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Cellular/Molecular
Vesicular Glutamate Transporter-Dependent Glutamate
Release from Astrocytes
Vedrana Montana,
1
Yingchun Ni,
1
Vice Sunjara,
2
Xue Hua,
1
and Vladimir Parpura
1
1
Department of Cell Biology and Neuroscience and Center for Nanoscale Science and Engineering,
2
International Scholars Program, University of
California, Riverside, California 92521
Astrocytes exhibit excitability based on variations of their intracellular Ca
2⫹
concentrations, which leads to glutamate release, that in
turn can signal to adjacent neurons. This glutamate-mediated astrocyte–neuron signaling occurs at physiological intracellular Ca
2⫹
levels in astrocytes and includes modulation of synaptic transmission. The mechanism underlying Ca
2⫹
-dependent glutamate release
from astrocytes is most likely exocytosis, because astrocytes express the protein components of the soluble N-ethyl maleimide-sensitive
fusion protein attachment protein receptors complex, including synaptobrevin 2, syntaxin, and synaptosome-associated protein of 23
kDa. Although these proteins mediate Ca
2⫹
-dependent glutamate release from astrocytes, it is not well understood whether astrocytes
express functional vesicular glutamate transporters (VGLUTs) that are critical for vesicle refilling. Here, we find in cultured and freshly
isolated astrocytes the presence of brain-specific Na
⫹
-dependent inorganic phosphate cotransporter and differentiation-associated
Na
⫹
-dependent inorganic phosphate cotransporter that have recently been identified as VGLUTs 1 and 2. Indirect immunocytochem-
istry showed a punctate pattern of VGLUT immunoreactivity throughout the entire cell body and processes, whereas pharmacological
inhibition of VGLUTs abolished mechanically and agonist-evoked Ca
2⫹
-dependent glutamate release from astrocytes. Taken together,
these data indicate that VGLUTs play a functional role in exocytotic glutamate release from astrocytes.
Key words: astrocytes; vesicular glutamate transporters; SNAREs; V-ATPase; calcium-dependent glutamate release; exocytosis; signaling;
synapse
Introduction
The ultrastructure of the CNS suggests that astrocytes might reg-
ulate synaptic neurotransmission. Astrocytes enwrap nerve ter-
minals (Peters et al., 1991), which makes them perfectly posi-
tioned to exchange information with synapses. Indeed, it has
been demonstrated that astrocytes can respond to synaptic acti-
vation (Dani et al., 1992) and that they can modulate synaptic
neurotransmission by releasing glutamate in a Ca
2⫹
-dependent
manner (Araque et al., 1998a; Kang et al., 1998). Because astro-
cytic internal Ca
2⫹
levels necessary for glutamate release are
within the physiological range (Parpura and Haydon, 2000), this
release can be used as a signaling pathway that may influence
synaptic neurotransmission and plasticity within the CNS.
Although the mechanism of Ca
2⫹
-dependent release of glu-
tamate from astrocytes is not fully defined, the protein compo-
nents of the neuronal exocytotic machinery are expressed in as-
trocytes (Parpura et al., 1995b; Jeftinija et al., 1997; Bezzi et al.,
1998; Hepp et al., 1999; Maienschein et al., 1999; Araque et al.,
2000; Pasti et al., 2001). Additionally, Clostridial toxins, which
cleave some of these exocytosis-related proteins, inhibit Ca
2⫹
-
dependent release of glutamate from astrocytes (Jeftinija et al.,
1997; Bezzi et al., 1998; Araque et al., 2000), indicating that reg-
ulated exocytosis most likely mediates glutamate release from
astrocytes.
Immunoelectron microscopic study indicated that exocytotic
proteins in astrocytes can be associated with electronlucent or
dense-core vesicular structures, the diameters of which are less
uniform than those reported in neurons (Maienschein et al.,
1999). The storage of glutamate in synaptic vesicles requires the
presence of V-type H
⫹
-ATPase (V-ATPase) and vesicular gluta-
mate transporters (VGLUTs). Hence, astrocytic Ca
2⫹
-
dependent glutamate release can be blocked with bafilomycin A
1
(Araque et al., 2000; Bezzi et al., 2001; Pasti et al., 2001), which
specifically interferes with V-ATPase, leading to alkalinization of
vesicular lumen and collapsing the proton gradient necessary for
VGLUTs to transport glutamate into vesicles. Brain tissue ex-
presses VGLUT 1 and 2 isoforms (Ni et al., 1994, 1995; Hisano et
al., 2000; Bai et al., 2001) in glutamatergic neurons (Bellocchio et
Received Aug. 12, 2003; revised Jan. 30, 2004; accepted Jan. 30, 2004.
This work was supported by a grant from the Department of Defense/Defense Advanced Research Planning
Agency/Defense Microelectronics Activity under Award DMEA90-02-2-0216 and the Whitehall Foundation (Award
2000-05-17). V.P. is an Institute for Complex Adaptive Matter Senior Fellow. We thank Dr. Karl Bauer (Max-Planck-
Institute for Experimental Endocrinology, Hannover, Germany) for kindly providing

-Ala-Lys-N
⑀
-AMCA peptide,
Dr. James E. Rothman (Memorial Sloan-Kettering Cancer Center, New York, NY) for generously providing a plasmid
encoding for super-ecliptic synapto-pHluorin, and Dr. Robert H. Edwards (University of California San Francisco, San
Francisco, CA) for graciously providing polyclonal antibodies against VGLUTs 1 and 3 and plasmids encoding for
GST-VGLUTs 1 and 3 fusion proteins. We are grateful for Dr. Glenn I. Hatton’s arrangements regarding supply of
“Bauer’s” peptide and for providing an Alexa Fluor 488 goat anti-mouse antibody. We also thank Dr. Hatton and
Todd A. Ponzio for comments on previous versions of this manuscript, for engaging discussions related to this work,
and help with brain dissection of adult rats.
Correspondenceshould beaddressed toDr. VladimirParpura, Departmentof CellBiology andNeuroscience,1208
Spieth Hall, University of California, Riverside, CA 92521. E-mail: vlad@citrus.ucr.edu.
V. Sunjara’s present address: School of Medicine, University of Zagreb, 10000 Zagreb, Croatia.
DOI:10.1523/JNEUROSCI.3770-03.2004
Copyright © 2004 Society for Neuroscience 0270-6474/04/232633-10$15.00/0
The Journal of Neuroscience, March 17, 2004 •24(11):2633–2642 • 2633
al., 1998; Fremeau et al., 2001; Fujiyama et al., 2001; Herzog et al.,
2001; Sakata-Haga et al., 2001; Kaneko et al., 2002; Varoqui et al.,
2002). Recently, there have been reports indicating the presence
of a VGLUT 3 isoform in subpopulations of GABAergic
(Fremeau et al., 2002), cholinergic, and monoaminergic neurons
(Fremeau et al., 2002; Gras et al., 2002; Schafer et al., 2002) and
some astrocytes (Fremeau et al., 2002). However, whether astro-
cytes can express the VGLUT 1 and 2 isoforms, which account for
the release of glutamate by all known excitatory neurons, is not
yet known.
In this study, we found the presence of VGLUTs 1 and 2 in
cultured and freshly isolated astrocytes from rat visual cortices,
exhibiting a subcellular localization pattern consistent with their
vesicular association. Because pharmacological inhibition of
VGLUTs greatly reduced Ca
2⫹
-dependent exocytotic release of
glutamate from astrocytes, these proteins can play a functional
role in astrocytic glutamate release in the CNS.
Some of these data appeared in preliminary form (Ni et al.,
2003).
Materials and Methods
Cell cultures. We prepared enriched astrocytic cultures using a modifica-
tion (Parpura et al., 1995a) of the originally described shaking procedure
(McCarthy and deVellis, 1980). Visual cortices isolated from 0- to 2-d-
old Sprague Dawley rats were treated enzymatically (papain, 20 IU/ml; 1
hr at 36.8°C). After subsequent treatment with trypsin inhibitor (10 mg/
ml; type II-O; 5 min at room temperature) to terminate the enzymatic
reaction, tissue was dispersed mechanically by triturating through a glass
pipette. Cells were initially plated into tissue culture flasks (25 cm
2
) and
maintained at 36.8°C in a humidified 5% CO
2
/95% air atmosphere in a
complete culture medium that consisted of
␣
-MEM (without phenol
red; Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated
FBS (HyClone, Logan, UT), L-glutamine (2 mM; Invitrogen), D-glucose
(20 mM; Sigma-Aldrich, St. Louis, MO), sodium pyruvate (1 mM; Invitro-
gen), penicillin (100 IU/ml), streptomycin (100
g/ml), and sodium
bicarbonate (14 mM; Invitrogen), pH 7.4.
After 6 –24 d in culture, the cells were shaken twice (260 rpm at
36.8°C), first for 1.5–2 hr and then, after exchange of complete medium,
again for 18 –20 hr. At that time, the remaining attached cells were de-
tached from flasks using trypsin [10,000 N
␣
-benzoyl-L-arginine ethyl
ester hydrochloride (BAEE) units/ml; Sigma-Aldrich] and replated onto
12 mm round glass coverslips precoated with polyethyleneimine (PEI; 1
mg/ml; Sigma-Aldrich). Resulting purified astrocytes were kept in cul-
ture for 1–10 d (8 –29 d after initial plating) until used in experiments.
The purity (⬎99%) of astrocytic culture was confirmed by anti-GFAP
antibody and indirect immunocytochemistry. In some experiments be-
fore GFAP immunocytochemisty, cells were incubated with a dipeptide

