Cerebral Cortex Advance Access published August 1, 2011
Cerebral Cortex
doi:10.1093/cercor/bhr203
Functional and Anatomical Identification of a Vesicular Transporter Mediating Neuronal
ATP Release
Max Larsson1, Keisuke Sawada2, Cecilie Morland1, Miki Hiasa2, Lasse Ormel1, Yoshinori Moriyama2 and Vidar Gundersen1,3
1
Department of Anatomy and Centre for Molecular Biology and Neuroscience, University of Oslo, N-0317 Oslo, Norway,
Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama 700-8530, Japan and 3Department of Neurology, Oslo University Hospital, N-0424 Oslo, Norway
2
Address correspondence to Vidar Gundersen, Department of Anatomy and Centre for Molecular Biology and Neuroscience, University of Oslo, PO
Box 1105 Blindern, N-0317 Oslo, Norway. Email: vidar.gundersen@medisin.uio.no.
Keywords: exocytosis, neurotransmitter, purinergic, ultrastructure, uptake
ATP exerts its transmitter effects through activation of
ionotropic (P2X) and metabotropic (P2Y) receptors. Both P2X
and P2Y receptors are widely distributed in neural tissue
(Nörenberg and Illes 2000; Fields and Burnstock 2006). P2X
receptor-mediated excitatory synaptic transmission has been
demonstrated in several regions of the central nervous system
(Edwards et al. 1992; Bardoni et al. 1997; Pankratov et al. 1998,
2002; Mori et al. 2001). In the hippocampus (Pankratov et al.
1998, 2006), as well as in cortex (Pankratov et al. 2002, 2003,
2007), results from electrophysiological studies have suggested
that ATP is coreleased with glutamate. A recent study showed
that ATP is released during stimulation of glutamatergic
neuronal pathways (Jourdain et al. 2007). However, it is not
clear whether ATP and glutamate reside in the same nerve
terminals, let alone in the same synaptic vesicle pools.
Identification of cellular elements that may release ATP has
previously been hampered by lack of ATP-specific antibodies.
However, such information can now be obtained using
antibodies against VNUT. Here, we use anatomical, biochemical, and functional approaches to investigate the vesicular
localization and release of ATP in hippocampal neurons.
Introduction
Materials and Methods
Extracellular ATP has a multifaceted role as a signaling molecule
in intercellular communication. In the nervous system, gliaderived ATP has been widely implicated as an extracellular
messenger and as a precursor of adenosine in glia-to-glia and gliato-neuron interaction (Koizumi et al. 2005). It is furthermore
known that ATP may act as an excitatory neurotransmitter in
several brain regions, including the hippocampus (Pankratov
et al. 1998, 2006; Mori et al. 2001; Fields and Burnstock 2006).
Considerable effort has been devoted to attempts at clarifying
the mechanism of release of ATP from glial cells (Stout et al. 2002;
Kang et al. 2008; Liu, Sabirov, et al. 2008; Liu, Touchiev, et al.
2008), although controversy regarding this issue remains
(Hamilton and Attwell 2010). By contrast, how ATP is released
from central neurons has not been well studied. A vesicular
nature of neuronal ATP release was first indicated by observations of calcium-dependent release of ATP from whole-brain
synaptosomes (White 1978) and several types of brain slice
preparation, including hippocampal slices (Wieraszko et al.
1989; Cunha et al. 1996). Furthermore, ATP is taken up by
isolated synaptic vesicles (Gualix et al. 1999). Recently,
a vesicular nucleotide transporter (VNUT) capable of transporting ATP into vesicles was identified (Sawada et al. 2008).
VNUT was shown to be present in the brain (Sawada et al. 2008),
but its cellular and subcellular distribution is not known.
Expression and Purification of Mouse VNUT
A cDNA-encoding mouse VNUT (mVNUT) was cloned (Sawada et al.
2008). Recombinant baculovirus containing mVNUT cDNA were
constructed using the Bac-to-Bac baculovirus expression system
(Invitrogen) according to the manufacturer’s protocol. mVNUT cDNA
was amplified by polymerase chain reaction (PCR) using the primers 5#CACCATGCCATCCCAGCGCTCTAGC-3# and 5#-TTAGAGTCCTCATGAGTGG-3#. High Five cells (1 3 107 cells/10 cm dish) were grown at
27 C in Express Five medium (Invitrogen) supplemented with 2 mM Lglutamine and 10 lg/mL gentamicin. Cells were infected by recombinant
baculoviruses at a multiplicity of infection of 2, cultured for 48 h for
High Five cells and harvested for membrane preparation. High Five cells
(1--2 3 108 cells) were suspended in a 20 mM Tris--HCl buffer (pH 8.0)
containing 0.1 M potassium acetate, 10% glycerol, 0.5 mM dithiothreitol,
10 lg/mL pepstatin A, and 10 lg/mL leupeptin and disrupted by
sonication with a TOMY UD200 tip sonifier. Cell lysates were centrifuged
at 700 3 g for 10 min to remove debris, and the resultant supernatant was
centrifuged at 160 000 3 g for 1 h. The pellet (membrane fraction) was
suspended in buffer containing 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS)--Tris (pH 7.0), 10% glycerol, 10 lg/mL pepstatin A, and
10 lg/mL leupeptin to a concentration of approximately 1.5 mg protein/
mL. The membrane fraction was solubilized with 2% octylglucoside. After
centrifugation at 260 000 3 g for 30 min, the supernatant was added to
1 mL of Ni-NTA Superflow resin (Qiagen). The resin was incubated for 4 h
at 4 C and washed with 10 mL of 20 mM MOPS--Tris (pH 7.0) containing
5 mM imidazole, 20% glycerol, and 1% octylglucoside in a column.
mVNUT was eluted from the resin with 3 mL of the same buffer
The Author 2011. Published by Oxford University Press. All rights reserved.
For permissions, please e-mail: journals.permissions@oup.com
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
ATP is known to be coreleased with glutamate at certain central
synapses. However, the nature of its release is controversial. Here,
we demonstrate that ATP release from cultured rat hippocampal
neurons is sensitive to RNAi-mediated knockdown of the recently
identified vesicular nucleotide transporter (VNUT or SLC17A9). In
the intact brain, light microscopy showed particularly strong VNUT
immunoreactivity in the cerebellar cortex, the olfactory bulb, and
the hippocampus. Using immunoelectron microscopy, we found
VNUT immunoreactivity colocalized with synaptic vesicles in
excitatory and inhibitory terminals in the hippocampal formation.
Moreover, VNUT immunolabeling, unlike that of the vesicular
glutamate transporter VGLUT1, was enriched in preterminal axons
and present in postsynaptic dendritic spines. Immunoisolation of
synaptic vesicles indicated presence of VNUT in a subset of
VGLUT1-containing vesicles. Thus, we conclude that VNUT
mediates transport of ATP into synaptic vesicles of hippocampal
neurons, thereby conferring a purinergic phenotype to these cells.
following primers were used: VNUT, TGTGGTAGGCGTGTGTCTAG
(forward), AGGTTGCTGACGATGGCCAC (reverse).
Reconstitution and ATP Transport
Reconstitution of purified mVNUT was carried out by the freeze--thaw
method as described previously (Sawada et al. 2008). In brief, 10 lg
mVNUT was mixed with liposomes (0.5 mg asolectin, Sigma type II-S),
frozen at –80 C. After 5 min, the mixture was thawed quickly and
diluted 60-fold with 20 mM MOPS--Tris (pH 7.0) with 0.5 mM
dithiothreitol, 0.15 M sodium acetate, and 5 mM magnesium acetate.
Reconstituted proteoliposomes were sedimented by centrifugation at
160 000 3 g for 1 h at 4 C and suspended in 0.4 mL of the same buffer.
For ATP transport assay, reconstituted proteoliposomes (0.5 lg
protein per assay) were suspended in 500 lL of 20 mM MOPS--Tris
(pH 7.0) containing 5 mM magnesium acetate, 4 mM KCl, and 0.15 M
potassium acetate and incubated for 2 min at 27 C. Valinomycin was
added to give a final concentration of 2 lM, and the mixture was
incubated for an additional 2 min. The assay was initiated by addition of
0.1 mM [a-32P]ATP (3.7 GBq/mmol). Aliquots (130 lL) were taken at the
times indicated and centrifuged through a Sephadex G-50 (fine) spin
column at 760 3 g for 2 min. Radioactivity and protein concentration of
the eluate were measured. To determine GTP or ADP transport,
[a-32P]GTP (3.7 GBq/mmol) or [2,8-3H]ADP (0.37 GBq/mmol) was used
as substrates instead of ATP.
