J Neurophysiol
88: 1279 –1287, 2002; 10.1152/jn.00163.2002.
GluR2-Dependent Properties of AMPA Receptors Determine the
Selective Vulnerability of Motor Neurons to Excitotoxicity
P. VAN DAMME,1 L. VAN DEN BOSCH,1 E. VAN HOUTTE,1 G. CALLEWAERT,2 AND W. ROBBERECHT1
Laboratory for 1Neurobiology and 2Physiology, University of Leuven, B-3000 Leuven, Belgium
Received 6 March 2002; accepted in final form 14 May 2002
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and
lower motor neurons. AMPA receptor-mediated excitotoxicity
has been implicated in this selective motor neuron loss (BarPeled et al. 1999; Carriedo et al. 1995, 1996; Hugon et al.
1989; Ikonomidou et al. 1996; Rothstein 1996; Rothstein et al.
1993; Shaw and Ince 1997).
This selective toxicity seems to be dependent on Ca2⫹ influx
through Ca2⫹-permeable AMPA receptors (Carriedo et al.
1995, 1996; Greig et al. 2000; Van Den Bosch et al. 2000). The
Ca2⫹ permeability of the AMPA receptor is largely determined
by the presence of the GluR2 subunit in the receptor complex.
Receptors containing GluR2 have a very low relative Ca2⫹
permeability compared with GluR2-lacking receptor channels
(Hollmann et al. 1991). The impermeability to Ca2⫹ is attributable to a positively charged arginine at position 586 (Q/R
site) instead of a genetically encoded neutral glutamine (Burnashev et al. 1992; Hume et al. 1991). This arginine residue at
the Q/R site is introduced by editing of the GluR2 pre-mRNA
(Sommer et al. 1991), which is virtually complete. Three other
functional properties of AMPA receptors (sensitivity to channel block by external polyamines, current rectification, and
single-channel conductance) also depend on the presence of
GluR2. GluR2-lacking channels are easily blocked by external
polyamines (Brackley et al. 1993; Herlitze et al. 1993), display
a strong inward rectification (Boulter et al. 1990; Hollmann et
al. 1991; Verdoorn et al. 1991), and have a higher single
channel conductance (Swanson et al. 1997). The inward rectification is due to the permeation of intracellular polyamines in
the channel pore of GluR2-lacking receptors at positive potentials (Donevan and Rogawski 1995; Kamboj et al. 1995; Koh
et al. 1995).
Although Ca2⫹ influx through Ca2⫹-permeable AMPA receptors appears to be critical for the selective motor neuron
vulnerability, conflicting evidence exists about the relative
expression of GluR2 in motor neurons, both at the mRNA
(Greig et al. 2000; Takuma et al. 1999; Tölle et al. 1993;
Tomiyama et al. 1996; Vandenberghe et al. 2000b; Virgo et al.
1996; Williams et al. 1997) and at the protein level (Bar-Peled
et al. 1999; Del Cano et al. 1999; Morrison et al. 1998; Shaw
et al. 1999). A critical role for GluR2 in the survival of motor
neurons in vivo is suggested by the fact that transgenic mice
overexpressing a GluR2 gene that encodes an asparagine at the
Q/R site (yielding Ca2⫹-permeable AMPA receptors) develop
a motor neuron disease later in life (Feldmeyer et al. 1999). On
the contrary, GluR2 knock-out mice display no overt motor
neuron disorder (Jia et al. 1996).
In this study, we used a co-culture system of either rat spinal
motor neurons or dorsal horn neurons grown on a pre-established astroglial feeder layer to study the role of GluR2 in
excitotoxic cell death. As previously shown (Van Den Bosch et
al. 2000), almost half of the motor neurons are killed by short
exposures to kainate (KA), whereas most dorsal horn neurons
survive such a treatment. This selective motor neuron death
can be prevented by antagonists of AMPA receptors, antagonists of Ca2⫹-permeable AMPA receptors, and by removal of
extracellular Ca2⫹ (Van Den Bosch et al. 2000, 2002b). Using
Address for reprint requests: P. Van Damme, Laboratory for Neurobiology,
Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium (E-mail:
philip.vandamme@med.kuleuven.ac.be).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Van Damme, P., L. Van Den Bosch, E. Van Houtte, G. Callewaert,
and W. Robberecht. GluR2-dependent properties of AMPA receptors
determine the selective vulnerability of motor neurons to excitotoxicity. J
Neurophysiol 88: 1279 –1287, 2002; 10.1152/jn.00163.2002. AMPA
receptor-mediated excitotoxicity has been implicated in the selective
motor neuron loss in amyotrophic lateral sclerosis. In some culture
models, motor neurons have been shown to be selectively vulnerable
to AMPA receptor agonists due to Ca2⫹ influx through Ca2⫹-permeable AMPA receptors. Because the absence of GluR2 in AMPA
receptors renders them highly permeable to Ca2⫹ ions, it has been
hypothesized that the selective vulnerability of motor neurons is due
to their relative deficiency in GluR2. However, conflicting evidence
exists about the in vitro and in vivo expression of GluR2 in motor
neurons, both at the mRNA and at the protein level. In this study, we
quantified electrophysiological properties of AMPA receptors, known
to be dependent on the relative abundance of GluR2: sensitivity to
external polyamines, rectification index, and relative Ca2⫹ permeability. Cultured rat spinal cord motor neurons were compared with dorsal
horn neurons (which are resistant to excitotoxicity) and with motor
neurons that survived an excitotoxic insult. Motor neurons had a
higher sensitivity to external polyamines, a lower rectification index,
and a higher relative Ca2⫹ permeability ratio than dorsal horn neurons. These findings confirm that motor neurons are relatively deficient in GluR2. The AMPA receptor properties correlated well with
each other and with the selective vulnerability of motor neurons
because motor neurons surviving an excitotoxic event had similar
characteristics as dorsal horn neurons. These data indicate that the
relative abundance of GluR2 in functional AMPA receptors may be a
major determinant of the selective vulnerability of motor neurons to
excitotoxicity in vitro.