-Ala-Lys conjugated to 7-amino-4methylcoumarin-3-acetic acid
(AMCA), kindly provided by Dr. Karl Bauer (Max-Planck-Institute for
Experimental Endocrinology, Hannover, Germany). This peptide selec-
tively accumulates in astrocytes after its uptake is mediated by the PepT2
peptide transporter (Dieck et al., 1999). Incubation with

-Ala-Lys-N
⑀
-
AMCA (20
Mat 36.8°C for 2 hr) labels astrocytes, because this peptide
accumulates only within GFAP-positive cells (n⫽68 of 68 tested).
Freshly isolated cells. Cells were freshly isolated from visual cortices of
1-, 2-, 8-, and 55-d-old Sprague Dawley rats using a modification of a
previously described procedure (Zhou and Kimelberg, 2000). Briefly,
visual cortices isolated from rats were subjected to papain treatment,
followed by a trituration as described for cell cultures. After trituration,
cells were applied onto PEI-coated coverslips and allowed to adhere while
they were incubated with

-Ala-Lys-N
⑀
-AMCA (20
Mat 36.8°C for 1
hr). After washing, cells were fixed for immunocytochemisty.
Immunocytochemistry and nuclear staining. For confirmation of astro-
cytic culture purity, cells were exposed to Dent’s fixative at room tem-
perature for 30 min (Parpura and Haydon, 2000). A monoclonal anti-
body (catalog #69110; 1:500 dilution; 1 hr at room temperature or
overnight at 4°C; ICN Biomedicals, Aurora, OH), followed by a
rhodamine-conjugated secondary antibody (1 hr at room temperature),
was used to probe for GFAP. In some experiments, we subsequently
colabeled astrocytic nuclei using 4⬘,6-diamidino-2-phenylindole dilac-
tate (DAPI dilactate; 3
M; Molecular Probes, Eugene, OR) for 5 min at
room temperature.
Subcellular localization of VGLUTs 1, 2, and 3, synaptobrevin 2, and
synaptosome-associated protein of 23 kDa (SNAP-23) was determined
by indirect immunocytochemistry, in which cells were incubated with
primary antibodies overnight at 4°C. A monoclonal antibody against
synaptobrevin 2 (catalog #104201; 1:250 dilution; Synaptic Systems,
Goettingen, Germany), a polyclonal antibody against SNAP-23 (catalog
#111202; 1:50; Synaptic Systems), and seven different antibodies against
VGLUTs (Table 1) were used. After washout of the primary antibody,
TRITC (rhodamine)-conjugated secondary antibodies were applied, and
preparation was incubated for 1 hr at room temperature. In double-
labeling experiments, an Alexa Fluor 488-conjugated secondary antibody
(Molecular Probes) was used for visualization of synaptobrevin 2 immu-
noreactivity. In all experiments, we performed controls in which primary
antibodies were omitted to test for nonspecific binding of secondary
antibodies. We classified cells as immunoreactive if the average fluores-
cence intensity for a probed protein, within a cellular region of interest
(at least 40 ⫻40 pixels), was at least 2 SDs above the average signals
acquired from control cells. In a subset of experiments, we performed
adsorption controls, in which primary antibodies were preincubated
overnight at 4°C with respective antigens, proteins, or peptides (Table 1),
to test for specificity of primary antibodies (Bellocchio et al., 1998;
Fremeau et al., 2001, 2002; Takamori et al., 2001). All imaging data were
background subtracted using fluorescence emission originating from a
region on the coverslip containing no cells.
Preparation of subcellular fractions and Western blotting. Synaptosomal
Table 1. Antibodies raised against VGLUTs and their respective antigens used in this study
Final dilutions
Antibody Source Type
Catalog number
or reference ICC WB Adsorption antigen
Catalog number
or reference
Final
concentration
(
g/ml)
Anti-VGLUT 1 Synaptic Systems Mouse monoclonal 135001 1:1000 1:1000 GST-rVGLUT1 (aa 523–560) 135– 0P 5
Synaptic Systems Rabbit polyclonal 135002 1:500 –1:1000 1:1000–1:2000 GST-rVGLUT1 (aa 523–560) 135– 0P 2.5–5
Dr. R. H. Edwards Rabbit polyclonal
a
Bellocchio et al.
(1998)
1:500 1:1000 GST-rVGLUT1 (aa 493–560) Bellocchio et al.
(1998)
10.5
Anti-VGLUT 2 Synaptic Systems Rabbit polyclonal
a
135102 1:500 1:1000 GST-rVGLUT2 (aa 510 –582) 135–1P 2–5
Chemicon Guinea pig polyclonal AB5907 1:2000 1:2000 rVGLUT2 (aa 565–582), Sp AG209 2.5
Anti-VGLUT 3 Chemicon Guinea pig polyclonal AB5421 1:5000 1:5000 rVGLUT3 (aa 569 –588), Sp
b
AG320 1–5
Dr. R. H. Edwards Rabbit polyclonal Fremeau et al.
(2002)
1:500 1:1000 GST-rVGLUT3 (aa 530 –588) Fremeau et al.
(2002)
11.5
ICC, Immunocytochemistry; WB, Western blots; GST, glutathione S-tranferase; r, rat; Sp, synthetic peptide. Parentheses indicate the portion of VGLUTs, based on rat amino acid sequences.
a
No cross-reactivity to VGLUT 3.
b
Used in cross-reactivity experiments.
2634 •J. Neurosci., March 17, 2004 •24(11):2633–2642 Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes
membrane-enriched [lysate pellet (LP-1)] preparations were done ac-
cording to the standard procedures (Huttner et al., 1983). We obtained
non-nuclear membranes and vesicular extracts from purified astrocytes
[astrocytic preparation (AsP)] as described previously (Parpura et al.,
1995b). LP-1 and AsP were subjected to 15% SDS-PAGE, followed by
transfer to nitrocellulose membranes that were probed with VGLUT
antibodies used in immunocytochemistry, but at different dilutions (Ta-
ble 1), and antibodies against synaptobrevin 2 and SNAP-23, which we
used at 1:1000 and 1:400, respectively. Additionally, we used monoclonal
antibodies against SNAP-25 (catalog #111001; 1:500; Synaptic Systems)
and syntaxin 1 (catalog #S0664; 1:1000; Sigma-Aldrich). Immunoreac-
tivity of bands was detected using ECL (Amersham Biosciences, Piscat-
away, NJ). In a subset of experiments, we performed adsorption controls
for primary antibodies against VGLUTs (Table 1) as described for im-
munocytochemistry (Bellocchio et al., 1998; Fremeau et al., 2001, 2002;
Takamori et al., 2001). In experiments testing for cross-reactivity of an-
tibodies raised against VGLUTs 1 and 2 to their heterologous VGLUT 3
antigen, we preincubated different VGLUT antibodies with VGLUT 3
antigen.
Reverse transcription-PCR. Total RNA was extracted from purified as-
trocytic culture and cortical tissue of postnatal Sprague Dawley rats (0 –
2-d-old) using TRIzol Reagent (Invitrogen) and protocols provided by
the manufacturer. Five micrograms of total RNA were used for reverse tran-
scription using Oligo(dT)
12–18
and superscript II reverse transcriptase (In-
vitrogen). Several pairs of primers were used to amplify cDNA using PCR
(each pair, 35 cycles). Primers for SNAP-25A (GenBank accession number
L19760) amplification were 5⬘-CTGGAAAGCACCCGTCGTATG-3⬘and
5⬘-GCACGTTGGTTGGCTTCATCA-3⬘, whereas for

