less efficiently than synaptic vesicles, astrocytic vesicles were able to actively uptake glutamate (Fig. 1C). The neurotransmitter was specifically transported into this vesicle fraction since there was no glutamate uptake above background in a fraction corresponding to dense core granules (Fig. 1C, fractions 15–17; 1.2–1.5 M sucrose). Furthermore the uptake of glutamate could be specifically reduced by the inhibitor of the vesicular H+-ATPase bafilomycin A1 (pM (mg of protein)–1: 49.3 ± 2.18, control; 5.97 ± 0.35, bafilomycin A1; P < 0.01). Residual glutamate uptake, detected in the presence of bafilomycin A1, was comparable to that obtained in synaptic vesicles upon bafilomycin treatment (Fig. 1C).
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A and B, sucrose equilibrium gradient analysis of relative densities of the vesicles containing Syb2, Syb3 and SgII in astrocytes. The post-nuclear supernatant from astrocyte cultures was centrifuged through a 0.4–1.8 M sucrose gradient. Twenty two fractions were collected and pelleted in 80% acetone at –20°C. Aliquots of each fraction were analysed by immunoblotting using antibodies against Syb2, cellubrevin/Syb3 and SgII. C, quantitative analysis of (3H)L-glutamate uptake in Syb2-enriched fractions (11–14) of astrocytic vesicles (n= 3) and crude synaptic vesicles (n= 3) (white and grey bars, respectively). In the presence of 1 μM bafilomycin A1 applied 2 min before measurements, the uptake of glutamate was significantly reduced to levels comparable in astrocytic (n= 2) and synaptic (n= 2) vesicles. P < 0.01. (3H)L-glutamate uptake was undetectable in a fraction corresponding to dense core granules (fractions 15–17).
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To perform a detailed characterization of this vesicle population, Syb2-positive vesicles were immunoisolated from a low speed supernatant (LSS) prepared from cultured hippocampal astrocytes (Fig. 2A). Electron microscopy of magnetic beads, carrying astrocytic vesicles bound to anti-Syb2 antibodies, revealed that these vesicles were of variable size ranging from 30 nm to above 110 nm (total number of vesicles examined: 300; Fig. 2B). The majority of these vesicles had diameters of 30–80 nm. Astrocytic, Syb2-positive vesicles appear thus more heterogeneous in size compared with small, clear synaptic vesicles (40–60 nm diameter) (De Camilli & Navone, 1987). Only sporadically, anti-Syb2-coated beads carried, in addition to clear vesicles, dense core vesicles that corresponded to 2.34% of the total number of vesicles examined (n= 513).
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A, electron microscopy images showing anti-Syb2 antibody-coated beads with bound astrocytic vesicles. Clear vesicles, heterogeneous in size represent the large majority of Syb2-positive organelles. Dense core vesicles were also sporadically observed. The highest extent of membrane contamination in Syb2 antibody-coated beads is shown in the bottom panel. Calibration bars, 140 nm. B, histogram showing the size distribution (percentage of total) of clear vesicles bound to Syb2 antibody-coated beads (n= 300). The actual number of vesicles analysed is reported within the bars. C, Western blot analysis of low speed supernatant (LSS) and immunoisolated organelles (IP vesicles) for Syb2 (VAMP2), cellubrevin/Syb3 (VAMP3), TI-VAMP/VAMP7 (VAMP7), v-Glut-2, rab7, kinesin (u-kin), the AMPA receptor subunits GluR1,2,3, Syt IV and the t-SNARE SNAP-23. D, Western blot analysis of brain synaptosomes, astrocyte homogenates, low speed supernatant (LSS) and immunoisolated organelles (IP vesicles) for Syt I. Note that Syt I immunoreactivity is detectable in synaptosomes but not astrocytes.