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VAN DAMME ET AL.
the perforated-patch-clamp technique, we studied GluR2-dependent properties of AMPA receptors currents [sensitivity to
the selective Ca2⫹-permeable AMPA receptor antagonist
1-naphthyl acetyl spermine (NAS), rectification index and
Ca2⫹ permeability] in both cell types and correlated these
findings to KA-induced cell death. In contrast to GluR2 protein
or mRNA detection, these properties relate only to the GluR2
content of functional AMPA receptors in the cell membrane.
METHODS
Cell cultures
Toxicity experiments
Motor neuron cultures after 8 days in culture were exposed to 300
␮M KA for 30 min at 37°C in a modified Krebs solution [which
contained (in mM) 122.3 NaCl, 5.9 KCl, 10 CaCl2, 1.2 MgCl2, 11.6
HEPES, and 11.5 glucose). The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 ␮M) was added during all KA exposures. After 24 h of recovery, the surviving cells were used for
electrophysiological recordings. This procedure is known to cause cell
death in 46% of motor neurons (Van Den Bosch et al. 2000).
Electrophysiology
The gramicidin perforated-patch-clamp technique (Kyrozis and
Reichling 1995) was used for electrophysiological recordings. Pipettes were back-filled with pipette solution containing 50 –75 ␮g/ml
gramicidin, after tip-filling with gramicidin-free solution. Gramicidin
was dissolved in DMSO (1 mg/20 ␮l) before each experiment. Pipettes had a resistance of 2– 4 M⍀ when filled with intracellular
solution. After seal formation, the progress of perforation was followed by evaluating the decrease in series resistance. Cells were
accepted for study if series resistance (Rs) dropped below 30 M⍀ and
remained stable during the experiment. Cells were held at a membrane
potential of ⫺60 mV and I-V relationships were generated using
voltage ramps from ⫺100 to ⫹50 mV. Signals were recorded using a
L/M-EPC7 List-Medical amplifier, filtered at 3 kHz, sampled at 2
kHz, and analyzed off-line (Digidata 1200, pClamp8, Axon Instruments). Rs was compensated off-line to estimate the voltage error due
to Rs. The error on the relative Ca2⫹ permeability ratio and on the
J Neurophysiol • VOL
Materials and statistics
Media and additives were obtained from Gibco BRL (Grand Island,
NY); TTX was from Calbiochem (San Diego, CA), MK-801 was from
Tocris Cookson (Bristol, UK). All other chemicals were from Sigma
(St. Louis, MO). For dose-response curves, a logistic equation was
used to fit and calculate EC50 and statistical difference was calculated
by difference of slope analysis. Student’s t-tests were used to calculate
significance of the GluR2-dependent properties between the different
cell populations. Because the distributions were not always Gaussian,
significance was confirmed by a nonparametric test (Wilcoxon).
