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INTRODUCTION |
Astrocytes are the support cells for neurons in the brain. The way
in which astrocytes protect neurons from various insults is under
continued investigation. In the cerebellum where glutamate is a major
neurotransmitter (1), the astrocyte population provides effective
protection against the excitotoxicity of glutamate (2). Astrocytes
clear the glutamate released by neurons via active uptake through
glutamate-transporting proteins such as GLAST and GLT-1 (3-6). Without
rapid clearance there is the possibility that glutamate will have a
toxic effect mediated through binding to NMDA receptors (7). The
presence of astrocytes in neuron-rich cultures has been shown to reduce
the toxic potency of glutamate (8, 9). When glutamate is not cleared,
levels of glutamate can easily rise activating NMDA receptors, causing
increased calcium entry, internal oxidative stress, mitochondrial
dysfunction, and eventually apoptosis (7, 10-13). Therefore,
understanding what regulates glutamate uptake by astrocytes is essential.
There is now strong evidence that factors from neurons alter the level
of expression of the astrocyte glutamate transporters (14-16). This
can result in an increased clearance of glutamate via these
transporters (17, 18). Thus, factors released by neurons signal to
astrocytes the presence of neurons and induce an alteration of
astrocyte protein expression that results in increased protection of
neurons by astrocytes against glutamate toxicity. We have identified
vasoactive intestinal peptide as one such factor released by neurons
(18). It has been shown that substances such as vasoactive intestinal
peptide not only alter the effectiveness of glutamate transport by
astrocytes but stimulate the release of protective factors, which also
help neurons resist the excitotoxicity of substances such as glutamate
and NMDA (19, 20). In understanding the mechanism by which glutamate toxicity is prevented in the normal brain, there is clearly a need to
understand not just how glutamate uptake by astrocytes is altered by
the presence of neurons but also how neuronal sensitivity to glutamate
is regulated by the presence of astrocytes other than their removal of
glutamate by active uptake.
Previous studies have shown that mouse cerebellar neurons become
dependent on astrocytes for protection from glutamate toxicity (17,
21). Cerebellar neurons co-cultured with astrocytes show an increased
sensitivity to the toxicity of glutamate as compared with cerebellar
neurons not co-cultured. This effect is not dependent on the regional
origin of the astrocytes used for co-culture (17). Region-specific
effects of astrocytes have been suggested to be contact-mediated (22),
and the changes to glutamate sensitivity were related to diffusable
factors, some of which have been defined (18). In the presence of
astrocytes, this increased sensitivity to the toxic effects of
glutamate is normally not noticeable because of the survival promoting
effects of astrocytes such as clearance of glutamate and release of
protective factors. Increased sensitivity to glutamate toxicity is only
of consequence if the protectiveness of astrocytes is compromised. We
have found several ways this compromise can be caused, which lead to
glutamate-induced neuronal death: 1) physical removal of astrocytes, 2)
addition of substances that inhibit astrocytic glutamate uptake (21),
3) substances that activate astrocytes (e.g. transforming
growth factor-
) (17), or 4) inactivation of protective factors such
as interleukin-6 (21). This increased sensitivity to glutamate is
induced in neurons by a factor released by astrocytes. Neurons treated
with conditioned medium
(NAM)1 from astrocytes
exposed to medium from neuronal cultures showed decreased survival.
This decreased survival was a result of glutamate toxicity, but it was
not caused by increased glutamate concentration (21). Thus, some
additional factor released by astrocytes into NAM makes neurons more
sensitive to glutamate toxicity. Release of this factor is enhanced by
the presence of neurons themselves or by conditioned medium from
neurons (17). Clearly, identification of this factor and its mechanism
of action are of great importance because of their possible role in
regulating neuronal sensitivity to excitotoxic death.
In the present report, we have examined regulation of neuronal (CGN)
sensitivity to glutamate. We have used the rat system and confirmed
that this displays the same characteristics as the mouse system. We
have identified changes in NMDA receptors subunit subtype composition
that parallel increased sensitivity to glutamate toxicity.