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To characterize their molecular composition, immunoisolated, Syb2-positive astrocytic vesicles were analysed by SDS PAGE and Western blotting. Syb2-positive vesicles were found to lack the vSNARE TI-VAMP/VAMP7 (Galli et al. 1998), but contained the ubiquitous v-SNARE cellubrevin/Syb3. Rab7, a marker for late endosomes/lysosomes, was totally absent from the immunoisolated vesicles, further confirming the specificity of the procedure. The ubiquitous form of kinesin was associated, although not enriched, with immunoisolated vesicles, thus suggesting that the isolated particles possess molecular motors specific for microtubule-based movement. In accordance with the ability of the vesicles to internalize glutamate, Syb2-positive vesicles were immunoreactive for the vesicular glutamate transporter v-Glut-2, which co-enriched together with Syb2 from the LSS. Furthermore, Syb2-positive vesicles were immunopositive for the AMPA receptor subunits GluR2,3 and, although to a lesser extent, for GluR1 (Fig. 2C). We did not detect synaptophysin on isolated vesicles (not shown). Furthermore, although clearly detectable in brain synaptosomes, synaptotagmin I immunoreactivity was absent from astrocyte homogenates and immunoisolated vesicles (Fig. 2D). Syb2-positive vesicles were instead immunoreactive for synaptotagmin IV. Finally, differently from neuronal synaptic vesicles which contain major pools of the t-SNARE SNAP-25 recycling from the plasma membrane (Walch-Solimena et al. 1995), they did not contain detectable amount of the t-SNARE SNAP-23 (Fig. 2C).
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Astrocytic Syb2–EGFP vesicles are highly mobile
confocal microscopy imaging of astrocytes transfected with the Syb2–EGFP construct a punctate pattern of fluorescence is evident throughout the cytoplasm of soma and processes (Fig. 3A).
A, confocal image of a Syb2–EGFP transfected astrocyte displaying a punctate pattern of fluorescence. Due to scattering of the fluorescent signal the size of the vesicles seem larger than that of Syb2-immunoisolated vesicles as measured by electron microscopy. Scale bar, 5 μm. B, the bar histogram shows the distribution of the velocities of non-directional (white bars) and directional (grey bars) Syb2–EGFP vesicles. C, vector tracings report the path of several vesicles. Examples of the non-directional (left) and directional (right) types of motion are illustrated. The value of maximal displacement for each vesicle is reported (±S.E.M., in the case of multiple measurements). The number on the path of the vesicle corresponds to the number that marks the same vesicle path in the Movie 1b in Supplemental material. Time frame, 1 s. D, sequence of imaging illustrating the movement of a vesicle along an astrocyte process towards the growth cone. The arrows in each frame indicate the position of the mobile vesicle. Time frame, 1 s; scale bar, 3 μm.
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Time-lapse confocal microscopy imaging revealed that, while a majority of vesicles are immobile, a subpopulation of Syb2–EGFP vesicles, corresponding to about 25% of the total number of vesicles, shows different types of motion (see Supplementary material, Movie 1a). Vesicles move at a mean velocity of 0.65 ± 0.04 μm s–1 (n= 81). The bar histogram that reports the velocity of each vesicle motion shows, however, a clear bimodal distribution suggesting the presence of two distinct populations of vesicles that move at different velocities (Fig. 3B). By measuring the maximal displacement for individual vesicles, two types of motion were also distinguishable. Vesicles with maximal displacement below and above 1 μm were classified as short-range, non-directional and long-range, directional vesicles, respectively (Potokar et al. 2005) (Supplementary material, Movie 1a). The motion of these latter vesicles involves occasionally rapid changes, or even reversals in direction as well as pauses. The path travelled by some vesicles through the two types of motion is reported in Fig. 3C (the path of some of these vesicles, labelled by various colours, is presented in Supplementary material, Movie 1b). The mean values of the maximal displacement of non-directional (0.6 ± 0.1 μm, n= 33) and directional (3.5 ± 0.4 μm, n= 48) vesicles was significantly different (P < 0.001). It appears that non-directional vesicles move preferentially at slow velocity (below 0.4 μm s–1) while directional vesicles move preferentially at a velocity above 0.6 μm s–1 (Fig. 3B).