-actin the primers
were 5⬘-TCATGAAGTGTGACGTTGACATCCGT-3⬘and 5⬘-CCTAGAA-
GCATTTGCGGTGCACGATG-3⬘(catalog #G5740; Promega, Madison,
WI). In some experiments, we used two rounds of amplification for SNAP-
25A (GenBank accession number NM_030991). In the first round, we used
5⬘-ATGGCCGAGGACGCAGACA-3⬘and 5⬘-ACCACTTCCCAGCA-
TCTTTGT-3⬘(618 bp product). Amplified DNA was used as the template
for the second round of PCR using nested primers 5⬘-CTGGAAAGCACCC-
GTCGCATG-3⬘and 5⬘-GCACGTTGGTTGGCTTCATCA-3⬘(521 bp
product). RT-PCR reaction in two subsequent rounds was also performed
for VGLUT 2 (GenBank accession number NM_053427) using 5⬘-
AGCAAGGTTGGCATGTTGTCTG-3⬘and 5⬘-CGGTCCTTATAGGAGT-
ACGCGT-3⬘(698 bp product), followed by amplification of the product
using nested primers 5⬘-TGGTGCAATGACGAAGAACAAG-3⬘and 5⬘-
TCC TTTTTCTCCCAGCCGTT-3⬘(294 bp product). Primers for VGLUT
1 (GenBank accession number NM_053859) amplification were 5⬘-
GAGAAACAGCCGTGGGCAGAG-3⬘and 5⬘-TCAGTAGTCCCGGACA-
GGGGGTGG-3⬘(207 bp product), whereas for VGLUT 3 (GenBank acces-
sion number NM_153725) the primers were 5⬘-ACCCGGGAAGAATGG-
C A GAATGTG-3⬘and 5⬘-ATGGGAAAAGCAATGGGTGTGGAG-3⬘(399
bp product).
Calcium measurements. We monitored astrocytic intracellular Ca
2⫹
levels using a Ca
2⫹
indicator, fluo-3 (Parpura et al., 1994). Cells were
loaded in a complete culturing medium containing an acetoxymethyl
(AM) ester derivative of fluo-3 (10
g/ml; Molecular Probes) and plu-
ronic acid (0.025% w/v; Molecular Probes), at 36.8°C for 30 min. After
washing in normal external solution, de-esterification of the dye was
permitted for 30 min at room temperature. The normal external solution
contained (in mM) 140 NaCl, 5 KCl, 2 CaCl
2
, 2 MgCl
2
, 5 glucose, and 10
HEPES, pH 7.4. Coverslips containing fluo-3-loaded cells were mounted
into a recording chamber and imaged. All data were background sub-
tracted and expressed as dF/Fo (percentage), where Fo represents the
fluorescent level before cell stimulation, and dF represents the change in
fluorescence.
Glutamate measurements. We optically monitored extracellular gluta-
mate levels using an L-glutamate dehydrogenase (GDH; Sigma-Aldrich)-
linked assay (Bezzi et al., 1998; Innocenti et al., 2000). GDH generates
NADH from NAD
⫹
(

-nicotinamide adenine dinucleotide; Sigma-
Aldrich) in the presence of glutamate. Provided that GDH and NAD
⫹
are added to the solution in which astrocytes are bathed, glutamate re-
leased in the extracellular space can be detected as an increase in NADH
fluorescence. Astrocytes were bathed in an enzymatic assay solution con-
taining normal external solution supplemented with NAD
⫹
(1 mM) and
GDH (⬃53 IU/ml; pH 7.4). Every experiment was preceded by a sham
run on cells bathed in solution lacking GDH and NAD
⫹
, which was used
to correct for photobleaching and background subtraction. The reduc-
tion of fluorescence because of photobleaching in the sham run exhibited
the same time course as in the matching experimental run. All imaging
data, corrected for photobleaching and background subtracted, were ex-
pressed as dF/Fo (percentage).
Transfection. After the purification of astrocytes, cells in flasks were
transfected with a plasmid encoding for super-ecliptic synapto-pHluorin
(Sankaranarayanan et al., 2000), kindly provided by Dr. James E. Roth-
man (Memorial Sloan-Kettering Cancer Center, New York, NY), each
flask receiving 6
g of plasmid premixed with 12
l of TransIT-293
transfection reagent (catalog #2700; Mirus, Madison, WI), which aids
plasmid entry to cells for 3–4 hr. At that time, we replaced culturing
medium and returned cells to the culture incubator. Cells were main-
tained at 36.8°C in a humidified 5% CO
2
/95% air atmosphere for 2 d,
when they were replated onto PEI-coated coverslips and kept in culture
until used in experiments.
Imaging acquisition and processing. All experiments were done at room
temperature (20 –24°C). We used an inverted microscope (TE 300; Ni-
kon, Melville, NY) equipped with wide-field epifluorescence. Visualiza-
tion of indirect immunocytochemistry of GFAP, VGLUTs, and SNARE
proteins was accomplished using a standard rhodamine/TRITC filter set
(Chroma Technology, Rockingham, VT), except in double-labeling ex-
periments in which immunoreactivity of synaptobrevin 2 was visualized
using a standard fluorescein/FITC filter set (Chroma Technology),
whereas for nuclear staining (DAPI) and