RESULTS
Motor neurons with a high sensitivity to NAS are vulnerable
to KA-induced cell death
As a first electrophysiological parameter of the relative
abundance of GluR2 in functional AMPA receptors, sensitivity
to NAS was studied. Application of 100 ␮M KA induced a
large inward current at negative membrane potentials that
could be blocked to a variable degree by NAS selective antagonist of GluR2-lacking AMPA receptors. Dose-response
curves of NAS were generated by applying increasing doses of
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Motor neurons were cultured as previously described (Vandenberghe et al. 1998; Van Den Bosch et al. 2000), following procedures
approved by the local ethical committee. In brief, ventral spinal cords
were dissected from 14-day-old Wistar rat embryos in Hanks’ balanced salt solution (HBSS), cut in pieces of about 1 mm and digested
for 15 min in 0.05% trypsin in HBSS at 37°C. After treatment with
DNase, the tissue was further dissociated by trituration. A motor
neuron-enriched neuronal population was purified from the ventral
spinal cord by centrifugation on a 6.5% metrizamide cushion and was
cultured on a glial feeder layer, which had been preestablished on
18-mm round glass coverslips coated with poly-L-ornithine and laminin. The culture medium consisted of L15 supplemented with sodium
bicarbonate (0.2%), glucose (3.6 mg/ml), progesterone (20 nM), insulin (5 ␮g/ml), putrescine (0.1 mM), conalbumin (0.1 mg/ml), sodium selenite (30 nM), penicillin (100 IU/ml), streptomycin (100
␮g/ml), and horse serum (2%). As previously described, most of the
cells in culture are motor neurons as shown by immunostainings with
the motor neuron marker peripherin (Van Damme et al. 2002).
Dorsal horn neurons were dissociated from the dorsal spinal cord
using the same protocol, except that the metrizamide gradient centrifugation was omitted.
Cultures were kept in a 7% CO2 humidified incubator at 37°C.
Neurons were used for experiments between 7 and 9 days in culture.
rectification index was minimal (0.6 ⫾ 1% on PCa/PNa, 2.6 ⫾ 3% on
rectification index, n ⫽ 16). On the inhibition of KA-induced currents
by NAS the estimated error was 6.6 ⫾ 2% (n ⫽ 16). This error led to
an underestimation of NAS sensitivity, particularly in cells with large
currents. Because motor neurons have larger current amplitudes than
dorsal horn neurons, this underestimation rather reinforces our findings. Before each measurement, the cell size was estimated and the
cell capacitance was measured. There was no correlation between
these parameters and the measured NAS sensitivity, rectification
index, or relative Ca2⫹ permeability. All recordings were performed
at room temperature.
The normal pipette solution consisted of (in mM) 125 CsCl, 1.2
MgCl2, 10 HEPES, 2 Na2ATP, and 1 EGTA, pH adjusted to 7.3 with
CsOH. The standard extracellular solution contained (in mM) 99.1
NaCl, 30 TEACl, 5.9 KCl, 3.2 CaCl2, 1.2 MgCl2, 11.6 HEPES, and
11.5 glucose, pH 7.3 with NaOH. TEACl was added to this solution
to avoid KA-induced inhibition of voltage-gated K⫹ channels (Van
Damme et al. 2002). The Na⫹-rich extracellular solution contained (in
mM) 105 NaCl, 30 TEACl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES,
and 15.8 glucose, pH adjusted to 7.3 with NaOH. The Ca2⫹-rich
extracellular solution consisted of (in mM) 30 CaCl2, 30 TEACl, 75
N-methyl-D-glucamine, 5 HEPES, and 5 glucose, pH adjusted to 7.3
with HCl. All experiments were carried out in the presence of 500 nM
tetrodotoxin (TTX), 10 ␮M MK-801, and 100 ␮M Cd2⫹ to block
voltage-gated Na⫹ channels, NMDA receptors, and voltage-operated
Ca2⫹ channels, respectively. NAS was applied as a selective antagonist of GluR2-lacking AMPA receptors (Blaschke et al. 1993; Koike
et al. 1997). Pentobarbital (PB), up to 100 ␮M, was used as a selective
blocker of GluR2-containing AMPA receptors (Taverna et al. 1994;
Yamakura et al. 1995).
The rectification of KA-induced currents was quantified using the
following expression (Ozawa et al. 1991): Rectification index ⫽
[I40/(40 ⫺ Erev)]/[I-60/(⫺60 ⫺ Erev)]
The Ca2⫹ permeability ratios, PCa/PNa were determined from the
reversal potentials obtained in a Na⫹-rich (VrevNa) and a Ca2⫹-rich
solution (VrevCa) according to the equation (Geiger et al. 1995): PCa/PNa ⫽
0.25 aNa/aCa {exp[(2VrevCa – VrevNa) F/RT] ⫹ exp[(VrevCa – VrevNa)
F/RT]}, where aNa and aCa are the ion activities of Na⫹ and Ca2⫹ in
the extracellular solution and F, R, and T have their conventional
meaning. Activity coefficients were estimated by interpolation of
tabulated values (0.75 for NaCl, 0.55 for CaCl2).
ROLE OF GLUR2 IN EXCITOTOXIC MOTOR NEURON DEATH
37.5% (21 of 56 cells) of motor neurons displayed a high NAS
sensitivity. On the contrary, almost no dorsal horn neurons
with a high sensitivity to NAS were found (Fig. 3B, 3 of 36 ⫽
8.3%). Motor neurons with a high sensitivity to NAS were
selectively killed by KA application, as these cells were no
longer encountered following a short KA exposure (Fig. 3C). If
motor neurons were divided into cells with a low and high
sensitivity to NAS, the dose-response curves of the resistant
motor neuron population almost fully matched with the dorsal
horn neurons and differed clearly from the vulnerable motor
neuron population (Fig. 1C).