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In astrocytic processes, the long-range, directional motion is largely dominant while the non-directional type of motion is only occasionally observed. An example of a vesicle that moves rapidly towards the growth cone of an astrocytic process is reported in Fig. 3D (arrows) and dynamically illustrated in the Movie 2 (in Supplementary material). In the Movie 1b, an additional example of the movement of a vesicle (the path of this vesicle is shown also in Fig. 3C, track 3) along a process is marked as a coloured path. Once the growth cone is reached, vesicles stabilize their motion. Indeed, average velocity of vesicles in the growth cone is 0.096 ± 0.006 μm s–1 (n= 16).
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Ca2+-dependent membrane fusion of Syb2–EGFP vesicles
To investigate the Ca2+-dependent exocytosis of Syb2–EGFP vesicles, we first used the potent secretory stimulus ionomycin that triggers in cultured astrocytes a large increase in the concentration of intracellular Ca2+ ([Ca2+]i). By means of a fluorescence enzymatic assay we confirmed previous observations and demonstrated the ability of ionomycin to trigger a significant release of glutamate from astrocytes. Specifically, we found that 10 μM ionomycin induced the release of 49.5 ± 2.1 nmol glutamate (mg of protein)–1 (n= 3). After binarization of the image, the number of Syb2–EGFP vesicles in the cytoplasm of astrocytes transfected with our Syb2–EGFP construct was counted at a single focal plane from successive confocal microscope images taken at 1 Hz. In these transfected astrocytes ionomycin resulted in a significant reduction of fluorescent puncta (Fig. 4A and D). The decrease in vesicle number after ionomycin was not due to a movement of vesicles out of focus. In two astrocytes five images were taken at different focal planes, before and 5 min after ionomycin stimulation, and the number of vesicles in each plane was counted. The values of the relative reduction in vesicle number in the different images taken at the same focal plane after ionomycin were similar. Specifically, in the two astrocytes analysed, after ionomycin vesicles counted from the five images taken at distinct focal planes were reduced to 62.3 ± 6.1 and 72.9 ± 6.0% (mean ±S.D.) with respect to vesicles counted from images taken at the same focal planes before ionomycin. To ensure that the parameters under study maintained stable values, astrocytes were monitored over a period of at least 15 min before ionomycin application. During this period, the number of vesicles measured at a single focal plane in transfected astrocytes (n= 8) did not change significantly. In additional control experiments, we confirmed the stability of this value over a period of 30 min by monitoring astrocytes for 30 s every 5 min. Furthermore, in control astrocytes the number of vesicles measured in the stacked images over a period of 10 min (n= 1) and 15 min (n= 2) did not change significantly (relative change, mean ±S.D., astrocyte 1, –4.5 ± 2.9%; astrocyte 2, 2.2 ± 9.9%; astrocyte 3, –3.8 ± 5.2%). In the example illustrated in Fig. 4, we counted a mean number of 42 ± 1 vesicles in 30 images (taken at 1 frame s–1) before ionomycin and 17 ± 0.6 vesicles in the 60 images taken immediately after ionomycin application (Fig. 4A and B; see also Movie 3a and b). Upon ionomycin stimulation, several vesicles (arrowheads in Fig. 4A) are seen to be associated with the membrane. The movement of these vesicles in the region immediately beneath the astrocytic membrane and their association with the membrane is also dynamically illustrated in the Movie 3b. As a consequence of this event, the fluorescence associated with the membrane increases in parallel to a decrease in the number of vesicles in the cytoplasm (Fig. 4C). Probably due to the association of a large number of vesicles with the membrane, we could observe that after ionomycin stimulation, processes from some astrocytes, as in the example illustrated in Fig. 4, underwent a change in morphology. Results obtained from a total of eight experiments are summarized in Fig. 4D. The time course of ionomycin effect shows a certain degree of variability. In 6 of the 8 astrocytes examined it acted rapidly resulting in a significant decrease in vesicle number within 2 min, while in the two remaining astrocytes its effect was delayed, being observed between 3 and 5 min after its application. The average values of the reduction in vesicle number at different times after ionomycin application are reported in Fig. 4D.
