Previous Article | Next Article 
The Journal of Neuroscience, April 1, 2001, 21(7):2224-2239
The Influence of Glutamate Receptor 2 Expression on
Excitotoxicity in GluR2 Null Mutant Mice
Koji
Iihara1,
Daisy T.
Joo2,
Jeffrey
Henderson3,
Rita
Sattler1,
Franco A.
Taverna3,
Sandra
Lourensen3,
Beverley A.
Orser2,
John C.
Roder3, and
Michael
Tymianski1
1 Toronto Western Hospital, University of Toronto,
Toronto, Ontario M5T-2S8, Canada, 2 Department of
Anesthesia, University of Toronto, Toronto, Ontario M5G-1X8, Canada,
and 3 Samuel Lunenfeld Research Institute, Mount Sinai
Hospital, Toronto, Ontario M5G-1X5, Canada
 |
ABSTRACT |
AMPA receptor (AMPAR)-mediated ionic currents that govern
gene expression, synaptic strength, and plasticity also can
trigger excitotoxicity. However, native AMPARs exhibit heterogeneous
pharmacological, biochemical, and ionic permeability characteristics,
which are governed partly by receptor subunit composition.
Consequently, the mechanisms governing AMPAR-mediated excitotoxicity
have been difficult to elucidate. The GluR2 subunit is of particular
interest because it influences AMPAR pharmacology,
Ca2+ permeability, and AMPAR interactions with
intracellular proteins. In this paper we used mutant mice lacking the
AMPAR subunit GluR2 to study AMPAR-mediated excitotoxicity in cultured
cortical neurons and in hippocampal neurons in vivo. We
examined the hypothesis that in these mice the level of GluR2
expression governs the vulnerability of neurons to excitotoxicity and
further examined the ionic mechanisms that are involved. In cortical
neuronal cultures AMPAR-mediated neurotoxicity paralleled the magnitude
of kainate-evoked AMPAR-mediated currents, which were increased in
neurons lacking GluR2. Ca2+ permeability, although
elevated in GluR2-deficient neurons, did not correlate with
excitotoxicity. However, toxicity was reduced by removal of
extracellular Na+, the main charge carrier of
AMPAR-mediated currents. In vivo, the vulnerability of
CA1 hippocampal neurons to stereotactic kainate injections and of CA3
neurons to intraperitoneal kainate administration was independent of
GluR2 level. Neurons lacking the GluR2 subunit did not demonstrate
compensatory changes in the distribution, expression, or function of
AMPARs or of Ca2+-buffering proteins. Thus GluR2
level may influence excitotoxicity by effects additional to those on
Ca2+ permeability, such as effects on agonist
potency, ionic currents, and synaptic reorganization.
Key words:
AMPA receptors; kainate; excitotoxicity; GluR2 subunit; calcium permeability; cortical neurons
| |
INTRODUCTION |
Ionic flux via NMDA and AMPA
glutamate receptors (NMDARs and AMPARs, respectively) can trigger
neuronal death after excitotoxic and hypoxic/ischemic insults (Choi,
1988
; Tymianski, 1996; Ying et al., 1997). NMDAR activity exhibits
relatively homogeneous macroscopic ionic currents characterized by a
high permeability to Na+,
K+, and Ca2+
ions for which the roles in NMDAR-mediated excitotoxicity are established (Choi, 1988; Tymianski, 1996; Sattler et al., 1999). Compared with NMDARs, native neuronal AMPARs exhibit more heterogeneous macroscopic ionic current properties and ionic permeability
characteristics. Their biophysical and pharmacological properties are
governed by four genes (GluR1 to GluR4 or GluR-A to GluR-D) that encode heteromeric receptors with high AMPA affinities that are permeable to
Na+ and K+
ions (Hollmann and Heinemann, 1994). However, the relative expression of these genes, as well as the splicing and editing of their mRNAs, imparts a diversity of pharmacological properties, gating
characteristics, and Ca2+ permeability
between cells (Geiger et al., 1995). Specifically, impermeability to
Ca2+ is determined by the presence of the
GluR2 subunit, which has a positively charged arginine at position 586 of transmembrane segment 2 (Q/R site) instead of a neutral glutamine
(Hume et al., 1991; Burnashev et al., 1992). Thus permeability to
Ca2+ ions is highest in AMPARs that lack GluR2.
However, the GluR2 subunit governs more than just
Ca2+ permeability. GluR subunits display
sequence divergence within the C-terminal (CT) cytoplasmic tail, and
this region has been shown to mediate subunit-specific interactions
with various cytoplasmic proteins (Dong et al., 1997; Lin and Sheng,
1998; Osten et al., 1998; Xia et al., 1999). These AMPAR CT-protein
interactions may govern the pharmacological properties of the receptor
(Mainen et al., 1998; Cotton and Partin, 2000), receptor turnover at
synapses (Man et al., 2000), clustering (Matsuda et al., 2000),
synaptic transmission, efficacy, and plasticity (Jia et al., 1996;
Nishimune et al., 1998; Luthi et al., 1999). Thus the influence of
GluR2 subunits on neuronal function and vulnerability to excitotoxicity may occur by mechanisms other than solely those attributable to the
effects of GluR2 on ionic permeability profiles.
GluR2 is expressed widely in mammalian neurons. For example, in
cultured dissociated cortical neurons, a preparation that commonly is
used to study excitotoxicity, only 8-15% of neurons express AMPA
channels lacking GluR2 (Pruss et al., 1991; Turetsky et al., 1994; Lu
et al., 1996). In vivo, GluR2 is expressed widely in
hippocampal pyramidal and granule neurons (Hollmann and Heinemann, 1994) and in cortical neurons (Kondo et al., 1997) that frequently are
damaged by ischemia. Thus the relative abundance and, yet, heterogeneity of GluR2 expression have made it more difficult to define
its role in AMPAR-mediated excitotoxicity.
Previous studies already have examined ionic mechanisms of
AMPAR-mediated excitotoxicity. Despite generally low calcium
permeability, AMPAR toxicity is likely to be, at least in part,
mediated by Ca2+ ions
(Pellegrini-Giampietro et al., 1992; Brorson et al., 1994; Turetsky et
al., 1994; Lu et al., 1996; Gorter et al., 1997; Carriedo et al.,
1998). However, difficulties arise in determining how GluR2 level and
Ca2+ permeability relate to AMPAR-mediated
toxicity because neurons that express GluR2 exhibit at least some
Ca2+ permeability (Brorson et al., 1999),
and measurements of whole-cell relative
Ca2+ permeability and GluR2 levels in
single cultured neurons do not necessarily correlate with their overall
vulnerability to AMPAR overactivation (Vandenberghe et al., 2000).
Further indication that Ca2+ permeability
alone may not be the sole predictor of vulnerability arose from studies
that used mice with GluR2 mutations producing AMPARs with high
Ca2+ permeability. These animals displayed
adverse changes in behavior and phenotype, underscoring the importance
of the GluR2 subunit. However, they did not exhibit neuropathological
lesions suggestive of excitotoxicity (Jia et al., 1996; Kask et al.,
1998; Feldmeyer et al., 1999). These raise the possibility that GluR2
is involved in governing neurological development and function by
subtler mechanisms than those related only to
Ca2+ permeability. We wondered whether
similar factors also could contribute to AMPAR-mediated neurotoxicity.