Oligonucleotide knockdown of expression of these subunit subtypes
inhibits glutamate toxicity markedly. Oligonucleotide therapy based on
these observations may provide an effective way to combat excitotoxic
neuron death in a variety of diseases.
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EXPERIMENTAL PROCEDURES |
Unless specified chemicals and pharmacological agents were from
Sigma. The rats used were Wistar from Harlan.
L-trans-Pyrrolidine-2,4-dicarboxylic acid (PDC)
was from Tocris.
Glial Culture--
Mixed glial cultures were prepared from
dissociating cerebral cortices of newborn rats. 4-5 cortices were
trypsinized in 0.05% trypsin (Sigma) and plated in a
75-cm2 culture flask (Falcon) in Dulbecco's minimal
essential medium (Life Technologies, Inc.) supplemented with 10% fetal
calf serum (Sigma) and 1% antibiotic solution
(penicillin/streptomycin, Life Technologies, Inc.). Cultures were
maintained at 37 °C with 5% CO2 for 14 days until glial
cultures were confluent.
Astrocytes were isolated and characterized following standard methods
as described previously (23). Microglia were dislodged into the medium
and discarded. Type 1 astrocytes were purified by taking the remaining
adhesive cells and trypsinizing. The cells were preplated for 30 min to
remove contaminating microglia. Astrocytes remaining in the medium were
then collected and plated for 2 h at 104 cells/well
into 24-well trays, after which time the medium was replaced to remove
less adhesive contaminating cells. Purified astrocytes were maintained
under the conditions described for mixed glial cultures. Purity of
cultures was determined as described previously (21, 23). Astrocyte
cultures were not used unless purity was close to 100%. Astrocyte
cultures were stained for glial fibrillary acidic protein (GFAP) using
a rabbit polyclonal antibody (Dako) and detected with a
fluorescein-conjugated secondary anti-rabbit IgG antibody (Roche
Molecular Biochemicals). Stained cells were examined using a Leitz
fluorescence microscope.
Conditioned media were prepared from type 1 astrocyte cultures in which
the cells had been plated at 104 cells/well. Cells were
kept in culture for 24 h before addition of fresh medium. Media
were collected after 2 days of exposure of the cells to the medium and
other agents, and the conditioned media were used without freezing by
direct application to neuronal cultures. NAM was prepared by exposing
the astrocytes for 2 days to neuronally conditioned medium.
Neuronal Cell Culture--
Preparation of cerebellar cells from
seven or 8 day old mice (P7-8) was as described previously (21).
Briefly, the cerebella were dissociated in Hanks' salt solution (Life
Technologies, Inc.) containing 0.5% trypsin and plated at 1-2 × 106 cells/cm2 in 24-well trays (Falcon) coated
with poly-D-lysine (50 µg/ml, Sigma). Cultures were
maintained in Dulbecco's minimal essential medium supplemented with
10% fetal calf serum and 1% antibiotics (penicillin, streptomycin).
Cultures were maintained at 37 °C with 7% CO2.
Neuronal cells were stained in cerebellar cell cultures with
anti-neuronal nuclei mouse (Chemicon), and detection with a
fluorescein-conjugated anti-mouse IgG antibody.
A neuronal conditioned medium was prepared by taking cerebellar cells
that had been in culture for 2 days and exposing them to fresh medium.
The conditioned medium was collected after 2 days and applied directly
to astrocyte cultures without freezing or storage. This medium was
centrifuged to removed cellular particles.
Co-culture--
For co-culture experiments, cerebellar cells
were plated as normal in 24-well trays. Astrocytes and/or microglia
were plated in tissue culture inserts (Falcon) with 3.0-µm pores.