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A, the two images show a transfected astrocyte at basal conditions (left panel) and 30 s after 10 μM ionomycin application (right panel). Stimulation causes a prompt reduction in the number of visible vesicles in the cytoplasm and a parallel increase in the fluorescence associated with the plasma membrane (arrowheads). B, time course of the vesicle number in the astrocyte shown in A, before (35 images; 1 s–1) and after ionomycin (60 images, starting 30 s after stimulation). C, bar graph showing the mean reduction in vesicle number in the cytoplasm (white bars) and the mean increase in the fluorescence of the plasma membrane (grey bars), as expressed in arbitrary units at basal conditions and after ionomycin stimulation. In this and in the following figures, P < 0.001. D, bar graph showing the average reduction in vesicle number with respect to control values occurring within 2 and 5 min of ionomycin application. Each bar reports the number of performed experiments. In this and in the following figures, P < 0.05.
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As an additional, powerful secretory stimulus for vesicle fusion we used -latrotoxin (-LTX). This toxin causes the release of neurotransmitters in neurones via both Ca2+-dependent and Ca2+-independent mechanisms (Davletov et al. 1998). As previously observed (Parpura et al. 1995b), the application of 3 nM-LTX triggered the release of glutamate from cultured astrocytes (34.8 nmol (mg of protein)–1). In transfected cells (n= 4), the number of Syb2–EGFP vesicles, following application of -LTX, was significantly reduced (average reduction, 52.2 ± 19.4% with respect to basal conditions, P < 0.05). All these observations are consistent with the view that astrocytes possess a regulated exocytosis of glutamate-containing vesicles.
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Additional experiments were then performed to investigate whether a more physiological activation of the [Ca2+]i response in astrocytes, such as the [Ca2+]i increase mediated by activation of mGluRs, can also trigger membrane fusion of Syb2–EGFP vesicles. We found that [Ca2+]i elevations in astrocytes mediated by the mGlu and AMPA receptor agonist L-quisqualate (100 μM), induced a glutamate release of 31 ± 2.4 nmol (mg of protein)–1 (n= 6) as well as a significant decrease in vesicle number. Figure 5 shows a Syb2–EGFP transfected astrocyte at basal conditions, and at 1 and 3 min after the application of L-quisqualate (Fig. 5A). In this astrocyte, at 1 and 3 min after stimulation the number of vesicles was reduced to 78.0 ± 2.5 and 58.0 ± 3.2%, respectively (Fig. 5B). From all the experiments, the average number of vesicles were significantly reduced at 3 min but not at 1 min after L-quisqualate stimulation (Fig. 5C). The change in morphology that we occasionally observed in the astrocyte upon ionomycin stimulation was never observed upon L-quisqualate stimulation. Taken together, these results demonstrate that stimuli that in astrocytes trigger both [Ca2+]i elevations and glutamate release also trigger the exocytosis of Syb2–EGFP vesicles.
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A, the three images show a transfected astrocyte at basal conditions (left panel), at 1 min (central panel) and at 3 min (right panel) after 100 μML-quisqualate application. The small decrease in vesicle number that can be observed at 1 min is significant at 3 min. Scale bar, 5 μm. B, time course of vesicle number as measured from each image (taken at 1 s intervals) of the astrocyte shown in A, before (19 images), 1 (20 images) and 3 min (19 images) after L-quisqualate. C, bar graph showing the average reduction in vesicle number with respect to control values occurring at 2 and 5 min after L-quisqualate application. Each bar reports the number of astrocytes analysed. D, bar graph showing the reduction in vesicle number at the level of cell body and processes following stimulation of astrocytes with either ionomycin (n= 4) or L-quisqualate (n= 3). The distinct values of vesicle number reduction at the cell body and processes are 63 ± 12% and 18 ± 14%, for ionomycin experiments, and 56 ± 9% and 39 ± 13%, for L-quisqualate experiments.
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In view of the possibility that glutamate release may preferentially occur at the level of astrocyte processes, we compared the reduction in the number of vesicles evoked by astrocyte stimulation at the processes and the soma. For this analysis we considered only cells (n= 7) in which the processes could be clearly visualized. The rate of vesicle fusion was found to be higher in the processes than in the cell body for each astrocyte analysed except one. As a mean, the reduction in the number of vesicles at the processes was significantly higher than that at the soma (Fig. 5D).
