 |
 Previous Article | Next Article 
The Journal of Neuroscience, January 1, 2000, 20(1):251-258
AMPA-Kainate Subtypes of Glutamate Receptor in Rat Cerebral
Microglia
Mami
Noda1,
Hiroshi
Nakanishi2,
Junichi
Nabekura1, and
Norio
Akaike1
1 Laboratory of Cellular and System Physiology,
Graduate School of Medical Science, and 2 Department of
Pharmacology, Faculty of Dentistry, Kyushu University, Fukuoka
812-8582, Japan
 |
ABSTRACT |
Microglial cells were isolated from rat cerebral cortex, and
kainate (KA)-induced inward current was measured at a holding potential
of  40 or 60 mV. 6-Cyano-7-nitroquinoxaline-2,
3-dione-sensitive KA-induced currents increased with increasing
KA concentration. The half-activation concentration and Hill
coefficient were 3.3 × 10 4 M and
1.4, respectively. Although glutamate (Glu) and AMPA-induced currents were much smaller than that induced by KA, all KA-, Glu-, and
AMPA-induced currents were greatly and consistently enhanced in the
presence of cyclothiazide (CTZ). On the other hand, KA-induced currents
were much less sensitive to potentiation by concanavain A, suggesting
that the KA-induced response in rat microglia is predominantly mediated
by AMPA-preferring receptors (subunits GluR1-GluR4). The
current-voltage relationships of KA- and AMPA-CTZ-induced currents
were almost linear or slightly outward rectifying. The reversal
potential of KA-induced current shifted to negative potentials (from +4
to 40 mV) on switching from high Na+ to high
Ca2+ external solution, indicating the low
Ca2+ permeability through the AMPA-KA receptor
channel complexes. AMPA-KA receptor expression was studied with
immunohistochemistry and reverse transcription-PCR, from which GluR2,
GluR3, GluR4, and GluR5 were identified. Lower levels of mRNAs for
GluR7 and KA-1-KA-2 were also indicated. Finally, activation of
these receptors with KA or Glu significantly enhanced the production of
tumor necrosis factor- . These results suggest that primary cultured rat microglia possesses functional Glu receptor, which may mediate neuron to microglia communication in the physiological and pathological states.
Key words:
microglia; whole-cell patch clamp; kainate; glutamate; AMPA; cyclothiazide; reverse transcription-PCR; tumor necrosis
factor-
| |
INTRODUCTION |
There is increasing evidence that
functional glutamate receptors (GluR) are not restricted to neurons but
are also expressed in glial cells. Among glial cells, macroglial cell
types, astrocytes, and oligodendrocytes express various types of Glu
receptors (for review, see Steinhäuser and Gallo, 1996 ). The
possible functional roles of Glu receptors have been analyzed (Bowman
and Kimelberg, 1984; Sontheimer et al., 1988; Gallo et al., 1989;
Usowicz et al., 1989; Cornell-Bell et al., 1990; Parpura et al., 1994;
Queiroz et al., 1997; Bezzi et al., 1998) and indicated the active
glial participation in information processing and plasticity, as well as in pathological states. Glial precursor cells, oligodendrocyte-type 2 astrocyte (O-2A) cells, also express various Glu receptors (Patneau et al., 1994; Wang et al., 1996). The activation of Glu receptors modulates the proliferation and differentiation of O-2A cells, suggesting that the excitatory transmitter might be one of the environmental signals that regulate glial cell development
(Steinhäuser and Gallo, 1996; Matute et al., 1997). Moreover,
another neuron-glia interaction between Purkinje cells and Bergman
glial cells via Glu receptors has been proposed in the cerebellum
(Müller et al., 1992).
The question then arises whether microglia have Glu receptors.
Microglial cells are rapidly activated even in response to minor
pathological changes so that they may be viewed as the cellular sensory
element of brain pathology (Kreutzberg, 1996) and may contribute to
neurodegenerative diseases and to the dementia of AIDS (Streit and
Kincaid-Colton, 1995). The most prominent feature of activated
microglia is the generation of both neurotrophic (Mallat et al., 1989;
Shimojo et al., 1991; Araujo and Cotman, 1992; Nakajima et al.,
1992a,b) and neurotoxic molecules (Meda et al., 1995; El Khoury et al.,
1996; Wyss-Coray et al., 1997; Noda et al., 1999). Furthermore,
microglial cells have metabotropic and ionotropic receptors, such as
endothelin and ATP receptors, that link to intracellular calcium
signaling (Verkhratsky and Kettenmann, 1996; Chessell et al., 1997;
Möller et al., 1997; Inoue et al., 1998). On the basis of these
characteristics of microglia, Glu appears to be also a good candidate
to mediate neuron-to-microglia communication in both physiological and
pathological states. So far, however, there is no evidence showing
ionotropic Glu receptors in microglia. In the present study, we
therefore conducted electrophysiological study using a "Y tube"
technique, which enables a rapid application of drugs (e.g., within 20 msec) (Min et al., 1996), in combination with benzothiazide
cyclothiazide (CTZ) and concanavalin A, which block desensitization of
AMPA-preferring receptors and kainate (KA)-preferring receptors,
respectively. In this paper, we provide evidence for the existence of
AMPA-KA receptors in primary cultured rat microglia by
electrophysiolgical, immunohistochemical, and reverse transcription-PCR
(RT-PCR) studies. We also show that activation of these receptors is
involved in the production of tumor necrosis factor- (TNF-) by microglia.
| |
MATERIALS AND METHODS |
Materials. Papain and DNase were purchased from
Worthington Biochemical (Freehold, NJ). DMEM was obtained from
Life Technologies (Grand Island, NY). Fetal calf serum (FCS) was
from HyClone (Logan, UT).
1,1'-Dioctadecyl-3,3,3',3'-tetramethyl-indo-carbocyanine bound to
acetylated low-density lipoprotein (DiI-ac-LDL) and
streptavidin-Alexia 488 were obtained from Molecular Probes (Eugene,
OR). Anti-glutamate receptor 2 and 3 antibody, recognizing both GluR2
and GluR3, was from Chemicon (Temecula, CA). Streptavidin-Cy3 was from
Amersham (Buckinghamshire, UK). KA, Glu, NMDA, AMPA, quisqualate
(QA), cremophore EL, Griffonia simplicifonia
B4-isolectin (GSA-I-B4), and concanavalin A were purchased from Sigma (St. Louis, MO). CNQX was
from Tocris Cookson (Bristol, UK). CTZ was obtained from Research
Biochemicals (Natick, MA).
Cell culture. Microglial cells were isolated from the mixed
cultures of cerebrocortical cells from postnatal day 3 Wistar rats
(Seac Yoshitomi, Fukuoka, Japan), as described previously (Sastradipura
et al., 1998; Noda et al., 1999). In brief, the cerebral cortex was
minced and treated with papain (90 U) and DNase (2000 U) at 37°C for
20 min. The dissociated cells were seeded into 300 cm2 plastic flasks at a density of
107/300 cm2
in DMEM with 0.37% NaHCO3, 50 U/ml penicillin,
100 mg/ml streptomycin, and 10% FCS, and maintained at 37°C in 10%
CO2-90% air with a change of medium twice per
week. After 10-14 d, floating cells and weakly attached cells on the
mixed primary culture cell layer were obtained by gently shaking for
3-5 min. The resulting cell suspension was seeded onto glass
coverslips and allowed to adhere for 30 min at 37°C. Then, microglia
were isolated as strongly adhering cells after unattached cells were
removed. The purity of microglia was ranged from 92 to 95% as
determined by immunostaining with OX42 directed against CR3 receptors
or OX6 directed against major histocompatibility complex class II
molecules. Microglial cells were maintained in the medium mentioned
above at 37°C in 10% CO2-90% air until the
electrophysiological experiments.