Antibodies
Site-specific polyclonal antibodies against mVNUT were prepared by
repeatedly injecting GST-fusion polypeptides encoding LMQPIPEETRKTPSAAAEDTRWSRPECQAWTGILLLGTCLLYCARVTMPVCTVAMSQDFGWNKKEAGIVLS SFFWGYCLTQVVGGHLGDR (L8--R97)
(Sawada et al. 2008). Antibodies against V-ATPase subunit A and
synaptotagmin 1 (kindly supplied by Prof. Masami Takahashi, Kitasato
University, Japan) have been described (Shoji-Kasai et al. 1992; Moriyama
et al. 1995). A mouse anti-synaptophysin antibody (clone SY-38;
Wiedenmann and Franke 1985) was obtained from PROGEN (Heidelberg,
Germany). A mouse monoclonal antibody against MAP2A-C (clone HM-2)
was purchased from Sigma. Two guinea pig anti-VGLUT1 antibodies from
Millipore (AB5905) and Synaptic Systems (cat. no. 135 304; Göttingen,
Germany), one rabbit anti-VGLUT1 (Synaptic Systems; cat. no. 135 302),
one mouse anti-VGLUT1 (UC Davis/NIH NeuroMab Facility; clone N28/
9), and a VGAT antibody raised in guinea pig (Synaptic Systems; cat. no.
131 004) were used. AB5905 has been used extensively for immunohistochemical detection of VGLUT1 (e.g., Persson et al. 2006). The VGLUT1
and VGAT antibodies from Synaptic Systems detect bands of appropriate
sizes by immunoblotting of rat brain tissue (manufacturer’s specification;
L. Ormel, V. Gundersen, unpublished data) and have previously been
successfully used for postembedding immunogold labeling (Zander et al.
2010). The mouse anti-VGLUT1 from NeuroMab detects a single band at
~50 kDa (manufacturer’s specification) and has been extensively used
for immunohistochemistry (e.g., Linhoff et al. 2009). Alexa Fluor 488labeled goat anti-rabbit IgG and Alexa Fluor 568- or Alexa Fluor 555labeled goat anti-mouse IgG were obtained from Invitrogen (Molecular
Probes). Biotinylated donkey anti-rabbit Ig was from GE Healthcare,
whereas 10 nm colloidal gold-conjugated goat anti-rabbit IgG, 5 nm
colloidal gold-conjugated goat F(ab)2 anti-rabbit IgG, and 15 nm colloidal
gold-conjugated goat anti-guinea pig IgG were obtained from British
Biocell International (Cardiff, UK).
RNAi-Mediated VNUT Knockdown
Neuronal cultures were prepared from the hippocampus of male Wistar
rats (E17). Briefly, after dissection, the hippocampi were dissociated by
treatment with trypsin (0.25% for 15 min at 37 C) in the presence of
DNase (0.01%). Cells (5 3 105 cells/35 mm dish) were plated on poly-Llysine and laminin-coated cell culture dish and grown in Neurobasal
medium (Invitrogen) containing B27 supplement (Invitrogen). The
culture medium was exchanged every 2--3 days. Experiments were
performed between 18 and 21 days in culture. For gene knockdown,
HiPerFect transfection reagent (Qiagen) was used for transfection of
10 nM AllStars negative control small interfering RNA (siRNA) or rat
SLC17A9 siRNA: UAUUCGAGAGAAUGUCACG. ATP secretion was
assayed after 48 h incubation.
Stimulation of Neuronal Cultures and Measurement of ATP
Release
ATP secretion was assayed as described (Sawada et al. 2008). Briefly,
the cells (5 3 105 cells/35 mm dish) were incubated in 1.5 mL of
medium consisting of 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH2PO4, 1.3 mM
MgSO4, 26 mM NaHCO3, 2.4 mM CaCl2, 10 mM glucose, 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/Tris (pH
7.4), and 0.2% bovine serum albumin (BSA) for 30 min at 37 C. Then,
the cultured neurons were incubated under control conditions (1.9 mM
KCl) or stimulated in depolarizing conditions (50 mM KCl) for 20 min
(37 C). The effect of preincubation with control and VNUT siRNA (48 h,
see above) and tetanus toxin (15 lg/mL, 36 h), as well as the effect of low
+
+
+
Ca2 (0 mM Ca2 or addition of EGTA-AM [50 lM]) and the Ca2 ionophore
A23186 (5 lM) on extracellular ATP concentration was measured under
control and depolarizing conditions. Assay medium (1 mL) was collected
under various experimental conditions, kept on ice, and 50 lL aliquots
used for measuring ATP concentration with a bioluminescence method
using a kit (Invitrogen). A 50 lL aliquot of the luciferin--luciferase reaction
medium was added to each cuvette. The resulting light signal was
measured with BLR-101C Luminescence Reader (Aloka).
Real-Time PCR
RNA was purified from cultured hippocampal neurons (8 3 106 cells)
using RNeasy kit (Qiagen) according to the manufacturer’s instructions.
Real-time quantitative PCR was performed using SYBR Premix Ex Taq
II2 (TARARA BIO, Japan) containing the double-stranded DNA-binding
fluorescent probe Sybr Green and all necessary components except
primers. Quantitative PCR conditions included an initial denaturation
step of 94 C for 10 min followed by 40 cycles of 94 C for 15 s, and
55 C for 15 s. Standards and samples were analyzed in triplicate. The
Page 2 of 12 VNUT-Mediated Neuronal ATP Release
d
Larsson et al.
Western Blotting on Cultured Neurons
The cells (2 3 106 cells) were suspended in the 20 mM MOPS--Tris (pH
7.0) containing 5 mM ethylenediaminetetraacetic acid (EDTA), 0.25 M
sucrose, 10 lg/mL pepstatin A, and 10 lg/mL leupeptin and disrupted by
sonication with TOMY UD200 tip sonifier. Cell lysates were centrifuged
at 700 3 g for 10 min to remove debris, and the resultant supernatant was
centrifuged at 160 000 3 g for 1 h. The pellet was suspended in the same
buffer, and proteins were dissolved with sample buffer containing 10%
sodium dodecyl sulfate (SDS) and 10% b-mercaptoethanol. Polyacrylamide gel electrophoresis (PAGE) in the presence of SDS and western
blotting were performed as described (Sawada et al. 2008).
Immunofluorescence Miscroscopy on Cultured Neurons
Indirect immunofluorescence microscopy was performed as previously
described (Sawada et al. 2008). Cultured cells on poly-L-lysine-coated
coverslips were fixed with 4% paraformaldehyde in phosphate-buffered
saline (PBS) for 30 min. After washing with PBS, cells were incubated
for 15 min in PBS containing 0.2% saponin, 2% normal goat serum, and
0.5% BSA. The specimens were incubated with the primary antibodies
(anti-VNUT diluted 1:200 and anti-synaptophysin diluted 1:50 or antisynaptotagmin diluted 1:500) in PBS containing 0.5% BSA for 1 h at
room temperature. Samples were washed 4 times with PBS and then
reacted with Alexa Fluor 568-labeled anti-mouse IgG (2 lg/mL) or
Alexa Fluor 488-labeled anti-rabbit IgG (4 lg/mL) for 1 h at room
temperature. Finally, immunoreactivity was examined in an Olympus
FV300 confocal laser microscope.
Tissue Preparation for Light and Electron Microscopy
Adult Wistar rats were anesthetized using pentobarbital (60 mg/kg,
intraperitoneally) and transcardially perfused with PBS containing 4%
paraformaldehyde (for immunoperoxidase and immunofluorescence
labeling; 3 rats) or 4% paraformaldehyde and 0.1 or 0.5% glutaraldehyde
(for electron microscopy; 3 rats). Animal use was in accordance with the
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
containing 60 mM imidazole. The eluate containing purified mVNUT was
stored at –80 C until use.
guidelines of the Norwegian Committees on Animal Experimentation
(Norwegian Animal Welfare Act and European Communities Council
Directive of 24 November 1986-86/609/EEC). Rat hippocampal tissue
was embedded for postembedding immunogold labeling essentially as
described (Bergersen et al. 2008). Briefly, tissue encompassing the
hippocampal CA1 region and the dentate gyrus was dissected from the
brain and cryoprotected in graded solutions of glycerol. The tissue was
subsequently freeze-substituted and embedded at low temperature in
Lowicryl HM20. Ultrathin sections were cut and collected on Ni mesh
grids (one section per grid).
