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The Journal of Neuroscience, December 1, 2000, 20(23):8831-8837
NMDA and Glutamate Evoke Excitotoxicity at Distinct Cellular
Locations in Rat Cortical Neurons In Vitro
Jeroo D.
Sinor1,
Shen
Du1,
Sriram
Venneti1,
Rachel C.
Blitzblau2,
Daniel N.
Leszkiewicz1,
Paul A.
Rosenberg2, and
Elias
Aizenman1
1 Department of Neurobiology, University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania 15261, and
2 Department of Neurology and Program in Neuroscience,
Children's Hospital and Harvard Medical School, Boston, Massachusetts
02115
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ABSTRACT |
The development of cortical neurons in vivo and
in vitro is accompanied by alterations in NMDA receptor
subunit expression and concomitant modifications in the pharmacological
profile of NMDA-activated ionic currents. For example, we observed that
with decreasing NR2B/NR2A subunit expression ratio, the block of NMDA receptor-mediated whole-cell responses by the NR2B-selective antagonist haloperidol was also decreased. In mature cultures (>22 d in
vitro), however, NMDA responses obtained from excised nucleated
macropatches, which comprised a large portion of the soma, remained
strongly antagonized by haloperidol. These results suggest that in more mature neurons NR1/NR2B receptors appear to be preferentially expressed
in the cell body. As predicted from the whole-cell recording pharmacological profile, NMDA-induced toxicity was largely unaffected by haloperidol in mature cultures. However, haloperidol effectively blocked glutamate toxicity in the same cultures, suggesting that the
neurotoxic actions of this amino acid were mostly due to the activation of somatic NMDA receptors. In experiments in which the
potency of glutamate toxicity was increased by the transport inhibitor
L-trans-pyrrolidine-2,4-dicarboxylic acid,
the neuroprotective effects of haloperidol were significantly
diminished. This was likely because of the fact that glutamate, now
toxic at much lower concentrations, was able to reach and activate
dendritic receptors under these conditions. These results strongly
argue that exogenous glutamate and NMDA normally induce excitotoxicity
at distinct cellular locations in mature mixed neuronal cultures and
that NR1/NR2B receptors remain an important component in the expression of glutamate, but not NMDA-induced excitotoxicity.
Key words:
haloperidol; development; excitotoxicity; NMDA receptors; glutamate; NR2B subunit; glutamate transport; cortical neurons; tissue
culture
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INTRODUCTION |
Neurons and glia grown in tissue
culture have been important experimental models for the study of the
cellular and molecular mechanisms underlying excitotoxicity (Rothman,
1983 ; Choi et al., 1987). Observations obtained with these in
vitro systems have had direct applications to the study of
neurodegenerative processes in vivo (Doble, 1999). An
important component of excitotoxicity that has been studied in detail
in culture systems is the glutamate uptake transport system and its
role in regulating NMDA receptor-mediated responses, including neuronal
injury (Garthwaite, 1985; Rosenberg and Aizenman, 1989; Sugiyama et
al., 1989; Rosenberg et al., 1992). Indeed, deficits in
glutamate uptake have been implicated in human neurological disease
(Rothstein et al., 1992; Lin et al., 1998). We previously determined
that a powerful glutamate uptake system in mixed cultures of astrocytes
and neurons influenced the potency of transported agonists in mediating
cell death and had a dramatic effect in altering the neuroprotective
properties of competitive NMDA receptor antagonists (Speliotes et al.,
1994). The avid uptake system present in these cultures, combined with
the fact that most synapses are buried beneath an astrocyte layer
(Harris and Rosenberg, 1993), led us to hypothesize that the action of
exogenous glutamate on neurons was restricted to the cell bodies. We
further proposed that nontransported agonists such as NMDA could
penetrate through the interstices of the tissue to reach receptors on
dendrites. In essence, we suggested that glutamate and NMDA induced
NMDA receptor-mediated toxicity at different cellular locations and possibly through different mechanisms (Speliotes et al., 1994).
