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The Journal of Neuroscience, January 15, 2001, 21(2):641-653
Distinct Localization of P2X Receptors at Excitatory Postsynaptic
Specializations
Maria E.
Rubio and
Florentina
Soto
Department of Molecular Biology of Neuronal Signals,
Max-Planck-Institute for Experimental Medicine, D-37075
Göttingen, Germany
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ABSTRACT |
ATP mediates fast excitatory synaptic transmission in some regions
of the central nervous system through activation of P2X receptors.
Nonetheless, the functional significance of ATP-mediated neurotransmission is not yet understood. Using postembedding
immunocytochemistry, we describe the distribution of
P2X2, P2X4, and P2X6
subunits in cerebellum and in the CA1 region of the hippocampus.
Dendritic spines of cerebellar Purkinje cells showed immunogold
labeling for all three subunits when apposed to parallel fiber (PF)
terminals. In contrast, no immunogold labeling was observed on
dendritic spines or cell bodies receiving inputs from climbing fibers
and basket cells, respectively. In CA1 pyramidal cells, postsynaptic membranes apposed to terminals of Schaffer collaterals were
immunogold-labeled for P2X2,
P2X4, and P2X6 subunits. Immunolabeling
was also observed perisynaptically and intracellularly in relation to
membranes of the endoplasmic reticulum. The analysis of the tangential
distribution of gold particles showed that they were preferentially
located at the peripheral portion of the postsynaptic specialization of both parallel fiber and Schaffer collateral synapses. By double imunogold labeling using antibodies for P2X receptor subunits and
GluR2/3 subunits of the AMPA glutamate receptors, we show that synapses
expressing P2X receptors are also glutamatergic. The present study
shows for the first time qualitatively and quantitatively the precise
localization of P2X receptors in brain. Moreover, our data indicate
that P2X receptors may play a significant role at glutamatergic synapses.
Key words:
ATP; P2X receptors; ligand-gated ion channels; excitatory
synapses; cerebellum; hippocampus; postembedding
immunocytochemistry
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INTRODUCTION |
The first indication of a
transmitter role for ATP in the nervous system was presented 40 years
ago, with the demonstration of ATP release from sensory nerves during
antidromic stimulation (Holton, 1959 ). It is now well accepted that ATP
is involved in cell to cell communication in the peripheral nervous
system (for review, see Dubyak and el-Moatassim, 1993; Burnstock,
1999). However, ATP-mediated fast neurotransmission has only recently
been described in the CNS, where its role is not yet well understood
(Edwards et al., 1992; Bardoni et al., 1997; Nieber et al.,
1997; Pankratov et al., 1998, 1999; for review, see Robertson,
1998).
Fast responses to extracellular ATP are mediated by the activation of
P2X receptors. Seven members of this family of ligand-gated ion
channels have been cloned
(P2X1-P2X7) (North and
Barnard, 1997; Soto et al., 1997). Native and cloned P2X receptors are permeable to monovalent cations such as
Na+ and K+ as
well as divalent cations such as Ca2+
(Burnashev, 1998). In contrast to NMDA receptors, influx of
Ca2+ through P2X receptors occurs at the
resting potential of the neuron, providing an additional source of
Ca2+ entry that could modulate the
performance of the postsynaptic cell (Edwards and Gibb, 1993).
Of the seven cloned P2X subunits, only P2X4 and
P2X6 mRNA transcripts are widely distributed with
overlapping patterns in rat brain (Collo et al., 1996; Soto et al.,
1996a,b). Light microscopy using an antibody against
P2X4 confirmed these results (Lê et al.,
1998b). The distribution of P2X6 protein in the
brain is unknown. mRNA and protein expression for
P2X2 in adult rat brain were originally found in
a subset of neurons (Kidd et al., 1995; Collo et al., 1996; Vulchanova
et al., 1996). In particular, no expression was detected in areas where
P2X4 and P2X6 subunits were
present at high levels, such as cerebellum and hippocampus. However,
recent work (Kanjhan et al., 1999) supports the notion that the
distribution of P2X2 receptor subunits is as
extended in brain as that described for P2X4 and
P2X6 subunits.
To elucidate the role of ATP as a neurotransmitter in the CNS, the
subcellular distribution of the three predominant P2X receptor subunits
(P2X2, P2X4, and
P2X6) in rat brain was analyzed by postembedding immunogold labeling. The analysis was performed in cerebellum and
hippocampus for the following reasons: (1) These two regions express
all three P2X subunits. (2) Fast synaptic currents mediated by ATP have
been detected in the CA1 region of the hippocampus (Pankratov et al.,
1998, 1999), and cultured cerebellar Purkinje cells respond to ATP with
an increase in intracellular Ca2+ evoked
by activation of P2X receptors (Mateo et al., 1998). (3) Hippocampal
pyramidal neurons and cerebellar Purkinje cells have well described
synaptic circuitry. Our results show the presence of P2X receptor
subunits at excitatory postsynaptic specialization of Schaffer
collaterals in hippocampus CA1 and parallel fibers (PFs) in cerebellum.
Immunolabeling for P2X receptor subunits was mainly found at the
periphery of the postsynaptic specialization. The physiological
relevance of this is discussed.
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MATERIALS AND METHODS |
Antibodies. Antibodies to the C terminus of
P2X2 and P2X4 were
generated using a synthetic peptide corresponding to the following sequences: P2X2, DSTSTDPKGLAQL;
P2X4, DYEQGLSGEMNQ. A cysteine residue was added
to the N terminus of the peptide to facilitate coupling to the carrier
protein. The peptides were conjugated to BSA and KLH, respectively, and
injected into New Zealand White rabbits. Antibodies were purified using
the same peptide coupled to SulfoLink resin (Pierce, Rockford, IL). The
antibody to GluR2/3 was a generous gift from R. J. Wenthold
(Wenthold et al., 1992). Commercial antibodies to
P2X2 and P2X4 were obtained
from Alomone Labs (Jerusalem, Israel).