Postembedding Immunogold Labeling
A modified standard postembedding immunogold protocol was used
(Larsson and Broman 2005; Bergersen et al. 2008). Ultrathin sections
were incubated in drops in a humid chamber and rinsed by dipping 3 3 5
s in beakers containing the rinsing solution. The sections were incubated
in 50 mM glycine in Tris-buffered saline (0.9% NaCl; pH 7.4) with 0.1%
Triton X-100 (TBST) for 10 min. After rinsing in TBST and incubation in
blocking buffer (10 min), the sections were incubated in primary
antibody in blocking buffer overnight at room temperature. In VNUT
single-immunolabeling and VNUT/VGLUT1 double-immunolabeling
experiments, TBST containing 2% human serum albumin was used as
blocking buffer; for VNUT/VGAT double immunolabeling, the blocking
buffer was supplemented with 5% normal goat serum. The sections were
subjected twice to a rinsing step consisting of dipping in TBST and a
10-min incubation in TBST, which was followed by incubation in
blocking buffer (10 min) and secondary antibody solution (2 h). After
rinsing in H2O, the sections were counterstained with uranyl acetate and
lead citrate (the latter omitted when using 5 nm gold) prior to
examination in a FEI Tecnai 12 or G2 Spirit electron microscope. The
rabbit anti-VNUT antibody was used at a concentration of 1:300 for all
experiments except the VNUT/VGAT double-immunolabeling experiment, where the concentration was 1:200. The 2 guinea pig antiVGLUT1 antibodies (Synaptic System and Millipore) were used in
combination, both at a concentration of 1: 2000, while guinea pig antiVGAT was used at a concentration of 1:200. Gold-conjugated secondary
antibodies were diluted in blocking buffer. In single VNUT immunogold
labeling experiments, goat anti-rabbit conjugated to 10 nm gold (at
a concentration of 1:20) was used, whereas in double-labeling experiments, the secondary antibodies were goat F(ab)2 anti-rabbit conjugated
to 5 nm gold (1:40) and goat anti-guinea pig conjugated to 15 nm gold
(1:20). Preabsorption of the VNUT antibody using the immunizing
peptide at a concentration of 40 lg/mL substantially reduced the
immunogold labeling, similar to what has been previously observed by
Quantitative Analysis of Immunogold Labeling
Neuropil from the outer two-thirds of molecular layer of the dentate
gyrus and from CA1 stratum radiatum as well as inhibitory terminals in
the dentate granule and CA1 pyramidal cell layers were subject to
quantitative analysis of single VNUT immunogold labeling in sections
from 3 different rats. Sections through the dentate granule cell layer
from 2 rats were used for quantitative analysis of double VNUT/VGLUT1
and VNUT/VGAT immunogold labeling. For each animal, analysis of all
regions was performed in the same section. Random electron micrographs containing profiles of interest were obtained at a magnification of
43 0003 using a Veleta digital camera. A plugin for ImageJ (http://
rsb.info.nih.gov/ij/) was used to outline the plasma membrane of the
profile and record the locations of the center of each gold particle in the
profile; in dendritic spines, the postsynaptic density (PSD) was also
outlined. Random points were placed over the micrograph. Recorded
coordinates were submitted to a program written in Python (http://
www.python.org) for computation of particle densities and particle-membrane and particle--particle distances (Larsson and Broman 2005).
The source code of the ImageJ plugin and the Python program is
available at http://www.neuro.ki.se/broman/maxl/software.html. Mitochondria and PSDs were excluded from the analysis. Gold particle
density over basal lamina of the capillary endothelium was used as
a measure of nonspecific background immunolabeling. Electron micrographs used for figures were adjusted for brightness and contrast in
Adobe Photoshop CS2.
Immunoisolation of Synaptic Vesicles
Male Wistar rats (4--5 weeks old) were anesthetized with halothane and
decapitated. This procedure was in accordance with the guidelines of
the Norwegian Committees on Animal Experimentation as above. The
forebrains were quickly removed, put on ice, and homogenized (10
strokes at 450 rpm) in 0.32 M sucrose. After centrifugation of the
homogenate at 1000 3 g for 10 min, the supernatant (S1) was collected
and centrifuged at 21 000 3 g for 30 min. The resulting pellet (P2) was
resuspended in ice-cold H2O, homogenized (7 strokes at 450 rpm), and
centrifuged at 21 000 3 g for 30 min. The supernatant (S3) from this
centrifugation was supplemented at a proportion of 9:1 with 1 M
dipotassium tartrate in 0.25 M HEPES (pH 7.4) and centrifuged at
120 000 3 g for 60 min. The resulting pellet (P4) resuspended in 0.32 M
sucrose was used as starting material for immunoisolation of synaptic
vesicles.
For immunoisolation, protein A-coated Dynabeads (Invitrogen) were
incubated with primary antibodies diluted 1:4 (guinea pig anti-VGLUT1
from Synaptic Systems) or 1:9 (anti-VNUT) in 0.1 M phosphate buffer
(pH 8; PB) at room temperature for 2 h. After washing in PB, the beads
were incubated in 1% BSA in PB at room temperature for 1 h. Vesicle
suspension prepared as above was added to the beads, and this mixture
was incubated overnight at 4 C. The beads were put on a magnet and
the supernatant (material not bound to the beads) collected and diluted
1:1 in 1% SDS and 5 mM EDTA in 10 mM phosphate buffer (pH 7.4).
After washing, material bound to the beads was eluted by incubation of
the beads in 1% SDS and 5 mM EDTA in 10 mM phosphate buffer (pH
7.4) at room temperature for 15 min, followed by brief centrifugation
and collecting of the eluate on magnet. The supernatants and eluates
were subjected to SDS--PAGE (3 lg protein per lane) and western
blotting according to standard protocol. The blots were incubated
overnight at 4 C in primary antibody (rabbit anti-VGLUT1 (from
Synaptic Systems) or anti-VNUT both diluted 1:1000) in 1% BSA in Trisbuffered saline (pH 7.4) with 0.05% Tween 20.
Results
Functional Characterization of mVNUT
Mammalian VNUT orthologues show some interspecies
sequence variation in the N-terminal domain but are rather
Cerebral Cortex Page 3 of 12
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Immunoperoxidase and Immunofluorescence Labeling of Brain
Tissue
Coronal or parasagittal 40 lm sections of the brain were cut on a freezing
microtome, and immunohistochemistry was performed on free-floating
sections according to standard immunofluorescence and immunoperoxidase protocols. The sections were incubated overnight, for 36 or 60 h
at room temperature in anti-VNUT antibody diluted 1:1000 or 1:1500 in
PBS with 3% normal goat serum, 0.5% BSA, and 0.5% Triton X-100. In
immunofluorescence experiments, the primary antibody solution was
supplemented with mouse anti-VGLUT1 (NeuroMab) diluted 1:500 or
1:1500 or mouse anti-MAP2 diluted 1:500. For immunofluorescent
detection, sections were incubated in goat anti-rabbit Alexa Fluor 488
and goat anti-mouse Alexa Fluor 555 (Invitrogen). For immunoperoxidase detection, the sections were incubated sequentially in biotinylated
donkey anti-rabbit and streptavidin-horseradish peroxidase. Vector SG
(Vector Laboratories, Burlingame, CA) or 3,3#-diaminobenzidine (Sigma)
was used as chromogen. Confocal microscopy of immunofluorescence
labeling was performed using a 633 oil immersion objective with a Zeiss
LSM 5 Pa confocal unit mounted on a Zeiss Axoplan 2 microscope.
Micrographs of immunoperoxidase-labeled sections were obtained with
a 203 objective in a Zeiss Axioplan 2 microscope or a 1003 oil immersion
objective in a Nikon Optiphot2 microscope. A Zeiss SteREO Lumar. V12
stereomicroscope was used to acquire low-power whole-brain micrographs. For presentation, brightness and contrast were adjusted using
Adobe Photoshop CS2.
western blot and immunofluorescence microscopy (Sawada et al. 2008;
Iwatsuki et al. 2009; Haanes and Novak 2010).
cyanatostilbene-2,2#-disulphonic acid and Evans Blue suppressed
ATP uptake by mVNUT. Finally, similar to human VNUT (Sawada
et al. 2008), mVNUT was found to transport ADP and GTP
(Supplementary Fig. S1).