Because of the importance of AMPARs, the GluR2 subunit, and
Ca2+ ions in neuronal function and
excitotoxicity (Turetsky et al., 1994; Lu et al., 1996; Tymianski,
1996; Carriedo et al., 1998), we examined the hypothesis that the level
of GluR2 expression governs the vulnerability of neurons to
AMPAR-mediated neuronal damage. Additional experiments also were
performed to determine whether Ca2+
permeability alone or additional GluR2-related factors participate in
governing AMPAR-mediated excitotoxicity. To this end, we studied mice
deficient in the GluR2 AMPAR subunit (Jia et al., 1996). AMPARs in
neurons of homozygous mice
[GluR2(
/)]
are uniformly Ca2+-permeable, providing an
unprecedented opportunity to examine the effect of
Ca2+ permeability on AMPAR function as
compared with wild-type [GluR2(+/+)] and
heterozygous
[GluR2(+/)]
controls. By controlling for GluR2 level, we eliminated the confounding
effects of uncertain Ca2+ permeability and
were able to determine its impact on excitotoxicity. Here we
demonstrate that AMPAR-mediated excitotoxicity cannot be attributed
solely to increased Ca2+ permeability.
AMPAR-mediated excitotoxicity is affected by GluR2 level because of the
influence of the GluR2 subunit on agonist affinity and the amplitude of
macroscopic ionic currents.
| |
MATERIALS AND METHODS |
Experimental animals. GluR2 mutant mice were
generated as described in Jia et al. (1996). In brief, for disruption
of the GluR2 locus, an isogenic targeting vector was designed to delete transmembrane region 1 and the pore loop, which are essential for
receptor function (Hollmann and Heinemann, 1994). R1 embryonic stem
(ES) cells (strain 129) were electroporated with this vector and
selected in G418 and ganciclovir (Nagy et al., 1993). Double-resistant clones were screened for the desired homologous recombination by
Southern blotting, using a probe 5' to exon 10. Four ES clones contained the targeting events and were used to produce aggregation chimeras with CD1 morulae (Wood et al., 1993). Only one ES clone transmitted the GluR2 mutation through the germline. Heterozygous mice
from a CD1 × 129 cross were intercrossed to produce 477 F2 offspring in a 1:2:1 Mendelian ratio of
GluR2(+/+):GluR2(+/):GluR2(/),
suggesting no embryonic lethality in the mutants. F2 littermates from
the same cross were used throughout.
Mixed cortical cell cultures. Cultures containing both
neurons and glia were prepared separately from each 1- to 2-d-old
postnatal mouse pup born of
GluR2(+/)
parents. Otherwise, the cultures were prepared as previously described
(Sattler et al., 1997, 1998). In brief, cerebral cortices from each pup
were incubated for 10-12 min in 0.05% trypsin in EDTA, dissociated by
trituration, and plated on poly-L-ornithine-coated 24-well
plates (Corning, Corning, NY) or glass coverslips at a density of
0.43 × 106 cells/well or 0.9 × 106 cells/coverslip. Plating medium
consisted of Eagle's minimum essential medium (MEM, Earle's salt)
supplemented with 10% heat-inactivated horse serum (ICN Biochemicals,
Montr
al, Canada) and (in mM) 2 glutamine, 25 glucose, and
26 bicarbonate. The cultures were maintained at 37°C in a humidified
5% CO2 atmosphere. After 3-5 d in
vitro the growth of non-neuronal cells was halted by a 24-48 hr
exposure to 10 µM FDU solution [5
µM uridine and 5 µM
(+)-5-fluor-2'-deoxyuridine]. The cultures were used for experiments
on day 11 (12-13 d postnatal). Embryonic cortical neuronal cultures
(used for Fig. 6A) were produced as above from
embryonic Swiss mice at 15 d of gestation and used on days 12-14
in vitro.
Because experiments were performed on cultures grown from pups born of
two
GluR2(+/)
parents, each data set was obtained from sister cultures that included
same-generation GluR2(+/+),
GluR2(+/),
and
GluR2(/) cultures.
Electrophysiology. Whole-cell patch-clamp recordings were
performed in the cultured neurons at room temperature (RT), as
previously described (Jia et al., 1996). The extracellular solution
contained (in mM): 140 NaCl, 5.4 KCl, 1.0 CaCl2, 25 HEPES, 33 glucose, and 0.0003 tetrodotoxin, pH 7.3-7.4, at 320-335 mOsm. A multi-barrel perfusion
system was used to exchange kainate-containing solutions rapidly. The
pipette solution contained (in mM): 140 CsF, 35 CsOH, 10 HEPES, 11 EGTA, 2 tetraethylammonium chloride (TEA), 1 CaCl2, and 4 MgATP, pH 7.3, at 300 mOsm. The
neurons were patch-clamped at a holding potential of 
60 mV. The
PCa2+/PCs+
permeability ratios for mutant and wild-type neurons, acutely isolated
from hippocampal slices, were calculated previously in our laboratory by studying the reversal potential of currents recorded in low or high
Ca2+ solutions. These solutions contained
the following (in mM): 140 NaCl, 0.2 or 20 CaCl2, 5.4 KCl, 25 HEPES, 33 or 13 glucose, and 0.0005-0.0001 TTX. A Na+-free solution
consisted of 10 mM CaCl2
and 25 mM HEPES with equi-osmotic glucose or
sucrose substituted for NaCl. The relative permeability ratios were
determined to be
PCa2+/PCs+ = 3.51 and 0.41 for mutant and wild-type neurons, respectively, as
calculated from the reversal potential of the constant field equation:
![P<SUB><UP>Ca<SUP>2+</SUP></UP></SUB>/P<SUB><UP>Cs<SUP>+</SUP></UP></SUB>=[<UP>Cs<SUP>+</SUP></UP>]<SUB><UP>i</UP></SUB>/<UP>Ca<SUP>2+</SUP></UP>]<SUB><UP>o</UP></SUB><UP>exp</UP>(EF/RT)[<UP>exp</UP>(EF/RT+1]/4,](/content/vol21/issue7/fulltext/2224/img001.gif)
|
|
where E is the reversal potential, F,
R, and room temperature T are standard
thermodynamic parameters, and PCa2+
and PCs+ represent permeability
coefficients for Ca2+ and
Cs+ (Lewis, 1979; Burnashev et al., 1995).
To determine whether the mutant cultured cortical neurons were also
relatively more permeable to Ca2+, we
changed the extracellular Ca2+
concentration from 1 to 20 mM, and we examined
the shift in reversal potential.