Polyclonal antibodies to P2X6 were raised using a
fusion protein. The cDNA corresponding to the C-terminal domain of rat
P2X6 (aa 355-379) subunit was obtained by PCR
from the rat P2X6 full length cDNA (Soto et
al., 1996b) using the following oligonucleotides: forward,
5'-CCGGATCCGATAGAGAGGCCGGTTTCT-3'; reverse,
5'-GGAATAAGCTTTGCACTGTTGGTAGTTGC-3'. The PCR product was
cloned in pBlue-script II KS(+) using the BamHI/HindIII sites inserted in the
oligonucleotides and sequenced. A PCR fragment containing the correct
sequence was subcloned (BamHI/SalI) in frame with
glutathione-S-transferase (GST) into pGEX-4T vector (Amersham Pharmacia Biotech, Uppsala, Sweden). The same fragment was
subcloned (BamHI/HindIII) in frame with
thioredoxin (Trx) in the pET32(a)+ vector (Novagen, Madison, WI) for
affinity purification. GST-P2X6 fusion protein
was expressed in Escherichia coli (DH5 strain) and
purified from the soluble fraction after bacterial lysis using
glutathione-agarose beads (Sigma, St. Louis, MO) as described
previously (Smith and Corcoran, 1995). The expected molecular weight of
33 kDa was confirmed by electrophoresis in a 12% SDS-PAGE. The
purified GST-P2X6 fusion protein was dialyzed and
injected in Chinchilla bastard rabbits for antibody production. The His-tagged Trx-P2X6 fusion protein was
overexpressed in E. coli (BL21 strain) and purified from the
soluble fraction using Ni-NTA agarose (Quiagen, Hilden, Germany)
following the manufacturer's recommendations. The
Trx-P2X6 fusion protein was covalently coupled to
POROS EP 20 (PerSeptive Biosystems, Framingham, MA) and used to
affinity purify the antiserum.
Western blot of brain homogenates. Brain homogenates were
obtained from postnatal day 5 (P5) Sprague Dawley rats. Ten micrograms of protein for P2X4 and
P2X6 and 35 µg for P2X2
were loaded on 12% SDS-PAGE gels and then transferred to
nitrocellulose. Blots were probed with 2 µg/ml of affinity-purified
rabbit anti-P2X2, P2X4, or
P2X6 antibodies. Preadsorption controls were
performed by incubating the antibodies to P2X2
and P2X4 with 50 µg/ml of the specific peptide
and the antibody to P2X6 with 15 µg/ml of the
Trx-P2X6 fusion protein at 4°C for 24 hr.
Anti-rabbit peroxidase-conjugated secondary antibody was used for
visualization with enhanced luminescence (ECL kit, Amersham-Pharmacia Biotech).
Immunostaining of human embryonic kidney-293 cells.
Permanent transfection of human embryonic kidney (HEK)-293 cells with a
full-length human P2X3, human
P2X4, rat P2X5, or rat
P2X6 cDNA cloned in the mammalian expression
vector pcDNA3 (Invitrogen, San Diego, CA) was performed using a calcium
phosphate precipitation method (Chen and Okoyama, 1987). Independent
foci were selected and expanded in the continuous presence of geneticin
(500 µg/ml; Sigma). HEK-293 cells transfected with rat
P2X1 and P2X2 cDNA were a
generous gift from A. Suprenant. All procedures were performed at room
temperature. Cultured cells were washed with PBS, fixed with 4%
paraformaldehyde in PBS for 5 min, and blocked with 10% normal goat
serum (NGS) in PBS for 1 hr. Permeabilization was achieved with 0.2%
Triton 100-X in PBS. Cells were incubated with a primary antibody (0.5 µg/ml) to P2X2, P2X4, or
P2X6 subunits in PBS for 1 hr. After washing,
cells were incubated with a goat anti-rabbit fluorescein-conjugated
secondary antibody (1:500) (Amersham, Arlington Heights, IL) in PBS
containing 0.3% NGS and analyzed with a Zeiss Axiophot microscope.
Light microscopic immunocytochemistry. Six P21 Sprague
Dawley rats were anesthetized with a mixture of ketamine HCl (Ketaset; 100 mg/ml; Fort Dodge Laboratories, Inc.) and xylazine (Rompun; 20 mg/ml; Miles, Elkhart, IN) at 0.1 ml/100 gm of body weight. The animals
were transcardially perfused with a fixative consisting of 4%
paraformaldehyde in 0.12 M phosphate buffer, pH
7.2. After perfusion, brains were removed, fixed for an additional hr
at 4°C, rinsed three times in PBS, and stored overnight at 4°C.
For peroxidase immunocytochemistry, brain coronal and sagittal sections
(40-50 µm) were cut in cold PBS using a Vibratome (Leica, Vienna,
Austria). Slices were incubated for 1 hr in PBS containing 10% NGS and
then with primary antibody to P2X2 (1 µg/ml), P2X4 (1 µg/ml), or P2X6
(0.9 µg/ml) subunits in PBS overnight at 4°C and processed using
the avidin-biotin-peroxidase system (Vectastain kit; Vector
Laboratories, Burlingame, CA). Antibody binding was visualized using
3'-3-diaminobenzidine tetrahydrochloride (DAB) (DAB substrate kit for
peroxidase, Vector Laboratories). Controls were performed either by
omitting the primary antibody or by preincubating the primary antibody
with the corresponding peptide (for P2X2 and
P2X4; 50 µg/ml final concentration) or fusion protein (for P2X6; 15 µg/ml final
concentration) at 4°C for 24 hr and then by following the procedure
described above. Sections were analyzed with a Zeiss Axiophot microscope.
For fluorescence immunocytochemistry, brain sagittal sections (20-25
µm) were incubated for 1 hr in PBS containing 10% NGS and then with
primary antibody (10 µg/ml) to P2X2,
P2X4, or P2X6 subunits in
PBS at 4°C for 24 hr. Afterward, sections were incubated with goat
anti-rabbit fluorescein-conjugated secondary antibody (1:400; Amersham)
in PBS containing 2% BSA, 0.3% NGS, and analyzed using a Bio-Rad MRC
1024 confocal microscope.
Freeze substitution. Four P21 Sprague Dawley rats were used
for the freeze-substitution procedure. Animals were anesthetized as
described above and transcardially perfused with a fixative consisting
of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.12 M phosphate buffer, pH 7.2. Brains were then
removed and fixed in the same fixative for 90 min at 4°C, rinsed in
three changes of 0.1 M phosphate buffer, pH 7.2, containing 4% glucose and stored overnight at 4°C in the same
buffer. Brain sagittal sections (300 µm) were cut in cold 0.1 M phosphate buffer, pH 7.2, containing 4%
glucose, using a Vibratome (Leica). The freeze substitution was
performed as described before for glutamate receptors (Rubio and
Wenthold, 1997, 1999).