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NAS during KA application. Motor neurons were significantly
more sensitive to NAS compared with dorsal horn neurons
(P ⫽ 0.001, Fig. 1A). The sensitivity to NAS was also determined in motor neurons that were exposed to 300 ␮M KA for
30 min. to induce cell death. Interestingly, motor neurons
surviving such an excitotoxic insult were less sensitive to NAS
to a level comparable to dorsal horn neurons. The calculated
EC50 for NAS was 5.57 ⫾ 0.52, 9.40 ⫾ 0.96, and 8.71 ⫾ 1.04
␮M in motor neurons (n ⫽ 32), dorsal horn neurons (n ⫽ 23),
and motor neurons surviving a KA exposure (n ⫽ 23), respectively. The mean inhibition of KA currents by 100 ␮M NAS
amounted to 43 ⫾ 3.4% (n ⫽ 56), 26 ⫾ 2.2 (n ⫽ 46, P ⫽
0.0001), and 27 ⫾ 2.3% (n ⫽ 30, P ⫽ 0.001) in motor neurons,
dorsal horn neurons and motor neurons surviving a toxic KA
exposure, respectively. Pentobarbital up to a concentration of
100 ␮M has been reported to be a selective antagonist of
GluR2-containing AMPA receptors (Taverna et al. 1994; Yamakura et al. 1995). As expected, dorsal horn neurons were
more sensitive to pentobarbital at low concentrations than
motor neurons (P ⫽ 0.001, Fig. 1B). Again, motor neurons
surviving a short KA exposure behaved like dorsal horn neurons. The estimated EC50 for pentobarbital was 513 ⫾ 65,
373 ⫾ 87, and 289 ⫾ 30 ␮M in motor neurons (n ⫽ 10), dorsal
horn neurons (n ⫽ 10), and motor neurons surviving a KA
exposure (n ⫽ 7), respectively.
Sensitivity to NAS appeared to divide motor neurons into
two populations, cells with a high (Fig. 2A) and cells with a
low (Fig. 2B) sensitivity to NAS. We therefore studied the
distribution of inhibition of AMPA receptor currents by 100
␮M NAS (Fig. 3A). Taking the maximum inhibition (51%) in
motor neurons surviving a toxic KA exposure as a limit value,
FIG. 1. Sensitivity of AMPA receptor currents to 1-naphthyl acetyl spermine (NAS) and pentobarbital (PB) in motor neurons and dorsal horn neurons.
A: NAS dose-response curve in motor neurons (■, —) and dorsal horn neurons
(●, 䡠 䡠 䡠 ), and motor neurons surviving a kainate (KA) exposure (Œ, - - -). Inset:
an example of inhibition of the KA current at ⫺60 mV by increasing doses of
NAS in a dorsal horn neuron. Each point represents the mean ⫾ SE of data
from 32 motor neurons, 13 dorsal horn neurons, and 19 motor neurons
surviving a KA exposure. Motor neurons are significantly more sensitive to
external NAS block of AMPA receptors (P ⫽ 0.001), whereas motor neurons
that survived an excitotoxic insult had values comparable to dorsal horn
neurons. By means of a logistic fit, EC50 were estimated to be 5.57 ⫾ 0.52,
9.40 ⫾ 0.96, and 8.71 ⫾ 1.04 ␮M for motor neurons, dorsal horn neurons, and
motor neurons surviving a KA exposure respectively. B: PB dose-response
curve in motor neurons (■, —), dorsal horn neurons (●, 䡠 䡠 䡠 ), and motor
neurons surviving a KA exposure (Œ, - - -). Inset: an example of inhibition of
the KA-current at ⫺60 mV by increasing doses of PB in a motor neuron. Each
point represents the mean ⫾ SE of data from 10 motor neurons, 10 dorsal horn
neurons, and 7 motor neurons surviving a KA exposure. High concentrations
of PB resulted in a nonselective block of AMPA receptors. At low concentrations however, motor neurons were significantly less sensitive to PB (P ⬍
0.001) than dorsal horn neurons. Again motor neurons that were resistant to an
excitotoxic insult behaved like dorsal horn neurons. Data were fit with a
logistic equation. EC50 values were 513 ⫾ 65, 373 ⫾ 87, and 289 ⫾ 30 ␮M
for motor neurons, dorsal horn neurons, and motor neurons surviving a KA
exposure, respectively. C: NAS dose-response curve in dorsal horn neurons
and in subpopulations of motor neurons. Data from the same neurons as shown
in A, but the motor neurons are divided in 2 subpopulations: cells with a high
(more than 51%, ■, —) and low (less than 51%, 䊐, —) sensitivity to NAS. The
dose-response curve of motor neurons with a low sensitivity almost fully
matched with the trace of motor neurons surviving a toxic KA exposure and
with the trace obtained from dorsal horn neurons. Each point represents the
mean value ⫾ SE from 11 motor neurons with a high sensitivity to NAS (■,
—), from 21 motor neurons with a low sensitivity to NAS (䊐, —), from 19
motor neurons surviving a KA exposure (Œ, - - -), and from 13 dorsal horn
neurons (●, 䡠 䡠 ).