Cell identification before electrophysiological
measurements. Microglial cells were identified by the fluorescent
probe DiI-ac-LDL. Cells were incubated with DiI-ac-LDL in a
concentration of 10 µg/ml for 12 hr at 37°C and visualized on an
inverted fluorescent microscope before electrophysiological
measurements. Most cells were round- or rod-shaped cell bodies with the
diameter of <10 µm, and some of them had few thick processes.
Electrophysiological measurements. Whole-cell recordings
were made as reported previously (Noda et al., 1998, 1999). Microglial cells were whole-cell clamped using a patch pipette containing (in
mM): NaCl or CsCl 100, Na2ATP 3, HEPES 5, CaCl2 1, MgCl2 4, EGTA 5, and
N-methyl-D-glucamine (NMDG) 10. The pH
of the solution was adjusted to 7.3 with 1N HCl. The pipette resistance
was 6-9 M . The external solution contained (in
mM): KCl 2.5, NaCl 110, CaCl2 3, BaCl2 6, glucose
15, and HEPES 5, and the pH was adjusted to 7.4 with NMDG. The high
Ca2+ solution contained (in
mM): CaCl2 80, glucose 15, and HEPES 5, and the pH was adjusted to 7.4 with NMDG. The external KA
or drugs were applied rapidly using the Y tube technique (Min et
al., 1996), which allows the complete exchange of the external solution
surrounding a cell within 20 msec. The temperature monitored in the
recording dishes was 33-34°C.
The electrophysiological data are presented as means ± SEM in the
text, and the SEM is indicated by a vertical bar in the figures.
Fluorescent immunohistochemistry. Primary cultured microglia
plated on the chamber slides were fixed with 4% paraformaldehyde for
30 min at room temperature, followed by a wash with PBS. For immunohistochemical staining of Glu receptors, the cells were treated
as follows: with 3% normal goat serum for 4 hr at room temperature;
with anti-glutamate receptor 2 and 3 antibody recognizing both GluR2
and GluR3 over night at 4°C; and stained with streptavidin-Alexia 488 for 2 hr at room temperature. Controls were incubated with nonimmune rabbit IgG instead of anti-GluR2 and 3 antibody. For the
identification of microglia, the same cells were treated with GSA-I-B4 overnight at 4°C and stained with
streptavidin-Cy3 for 2 hr at room temperature. Every treatment was
followed by washing with PBS. Then, the cells were mounted in the
anti-fading medium Vectashield (Vector Laboratories, Burlingame, CA)
and were examined with a confocal laser scanning microscope MRC-1000
(Bio-Rad, Herz, UK) equipped with a krypton-argon ion laser and
mounted on a light microscope (Nikon Optiphoto, Tokyo, Japan).
RT-PCR analysis. General methods were similar to those
described previously (Stefani et al., 1998). Cells were cultured in 100 mm dishes and harvested, and cellular mRNA was extracted using TRIZOL
(Life Technologies), according to the protocol provided by the
manufacturer. Single-strand cDNA was synthesized from the cellular mRNA
by adding SuperScript II RT (1 µl; 200 U/µl), buffer [10 µl; 5×
First Strand Buffer (in mM): contains 250 Tris-HCl, 375 KCl, and 15 MgCl2], DTT (5 µl; 0.1 M), and mixed dNTPs (18 µl; 2.5 mM). The mixture (45 µl) was incubated for 50 min at 42°C. The reaction was terminated by heating the mixture to
70°C for 2 min and then icing. The RNA strand in the RNA-DNA hybrid
was removed by adding 5 µl of RNase H (2 U/µl) and incubating for 1 hr at 37°C. All reagents were obtained from Life Technologies. The
DNA from the RT of RNA in microglia was subjected to PCR to detect the
expression of GluR mRNAs.
Amplification was performed on a thermal cycler (PC800; Astec, Fukuoka,
Japan) using DNA polymerase, LA Taq (TaKaRa, Tokyo, Japan) under the following cycle conditions: denaturation at 94°C for
1 min, annealing at 60°C, and extension at 72°C for 1 min and 30 sec (repeated for a total of 40 cycles). The first round PCR product
was purified and then used as a template for second round PCR
amplification (28 cycles) using the same primers in the first round.
After PCR amplification, 8.5 µl aliquot of reaction product was
analyzed by electrophoresis on ethidium bromide-stained agarose gel
(1.5%). The primers used for AMPA receptor subunit cDNA
amplification have been published previously (Lambolez et al., 1992).
The primers for the detection of AMPA receptor subunit (GluR1-GluR4)
cDNA were 5'-CCTTTGGCCTATGA-GATCTGGATGTG-3' and 5'-TCGTACCACCATTTGTTTTTCA-3'. This set of primers is common to GluR1-GluR4 mRNA and coamplified all four AMPA receptor subunits (Lambolez et al., 1992). The size of amplified products is 749 bp for
GluR1, GluR2, and GluR4, and 755 bp for GluR3, and they appear as a
single band on electrophoresis gel. To determine the presence of specific GluR1-GluR4 mRNA, the amplified PCR products were
cut with subunit-specific restriction enzymes. Aliquots (10 µl) of
the PCR product were digested with Bg/I (for GluR1),
Bsp1286I (for GluR2), Eco47III (for GluR3), and
EcoRI (for GluR4) at 37°C for 2 hr. The enzyme-cleaved
products were then analyzed with agarose gel electrophoresis and
stained with ethidium bromide. The predicted restriction fragments were
300 and 449 bp for GluR1, 478 and 271 bp for GluR2, 359 and 396 bp for
GluR3, and 411 and 338 bp for GluR4.
RT-PCR analysis of GluR5-GluR7 subunit mRNA used subunit-specific
primer sets. Two rounds of amplification (45 cycles total) were
performed using conditions similar to those described above (annealing
temperature of 61°C). Primer sets were derived from GenBank sequence using commercial primer design software (Oligo; National Biosciences, Plymouth, MN). The primers for detection of GluR5
cDNA (GenBank accession number M83560) (Bettler et al., 1990) were
5'-GCCCCTCTCACCATCACATAC-3' and 5'-ACCTCGCAATCACAAACAGTACA-3'. The
predicted product length was 208 bp. The primer for detection of GluR6
cDNA (GenBank accession number Z11548) were 5'-TTCCTGAATCCTCTCTCCCCT-3' and 5'-CACCAAATGCCTCCCACTATC-3'. The predicted product length was 260 bp. The primers for detection of GluR 7 cDNA (GenBank accession number
M83552) (Bettler et al., 1992) were 5'-TGGGCCTTCACCTTGATCATCA-3' and
5'-ACTCCACACCCCGACCTTCT-3'. The predicted product length was 423 bp.