Recently published results by Tovar and Westbrook (1999) and Rumbaugh
and Vicini (1999) led us to revisit and test this hypothesis. Using the
NR1/NR2B subunit-specific blockers ifenprodil (Williams, 1993) and CP
101,606 (Boeckman and Aizenman, 1996), these investigators reported
that NR1/NR2B receptors segregate to extrasynaptic regions as
hippocampal and cerebellar neurons mature developmentally both in
tissue culture and in vivo. Importantly, through the use of excised "nucleated" macropatches, Rumbaugh and Vicini (1999)
observed that the NR1/NR2B NMDA receptor configuration was selectively expressed in the soma of cerebellar granule neurons at late
developmental stages. In the present study, we have used haloperidol,
another antagonist with a high selectivity for the NR1/NR2B subunit
configuration (Ilyin et al., 1996; Lynch and Gallagher, 1996), to
confirm that NR1/NR2B receptors also segregate to the cell body in
mature rat cortical neurons in culture. The somatic segregation of
these receptors and their distinctive pharmacology allowed us to
evaluate whether glutamate toxicity was restricted to the cell body at advanced developmental stages.
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MATERIALS AND METHODS |
Materials. All drugs and tissue culture reagents were
obtained from Sigma (St. Louis, MO) with the exception of
Fe-supplemented bovine calf serum (HyClone, Logan, UT), tetrodotoxin
(Calbiochem, La Jolla, CA),
L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC; Tocris Neuramin, Bristol, England), CP 101,606 (a gift from Dr. W. F. White, Pfizer Central Research, Groton, CT), and CGS-19755 (a gift
from Dr. P. Ornstein, Eli Lilly, Indianapolis, IN). RNA extraction from
cortical cultures was achieved by using a commercially available kit
(RNeasy; Qiagen, Santa Clarita, CA). Components for the RNase
protection assay were purchased from Promega (Madison, WI) and Ambion
(Austin, TX). NMDA subunit probes were generous gifts from Drs. Jie
Zhong and Perry Molinoff (Bristol-Myers, Squibb, Wallingford, CT).
Probes were labeled using [32P]UTP from
DuPont-New England Nuclear (Boston, MA).
Astrocyte-rich cortical cultures. Cerebral cortices were
obtained from embryonic day 16 (E16) Sprague Dawley rats and
dissociated by previously described methods (Hartnett et al., 1997).
Cells were plated onto poly-L-lysine-coated glass
coverslips in either 6-well or 24-well culture dishes at a density of
300,000-500,000 cells/ml of growth media (v/v mixture of 80%
DMEM, 10% Ham's F12, 10% heat-inactivated iron-supplemented
bovine calf serum, 25 mM HEPES, 24 U/ml penicillin, 24 µg/ml streptomycin, and 2 mM L-glutamine) and
maintained at 37°C in 5% CO2. A mitotic
inhibitor (cytosine arabinoside; 2 µM) was added once at
15 DIV after which the growth medium contained no F-12 and only 2%
serum. Media was partially replaced with fresh growth medium 3 times
per week. At 3-5 weeks in vitro these cultures contain
~10-20% neurons (Rosenberg and Aizenman, 1989; Rosenberg,
1991).
RNase protection assay. RNA was harvested from the cultures
and tested for degradation with an agarose-formaldehyde gel; the 18 S
and 28 S rRNA bands were visualized with ethidium bromide. The
concentration of total RNA was measured for every preparation, with a
yield between 30 and 50 µg of RNA per two to three 6-well plates. One
microgram of linearized NR subunit plasmid was added to a 20 µl
solution containing [32-P]UTP (800 Ci/mmol; 10 mCi/ml), nucleotides (ATP, GTP, and CTP; 10 mM
each), unlabeled UTP (100 µM), SP6 or T7 RNA polymerase (20 U), transcription buffer, and RNasin RNase Inhibitor (1 U). The
mixture was incubated for 1.5 hr at 30°C. DNA template was then
degraded with 5 U of DNase I. The probes were removed from unincorporated nucleotides using phenol chloroform extraction and
pelleted by ethanol precipitation. Total RNA samples (10 µg) were
incubated with labeled probes (5 × 105 cpm) and hybridized overnight at
50°C. The solution was digested with RNase A and RNase T1 at 30°C
for 1 hr followed by a proteinase K-1% SDS incubation for 30 min at
37°C. The hybridized RNA species was extracted using phenol
chloroform and pelleted by ethanol precipitation. The protected RNA
species were separated by electrophoresis (8 M urea, 5%
acrylamide gel). Bands were visualized and quantified using a
PhosphorImager (Storm; Molecular Dynamics, Sunnyvale, CA).