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Motor neurons with a strong inward rectification are
selectively killed by KA
As a second GluR2-dependent property of AMPA receptors, the current rectification was studied. Most motor neurons displayed a clear inward rectification, with a mean
rectification index (Ozawa et al. 1991) of 0.78 ⫾ 0.026 (n ⫽
48). In Fig. 2, an example of an I-V relation with (Fig. 2C)
and without (Fig. 2D) a clear inward rectification is shown.
In the presence of 100 ␮M NAS, the inward rectification
was completely lost, whereas 100 ␮M pentobarbital did not
affect the rectification index (Table 1). These results support
J Neurophysiol • VOL
the idea that 100 ␮M NAS and 100 ␮M PB act as selective
antagonists of GluR2-lacking and -containing AMPA receptors, respectively. The rectification index determined in
dorsal horn neurons was significantly higher than the value
obtained in motor neurons (0.94 ⫾ 0.027, n ⫽ 34, P ⬍
0.0001), but was close to the value in motor neurons surviving a toxic KA exposure (0.90 ⫾ 0.018, n ⫽ 24). The
rectification index in motor neurons surviving a toxic KA
exposure was significantly higher than the rectification index in motor neurons (P ⫽ 0.002). As indicated in Fig. 4A,
there was a good inverse correlation between the rectifica-
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FIG. 2. Properties of the 2 populations of
motor neurons. A, C, and E: motor neuron with
a high sensitivity to NAS, low rectification index, and high relative Ca2⫹ permeability, suggesting a low GluR2 content. B, D, and F: motor
neuron with a low sensitivity to NAS, high
rectification index, and low relative Ca2⫹ permeability, suggesting a high GluR2 content.
Top: the inward current induced by 100 ␮M KA
is shown in the absence and presence of 100 ␮M
NAS at ⫺60 mV. Middle: the I-V relation of the
KA-induced current in the absence and presence
of 100 ␮M NAS. Note that the inward rectification changes to outward rectification in the
presence of NAS in the motor neuron with a
high NAS sensitivity (C). Bottom: the I-V relations of the KA-induced current in the Na⫹- and
Ca2⫹-rich solution. The large shift in reversal
potential in the motor neuron with a low NAS
sensitivity (F) is indicative for a low PCa/PNa
value.
ROLE OF GLUR2 IN EXCITOTOXIC MOTOR NEURON DEATH
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Motor neurons with a high relative Ca2⫹ permeability are
lost during KA application
DISCUSSION
Motor neurons are particularly vulnerable to KA-induced
cell death (Bar-Peled et al. 1999; Carriedo et al. 1995, 1996;
Hugon et al. 1989; Ikonomidou et al. 1996; Rothstein et al.
1993). Ca2⫹ influx through Ca2⫹-permeable AMPA receptors
appears to play a key role in this selective vulnerability of
1. Rectification index in motor neurons and
dorsal horn neurons
TABLE
FIG. 3. Relation between NAS sensitivity and excitotoxic cell death. A: the
proportion of KA current blocked by 100 ␮M NAS at ⫺60 mV was determined in 56 motor neurons. - - -, the highest value obtained in motor neurons
surviving a short KA exposure (cf. Fig. 3C). Twenty-one of 56 motor neurons
(37.5%) had a high sensitivity to NAS. B: sensitivity to 100 ␮M NAS
determined in 36 dorsal horn neurons. Only 3 of 36 cells (8.3%) displayed a
high sensitivity to NAS. C: sensitivity to 100 ␮M NAS in 33 motor neurons
surviving a short KA exposure.
tion index and sensitivity to NAS in motor neurons (r ⫽
⫺0.697, P ⬍ 0.0001). In the dorsal horn neuron population,
only 4 of 34 cells (11.8%) had a rectification index below
0.72, the lowest value observed in the resistant motor neuron population (Fig. 4B). KA application selectively killed
motor neurons with a low rectification index (Fig. 4C).