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After 45 min incubation of astrocytes with the calcium chelator BAPTA-1 AM (20 μM), L-quisqualate, which in control astrocytes caused a prompt [Ca2+]i increase, failed to trigger [Ca2+]i elevations (Fig. 6A and B). Under these conditions, L-quisqualate triggered a low glutamate release (7.8 ± 1.3 nmol (ng of protein)–1, n= 4; Fig. 6D). Accordingly, in all the transfected astrocytes examined (n= 10), the number of Syb2–EGFP vesicles was not significantly changed after L-quisqualate stimulation (98.5 ± 6.7%, Fig. 6E).
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A, sequence of pseudocolour images illustrating the [Ca2+]i change in Indo-1-loaded astrocytes upon 100 μM quisqualate stimulation. Astrocytes display a prompt [Ca2+]i elevation upon a first L-quisqualate stimulation (a and b), and failed to respond upon a second L-quisqualate challenge that was performed after BAPTA-1 AM incubation (c and d). Scale bar, 10 μm. B, time course of the [Ca2+]i changes in the astrocytes labelled 1 and 2. R corresponds to the ratio of the intensity of the light emitted by Indo-1 at the two wavelengths (405 and 485 nm). C, average [Ca2+]i changes following L-quisqualate application that was performed either before and after BAPTA-1 AM incubation. Each bar reports the number of astrocytes analysed in four different experiments. D, glutamate released from astrocytes as measured by a fluorimetric assay. After astrocyte incubation with BAPTA-1 AM, the release of glutamate triggered by L-quisqualate is drastically reduced. Each bar reports the number of experiments. E, bar graph showing the average number of vesicles in the cytoplasm of transfected astrocytes after L-quisqualate stimulation (n= 10) in control slices and in slices preincubated with BAPTA-1 AM (n= 10).
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These results indicate that Ca2+-dependent release of glutamate is accompanied by a disappearance of Syb2–EGFP vesicles from astrocyte processes, further supporting the idea that Syb2-positive vesicles are indeed the source of glutamate in astrocytes.
Staining with acridine orange (AO) has been used recently to tag vesicles in cultured astrocytes (Bezzi et al. 2004). Following accumulation into acidic compartments, such as vesicles, this dye changes its spectral properties and shifts its emission from green to red light. By combining this approach with the total internal reflection fluorescence microscope technique (TIRF), following astrocyte stimulation a subpopulation of AO-loaded vesicles have been observed to undergo rapid membrane fusion (Bezzi et al. 2004). We thus investigated whether and to what extent our Syb2–EGFP vesicles could be labelled by AO and whether their number in the cytoplasm is reduced rapidly upon L-quisqualate stimulation.
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We first found that the number of Syb2 vesicles in transfected astrocytes was significantly higher than that of vesicles that were loaded with AO in the same astrocytes (n= 14) and only part of these AO vesicles were Syb2-positive (36%, Fig. 7A). Figure 7B–D shows an example of these different populations of vesicles in a transfected astrocyte. What is noteworthy is that the mean number of AO vesicles in transfected cells was not significantly different from that of AO vesicles in non-transfected cells (87 ± 12, n= 14, versus 70 ± 5, n= 21).
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A, bar graph showing the mean number of the different subpopulations of vesicles. P < 0.0001. B, AO-labelled vesicles in a Syb2–EGFP transfected astrocyte. From this image 61 AO-loaded vesicles were counted Scale bar, 5.5 μm. C and D, higher magnifications of the boxed region in B showing the green (C, Syb2–EGFP-positive vesicles) image and the merge (D) of the green and red signals. Arrows indicate yellow vesicles double fluorescent for AO and Syb2–EGFP. The total number of double fluorescent vesicles in this astrocyte was 50, while that of Syb2–EGFP vesicles, measured before loading with AO, was 151. Scale bar, 1.5 μm.
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AO-labelled vesicles were then monitored in astrocytes after astrocyte stimulation with L-quisqualate. In these experiments, the number of AO-labelled vesicles was not significantly reduced 30 s after the onset of stimulation (93.4 ± 6%, n= 22).