Electrophysiology. Electrophysiological recordings were
conducted using the whole-cell configurations of the patch-clamp
technique as described earlier (Tang and Aizenman, 1993; Brimecombe et
al., 1998). The extracellular recording solution contained (in
mM): NaCl, 150; KCl, 2.8; CaCl2, 1.0;
HEPES, 10, 0.3 µM tetrodotoxin, 10 µM
glycine; pH adjusted to 7.2 with 0.3 M NaOH. The
intracellular (pipette) solution contained (in mM): CsF,
140; EGTA, 10; CaCl2, 1.0; pH adjusted to 7.2 with CsOH. Recordings were performed with 2-3 M electrodes. Partial
compensation for series resistance (80%) was performed in some
experiments. Currents were filtered at 0.5-1 kHz and digitized at 1-2
kHz. Drugs were dissolved in external solution and applied to cells
using a multibarrel fast perfusion system (Warner Instruments, Hamden,
CT). The current amplitude measurement to calculate haloperidol block
was performed on the portion of the trace immediately before the
removal of this drug. The time course of haloperidol application used
was chosen from preliminary experiments with longer applications in which we noted the time required to reach steady state and selected the
briefest drug application that was near this level.
Toxicity assays. Cell survival was assessed on the cortical
cells plated or transferred to 24-well culture plates. Cells were exposed to either control solution [minimum essential medium (MEM) plus 0.01% BSA and 25 mM HEPES, with 10 µM
glycine], NMDA or glutamate for 30 min at 37°C and 5%
CO2 after which the treatment was terminated by
two complete rinses with MEM (a 200:1 dilution). Cells were maintained
for 18-20 hr after the exposure and the amount of lactate dehydrogenase released into the medium was measured
spectrophotometrically using a previously described protocol (Hartnett
et al., 1997). Neuroprotection provided by the various agents tested
was derived from the following equation:
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RESULTS |
Changes in NMDA receptor subunit expression in cultured cortical
neurons with development
Total RNA was isolated from the cortical cultures on a weekly
basis for up to 4 weeks. Using an RNase protection assay, we determined
the levels of expression of the NR1, NR2A, and NR2B subunits (Fig.
1A). NR1 message was
present at week one, and its levels increased throughout the maturation
of the cultures. NR2B message was also reliably detected starting at
week one, and its expression remained relatively constant. In contrast,
NR2A message was very low relative to NR1 and NR2B during the first 2 weeks in vitro. However, expression of NR2A nearly doubled
by the third week, and doubled again by week four. We observed that the
ratio of NR2B to NR2A message, averaged across three independent
experiments, decreased significantly (p < 0.05, ANOVA) from ~6.5 at 8 d in vitro (DIV) to nearly 2 at
29 DIV (Fig. 1B). These data essentially reproduce
earlier in vitro and in vivo findings that show
that NR2A expression occurs later in cerebral cortex development when compared with NR1 and NR2B (Monyer et al., 1994; Zhong et al., 1994,
1995; Portera-Cailliau et al., 1996; Wenzel et al., 1997).
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Figure 1.
Onset of NMDA receptor subunit expression in rat
cortex in vitro. A, Representative gel from an RNase
protection assay demonstrating the time course of NMDA receptor subunit
expression in cortical neurons grown in astrocyte-rich cultures for up
to 4 weeks. The expression of NMDA receptor subunits was determined by
using 32P-lableled probes specific for NR1, NR2A, and NR2B.