-Ala-Lys-N
⑀
-AMCA we used a
standard DAPI filter set (Chroma Technology). For calcium imaging and
synapto-pHluorin, we used a standard fluorescein/FITC filter set. Images
were captured through a 60⫻plan-achromatic oil-immersion objective
[numerical aperture (NA), 1.4; Nikon) using either a CoolSNAP-HQ
cooled CCD camera (Roper Scientific, Tucson, AZ) or an intensified
CCD camera (IC-300; Photon Technology International, Lawrenceville,
NJ) driven by V⫹⫹ imaging software (Digital Optics, Auckland, New
Zealand) or LabView/IMAQ (National Instruments, Austin, TX), re-
spectively. For glutamate imaging experiments, we used a 40⫻SFluor
objective (NA, 1.3; Nikon) and a DAPI filter set (Chroma Technology).
For time-lapse image acquisition, a camera and an electronic shutter
(Vincent Associates, Rochester, NY) inserted in the excitation pathway
were controlled by software. A Xenon arc lamp (100 W) was used as a
light source. In a subset of double-labeling experiments, we used a C1
modular confocal microscope system (Nikon) configured with an in-
Figure 1. Purified astrocytic cultures are immunopositive for GFAP but do not express SNAP-
25. A, GFAP was used to identify astrocytes (red), with nuclei that are counterstained with DAPI
(blue).B,Top, AfterSDS-PAGE (1
gperlane forLP-1 and15
gperlane forAsP), immunoblots
indicate the absence of SNAP-25 in the astrocytic non-nuclear cell membrane extract AsP, while
this protein was detected in synaptosomal membrane-enriched LP-1 preparations. Bottom,
RT-PCR using mRNA isolated from purified astrocytes (As) reveals the presence of

-actin and
lack of SNAP-25 PCR products.

-Actin and SNAP-25 PCR products (285 and 521 bp, respec-
tively) were present when mRNA isolated from rat brain (Br) was used in RT-PCR. L represents a
molecular size marker (100 bp DNA ladder), whereas NC represents a negative control for reac-
tion. Scale bar, 20
m.
Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes J. Neurosci., March 17, 2004 •24(11):2633–2642 • 2635
verted microscope (TE 2000; Nikon) and equipped with a VioFlame Plus
solid state laser (405 nm; for DAPI and AMCA excitation), an argon laser
(488 nm; for Alexa Fluor 488 excitation; Melles Griot, Carlsbad, CA) and
a Spectra Physics green helium neon laser (543 nm; for TRITC excita-
tion). All images shown in the figures represent raw data.
Stimulation of astrocytes. To evoke an increase in the internal Ca
2⫹
concentrations in astrocytes and consequential glutamate release, we ei-
ther bath applied agonists (duration, 55 sec) bradykinin (90 nM) and ATP
(9
M) or mechanically stimulated astrocytes using patch pipettes (Par-
pura et al., 1994; Araque et al., 2000). To control the establishment of the
contact between the pipette and an astrocyte, we monitored pipette re-
sistance during delivery of ⫺20 mV, 10 msec square pulses by a patch-
clamp amplifier (PC-ONE; Dagan, Minneapolis, MN) equipped with a
whole-cell headstage (PC-ONE-30; 1 G⍀). Pipette resistances measured
2.3–3.3 M⍀when pipette tips were immersed in an external solution,
which increased to 2.7–4.0 M⍀(7–21% increase) during transient con-
tacts with astrocytes, lasting ⬍1 sec.
Pharmacological agents. In experiments using a Ca
2⫹
chelator,
BAPTA, cells were loaded in a complete culturing medium containing
BAPTA-AM (50
M; Molecular Probes) and pluronic acid (0.025% w/v)
at 36.8°C for 30 min. After washing in normal external solution, de-
esterification of the chelator was permitted for 30 min at room temper-
ature before use of cells in imaging experiments. Holoprotein of tetanus
toxin (15
g/ml; List Biological Laboratories, Campbell, CA) was applied
to the cells in a complete culturing medium for 24 –48 hr at 36.8°C, at
which time cells were rinsed three times with an external solution and
used in imaging experiments. Astrocytes were preincubated with bafilo-
Figure 2. Astrocytes in culture express VGLUTs 1 and 2. A, After SDS-PAGE, immunoblots
indicate the presence of VGLUTs 1, 2, and 3 in astrocytes (AsP) as well as SNARE proteins,
synaptobrevin 2, syntaxin, and SNAP-23. In LP-1 preparations, SNAP-23 was not detected.
When probing SNARE proteins, LP-1 was loaded 1–2
g per lane, whereas AsP was loaded
10 –15
g per lane. However, when probing VGLUTs, LP-1 and AsP were equally loaded at 15
g per lane for VGLUTs 1 and 2 or 30
g per lane for VGLUT 3. B, The immunoreactive bands in
AsPrecognizedby antibodiesraisedagainst VGLUT1,2, and3isoforms (Ag⫺)werecompletely
abolished when antibodies were preincubated with their homologous antigens (Ag⫹). C, An-
tibodies raised against VGLUTs 1 and 2 do not cross-react with VGLUT 3 because their pread-
sorption with this heterologous antigen does not abolish the immunoreactive bands, although
the same VGLUT 3 antigen in preadsorption with anti-VGLUT 3, as seen in B, abolishes its ability
for detection. The arrows in Band Cindicate molecular weight markers. D, RT-PCR using mRNA
isolated from purified astrocytes (As) or from brain (Br) reveals the presence of VGLUT 1, 2, and
3 PCR products (207, 294, and 399 bp, respectively). L and arrows indicate molecular size
markers (100 bp DNA ladder).
Figure 3. Subcellular localization of synaptobrevin 2 ( A), SNAP-23 ( B), VGLUT 1 (C), VGLUT
2(D), and VGLUT 3 ( E). VGLUT immunoreactivity was completely abolished when primary
antibodies (1
o
Ab) were preadsorbed with their respective antigens (Ag) in cultured astrocytes
(C⬘–E⬘,F). Scale bar: A,B,20
m; C–E,C⬘–E⬘,10
m. Fluorescent immunoreactivity is ex-
pressed in intensity units (i.u.). Bars represent means ⫾SEMs of measurements from the
number of individual astrocytes (n). Asterisks indicate a significant change of measurements
compared with the control group (1
o
Ab⫹,Ag⫺;one-way ANOVA, followed by post hoc Fish-
er’s LSD test; **p⬍0.01). We found no difference between measurements in preadsorption
controls(1
o
Ab⫹,Ag⫹)when comparedwith controls inwhich primaryantibodieswere omit-
ted (1
o
Ab⫺,Ag⫺).
2636 •J. Neurosci., March 17, 2004 •24(11):2633–2642 Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes
mycin A
1
(5
M; 30 or 60 min; Sigma-Aldrich) and Rose Bengal (0.1 or
0.5
M; 30 min; Sigma-Aldrich) at room temperature.
Statistical analysis. The effect of agonists and mechanical stimulation
on intracellular Ca
2⫹
levels and consequential glutamate release, as well
as the effects of Rose Bengal on synapto-pHluorin fluorescence, were
assessed using a paired ttest. When testing the time-dependent effects of
bafilomycin A
1
on synapto-pHluorin fluorescence, we used a paired ttest
with Bonferroni adjustment for multiple comparisons. The effects of
pharmacological agents on glutamate and Ca
2⫹
measurements, as well as
the effects of preadsorption of primary antibodies with respective anti-
gens on VGLUT immunoreactivity, were determined using one-way
ANOVA, followed by Fisher’s least significant difference (LSD) test. Stu-
dent’sttest was used for assessing the effects of Rose Bengal on ATP- or
bradykinin-induced Ca
2⫹
increases and glutamate release.
Results
Differential interference contrast microscopy of astrocyte-
enriched cultures indicated that these cultures were neuron free.
This was confirmed using indirect immunocytochemistry, im-
munoblotting, and RT-PCR. Astrocytic cell cultures were immu-
nopositive for GFAP (245 of 245 cells tested), a characteristic
astrocytic marker, revealing the purity of this culture to be ⬎99%
(Fig. 1A). To assess the extent of putative neuronal contamina-
tion, we used immunoblots and RT-PCR. We prepared a non-
nuclear membrane extract from cultured astrocytes (AsP) that
was subjected to SDS-PAGE (Fig. 1B, top). We used synaptoso-
mal plasma membrane-enriched prepara-
tion (LP-1) as a positive control. Immuno-
blot analysis revealed the absence of a
neuron-specific protein, SNAP-25 (Par-
pura et al., 1995b; Latour et al., 2003), in
astrocytic culture, although SNAP-25 was
reliably detected in LP-1 preparations.
Next, we performed RT-PCR using mRNA
isolated from rat brain and from astrocytic
cultures (Fig. 1 B, bottom). The PCR prod-
uct for SNAP-25 was not detected in astro-
cytic mRNA, whereas the positive control
was obtained from brain mRNA. Taken
together, immunocytochemisty, Western
blots, and RT-PCR demonstrate that the
cultures used in this study were devoid of
neurons and contain GFAP-positive po-
lygonal astrocytes.
To determine for the presence of
VGLUTs 1 and 2, we submitted AsP and
LP-1 to SDS-PAGE, followed by Western
blots (Fig. 2A). We found that astrocytic
preparations showed expression of
VGLUTs 1 and 2 and also VGLUT 3, the
presence of which had been recently re-
ported in astrocytes in vivo (Fremeau et al.,
2002). VGLUTs 1 and 2 were much less
abundant in AsP than in LP-1, whereas
VGLUT3 was more abundant in AsP. Ad-
ditionally, astrocytes contained SNARE
proteins, synaptobrevin 2, syntaxin, and
SNAP-23 (Parpura et al., 1995b; Hepp et
al., 1999). To assess the specificity of anti-
bodies raised against VGLUTs (Table 1),
we first performed preadsorption controls
(Fig. 2B). We subjected AsP to the SDS-
PAGE, followed by Western blot analysis
using antibodies alone or antibodies that
were preincubated with their respective
antigens (Table 1). We detected VGLUTs only when probing
with antibodies alone but not in preadsorption controls. Next, we
performed controls for cross-reactivity of antibodies raised
against VGLUTs 1 and 2 to a VGLUT 3 antigen (Fig. 2C). Lack of
cross-reactivity of anti-VGLUTs 1 and 2 to VGLUTs 2 and 1,
respectively, had been demonstrated previously (Bellocchio et al.,
1998; Takamori et al., 2001), but not their possible cross-
reactivity to VGLUT 3. Because astrocytes in vivo express VGLUT
3, it is possible that the antibodies we used against VGLUTs 1 and
2 cross-react with VGLUT 3. To address this issue, we preincu-
bated antibodies against VGLUTs 1, 2, and 3 with a VGLUT 3
antigen. We used antibodies alone or preincubated antibodies to
probe membranes containing AsP. We found that preadsorption
with VGLUT 3 antigens abolished VGLUT detection on Western
blots only when using anti-VGLUT 3 (⫺99% of control; n⫽3)
but not for anti-VGLUT 1 (⫹32% of control; n⫽3) and anti-
VGLUT 2 (⫹11% of control; n⫽3), indicating that these anti-
bodies to not exhibit cross-reactivity to VGLUT 3 (Fig. 2C). After
assessment of the antibody specificity, we performed RT-PCR
using mRNA isolated from rat brain and from astrocytic cultures
(Fig. 2 D). PCR products for VGLUTs 1, 2, and 3 were detected in
astrocytic mRNA. Thus, Western blots and RT-PCR demonstrate
that the purified astrocytic cultures express all three known iso-
forms of VGLUTs.
Figure 4. VGLUT immunoreactivity colocalizes with vesicular marker synaptobrevin 2. A, Astrocytes were identified based on
their ability to intracellularly accumulate