Histological techniques. Kainate-activated cobalt labeling
(see Fig. 1) was performed as previously described (Pruss et al., 1991;
Turetsky et al., 1994). In brief, the cells were exposed to 100 µM kainate plus 5 mM
CoCl2 in uptake buffer (in mM): 139 sucrose, 57.7 NaCl, 5 KCl, 2 MgCl2, 1 CaCl2, 12 glucose, and 10 HEPES, pH 7.6, for 30 min at RT. Then the cultures were washed in uptake buffer containing 5 mM EDTA to chelate any extracellular cobalt. After a 5 min
incubation in 0.12%
(NH4)2S, the cells were washed three times in uptake buffer and finally fixed in 4%
paraformaldehyde for 30 min at RT. Enhancement of the CoS precipitation
was performed by washing the fixed cells three times in development
buffer (in mM): 292 sucrose, 15.5 hydroquinone, and 42 citric acid and then by incubating them in 0.1%
AgNO3 in development buffer at 50°C. This
solution was changed every 15 min until the silver enhancement was
complete (usually four changes). The reaction was terminated by washing
the cultures three times with development buffer.
Immunolabeling for GluR1 was performed in the cultured cells as
described previously (Allison et al., 1998; Sattler et al., 2000). In
brief, the cells were fixed first with 4% paraformaldehyde in PBS plus
4% sucrose for 20 min at 4°C. Cultures subsequently were fixed in
ice-cold 100% methanol for 10 min at 4°C. After repeated washing,
they were permeabilized with 0.02% Triton X-100 in PBS for 10 min at
4°C, blocked in 10% goat serum in PBS for 45 min at RT, followed by
incubation with a rabbit affinity-purified anti-rat GluR1 IgG (1:3000
dilution; Upstate Biotechnology, Lake Placid, NY) primary antibody in
10% goat serum in PBS for 3 hr at RT or 37°C. Then the cultures were
washed and incubated with secondary antibody (Cy5.5-tagged goat
anti-rabbit IgG; 1:500 dilution; Jackson ImmunoResearch, West Grove,
PA) for 1.5 hr at RT. Immunostaining was visualized with a
laser-scanning confocal microscope (Bio-Rad MRC 1000, Hercules, CA)
through a 60× oil immersion lens.
For calbindin staining, paraformaldehyde-fixed brain sections (30 µm)
from four 1-month-old GluR2 mutant mice were labeled with a
monoclonal anti-calbindin-D mouse IgG1 (1:200 dilution; Sigma, St.
Louis, MO) and then with the Vectastatin elite kit (Vector Labs,
Burlingame, CA), using diaminobenzidine as the chromogen.
Immunoblotting. Immunoblotting was done as described (Jia et
al., 1996; Sattler et al., 1999, 2000) by using cells harvested from
two cultures per genotype per lane or from three brains of each GluR2
genotype. The blotted proteins were probed with a rabbit affinity-purified anti-rat GluR1 IgG (1:3000 dilution; Upstate Biotechnology) or a monoclonal anti-calbindin-D mouse IgG1 (1:200 dilution; Sigma). Then the blots were probed with sheep anti-mouse or
donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham,
Arlington Heights, IL), and the proteins were detected by enhanced
chemiluminescence (Amersham).
Neuronal cell death measurements. These measurements were
performed by serial quantitative measurements of propidium iodide (PI)
fluorescence, using a multiwell plate fluorescence scanner (Cytofluor
II, PerSeptive Biosytems, Framingham, MA) as described and previously
validated (Sattler et al., 1997, 1998). In brief, the culture medium in
each tissue culture well was replaced with control solution containing
50 µg/ml PI, and a baseline fluorescence reading was taken. Then
sequential readings were taken up to 24 hr after the experimental
manipulations. The fraction of dead neurons in each culture at a given
time was calculated as:
where Ft = PI fluorescence at
time t, Fc = PI
fluorescence of controls at 24 hr, and
FNMDA = background-subtracted PI
fluorescence of identical cultures from the same dissection and plating
24 hr after a 60 min exposure to 100 µM NMDA at
37°C. Based on manual observations made at the time of validation of
this technique, this NMDA exposure routinely produced near-complete
neuronal death in each culture but had no effect on surrounding glia
(also see Bruno et al., 1994; David et al., 1996; Sattler et al.,
1997). The control solution consisted of MEM supplemented with 50 µg/ml PI. For kainate exposures the solution also contained MK-801
(10 µM; Research Biochemicals, Natick, MA) and
nimodipine (2 µM; Miles Pharmaceuticals,
Elkhart, IN), whereas for NMDA exposures the solution contained
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM; Research Biochemicals) and nimodipine. All
experiments were performed at 37°C.
In vivo kainate injections. For intrathecal injection
studies, kainic acid was injected stereotactically into mice 7-9 weeks of age. Kainate was administered through a pulled glass capillary needle (60 µm diameter) that was inserted halfway between the bregma
and lambda sutures, 2 mm lateral to the midline, at a depth of 1.5 mm
(see Fig. 4C). Kainic acid or saline (200 nl) was introduced over 2 min. The needle was withdrawn after an additional 1 min wait.
Intraperitoneal kainate injections (15-25 mg/kg) were performed by
using kainate dissolved in 200-300 µl of saline. Animals were monitored for 2 hr for the onset of seizures, and the extent of injury
was determined after 48 hr or 7 d from 10 µm sections taken over
a 250 µm interval between 200 and 450 µm rostral to the needle tract. Estimates of cell death estimates were obtained by manually counting Nissl-stained sections from the central portion of the CA1 or
CA3 sector from every fifth section.
Fluorescence imaging. All experiments were performed on
dissociated cultures grown on glass coverslips. Immunostained GluR1 clusters were visualized with the 647 nm laser line of a confocal microscope (Bio-Rad MRC 1000) through a 60× oil immersion lens [numerical aperture (NA) 1.4; Nikon]. Fluorescent clusters were counted by two independent observers in randomly selected dendrites in
imaged neurons and were expressed as the numbers of clusters per unit
of dendrite length (Allison et al., 1998; Sattler et al., 2000).
Fura-2 [Ca2+]i
imaging was performed in the neuronal cultures identically to methods
previously described (Tymianski et al., 1993; Sattler et al., 1998). In
brief, neurons were loaded with fura-2 AM (2 µM;
Molecular Probes, Eugene, OR) and viewed with an inverted microscope
(Nikon Diaphot, xenon epifluorescence optics) through a fluorite oil
immersion lens (Nikon CF UV-F 40×, NA = 1.3). Fura-2 excitation
was evoked through narrow bandpass filters (340 ± 5/380 ± 6.5 nm), and fluorescence emissions >510 nm were recorded with an
intensified CCD array camera (Quantex Model QX-100) interfaced to a
PC-based personal computer. Four to eight images were averaged at each
excitation wavelength and corrected for background fluorescence and
camera dark current by subtracting a frame taken at the beginning of
each experiment at each excitation wavelength from an area of the
coverslip devoid of cells. Changes in
[Ca2+]i were
expressed as the background-subtracted 340/380 nm fura-2 fluorescence
ratio. Fluo-3
[Ca2+]i imaging
was performed with the confocal microscope in cultures loaded with
fluo-3 AM (5 µM) with a 40× oil immersion lens (Nikon, 1.3 NA), using identical settings for all experiments (excitation 488 nm; emission 515 LP; iris 6.7 mm; gain 1440; laser intensity 3%; zoom
3.0), and a multi-barrel perfusion system to exchange solutions.