B, Quantification of the ratio of NR2B to NR2A band
densities during the maturation of the cultures. Values represent the
mean ± SEM of three separate experiments performed on three
independent cultures. The decrease in band density ratio during
development was statistically significant (p < 0.05; ANOVA). C, Whole-cell voltage-clamped ( 60 mV)
currents obtained from cortical neurons in astrocyte-rich cultures at
two different developmental stages in vitro. Currents were recorded during fast
application of 30 µM NMDA in the absence or presence of
10 µM haloperidol (Hal). The
lines below the traces indicate drug
applications. Calibration: 150 pA, 1 sec. D, Responses
such as those shown in C were used to measure the
steady-state block by haloperidol of NMDA-elicited currents at various
developmental time points. The measurements of current inhibition were
corrected for the desensitization observed when agonist was applied
alone (C, light traces). Points represent individual
cells (n = 118); the line represents
a regression through the points (r = 0.55). The
decrease in haloperidol block with development was highly significant
(p < 0.0001; ANOVA). Inset
represents the degree of block produced by 0.7 µM
CGS-19755 of 30 µM NMDA-induced responses recorded under
similar circumstances (n = 69);
r = 0.35. The increase in block with development
was statistically significant (p < 0.05;
ANOVA).
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To confirm that the observed changes in the NR2B to NR2A expression
ratio reflected a functional change in NMDA receptor properties, we
performed electrophysiological recordings. Whole-cell currents evoked
by 30 µM NMDA in the absence or presence of 10 µM haloperidol were elicited in a total of 118 neurons
from developmental ages ranging from 13 to 30 DIV (Fig. 1C).
Haloperidol and other noncompetitive ifenprodil-like drugs have been
shown to be highly selective antagonists of NR1/NR2B receptors, having
a much lower affinity for receptors assembled with the NR1/NR2A subunit
configuration (Ilyin et al., 1996; Lynch and Gallagher, 1996) or on
putative NR1/NR2A/NR2B-containing receptors (Boeckman and Aizenman,
1996; Brimecombe et al., 1997; Vicini et al., 1998; Tovar and
Westbrook, 1999). The concentration of haloperidol chosen for these
studies (10 µM) is near the
EC50 reported for recombinant NR1/NR2B receptors
and less than the EC5 for the NR1/NR2A receptor
configuration (Ilyin et al., 1996; Lynch and Gallagher, 1996). We
observed a highly significant (p < 0.0001, ANOVA) decrease in the sensitivity to haloperidol with NMDA-induced
responses in neurons at later developmental stages (Fig.
1D), similar to what had previously been described
(Ilyin et al., 1996; Lynch and Gallagher, 1996). This result indicates that the decrease in NR2B relative to NR2A during development can be
functionally detected.
In contrast to this finding, the block induced by 0.7 µM
CGS-19755, a competitive antagonist at the glutamate recognition site
of the NMDA receptor (Murphy et al., 1988; Aizenman and Hartnett, 1992), was observed to increase slightly but significantly
(p < 0.01, ANOVA) with development in a total
of 69 additional neurons tested at 13-30 DIV (Fig.
1D, inset). This increase in block could be
reflective of the fact that CGS-19755 has a slightly higher affinity
for NR1/NR2A receptors when compared with NR1/NR2B (Laurie and
Seeburg, 1994; Boeckman and Aizenman, 1996).
Developmental changes in NMDA receptor-mediated toxicity
We next evaluated whether the changes in NMDA receptor subunits
during development influenced the pharmacological profile of
excitotoxicity in our cultures. Sister cultures at two
developmental stages (15-18 and 25 DIV) were exposed for 30 min to 100 and 300 µM NMDA and assayed for neuronal cell death a day
later. NMDA receptor-mediated injury was normalized to the nearly
complete neuronal death produced by an overnight exposure to 1 mM kainate (Koh and Choi, 1988). As seen previously in our
culture system (Sinor et al., 1997) and in others (Choi, 1985), younger
cultures were substantially less sensitive to NMDA toxicity than mature cultures, although a considerable amount of cell death could be produced with the higher NMDA concentration in the immature neurons (Fig. 2A). The extent
of NMDA toxicity in these young cultures was of sufficient magnitude to
allow us to test the neuroprotective effects of haloperidol (10 µM) at this developmental stage. This antagonist rescued ~50% of 13-19 DIV neurons after exposure to 300 µM NMDA. In contrast, haloperidol was much less
effective in protecting 25-32 DIV cells (~15% survival; Fig.
2B). This observation suggests that the decrease in
NR2B expression relative to NR2A during the maturation of the cultures
influences the effectiveness of haloperidol in blocking NMDA-induced
neurotoxicity. By comparison, 10 µM CGS-19755
was equally effective in protecting young and mature cells exposed to
300 µM NMDA (Fig. 2B). Hence,
the modest increase in CGS-19755 block observed with the
electrophysiological measurements during development (Fig.