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Rectification index
n
Rectification index
in the presence
of NAS
n
Rectification index
in the presence
of PB
n
Motor
Neurons
Dorsal Horn
Neurons
Motor Neurons
Surviving KA
Exposure
0.78 ⫾ 0.03
48
1.22 ⫾ 0.08
0.94 ⫾ 0.03
36
1.18 ⫾ 0.04
0.90 ⫾ 0.02
24
0.99 ⫾ 0.05
38 (⬍0.001)
0.79 ⫾ 0.04
34 (⬍0.001)
1.01 ⫾ 0.03
24 (0.046)
—
10 (0.85)
12 (0.25)
The rectification index of kainate (KA)-induced currents was determined in
motor neurons, dorsal horn neurons, and motor neurons surviving a short KA
exposure. Motor neurons have a significantly lower rectification index than
dorsal horn neurons (P ⬍ 0.0001) and than motor neurons surviving a short
KA exposure (P ⫽ 0.0029). The influence of 100 ␮M pentobarbital (PB) and
100 ␮M 1-naphthyl acetyl spermine (NAS) on the rectification index was also
checked in motor neurons and dorsal horn neurons. Results are shown as
means ⫾ SE. P values (in parentheses) in the table are relative to the value
without NAS or PB.
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The relative Ca2⫹ permeability ratios PCa/PNa were calculated from the reversal potentials in a Na⫹-rich and a Ca2⫹-rich
solution, both in motor neurons before and after a short KA
exposure and in dorsal horn neurons. In Fig. 2, an example of
a motor neuron with a high (Fig. 2E) and a low (Fig. 2F)
PCa/PNa is shown. PCa/PNa correlated with sensitivity to NAS
in motor neurons (Fig. 5A, r ⫽ 0.83, P ⬍ 0.0001, n ⫽ 20). The
mean value of PCa/PNa in motor neurons was significantly
higher compared with dorsal horn neurons (0.80 ⫾ 0.14 and
0.36 ⫾ 0.07, n ⫽ 20 and 13, respectively, P ⫽ 0.025). Motor
neurons surviving an excitotoxic insult had a value of 0.41 ⫾
0.07 (n ⫽ 14, P ⫽ 0.041). In the dorsal horn neuron population
and in motor neurons surviving a toxic KA exposure, no cells
with a PCa/PNa value above 0.86 were found (Fig. 5, B and C).
In accordance with previous findings (Vandenberghe et al.
2000a), we also found higher current densities in motor neurons (22.1 ⫾ 1.3 pA/pF at ⫺80 mV, n ⫽ 22) than in dorsal
horn neurons (13.5 ⫾ 1.6 pA/pF, n ⫽ 23, P ⫽ 0.0001). Motor
neurons surviving a short KA exposure had current densities
similar to dorsal horn neurons (12.2 ⫾ 0.6 pA/pF, n ⫽ 19, P ⬍
0.0001).
Taken together, motor neurons had a higher NAS sensitivity,
a lower rectification index, a higher Ca2⫹ permeability, and a
higher current density than dorsal horn neurons. Motor neurons
with a high NAS sensitivity, a low rectification index, a high
Ca2⫹ permeability, and a high current density were selectively
lost during a short KA exposure.
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FIG. 4. Relation between rectification index and NAS sensitivity in
motor neurons (A), dorsal horn neurons (B), and motor neurons surviving
an excitotoxic insult (C). A: the rectification index (for calculation of
rectification index see METHODS) and the inhibition of KA currents by 100
␮M NAS at ⫺60 mV were determined in 38 motor neurons. A wide range
of rectification indices was found, and there was an inverse correlation
between the rectification index and NAS sensitivity (r ⫽ ⫺0.697, P ⬍
0.0001). - - -, the lowest value obtained in motor neurons surviving a toxic
KA exposure (0.72). Of 38 motor neurons tested, 12 (31.6%) are predicted
to be killed during an excitotoxic insult. B: relation between the rectification index and NAS sensitivity determined in 34 dorsal horn neurons. The
fitted linear curve represents the correlation found in motor neurons (A).
Only 4 of 34 (11.7%) dorsal horn neurons were situated below the cutoff
value for excitotoxic death. C: relation between the rectification index and
NAS sensitivity from 24 motor neurons surviving a toxic KA exposure.
Motor neurons with a low rectification index and a high NAS sensitivity
were no longer encountered.
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FIG. 5. Relation between PCa/PNa and NAS sensitivity. A: from the reversal
potentials in a Na⫹- and Ca2⫹-rich solution the PCa/PNa was calculated in 20
motor neurons (for calculation of PCa/PNa and composition of Na⫹-rich and
Ca2⫹- solution, see METHODS). A good correlation between PCa/PNa and NAS
sensitivity was found (r ⫽ 0.83, P ⬍ 0.0001). - - -, the maximal value obtained
in motor neurons surviving a toxic KA exposure (0.86). Eight of 20 motor
neurons (40%) had a PCa/PNa above this value. B: determination of PCa/PNa in
13 dorsal horn neurons revealed no cells with a high PCa/PNa. The fitted linear
curve corresponds to the correlation found in motor neurons (A). C: relation of
PCa/PNa and NAS sensitivity in 14 motor neurons surviving a toxic KA
exposure. No cells with a high PCa/PNa were found. The maximal value
obtained (0.86) was used as the cutoff value for prediction of cell death.