Discussion
Over the last decade the ability of astrocytes to release glutamate through a Ca2+-dependent, vesicle-mediated mechanism has been the subject of intense debate. Only recent studies were able to provide direct evidence for the presence in astrocytes of a regulated exocytosis of glutamate-containing vesicles (Bezzi et al. 2004; Zhang et al. 2004b).
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In our study we demonstrated that subcellular fractions enriched in the SNARE protein Syb2 are able to specifically take up glutamate via a bafilomycin-sensitive mechanism. The expression of a functional glutamate transporter of the v-Glut family that we report here follows the immunocytochemical demonstration that v-Gluts are expressed in astrocytes in situ (Bezzi et al. 2004; Montana et al. 2004; Zhang et al. 2004b) and colocalized with glutamate in vesicles from cultured astrocytes (Anlauf & Derouiche, 2005). These results represent the first direct evidence that astrocytes possess a functional machinery devoted to glutamate loading into vesicular compartments containing Syb2.
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Immunoisolation of vesicles, endogenously expressing Syb2, using magnetic beads bound to specific antibodies allowed the molecular characterization of these organelles. The Western blot analysis of the Syb2 vesicles revealed the presence of the vesicular glutamate transporter v-Glut-2, thus indicating that Syb2 vesicles are endowed with the molecular machinery necessary for glutamate loading.
In line with the recent evidence that synaptotagmin IV, and not the neuronal isoform synaptotagmin I, regulates glial glutamate release (Zhang et al. 2004b), synaptotagmin IV was enriched in the Syb2-positive vesicle population immunoisolated from astrocytes. This result further supports its possible role as a Ca2+ sensor for gliotransmission. As an additional difference from neuronal vesicles, which contain SNAP-25 (Walch-Solimena et al. 1995), astrocytic vesicles do not contain detectable amounts of the non-neuronal homologue SNAP-23, nor do they contain synaptophysin. These subtle differences may reflect differences in the molecular mechanism at the basis of vesicle fusion in neurones and astrocytes.
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Interestingly, Syb2-positive vesicles appeared to be also positive for the ubiquitous SNARE cellubrevin/Syb3. Our data open up the possibility that a redundancy of v-SNAREs might occur in astrocytic vesicles, as previously described in other experimental models (Chilcote et al. 1995). Whereas the possibility that cellubrevin/Syb3 may support ‘per se’ regulated secretion could also be taken into consideration (Chilcote et al. 1995; Regazzi et al. 1996; Bezzi et al. 2004), the presence of Syb2 in v-Glut1-2-positive astrocytic vesicles provides a clear molecular basis for regulated exocytosis (Randhawa et al. 2000; Chen & Scheller, 2001; Jahn et al. 2003).
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Finally, we show here that Syb2-positive vesicles contain the AMPA receptor subunits GluR2,3 and, to a lesser extent, GluR1. This observation is in line with the recent demonstration that a major pool of presynaptic GluR2,3 and GluR1 subunits is associated with synaptic vesicle membranes and is recruited to the presynaptic membrane in response to depolarization (Schenk et al. 2003). The predominant presence of GluR2,3 subunits of AMPA receptors on Syb2-positive vesicles suggests the occurrence in astrocytes of a subunit-specific regulation of AMPA receptor delivery to the plasma membrane, with receptors cycling between intracellular pools and the membrane surface, as previously described in neurones (Passafaro et al. 2001; Shi et al. 2001; Schenk et al. 2003). Astrocytic glutamate receptors are involved, upon activation, in the regulation of proliferation and differentiation, as well as in the modulation of synaptic efficacy (reviewed in Gallo & Ghiani, 2000). Syb2-positive vesicles fusing with the astrocytic plasma membrane as a consequence of intracellular Ca2+ elevations may provide the pathway responsible for a regulated delivery of AMPA receptor subunits to the surface of astrocytic cells.