1D, inset) may not have been of sufficient magnitude
to be detected with the toxicity assay. A series of comparative
experiments were also performed using ifenprodil (1 µM), the prototypical NR1/NR2B-selective
antagonist (Williams, 1993), to ensure that the neuroprotective effects
of haloperidol were indeed because of NMDA receptor block. Results from
representative experiments performed on young (18 DIV) and mature (27 DIV) cultures showing typical LDH values obtained are shown (Fig.
2C,D). The effects of ifenprodil were virtually
indistinguishable from those of haloperidol.
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Figure 2.
Developmental changes in NMDA toxicity.
A, Neuronal viability of cortical neurons in
vitro 24 hr after a 30 min exposure to either 100 or 300 µM NMDA. Experiments were performed on sister cultures at
two developmental ages. Results were normalized to the death produced
by an overnight exposure to 1 mM kainate.
Asterisks denote significant differences in viability
between the two developmental ages (p < 0.005; paired t test). Values represent the mean ± SEM of seven (15-18 DIV) and four (25 DIV) independent experiments,
each performed in quadruplicate. B, Degree of
neuroprotection afforded by either haloperidol (10 µM) or
CGS- 19755 (10 µM) against 300 µM
NMDA (30 min exposure) in cortical neurons at two developmental periods
in vitro. Haloperidol was significantly less effective
in protecting mature neurons compared with younger cells
(p < 0.05; unpaired t test).
CGS-19755 protected neurons equally regardless of developmental age.
Values represent the mean ± SEM of five independent experiments
performed in quadruplicate. C, A representative
experiment performed on a young culture (18 DIV) using 1 µM ifenprodil (Ifen) for comparison
purposes. This figure shows LDH release values obtained; note the
substantial neuroprotection against 300 µM NMDA produced
by the NR1/NR2B-selective blocker. Results are the mean ± SD of a
single experiment performed in quadruplicate. A total of four
independent experiments were performed on 18-20 DIV cultures with
essentially identical results. KA represents an overnight exposure to 1 mM kainate. D, A similar experiment
performed on a mature culture (27 DIV). Note the relative lack of
neuroprotection produced by ifenprodil (1 µM). Results
are the mean ± SD of a single experiment performed in
quadruplicate. A total of four independent experiments were performed
on 26-32 DIV cultures with essentially identical results.
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NMDA receptor subunits are functionally segregated in
mature neurons
As indicated earlier, NMDA-evoked whole-cell currents at late
in vitro stages were poorly antagonized by 10 µM haloperidol (Fig. 1C,D). In fact,
the average inhibition produced by haloperidol in cells 22 DIV or older
was only 9.6 ± 1.1% (n = 57). This is the same
period of development that we had previously showed to closely coincide
with the full expression of sensitivity to NMDA-induced toxicity (Fig.
2A; Sinor et al., 1997). Hence, the NR1/NR2B
component of the total receptor population in cortical neurons appears
to diminish at a time when NMDA receptor-mediated excitotoxicity becomes fully expressed. Interestingly, mice deficient in the NR2A
subunit are more resistant to stroke-induced CNS damage when compared
with wild-type controls (Morikawa et al., 1998).
Recent reports have suggested that any remaining NR1/NR2B receptors in
mature hippocampal or cerebellar neurons are, for the most part,
restricted to extrasynaptic or somatic regions (Rumbaugh and Vicini,
1999; Tovar and Westbrook, 1999). We confirmed that this also occurred
in cortical neurons at 22-30 DIV by using a similar strategy to that
described by Rumbaugh and Vicini (1999). The degree of haloperidol (10 µM) block of 30 µM NMDA-induced currents
was compared between whole-cell recordings, and responses obtained
after excision of nucleated patches from the same neurons. These
macrovesicles essentially represent the excised soma, or a large
portion of it. Haloperidol blocked the whole-cell NMDA response by an
average of 5.9 ± 2.5% in four of these neurons, which is a value
very similar to that obtained earlier for cells older than 22 DIV. In
contrast, the block was significantly greater (p < 0.005, paired t test) in the nucleated patches obtained
from these cells, averaging 51.5 ± 5.9% (Fig.