The primers for detection of KA-1-KA-2 cDNA (GenBank accession number
59996) were 5'-TGGGCCTTCACCTTGATCATCA-3' and
5'-CTGTGGTCCTCCTCCTTGGG-3'. The predicted product length was 512 bp.
Care was taken to ensure that PCR signal arose from cellular mRNA.
Negative controls for contamination from extraneous and genomic DNA
were run. To ensure that genomic DNA did not contribute to the PCR
products, the cells were processed in the normal manner except that the
reverse transcriptase was omitted. Contamination from extraneous
sources was checked by replacing the cellular template with water. Both
controls were consistently negative in these experiments.
Assay of TNF-. The isolated microglial cells were seeded
in a 24-well plate at a density of 5.6 × 107 cells per well and were cultured for
3 d. Then, the cells were treated with
103 M KA, Glu,
NMDA, and AMPA. Most of the identified microglial cells displayed round
or spindle shape. After treatment for 2 hr, the amount of rat TNF-
released into the culture medium was measured by an ELISA kit
(Biosource, Camarillo, CA) having a detection limit of 2.3 pg/ml
following the protocol provided by the manufacturer. The absorbency
at 450 nm was performed by a Microplate Reader (model 450; Bio-Rad).
The data are presented as mean ± SD of four to six experiments.
The significance of differences between groups was determined with a
two-way ANOVA, followed by Scheffé's post hoc
test for multiple comparison when F ratios reached significance.
| |
RESULTS |
Identification of microglial cells
Isolated microglial cells showed phase-bright small round- or
rod-shaped cell bodies with no or few thick processes under the
phase-contrast microscope (Fig.
1A). In some
experiments, the identity of isolated cells used for
electrophysiological measurements were confirmed by using DiI-ac-LDL, a
fluorescent probe for scavenger receptors. As shown in Figure
1B, microglia identified by staining with DiI-ac-LDL
(23 of 25 cells in this microscopic field) were clearly visualized
under the inverted fluorescent microscope.
View larger version (75K):
[in this window]
[in a new window]
|
Figure 1.
Identification of microglial cells using
fluorescent probe DiI-ac-LDL. Microglial cells were visualized under
the fluorescent microscope combined with phase-contrast modes
(A) and the fluorescent modes
(B) that were used for whole-cell patch-clamp
recordings. Scale bar, 50 µm.
|
|
Electrophysiological measurement of kainate-induced response
Twenty percent (27 of 137) of the whole-cell patched microglial
cells showed a response to 3 × 104
to 103 M KA.
Application of KA induced an inward current under voltage-clamp conditions at the holding potential of 60 mV (Fig.
2A). The amplitude of
3 × 104 M
KA-induced inward current was 28 ± 5 pA (n = 17).
However, in the same cell that responded to KA, application of AMPA
between 104 and
103 M induced
apparently no or little inward current (Fig. 2Aa). Because AMPA responses showed very fast activation and inactivation (Partin et al., 1993; Patneau et al., 1994; Akaike and Rhee, 1997), we
might not be able to measure such a transient current, even with our
fast perfusion system at the temperature of 33-34°C. The response to
KA was reversibly cross-inhibited by addition of AMPA (Fig.
2Aa), thus suggesting that KA and AMPA activated the
same Glu receptor channel as found in the basket cells of rat
hippocampal dentate gyrus (Koh et al., 1995) and Meynert neurons of rat
nucleus basalis (Akaike and Rhee, 1997). Figure 2Ab
shows the responses to other agonists for Glu receptor channels in a KA-responsive cell. Similar to AMPA responses, 3 × 104 M Glu or
3 × 104 M QA
induced a little current. There was no response to 3 × 104 M NMDA in
the presence of 3 × 106
M glycine and without extracellular
Mg2+, suggesting microglia, unlike O-2A
progenitor cells, have no NMDA receptor channel complexes (Wang et al.,
1996). The response to 3 × 104
M KA was totally suppressed in the presence of
105 M CNQX (Fig.
2Ac).
View larger version (13K):
[in this window]
[in a new window]
|
Figure 2.
A, KA-induced current in rat
microglia. a, KA (3 × 104
M) induced an inward current, but apparently 3 × 104 M AMPA induced very small
inward current. There was cross-inhibition between KA and AMPA
responses. b, In the same microglial cell, 3 × 104 M KA but not 3 × 104 M NMDA (with 3 × 106 M glycine) induced an inward
current. Glu (3 × 104 M) and
3 × 104 M QA induced a small
inward current. c, KA-induced (3 × 104 M) current was blocked
by 105 M CNQX. The holding potential
was 60 mV. There are five other cells with similar results in
a-c each. B, Concentration-dependent
effect of KA. The holding potential was 40 mV. All currents were
normalized to the response induced by 103
M KA. Each point represents the mean of three
cells. The curve shows the least-squares fit to
the equation a = bn/(bn + Kn), where a is
relative activation, b is agonist concentration
(M), n is Hill coefficient (1.4), and
K is the half-activation concentration (0.33 × 103 M).
|
|
The KA-induced inward currents were concentration-dependent (Fig.
2B). Below 3 × 105 M, KA did not
induce any apparent inward current. In the KA-responsive cells, the
amplitudes of inward current induced by each concentration of KA
between 105 and 3 × 103 M were
normalized to the one induced by 103
M KA in the same cell. The
concentration-response relationship showed that the normalized maximum
current amplitudes, half-activation concentration, and Hill coefficient
were 1.19 ± 0.05, 0.33 × 103
M, and 1.4 ± 0.2 (n = 3), respectively.
Effects of cyclothiazide and concanavalin A on KA, Glu, and
AMPA responses
CTZ blocks desensitization of AMPA-preferring receptors and
produces marked potentiation of the response to KA or Glu (Patneau et
al., 1993; Trussell et al., 1993; Yamada and Tang, 1993; Akaike and
Rhee, 1997). Studies using KA-preferring receptors and recombinant AMPA- and KA-preferring receptors show that the action of CTZ is
selective for AMPA-preferring receptors (subunits GluR1-GluR4) and
that concanavalin A is selective for KA-preferring receptors (subunits
GluR5-GluR7 and KA-1 or KA-2) (Huettner, 1990; Partin et al., 1993;
Wong and Mayer, 1993).
In KA-responsive microglial cells, CTZ greatly potentiated KA-, Glu-,
and AMPA-induced inward currents (Fig.
3A) in all five cells tested.
Furthermore, the relatively small response to Glu and AMPA became
greater than the response to KA in the presence of CTZ. After
application of CTZ, the desensitization after the peak Glu- and
AMPA-induced current diminished in a time-dependent manner, finally
showing the steady-state current like KA-induced current (~20 min;
data not shown). These results suggest that KA-responsive cells all
express AMPA-preferring receptors. On the other hand, in only two of
seven KA-responsive cells, KA-induced current was affected by
application of concanavalin A (0.3 mg/ml; >3 min) (Fig.