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ROLE OF GLUR2 IN EXCITOTOXIC MOTOR NEURON DEATH
J Neurophysiol • VOL
ence of 30 mM external TEA⫹ because KA has no additional
inhibitory effect on K⫹ channels under these circumstances.
The mean value of rectification index in response to KA
application was 0.78 ⫾ 0.03, a value close to the value previously reported in motor neurons of rat spinal cord slices (Abdrachmanova et al. 2000) but was significantly lower than the
value obtained in dorsal horn neurons (0.94 ⫾ 0.03). There was
a good correlation between the rectification index and NAS
sensitivity and motor neurons with a low rectification index
were selectively lost during a short KA exposure.
Similar data were obtained by comparing PCa/PNa between
the different cell groups. The mean value of PCa/PNa obtained
in motor neurons amounted to 0.80 ⫾ 0.15. This value is
higher than the PCa/PCs value of about 0.4 found in cultured rat
spinal cord neurons by others (Greig et al. 2000; Vandenberghe
et al. 2000b). The fact that we used the perforated-patch-clamp
technique whereas the two other groups used the whole cell
configuration most likely does not contribute to this difference
because we found a similar high PCa/PCs value using the whole
cell configuration (data not shown). Differences in culture
conditions might explain this discrepancy. Alternatively, strain
differences can be involved as some investigators used Holtzman rats, whereas we used Wistar rats. For mice, it has been
shown that different strains can differ substantially in their
vulnerability to excitotoxicity (Schauwecker and Steward
1997; Shuttleworth and Connor 2001). The value of PCa/PNa
obtained in dorsal horn neurons amounted to 0.36 ⫾ 0.07 and
was lower than the values found in previous studies (Goldstein
et al. 1995; Vandenberghe et al. 2000b). Motor neurons in this
study thus have a much higher relative Ca2⫹ permeability ratio
compared with dorsal horn neurons. PCa/PNa values correlated
well with NAS sensitivity and consequently motor neurons
with the highest Ca2⫹ permeability were selectively killed
during a toxic KA exposure.
As has been reported previously (Vandenberghe et al.
2000a), we also found higher current densities in motor neurons compared with dorsal horn neurons. Furthermore, motor
neurons with the highest current amplitudes were selectively
lost during a short KA exposure. The higher current amplitudes
in motor neurons is compatible with a relatively low expression
of GluR2 because GluR2-lacking AMPA receptors are known
to have a higher single-channel conductance (Swanson et al.
1997).
In short, the studied properties of AMPA receptors, all
thought to be dependent on the relative abundance of GluR2,
correlated well with each other and with KA-induced motor
neuron death.
Using the limit values in the surviving motor neuron population (51% inhibition of AMPA receptor currents by 100 ␮M
of NAS, a rectification index of 0.72 and a PCa/PNa of 0.86), we
estimated the proportion of vulnerable motor neurons between
31.6 and 40%. As previously demonstrated in our culture
model (Van Den Bosch et al. 2000), 46 ⫾ 2% (n ⫽ 30) of
motor neurons are killed during a short KA exposure, whereas
only 7.3 ⫾ 2% (n ⫽ 5) of dorsal horn neurons are lost during
a KA exposure. Thus our estimate of vulnerable motor neurons
correlates well with the proportion of motor neuron death
during a short KA exposure (Table 2). Using Co2⫹ staining as
a histochemical marker for the presence of Ca2⫹-permeable
AMPA receptor, about 55% of motor neurons are marked as
positive. If the number of Co2⫹ positive cells that are found in
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motor neurons (Carriedo et al. 1995, 1996; Greig et al. 2000;
Van Den Bosch et al. 2000). AMPA receptors are assembled
from GluR1– 4 subunits, and their properties largely depend on
the presence of the GluR2 subunit. AMPA receptors lacking
GluR2 or containing the unedited GluR2 subunit are characterized by a high Ca2⫹ permeability, inward rectification, and
sensitivity to polyamine block (Boulter et al. 1990; Brackley et
al. 1993; Herlitze et al. 1993; Hollmann et al. 1991; Jonas and
Burnashev 1995; Verdoorn et al. 1991). Motor neurons are
therefore expected to have a low GluR2 expression or a low
GluR2 editing. Because GluR2 appears to be fully edited in
most cell types (Paschen et al. 1994; Puchalski et al. 1994),
including motor neurons (Greig et al. 2000; Vandenberghe et
al. 2000b), most attention has been focused on the level of
GluR2 expression. However, conflicting evidence exists about
the relative expression of GluR2 in motor neurons, both at the
mRNA (Greig et al. 2000; Tölle et al. 1993; Tomiyama et al.
1996; Vandenberghe et al. 2000b; Virgo et al. 1996; Williams
et al. 1997) and at the protein level (Bar-Peled et al. 1999; Del
Cano et al. 1999; Morrison et al. 1998; Shaw et al. 1999).