3A,C).
In five other cells we measured the block of NMDA-induced currents by
0.7 µM CGS-19755, which was not significantly
different (p > 0.05, paired t test)
in the intact cells (34.9 ± 5.5%) and in nucleated patches (45.2 ± 10.8%; Fig. 3B,C). These
results confirm that NR1/NR2B receptors are present in mature cortical
neurons, and that the majority of them segregate to the cell body.
Conversely, the density of NR1/NR2A or putative NR1/NR2A/NR2B appears
to be equally distributed throughout somatic and extrasomatic regions
at late developmental stages.
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Figure 3.
Haloperidol block reveals segregation of NR1/NR2B
receptors to the soma in mature cortical neurons. A,
NMDA (30 µM)-induced responses obtained from a mature
cortical neuron (25 DIV) before (left trace) or after
excision of a nucleated patch (right trace). Recordings
were obtained in the absence and presence of 10 µM
haloperidol as indicated by the lines
above the traces. A more substantial block was observed
in the nucleated patch when compared with the whole cell. Similar
results were obtained in a total of four cells. B, NMDA
(30 µM)-induced responses obtained from a second cell (28 DIV) before (left trace) or after excision of a
nucleated patch (right trace). Recordings were obtained
in the absence and presence of 0.7 µM CGS-19755, as
indicated by the lines above the traces. A similar
degree of block was observed in both recording configurations.
Calibration: 500 pA whole-cell, 50 pA patch, 2 sec. Similar results
were obtained in a total of five cells. C, Pooled block
data showing a significant difference in haloperidol block between
whole-cell recordings and nucleated patches
(p < 0.005; paired t
test).
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Figure 4.
Haloperidol block distinguishes between glutamate
and NMDA toxicity. Photomicrographs obtained from cortical cultures 24 hr after a 30 min exposure to vehicle (MEM; A), 10 µM haloperidol (B), 300 µM NMDA (C), NMDA plus haloperidol
(D), 300 µM glutamate
(E), or glutamate plus haloperidol
(F). Note the relative lack of neuroprotection by
haloperidol against NMDA toxicity compared with glutamate. Scale bar,
100 µm. Please see Figure 5 for quantification of the results.
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NMDA and glutamate induce excitotoxicity at different
cellular locations
In previous work we proposed that glutamate and NMDA induce NMDA
receptor-mediated excitotoxicity at different cellular locations (Speliotes et al., 1994). We suggested that glutamate injures neurons
by acting directly on the cell body, whereas NMDA killed by stimulating
receptors on cell processes. The observed segregation of NR1/NR2B
receptors to the soma at late developmental stages provided us with the
opportunity to test this hypothesis. We exposed mature sister cultures
(>25 DIV) to either 300 µM NMDA or 300 µM
glutamate in the absence or presence of 10 µM
haloperidol. Glutamate toxicity in these cultures at late developmental
stages is mediated solely by NMDA receptors activation (Aizenman and Hartnett, 1992). Once again, haloperidol was marginally effective in
blocking NMDA-mediated damage in these cells (Figs. 4,
5A,C). Strikingly,
glutamate-mediated toxicity was significantly antagonized (56.1 ± 9.6% neuroprotection; n = 7; p < 0.05; paired t test) by the NR1/NR2B receptor blocker (Figs.
4, 5A,C). In accordance with this finding, whole-cell
currents generated by 30 µM glutamate in 25 DIV
cells were inhibited nearly 40% by 10 µM
haloperidol (38.2 ± 3.2% block; n = 5). This
block is not significantly different (p > 0.05, unpaired t test) from the inhibition induced by haloperidol of NMDA responses in the nucleated patches. Furthermore, the difference between the block of glutamate toxicity and glutamate-induced currents
by haloperidol was not statistically significantly
(p > 0.05, unpaired t test). These
results are consistent with our hypothesis. Similar results were
obtained using 1 µM ifenprodil as the
antagonist. In 26-32 DIV cultures, this drug induced 51.6 ± 14.7% neuroprotection against glutamate and only 20.6 ± 7.9% neuroprotection versus NMDA (p < 0.05, paired
t test; n = 4).
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Figure 5.