Kainate solutions contained 10 µM MK-801, 2 µM nimodipine, and (in mM): 121 NaCl, 5 KCl,
20 D-glucose, 10 HEPES acid, 7 HEPES-Na salt, 3 NaHCO3, and 1.8 CaCl2.
Data analysis. All data were analyzed by ANOVA, with a
post hoc Student's t test, using the Bonferroni
correction for multiple comparisons. All means are presented with their
standard errors. Cell death measurements reported in all figures are
baseline-subtracted to reflect only the cell death produced by the
experimental insult (e.g., kainate or NMDA application). Baseline cell
death in the absence of an insult ranged from 13 to 28% of the neurons
at the 24 hr observation time point.
| |
RESULTS |
We first performed a series of control experiments that identified
the glutamate receptor subtype (AMPA vs kainate) responsible for
kainate-induced injury, the effects of genetic background on kainate
toxicity, and the calcium permeability characteristics of mutant AMPA
receptors in cortical neurons maintained in dissociated cultures (Figs.
1, 2).
View larger version (82K):
[in this window]
[in a new window]
|
Figure 1.
Characterization of kainate toxicity in
GluR2 mutant cultures. A, Kainate toxicity is mediated
by AMPARs. Wild-type neurons were exposed to 1 mM kainate
for 24 hr in solution containing 10 µM MK-801, 2 µM nimodipine, and (in mM): 121 NaCl, 5 KCl,
1 Na-pyruvate, 1.8 CaCl2, 25 NaHCO3, and 20 D-glucose, pH 7.4. Kainate toxicity was abolished by 10 µM ()-GYKI 53784, a selective AMPAR antagonist (ANOVA, F = 223;
p < 0.0001; n = 6 cultures per
condition). *p < 0.05, **p < 0.01 differences from controls. B, No differences in
vulnerability to kainate toxicity between CD1 and 129 strains at 100 µM kainate (t24 = 0.52;
p = 0.61) and at 1 mM kainate
(t25 = 0.26; p = 0.80). Cultures were exposed to kainate (0.1-1 mM) as
above. Numbers in bars indicate
n cultures per condition. C,
Representative staining for kainate-activated cobalt uptake in neuronal
clusters from GluR2 mutant mice. Although clusters were uncommon in the
cultures, these pictures are provided to illustrate the striking
differences between GluR2(+/+) and
GluR2(/)
cultures. Arrowheads, Cobalt-positive neurons in
GluR2(+/+) cultures. Scale bar, 100 µm.
|
|
View larger version (37K):
[in this window]
[in a new window]
|
Figure 2.
Enhanced Ca2+
permeability and increased kainate potency in GluR2-deficient cortical
pyramidal neurons. A-C, Representative kainate-evoked
(100 µM) whole-cell currents and the I-V
relationships for averaged steady-state currents in GluR2 mutant
neurons recorded in low (1 mM, open symbols)
and high (20 mM, filled symbols)
extracellular Ca2+. Curves were fit by a
fourth order polynomial equation from which interpolated reversal
potentials were calculated.
Erev(+/+), +1.1 ± 0.9 and +0.6 ± 1.1 mV;
Erev(+/), 0.4 ± 1.2 and 0.3 ± 1.0 mV;
Erev(/),
+4.5 ± 2.5 and +11.8 ± 2.3 mV, for low and high
Ca2+, respectively. D, Representative
kainate-evoked whole-cell currents and concentration-response
relationships for peak kainate-evoked currents recorded in GluR2 mutant
cortical pyramidal neurons. Concentration-response curves at 10, 30, 100, 300, 1000, and 3000 µM kainate were constructed and
normalized to the maximal response in GluR2(+/+)
( ), GluR2(+/) ( ), and
GluR2(/) ( ) neurons. The potencies of kainate
(EC50) and Hill coefficients
(nH) were determined by fitting the
curves to the equation: I = Imax × 1/(1 + (EC50/[kainate])n), where
Imax in the response at 3 mM kainate. GluR2(+/+)
EC50, 142.252 ± 15.672 µM;
nH, 1.330 ± 0.027 (n = 19).
GluR2(+/)
EC50, 131.286 ± 26.692 µM;
nH, 1.303 ± 0.062 (n = 11).
GluR2(/)
EC50, 56.511 ± 7.480 µM;
nH, 1.159 ± 0.048 (n = 15). *Differences from
GluR2(+/) and
GluR2(+/+), one-way ANOVA (F = 8.155; p = 0.001) with post hoc
Bonferroni t tests; p < 0.05. E, Currents from D plotted without
normalization to Imax.
|
|
Characterization of kainate toxicity in cortical cultures of
wild-type and parental strains
Kainic acid often is used for studies of AMPAR-mediated
excitotoxicity (Brorson et al., 1994; Turetsky et al., 1994; Bindonkas and Miller, 1995; Lu et al., 1996; Carriedo et al., 1998). It activates
both kainate- and AMPA-preferring receptors, the latter as an
incompletely desensitizing agonist (Burnashev et al., 1992). We first
examined which receptor subtype (AMPA or kainate) mediated kainic acid
neurotoxicity in this study. Kainate was applied to the cultured
cortical neurons for 24 hr in the presence of 10 µM
MK-801 and 2 µM nimodipine, antagonists of NMDA and
Ca2+ channels, respectively, to prevent
Ca2+ entry through these alternative
pathways (Brorson et al., 1994; Sattler et al., 1998; Jensen et al.,
1999). This approach isolates ionic influx to AMPA/kainate channels
(Sattler et al., 1998) and causes minimal
Ca2+ accumulation in cortical neurons from
nonmutant mouse strains that express the GluR2 subunit (Sattler et al.,
1999). Kainate was applied throughout the 24 hr observation period in
all studies. This treatment protocol maximizes the ionic disturbance
produced by the activation of kainate-sensitive receptors to reduce the proportional impact of other potentially toxic events initiated after
terminating the stimulus, such as the activation of the reverse
operation of the
Na+/Ca2+
exchanger (Yu and Choi, 1997). In wild-type cultures the kainate (0.3-1.0 mM) produced rapid swelling and the death of
>70% of neurons by 24 hr (Fig. 1A). A selective
noncompetitive AMPAR antagonist ()-GYKI 53784 (3-10
µM; Bleakman et al., 1996) completely blocked neuronal damage (Fig. 1A), indicating thatkainate
neurotoxicity in these cultures is predominantly AMPAR-mediated (Ohno
et al., 1997; Jensen et al., 1999).
Next, we examined the influence of genetic background on kainate
toxicity. Mutant mice used in the experiments that are described below
were generated by GluR2 gene targeting in ES cells of the 129 strain
origin. Chimeric offspring were mated with the CD1 strain to obtain
offspring that were tested for the presence or absence of the GluR2
null allele. F2 littermates from the same cross were used throughout.