Postembedding immunocytochemistry. Colloidal gold-coupled
goat anti-rabbit IgG (5 nm GAR G5 and 10 nm GAR G10; Amersham)
was used to detect rabbit polyclonal antibodies, as described
previously (Rubio and Wenthold, 1997, 1999). All procedures were
performed at room temperature. Ultrathin sections (60-70 nm) on nickel
grids (400 mesh) were incubated sequentially in the following
solutions: (1) 10 min in 0.1% sodium borohydride and 50 mM glycine in Tris-buffered saline containing
0.1% Triton X-100 (TBST); (2) 10 min in TBST containing 10% NGS; (3)
2 hr with primary antibodies to P2X2, P2X4 (5 µg/ml), P2X2, and
P2X4 (Alomone) (2 µg/ml), and
P2X6 (9 µg/ml) in TBST containing 10% NGS; (4)
10 min in TBST; (5) 10 min in TBST containing 10% NGS; and (6) 1 hr
with colloidal gold-coupled secondary antibody diluted 1:20 in TBST
containing 10% NGS and polyethylene glycol 20,000 (5 mg/ml). Ultrathin
sections were counterstained with 1% uranyl acetate and 0.3% lead
citrate and studied with a Zeiss 100CX II transmission electron
microscope at 50 kV. Controls were performed either by omitting the
primary antibody or by preincubating the primary antibody with the
corresponding peptide (for P2X2 and
P2X4; 50 µg/ml final concentration) or fusion protein (for P2X6; 15 µg/ml final
concentration) at 4°C for 24 hr, and then following the procedure
described above.
Double immunogold labeling using polyclonal antibodies for
P2X4 and GluR2/3 or P2X6
and GluR2/3 was performed as described previously (Rubio and Wenthold,
1999) using paraformaldehyde vapors between two sequential
immunogold-labeling procedures. All double immunogold labeling was
repeated after reversing the size of the gold particles. Control
experiments were performed by omitting the primary antibody. An
additional control consisted of omitting the primary antibody in the
sequential immunogold labeling after paraformaldehyde vapors.
Electron micrographs were taken at 30,000× magnification and scanned
at a resolution of 3600 dpi using a Linotype-Hell scanner (Heidelberg,
Germany). Image processing was performed with Adobe Photoshop, using
only the brightness and contrast commands to enhance the gold particles.
Areas survey. Cerebellar sections were taken from the
cerebellar folia III-V to eliminate variations caused by regional
differences in cerebellar structure/function and differences in timing
of ontogenesis. Posterior folia develop more rapidly than do anterior ones in early postnatal times, and this could affect the level of P2X
receptor expression as has been shown to occur for glutamate receptors
(Takayama et al., 1996).
Electron microscopic identification of parallel fiber, climbing fiber
(CF), and basket cell (BC) synapses on Purkinje cells was based on
defined criteria, as reviewed previously (Mugnaini, 1972; Palay and
Chan-Palay, 1974; Altman and Bayer, 1997). Parallel fiber-Purkinje
cell synapses have small and globular axonal varicosities containing a
loose collection of round synaptic vesicles. These varicosities form
asymmetrical synapses (Gray Type I) with the spines of spiny branchlets
of Purkinje cells dendrites. Climbing fiber varicosities are large and
filled with round clear synaptic vesicles, and they form asymmetrical
synapses with dendritic spines and larger dendritic shafts of Purkinje
cells. They differ from parallel fibers not only in the locus of
termination but also in that synaptic vesicles are less regular in
their shape, size, and distribution (Altman and Bayer, 1997). Basket
cell synaptic terminals contain flattened-pleomorphic synaptic vesicles
and make symmetric synaptic contacts (Gray Type II) with the cell bodies of Purkinje cells. Hippocampal sections were taken from the CA1
stratum radiatum. All of the synapses showed the characteristic features of asymmetric synapses (Gray Type I): postsynaptic density, cleft, and round presynaptic vesicles. Synapses that could not be
clearly identified by the above criteria were not included in the analysis.
Thin sections were examined from one block from two animals at P21 for
P2X2, P2X4, and
P2X6 and for double labeling of
P2X4 or P2X6 and GluR2/3.
All of the experiments shown have been performed with the antibodies to
P2X2, P2X4, and
P2X6 developed in our laboratory; characterization of the antibodies is presented here. As
control, qualitative light and electron microscopy were also performed with the P2X2 and P2X4
antibodies obtained from Alomone, with essentially the same results
(data not shown). Qualitative analysis of the distribution of P2X
subunits at the electron microscopic level was performed using
secondary antibodies coupled to 5 and 10 nm gold particles. To
get the highest possible signal, 5 nm colloidal gold was used for the
semiquantitative analysis (Hayat, 1989).
Quantitative evaluation of P2X receptor immunolabeling. Data
were collected from cases in which the postsynaptic specializations were well defined. The length of the postsynaptic specializations was
measured using NIH Scion Image software, and the number of associated
gold particles was counted. Only gold particles (5 nm) clearly seen at
the postsynaptic membranes and within the synaptic cleft were counted.
The maximum distance allowed between the postsynaptic specialization
and a gold particle was 18 nm, based on the spatial resolution of the
immunogold technique (Merighi and Polak, 1993).
To determine axodendritic distribution of gold particles, 20-25
synapses were analyzed. In these synapses, the distance was measured
between the center of each gold particle and the outer leaflet of the
postsynaptic specialization. The axodendritic axis was divided into 5 nm bins, and each gold particle was assigned to one bin. All gold
particles located 100 nm from the outer leaflet of the postsynaptic
specialization to the presynaptic and postsynaptic sides were included
in the analysis.
To define tangential distribution of labeling along the postsynaptic
specialization, the distance of each gold particle from the center of
the synaptic profile was measured and normalized. The radial length was
divided into five equal bins, and each gold particle was assigned to a
bin. An additional bin of 20% (i.e., 100-120%) was included to
sample the perisynaptic region (Matsubara et al., 1996; Petralia et
al., 1998). The tangential distribution was measured independently in
two animals, and both of them showed the same distribution along the
postsynaptic specialization.
The relative density of P2X receptor imunolabeling in cerebellum and
hippocampus was determined in a sample of 133 and 124 presynaptic
terminals and 110 and 83 glial profiles, respectively. The sample
included >20 areas for each of the profiles and for each of the
antibodies analyzed. All Bergmann glia (cerebellum) and astrocyte
(hippocampus) profiles were identified following described criteria
(Palay and Chan-Palay, 1974; Peters et al., 1991; Ventura and Harris,
1999). Briefly, astrocytic processes were identified by their
irregular, stellate shape and by the presence of glycogen granules and
bundles of intermediate filaments in a relatively clear cytoplasm. Only
identified glial profiles located at 1 µm2 around the synapse were measured.