3B). This result suggests that only some of the KA-responsive cells express KA-preferring receptors and not only AMPA-preferring receptors. When we treated cells with concanavalin A
before determining whether they respond to KA, the KA-responsive cells
were 4 of 10 cells (data not shown). These results also indicate the
heterogeneous distribution of AMPA- and KA-preferring receptors among
microglial cells; some cells express predominantly AMPA-preferring
receptors, some cells express predominantly KA-preferring receptors,
and some cells express both.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 3.
Cyclothiazide strongly potentiated KA-,
Glu-, and AMPA-induced inward currents, but concanavalin A did not
always potentiate KA-induced current. A, Inward current
evoked by 3 × 104 M KA, Glu, and
AMPA before (left) and during application of
104 M cyclothiazide (>3 min)
(right) in the same cell. B, In some
cells, inward current evoked by 3 × 104
M KA was potentiated by adding 0.3 mg/ml concanavalin A
(>3 min).
|
|
Figure 4 shows the I-V
relationships of the responses to 103
M KA and 104
M AMPA. The I-V relationships
(normalized to the respective peak current amplitudes at 40 mV)
reversed at ~0 to 10 mV. The rather linear shape of the
I-V relationship of the KA response in
Na+-containing solution pointed to the
possible expression of glial AMPA receptor with low
Ca2+ permeability (Hollmann et al., 1991;
Burnashev et al., 1992).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 4.
Current-voltage relationships of KA- and
AMPA-induced currents. A, KA-induced
(103 M) currents at different holding
potentials and the I-V relationships. B,
AMPA-induced (104 M) currents in the
presence of 104 M CTZ. All current
amplitudes were normalized to the peak current at a holding potential
of 40 mV. Each point represents the mean of three to four
cells. Some error bars are within the circles.
|
|
To estimate the divalent permeability of the receptor channel complexes
activated by KA, the external solutions were switched from high
Na+ (110 [Na+]/3
[Ca2+]) to high
Ca2+ (80 [Ca2+]/0
[Na+]) concentrations (Fig.
5). Under these conditions, KA-induced inward current at 40 and 0 mV turned to outward current (Fig. 5A), and the mean reversal potential shifted to negative
potential of approximately 40 mV (n = 3) (Fig.
5B). Nevertheless, the activation of receptor currents in
external solutions with Ca2+ being the
charge carrier for the GluR channel, together with a rather positive
deviation of the reversal potential from that expected for channels
with an exclusive monovalent permeability (far negative because no
monovalent cation was included in high Ca2+ solution), indicated an intermediate
Ca2+ permeability of this GluR channel
(Seifert and Steinhäuser, 1995; Washburn et al., 1997). To
estimate the relative permeability of the GluR channels to
Ca2+, the following constant field
equation was used:
![P<SUB><UP>Ca</UP></SUB>/P<SUB><UP>Na</UP></SUB>=([<UP>Na<SUP>+</SUP></UP>]<SUB><UP>i</UP></SUB><UP>/</UP>[<UP>Ca<SUP>2+</SUP></UP>]<SUB><UP>o</UP></SUB>)(<UP>exp</UP>(VF/RT)[<UP>exp</UP>(VF/RT)+1]/4)](/content/vol20/issue1/fulltext/251/img001.gif)
|
|
where V is the reversal potential, F,
R, and T have their usual meanings,
[Ca2+]o is the
extracellular Ca2+ concentration, and
PCa and
PNa represent the permeability
coefficient of Ca2+ and
Na+, respectively (Iino et al., 1990).
Then, we obtained a permeability ratio of
PCa/PNa = 0.26.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 5.
KA-induced currents in high
Na+ and high Ca2+ external
solutions. A, KA-induced (103
M) currents at different holding potentials in high
Na+ and high Ca2+ external
solutions. B, I-V relationships in high
Na+ and high Ca2+ external
solutions. All current amplitudes were normalized to the peak current
at a holding potential of 40 mV. Each point represents the
mean of three to four cells. Some error bars are within the
circles.
|
|
Immunocytochemical study of AMPA receptor
The low Ca2+ permeability of AMPA
receptors in microglial cells suggests that they include GluR2
(Hollmann et al., 1991; Hume et al., 1991; Verdoorn et al., 1991;
Burnashev et al., 1992). Therefore, the existence of AMPA receptors on
microglia was examined by a double-staining technique using anti-GluR2
and 3 antibody (Alexia 488 labeling; green) and
GSA-I-B4 (Cy3 labeling; red) (Fig.
6). Spindle-shaped cells were moderately
stained by anti-GluR2 and 3 and identified to be microglia by
GSA-I-B4 staining. A small population of cells
(5-8%), which had fusiform cell bodies with long processes and
assumed to be O-2A cells, were intensely stained by anti-GluR2 and 3 antibody but devoid of GSA-I-B4 staining. This
staining pattern clearly demonstrated that microglia possessed moderate
amount of GluR2 and/or GluR3. No immunoreaction was detected in control
experiments with nonimmune IgG.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 6.
Localization of AMPA-preferring subunits
of Glu receptor on microglia. Staining with anti-Glu receptor 2 and 3 antibody (green) and with GSA-I-B4
(red) were visualized by confocal laser scanning
microscope images. Scale bars, 10 µm.
|
|
Identification of AMPA-KA receptor subunits by RT-PCR
Our RT-PCR study of AMPA receptor subunits showed that rat
microglia contained GluR2, GluR3, and GluR4 mRNAs (Fig.
7). The amplified PCR products, which
contained all GluR1-GluR4 and appeared as a single band on
electrophoresis gels, were cut with subunit-specific restriction
enzymes, except for GluR1 (Fig. 7, lane 1). The predicted restriction fragments were found for GluR2 (Fig. 7, lane 2),
GluR3 (Fig. 7, lane 3), and GluR4 (Fig. 7, lane
4), respectively.
View larger version (89K):
[in this window]
[in a new window]
|
Figure 7.
RT-PCR was performed on mRNA harvested from rat
microglia to determine the subunits of AMPA-KA receptor mRNA. The AMPA
subunit content was determined by a two-step procedure. First, cDNA for
GuR1-GluR4 subunits were PCR-amplified simultaneously using primers
common to all four subunits. Separate fractions of these PCR products
were then treated with subunit-specific restriction enzyme for GluR1
(lane 1), GluR2 (lane 2), GluR 3 (lane 3), GluR4 (lane 4), or
without treatment (lane 0). The PCR products and/or
restriction enzyme products were visualized using gel electrophoresis
and ethidium bromide staining and were identified by their size. It
shows that rat microglia contain GluR2-GluR4, whereas GluR1 is barely
detectable. For PCR analysis of KA receptor mRNAs, the subunit-specific
primer sets were used. The PCR products showed detectable GluR5
(lane 5) but not GluR6 (lane
6).
|
|
Our RT-PCR studies of KA receptor subunits are more preliminary. GluR5
mRNA was detected (Fig. 7, lane 5), but GluR6 mRNA was not
expressed at detectable levels (Fig. 7, lane 6).
GluR7 and KA-1-KA-2 mRNAs were detected in a subpopulation of these cells, indicative of lower mRNA abundance (data not shown).