VNUT-Mediated ATP Release from Hippocampal Neurons
Expression of VNUT in cultured rat hippocampal neurons was
detected by western blot analysis and indirect immunofluorescence (Fig. 2A,B). RNAi-mediated knockdown of VNUT in
hippocampal neuronal cultures robustly decreased VNUT mRNA
and protein levels as determined by real-time PCR analysis,
western blot and immunocytochemistry (Fig. 2A--C). VNUT
immunofluorescence showed a somatoneuritic staining pattern,
only partly overlapping with the presynaptic terminal markers
synaptophysin and synaptotagmin 1 (Fig. 2B), suggesting that
VNUT was located in several neuronal compartments
(see below). VNUT knockdown attenuated K+-evoked but not
basal ATP release (Fig. 2D). K+-evoked ATP release was abolished
in the absence of extracellular Ca2+ or in the presence of the cellpermeable Ca2+ chelator EGTA-AM. Tetanus toxin prevented
K+-evoked ATP release (Fig. 2D,E). Furthermore, the Ca2+
ionophore A23187 induced ATP release from hippocampal
neuronal cultures (Fig. 2D). These observations suggest that
evoked release of ATP from hippocampal neurons occurs by
exocytosis of vesicles loaded with ATP by a VNUT-mediated
mechanism.
Figure 1. Functional characterization of mVNUT. (A) Purification of mVNUT. Purified mVNUT (10 lg protein) was run on SDS-gel electrophoresis in the presence of SDS and
stained with Coomassie brilliant blue. The positions of molecular standards are indicated. (B) Time course of [a-32P]ATP uptake. Naþ-trapped proteoliposomes were suspended in
the presence or absence of valinomycin. Upon the addition of [a-32P]ATP, samples were taken at the indicated times, and radioactivity taken up by the proteoliposomes was
counted. (C) Dose dependence of [a-32P]ATP uptake. ATP uptake at 1 min was determined under the indicated ATP concentrations. (D) [a-32P]ATP uptake was determined in the
presence of various nucleotides at 1 mM. Each experiment was repeated twice. Error bars indicate standard error of the mean. **P \ 0.01, ***P \ 0.001.
Page 4 of 12 VNUT-Mediated Neuronal ATP Release
d
Larsson et al.
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
highly conserved in other regions (Sawada et al. 2008). We
first assessed whether rodent VNUT exhibits similar functional properties to the human orthologue that was previously characterized (Sawada et al. 2008). Heterologously
expressed mVNUT was purified and reconstituted into
liposomes (Fig. 1A). As expected, liposomes possessing
mVNUT accumulated [a-32P]ATP in a manner dependent on
Dw (inside positive; established using the K+ ionophore
valinomycin) (Fig. 1B; Supplementary Table S1) but not on
DpH (Supplementary Table S1). mVNUT-mediated ATP uptake
into liposomes exhibited a Km of 230 lM and a Vmax of
23.8 nmol/min/mg of protein (Fig. 1C). The values of these
parameters were somewhat lower than for human VNUT
(Sawada et al. 2008). The intracellular ATP concentration in
dorsal root ganglion nerve cells and the squid giant axon has
been estimated to be ~1 to 5 mM (Mullins and Brinley 1967;
Fukuda et al. 1983). Thus, although ATP concentrations in
different compartments of central neurons and activitydependent fluctuations of cytosolic ATP levels are unknown,
it is likely that mVNUT-mediated vesicular transport is
saturated during basal conditions. Similar to human VNUT,
[a-32P]ATP uptake by mVNUT was inhibited by ATP, ADP, GTP,
and UTP, as well as by diadenosine triphosphate and the ATP
analogues AMP--PNP and ATP-c-s. ATP transport was less
sensitive to the presence of adenosine or adenine (Fig. 1D).
Like human VNUT, mVNUT was dependent on Cl– ions but not
on divalent cations (Supplementary Fig. S1). Both 4,4#-diisothio-
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Figure 2. VNUT- and Ca2þ-dependent ATP release from hippocampal neurons. (A) Western blot detection of VNUT in homogenate of cultured hippocampal neurons. RNAimediated knockdown strongly attenuated the VNUT immunoreactive band, whereas synaptophysin, synaptotagmin, and v-ATPase immunoreactivities were unchanged. (B) The
VNUT immunofluorescence (green) observed in soma and neurites of cultured hippocampal neurons was attenuated by RNAi-mediated knockdown. Synaptophysin (red) and
synaptotagmin 1 (red) were used as presynaptic terminal markers. Insets, higher magnification showing colocalization (yellow) of VNUT (green) and synaptophysin (red) or
synaptotagmin 1 (red). Arrowheads indicate colocalized immunoreactivity for VNUT and synaptophysin/synaptotagmin 1. Arrow indicates a VNUTþ punctum that is not
colocalized with synaptotagmin 1 immunolabeling. Differential interference contrast microscopy (DIC) shows the morphology of cultured neurons. Scale bar: 10 lm. (C) Effect of
RNAi-mediated knockdown of VNUT on evoked ATP release from cultured hippocampal neurons. Left panel: Quantitative analysis for VNUT mRNA levels was performed by realtime PCR. VNUT expression was reduced to ~20% of original expression by RNAi in cultured neurons. Right panel: Neuronal ATP release after 20-min incubation upon Kþ
stimulation from control siRNA-treated or VNUT siRNA-treated cultured hippocampal neurons. Kþ-evoked ATP release was strongly impaired in RNAi-treated cultures. (D)
ATP release from cultured hippocampal neurons was dependent on exocytotic mechanisms. Kþ-evoked ATP release was attenuated in the absence of extracellular Ca2þ, in the
presence of the cell-permeable Ca2þ buffer EGTA-AM (50 lM). The calcium ionophore A23186 (5 lM) also elicited ATP release. (E) Neurons were treated in the presence or
absence of 15 lg/mL tetanus toxin (TeTN) (Invitrogen) for 36 h, after which KCl-mediated ATP release was assayed. Each experiments were repeated twice. Error bars indicate
standard error of the mean. **P \ 0.01, ***P \ 0.001, 2-way analysis of variance followed by Bonferroni post hoc test.
Localization of VNUT in the Brain
Light Microscopy
We proceeded to assess the localization of VNUT in the
hippocampus in vivo. In the light microscope, immunoperoxidase histochemistry showed that VNUT was widely distributed
throughout the brain (Fig. 3). In particular, the cerebellum and
the olfactory bulb were heavily immunolabeled. Considerable
immunolabeling was also observed in the hippocampus and the
superficial superior colliculus (Fig. 3). In the cerebellum, strong
immunoreactivity was evident in the somatodendritic compartment of Purkinje cells, but labeling was also prominent
Cerebral Cortex Page 5 of 12
Furthermore, punctate VNUT immunofluorescent labeling
was often localized to MAP2-immunolabeled dendrites in
hippocampal sections double labeled for these proteins (Fig.
3F).
throughout the molecular layer (Fig. 3B). VNUT immunolabeling was present in all hippocampal layers and the dentate gyrus.
Somewhat stronger somatic or perisomatic immunolabeling
was found in the principal cell layers (Fig. 3C,D). Furthermore,
some interneuronal somata throughout the hippocampal
formation were strongly immunoreactive. Confocal microscopy
of sections immunofluorescence labeled for VNUT showed
punctate immunoreactivity intermingled with a more diffuse
staining in the hippocampus (Fig. 3E). In hippocampal sections,
double labeled for VNUT and VGLUT1, VNUT+ and VGLUT1+
puncta were frequently colocalized. However, VNUT+ and
VGLUT1+ puncta that did not coincide with punctate staining
for the other transporter were also common (Fig. 3E).
Page 6 of 12 VNUT-Mediated Neuronal ATP Release
d
Larsson et al.
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Figure 3. Immunohistochemical labeling of VNUT in rat brain. (A--D) Immunoperoxidase labeling. (A) Parasagittal section showing strong immunoperoxidase
labeling in the olfactory bulb, cerebellar cortex, and hippocampus. (B) Cerebellar
cortex. ml, molecular layer; pc, Purkinje cell layer; gc, granule cell layer. (C) Dentate
gyrus. ml, molecular layer; gc, granule cell layer; hi, hilus. (D) CA1. so, stratum
oriens; py, pyramidal cell layer; sr, stratum radiatum. Inset shows a portion of
proximal stratum radiatum (located as indicated by the dashed frame in the large
micrograph) imaged using a 1003 objective, showing punctate VNUT staining.
Micrographs shown in B--D were obtained at 203 magnification. (E) Confocal
images of VNUT (green) and VGLUT1 (magenta) immunofluorescent labeling in CA1
stratum radiatum. Some VNUTþ and VGLUT1þ puncta colocalize (white; examples
indicated by arrowheads), but VNUTþ/VGLUT1 (simple arrow) and VNUT/
VGLUTþ (double arrow) puncta are also observed. (E) Confocal micrographs of
VNUT (green) and MAP2 (magenta) immunofluorescence in CA1 stratum radiatum.
Two VNUTþ puncta that localize to a MAP2-immunolabeled dendrite are indicated
by arrowheads. Scale bar in D: 50 lm, valid for B--D (except inset in D). Scale bar in
E: 2 lm, valid also for F.