After counting the gold particles for each presynaptic or glial
profile, the perimeter of each segment was measured using NIH Scion
Image software. Once the number of gold particles and the area of each
profile were known, the average density of gold particles was computed
automatically for each type of dendritic or presynaptic profile.
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RESULTS |
Specificity of antibodies to P2X2,
P2X4, and P2X6 subunits
We have generated rabbit polyclonal antibodies against C-terminal
sequences of rP2X2, rP2X4,
and rP2X6 subunits, which show little
intersubunit homology (Soto et al., 1997). However, to demonstrate the
subunit specificity of the corresponding antibody, we used
immunofluorescence labeling of cells expressing
rP2X2, hP2X4, and
rP2X6 subunits (Fig.
1A). Immunofluorescence
signals were detected only with the antibody raised against the
specific subtype. Affinity-purified antibody to rat
P2X4 subunit showed cross-reactivity with the
human homolog, something that could be expected because of the high
percentage of amino acid identity between the rat and the human
sequence (Garcia-Guzman et al., 1997). For cells transfected with
P2X1, P2X3, and
P2X5 subunits, no immunofluorescence labeling was
visible (data not shown).
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Figure 1.
A, Immunofluorescence of HEK-293
cells transfected with P2X2,
P2X4, and P2X6 cDNAs using the
affinity-purified polyclonal antibodies for P2X2,
P2X4, and P2X6. Only cells transfected
with the corresponding cDNAs present immunofluorescence signal.
B, Immunoblot analysis of SDS-PAGE gels of brain
homogenates immunostained with an antibody to P2X2,
P2X4, and P2X6. The polyclonal
antibodies recognize only a band migrating at 64, 57, and 49 kDa
corresponding to P2X2, P2X4, and
P2X6 subunits, respectively. When the immunoblots were
performed using preabsorbed antibodies, no bands were observed. Scale
bar, 100 µm.
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To demonstrate the specificity of the generated antibodies in native
tissue, Western blots of crude membranes isolated from rat brain were
performed. Single bands of 64, 57, and 49 kDa were detected with
affinity-purified antibodies to P2X2,
P2X4, and P2X6 subunits,
respectively (Fig. 1B). No bands could be detected when the primary antibody used for Western blot was preincubated with
the corresponding peptide or fusion protein, indicating the specificity
of the antibody for the epitope (Fig. 1B). The
molecular weight of 57 kDa detected for P2X4
subunits is in good agreement with the value previously reported by
Lê and coworkers (Lê et al., 1998b), and a molecular weight
of 65 kDa has been described for P2X2 subunits
expressed in Xenopus oocytes (Newbolt et al., 1998).
The molecular weights obtained for the three subunits were always
higher than those predicted from the amino acidic sequence (53, 44, and
42 kDa for P2X2, P2X4, and
P2X6, respectively). This might be explained by
N-glycosylation occurring at consensus sequences in the extracellular
loop (Brake et al., 1994; Collo et al., 1996). A different degree of
N-glycosylation could account for the smear observed under the band for
P2X6 in Figure 1B. Modification
of P2X receptors by N-glycosylation has been demonstrated
experimentally for P2X1 (Valera et al., 1994) and
P2X2 (Newbolt et al., 1998; Torres et al.,
1998a).
Light microscopic immunocytochemistry of P2X2,
P2X4, and P2X6 in cerebellum and
hippocampus
At the level of light microscopy (Figs.
2, 3), all
three subunits of P2X receptors analyzed (P2X2,
P2X4, and P2X6) were
detected in cerebellum and hippocampus. We obtained the same patterns
of expression using the antibodies developed in our laboratory and the
commercial antibodies for P2X2 and
P2X4.
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Figure 2.
Light-microscopic micrographs showing the
immunohistochemical reactions for P2X2,
P2X4, and P2X6 in cerebellum and
hippocampus. A, Low-magnification views of cerebellum
and hippocampus plus preadsorption controls. B,
High-magnification views of Purkinje cells (PC) in
cerebellum and CA1 pyramidal cells (Py) in hippocampus
showing immunohistochemical reactions for P2X2,
P2X4, and P2X6. Both cell types present
immunohistochemical reactions extending from the cell body to the most
distal dendrites. A punctate pattern resembling dendritic spines
labeling is observed. BG, Bergmann glia. Scale bars:
A, 2 mm; B, 50 µm.
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Figure 3.
Confocal images showing immunofluorescence for
P2X2, P2X4, and P2X6
in cerebellum and hippocampus. Immunofluorescence signal is observed in
cell bodies, dendrites, and dendritic spines of cerebellar Purkinje
cells and hippocampal CA1 pyramidal neurons. Insets show
a magnification of apical dendrites. Scale bars: 100 µm;
insets, 25 µm.
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In cerebellum, Purkinje cells were the only cell type showing
immunoreactivity for the three subunits, with different intensities: from weak for P2X2 to strong for
P2X4 and P2X6. Labeling
extended from the cell body to dendrites (Figs. 2B,
3). Dendritic labeling was strong for P2X4 and
P2X6 and weak for P2X2.
Also, a punctate pattern resembling dendritic spine labeling was
detected for P2X2, P2X4,
and P2X6 (Fig. 3). Antibodies for
P2X4 also labeled basket and stellate cells in
the molecular layer (data not shown) and strongly labeled granule cells
in the granular layer(Figs. 2B, 3). Additionally,
P2X2 immunoreactivity was detected in basket cells, in Bergmann glia, and in synaptic boutons in the granular cell
layer. Immunostaining for P2X6 was only observed
on Purkinje cells. In the hippocampus, pyramidal cells in the CA1 and
CA3 region and granule cells in the dentate gyrus were stained with antibodies for P2X2 and
P2X4. Immunohistochemical reaction for the
P2X6 antibody was predominantly detected in CA1
and to a lesser extent in CA3 pyramidal cells. In CA1 pyramidal cells,
labeling for the three subunits extended from the cell body to the most distal dendrites, including dendritic spines in the stratum radiatum (Figs. 2B, 3). Immunohistochemical reaction was not
observed in synaptic boutons with any of the antibodies for the P2X
subunits analyzed. Both omission of primary antiserum (data not shown) and preadsorption of purified P2X2,
P2X4, and P2X6 antibodies with the corresponding peptide or fusion protein (Fig.
2A) resulted in the lack of DAB immunoreaction,
confirming the specificity of the assay.