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Electron microscopy analysis of Syb2-positive vesicles revealed a heterogeneous size of these organelles. This feature adds to the demonstration that, whereas synaptic vesicles are organized in tight clusters inside the nerve terminal, astrocytic vesicles show a more diffuse distribution inside processes (Bezzi et al. 2004). The size heterogeneity of astrocytic vesicles that we report here appears to be in contrast with the recent demonstration that vesicles detected by electron microscopy in astrocytes, both in situ and in culture, are homogeneous in size (around 30 nm) (Bezzi et al. 2004). The use of different experimental approaches (electron microscopy of vesicles in astrocyte processes in situversus isolation of the whole population of vesicles endogenously expressing Syb2) may at least partially explain this discrepancy. Interestingly, the lack, in the Syb2-positive vesicles, of endosomal markers such as rab7, and of other v-SNAREs, such as TI-VAMP/VAMP7, indicate that vesicle heterogeneity is not due to a contamination of this fraction by other organelles.
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Our approach to target vesicles with the Syb2–EGFP chimera allows vesicle movement to be monitored readily with confocal microscopy. Using different stimuli, we provide indirect, though compelling, evidence that, upon [Ca2+]i elevations in transfected astrocytes, Syb2–EGFP vesicles fuse to the membrane and release glutamate. By time-lapse imaging we could also determine the route taken, the distance travelled and the velocity of individual Syb2–EGFP vesicles in living astrocytes. Results obtained revealed that the behaviour of Syb2–EGFP vesicles is similar to that of synaptic vesicles in neurones and secretory granules in neuroendocrine cells (Steyer et al. 1997; Pouli et al. 1998). We also found that Syb2–EGFP vesicles moved in a chaotic fashion while others moved back and forth along linear routes. These two distinct patterns of mobility in a similar percentage of vesicles have been previously observed in astrocytic vesicles tagged with a DNA construct encoding the proatrial natriuretic peptide fused with GFP (Potokar et al. 2005). Interestingly, vesicles in astrocytic processes moved almost exclusively along linear pathways and upon reaching the distal end become immobile. This linear motion and the expression in astrocytes of kinesin are consistent with a model of motor-assisted diffusion in which vesicles are transported along microtubules to distal portions of astrocytic processes until they reach a position that may be adjacent to release sites. The existence of potential release sites in the astrocytic membrane is also supported by a recent high-resolution immunofluorescence study (Anlauf & Derouiche, 2005). This study reveals that the vesicles immunopositive for either glutamate and/or v-Gluts are concentrated in processes and at distinct sections of the cell boundary. The higher reduction in vesicle number that we detected upon stimulation at the level of processes than at the cell body provides further support for the existence of specific sites where exocytosis may preferentially occur.
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After BAPTA-1 AM incubation, astrocyte stimulation with L-quisqualate failed to trigger in astrocytes the release of glutamate as well as the reduction in the number of vesicles otherwise observed in Syb2–EGFP transfected astrocytes, confirming the dependency of both glutamate release and vesicle fusion on [Ca2+]i elevations.
By using total internal reflection fluorescence (TIRF) microscopy and AO staining, the exocytosis of glutamate-containing vesicles has been recently demonstrated in cultured astrocytes (Bezzi et al. 2004). The great majority of vesicle fusion events was observed to occur within 150 ms after the local application of the mGluR agonist (S)-3,5-dihydroxyphenylglcyine (DHPG), whereas rare events were observed thereafter. Our results confirm the ability of astrocyte vesicles to fuse with the membrane upon stimuli that trigger [Ca2+]i elevations, although they fail to reveal a significant decrease in cytoplasmic Syb2–EGFP vesicles shortly after stimulation. This latter result does not necessarily mean that Syb2–EGFP vesicles cannot fuse rapidly with the membrane upon astrocyte stimulation. In previous experiments in cultured astrocytes we have already provided evidence that glutamate can be released rapidly from astrocytes through an exocytotic mechanism, and trigger fast NMDA receptor-mediated inward currents in glutamate sniffer cells (Pasti et al. 2001). By estimating fusion events indirectly from the counting of cytoplasmic vesicles, our experimental approach cannot allow us to detect the initial, rapid phase of exocytosis described by Bezzi et al. (2004). After fusion, Syb2–EGFP vesicles can rapidly re-enter into the pool of releasable vesicles in the cytoplasm. A reduction in the number of cytoplasmic vesicles can thus be detected only after a relatively long-lasting stimulation of the exocytosis that may change the equilibrium between exocytotic and endocytotic events or the kinetics of one or both events.