-Ala-Lys-N
⑀
-AMCA (blue). Double labeling using indirect immunocytochemistry
against VGLUT 2 (B; red) reveals punctate immunoreactivity that colocalizes with

-Ala-Lys-N
⑀
-AMCA (C; overlay of Aand B; pink
indicates colocalization), as shown in X-Z (C; bottom) and Y-Z (C; right) projections from the regions identified by dotted lines.
Double labeling of astrocytes with synaptobrevin 2 (D; green) and a mixture of antibodies labeling all three isoforms of VGLUTs (E;
red) indicates that VGLUT immunoreactivity substantially overlaps with vesicular marker synaptobrevin 2 (F; overlay of Dand E;
yellow indicates colocalization). Scale bars: C, X-Z and Y-Z projections, 5
m; A–C,10
m; D–F,20
m.
Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes J. Neurosci., March 17, 2004 •24(11):2633–2642 • 2637
The intracellular localization of proteins was studied using
indirect immunocytochemistry (Fig. 3). Staining with anti-
VGLUT 1 (Fig. 3C), anti-VGLUT 2 (Fig. 3D), or anti-VGLUT 3
(Fig. 3E) showed a punctate pattern of immunoreactivity that was
present throughout the entire cell body and processes, and it was
completely abolished when antibodies were preadsorbed with
their respective antigens (Fig. 3C⬘–E⬘,F). However, consistent
with Western blot data, staining of astrocytes with anti-VGLUT 1
(n⫽68) or anti-VGLUT 2 (n⫽46) showed no reduction in
fluorescence intensities, when these antibodies were preincu-
bated with a VGLUT3 antigen. A similar pattern of punctate
immunoreactivity was observed using anti-SNAP-23 (Fig. 3B).
Synaptobrevin 2, however, was primarily located at the leading
edge of the cells, although there were puncta present throughout
the entire cell body (Figs. 3A,4D).
To further study intracellular distribution of VGLUTs, we
used double labeling. First, we used

-Ala-Lys-N
⑀
-AMCA (Dieck
et al., 1999) (also see Materials and Methods) to label astrocytes,
followed by colabeling using indirect immunocytochemistry
against VGLUTs 1, 2, or 3. In Figure 4, we show a typical distri-
bution of VGLUT 2 immunoreactivity that colocalizes with in-
tracellular astrocytic tag

-Ala-Lys-N
⑀
-AMCA, as revealed by
volume rendering after image acquisition using a laser confocal
scanning microscope (Fig. 4 A–C). Similar distributions were ob-
served for colabeling VGLUT 1 or 3 with