Immunogold labeling for P2X2,
P2X4, and P2X6 at synapses of
parallel fibers onto Purkinje cells in cerebellum
The distribution of P2X2,
P2X4, and P2X6 subunits in
cerebellum was analyzed in 306 synapses. The sample included previously described excitatory synapses of PFs (n = 159) and CFs
(n = 72) and inhibitory synapses of BCs
(n = 80) on cell bodies of Purkinje cells. Only PF
synapses showed immunogold labeling for P2X2,
P2X4, and P2X6, with no
labeling at CF and BC synapses.
Examples of postembedding immunogold labeling of PF/Purkinje cell
synapses are shown in Figure 4. All 159 PF synapses analyzed (Table
1) were localized in the molecular layer
and enwrapped by processes of Bergmann glia. In our sample of PF
synapses analyzed, we found that 55% (n = 29), 57%
(n = 30), and 68% (n = 38) were immunogold-labeled for P2X2,
P2X4, and P2X6,
respectively. Qualitatively, all antibodies used gave a similar pattern
of immunogold labeling on PF/Purkinje cells (Fig.
4). Gold particles were mainly present at
the postsynaptic specializations (Fig. 4) and extended at lower levels
perisynaptically and intracellularly with relation to endoplasmic reticulum membranes (Fig. 5). The
location of gold particles was quantitatively assessed by analyzing
their axodendritic distribution in 20 synapses (per antibody). The
distribution profile of P2X2, P2X4, and P2X6 gold
particles shows a preferentially postsynaptic localization of these
receptor subunits (Fig. 6) (peaks extend from ~5 to 35 nm inside the postsynaptic specialization). The density
of gold particles in relation to the length of the postsynaptic specialization was also analyzed, and the results are shown in Table
1.
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Figure 4.
Electron micrographs showing postembedding
immunogold labeling for P2X2 (A-C),
P2X4 (D-F), and P2X6
(G-I) at postsynaptic specializations of
parallel fibers (PF) on Purkinje cells in the
molecular layer of cerebellum. Gold particles (5 nm;
arrows) are observed at the peripheral portions of the
postsynaptic specialization. I, A PF and a climbing
fiber synapse (CF) making synaptic contact with
dendritic spines of Purkinje cells. Immunogold labeling is observed
only at the postsynaptic specialization of PF. G,
Bergmann glia; s, spine. Scale bars: 0.250 µm;
inset, 0.125 µm.
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Figure 5.
Electron micrographs showing the intracellular
immunogold labeling for P2X2 (C),
P2X4 (A), and P2X6
(B) in Purkinje cells of cerebellum
(A, B) and hippocampal CA1 pyramidal
cells (C). Gold particles (arrows)
are observed intracellularly in relation to endoplasmic reticulum
membranes. A, A basket cell (BC)/Purkinje
cell (PC) synapse lacking gold labeling at the
postsynaptic specialization. Py, Pyramidal cell. Scale
bar, 0.250 µm.
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Figure 6.
Axodendritic (left) and tangential
(right) distribution of gold particles representing
P2X2, P2X4, and P2X6
at PF-Purkinje cell synapses in cerebellum. The axodendritic
distribution of gold particles was analyzed in 20-25 synapses. The
distance was measured between the center of each gold particle and the
outer leaflet of the postsynaptic specialization. Labeling density of
particles at each distance is plotted. P2X receptor immunogold
distribution peaks at 5-40 nm postsynaptic to the plasma membrane. For
the tangential distribution, each bin in the histogram represents
one-fifth of the radial length of the postsynaptic specialization, with
zero defined as the center. The data were pooled from 21 (P2X2), 28 (P2X4), and 33 (P2X6) synapses. Only synaptic profiles of at least
200 nm diameter were included in the analysis. Immunolabeling for all
subunits occurs along the mediolateral extent of the postsynaptic
specialization, with a dramatic increase at the peripheral portion.
Gold particles were also present at the perisynaptic region of the
postsynaptic specialization.
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The analysis of the tangential distribution of gold particles (66 particles in 21 synapses for P2X2, 98 particles
in 28 synapses for P2X4, and 129 particles in 33 synapses for P2X6) showed that the labeling was
relatively low in the central part of the synapse and dramatically
increased in the peripheral portions (Fig. 6).
We also analyzed the density of gold particles for
P2X2, P2X4, and
P2X6 in presynaptic terminals of cerebellar PF
synapses (n = 133) and found very low levels
[particles per squared micrometer (µm2) ± SEM = 1.4 ± 0.5 for
P2X2, 0.6 ± 0.4 for
P2X4, and 0.9 ± 0.3 for
P2X6].
Immunogold labeling for P2X2,
P2X4, and P2X6 at synapses of Schaffer
collaterals onto CA1 pyramidal cells in hippocampus
Examples of postembedding immunogold labeling of excitatory
synapses on pyramidal cells in CA1 stratum radiatum are shown in Figure
7. Excitatory synapses (n = 184) were used to analyzed the distribution of
P2X2 (n = 55),
P2X4 (n = 63), and
P2X6 (n = 66) subunits in this
region. Of these synapses, 25 (45%), 29 (46%), and 37 (56%)
presented immunogold labeling for P2X2,
P2X4, and P2X6,
respectively. Qualitatively, all antibodies gave a similar pattern of
immunogold labeling on dendrites and spines of the stratum radiatum
(Fig. 7). Gold particles were mainly present at the postsynaptic
specializations and extended at lower levels perisynaptically and
intracellularly with relation to endoplasmic reticulum membranes (Fig.
6). The location of gold particles was quantitatively assessed by
analyzing their axodendritic distribution in 25 synapses (per
antibody). The distribution profile of P2X2, P2X4, and P2X6 gold
particles shows a preferentially postsynaptic localization of these
receptor subunits (Fig. 8) (peaks extend from ~5 to 40 nm inside the postsynaptic specialization). The density
of gold particles in relation to the length of the postsynaptic specialization was also analyzed, and the results are shown in Table
2. In our analysis of P2X receptor
subunit distribution in CA1, no variation in the number of gold
particles between the populations of big and small spines (Takumy et
al., 1999) was observed.
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Figure 7.
Electron micrographs showing postembedding
immunogold labeling for P2X2 (A-C),
P2X4 (D-E), and P2X6
(F-H) in CA1 stratum radiatum of the
hippocampus. Gold particles (5 nm; arrows) are observed
at the peripheral portions of the postsynaptic specialization. In two
spines (C, F), immunogold labeling
was also observed at the plasma membrane (arrowhead).
C, A putative perforated synapse; gold particles were
located at the periphery of one of the two postsynaptic specializations
observed. G, Astrocytes; p, presynaptic;
s, spine. Scale bars: 0.250 µm; inset,
0.125 µm.