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On the other hand, in experiments based on AO-labelling of vesicles, a long-lasting exocytotic process may not be revealed. In these experiments, fusion events are detected as green fluorescent light flashes accompanied by the disappearance of red-labelled vesicles. As such, this experimental approach may not allow the following of repetitive episodes of fusion from the same vesicle and thus a prolonged process of continuous exocytosis that may ultimately lead to the reduction of cytoplasmic vesicles that we detected.
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The ability of astrocytes to support a long-lasting exocytosis of glutamate-containing vesicles was previously demonstrated. Indeed, repetitive episodes of fast activation of the NMDA receptor in glutamate sniffer cells, reflecting the exocytosis of glutamate-containing vesicles, were observed for several minutes after stimulation of astrocyte [Ca2+]i oscillations with L-quisqualate (Pasti et al. 2001).
Data reported here also demonstrate that subpopulations of Syb2–EGFP vesicles load AO differently. Indeed, the number of AO vesicles is consistently lower than that of Syb2–EGFP vesicles and only part of the AO vesicles are also Syb2–EGFP positive. This could be related to different stages of vesicle functional maturation. The low accumulation of AO in some Syb2–EGFP vesicles may also be due to a different pH in these vesicles as well as to other factors that are recognized to play important roles in AO accumulation, such as the total internal volume and the nature of the anions that follow the movements of the protons into the vesicle (Palmgren, 1991; Clerc & Barenholz, 1998).
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Given that Syb2–EGFP and AO vesicles represent different populations that overlap only partially, we investigated whether a decrease in the number of AO vesicles could be detected rapidly after stimulation. When L-quisqualate was applied to trigger exocytosis, the number of AO vesicles in the cytoplasm did not change significantly within 30 s of the onset of stimulation. These data are not necessarily in disagreement with those obtained in TIRF experiments that detected a rapid fusion of AO vesicles after astrocyte stimulation. In these experiments, only a subpopulation of total AO vesicles – those that are just beneath the plasma membrane – could be monitored, and only part (34%) of these vesicles undergo a rapid fusion after maximal stimulation of astrocytes (Bezzi et al. 2004). Thus, our results indirectly support the view that vesicles ready to fuse with the membrane represent a small subpopulation of vesicles and their disappearance from the cytoplasm could not result in a significant reduction in the total number of AO vesicles.
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The observations that vesicles in cultured astrocytes are highly mobile and fuse with the membrane upon stimuli that trigger [Ca2+]i elevations give the opportunity to explore in future experiments many aspects in the secretory behaviour of these cells at the cellular and subcellular level. Glutamate release from astrocytes activated by stimuli that evoke Ca2+ elevations in these cells is emerging as a functionally relevant signalling pathway that modulates neuronal transmission in the hippocampus. In this brain region astrocytic glutamate has been reported to act on (i) interneurones to potentiate inhibitory transmission (Kang et al. 1998; Liu et al. 2004), (ii) extrasynaptic mGluR receptors to increase the probability of spontaneous glutamate release from axon terminals (Fiacco & McCarthy, 2004), and (iii) extrasynaptic NMDA receptors to promote synchronized activity in CA1 pyramidal neurones (Fellin et al. 2004). The results that we report here, i.e. the molecular characterization of astrocyte vesicles, the evidence for the functional expression of a glutamate transporter of the v-Glut family and for the ability of these vesicles to fuse with the membrane, provide significant support for the hypothesis that a regulated, sustained release of glutamate-containing vesicles represents a functionally relevant signalling pathway at the basis of astrocyte-to-neurone communication.
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Supplemental material
The online version of this paper can be accessed at: 10.1113/jphysiol.2005.094052
http://jp.physoc.org/cgi/content/full/jphysiol.2005.094052/DC1
and contains supplemental material entitled: Vesicle Movement.
This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com
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Footnotes
D. Crippa and U. Schenk contributed equally to this work.
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