-Ala-Lys-N
⑀
-AMCA,
indicating predominant intracellular, rather than plasma mem-
brane, association of VGLUTs. Because VGLUTs show synaptic
vesicular localization in neurons, next we used indirect immuno-
cytochemisty to label astrocytic vesicular pool using a synaptic
vesicle marker synaptobrevin 2, while counterstaining VGLUTs
using a mixture of antibodies encompassing all three isoforms
(Fig. 4D–F). We found a substantial colocalization of VGLUTs
with synaptobrevin 2, although some of the synaptobrevin 2 im-
munoreactivity was devoid of VGLUT immunoreactivity. Be-
cause astrocytes, in addition to glutamate, can exocytotically re-
lease ATP (Coco et al., 2003) and atrial natriuretic peptide (Krzan
et al., 2003), this is possibly attributable to vesicular packaging of
these segretagogues in the absence of intravesicular glutamate,
hence some synaptobrevin 2-positive vesicles lacking VGLUTs.
Because astrocytes could de-differentiate while cultured, pos-
sibly leading to gene expression of VGLUTs, we next used freshly
(acutely) isolated astrocytes that provide information of proper-
ties without the changes in gene expression (Kimelberg et al.,
2000a,b, 2001). Astrocytes were freshly isolated from rat visual
cortices of 1-, 2-, 8-, and 55-d-old rats. To determine the identity
of astrocytes in cell mixture, we used

-Ala-Lys-N
⑀
-AMCA. We
colabeled cells using indirect immunocytochemistry against ei-
ther VGLUT 1, VGLUT 2, or VGLUT 3. Once we localized astro-
cytes of interest based on their ability to accumulate

-Ala-Lys-
N
⑀
-AMCA (Fig. 5A–C), we checked for expression of VGLUTs in
these cells. We found that freshly isolated astrocytes contained
VGLUTs in a punctate pattern similar to that seen in cultured
astrocytes (Fig. 5 A⬘–C⬘). This immunoreactivity was completely
abolished when antibodies were preadsorbed with their respec-
tive antigens (Fig. 5D) but not in astrocytes probed with anti-
VGLUT 1 (n⫽54) or anti-VGLUT 2 (n⫽56) antibodies when
these antibodies were preincubated with a VGLUT 3 antigen. The
positive immunoreactivity exhibited fluorescence intensity that
was five (for VGLUTs 1 and 2) to six (for VGLUT 3) times higher
than the intensity recorded from control astrocytes in which pri-
mary antibodies were omitted. This signal/noise ratio may indi-
cate that the expression of VGLUTs in freshly isolated astrocytes
is low. The proportion of astrocytes expressing VGLUTs declined
with the increased age of the animals (Table 2). Together, these
data demonstrate that astrocytes express VGLUTs.
Having determined that astrocytes in culture as well as freshly
isolated astrocytes possess VGLUTs, we asked whether VGLUTs
play a role in Ca
2⫹
-dependent exocytotic release of glutamate
from astrocytes. We optically monitored glutamate release into
extracellular space surrounding cultured astrocytes using GDH-
linked assay, based on accumulation of the fluorescent product
NADH. To evoke Ca
2⫹
-dependent glutamate release from astro-
Figure 5. Freshly isolated astrocytes contain VGLUTs. A–C, Astrocytes were identified based
on their ability to accumulate