Enhanced production of TNF- from microglia by treatment
with KA
To elucidate whether AMPA-KA receptors on microglia have
functional consequences because microglia are known to produce
cytokines after cellular activation (McGeer et al., 1993; Meda et al.,
1995), the release of TNF- before and after treatment of KA and
other Glu agonists was measured. As shown in Figure
8, microglia constitutively produced
small amounts of TNF-. When microglia were treated with 103 M KA, the amount of
TNF- released in the culture medium at 2 hr after the treatment was
significantly increased (Fig. 8A). Similar
enhancement was also observed after the treatment with 103 M Glu. On the
other hand, neither NMDA nor AMPA had any significant effect on the
production of TNF- by microglia. Both Glu- and KA-induced
productions of TNF- were significantly depressed by adding 2 × 105 M CNQX (Fig.
8B).
View larger version (13K):
[in this window]
[in a new window]
|
Figure 8.
Effects of KA on the production of
TNF- by microglia. The amount of TNF- released in the culture
medium of microglia at 2 hr after stimulation was measured by ELISA.
A, Effects of 103 M
Glu, NMDA, KA, and AMPA on the production of TNF- by microglia.
**p < 0.01, ***p < 0.001, compared with control (two-way ANOVA). B, Effects of
CNQX (2 × 105 M) on the
103 M Glu-induced and
103 M KA-induced TNF- production.
**p < 0.05, ***p < 0.001, compared with control;  p < 0.05, p < 0.01, compared with the data
obtained in the absence of CNQX (two-way ANOVA).
|
|
| |
DISCUSSION |
The present studies provide the first evidence for the existence
of AMPA-KA subunits of Glu receptor in primary cultured rat microglia.
Although the present immunohistochemical and RT-PCR analyses clearly
showed that microglia have a substantial amount of GluR2-GluR5, the
net inward current induced by either Glu or AMPA was very small
compared with that induced by KA at the holding potential of 40 or
60 mV. One possible explanation for this apparent discrepancy is that
AMPA- and Glu-activated currents are desensitized very fast and the
fast response could not be detected, even with our fast perfusion
system using the Y tube technique. To test this possibility, we
examined the effects of CTZ and concanavalin A, agents that block the
desensitization of AMPA- and KA-preferring receptors, respectively. We
found that CTZ markedly enhanced KA-, AMPA-, and Glu-activated currents
in all of the KA-responsive microglia. Concanavalin A not only
augmented the amplitude of KA-induced current in some of the
KA-responsive microglia but also seemed to unmask KA receptors, which
were supposedly expressed predominantly in some cells and were missed
without concanavalin A. Thus, the extremely fast desensitization or
very small response of AMPA- and KA-preferring receptors (Partin et al., 1993; Swanson and Heinemann, 1998) is likely to be mainly responsible for the fact that their existence in microglia has long
gone unnoticed. According to Raman and Trussell (1992),
glutamate-evoked currents in neurons of the chicken cochlea nucleus
desensitizes very rapidly, and the high temperature accelerates
desensitization. However, it is not unreasonable to speculate that the
transient and localized activation of an ionotropic receptor could be
greatly amplified when assaying its downstream effects, because many
receptors can be tightly linked to other proteins that may be involved
in initiation of intracellular cascade. In addition, we also speculate that there might exist an endogenous active factor that can augment the
response of AMPA- and KA-preferring receptors in microglia, such as
glycine for NMDA receptors as reported by Johnson and Ascher (1987).
Alternatively, pathological states, for example extracellular acidity
as suggested by McDonald et al. (1998), may potentiate AMPA and KA
receptor-mediated responses. Further investigations are necessary to
clarify this point.
Although the subtype composition of Glu receptor expressed in rat
microglia was not yet known, we hypothesized that at least GluR2 should
be expressed, because either KA- or AMPA-activated receptor channels
have poor Ca2+ permeability (Fig. 5). This
expectation was preliminarily confirmed from double-labeling
immunohistochemical studies using anti-GluR2 and 3 antibody (Fig. 6).
Then, the precise expression of AMPA receptor subunits in microglia was
analyzed by RT-PCR, which showed that rat microglia contained
GluR2-GluR4 (Fig. 7). Because it was clear that KA receptors were also
present in some cells from the electrophysiological data using
concanavalin A, we further analyzed the expression of mRNAs for KA
receptor subunits. The results clearly showed the expression of mRNA
for GluR5 (Fig. 7) and indicated the lower expression levels of mRNAs
for GluR7 and KA-1-KA-2. On the other hand, O-2A lineage cells express
mRNAs for GluR2-GluR4, in addition to GluR6, GluR7, KA-1, and KA-2
(Patneau et al., 1994). Our microglial cells, especially for RT-PCR,
were carefully isolated without any contamination of other cells, i.e., O-2A cells, so that we could exclude the possibility that mRNA for GluR
from other cell types were amplified.
The physiological role of these AMPA-KA receptors in rat microglia
also remains unclear. However, we found that Glu and KA can enhance
production of TNF- (Fig. 8). It is possible that elevated levels of
Glu in pathological sites may directly activate AMPA-KA receptors on
microglia contributing enhanced production of TNF-. Recently,
expression and signaling of metabotropic glutamate receptor in
microglia has been reported (Biber et al., 1999), which might also
contribute to the functional role of Glu in microglia. The mechanism
and the signal transduction pathways mediating from GluR to TNF-
production in microglia are not known. However, it may be common to
those used by these cells in response to inflammatory stimuli, i.e.,
tyrosine kinase-based signaling cascade (Combs et al., 1999). Because
the cascade reported so far contains
Ca2+-sensitive pathway, TNF- production
by Glu and KA is also suggested to be dependent on intracellular
Ca2+, indicating that low
Ca2+ permeability of KA-mediated response
might have been enough for it. It might also be possible that KA
receptors rather than AMPA receptors are more likely mediators of the
increase in TNF- production because of the lack of effect of AMPA on
TNF-. Whether or not AMPA receptors and KA receptors exhibit
different Ca2+ permeability, which
receptors are dominating the responses we observed and their signaling
cascade are now under investigation. As for the function of TNF-,
although TNF- production rapidly increases after excitotoxic,
ischemic, and traumatic brain injuries (Minami et al., 1991; Taupin et
al., 1993; Liu et al., 1994), the effects of TNF- on neurons remains
controversial. For example, Chao and Hu (1994) have reported that
TNF- potentiates Glu neurotoxicity, whereas Cheng et al. (1994) have
shown that TNF- protects neurons against excitotoxic insults. On
the other hand, TNF-, as well as other cytokines, are also known to
work on glial cells and to kill oligodendrocytes (Selmaj and Raine,
1988; Louis et al., 1993), leading to destruction of myelin and the
dysfunction of axons (Merrill and Benveniste, 1996). Based on these
assumptions, more precise analysis of the functions and the
characteristics of Glu receptors in microglia will contribute to a
better understanding of the physiological and pathological events in
the CNS.
| |
FOOTNOTES |
Received April 30, 1999; revised Oct. 13, 1999; accepted Oct. 18, 1999.