NMDA and glutamate toxicity have distinct
pharmacological properties. A, Single representative
experiment that shows contrast between the effect of haloperidol
against NMDA toxicity and glutamate toxicity in mature neurons (25-27
DIV). Values represent the mean ± SD of LDH released into the
medium by cortical cultures 24 hr after a 30 min exposure to vehicle
(Control), 10 µM haloperidol, 300 µM NMDA, NMDA plus haloperidol, 300 µM
glutamate, glutamate plus haloperidol, or haloperidol alone. The
experiment was performed in quadruplicate. B, Addition
of the glutamate transport inhibitor PDC (100 µM)
increased the potency of glutamate in mature cultures (25-27 DIV). Values
represent the mean ± SD of LDH released into the medium by
cultures 24 hr after exposure to vehicle (Con), 30 µM glutamate, glutamate plus PDC, glutamate plus PDC and
10 µM haloperidol, and PDC or haloperidol alone. Note
that under these conditions, haloperidol no longer saves against
glutamate toxicity. This single representative experiment was performed
in quadruplicate. C, Data such as those shown in
A and B were normalized and averaged
across four to seven independent experiments performed in quadruplicate
to illustrate the degree of neuroprotection afforded by 10 µM haloperidol against 300 µM NMDA, 300 µM glutamate, or 30 µM glutamate in the
presence of 100 µM PDC. Asterisk denotes
significant difference between Glu + Hal group and the other two
experimental groups (p < 0.01; ANOVA with
Tukey-Kramer multiple comparisons test). Values represent the
mean ± SEM (n = 5 for NMDA + Hal;
n = 7 for Glu + Hal; n = 4 for
Glu + PDC + Hal).
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In accordance with our model, increasing the potency of glutamate by
using a glutamate transporter blocker (Robinson et al., 1993; Dugan et
al., 1995) should be the direct consequence of glutamate gaining access
to NMDA receptors on dendrites. Under these conditions, haloperidol
should lose its neuroprotective properties if our hypothesis is
correct. We first confirmed that the glutamate transport inhibitor PDC
did in fact shift the EC50 for glutamate toxicity
without altering the NMDA concentration-response relationship. As
reported by others (Robinson et al., 1993; Dugan et al., 1995), we
observed that the EC50 for glutamate in inducing cell death shifted approximately eightfold (p < 0.05; paired t test) in the presence of 100 µM PDC (a concentration of the transport inhibitor that is minimally toxic; Blitzblau et al., 1996), without affecting the NMDA EC50 in mature neurons (>25
DIV; Table 1). Hence, concentrations of
glutamate that were marginally or nontoxic under normal conditions now
became significantly lethal. Under these circumstances, the
neuroprotective effects of haloperidol (10 µM)
against glutamate disappeared (Figs. 5B,C). These results provide conclusive evidence that the model predicted by our previous investigations (Speliotes et al., 1994) was correct.
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DISCUSSION |
Mixed cultures of rat cerebral cortex contain various non-neuronal
cells types, including astrocytes, oligodendrocytes, microglia, and
ependymal cells (Dichter, 1978). These cells, especially astrocytes, represent the largest component of the cultures, contributing up to
~80-90% of the total cellular mass at 3-5 weeks in
vitro (Rosenberg, 1991). Previous work by our group and by other
investigators had suggested that the presence of a glutamate transport
system in glia strongly influenced the ability of glutamate to kill
neurons in culture (Garthwaite, 1985; Rosenberg and Aizenman, 1989;
Sugiyama et al., 1989; Rosenberg et al., 1992). We also reported
that the presence of the transport system could distort the
pharmacological profile of competitive antagonists in protecting
against the neurotoxic actions of transported agonists such as
glutamate, but not against nontransported agonists like NMDA (Speliotes
et al., 1994). These observations, coupled with the fact that dendrites
are shielded from the external milieu by an astrocyte layer (Harris and
Rosenberg, 1993), led us to propose that exogenous glutamate never
reaches the sites that are accessible to NMDA to injure neurons. We
hypothesized that exogenous glutamate is toxic neurons under these
circumstances by activating somatic receptors exclusively (Speliotes et
al., 1994). In the present study, we provide experimental evidence that
directly supports this hypothesis. The fact NR1/NR2B receptors appear
to segregate to the cell body as neurons mature in mixed cultures
allowed us to demonstrate that haloperidol could effectively block
glutamate-induced neurotoxicity without substantially altering NMDA-mediated cell death. Furthermore, a glutamate transport inhibitor was used to allow glutamate to reach dendritic receptors and become a
more potent toxin. Under these circumstances, haloperidol became ineffective in blocking neuronal death, similar to what was observed when NMDA was used as the stimulus.