It is possible that the different offspring littermates tested here
might contain a different complement of 129 genes linked to the GluR2
locus (Gerlai, 1996; Stryker et al., 1997). To determine whether this
might affect excitotoxic vulnerability, we obtained cultures from each
parental strain (129 and CD1) and tested them separately for their
sensitivity to kainate toxicity. Neuronal death in response to kainate
(at both 100 µM and 1 mM concentrations) was
the same in neurons derived from the 129 or CD1 strain. Therefore, any
anticipated differences in the excitotoxic response in
GluR2(+/+),
GluR2(+/),
and
GluR2(/)
mice could be attributed to the absence of the GluR2 receptor subunit
and not to any differential inheritance of background genes.
Ionic currents and Ca2+ permeability of AMPARs
in cultured GluR2 mutant neurons
Kainate-activated cobalt uptake is a staining technique that
identifies neurons bearing Ca2+-permeable
AMPARs (Pruss et al., 1991; Turetsky et al., 1994; Yin et al., 1999).
We first stained cortical neuronal cultures from GluR2 mutants by this
method to confirm that our cultures maintained
Ca2+-permeable AMPARs after 2 weeks
in vitro. No attempt was made to quantify the intensity of
staining, because the significance of this measure to the physiological
function of neurons is controversial. Figure 1C illustrates
cobalt staining of neuronal clusters containing hundreds of neurons
from the three mutant groups. Large clusters were uncommon in these
cultures, and all other experiments in this paper, including the counts
of Co2+-positive cells, were performed in
dispersed (nonclustered) cultures. However, the many neurons in each
cluster illustrate the striking paucity of cobalt staining in
GluR2(+/+) neurons as compared with
GluR2(+/)
and
GluR2(/).
We also counted cobalt-positive cells in three cultures per mutant
group. All
GluR2(/)
neurons (100%) were stained intensely, as compared with only 4.47 ± 1.76% of GluR2(+/+) neurons
(n = 3 cultures per group;
t(4) = 54.13; p < 0.0001). Most (>80%)
GluR2(+/)
neurons also were cobalt-stained, showing that this method also detects
neurons expressing a heterogeneous population of AMPARs, of which only
a fraction may be Ca2+-permeable.
The potency of kainate in activating inward currents and calcium
permeability in GluR2(+/+) and
GluR2(/)
neurons has been examined in acutely isolated CA1 pyramidal cells (Jia
et al., 1996; Joo et al., 1999), but not in cultured hippocampal nor in
cortical cells. Therefore, we examined whether AMPAR currents with high
Ca2+ permeability were maintained when
cortical neurons were cultured from GluR2 mutant mice. The
current-voltage relationships for GluR2(+/+) and
GluR2(+/)
neurons exhibited little or no inward rectification, and their reversal
potentials were insensitive to change from low (1 mM) to
high (20 mM) extracellular
Ca2+ (Fig.
2A,B). This suggests that most
AMPARs in GluR2(+/+) neurons contain the
edited GluR2 subunit that confers a low permeability to
Ca2+. Furthermore,
GluR2(+/)
neurons also must express sufficient numbers of GluR2 subunits so that
their macroscopic currents also exhibit linear I-V
relationships insensitive to extracellular
Ca2+. Currents from
GluR2(/)
neurons exhibited both an enhanced inward rectification and a Ca2+-dependent shift of the reversal
potential (Fig. 2C), as predicted for the loss of the GluR2
subunit (Hollmann et al., 1991; Jonas et al., 1994; Burnashev et al.,
1995). The reversal potential for current recorded from mutant neurons
was shifted to the right when currents were recorded with low (1 mM) versus high (20 mM) extracellular concentrations of Ca2+:
Erev(/),
+4.5 ± 2.5 and +11.8 ± 2.3 mV (p < 0.05). Thus similar to neurons acutely dissociated from mouse brains,
cortical neurons cultured from GluR2 mutant mice retain robust
AMPAR-mediated currents and characteristic
Ca2+ permeability that renders them
suitable for examining the effect of GluR2 level on AMPAR-mediated excitotoxicity.
GluR2(/)
neurons exhibit increased kainate potency and macroscopic currents
The subunit composition of ligand-gated receptors influences the
EC50 value of the receptor for agonists as well
as their sensitivity to pharmacological agents. Therefore, to determine the equi-effective concentration of kainate that could be used for
eliciting excitotoxicity, we first examined the magnitudes and the
concentration-response relationships for kainate-evoked ionic currents
in GluR2(+/+),
GluR2(+/),
and
GluR2(/)
neurons. Applications of kainate (>10 µM) activated an
inward current in all of the neurons that were tested. Recordings
revealed a higher potency of kainate in
GluR2(/)
neurons as compared with
GluR2(+/)
and GluR2(+/+) neurons (Fig.
2D). The potency of kainate
(EC50) in
GluR2(/)
neurons was approximately threefold higher than in the
GluR2(+/+) controls
(EC50(/),
57 ± 7.5 µM versus
EC50(+/+), 142 ± 16 µM; Bonferroni t test,
p < 0.01).
Our previous studies in acutely dissociated neurons revealed that
membrane capacitance and the maximum current evoked by a saturating
concentration of kainate (Imax) was
unaffected by the presence of the GluR2 subunit (Joo et al., 1999).
However, the acute dissociation obliterates dendritic arbors to which
functional AMPARs are localized and which are highly developed in
cultured neurons (Sattler et al., 1998, 2000). In the cultured neurons used in the present study,
GluR2(/)
cells exhibited increased peak kainate currents as compared with GluR2(+/)
and GluR2(+/+) (Table
1). Although this is consistent with the
influence of the GluR2 subunit on increasing the single channel
conductance of AMPA channels (Swanson et al., 1997), it also may
indicate an influence of GluR2 on dendritic development (Feldmeyer et
al., 1999) or on the subcellular distribution or functionality of
AMPARs in dendrites. Also,
GluR2(/)
neurons exhibited a reduced membrane capacitance, resulting in a
significantly higher current density as compared with their GluR2(+/+) and
GluR2(+/)
counterparts (Table 1). The reduced membrane capacitance implies smaller neurons in
GluR2(/)
cultures, although this was not apparent on light microscopic examination (Fig. 3B).
View larger version (54K):
[in this window]
[in a new window]
|
Figure 3.
Kainate toxicity in vitro.
A, Use of the concentration-response data from Figure
2E for selecting kainate concentrations for
excitotoxicity experiments (i-iv). B,
Appearance of
GluR2(/)
and GluR2(+/+) cultures at baseline (0 hr) and at
the end (24 hr) of a challenge with 1 mM kainate, using
phase contrast (top) and PI fluorescence optics
(bottom). C, Effects of kainate insults
on 24 hr neuronal survival, using equipotent (i, ii) and
nonequipotent (iii, iv) kainate concentrations.
i, EC 50 (+/+);
ii, EC 90 (+/+);
iii, 100 µM for all groups,
iv, 1 mM for all groups; v,
toxicity of NMDA (100 µM × 60 min) at 24 hr.