Electron Microscopy—VNUT Single Immunogold Labeling
To delineate the ultrastructural distribution of VNUT immunoreactivity in neuropil, we used postembedding immunogold
labeling and electron microscopy. As we had demonstrated
VNUT-dependent release of ATP from cultured hippocampal
neurons, we focused here on the hippocampal region. Qualitative examination revealed immunolabeling of a variety of
neuronal compartments. Gold particles--signaling VNUT were
located over synaptic vesicles in nerve terminals forming
asymmetric synaptic specializations with postsynaptic spines
(presumed excitatory synapses; Fig. 4A--C) as well as in terminals
forming symmetric axosomatic synapses (presumed inhibitory
synapses) (Fig. 4D,E). Quantitative analysis of the immunolabeling in presumed excitatory terminals was performed in the outer
two-thirds of the molecular layer of the dentate gyrus (MLo) and
in the stratum radiatum (SR) of CA1. Labeling in inhibitory
terminals was analyzed in the dentate granule and CA1 pyramidal
cell layers (GCL and PCL, respectively). The pattern of
immunolabeling was very similar between the examined regions.
In the MLo, 44 ± 10% (n = 3 animals; mean ± standard deviation) of
terminals forming asymmetric synapses were immunopositive
for VNUT (Table 1). In the SR, 43 ± 5% (n = 3 animals) of such
terminals contained VNUT immunoreactivity. VNUT immunolabeling was detected in 55 ± 27% (n = 2 animals) of presumed
inhibitory terminals in the GCL and in 74 ± 6% (n = 2) of
presumed inhibitory terminals in the CA1 PCL (Table 1).
However, this apparent difference between GCL and PCL
inhibitory terminals was partly attributed to differences in
overall immunolabeling in the analyzed tissue sections. Many
unmyelinated preterminal axons exhibited immunoreactivity for
VNUT (Fig. 4F--H). In single ultrathin sections, 15 ± 4% and 13 ±
5% of transversely cut unmyelinated axons in MLo and SR,
respectively, were immunopositive (Table 1). Myelinated axons
were also often strongly VNUT immunoreactive (Fig. 4F,G), but
the immunolabeling in such axons was not subject to quantitative analysis. VNUT immunogold labeling was present not only in
presynaptic profiles but also in a proportion (33 ± 8% in MLo and
30 ± 6% in SR) of postsynaptic dendritic spines (Fig. 4I--K and
Table 1). In both the MLo and the SR, the density of gold
particles--signaling VNUT was higher in unmyelinated axons than
in excitatory nerve terminals and dendritic spines (Table 1 and
Fig. 4L). In dendritic spines, VNUT gold particles were observed
in the vicinity of small vesicle-like structures (insets in Fig. 4I,J).
However, in order to increase immunogold sensitivity, the tissue
used here was fixed with low-concentration glutaraldehyde,
treated with uranyl acetate rather than osmium tetroxide and
embedded at low temperature in a methacrylate resin. Because
this leads to suboptimal morphological preservation as compared to conventional embedding protocols, vesicular profiles in
dendritic spines were often not readily discernible (Fig. 4K).
Moreover, the distance from the center of each VNUT gold
particle to the plasma membrane was determined in excitatory
terminals, postsynaptic dendritic spines, and unmyelinated axons
in the MLo and SR. The frequency distributions of such distances
were compared with the distribution of distances between the
center of points randomly distributed over the above mentioned
profiles and the plasma membrane. In excitatory nerve terminals,
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Figure 4. Postembedding immunogold labeling of VNUT in the dentate gyrus and CA1. (A--C) Examples of VNUT-immunopositive terminals (t) establishing asymmetric
axospinous synapses in the dentate molecular layer (A,B) or CA1 stratum radiatum (C). Note that the labeling is concentrated over synaptic vesicles. (D,E) Examples of VNUTimmunolabeled terminals forming symmetric axosomatic synapses in the granule cell layer. (F--H) Unmyelinated (ax) and myelinated (my) axons exhibiting strong VNUT
immunoreactivity in CA1 stratum radiatum (F) and the dentate molecular layer (G,H). In F, t indicates an immunolabeled terminal. (I--K) Examples of VNUT-immunopositive
dendritic spines (s) postsynaptic to presumed excitatory terminals (t) in the dentate molecular layer (I,J) and CA1 stratum radiatum (K). (L) VNUT labeling density in presumed
excitatory terminals, dendritic spines, and unmyelinated axons of the molecular layer of the dentate gyrus (DG) and CA1 stratum radiatum (see also Tables 1). The summed gold
particle density (number of gold particles/lm2) was determined for each sample and normalized against background labeling density over endothelial basal lamina. Error bars
indicate standard error of the mean. *P \ 0.05, **P \ 0.01, ***P \ 0.001, one-way analysis of variance followed by Bonferroni post hoc test (n 5 3 animals).
VNUT gold particles were located significantly closer to the
plasma membrane than random points (Fig. 5). The largest
fraction of VNUT gold particles was situated within 60 nm from
the plasma membrane. However, in unmyelinated axons, the
reverse pattern was observed: the proportion of gold particles
found immediately below the membrane was significantly smaller
than would be expected if the distribution was random (Fig. 5). In
dendritic spines, there was no significant difference between the
distribution of VNUT immunogold labeling relative to the plasma
membrane and that of random points.
Electron Microscopy—VNUT--VGLUT1 Double Immunogold
Labeling
In tissue double immunogold labeled for VNUT and VGLUT1,
we observed many terminals that labeled for both vesicular
transporters (Fig. 6A; Supplementary Table S2). About half (51 ±
Cerebral Cortex Page 7 of 12
3%; n = 2 animals) of the VGLUT1-immunopositive nerve
terminals in the MLo also contained VNUT labeling. This was in
accordance with the proportion of presumed excitatory
terminals that was VNUT immunopositive in single-labeling
experiments. Moreover, the same axonal enrichment of VNUT
immunogold particles was observed as in single-labeled
sections. By contrast, VGLUT1 immunoreactivity was very
sparse in unmyelinated axons as compared with excitatory
terminals (Fig. 6B). Next, we determined the distance between
the center of each VGLUT1-signaling gold particle to the
center of the nearest VNUT gold particle (Fig. 6C). Given
a lateral resolution of the immunogold labeling of about 25 nm
(determined by the radius of the gold particle and the
dimension of the antibody bridge) (Bergersen et al. 2008)
and a synaptic vesicle diameter of ~40 nm, 2 gold particles
located closer together than 90 nm could signal antigens on the
same vesicle. A minority (15 ± 0%; n = 2 animals) of VGLUT1-VNUT interparticle distances was less than 90 nm (Fig. 6C).
Dentate gyrus
Excitatory terminals
Inhibitory terminals
Dendritic spines
Unmyelinated axons
CA1
Excitatory terminals
Inhibitory terminals
Dendritic spines
Unmyelinated axons
Animals
Total
n
profiles
Total
profile
area (lm2)
%
Labeled
Labeling
density
(lm2)
3
2
3
3
242
90
262
391
38.3
20.1
26.1
6.0
44 ± 10
55 ± 27
33 ± 8
15 ± 4
6.1
6.7
9.1
24.2
±
±
±
±
1.3
4.8
1.3
9.4
3
2
3
3
178
67
198
342
26.3
23.1
16.8
4.0
8.1
9.1
9.5
19.2
±
±
±
±
1.4
0.2
0.6
4.2
43
74
30
13
±
±
±
±
5
6
6
5
Note: Quantitative data were obtained from the outer two-thirds of the dentate molecular layer or
CA1 stratum radiatum, except for inhibitory terminals, which were sampled from the dentate
granule cell layer or the CA1 pyramidal cell layer. Labeling percentages and summed densities
were calculated for each animal and are given as mean ± standard deviation. Mitochondria and
the PSD of spines were excluded from the analysis.
Electron Microscopy—VNUT--VGAT Double Immunogold
Labeling
Double immunogold labeling for VNUT and VGAT demonstrated coexistence of these transporters in many presumed
inhibitory terminals (Fig. 7). Quantitative analysis of the VNUT/
VGAT double immunolabeling revealed no clear colocalization
between VNUT and VGAT immunolabeling or any difference
between VNUT and VGAT immunogold labeling with respect
to their distribution relative to the plasma membrane.