Pharmacological agents were added to both wells. The volume of the dual
well system was maintained at 1 ml. Cerebellar cells were maintained in
culture for 1 day before addition of astrocytes. For co-culture
experiments, astrocytes were plated into inserts at 104
cells/insert. Treatment with agents followed 2 days of co-culture and
continued for 4 days afterward. The medium was exchanged 2 days later,
and the pharmacological agents re-applied with the fresh medium. Before
assaying cerebellar cell survival, the inserts were removed and
3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma) assays carried out on the cerebellar cells. Fractionation of NAM
was performed using Microcon spin concentrators (Amersham Pharmacia
Biotech). NAM was passed sequentially through a series of concentrator
filters with molecular mass cut-offs of 3, 10, 30, and 50 kDa. The
filtrated was collected, and the retained material was reconstituted to
the same volume to pass through the next filter. This produced
fractions with solutes approximately <3, 3-10, 10-30, 30-50, and
>50 kDa.
Survival Assay--
The assay used for testing the survival of
cerebellar cell cultures was as described previously (24) and was based
on the conversion of MTT to a formazan product by respiring cells. MTT was diluted to 200 µM in Hanks' solution (Life
Technologies, Inc.) and added to cultures for 1 h at 37 °C. The
MTT formazan product was released from cells by addition of dimethyl
sulfoxide (Sigma) and measured at 570 nm in an Unicam Helios
spectrophotometer (ATI Unicam). Relative survival in comparison to
untreated controls could then be determined.
PCR Analysis--
Total RNA was extracted from cultured
astrocytes grown under various conditions using a kit (Qiagen). The
cerebellar neurons were either 1) untreated, 2) treated with astrocyte
condition medium, 3) exposed to NAM for 2 days, or 4) co-cultured with
cerebellar neurons. The RNA was reverse transcribed with Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Inc.).
Equivalent amounts of cDNA were used for PCR analysis of NMDA
receptor type 1 (NR1) and NMDA receptor type 2 (NR2) subtypes. The
method used was as previously described (25, 26). For the NR1 subtypes NR1a and NR1b were detected using a single PCR reaction using the
primers CTCCCACCAGTCCAGCGTCT (forward) and GTCATGTTCAGCATTGCGGC (reverse) where the product for NR1b is the larger of the two produced.
For NR2 subtypes NR2a, NR2b, and NR2c products are first amplified with
the primers GGGGTTCTGCATCGACATCC (forward) and GACAGCAAAGAAGGCCCACAC
(reverse). The product is then digested with one of three restriction
enzymes, which can distinguish the different subtypes Bpm1
(NR2a), Bfa1 (NR2b), or ScaI (NR2c). The primers
for PCR were generated in house by the PNAC Facility. PCR for the
glyceraldehyde-3-phosphate dehydrogenase housekeeping gene was carried
out using the specific primers (sense, 5'- GGTGAAGGTCGGTGTGAACGG -3';
antisense, 5'-CGACGGACACATTGGGGGTAGG -3') to ensure equivalence of the
samples. Additionally, RT-PCR was used to assess the level of
5-lipoxygenase expression. The primers for this analysis were 5'-TGGAACCCCGGCTTCCCTTTGAG-3' (senses) and 5'-AAAAGCCAGTCGTACTTTGAA-3'. Densitometric analysis of the bands was carried out using NIH Image.