*p < 0.05, **p < 0.01;
Bonferroni t test indicating differences from
GluR2(+/+). The fraction of dead neurons in
i-v was obtained by averaging measurements from four
cultures per mutant mouse. The digits on each
bar indicate the number of mice per group. Data for each
bar were replicated from at least two litters of mutant pups.
|
|
Vulnerability to AMPAR toxicity in vitro
We next studied AMPAR-mediated excitotoxicity in the GluR2 mutant
cortical cultures.
GluR2(/)
neurons appeared morphologically similar to
GluR2(+/+) controls and exhibited a low
basal propidium iodide (PI) fluorescence (Fig. 3B,
leftmost two panels). First, it was our goal to use equi-effective kainate concentrations that would expose neurons in the
different mutant groups to similar ionic loads. To this end, it was
necessary to take into account that
GluR2(+/+),
GluR2(+/),
and
GluR2(/)
neurons exhibited differences both in EC50 (see
Fig. 2D) and in peak currents (Table 1). Thus
equi-effective concentrations were calculated from the absolute rather
than from normalized kainate-evoked currents (see Fig.
2E), because the former represent the actual ionic
current incurred in the cell. Using this approach, we determined the
kainate concentration needed to elicit, in each GluR2 group, currents
measuring 50 and 90% of the maximum current attainable in
GluR2(+/+) neurons
[Imax(+/+)].
The effective concentrations are termed
EC50(+/+) and
EC90(+/+), respectively,
and are as listed in Table 2 and
illustrated in Figure 3A, i and ii. In
addition to these two equi-effective concentrations, toxicity was
assessed by using 100 µM and 1 mM kainate, which evoke different inward currents
in the different groups (Fig. 3Aiii, Aiv).
The cultures were exposed to kainate for 24 hr in MK-801 (10 µM) and nimodipine (2 µM), antagonists of
NMDARs and voltage-sensitive Ca2+ channels
(VSCCs), respectively (Sattler et al., 1998, 1999). Sister cultures
were exposed to NMDA (100 µM) in the presence of
nimodipine (2 µM) and CNQX (10 µM), an
AMPA/kainate antagonist, to isolate Ca2+
influx to NMDA receptors (Sattler et al., 1998).
The use of equi-effective kainate concentrations is anticipated to
control for confounding effects of kainate potency between mutant
groups, thus leaving the GluR2 level as the only variable. We first
exposed the cells to kainate concentrations that evoked 50% of
Imax(+/+)
[EC50(+/+); Table 2;
Fig. 3Ai]. These insults produced 10-20% cell death in
all groups but revealed no effect of the GluR2 level on AMPAR-mediated excitotoxic vulnerability (Fig. 3Ci; one-way ANOVA,
F = 0.24; p = 0.79). These experiments
produced relatively low neuronal mortality. Consequently, they were
repeated by using equi-effective kainate concentrations that evoked
90% of
Imax(+/+)
[EC90(+/+); Table 2;
Fig. 3Aii]. Although these higher kainate concentrations caused ~50% of the neurons to die, there was no apparent effect of
GluR2 level on excitotoxic vulnerability (Fig. 3Cii; one-way ANOVA, F = 0.02; p = 0.98).
Next, we used 100 µM kainate, an intermediate agonist
concentration that evokes larger kainate-activated currents in
GluR2(/)
as compared with GluR2(+/+) neurons (see
Fig. 2E). As anticipated, this challenge triggered more toxicity in neurons lacking GluR2 than in wild-type controls (Fig.
3Aiii; one-way ANOVA, F = 25.7;
p < 0.0001). We then treated the cultures with 1 mM kainate (Fig. 3Aiv). This is a
near-saturating agonist concentration that produced near-maximal
currents in all mutant groups (see Fig. 2D), although
the actual current remained highest in the
GluR2(/)
group (see Fig. 2E). Kainate (1 mM) caused neuronal swelling and a rise in PI
fluorescence that peaked at 24 hr (Fig. 3B, rightmost two panels). However, even at this highly toxic kainate
concentration, GluR2(/)
neurons were no more vulnerable than the
GluR2(+/)
or GluR2(+/+) controls (Fig.
3Aiii; one-way ANOVA, F = 0.11;
p = 0.90).
The data in Figure 3, Ci-Civ, suggest that vulnerability to
AMPAR-mediated toxicity parallels the magnitude of the kainate-evoked ionic current. When equipotent kainate concentrations were used, mortality was similar between the mutant groups and rose with increasing kainate concentration [compare
EC50(+/+) versus
EC90(+/+)]. When
nonequipotent concentrations were used, neuronal loss evoked by 100 µM kainate also paralleled the size of the
anticipated current, with the highest mortality in
GluR2(/).
This effect disappeared at 1 mM, a
near-saturating kainate insult (see Fig. 2D) that
causes 70-80% neuronal death, the maximum achievable with kainic acid
in these cultures. This suggests that with 1 mM
kainate the current that triggers excitotoxicity had reached a
threshold level sufficient to trigger maximal neurotoxicity.
As a control we treated the cultures with 100 µM NMDA for
60 min, an insult that is highly toxic to cortical neurons in culture (Sattler et al., 1999, 2000).
GluR2(/),
GluR2(+/),
and GluR2(+/+) neurons were equally
vulnerable to NMDA toxicity at these concentrations (Fig.
3Cv; one-way ANOVA, F = 0.04;
p = 0.96), indicating that mechanisms of NMDA-mediated
toxicity remain in these cells.
Calcium dynamics in GluR2 mutant neurons
To probe further the impact of increased
Ca2+ permeability on
Ca2+ homeostasis and excitotoxicity in
GluR2-deficient cells, we measured kainate-evoked changes in free
intracellular Ca2+ concentration
([Ca2+]i). As
Ca2+ ions are sequestered into
intracellular organelles, buffered by
Ca2+-binding proteins, or extruded through
membrane pumps and exchangers (Blaustein, 1988; Pozzan et al., 1994),
the kainate-evoked
[Ca2+]i rise
reflects the result of AMPAR-mediated Ca2+
influx, efflux, and buffering. All kainate applications were performed
in the presence of NMDAR and Ca2+ channel
blockers (Sattler et al., 1998).
Baseline [Ca2+]i
was similar in
GluR2(/)
and GluR2(+/+) neurons loaded with the
ratiometric Ca2+ indicator fura-2 (see
Fig. 4A-C; t(22) = 0.59; p = 0.56). However, kainate exposure (100 µM) elicited significantly greater
[Ca2+]i elevations
in
GluR2(/)
neurons as compared with GluR2(+/+)
controls (Fig. 4A-C;
t(22) = 3.3; p = 0.003). The kainate-evoked change in
[Ca2+]i was only
detectable in
GluR2(/),
but not in GluR2(+/+), neurons loaded
with fura-2 FF, a low Ca2+-affinity
indicator (KD ~35
µM) (Golovina and Blaustein, 1997; Carriedo et
al., 1998) (data not shown). Thus fura-2
(KD ~224 nM)
may be more sensitive to the relatively small
[Ca2+]i changes
evoked in the controls. The rise and persistence of higher
[Ca2+]i levels in
GluR2(/)
neurons during kainate exposure (Fig. 4B,C) suggest
that
[Ca2+]i-lowering
mechanisms did not compensate for increased
Ca2+ permeability in
GluR2(/)
neurons.