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Figure 8.
Axodendritic (left) and tangential
(right) distribution of gold particles representing
P2X2, P2X4, and P2X6
at excitatory synapses in the CA1 stratum radiatum of the hippocampus.
The axodendritic distribution of gold particles was analyzed in 20-25
synapses. The distance was measured between the center of each gold
particle and the outer leaflet of the postsynaptic specialization.
Labeling density of particles at each distance is plotted. P2X receptor
immunogold distribution peaks at 5-40 nm postsynaptic to the plasma
membrane. For the tangential distribution, each bin in the histogram
represents one-fifth of the radial length of the postsynaptic
specialization, with zero defined as the center. The data were pooled
from 24 (P2X2), 25 (P2X4), and 35 (P2X6) synapses. Only synaptic profiles with at
least 200 nm of diameter were included in the analysis. Immunolabeling
for all subunits occurs along the mediolateral extent of the
postsynaptic specialization, with a dramatic increase at the peripheral
portion. Gold particles were also present at the perisynaptic region of
the postsynaptic specialization.
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Table 2.
Summary of the postembedding immunoreactivity for
P2X2, P2X4, and P2X6 in CA1 stratum
radiatum of the hippocampus
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The analysis of the tangential distribution of gold particles (76 particles in 24 synapses for P2X2, 80 particles
in 25 synapses for P2X4, and 125 particles in 35 synapses for P2X6) showed that the labeling was
relatively low in the central part of the synapse and dramatically
increased in the peripheral portion (Fig. 8). This tangential
distribution is similar to the one at the postsynaptic specialization
of the PF/Purkinje cell synapses (see above).
The density of gold particles obtained for P2X2,
P2X4, and P2X6 in
presynaptic terminals of Schaffer collaterals (n = 144) in hippocampus was very low [particles per squared micrometer (µm2) ± SEM = 0.6 ± 0.2 for
P2X2, 0.2 ± 0.1 for
P2X4, and 0.6 ± 0.2 for
P2X6].
It has been shown that in CA1 stratum radiatum ~58% of the synapses
have astrocytic processes at their perimeters (Ventura and Harris,
1999). Because of the predominance of P2X receptor immunogold labeling
at the outer portions of the postsynaptic specialization, we wanted to
know from our sample of synapses analyzed (n = 184) in
this brain region how many had astrocytic profiles at their perimeters
and of these how many were immunogold-labeled for P2X subunits. We
found that 98 synapses (53%) had well defined astrocytic profiles at
their perimeters, from which 62 (63%) presented gold particles for P2X
receptors at the postsynaptic specialization.
Immunogold labeling of P2X2,
P2X4, and P2X6 subunits in glia
processes in cerebellum and hippocampus
Light-microscopy and pre-embedding immunocytochemistry studies
have reported the presence of several P2X receptor subunits on glial
cells (Loesch and Burnstock, 1998; Kanjhan et al., 1999; this study).
To know the levels of expression for P2X2,
P2X4, and P2X6 subunits in
glial cells, we analyzed the relative density of gold particles in
processes of cerebellar Bergmann glia and CA1 hippocampal astrocytes 1 µm2 around the synapse. Relatively low
levels of gold particles for the three subunits both in the Bergmann
glia [gold particles per squared micrometer (µm2) ± SEM = 1.3 ± 0.3 for P2X2, 1.1 ± 0.4 for P2X4 and 1.4 ± 0.7 for
P2X6] and in the CA1 astrocytes [gold particles
per squared micrometer (µm2) ± SEM = 0.2 ± 0.2 P2X2, 0.2 ± 0.1 P2X4, and 0.7 ± 0.5 P2X6] were obtained.
Colocalization of P2X receptors with AMPA receptors at excitatory
postsynaptic membranes in cerebellum and hippocampus
In addition to the well described ultrastructural characteristics
used to identify excitatory synapses in the CNS, we verified that P2X
receptor subunits were localized at glutamatergic synapses in
cerebellum and hippocampus by double postembedding immunocytochemistry. We combined antibodies against P2X4,
P2X6 (Fig. 9), or
P2X2 (data not shown) with an antibody against
the GluR2/3 subunits of AMPA receptors using sequential immunogold
labeling. Postsynaptic specializations of PF synapses in cerebellum and
Schaffer collateral synapses in hippocampus presented gold particles
for both P2X and AMPA receptors. From the 25 synapses analyzed for each
antibody in both cerebellum and hippocampus, approximately half
presented gold particles for both P2X and AMPA subunits. Synapses that
were immunogold-labeled for only one type of receptor
(n = 5-7) or without gold particles (n = 5-7) were also observed. However, this must be considered only an
approximation because of the limitations of the sequential
immunogold-labeling technique (Rubio and Wenthold, 1999; Racca et al.,
2000; Sassoè-Pognetto and Ottersen, 2000).
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Figure 9.
Electron micrographs showing double immunogold
labeling for P2X receptors subunits (5 nm) with GluR2/3 subunit of the
AMPA type of glutamate receptors (10 nm) in cerebellum and
hippocampus. Postsynaptic specializations of PF
(A-C) in cerebellum and excitatory synapses in
CA1 (B-D) presented gold particles for both
P2X4 (A-B) or P2X6
(C-D) with GluR2/3 (A-D).
PF, Parallel fibers; p, presynaptic;
s, spine. Scale bar, 0.125 µm.
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Controls of immunogold labeling
Several controls were performed at the light and electron
microscopic level to ensure that the observed patterns of P2X receptor distribution were specific. (1) The use of two primary antibodies to
some subunits for both light and electron microscopy: antibodies to the
same subunit always gave the same pattern of labeling. (2) The use of
secondary antibodies coupled to two different sizes of gold particles
(5 and 10 nm) to determine ultrastructurally the distribution of
P2X receptors in cerebellum and hippocampus (Figs. 4, 7, and
10): the pattern of immunogold labeling
was the same for both sizes of gold particles. (3) Omission of primary antibody in the first and sequential immunogold labeling and (4) preadsorption of primary antibody with the corresponding peptide conjugate or protein were performed for the antibodies to
P2X2, P2X4, and
P2X6 subunits. Both procedures resulted in
undetectable labeling in all cases. (5) To discard the possibility that
the predominance of immunogold labeling at the peripheral portion of
the postsynaptic specialization might be caused by unspecific clustering of the secondary antibody coupled to 5 nm gold particles, the distribution GluR2/3 subunits of AMPA receptors in cerebellum and
hippocampus was determined (Fig. 11).