Isolated Synaptic Vesicles
To determine whether VNUT and VGLUT1 may exist in the
same synaptic vesicle, first, we used VNUT and VGLUT1
antibodies to immunoisolate VNUT- and VGLUT1-containing
vesicles from crude synaptic vesicles obtained from whole
forebrain. Then, we performed western blots to examine the
presence of VGLUT1 and VNUT in the resulting eluates
(the immunoisolated vesicle fraction) and supernatant (the
Figure 5. Subcellular distribution of VNUT immunolabeling in neuronal compartments of the neuropil in the molecular layer of the dentate gyrus (DG) and the CA1 stratum
radiatum. Shown are the distributions of the distance of the center of each VNUT-signaling gold particle and of random points to the plasma membrane in presumed excitatory
terminals, dendritic spines and unmyelinated axons (n 5 3 animals). Error bars indicate standard error of the mean. **, P \ 0.01, ***, P \ 0.001, 2-way analysis of variance
followed by Bonferroni post hoc test.
Page 8 of 12 VNUT-Mediated Neuronal ATP Release
d
Larsson et al.
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Table 1
Summary of VNUT immunogold labeling
However, in nerve terminals, where synaptic vesicles are spaced
close together, the immunogold method cannot precisely
ascribe a gold particle to a particular synaptic vesicle. Nevertheless, the VGLUT1--VNUT interparticle distances were significantly shorter than distances between randomly distributed
points and VNUT gold particles (Fig. 6C), suggesting that vesicles
possessing VNUT and vesicles with VGLUT1 to some extent
colocalize in the terminal. Importantly, the majority (85 ± 0%;
n = 2 animals) of VNUT and VGLUT1 immunogold particles were
localized above 90 nm from each other (Fig. 6C) and thus cannot
signal VNUT and VGLUT1 in the same vesicle. Analysis of the
perpendicular distance from gold particles representing VNUT
and VGLUT1 to the plasma membrane in double-labeled
excitatory nerve terminals showed that VNUT immunogold
particles were significantly closer to the plasma membrane than
were gold particles indicating VGLUT1 (Fig. 6D). Further this
suggests that VNUT and VGLUT1 are segregated into different
vesicular pools.
Figure 7. Colocalization of VNUT and VGAT immunolabeling in inhibitory nerve
endings in the dentate granule cell layer in double immunogold--labeled tissue.
A terminal (t) double labeled for VGAT (large gold particles) and VNUT (small gold
particles; arrowheads) forming a symmetric synapse (arrow) with a cell body (cb).
Scale bar: 200 nm.
vesicle fraction left after immunoisolation) (Fig. 8). Both the
VNUT-immunoisolated vesicles and the non-VNUT vesicle
fraction contained VGLUT1 (Fig. 8), suggesting that some, but
not all VGLUT1-containing vesicles possess VNUT. VNUT
immunoreactivity was not observed in the non-VNUT vesicle
fraction, indicating that the VNUT immunoisolation efficiently
depleted VNUT-containing vesicles from the crude synaptic
vesicle fraction. Furthermore, VNUT was not found in VGLUT1
immunoisolated vesicles or in the corresponding supernatant.
The failure to detect VNUT in vesicles isolated with anti-VGLUT1
is difficult to fully explain. However, it may be attributed partly to
a low abundance of VNUT in VGLUT1-containing vesicles in
combination with masking of VNUT antigenicity by other
proteins of similar electrophoretic mobility. Moreover, the
VNUT antibodies appear to have a relatively low affinity or titer,
resulting in comparatively low sensitivity of the immunoblotting
procedure for this antigen. This suggests that VNUT is restricted
to a small proportion of VGLUT1-containing vesicles.
Discussion
The present study shows that VNUT is present in synaptic
vesicles in a considerable proportion of glutamatergic and
inhibitory nerve terminals in the hippocampal formation, that
VNUT is localized to subpopulations of VGLUT1-containing
vesicles, and that vesicular ATP release from cultured hippocampal neurons depends on VNUT. Thus, our data suggest that
VNUT may confer a purinergic transmitter phenotype to
neurons by establishing a vesicular pool of ATP that is
releasable by regulated exocytosis (Sawada et al. 2008). This
supports previous results suggesting that ATP is released by
way of exocytosis from neurons (White 1978).
The quantitative data on the subcellular localization of VNUT
in hippocampal neuropil presented here were obtained using
the postembedding immunogold labeling technique. This
method has distinct advantages for such analysis as compared
with preembedding immunolabeling methods, including clearcut measurement of labeling and a comparatively unbiased
access to different epitopes in the tissue. On the other hand,
the sensitivity of postembedding immunolabeling is relatively
low, which may result in false-negative profiles. Therefore, the
Cerebral Cortex Page 9 of 12
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Figure 6. Colocalization of VNUT and VGLUT1 in double immunogold--labeled
hippocampal tissue (A) A terminal (t) double labeled for VGLUT1 (large gold particles)
and VNUT (small gold particles; arrowheads) apposing an immunonegative spine (s)
in the dentate molecular layer. Scale bar: 200 nm. (B) Ratio of VNUT and VGLUT1
immunogold labeling densities in unmyelinated axons versus excitatory nerve
terminals in the dentate molecular layer (n 5 2 rats). Error bars indicate standard
error of the mean. *P \ 0.05, Student’s t-test. Scale bar in K: 200 nm, valid for A--K.
(C) Boxplot (0, 25, 50, 75, and 100 percentiles) of distances of VGLUT1 gold particles
(n 5 279 particles) and of random points (n 5 1848 points) to the nearest VNUT
gold particle (n 5 125 particles). ***P \ 0.001, Mann--Whitney U-test. Similar
observations were made in 2 animals. (D) The distributions of the distance of the
center of each VNUT or VGLUT1 indicating gold particle to the plasma membrane in
VNUTþ/VGLUT1þ terminals. The P value indicates significant difference in distance
distributions between VNUT and VGLUT1 immunolabeling as determined by v2 test.
Analysis of 2 animals yielded similar results.
Figure 8. Western blots of VGLUT1 and VNUT of forebrain vesicle fractions
immunoisolated using VNUT or VGLUT1 antibody-coupled magnetic beads. Antigens
that were targets for immunoisolation are indicated below the blots, whereas the
antigens detected by immunoblotting are indicated to the left of each blot. eluate,
material isolated with beads coupled to antibody reactive to the respective antigen.
super, supernatant material that did not bind beads coupled to the respective
antibody. The blots are representative of at least 3 separate immunoisolation
experiments.
Page 10 of 12 VNUT-Mediated Neuronal ATP Release
d
Larsson et al.
VNUT may be present also in axons that do not express VGLUT1,
it is not likely to explain the observed difference in axonal
enrichment of VNUT versus VGLUT1 immunolabeling. Thus, the
distinct axonal localization of VNUT immunolabeling as compared with that of VGLUT1 clearly indicates that these transporters may be differentially distributed between different
vesicular populations. Thus, the present analysis of immunogold
labeling suggests that VNUT is at least partly segregated from
VGLUT1+ vesicles. Whether VNUT is present on a subpopulation
of VGLUT1+ vesicles could not be firmly determined from the
immunogold data. However, our immunoisolation experiments
suggest that some, but far from all, VGLUT1+ vesicles also possess
VNUT, whereas a considerable proportion of VNUT+ vesicles also
contains VGLUT1. Taken together, our observations indicate
that VNUT likely is present in a subpopulation of VGLUT1+
vesicles and that many VGLUT1+ vesicles do not have VNUT.
Analysis of immunogold labeling suggests that a similar partial
segregation exists between VGAT- and VNUT-containing
vesicles in inhibitory terminals. Notably, whether some VNUTcontaining vesicles in VGLUT1- or VGAT-expressing neurons
lack either of the latter transporters and thus store ATP but not
glutamate or ?-aminobutyric acid could not be inferred from the
present data.
The VNUT immunoreactivity in preterminal axons was
preferentially located to the axoplasm as opposed to near the
plasma membrane, suggesting that VNUT-containing vesicles in
the axon are undergoing axonal transport. However, such
vesicles may be releasable under some conditions. It has been
suggested that axonal activity induces release of ATP from the
axons (Stevens and Fields 2000) possibly via an exocytotic
mechanism (Thyssen et al. 2010; but see Fields and Ni 2010),
activating purinergic receptors on NG2 glia and astrocytes
(Ishibashi et al. 2006; Hamilton et al. 2008, 2010). This forms an
interesting parallel to vesicular glutamate, which has recently
been shown to be liberated from unmyelinated axons onto
NG2 glia in the corpus callosum (Kukley et al. 2007; Ziskin
et al. 2007).