View larger version (42K):
[in this window]
[in a new window]
|
Figure 4.
Increased Ca2+ entry into
GluR2(/)
neurons. Kainate was applied in the presence of MK-801 and nimodipine.
A-C, Experiments with fura-2. Kainate was applied for
10 min. A, Representative time course of
[Ca2+]i averaged from
n = 4 GluR2(+/+) neurons.
B, Representative time course of
[Ca2+]i averaged from
n = 5 GluR2(/)
neurons. C, Pooled baseline and peak
[Ca2+]i measurements from three
separate cultures per group. *p < 0.05 between
wild-type and homozygous neurons. D, E,
Confocal imaging of [Ca2+]i with
fluo-3. Kainate (100 µM) was applied for 25 sec.
D, Representative time course of
[Ca2+]i averaged from
n = 4 GluR2(+/+) neurons.
E, Representative time course of
[Ca2+]i averaged from
n = 4 GluR2(/)
neurons. F, Peak
[Ca2+]i transients in the soma and
dendrites of neurons measured with fluo-3 and evoked by 30 sec
applications of NMDA (100 µM) in the presence of CNQX and
nimodipine. Data were pooled from 13-18 cultures from two dissections.
G, Peak [Ca2+]i
transients measured with fluo-3 and evoked by 25 sec applications of
kainate, using equipotent (i, ii) and nonequipotent
(iii, iv) concentrations. i, EC 50
(+/+); ii, EC 90
(+/+); iii, 100 µM for all groups; iv, 1 mM
for all groups. *p < 0.01; Bonferroni
t test indicating differences from
GluR2(+/) and
GluR2(/).
Numbers in legends indicate numbers of
cultures per group. Data were pooled from at least two dissections.
Dendritic [Ca2+]i was measured 50-100
µm from the cell soma.
|
|
Our excitotoxicity studies (see Fig. 3) indicated a dependence of
AMPAR-mediated toxicity on the magnitude of the anticipated ionic
current but did not indicate which ions were responsible. Given that
GluR2(/)
cells are highly permeable to Ca2+, it was
surprising that equipotent kainate insults failed to demonstrate
increased toxicity in this group, because these cells are expected to
incur larger Ca2+ loads. We therefore
examined kainate-evoked changes in
[Ca2+]i in the
GluR2 mutant neurons under the same conditions as in the excitotoxicity
experiments. Because kainate excitotoxicity also may depend on
dendritic AMPARs (Bindonkas and Miller, 1995), we studied
kainate-evoked
[Ca2+]i changes in
the neurons by confocal imaging of both soma and dendrites (Fig.
5). On the basis of our experience with
fura-2 (KD for
Ca2+ ~224 nM)
versus fura-2 FF (KD for
Ca2+ ~35 µM; see
above), we used fluo-3, an indicator with a
Ca2+ affinity more similar to that of
fura-2 (KD for
Ca2+ ~500 nM).
Thus we anticipated to better resolve
[Ca2+]i changes in
GluR2(+/+) neurons versus
GluR2(+/)
and
GluR2(/)
cells.
View larger version (140K):
[in this window]
[in a new window]
|
Figure 5.
Representative confocal images of fluo-3
fluorescence from GluR2 mutant neurons at baseline and at the peak of a
[Ca2+]i transient evoked with 100 µM kainate. Scale bar, 50 µm.
|
|
[Ca2+]i changes
were measured in the soma and dendrites of neurons during 25-30 sec
applications of kainic acid in the presence of MK-801 and nimodipine.
Representative experiments (Fig. 4D,E) that used 100 µM kainate produced results similar to those
obtained with fura-2 (Fig. 4A,B), with larger
[Ca2+]i changes
occurring in
GluR2(/)
cells as compared with GluR2(+/+) controls.
Next we used equi-effective kainate concentrations at
EC50(+/+) and
EC90(+/+) (Table 2),
which produce similar degrees of AMPAR-mediated cell death (see Fig.
3Ci,Cii). Consistent with the results obtained with cobalt staining (see Fig. 1C) and electrophysiology
(see Fig. 2A-C),
GluR2(+/+) cells, having AMPARs with low
Ca2+ permeability, exhibited significantly
smaller increases in
[Ca2+]i in both
soma and dendrites as compared with
GluR2(+/)
and
GluR2(/)
mutants (Fig. 4Gi,Gii). Thus when the anticipated
overall ionic currents are similar between groups, neurons that incur
higher increases in
[Ca2+]i (Fig.
4Gi,Gii) do not necessarily exhibit increased
mortality (see Fig. 3Ci,Cii). Exposing the
cultures to 100 µM and to 1 mM kainate also revealed that
GluR2(+/+) cells exhibited significantly
smaller increases in
[Ca2+]i in both
soma and dendrites as compared with
GluR2(+/)
and
GluR2(/)
mutants (Fig. 4Giii,Giv). These
[Ca2+]i imaging
data indicate that the overall size of the anticipated ionic current,
but not necessarily the Ca2+ permeability,
is the property of AMPARs that more closely predicts excitotoxic vulnerability.
Next, to determine whether GluR2 mutant neurons maintain normal
[Ca2+]i responses
via pathways other than AMPARs, we examined the effects of 30 sec
applications of NMDA (100 µM) in the presence of CNQX and
nimodipine to isolate Ca2+ influx to
NMDARs (Sattler et al., 1998). All three groups exhibited similar
changes in [Ca2+]i
both in the soma (ANOVA, F = 0.06; p = 0.94) and the dendrites (ANOVA, F = 0.22;
p = 0.80; Fig. 4F). This indicates
that the differences in the
[Ca2+]i responses
observed after AMPAR stimulation were attributable to the GluR2 level,
not to differences in Ca2+ buffering or
extrusion, because these factors also would have influenced the
NMDAR-mediated responses. Furthermore, because [Ca2+]i transients
were significantly larger in both soma and dendrites of
GluR2(+/)
and
GluR2(/)
neurons as compared with GluR2(+/+)
controls, it is unlikely that GluR2-deficient neurons had upregulated their
[Ca2+]i-lowering
mechanisms to compensate for increased
Ca2+ permeability.
Ionic dependence of AMPAR-mediated excitotoxicity
The majority of the AMPAR-mediated ionic current is carried by
Na+ ions. Because excitotoxic
vulnerability was predicted by the size of the ionic current (see Fig.
3), not Ca2+ permeability (see Fig. 4), we
investigated further the ionic dependence of kainate-evoked AMPAR
neurotoxicity. AMPAR-evoked neurotoxicity might be mediated by
Na+ influx (Kato et al., 1991; Bindonkas
and Miller, 1995; Itoh et al., 1998) or by
K+ efflux, because
K+ depletion promotes both necrosis and
apoptosis (Miller and Johnson, 1996; Villalba et al., 1997; Yu et al.,
1997) and K+ supplementation promotes
neuronal survival (Gallo et al., 1987; Tymianski et al., 1994).