Immunogold labeling was observed along excitatory postsynaptic
specializations, presenting a distribution pattern similar to what has
been shown previously for GluR2/3 receptors (Landsend et al., 1997;
Rubio and Wenthold, 1997; Petralia et al., 1998; Takumy et al.,
1999).
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Figure 10.
Electron micrographs showing postembedding
immunogold labeling for P2X4 subunits in cerebellum and
hippocampus using 10 nm gold particles. Gold particles were observed at
peripheral portions of cerebellar PF and CA1 hippocampal Schaffer
collateral postsynaptic specializations. PF, Parallel
fibers; p, presynaptic; s, spine. Scale
bar, 0.250 µm.
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Figure 11.
Electron micrographs showing postembedding
immunogold labeling for GluR2/3 (5 nm gold particles) subunits of the
AMPA glutamate receptors in cerebellum and hippocampus. Gold particles
are observed distributed along the entire postsynaptic specialization
of excitatory synapses (A-D). A,
A putative basket cell dendrite in the molecular layer of the
cerebellum receiving inhibitory (1) and
excitatory (2) synaptic inputs. Only the
excitatory postsynaptic specialization (2)
appears decorated with gold particles. Scale bars: 0.250 µm;
inset, 0.125 µm.
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DISCUSSION |
In this study we show that typical synapses that are considered
glutamatergic on the basis of immunocytochemistry and ultrastructural characteristics express P2X receptors. We describe for the first time,
using a high-resolution anatomical technique, the precise subcellular
localization of P2X receptors in the CNS. P2X subunits are present at
postsynaptic specializations of PF synapses in cerebellum and of
Schaffer collateral synapses in hippocampus and may have a functional
role in long-term depression and long-term potentiation, respectively.
Analysis of their tangential distribution at the synapse showed that
P2X receptors are localized at peripheral portions of the postsynaptic
specialization, where ionotropic glutamate receptor density drops off.
Presence of P2X receptor subunits at these excitatory synapses suggests
an interaction between glutamatergic and purinergic transmission.
Distribution of P2X subunits in cerebellum and hippocampus
The expression of P2X2,
P2X4, and P2X6 mRNA
transcripts in adult rat hippocampus and cerebellum has been documented
(Buell et al., 1996; Collo et al., 1996; Séguéla et al.,
1996; Soto et al., 1996a,b). Light microscopic immunostaining for
P2X2 and P2X4 on the cell
bodies and dendrites of Purkinje and CA1 pyramidal cells has been
presented (Lê et al., 1998b; Kanjhan et al., 1999; this study).
Additionally, a presynaptic localization for P2X2 was reported for synapses on the cell body and on dendrites of Purkinje
cells (Kanjhan et al., 1999). No immunocytochemical data has been
published on the distribution of P2X6 subunits.
With light microscopy, we found that immunolabeling for the three
subunits is confined to cell bodies and dendrites, including dendritic spines of Purkinje and CA1 neurons, without indication of presynaptic labeling. Analysis of the axodendritic distribution of gold particles confirmed the preferential postsynaptic localization of the three subunits in both areas of the brain. No peak was detected indicating the existence of a presynaptic pool of receptors. Also, these data
indicate that the epitopes are located at the cytoplasmic side of the
postsynaptic specialization, pointing toward an intracellular localization of the C terminus, in agreement with the proposed topological model for P2X receptors (Newbolt et al., 1998;
Torres et al., 1998b).
There is remarkable selectivity in the expression of
P2X2, P2X4, and
P2X6 receptors in the cerebellar cortex. We found
labeling on ~50% of the Purkinje cell synapses that were apposed to
PF, whereas no labeling could be detected in CF and BC synapses,
suggesting selective targeting of P2X subunits. A similar pattern of
expression has been described for the  2 subunit of glutamate
receptors (Landsend et al., 1997; Zhao et al., 1998).
Overlapping patterns of expression for P2X2,
P2X4, and P2X6 in the CNS
and synaptic localization of the three subunits in dendritic spines of
the same cell type suggest heteromultimerization. P2X2 and P2X4 do not
co-assemble when expressed in heterologous systems (Lewis et al., 1995;
Torres et al., 1999). However, P2X6 can assemble
with both P2X2 and P2X4
subunits but cannot form functional homomultimeric receptors (Soto et
al., 1996b; Lê et al., 1998a; Torres et al., 1999). Therefore,
P2X receptors at excitatory synapses in hippocampus and cerebellum
might be formed by homomeric P2X2 or
P2X4 receptors or by heteromultimeric
P2X2/P2X6, P2X4/P2X6 and
P2X2/P2X4/P2X6
combinations. The presence of P2X1 receptors
described in adult Purkinje cells by pre-embedding immunocytochemistry could add further complexity to the receptor composition (Loesch and
Burnstock, 1998). However, the scarcity of P2X1
labeling in dendritic spines of Purkinje cells argues against a high
percentage of P2X receptors containing this subunit. Functional
characterization of heterologously expressed
P2X2/P2X4/P2X6
receptors would help to correlate expression of subunits with
physiological responses.
Functional implications of P2X receptors in cerebellum
and hippocampus
P2X receptors permeable to Ca2+ and
with a pharmacological behavior that resembles
P2X2 homomeric receptor have been described in
Purkinje cells isolated from 7-d-old rats (Mateo et al., 1998). Studies
of the single-channel properties of P2X receptors in rat cerebellar
slices (3- to 7-d-old rats) suggest that receptors on Purkinje cells
could be homomeric P2X2 or
P2X4 receptors (Halliday and Gibb, 1997).
However, possible differences in subunit composition between young and
adults rats (Vulchanova et al., 1996; Kidd et al., 1998) and the lack
of a dendritic tree of Purkinje neurons at that age (Altman and Bayer,
1997) make it difficult to compare the pharmacological data and the
localization of P2X subunits described here.
ATP induces fast synaptic currents and an intracellular
Ca2+ increase in cultured hippocampal
neurons (Inoue et al., 1992, 1995). Moreover, ATP at low concentrations
increases the amplitude of the population spike following the
stimulation of Schaffer collaterals (Wieraszko and Seyfried, 1989).
Experiments on hippocampal slices of 17- to 19-d-old rats have shown
fast ATP-mediated responses in CA1 neurons (Pankratov et al., 1998,
1999). EPSCs evoked by stimulation of Schaffer collaterals were not
completely blocked by a combination of AMPA and NMDA receptor
antagonists in 70% of the neurons tested. Low concentrations of the
antagonist pyridoxal 5-phosphate 6-azophenyl-2',4' disulfonic acid
(PPADS) blocked the remaining EPSCs in 60% of the neurons,
indicating the presence of P2X receptor-mediated responses.