Endogenously released ATP can activate presynaptic P2X
receptors which induces or enhances glutamate release at
many synapses (Gu and MacDermott 1997; Khakh and
Henderson 1998; Nakatsuka and Gu 2001; Khakh et al. 2003;
Rodrigues et al. 2005; Sperlágh et al. 2007; Khakh 2009),
whereas presynaptic P2Y receptors may negatively modulate
transmitter release (Rodrigues et al. 2005). Furthermore,
adenosine derived from extracellular ATP may bidirectionally
modulate transmitter release via presynaptic adenosine receptors (Goncxalves and Queiroz 2008). The ATP involved in
presynaptic modulation could originate from, for example,
astrocytes, neighboring nerve terminals, or the terminal itself.
Indeed, in a parallel study, evidence of VNUT-mediated release
of ATP from astrocytes has been obtained (Y. Moriyama,
S. Koizumi, unpublished data), suggesting that also astrocytes
are capable of exocytotic ATP release (see Pascual et al. 2005).
An additional potential source of transmitter ATP that has
received little attention is postsynaptic dendrites. In fact, we
observed VNUT immunoreactivity in association with vesicular
structures in postsynaptic dendritic spines. In line with this, in
the cell body of dorsal root ganglion neurons it has been
suggested that small and clear vesicles exocytose ATP, leading
to activation of surrounding satellite cells (Zhang et al. 2007).
The function of the postsynaptically localized VNUT needs to
be addressed in future studies. However, it is tempting to
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
fractions of VNUT-immunopositive profiles of different types
reported here are most likely underestimates of the true
proportions of profiles containing antigen recognized by the
antibody. This is especially true for small profiles such as the
transversely cut unmyelinated axons and dendritic spines.
Another potential problem with the postembedding immunogold technique, as with other immunochemical methods, is
that of nonspecific background labeling. However, RNAimediated knockdown of VNUT or preabsorption with the
peptide against which the VNUT antibody was raised, strongly
attenuate VNUT immunolabeling as judged by western blot,
light microscopic immunocytochemistry, and postembedding
immunogold histochemistry, indicating that the observed
VNUT labeling is specific (present study; Sawada et al. 2008;
Iwatsuki et al. 2009; Haanes and Novak 2010). Furthermore, the
nonrandom distribution of immunogold labeling, the clear
association with synaptic vesicles and the distinct pattern of
axonal and terminal labeling as compared to that of VGLUT1
and VGAT suggest that the postembedding VNUT immunogold
labeling observed here reflected specific labeling.
Purinergic excitatory postsynaptic currents (EPSCs), indicative of quantal ATP release, have been described in several CNS
regions (Edwards et al. 1992; Bardoni et al. 1997; Pankratov et al.
2002, 2003, 2007), including in the hippocampus (Pankratov
et al. 1998, 2006; Mori et al. 2001). Furthermore, electrically
evoked ATP release has been reported in the hippocampus.
However, although this ATP was believed to at least partly
originate from nerve terminals, the precise source could not be
ascertained (Wieraszko et al. 1989; Cunha et al. 1996). The
present findings complement and extend these studies by
providing evidence at the ultrastructural level for presynaptic
storage of transmitter ATP at hippocampal synapses and by
identifying VNUT as the protein responsible for such storage.
Moreover, previous immunogold studies have shown that there
are postsynaptic purinergic receptors at the hippocampal
synapses examined here (Rubio and Soto 2001; Tonazzini et al.
2007).
Whether ATP is always colocalized with other neurotransmitters in synaptic vesicles or is transported into a distinct subset of
ATP-only vesicles is unclear. Pankratov et al. (2006, 2007) have
shown that a proportion of low-amplitude miniature EPSCs in
CA1 or neocortex layer 2/3 pyramidal cells is mediated by P2X
but not glutamate receptors. Nevertheless, stimulation of single
glutamatergic fibers often resulted in mixed glutamate/ATPmediated EPSCs. These results are in line with the existence of
a vesicular population containing ATP in glutamatergic terminals. However, whether ATP is located in the same vesicle pool as
glutamate could not be definitely determined. The observations
presented here suggest that ATP is stored in only a subset of
glutamatergic VGLUT1-possessing vesicles. First, only a weak
spatial relation was detected between VNUT-signaling immunogold particles and those indicating VGLUT1, although this could
be attributed to inadequate sensitivity of the immunogold
labeling. Second, in excitatory nerve terminals, VNUT immunolabeling was enriched near the plasma membrane (possibly
indicating preferred labeling of docked vesicles), whereas no
such pattern was seen for VGLUT1 labeling. Indeed, the
distribution of the distance of VNUT gold particles to the plasma
membrane was significantly different from that of VGLUT1.
Third, whereas VGLUT1 is present at low levels in unmyelinated
preterminal axons, such axons showed high levels of VNUT
immunogold labeling compared with nerve terminals. Although
speculate that certain spines have the ability to release ATP that
may act as a retrograde signal to modulate presynaptic
transmitter release.
In conclusion, we have found that hippocampal neurons in the
rat are capable of releasing ATP transported into synaptic vesicles
by VNUT. Furthermore, VNUT is broadly expressed in the brain,
including in the cerebellar cortex, the olfactory bulb, and the
hippocampus. At the ultrastructural level, VNUT is present in
a wide variety of neuronal compartments in the hippocampal
neuropil, including excitatory and inhibitory nerve terminals,
postsynaptic dendrites and preterminal axons. Thus, multiple
neuronal sources of ATP may contribute to a complex pattern of
presynaptic and postsynaptic purinergic modulation of synaptic
transmission.
Supplementary Material
Supplementary material
oxfordjournals.org/
can
be
found
at:
http://www.cercor.
This work was supported by the Norwegian Research Council
and by a Grant-in-Aid from the Japanese Ministry of Education,
Science, Sport and Culture.
Notes
Conflict of Interest : None declared.
References
Bardoni R, Goldstein PA, Lee CJ, Gu JG, MacDermott AB. 1997. ATP P2X
receptors mediate fast synaptic transmission in the dorsal horn of
the rat spinal cord. J Neurosci. 17:5297--5304.
Bergersen LH, Storm-Mathisen J, Gundersen V. 2008. Immunogold
quantification of amino acids and proteins in complex subcellular
compartments. Nat Protoc. 3:144--152.
Cunha RA, Vizi ES, Ribeiro JA, Sebastiao AM. 1996. Preferential release of
ATP and its extracellular catabolism as a source of adenosine upon
high- but not low-frequency stimulation of rat hippocampal slices.
J Neurochem. 67:2180--2187.
Edwards FA, Gibb AJ, Colquhoun D. 1992. ATP receptor-mediated
synaptic currents in the central nervous system. Nature. 359:144--147.
Fields RD, Burnstock G. 2006. Purinergic signalling in neuron-glia
interactions. Nat Rev Neurosci. 7:423--436.
Fields RD, Ni Y. 2010. Nonsynaptic communication through ATP
release from volume-activated anion channels in axons. Sci Signal.
3:ra73.
Fukuda J, Fujita Y, Ohsawa K. 1983. ATP content in isolated mammalian
nerve cells assayed by a modified luciferin-luciferase method.
J Neurosci Methods. 8:295--302.
Gonc
xalves J, Queiroz G. 2008. Presynaptic adenosine and P2Y receptors.
Handb Exp Pharmacol. 184:339--372.
Gu JG, MacDermott AB. 1997. Activation of ATP P2X receptors elicits
glutamate release from sensory neuron synapses. Nature. 389:
749--753.
Gualix J, Pintor J, Miras-Portugal MT. 1999. Characterization of nucleotide
transport into rat brain synaptic vesicles. J Neurochem. 73:
1098--1104.
Haanes KA, Novak I. 2010. ATP storage and uptake by isolated
pancreatic zymogen granules. Biochem J. 429:303--311.
Hamilton N, Vayro S, Kirchhoff F, Verkhratsky A, Robbins J, Gorecki DC,
Butt AM. 2008. Mechanisms of ATP- and glutamate-mediated
calcium signalling in white matter astrocytes. Glia. 56:734--749.
Hamilton N, Vayro S, Wigley R, Butt AM. 2010. Axons and astrocytes
release ATP and glutamate to evoke calcium signals in NG2-glia.
Glia. 58:66--79.
Cerebral Cortex Page 11 of 12
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Funding
Hamilton NB, Attwell D. 2010. Do astrocytes really exocytose neurotransmitters? Nat Rev Neurosci. 11:227--238.
Ishibashi T, Dakin KA, Stevens B, Lee PR, Kozlov SV, Stewart CL,
Fields RD. 2006. Astrocytes promote myelination in response to
electrical impulses. Neuron. 49:823--832.
Iwatsuki K, Ichikawa R, Hiasa M, Moriyama Y, Torii K, Uneyama H. 2009.
Identification of the vesicular nucleotide transporter (VNUT) in
taste cells. Biochem Biophys Res Commun. 388:1--5.