Rather than determining the relative abundance of GluR2, we
have focused in this study on properties of AMPA receptors,
which are known to be dependent on the relative abundance of
the GluR2 protein. These properties relate only to the GluR2
content in functional AMPA receptors in the cell membrane.
The three different properties were concordant in all experiments, suggesting that they reliably reflect the GluR2 content.
These properties were studied in motor neurons (which are
vulnerable to excitotoxicity), dorsal horn neurons (which are
resistant to excitotoxicity), and in motor neurons that survived
a toxic KA exposure. To avoid washout of intracellular polyamines, which are responsible for the inward rectification of
GluR2-lacking AMPA receptor, the gramicidin perforatedpatch-clamp technique was used.
We found that motor neurons had a significantly higher
sensitivity to AMPA receptor block by NAS, a selective antagonist of GluR2-lacking AMPA receptors (Blaschke et al.
1993; Koike et al. 1997), compared with dorsal horn neurons.
Inversely, dorsal horn neurons displayed a larger inhibition of
AMPA receptor currents by low concentrations of PB than
motor neurons. PB in concentrations up to 100 ␮M is believed
to be a selective antagonist of GluR2-containing AMPA receptors (Taverna et al. 1994; Yamakura et al. 1995). Interestingly,
the nonvulnerable subpopulation of motor neurons, which was
defined as the population surviving a short KA exposure,
displayed a similar sensitivity to NAS and PB as dorsal horn
neurons. The fact that no motor neurons with a high sensitivity
to NAS were encountered in the cells that survived a KA
exposure suggests that these cells were selectively killed by
KA. The similarity between the proportion of motor neuron
death and the proportion of motor neurons with a high NAS
sensitivity makes it less likely that major changes in the GluR2
content during the KA exposure (e.g., by downregulation of
GluR2 or internalization of GluR2-containing receptors) are
involved.
This idea was further supported by the analysis of rectification indices. As previously shown (Van Damme et al. 2002),
application of KA gives rise to an inhibition of TEA⫹-sensitive
voltage-gated K⫹ channels. This inhibition leads to an apparent
inward rectification of the KA-induced current. Reliable measurements of the KA-induced current are obtained in the pres-
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VAN DAMME ET AL.
2. Summary of relation between cell death data and
parameters of GluR2-lacking AMPA receptors
REFERENCES
TABLE
KA-induced cell death
Co2⫹-positivity
NAS sensitivity ⬎51%
Rectification index ⬍0.72
PCa/PNa ⬎ 0.86
Motor Neurons, %
Dorsal Horn
Neurons, %
46 ⫾ 2
55.1 ⫾ 1.5
37.5
31.6
40
7.3 ⫾ 4.1
8.2 ⫾ 2.3
8.3
11.8
0
Summary of data obtained from motor neurons and dorsal horn neurons. The
survival of neurons was quantified by cell count before and following a short
exposure of 300 ␮M KA (n ⫽ 30 for motor neurons, n ⫽ 5 for dorsal horn
neurons), as previously described (Van Den Bosch et al. 2000). Co2⫹ stainings,
which are histochemical markers for the presence of Ca2⫹-permeable AMPA
receptors, were previously carried out in motor neuron cultures (n ⫽ 5), and
dorsal horn neuron cultures (n ⫽ 4) (Van Den Bosch et al. 2000).
We thank P. Theys, MD, PhD for help with statistical analysis of the data.
This work was supported by grants from the Fund for Scientific Research
Flanders (F.W.O. Vlaanderen) and the University of Leuven. P. Van Damme
is a Research Assistant, L. Van Den Bosch is a Postdoctoral Fellow, and W.
Robberecht is a Clinical Investigator of the Fund For Scientific Research
Flanders. This research project is part of the IUAP Phase V (Molecular
Genetics and Cell Biology).
J Neurophysiol • VOL
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motor neurons surviving a short KA exposure (11.5 ⫾ 1.1%,
n ⫽ 3) is taken into account, the proportion of vulnerable
motor neurons becomes 43.6%, a value close to our estimation
of vulnerable cells.
This study provides evidence that the relative abundance of
GluR2 in AMPA receptors is critical for the selective vulnerability of motor neurons to excitotoxic cell death in vitro. Other
factors that contribute to the selective vulnerability of motor
neurons have been described. Motor neurons are known to
have a low Ca2⫹ buffering capacity (Alexianu et al. 1994; Ince
et al. 1993; Lips and Keller 1998, 1999; Palecek et al. 1999;
Vanselow and Keller 2000), which might render them more
susceptible to Ca2⫹-mediated cell death. Furthermore, overexpression of the Ca2⫹-binding protein parvalbumin was shown
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the desensitized state (Harvey et al. 2001). Further research is
necessary to clarify the role of GluR2-lacking AMPA receptors
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