-Ala-Lys-N
⑀
-AMCA. Double labeling using indirect immunocy-
tochemistry against VGLUT1 (A⬘), VGLUT2 (B⬘), or VGLUT 3 (C⬘) revealed punctate immunore-
activity that was completely abolished ( D) when primary antibodies (1
o
Ab) were preadsorbed
with their respective antigens (Ag⫹). Fluorescent immunoreactivity is expressed in intensity
units(i.u.).Bars representmeans ⫾SEMs ofmeasurements fromanumber ofindividual freshly
isolated astrocytes (n). Asterisks indicate a significant change of measurements compared with
the control group (1
o
Ab⫹,Ag⫺; one-way ANOVA, followed by post hoc Fisher’s LSD test;
**p⬍0.01). We found no difference between measurements in preadsorption controls
(1
o
Ab⫹,Ag⫹) when compared with controls in which primary antibodies were omitted
(1
o
Ab⫺,Ag⫺).Scale bar, 10
m.
Table 2. Expression of VGLUTs 1, 2, and 3 in cultured and freshly isolated (FIA)
astrocytes
Astrocytes Cultured FIA
Animal age (days) 0 –1 1–2 8 55
VGLUT 1
Tested 335 156 22 22
Positive 316 134 16 7
Percentage positive 94 86 73 32
VGLUT 2
Tested 404 113 25 26
Positive 381 112 17 9
Percentage positive 94 99 68 35
VGLUT 3
Tested 144 74 19 22
Positive 136 55 10 9
Percentage positive 94 74 53 41
2638 •J. Neurosci., March 17, 2004 •24(11):2633–2642 Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes
cytes, we used mechanical stimulation, known to cause an in-
crease in astrocytic intracellular Ca
2⫹
levels (Nedergaard, 1994;
Araque et al., 1998a), leading to glutamate release (Parpura et al.,
1994; Araque et al., 1998a). To apply mechanical stimulus, we
established a contact between an astrocyte and a patch pipette
under control of a patch-clamp amplifier. This direct stimulation
reliably caused glutamate release from as-
trocytes as indicated by a transient increase
in NADH fluorescence surrounding astro-
cytes (Fig. 6A-D) (control group, n⫽41;
peak response dF/Fo ⫽90 ⫾12%; paired t
test; p⬍0.01).
We confirmed the Ca
2⫹
and SNARE
dependency of this glutamate release (Par-
pura et al., 1994, 1995a,b; Jeftinija et al.,
1997; Bezzi et al., 1998; Araque et al., 2000;
Parpura and Haydon, 2000; Pasti et al.,
2001). We monitored the intracellular
Ca
2⫹
levels using the Ca
2⫹
indicator
fluo-3. Mechanical stimulation caused
transient Ca
2⫹
elevations (Fig. 6 E–H)
(control group, n⫽7; peak response dF/
Fo ⫽208 ⫾58%; paired ttest; p⬍0.015),
which was greatly reduced when we prein-
cubated astrocytes with BAPTA-AM (50
M; 30 min; 23% of control; n⫽8; peak
response dF/Fo ⫽47 ⫹11%; Fisher’s LSD;
p⬍0.01) (Fig. 6H). Consistent with Ca
2⫹
dependency of mechanically induced glu-
tamate release, this Ca
2⫹
chelator greatly
reduced the extracellular NADH fluores-
cence accumulation reporting on gluta-
mate release (29% of control; n⫽4; peak
response dF/Fo ⫽26 ⫾8%; Fisher’s LSD
test; p⬍0.05) (Fig. 6D).
Growing evidence indicates that the
mechanism underlying the Ca
2⫹
-
dependent glutamate release from astro-
cytes is exocytosis (Jeftinija et al., 1997;
Bezzi et al., 1998; Araque et al., 2000; Pasti
et al., 2001). Consequently, we tested the
sensitivity of mechanically induced gluta-
mate release from astrocytes to tetanus
toxin and bafilomycin A
1
.Ca
2⫹
-
dependent glutamate release from astro-
cytes can be reduced when astrocytes are
exposed to tetanus toxin that specifically
cleaves synaptobrevin 2 (Jeftinija et al.,
1997; Bezzi et al., 1998; Pasti et al., 2001).
We confirmed the presence of synaptobre-
vin 2 (Parpura et al., 1995b) in astrocytes
using Western blots (Fig. 2) and indirect
immunocytochemistry (Fig. 3). Addition-
ally, we tested functional involvement of
synaptobrevin 2 in Ca
2⫹
-dependent glu-
tamate release from astrocytes by pretreat-
ing these cells with holoprotein of tetanus
toxin (15
g/ml for 24 –48 hr). After incu-
bation with this toxin, astrocytes had re-
duced ability to release glutamate in re-
sponse to mechanical stimulation (41% of
control; n⫽13; peak response dF/Fo ⫽
37 ⫾5%; Fisher’s LSD test; p⬍0.01) (Fig.
6D), although toxin treatment did not affect mechanically in-
duced Ca
2⫹
elevations (Fig. 6H)(n⫽6).
Bafilomycin A
1
, a specific inhibitor of V-ATPase (Bowman et
al., 1988), has been shown to reduce the extent of Ca
2⫹
-
dependent glutamate release from astrocytes, by collapsing the
proton gradient necessary for vesicular storage of glutamate
Figure 6. VGLUTs mediate exocytotic glutamate release from cultured astrocytes. Mechanical stimulation evokes glutamate
release ( A–D) caused by the internal Ca
2⫹
elevations in astrocytes ( E–H). Glutamate release from astrocyte is shown (raw data)
in A(rest) and B(after stimulation). The pseudocolor scale is a linear representation of the fluorescence intensities ranging from
1240 to 1350. C, Time lapse of NADH fluorescence, reporting on glutamate release. Mechanical stimulation caused glutamate
release (black squares) that was greatly reduced when cells were preincubated with Rose Bengal (open circles; 0.5
M; 30 min),
an allosteric site modulator of VGLUTs. D, Peak values of mechanically induced glutamate release. Incubation of astrocytes with
BAPTA-AM (50
M; 30 min), a membrane-permeable Ca
2⫹
chelator, reduced the mechanically induced glutamate release.
Pretreatment of astrocytes with a holoprotein of tetanus toxin (TeTx; 15
g/ml; 24 –48 hr), which cleaves synaptobrevin 2, or
bafilomycin A
1
(5
M, 1 hr), a specific inhibitor of V-ATPase, reduces mechanically induced glutamate release consistent with
exocytoticmechanismunderlying Ca
2⫹
-dependentglutamaterelease fromastrocytes.Changes inNADHfluorescence areshown
as dF/Fo (percentage) after background subtraction and correction for bleaching. E–H, In experiments parallel to those in A–D,
astrocytes were mechanically stimulated while measuring intracellular Ca
2⫹
levels using fluo-3. This stimulation reliably in-
creasestheintracellular Ca
2⫹
levels ( F) from its resting level ( E)(rawdata). Thepseudocolor scale isa linearrepresentationof the
fluorescence intensities ranging from 0 to 255. G, Time lapse of fluo-3 fluorescence, reporting on Ca
2⫹
levels in astrocytes.
Mechanical stimulation caused an increase in astrocytic intracellular Ca
2⫹
levels (black squares) that were unaffected when cells
were preincubated with Rose Bengal (open circles; 0.5
M; 30 min). H, Peak values of mechanically induced Ca
2⫹
elevations are
greatlyreducedwhen cellsare preincubatedwithBAPTA butnot whentreatedwith TeTx,bafilomycin A
1
,orRose Bengal.Changes
in fluo-3 fluorescence are expressed as dF/Fo (percentage) after background subtraction. Arrows (C,G) indicate the time when the
pipette–astrocytecontactoccurred. Pointsand barsrepresentthe meansand SEMs. InCand G,SEMsare shownin singledirections
for clarity. Asterisks indicate a significant change of measurements compared with the control group (one-way ANOVA, followed
by post hoc Fisher’s LSD test; *p⬍0.05; **p⬍0.01). We found no significant difference between various treatments in D,
whereas in H, preincubation with BAPTA, in addition to a significant change in measurement compared with the control group,
showed a statistically significant difference when compared with all other treatments ( p⬍0.05).
Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes J. Neurosci., March 17, 2004 •24(11):2633–2642 • 2639
(Araque et al., 2000; Pasti et al., 2001). Consistent with previous
observations, incubation of astrocytes with bafilomycin A
1
(5
M; 1 hr) greatly reduced mechanically induced glutamate release
(22% of control; n⫽9; peak response dF/Fo ⫽20 ⫾5%; Fisher’s
LSD test; p⬍0.01) (Fig. 6D), without significantly affecting
Ca
2⫹
elevations (Fig. 6H;n⫽6).
After demonstration that mechanically induced glutamate re-
lease is mediated by exocytosis, we incubated astrocytes with Rose
Bengal (0.5
M; 30 min), an inhibitor of vesicular glutamate up-
take in synaptic vesicles via allosteric modulation of VGLUTs
(Ogita et al., 2001; Schafer et al., 2002). We found that this treat-
ment abolished mechanically induced glutamate release from as-
trocytes (Fig. 6C,D) (3% of control; n⫽6; dF/Fo ⫽2⫾2%;
Fisher’s LSD; p⬍0.01) without affecting Ca
2⫹
elevations (Fig.
6G;n⫽7). Together, these data indicate that VGLUTs mediate
mechanically induced exocytotic release from astrocytes.
Although the presented data are consistent with the notion
that glutamate released from astrocyte is stored in acidic com-
partments that are sensitive to bafilomycin A
1
and Rose Bengal, it
is important to confirm specificity of these pharmacological
agents. To address this issue, we transfected the cultured cortical
astrocytes using a plasmid encoding for synapto-pHluorin (San-
karanarayanan et al., 2000), a fusion protein that contains a pH-
sensitive and fluorescent protein, pHluorin, attached to the C