Western Blot Analysis--
Protein was extracted from CGNs and
astrocytes by homogenization in an extraction buffer (20 mM
Tris acetate, pH 7.5, 0.27 M sucrose, 1 mM
EDTA, 1 mM EGTA, 10 mM sodium
-glycerophosphate, 50 mM sodium fluoride, 5 mM pyrophosphate, 1% Triton X-100, 1 mM sodium
orthovanadate, 0.1%
-mercaptoethanol, 0.2 mM
phenylmethylsulfonyl fluoride, and 1 mM benzamidine). After
20 min of incubation, the homogenate was centrifuged at 14,000 rpm for
10 min. The supernatant was taken as the extract. A protein
determination was carried out using a bicinchoninic acid (BCA) protein
assay kit (Sigma) to ensure identical amounts of protein were loaded
onto gels. After boiling, extracts were electrophoresed on a 10%
acrylamide gel with SDS in a Bio-Rad Mini-Protean 2 system. After
electrophoresis, the protein was transferred to a nylon membrane
(Immobilon, Millipore) with a semidry blotter (Bio-Rad). Detection of
specific antigens was carried out following blocking the membrane with
5% milk powder in Tris-buffered saline (pH7.4). Primary antibodies
included anti-NMDA receptor type 1 (NR1, mouse, Affinity Bioreagents),
anti-NMDA receptor type 2 (NR2, mouse, Affinity Bioreagents),
anti-synaptophysin (mouse, Sigmna), anti-GFAP (rabbit, Dako), and
anti-
-tubulin (mouse, Sigma). Detection was via binding of an
appropriate HRP-conjugated secondary antibody and detection using the
ECL chemiluminescence reagent (Amersham Pharmacia Biotech) and exposure
to x-ray film (Eastman Kodak Co.).
Oligonucleotide Knockdown--
Antisenses oligonucleotides
specific to NR1 and NR2 subtypes were prepared as described by
others (27): NR1a, GGGTCCGCGCTTGTTGTCAT; NR1b, CTGCAGCACCTTCTCTGCCT;
NR2a, CATAGCCCAATCTGCCCAT; NR2b, CTTCATCTTCAGCTAGTCGG; NR2c, CCCACCCATGTCACCTGGAG.
Oligonucleotides were applied daily at 100 nM to CGN
cultures and cultures treated with NAM or astrocyte conditioned medium for 4 days. At the end of that time, a survival assay was carried out
and the values compared with those of untreated cultures.
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RESULTS |
Astrocytes Influence Glutamate Neurotoxicity--
For the
experiments to be described here, CGN from 7-8-day-old rats were
prepared identically but treated four different ways. (a)
control cultures were untreated, (b) cultures were
co-cultured with astrocytes placed in removable inserts, (c)
cultures were exposed to astrocyte conditioned medium (AM), and
(d) cultures were exposed to NAM. Unless stated AM and NAM
were applied at 20% of the total volume of medium. For some
experiments neuronal conditioned medium was used to treat CGN cultures,
but as reported previously (21) this medium had no effect on the
survival of CGNs (data not shown). Cultures grown under the four
conditions for 4 days were evaluated using immunostaining to detect
neurons and astrocytes. All cultures contained neurons and astrocytes (Fig. 1) but treatment of cultures with
NAM resulted in an apparent reduction in neuronal number. Using Hoechst
staining, it was apparent that only cultures treated with NAM contained
cells with fragmented or shrunken nuclei beyond those of controls
(control = 6 ± 3, co-culture = 5 ± 3, AM = 3 ± 4, NAM = 17 ± 4, average cells per field).

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Fig. 1.
Immunostaining of cerebellar neurons and
astrocytes in CGN cultures. Neurons were detected with
anti-neuronal nuclei (a nuclear stain) and astrocytes with GFAP. In
addition cultures were labeled with the Hoechst reagent. Shown are
untreated cultures (control), CGNs co-cultured with
astrocytes (Co-Culture), AM-treated cultures
(AM), and NAM-treated cultures (NAM).
Scale bar, 50 µM for all
parts.
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The survival of the cultures was also determined using an MTT assay.
Only NAM reduced the survival of CGN cultures after 4 days of treatment
(Fig. 2A). NAM was also
applied to CGN cultures at increasing concentrations and was also
applied to CGNs co-cultured with astrocytes. NAM but not AM killed CGN
cultures in a dose-dependent manner (Fig. 2B).
Co-culture of CGN cultures with astrocytes inhibited NAM toxicity. NAM
toxicity could also be inhibited with MK801 (Fig. 2A). The
glutamate transport inhibitor PDC was applied to CGN cultures alone or
co-cultured with astrocytes for 4 days. PDC had no effect on CGNs not
co-cultured but was toxic to CGNs co-cultured with astrocytes (Fig.