To examine further the ionic mechanisms of kainate toxicity, we
performed experiments according to the same protocols as in Figure 3.
In wild-type cortical neuronal cultures grown from Swiss mice, kainate
toxicity was abolished completely by substituting extracellular
Na+ with
N-methyl-D-glucamine (NMDG) to prevent
Na+ influx (Fig.
6A). However, toxicity
was unaffected by increasing extracellular
K+ to 20 mM (Fig.
6A) or to 50 mM (data not
shown) to reduce K+ depletion (Yu et al.,
1997) or by cycloheximide (Fig. 6A), a protein
synthesis inhibitor that inhibits neuronal apoptosis caused by
potassium depletion (Yu et al., 1997). Thus in wild-type neurons from
the Swiss mouse strain, kainate toxicity was determined primarily by
Na+ ions.
View larger version (37K):
[in this window]
[in a new window]
|
Figure 6.
Effects of ion substitution and of
GluR2 levels on kainate toxicity. A, Effect of
Na+ removal, K+ supplementation,
and protein synthesis inhibition on kainate toxicity in wild-type
neurons. For low Na+, NMDG was substituted for NaCl
in the control solution. For high-K+, 15 mM KCl was substituted for 15 mM NaCl, for a
total of 20 mM K+. Cycloheximide
(CHX) was applied at 1 µg/ml.
*t(14) = 6.06; p < 0.0001. **t(22) = 5.83;
p < 0.0001. n = 8-12 cultures
per condition. B, Effect of Na+
removal on the toxicity of equi-effective kainate concentrations in
GluR2 mutant neurons. Data for controls are from Figure
3Cii. Na+ removal had equal effects
on all mutant groups (ANOVA, F = 0.16;
p = 0.85) and reduced kainate toxicity by ~50%.
*Difference from same GluR2 group control.
t16(+/+) = 2.83;
p = 0.01. t15(+/) = 2.30; p = 0.04. t15(/) = 3.59; p = 0.002. C, Effect of
Na+ removal on the toxicity of 100 µM
kainate in GluR2 mutants. Data for controls are from Figure
3Ciii.
GluR(/)
neurons were more vulnerable to kainate toxicity than
Glu(+/+) neurons both in the presence of
Na+ (see Fig. 3) and in its absence (ANOVA,
F = 6.4; p = 0.007 for NMDG
group). *Difference from GluR(+/+), Bonferroni
t test; p < 0.01. Na+ removal was protective in
GluR2(/)
neurons. **Difference from controls,
t22(/) = 3.88; p = 0.0008. D, Effect of
Ca2+ removal on the toxicity of 100 µM
kainate in GluR2 mutants. The control solution was modified by omitting
CaCl2 and by adding 100 µM EGTA, a
Ca2+ chelator. The rank order of vulnerability to
kainate toxicity was proportional to the GluR2 level, with
GluR(/)
remaining more vulnerable to kainate toxicity than
Glu(+/+) neurons (ANOVA, F = 3.7; p = 0.039). *Difference from
GluR(+/+), Bonferroni t test;
p < 0.05. N.S., No significant
difference from controls
(t13(/) = 1.55; p = 0.144).
|
|
We next examined the ionic dependence of kainate toxicity in the GluR2
mutants. Concurrently with the AMPAR-mediated excitotoxicity experiments shown in Figure 3C, we also studied the effect
of Na+ removal. In recombinant AMPARs
Na+ ions are the main charge carriers and
are responsible for >95% of the charge transfer regardless of GluR2
level (Burnashev et al., 1995). Thus we examined whether
Na+ removal affects AMPAR-mediated
excitotoxicity. First, we applied kainate at the equi-effective
concentration that produces 90% of
Imax(+/+)
(Table 2) and that kills approximately one-half of the neurons in each
GluR2 group (see Fig. 3Cii). Removing
Na+ reduced the mortality of neurons by
>50% in each group challenged with kainate (Fig.
6B). Next, we applied kainate at 100 µM (a nonequipotent concentration) to each
group. Removing Na+ also reduced neuronal
mortality in each group as compared with the same group controls (Fig.
6C). However, cell death in the GluR2(/)
group was not abolished completely by removing
Na+ under any conditions (Fig.
6B,C), suggesting either that AMPAR-mediated neurotoxicity may depend on factors additional to the
Na+ component of the ionic current or that
Na+ removal has inherent deleterious
effects on GluR2 mutant neurons.
Because Na+ removal did not abolish
kainate-mediated AMPAR toxicity completely (Fig.
6B,C), we investigated the effect of removing extracellular Ca2+ ions on kainate
toxicity in the GluR2 mutants. Experiments by other authors already
have suggested that this maneuver reduces kainate-mediated toxicity
(Brorson et al., 1994), thus causally implicating
Ca2+ ions in the process. Figure
6D shows that removing
Ca2+ was well tolerated by the cultures in
the absence of a kainate challenge. Kainate (100 µM) still produced toxicity in the absence of
Ca2+ influx, apparently to a lesser but
not statistically significant degree than in the presence of
Ca2+ (compare with Fig. 6C;
t21 = 1.89; p = 0.073). The rank order of vulnerability to kainate toxicity was
proportional to GluR2 level, with
GluR(/)
remaining more vulnerable to kainate toxicity than
GluR(+/+) neurons. This experiment
suggests, as have previous studies, that
Ca2+ ions may be implicated, at least in
part, in mediating cell death in AMPAR-mediated excitotoxicity.
However, consistent with the other findings in this paper, the data
also indicate that the GluR2 level may dictate excitotoxic
vulnerability by mechanisms other than those dependent solely on the
magnitude of Ca2+ loading.
AMPAR localization in GluR2 mutant neurons
The AMPAR GluR2 subunit interacts via its cytoplasmic C terminus
with PDZ domain-containing proteins such as GRIP (glutamate receptor-interacting protein) that serve to cluster AMPARs at excitatory synapses (Dong et al., 1997; Matsuda et al., 2000). Thus the
GluR2 level could impact receptor targeting and localization, which may
affect function. For example, if GluR2 deficiency were to decrease the
trafficking of AMPARs to dendrites, this could result in a larger
number of receptors at the cell soma and could be reflected in our
finding of larger kainate-evoked whole-cell currents
(Imax) and current density in the
GluR2(/)
cultures (see Table 1). Alternately, these same findings could be
explained by an effect of GluR2 level on receptor expression levels.
Thus we examined the expression, localization, and clustering of AMPARs
in our cultures.
Western blot analyses of whole brain homogenates from GluR2 mutant mice
have already revealed no alterations in the levels of GluR1 and GluR4
AMPAR subunits, nor in GluR6 and 7 kainate subunits, nor in NR1, NR2A,
and NR2B NMDAR subunits (Jia et al., 1996
TOP
ABSTRACT
INTRODUCTION