This work was supported by Grants-in-Aid for Scientific Research Grants
11671845 (H.N.), 11170240 (J.N.), and 10470009 (N.A.) from the Ministry
of Education, Science, and Culture, Japan, and the Hayashi Memorial
Foundation for Female Natural Scientists (M.N.). We thank Dr. N. Takai
(Kyushu University) for assistance with the preparation of the primary
cultured microglia, Dr. T. Fukuda (Kyushu University) for helping us
with the confocal laser scanning microscope, and Prof. D. A. Brown
(University College, London, UK) and Dr. M. Brodwick (University of
Texas Medical Branch, Galveston, TX) for reading this manuscript.
Correspondence should be addressed to Dr. Norio Akaike, Laboratory of
Cellular and System Physiology, Graduate School of Medical Science,
Kyushu University, Fukuoka 812-8582, Japan. E-mail:
akaike{at}mailserver.med.kyushu-u.ac.jp.
Dr. Noda's present address; Laboratory of Pathophysiology, Graduate
School of Pharmaceutical Science, Kyushu University, Fukuoka 812-8582, Japan
| |
REFERENCES |
-
Akaike N,
Rhee JS
(1997)
Age-related functional changes of the glutamate receptor channels in rat Meynert neurones.
J Physiol (Lond)
504:665-681[Abstract/Free Full Text].
-
Araujo DM,
Cotman CW
(1992)
Basic FGF in astroglial, microglial, and neuronal cultures: characterization of binding sites and modulation of release by lymphokines and trophic factors.
J Neurosci
12:1668-1678[Abstract].
-
Bettler B,
Boulter J,
Hermans-Borgmeyer I,
O'Shea-Greenfield A,
Deneris ES,
Moll C,
Borgmeyer U,
Heinemann S
(1990)
Cloning of a novel glutamate receptor subunit, GluR5: expression in the nervous system during development.
Neuron
5:583-595[Web of Science][Medline].
-
Bettler B,
Egebjerg J,
Sharma G,
Pecht G,
Hermans-Borgmeyer I,
Moll C,
Stevens CF,
Heinemann S
(1992)
Cloning of a putative glutamate receptor: a low affinity kainate-binding subunit.
Neuron
8:257-265[Web of Science][Medline].
-
Bezzi P,
Carmignoto G,
Pasti L,
Vesce S,
Rossi D,
Rizzini BL,
Pozzan T,
Volterra A
(1998)
Prostaglandins stimulate calcium-dependent glutamate release in astrocytes.
Nature
391:281-285[Medline].
-
Biber K,
Laurie DJ,
Berthele A,
Sommer B,
Tölle TR,
Gebicke-Härter PJ,
Van Calker D,
Boddeke HWGM
(1999)
Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia.
J Neurochem
72:1671-1680[Web of Science][Medline].
-
Bowman CL,
Kimelberg HK
(1984)
Excitatory amino acids directly depolarize rat brain astrocytes in primary culture.
Nature
311:656-659[Medline].
-
Burnashev N,
Monyer PH,
Seeburg B,
Sackmann B
(1992)
Divalent ion permeability of AMPA receptor channels is dominated by edited of a single subunit.
Neuron
8:189-198[Web of Science][Medline].
-
Chao CC,
Hu S
(1994)
Tumor necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures.
Dev Neurosci
16:172-179[Web of Science][Medline].
-
Cheng B,
Christakos S,
Mattson MP
(1994)
Tumor necrosis factors protect neurons against excitotoxic/metabolic insults and promote maintenance of calcium homeostasis.
Neuron
12:139-153[Web of Science][Medline].
-
Chessell IP,
Michel AD,
Humphrey PPA
(1997)
Properties of the pore-forming P2X7 purinoceptor in mouse NTW8 microglial cells.
Br J Pharmacol
121:1429-1437[Web of Science][Medline].
-
Combs CK,
Johnson DE,
Cannady SB,
Lehman TM,
Landreth GE
(1999)
Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of
 -amyloid and prion proteins.
J Neurosci
19:928-939[Abstract/Free Full Text]. -
Cornell-Bell AH,
Finkbeiner SM,
Cooper MS,
Smith SJ
(1990)
Glutamate induces calcium waves in cultured astrocytes: long range glial signalling.
Science
247:470-474[Abstract/Free Full Text].
-
El Khoury J,
Hickman SE,
Thomas CA,
Cao L,
Silverstein SC,
Loike JD
(1996)
Scavenger receptor mediated adhesion of microglia to -amyloid fibrils.
Nature
382:716-719[Medline].
-
Gallo V,
Giovannini C,
Suergiu R,
Levi G
(1989)
Expression of excitatory amino acid receptors by cerebellar cells of the type-2 astrocyte lineage.
J Neurochem
52:1-9[Web of Science][Medline].
-
Hollmann M,
Hartley S,
Heinemann S
(1991)
Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition.
Science
252:851-853[Abstract/Free Full Text].
-
Huettner JE
(1990)
Glutamate receptor channels in rat DRG neurons: activation by kainate and quisqualate, and blockade of desensitization by Con A.
Neuron
5:255-266[Web of Science][Medline].
-
Hume RI,
Dingledine DR,
Heinemann SF
(1991)
Identification of a site in glutamate receptor subunits that controls calcium permeability.
Science
253:1028-1031[Abstract/Free Full Text].
-
Iino M,
Ozawa S,
Tsuzuki K
(1990)
Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurons.
J Physiol (Lond)
424:151-165[Abstract/Free Full Text].
-
Inoue K,
Nakajima K,
Morimoto T,
Kikuchi Y,
Koizumi S,
Illes P,
Kohsaka S
(1998)
ATP stimulation of Ca2+-dependent plasminogen release from cultured microglia.
Br J Pharmacol
123:1304-1310[Web of Science][Medline].
-
Johnson JW,
Ascher P
(1987)
Glycine potentiates the NMDA response in cultured mouse brain neurons.
Nature
325:529-531[Medline].
-
Koh DS,
Geiger JR,
Jonas P,
Sakmann B
(1995)
Ca2+-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus.
J Physiol (Lond)
485:383-402[Abstract/Free Full Text].
-
Kreuzberg GW
(1996)
Microglia: a sensor for pathological events in the CNS.
Trends Neurosci
19:312-318[Web of Science][Medline].
-
Lambolez B,
Audinat E,
Bochet P,
Crepel F,
Rossier J
(1992)
AMPA receptor subunits expressed by single Purkinje cells.
Neuron
9:247-258[Web of Science][Medline].
-
Liu T,
Clark RK,
McDonnell PC,
Young PR,
White RF,
Barone FC,
Feuerstein GZ
(1994)
Tumor necrosis factor- expression in ischemic neurons.
Stroke
25:1481-1488[Abstract].
-
Louis JC,
Magal E,
Takayama S,
Varon S
(1993)
CNTF protection of oligodendrocytes against natural and tumor necrosis factor-induced death.
Science
259:689-692[Abstract/Free Full Text].
-
Mallat M,
Houlgatte R,
Brachet P,
Prochiantz A
(1989)
Lipopolysaccharide-stimulated rat brain macrophages release NGF in vitro.
Dev Biol
133:309-311[Web of Science][Medline].
-
Matute C,
Sánches-Gómez MV,
Martínez-Millán L,
Miledi R
(1997)
Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes.
Proc Natl Acad Sci USA
94:8830-8835[Abstract/Free Full Text].
