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The Journal of Neurophysiology Vol. 88 No. 3 September 2002, pp. 1279-1287
Copyright ©2002 by the American Physiological Society
Laboratory for 1Neurobiology and 2Physiology, University of Leuven, B-3000 Leuven, Belgium
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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. 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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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
(Bar-Peled 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 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.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell cultures
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 µg/ml), putrescine (0.1 mM),
conalbumin (0.1 mg/ml), sodium selenite (30 nM), penicillin (100 IU/ml), streptomycin (100 µg/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.
Toxicity experiments
Motor neuron cultures after 8 days in culture were exposed to
300 µM 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 µM) 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 µg/ml gramicidin, after tip-filling with gramicidin-free
solution. Gramicidin was dissolved in DMSO (1 mg/20 µl) 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 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 µM MK-801, and 100 µM
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 µM, 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).
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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 µM 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 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 µM 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 µM 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 µM 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 µM 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 µM in motor neurons
(n = 10), dorsal horn neurons (n = 10),
and motor neurons surviving a KA exposure (n = 7),
respectively.
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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 µM NAS (Fig. 3A). Taking the maximum inhibition (51%) in motor neurons surviving a toxic KA exposure as a limit value, 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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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 µM NAS, the inward rectification was completely lost, whereas 100 µM
pentobarbital did not affect the rectification index (Table
1). These results support the idea that
100 µM NAS and 100 µM 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 rectification 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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Motor neurons with a high relative Ca2+ permeability are lost during KA application
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).
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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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 perforated-patch-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 µM 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 presence 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 µM 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 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.
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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 to diminish excitotoxic motor neuron death
(Van Den Bosch et al. 2002a) and to prolong the survival
of transgenic mutant SOD1 mice (Beers et al. 2001). Increased Ca2+-uptake in mitochondria and
subsequent free radical formation is another factor that can account
for the selective vulnerability of motor neurons to excitotoxicity
(Carriedo et al. 2000).
Evidence for the importance of GluR2 in motor neuron viability in vivo
came from a transgenic mice model with overexpression of a GluR2 gene
that encodes an asparagine (GluR2-N) at the Q/R site (Feldmeyer
et al. 1999). AMPA receptor channels incorporating GluR2-N are
permeable to Ca2+ (Burnashev et al.
1992), and these mice were shown to develop a motor neuron
degeneration later in life. However, transgenic mice that lack the
GluR2 subunit, do not suffer from a motor neuron disease (Jia et
al. 1996). This might be due to some adaptation during
development to limit excessive AMPA receptor stimulation, such as a
long latency for recovery from the desensitized state (Harvey et
al. 2001). Further research is necessary to clarify the role of
GluR2-lacking AMPA receptors in the selective vulnerability of motor
neurons in in vivo models for ALS.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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Address for reprint requests: P. Van Damme, Laboratory for Neurobiology, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium (E-mail: philip.vandamme{at}med.kuleuven.ac.be).
Received 6 March 2002; accepted in final form 14 May 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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, 
) and dorsal horn neurons (
, · · · ), and motor neurons surviving a kainate (KA) exposure
(
, - - -). Inset: an example of
inhibition of the KA current at 
, 