An alternative scenario, which may result in the receptor segregation
we observe in the mature cultures, is that NR1/NR2B-containing neurons
simply perish during development. Although we normally do not see
widespread natural neuronal cell death even after 6 weeks in culture,
and NR2B message does not decline with development, we cannot totally
rule out this possibility. Nonetheless, whatever population of neurons
becomes prominent at later stages in development, it still has NR1/NR2B
receptors segregated to the soma, which allows us to test our hypothesis.
Glutamate-induced toxicity triggered by activation of somatic receptors
requires a high concentration of agonist, and therefore, protection by
competitive antagonists like D-APV or CGS-19755 can only be
achieved by very high concentrations of these drugs (Speliotes et al.,
1994). The high concentration of glutamate required to produce toxicity
via somatic receptors suggests that the mechanisms by which glutamate
and NMDA injure neurons in mixed cultures may be quite different.
Results from several studies have strongly argued that the
compartmentalization and association of NMDA receptors with other
proteins or subcellular organelles may be critical for triggering
excitotoxicity (Furukawa et al., 1997; Peng and Greenamyre, 1998;
Sattler et al., 1998, 1999). As such, it is possible that the coupling
between the NMDA receptor and the intracellular processes necessary to
trigger cell death vary between the dendrites and the cell body. The
high concentration of glutamate that is required to kill neurons in
mixed cultures cannot be accounted for by differences in agonist
affinity between NMDA receptor subtypes (Laurie and Seeburg, 1994;
Boeckman and Aizenman, 1996), and is reminiscent of the levels of
glutamate needed to elicit oxidative stress and cell death via
inhibition of cystine transport in cell lines, neurons and
oligodendrocytes (Murphy et al., 1989,, 1990; Oka et al., 1993; Ratan
et al., 1994; Li et al., 1997). We currently favor the hypothesis that
glutamate toxicity in mature astrocyte-rich cortical cultures requires
not only NMDA receptor activation, but also at least a second injurious process, such as oxidative stress (Coyle and Puttfarcken, 1993). Future
experiments will be designed to test this hypothesis.
Glutamate-induced neurotoxicity in mixed cultures is a commonly used
model of neuronal injury despite the fact that the NMDA receptors that
cluster in dendritic spines (O'Brien et al., 1998) are likely to be
critical in mediating more relevant pathophysiological processes, such
as those that are initiated during ischemia (Sattler et al., 2000).
Indeed, synaptically released glutamate appears to mediate a large
component of the excitotoxic damage under these circumstances (Rothman,
1983, 1984; Monyer et al., 1992; Sattler et al., 2000). Hence, the
demonstration that NMDA and glutamate act at different cellular
locations presented in this study warrants the reassessment of previous
work focusing on glutamate-mediated intracellular alterations that are
limited to nonsynaptic regions, including imaging studies at the soma.
| |
FOOTNOTES |
Received June 30, 2000; revised Sept. 18, 2000; accepted Sept. 19, 2000.
This work was funded in part by grants from the National Association
for Research in Schizophrenia and Depression (E.A.), Ron Shapiro
Charitable Foundation (P.A.R.), and the Muscular Dystrophy Association
(P.A.R.) and by National Institutes of Health Grants NS29365 (E.A.) and
NS38475 (P.A.R.). E.A. and D.N.L. are supported by the American Heart
Association. We thank B. A. McLaughlin, K. Hartnett, K. Kandler,
and G. A. Herin for helpful discussions and suggestions, and
Koichi Takimoto for advice with the RNase protection assay.
Correspondence should be addressed to Dr. Elias Aizenman, Department of
Neurobiology, University of Pittsburgh School of Medicine, E-1456 BST,
Pittsburgh, PA 15261. E-mail: redox+{at}pitt.edu.
| |
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ABSTRACT
INTRODUCTION
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