-
McDonald JW,
Bhattacharyya T,
Sensi SL,
Lobner D,
Ying HS,
Canzoniero LM,
Choi DW
(1998)
Extracellular acidity potentiates AMPA receptor-mediated cortical neuronal death.
J Neurosci
18:6290-6299[Abstract/Free Full Text].
-
McGeer PL,
Kawamata T,
Walker DG,
Akiyama H,
Tooyama I,
McGeer EG
(1993)
Microglia in degenerative neurological disease.
Glia
7:84-92[Web of Science][Medline].
-
Meda L,
Cassarella MA,
Szendrei GI,
Otvos Jr L,
Baron P,
Villalba M,
Ferrari D,
Rossi F
(1995)
Activation of microglial cells by -amyloid protein and interferon-
 .
Nature
374:647-650[Medline]. -
Merrill JE,
Benveniste EN
(1996)
Cytokines in inflammatory brain lesions: helpful and harmful.
Trends Neurosci
19:331-338[Web of Science][Medline].
-
Min BI,
Kim CJ,
Rhee JS,
Akaike N
(1996)
Modulation of glycin-induced chloride current in acutely dissociated rat periaqueductal gray neurons by µ-opioid agonist DAGO.
Brain Res
734:72-78[Web of Science][Medline].
-
Minami M,
Kuraishi Y,
Satoh M
(1991)
Effects of kainic acid on messenger RNA levels of IL-1 beta, IL-6, TNF- and LIF in the rat brain.
Biochem Biophys Res Commun
176:593-598[Web of Science][Medline].
-
Möller T,
Kann O,
Prinz M,
Kirchhoff F,
Verkhratsky A,
Kettenmann H
(1997)
Endothelin-induced calcium signaling in cultured mouse microglial cells is mediated through ETB receptors.
NeuroReport
8:2127-2131[Web of Science][Medline].
-
Müller T,
Möller T,
Berger T,
Schnitzer J,
Kettenmann H
(1992)
Calcium entry through kainate receptors and resulting potassium-channel blockade in Bergmann glial cells.
Science
256:1563-1565[Abstract/Free Full Text].
-
Nakajima K,
Tsuzaki N,
Shimojo M,
Hamanoue M,
Kohsaka S
(1992a)
Microglia isolated from rat brain secrete a urokinase-type plasminogen activator.
Brain Res
577:285-292[Web of Science][Medline].
-
Nakajima K,
Tsuzaki N,
Nagata K,
Takemoto N,
Kohsaka S
(1992b)
Production and secretion of plasminogen in cultured rat brain microglia.
FEBS Lett
308:179-182[Web of Science][Medline].
-
Noda M,
Obana M,
Akaike N
(1998)
Inhibition of M-type K+ current by linopirdine, a neurotransmitter release enhancer, in NG108-15 neuronal cells and rat cerebral neurons in culture.
Brain Res
794:274-280[Web of Science][Medline].
-
Noda M,
Nakanishi H,
Akaike N
(1999)
Glutamate release from microglia via glutamate transporter is enhanced by amyloid- peptide.
Neuroscience
92:1465-1474[Web of Science][Medline].
-
Parpura V,
Basarsky TA,
Liu F,
Jeftinija K,
Jeftinija S,
Haydon PG
(1994)
Glutamate-mediated astrocyte-neuron signalling.
Nature
369:744-747[Medline].
-
Partin KM,
Patneau DK,
Winters CA,
Mayer ML,
Buonanno A
(1993)
Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A.
Neuron
11:1069-1082[Web of Science][Medline].
-
Patneau DK,
Vyklicky Jr L,
Mayer ML
(1993)
Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate.
J Neurosci
13:3496-3509[Abstract].
-
Patneau DK,
Wright PW,
Winters C,
Mayer ML,
Gallo V
(1994)
Glial cells of the oligodendrocytes lineage express both kainate- and AMPA-preferring subtypes of glutamate receptor.
Neuron
12:357-371[Web of Science][Medline].
-
Queiroz G,
Gebicke-Haerter PJ,
Schobert A,
Starke K,
Von Kugelgen I
(1997)
Release of ATP from cultured rat astrocytes elicited by glutamate receptor activation.
Neuroscience
78:1203-1208[Web of Science][Medline].
-
Raman IM,
Trussell LO
(1992)
The kinetics of the response to glutamate and kainate in neurons of the avian cochlear nucleus.
Neuron
9:173-186[Web of Science][Medline].
-
Sastradipula DF,
Nakanishi H,
Tsukuba T,
Nishishita K,
Sakai H,
Kato Y,
Gotow T,
Uchiyama Y,
Yamamoto K
(1998)
Identification of cellular compartments involved in processing of cathepsin E in primary cultures of rat microglia.
J Neurochem
70:2045-2056[Web of Science][Medline].
-
Seifert G,
Steinhäuser C
(1995)
Glial cells in the mouse hippocampus express AMPA receptors with an intermediate Ca2+ permeability.
Eur J Neurosci
7:1872-1881[Web of Science][Medline].
-
Selmaj K,
Raine CS
(1988)
Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro.
Ann Neurol
21:339-346.
-
Shimojo M,
Nakajima K,
Takei N,
Hamanoue M,
Kohsaka S
(1991)
Production of basis fibroblast growth factor in cultured brain microglia.
Neurosci Lett
155:11-14.
-
Sontheimer H,
Kettenmann H,
Backus KH,
Schachner M
(1988)
Glutamate opens Na+/K+ channels in cultured astrocytes.
Glia
1:328-336[Web of Science][Medline].
-
Stefani A,
Chen Q,
F-Hernandez J,
Jiao Y,
Reiner A,
Surmeier D J
(1998)
Physiological and molecular properties of AMPA/kainate receptors expressed by striatal medium spiny neurons.
Dev Neurosci
20:242-252[Web of Science][Medline].
-
Steinhäuser C,
Gallo V
(1996)
News on glutamate receptors in glial cells.
Trends Neurosci
19:339-345[Web of Science][Medline].
-
Streit WJ,
Kincaid-Colton CA
(1995)
The brain's immune system.
Sci Am
273:38-43.
-
Swanson GT,
Heinemann SF
(1998)
Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells.
J Physiol (Lond)
513:639-646[Abstract/Free Full Text].
-
Taupin V,
Toulmond S,
Serrano A,
Benavides J,
Zavala F
(1993)
Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864: a peripheral-type (p site) benzodiazepine ligand.
J Neuroimmunol
42:177-185[Web of Science][Medline].
-
Trussell LO,
Zhang S,
Raman I
(1993)
Desensitization of AMPA receptors upon multiquantal neurotransmitter release.
Neuron
10:1185-1196[Web of Science][Medline].
-
Usowicz MM,
Gallo V,
Cull-Candy SG
(1989)
Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids.
Nature
339:380-383[Medline].
-
Verdoorn TA,
Burnashev H,
Monyer PH,
Seeburg B,
Sackmann B
(1991)
Structural determinations of ion flow through recombinant glutamate receptor channels.
Science
252:1715-1718[Abstract/Free Full Text].
-
Verkhratsky A,
Kettenmann H
(1996)
Calcium signalling in glial cells.