Jourdain P, Bergersen LH, Bhaukaurally K, Bezzi P, Santello M,
Domercq M, Matute C, Tonello F, Gundersen V, Volterra A. 2007.
Glutamate exocytosis from astrocytes controls synaptic strength.
Nat Neurosci. 10:331--339.
Kang J, Kang N, Lovatt D, Torres A, Zhao Z, Lin J, Nedergaard M. 2008.
Connexin 43 hemichannels are permeable to ATP. J Neurosci.
28:4702--4711.
Khakh BS. 2009. ATP-gated P2X receptors on excitatory nerve
terminals onto interneurons initiate a form of asynchronous
glutamate release. Neuropharmacology. 56:216--222.
Khakh BS, Gittermann D, Cockayne DA, Jones A. 2003. ATP modulation
of excitatory synapses onto interneurons. J Neurosci. 23:7426--7437.
Khakh BS, Henderson G. 1998. ATP receptor-mediated enhancement of
fast excitatory neurotransmitter release in the brain. Mol Pharmacol.
54:372--378.
Koizumi S, Fujishita K, Inoue K. 2005. Regulation of cell-to-cell
communication mediated by astrocytic ATP in the CNS. Purinergic
Signal. 1:211--217.
Kukley M, Capetillo-Zarate E, Dietrich D. 2007. Vesicular glutamate
release from axons in white matter. Nat Neurosci. 10:311--320.
Larsson M, Broman J. 2005. Different basal levels of CaMKII
phosphorylated at Thr286/287 at nociceptive and low-threshold
primary afferent synapses. Eur J Neurosci. 21:2445--2458.
Linhoff MW, Laurén J, Cassidy RM, Dobie FA, Takahashi H, Nygaard HB,
Airaksinen MS, Strittmatter SM, Craig AM. 2009. An unbiased
expression screen for synaptogenic proteins identifies the LRRTM
protein family as synaptic organizers. Neuron. 61:734--749.
Liu HT, Sabirov RZ, Okada Y. 2008. Oxygen-glucose deprivation induces
ATP release via maxi-anion channels in astrocytes. Purinergic Signal.
4:147--154.
Liu HT, Toychiev AH, Takahashi N, Sabirov RZ, Okada Y. 2008. Maxianion channel as a candidate pathway for osmosensitive ATP release
from mouse astrocytes in primary culture. Cell Res. 18:558--565.
Mori M, Heuss C, Gahwiler BH, Gerber U. 2001. Fast synaptic
transmission mediated by P2X receptors in CA3 pyramidal cells of
rat hippocampal slice cultures. J Physiol. 535:115--123.
Moriyama Y, Yamamoto A, Yamada H, Tashiro Y, Tomochika K,
Takahashi M, Maeda M, Futai M. 1995. Microvesicles isolated from
bovine posterior pituitary accumulate norepinephrine. J Biol Chem.
270:11424--11429.
Mullins LJ, Brinley FJ, Jr.. 1967. Some factors influencing sodium
extrusion by internally dialyzed squid axons. J Gen Physiol.
50:2333--2355.
Nakatsuka T, Gu JG. 2001. ATP P2X receptor-mediated enhancement of
glutamate release and evoked EPSCs in dorsal horn neurons of the
rat spinal cord. J Neurosci. 21:6522--6531.
Nörenberg W, Illes P. 2000. Neuronal P2X receptors: localisation and
functional properties. Naunyn Schmiedebergs Arch Pharmacol.
362:324--339.
Pankratov Y, Castro E, Miras-Portugal MT, Krishtal O. 1998. A purinergic
component of the excitatory postsynaptic current mediated by P2X
receptors in the CA1 neurons of the rat hippocampus. Eur
J Neurosci. 10:3898--3902.
Pankratov Y, Lalo U, Krishtal O, Verkhratsky A. 2002. Ionotropic P2X
purinoreceptors mediate synaptic transmission in rat pyramidal
neurones of layer II/III of somato-sensory cortex. J Physiol.
542:529--536.
Pankratov Y, Lalo U, Krishtal O, Verkhratsky A. 2003. P2X receptormediated excitatory synaptic currents in somatosensory cortex. Mol
Cell Neurosci. 24:842--849.
Pankratov Y, Lalo U, Verkhratsky A, North RA. 2006. Vesicular release of
ATP at central synapses. Pflugers Arch. 452:589--597.
Page 12 of 12 VNUT-Mediated Neuronal ATP Release
d
Larsson et al.
Thyssen A, Hirnet D, Wolburg H, Schmalzing G, Deitmer JW, Lohr C.
2010. Ectopic vesicular neurotransmitter release along sensory
axons mediates neurovascular coupling via glial calcium signalling.
Proc Natl Acad Sci U S A. 107:15258--15263.
Tonazzini I, Trincavelli ML, Storm-Mathisen J, Martini C, Bergersen LH.
2007. Co-localization and functional cross-talk between A1 and
P2Y1 purine receptors in rat hippocampus. Eur J Neurosci. 26:
890--902.
White TD. 1978. Release of ATP from a synaptosomal preparation by
+
elevated extracellular K and by veratridine. J Neurochem. 30:
329--336.
Wiedenmann B, Franke WW. 1985. Identification and localization of
synaptophysin, an integral membrane glycoprotein of Mr 38,000
characteristic of presynaptic vesicles. Cell. 41:1017--1028.
Wieraszko A, Goldsmith G, Seyfried TN. 1989. Stimulation-dependent
release of adenosine triphosphate from hippocampal slices. Brain
Res. 485:244--250.
Zander JF, Munster-Wandowski A, Brunk I, Pahner I, Gomez-Lira G,
Heinemann U, Gutierrez R, Laube G, Ahnert-Hilger G. 2010.
Synaptic and vesicular coexistence of VGLUT and VGAT in
selected excitatory and inhibitory synapses. J Neurosci. 30:
7634--7645.
Zhang X, Chen Y, Wang C, Huang LY. 2007. Neuronal somatic ATP
release triggers neuron-satellite glial cell communication in dorsal
root ganglia. Proc Natl Acad Sci U S A. 104:9864--9869.
Ziskin JL, Nishiyama A, Rubio M, Fukaya M, Bergles DE. 2007. Vesicular
release of glutamate from unmyelinated axons in white matter. Nat
Neurosci. 10:321--330.
Downloaded from cercor.oxfordjournals.org at University of Oslo Library on August 4, 2011
Pankratov Y, Lalo U, Verkhratsky A, North RA. 2007. Quantal release of
ATP in mouse cortex. J Gen Physiol. 129:257--265.
Pascual O, Casper KB, Kubera C, Zhang J, Revilla-Sanchez R, Sul JY,
Takano H, Moss SJ, McCarthy K, Haydon PG. 2005. Astrocytic purinergic
signalling coordinates synaptic networks. Science. 310:113--116.
Persson S, Boulland JL, Aspling M, Larsson M, Fremeau RT, Edwards RH,
Storm-Mathisen J, Chaudhry FA, Broman J. 2006. Distribution of
vesicular glutamate transporters 1 and 2 in the rat spinal cord, with
a note on the spinocervical tract. J Comp Neurol. 497:683--701.
Rodrigues RJ, Almeida T, Richardson PJ, Oliveira CR, Cunha RA. 2005.
Dual presynaptic control by ATP of glutamate release via facilitatory
P2X1, P2X2/3, and P2X3 and inhibitory P2Y1, P2Y2, and/or P2Y4
receptors in the rat hippocampus. J Neurosci. 25:6286--6295.
Rubio ME, Soto F. 2001. Distinct localization of P2X receptors at
excitatory postsynaptic specializations. J Neurosci. 21:641--653.
Sawada K, Echigo N, Juge N, Miyaji T, Otsuka M, Omote H, Yamamoto A,
Moriyama Y. 2008. Identification of a vesicular nucleotide transporter. Proc Natl Acad Sci U S A. 105:5683--5686.
Shoji-Kasai Y, Yoshida A, Sato K, Hoshino T, Ogura A, Kondo S,
Fujimoto Y, Kuwahara R, Kato R, Takahashi M. 1992. Neurotransmitter release from synaptotagmin-deficient clonal variants of PC12
cells. Science. 256:1821--1823.
Sperlágh B, Heinrich A, Csölle C. 2007. P2 receptor-mediated modulation
of neurotransmitter release-an update. Purinergic Signal. 3:269--284.
Stevens B, Fields RD. 2000. Response of Schwann cells to action
potentials in development. Science. 287:2267--2271.
Stout CE, Costantin JL, Naus CC, Charles AC. 2002. Intercellular calcium
signalling in astrocytes via ATP release through connexin hemichannels. J Biol Chem. 277:10482--10488.