2C). This effect could also be inhibited with MK801 (data
not shown). These results suggest that NAM makes neurons more sensitive
to glutamate toxicity and that co-culture protects CGNs from glutamate
toxicity by active clearance of glutamate. These results are similar to
those from work investigating the toxicity of NAM to mouse CGNs (21).
This implies that the system previously investigated in the mouse and this system in the rat are interchangeable.

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Fig. 2.
Toxicity of NAM to rat cerebellar
cells. A, CGN cultures were grown under various
conditions with (open bars) or without
(gray bars) the addition of 50 µM
MK801. The conditions were either no treatment (control), co-culture
with astrocytes, or treatment with medium containing 20% AM or NAM for
5 days. At the end of that time, inserts containing astrocytes were
discarded and an MTT assay was carried out on the CGNs. The values were
compared with those of untreated cultures as a percentage.
B, NAM ( ) or AM ( ) was applied to CGNs for 4 days at
increasing concentrations. At the end of the 4 days, the survival of
CGNs was determined using an MTT assay. Values were compared with those
of untreated cultures as a percentage. Additionally, NAM was also
applied to cerebellar cells in co-culture with astrocytes at the same
concentrations ( ). C, CGN cultures maintained in
co-culture with astrocytes ( ) or without co-culture ( ) were
treated with PDC at varying concentrations for 4 days. At the end of
that time the survival of the CGN cultures were measured with an
MTT assay. Before assay the inserts in the culture dish containing the
astrocytes were discarded and the MTT assay was performed in the
absence of the astrocytes. Shown are the mean and S.E. for four
experiments with three determinations each.
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Direct deprivation of CGNs from astrocytic protection against glutamate
can be achieved in our co-culture system by growing CGNs with
astrocytes in co-culture for 4 days and then withdrawing the astrocytes
in the co-culture insert. Although this method does not remove all the
astrocytes in the culture, it does diminish them considerably. CGNs
treated in this manner were treated with increasing concentrations of
glutamate or NMDA. Both glutamate and NMDA were more toxic to CGNs
removed from co-culture than to those that had not experienced
co-culture or to those maintained in co-culture (Fig.
3). These results suggest that co-culture with astrocytes causes specific changes in neurons that make them more
sensitive to glutamate and NMDA toxicity.

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Fig. 3.
Withdrawal of astrocytic protection makes
neurons more sensitive to the toxicity of glutamate and NMDA. CGNs
were co-cultured with astrocytes for 4 days; after this time, the
inserts containing the astrocytes were discarded and glutamate
(A) or NMDA (B) added at various concentrations.
In parallel, cultures of CGNs that had not been co-cultured were also
treated with glutamate of NMDA. After 2 days survival was determined
using an MTT assay. Shown are CGNs not co-cultured ( ) and CGNs
withdrawn from co-culture ( ). C, control CGN cultures
were maintained in serum-free conditions. Some cultures were treated
with NAM that had been prepared using serum-free medium. Other cultures
were treated with heat-inactivated serum-free NAM. Additionally,
cultures were either treated with 50 µM NMDA
(gray bars) or 50 µM NMDA and 50 µM MK801 (black bars) or neither
(open bars). At the end of 4 days of treatment,
survival was determined using an MTT assay. D, toxicity of
NAM after partition of solutes according to molecular size. NAM was
passed through a series of filters with molecular mass cut-offs of 3, 10, 30, and 50 kDa. The filtered material was collected, and the
retained material was reconstituted to the same volume to pass through
the next filter. This produced fractions with solutes approximately
<3, 3-10, 10-30, 30-50, and >50 kDa. These fractions were then
tested for toxicity on CGNs as compared with unfiltered NAM. Shown are
the mean and S.E. for four experiments with three determinations
each.