Trends Neurosci
19:346-352[Web of Science][Medline].
-
Wang C,
Pralong W-F,
Schulz M-F,
Rougon G,
Aubry J-M,
Pagliusi S,
Robert A,
Kiss JZ
(1996)
Functional N-methyl-D-aspartate receptors in O-2A glial precursor cells: a critical role in regulating polysialic acid-neural cell adhesion molecule expression and cell migration.
J Cell Biol
135:1565-1581[Abstract/Free Full Text].
-
Washburn MS,
Numberger M,
Zhang S,
Dingledine R
(1997)
Differential dependence on GluR2 expression of three characteristic features of AMPA receptors.
J Neurosci
17:9393-9406[Abstract/Free Full Text].
-
Wong L,
Mayer ML
(1993)
Differential modulation by cyclothiazide and concanavalin A of desensitization at native -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate-preferring glutamate receptors.
Mol Pharmacol
44:504-510[Abstract].
-
Wyss-Coray T,
Masliah E,
Mallory M,
McConlogue L,
Johnson-Wood K,
Lin C,
Mucke L
(1997)
Amyloidogenic role of cytokine TGF-1 in transgenic mice and in Alzheimer's disease.
Nature
389:603-606[Medline].
-
Yamada KA,
Tang C-M
(1993)
Benzothiadiazides inhibit rapid glutamate receptor desensitization and enhance glutamatergic synaptic currents.
J Neurosci
13:3904-3915[Abstract].
Copyright © 2000 Society for Neuroscience 0270-6474/0/201251-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
H. Wake, A. J. Moorhouse, S. Jinno, S. Kohsaka, and J. Nabekura
Resting Microglia Directly Monitor the Functional State of Synapses In Vivo and Determine the Fate of Ischemic Terminals
J. Neurosci.,
April 1, 2009;
29(13):
3974 - 3980.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I.-H. Cho, J. Hong, E. C. Suh, J. H. Kim, H. Lee, J. E. Lee, S. Lee, C.-H. Kim, D. W. Kim, E.-K. Jo, et al.
Role of microglial IKK{beta} in kainic acid-induced hippocampal neuronal cell death
Brain,
November 1, 2008;
131(11):
3019 - 3033.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L.-J. Wu and M. Zhuo
Resting Microglial Motility Is Independent of Synaptic Plasticity in Mammalian Brain
J Neurophysiol,
April 1, 2008;
99(4):
2026 - 2032.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Beck, R. Penner, and A. Fleig
Lipopolysaccharide-induced down-regulation of Ca2+ release-activated Ca2+ currents (ICRAC) but not Ca2+-activated TRPM4-like currents (ICAN) in cultured mouse microglial cells
J. Physiol.,
January 15, 2008;
586(2):
427 - 439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Porcheray, C. Leone, B. Samah, A.-C. Rimaniol, N. Dereuddre-Bosquet, and G. Gras
Glutamate metabolism in HIV-infected macrophages: implications for the CNS
Am J Physiol Cell Physiol,
October 1, 2006;
291(4):
C618 - C626.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. J. Liu, A. Kalous, E. L. Werry, and M. R. Bennett
Purine Release from Spinal Cord Microglia after Elevation of Calcium by Glutamate
Mol. Pharmacol.,
September 1, 2006;
70(3):
851 - 859.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Dallas, D. S. Miller, and R. Bendayan
Multidrug resistance-associated proteins: expression and function in the central nervous system.
Pharmacol. Rev.,
June 1, 2006;
58(2):
140 - 161.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kajitani, H. Yamaguchi, Y. Dan, M. Furuichi, D. Kang, and Y. Nakabeppu
MTH1, an Oxidized Purine Nucleoside Triphosphatase, Suppresses the Accumulation of Oxidative Damage of Nucleic Acids in the Hippocampal Microglia during Kainate-Induced Excitotoxicity
J. Neurosci.,
February 8, 2006;
26(6):
1688 - 1698.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. E. Jensen
Role of Glutamate Receptors in Periventricular Leukomalacia
J Child Neurol,
December 1, 2005;
20(12):
950 - 959.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Y. S. Shibakawa, Y. Sasaki, Y. Goshima, N. Echigo, Y. Kamiya, K. Kurahashi, Y. Yamada, and T. Andoh
Effects of ketamine and propofol on inflammatory responses of primary glial cell cultures stimulated with lipopolysaccharide
Br. J. Anaesth.,
December 1, 2005;
95(6):
803 - 810.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. J. Stuckey, D. C. Anthony, J. P. Lowe, J. Miller, W. M. Palm, P. Styles, V. H. Perry, A. M. Blamire, and N. R. Sibson
Detection of the inhibitory neurotransmitter GABA in macrophages by magnetic resonance spectroscopy
J. Leukoc. Biol.,
August 1, 2005;
78(2):
393 - 400.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Suo, M. Wu, B. A. Citron, G. T. Wong, and B. W. Festoff
Abnormality of G-Protein-Coupled Receptor Kinases at Prodromal and Early Stages of Alzheimer's Disease: An Association with Early {beta}-Amyloid Accumulation
J. Neurosci.,
March 31, 2004;
24(13):
3444 - 3452.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Glezer, H. Zekki, C. Scavone, and S. Rivest
Modulation of the Innate Immune Response by NMDA Receptors Has Neuropathological Consequences
J. Neurosci.,
December 3, 2003;
23(35):
11094 - 11103.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. E. Ayoub and A. K. Salm
Increased Morphological Diversity of Microglia in the Activated Hypothalamic Supraoptic Nucleus
J. Neurosci.,
August 27, 2003;
23(21):
7759 - 7766.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Furuta, M. Noda, S. O. Suzuki, Y. Goto, Y. Kanahori, J. D. Rothstein, and T. Iwaki
Translocation of Glutamate Transporter Subtype Excitatory Amino Acid Carrier 1 Protein in Kainic Acid-Induced Rat Epilepsy
Am. J. Pathol.,
August 1, 2003;
163(2):
779 - 787.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Hoffmann, O. Kann, C. Ohlemeyer, U.-K. Hanisch, and H. Kettenmann
Elevation of Basal Intracellular Calcium as a Central Element in the Activation of Brain Macrophages (Microglia): Suppression of Receptor-Evoked Calcium Signaling and Control of Release Function
J. Neurosci.,
June 1, 2003;
23(11):
4410 - 4419.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Lee, S. Dallas, M. Hong, and R. Bendayan
Drug Transporters in the Central Nervous System: Brain Barriers and Brain Parenchyma Considerations
Pharmacol. Rev.,
December 1, 2001;
53(4):
569 - 596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. M. Tikka and J. E. Koistinaho
Minocycline Provides Neuroprotection Against N-Methyl-D-aspartate Neurotoxicity by Inhibiting Microglia
J. Immunol.,
June 15, 2001;
166(12):
7527 - 7533.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Tikka, B. L. Fiebich, G. Goldsteins, R. Keinanen, and J. Koistinaho
Minocycline, a Tetracycline Derivative, Is Neuroprotective against Excitotoxicity by Inhibiting Activation and Proliferation of Microglia
J. Neurosci.,
April 15, 2001;
21(8):
2580 - 2588.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