terminus of synaptobrevin 2. Because pHluorin is attached to the
lumenal (intravesicular) portion of synaptobrevin, it is sensing
intravesicular pH with its fluorescence mainly quenched at rest-
ing intravesicular pH (⬃5.5). However, blockade of V-ATPase
using bafilomycin A
1
can collapse proton gradient, leading to
alklinization of intravesicular lumen and increase in pHluorins
fluorescence (Sankaranarayanan and Ryan, 2001). Incubation of
astrocytes with bafilomycin A
1
(5
M; 30 and 60 min) caused a
time-dependent increase in synapto-pHluorin fluorescence
(each group, n⫽8; paired ttest with Bonferroni adjustment for
multiple comparisons; p⬍0.01) (Fig. 7 A,B,E). Furthermore,
synapto-pHluorin fluorescence showed a punctate pattern con-
sistent with the vesicular nature of exocytosis (Fig. 7B). Although
it has been reported that Rose Bengal, at submicromolar concen-
trations used in the present study, has a much higher affinity for
VGLUTs than for V-ATPase (Ogita et al., 2001), it is necessary to
confirm that Rose Bengal does not reduce glutamate release as a
result of inhibition of V-ATPase, thereby reducing the proton
gradient necessary for glutamate uptake into vesicular lumen, as
opposed to its action on allosteric site of VGLUTs. Vesicular
proton shunt through VGLUTs reduces the intravascular proton
concentration created by V-ATPase. By inhibiting VGLUTs and
their proton-shunting activity, Rose Bengal would lead to vesic-
ular acidification and the reduction of synapto-pHluorin fluores-
cence. Incubation of astrocytes with Rose Bengal (0.1 or 0.5
M;
30 min) caused a reduction of pHluorin fluorescence (0.1
M,
n⫽6; paired ttest; p⬍0.05; 0.5
M,n⫽10; paired ttest; p⬍
0.01), consistent with its action on VGLUTs (Fig. 7C–E).
Whereas mechanical stimulation offers direct stimulation of
astrocytes without receptor activation and mimics the action of
endogenous ligands (Sanzgiri et al., 1999), it is not a physiological
stimulus. Consequently, to further study VGLUT-dependent
exocytotic glutamate release from astrocytes, we determined the
effects of Rose Bengal on astrocytes stimulated by bradykinin (90
nM; 55 sec) or ATP (9
M; 55 sec), endogenous ligands known to
stimulate Ca
2⫹
-dependent glutamate release (Parpura et al.,
1994, 1995a; Jeftinija et al., 1997; Jeremic et al., 2001). Both bra-
Figure 7. Bafilomycin A
1
alkalinizes vesicular lumen, whereas Rose Bengal acidifies it, con-
sistent with the actions of these agents on V-ATPase and VGLUTs, respectively. A–D, Astrocytes
expressing synapto-pHluorin (raw data). The addition of bafilomycin A
1
to the astrocytes at rest
expressing synapto-pHluorin ( A) causes an increase in pHluorin fluorescence (B,E). Quite the
contrary, the addition of Rose Bengal to synapto-pHluorin-expressing astrocytes at rest (C)
causes decrease of pHluorin fluorescence (D,E). The image in Bis acquired after incubation with
bafilomycin A
1
(5
M; 60 min), whereas the image in Dis acquired after incubation with Rose
Bengal (0.5
M; 30 min). The pseudocolor scale is a linear representation of the fluorescence
intensities ranging from 0 to 255. Changes in synapto-pHluorin fluorescence are expressed as
dF/Fo (percentage) after background subtraction. Bars represent the means and SEMs. Concen-
trations (c) and exposure times (t) of pharmacological agents are given in micromolars and
minutes, respectively.
Figure 8. Rose Bengal reduces bradykinin- and ATP-evoked Ca
2⫹
-dependent glutamate
release from astrocytes. A, Stimulation of astrocytes with bath-applied (55 sec) bradykinin (90
nM) or ATP (9
M) causes glutamate release (A, black bars) and the increase in internal Ca
2⫹
levels (B, black bars). A,B, This agonist-induced glutamate release from astrocytes was greatly
reduced when astrocytes were preincubated with Rose Bengal (0.1
Mfor 30 min; A, open
bars),althoughastrocytic internalCa
2⫹
elevationswerenot affected(B, open bars).Changes in
NADH fluorescence, reporting on glutamate release, are shown as dF/Fo (percentage) after
background subtraction and correction for bleaching. Changes in fluo-3 fluorescence ( B), re-
porting on internal Ca
2⫹
levels, are expressed as dF/Fo (percentage) after background subtrac-
tion. Bars represent the means and SEMs. Asterisks indicate a significant change of measure-
ments compared with the matching control group (Student’s ttest; **p⬍0.01).
2640 •J. Neurosci., March 17, 2004 •24(11):2633–2642 Montana et al. •VGLUT-Dependent Glutamate Release from Astrocytes
dykinin (n⫽19) and ATP (n⫽33) caused glutamate release
from astrocytes (paired ttests; p⬍0.01); release magnitudes were
reduced to 44% (n⫽15) and 47% (n⫽21) of control, respec-
tively (Student’sttests; p⬍0.01), when we preincubated astro-
cytes with Rose Bengal (0.1
M; 30 min) (Fig. 8A). Consistent
with preservation of Ca
2⫹
mobilization by bradykinin or ATP,
Rose Bengal did not affect ligand-induced Ca
2⫹
elevations in
astrocytes (Fig. 8 B)(n⫽10 for bradykinin alone and n⫽9 for
bradykinin with Rose Bengal; n⫽26 for ATP alone and n⫽18
for ATP with Rose Bengal; Student’sttests; p⬎0.4). Thus, three
distinct stimuli, mechanical contact, bradykinin, and ATP, can
raise intracellular Ca
2⫹
levels and cause glutamate release from
astrocytes; release magnitudes can be significantly reduced when
we pharmacologically interfered with VGLUTs.
Discussion
Our findings indicate that the expression of VGLUTs 1 and 2 is
not restricted to neurons but that these proteins necessary for
glutamate accumulation in synaptic vesicles are also found in
astrocytes, as determined by using purified astrocytic culture and
Western blots. Immunocytochemisty performed on both cul-
tured and freshly isolated astrocytes reveals punctate pattern of
immunoreactivity consistent with the possible role of VGLUTs in
exocytotic glutamate release from astrocytes. Furthermore, me-
chanically, bradykinin-, and ATP-induced glutamate release
from astrocytes was greatly reduced when astrocytes were prein-
cubated with Rose Bengal, a broad spectrum modulator of
VGLUTs allosteric site, indicating a functional role of VGLUTs in
this release.
Interestingly, in freshly isolated astrocytes originating from 1-
to 2-d-old rat pups, there is a high likelihood that VGLUTs 1, 2,
and 3 are co expressed in a single cell (Table 2). Although we did
not directly demonstrate the presence of these proteins in single
cells, the proportion of cells expressing individual proteins (86%
for VGLUT 1, 99% for VGLUT 2, and 74% for VGLUT 3) sup-
ports this inference. However, the probability of dual or triple
expression of VGLUTs in individual astrocytes was greatly re-
duced when we studied astrocytes isolated from 55-d-old ani-
mals, as was the proportion of astrocytes expressing individual
VGLUTs. Why would individual astrocytes in the developing
brains of postnatal animals simultaneously express two or three
different VGLUTs? One possible explanation is that the Ca
2⫹
-
dependent release of glutamate is important for astrocytic roles in
modulation of synaptic transmission and that it is necessary to
have protein redundancy to ensure astrocytic contribution to the
physiology in the CNS. An alternative explanation could be that
different VGLUTs have specialized functions in astrocytic gluta-
mate metabolism and/or release. For example, it is entirely pos-
sible that VGLUT isoforms are exclusively expressed in a different
subpopulation of vesicles within individual astrocytes. These
subpopulations of vesicles could represent distinct releasable
pools that could control spatiotemporal characteristics of gluta-
mate release. Such a possibility could explain different forms of
neuronal responses to glutamate released from astrocytes, in-
cluding internal Ca
2⫹
increases (Parpura et al., 1994), slow-
inward currents (Araque et al., 1998a; Parpura and Haydon,
2000), and modulation of spontaneous and action potential-
evoked synaptic transmissions (Araque et al., 1998b; Kang et al.,
1998). In mature animals, however, VGLUT isoforms 1 and 2 are
each expressed in approximately one-third of the astrocytic pop-
ulation (32 and 35%, respectively). Because VGLUT 3 is ex-
pressed in a similar proportion (41%), it is tempting to suggest
that astrocytes could show highly complementary distributions
of these three proteins, such as is found in neurons (Herzog et al.,
2001; Fremeau et al., 2002; Schafer et al., 2002). An alternative
extreme possibility is that only one-third of astrocytes express
VGLUTs but all isoforms. Subsequent work will be necessary to
resolve these issues. Consequently, it should become possible to
selectively manipulate individual VGLUTs in astrocytes to test
for their roles in glutamate-mediated astrocyte-neuron signaling
in health and disease.
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