Pharmacological characterization of the response to ATP using local
application revealed a high variability of blockade by PPADS. Homomeric
P2X2 receptors are sensitive to PPADS (Evans et
al., 1995), whereas homomultimeric P2X4 and
heteromultimeric P2X4/P2X6
receptors are insensitive (Buell et al., 1996; Soto et al., 1996a;
Lê et al., 1998a). Thus, variability in the subunit composition
of the receptors might explain these differences in the pharmacological
properties. The purinergic component of the response is
apparently postsynaptic (Pankratov et al., 1998, 1999), notwithstanding
a previous report in which ATP was proposed to act presynaptically to
increase glutamate release from Schaffer collaterals (Motin and
Bennett, 1995). Our data are in concordance with a postsynaptic action
of ATP at Schaffer collateral synapses.
Heterologously expressed homomeric P2X2 and
P2X4 receptors are more permeable to
Ca2+ than to monovalent cations. Likewise,
P2X receptors present in neurons have a significant permeability to
Ca2+ (Burnashev, 1998). For instance, P2X
receptors in sympathetic ganglion cells have a permeability ratio
(PCa2+/PNa+) of 5, whereas a permeability ratio of >10 has been described for some
neurons of the medial habenula nucleus (Edwards et al., 1997). An
increase on
[Ca2+]i has been
related to hippocampal long-term potentiation and cerebellar long-term
depression (Denk et al., 1995; Nicoll and Malenka, 1995). In cerebellum
it was shown recently that high-frequency stimulation of PF can change
the composition of AMPA receptors at stellate cell dendrites by
controlling the expression or targeting of edited subunits (Liu and
Cull-Candy, 2000). Increases in
[Ca2+]i produced
by the activation of Ca2+-permeable AMPA
receptors may be necessary for the alteration in receptor composition.
We have found immunogold labeling for P2X receptor subunits on stellate
cell dendrites postsynaptic to PF (our unpublished results). The slow
decay kinetics of ATP-mediated EPSCs (time constant 30 msec) (Edwards
et al., 1992; Evans et al., 1992; Pankratov et al., 1998, 1999), the
high Ca2+ permeability of P2X receptors,
and their localization at appropriate sites suggest a further role for
P2X receptors in the synaptic plasticity of these cells.
P2X receptors at excitatory postsynaptic specializations
In the present study we report the presence of P2X receptors as a
new component at glutamatergic postsynaptic specializations. Moreover,
by analyzing the tangential distribution of gold particles we found
that ~90% of P2X receptors are located at the peripheral portions of
the synaptic disk. With few exceptions [cochlea: Matsubara et al.
(1996); neostriatum: Bernard et al. (1997)], the density of ionotropic
glutamate receptors decreases at the peripheral portions of the
postsynaptic membrane (Kharazia and Weinberg, 1997; Landsend et al.,
1997; Petralia et al., 1998; Nusser, 1999). This distribution of
glutamate receptors could be necessary for an effective response to
glutamate and may be modified through synaptic plasticity (Edwards,
1995). Similarly, the location of P2X receptors at the peripheral
portion of the postsynaptic specialization might be necessary to assure
the response to ATP in two ways. First,
Ca2+ entry through P2X receptors might
activate a subset of intracellular second messengers by being located
at the peripheral portion of the postsynaptic specialization. Second,
P2X receptors could be close to the ATP releasing sites. In CNS, ATP
can be co-released with GABA in the spinal cord (Jo and Schlichter,
1999) and with noradrenaline in the locus coeruleus (Nieber et al.,
1997). ATP is not co-released with glutamate in the medial habenula
(Robertson and Edwards, 1997). However, co-release of glutamate and ATP
from Schaffer collaterals and/or PF, or both, cannot be
discarded. Glia can influence neuronal activity by releasing
biologically active substances (Kang et al., 1998). For instance, ATP
is released during Ca2+ waves (Cotrina et
al., 1998; Guthrie et al., 1999) and after activation of glutamate
receptors (Clark and Barbour, 1997; Querioz et al., 1997; Dzubay and
Jahr, 1999; Bergles et al., 2000). As we have shown in this study
P2X2, P2X4, and
P2X6 subunits are concentrated at peripheral
portions of postsynaptic specializations of PF and of Schaffer
collaterals, in close relationship with glial processes. The distance
between glial processes and P2X receptors is in the range of 20-100
nm, and the theoretical distance at which diffusing ATP would remain
above 100 nM is 100 µm [see discussion by Guthrie et al.
(1999)]. Thus, our data suggest that postsynaptic P2X receptors are
close enough to glial processes to be activated by ATP released from
glial cells.
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FOOTNOTES |
Received June 12, 2000; revised Oct. 3, 2000; accepted Oct. 23, 2000.
M.E.R. is supported by the Alexander von Humboldt Foundation. We are
grateful to Prof. W. Stühmer for support, lab space, equipment,
and reading of this manuscript. We thank Dr. R. J. Wenthold for
valuable comments on this manuscript and for helping with the
generation of the antibodies for P2X2 and P2X4.
We acknowledge the expert technical assistance of K. Borchardt. We
thank Dr. R. J. Wenthold and Prof. A. Surprenant for kindly
providing us with the antibody for GluR2/3 subunits and the HEK-293
cells transfected with P2X1 and P2X2,
respectively. We acknowledge Dr. Martin Stocker for technical advice on
the purification of GST-fusion proteins and Dr. Luis Pardo for his
great help with the confocal microscope. Additionally, we thank D. Kerschensteiner for valuable scientific discussion and critical reading
of this manuscript.
Correspondence should be addressed to Dr. Maria E. Rubio,
Max-Plank-Institute for Experimental Medicine, Department of Molecular Biology of Neuronal Signals, Hermann-Rein Strasse 3, D-37075
Göttingen, Germany. E-mail:mrubio{at}gwdg.de.
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15752 - 15757.
[Abstract]
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M. Mori, C. Heuss, B. H Gahwiler, and U. Gerber
Fast synaptic transmission mediated by P2X receptors in CA3 pyramidal cells of rat hippocampal slice cultures
J. Physiol.,
August 15, 2001;
535(1):
115 - 123.
[Abstract]
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B. S. Khakh, W. B. Smith, C.-S. Chiu, D. Ju, N. Davidson, and H. A. Lester
Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors
PNAS,
April 24, 2001;
98(9):
5288 - 5293.
[Abstract]
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[PDF]
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TOP
ABSTRACT
INTRODUCTION
Gene