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Astrocytes were exposed to conditioned medium from neurons grown in
serum-free medium. Serum-free AM was also produced (data not shown).
The serum-free AM or NAM were applied to CGNs. Neither medium produced
in this manner was toxic to CGNs. However, when NMDA was applied in
parallel, it was far more toxic to CGNs exposed to serum-free NAM. This
toxicity was fully blocked by MK801, suggesting that the toxicity is
mediated through the NMDA receptor (Fig. 3C). The
implication of this result is that NAM alters NMDA receptor response to
NMDA or glutamate. Serum-free NAM was produced and heated to 75 °C
to denature proteins that might have been secreted by astrocytes. NAM
produced this way had no effect on the toxicity of NMDA (Fig.
3C). NAM was also fractionated according to molecular weight
of its solutes. These fractions were tested for toxicity on CGNs. The
majority of the toxicity was associated with the 10-30-kDa fraction.
These results imply that the factor from astrocytes in NAM making CGNs
more susceptible to the toxicity of NMDA or glutamate is heat-sensitive
and thus likely to be a protein of 10-30 kDa.
NMDA Receptor Subunits--
We investigated whether our four
culture conditions alter the expression of NMDA receptors subunits
(NR1, NR2) using Western blotting. As controls synaptophysin and
tubulin were examined. Co-culture of CGNs with astrocytes did not alter
the levels of any of these proteins as compared with untreated CGNs
(Fig. 4). Treatment with AM enhanced the
expression of synaptophysin dramatically. This suggests that AM greatly
enhances the formation of synaptic structures in cultures of CGNs.
Furthermore, levels of NR1 and NR2 were also elevated by AM treatment.
In comparison tubulin was not significantly elevated (n = 4, Student's t test on densitometric analysis of blots,
p > 0.05). Interestingly, NAM did not show the same
effect as AM. Although NAM produced a slight elevation of
synaptophysin, there was no increase in the level of NR1or NR2. The
implication of this is that some factor additionally released by
astrocytes into NAM and not into AM suppressed the action of AM or a
neuronal factor suppressed astrocytic release of a factor that would
otherwise stimulate expression of synaptophysin and NMDA receptors.

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Fig. 4.
Changes in NMDA receptor subunits.
Extracts from CGNs grown as controls (C), in co-culture
(Co), in the presence of AM or NAM were prepared and run on
a 7% acrylamide SDS-PAGE gel. Following Western blot, immunodetection
of synaptophysin, tubulin, NR1, and NR2 using specific monoclonal
antibodies was carried out.
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As it was difficult to ascertain accurately by Western blotting the
proportion of each subtype of NR1 and NR2 expressed by CGNs grown under
the four conditions, quantitative PCR was used to examine the
expression of receptor subunit subtypes. The analysis is shown in Fig.
5, and the quantitation shown in Table
I. Analysis of NR1 again shows an
increased total level of NR1 subtypes in AM-treated CGNs with a lesser
effect on NAM-treated cultures. Additionally, both co-cultured CGNs and
NAM-treated CGNs showed a higher proportion of NR1b subunit than
controls of AM-treated cultures. Analysis of NR2 showed little change
in total levels of NR2 under any condition. However, NAM-treated
astrocytes showed virtually no NR2b, implying a higher level of NR2a
and NR2c present in NAM-treated CGNs. Co-culture of CGNs with
astrocytes resulted in an increase in the amount of NR2c. The
implication is that NAM might have the effect of elevating the
percentage of NR2a and NR1b subunits while co-culture elevates NR2c and
NR1b.

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Fig. 5.
PCR analysis of changes to they ratio of NMDA
receptor subunits. Quantitative RT-PCR was carried out to
determine the relative levels of NR1 and NR2 subtypes. Different cycles
were used to optimize the level of product formation without reaching
saturation. Glyceraldehyde-3-phosphate dehydrogenase was used as a
control. For NR1 two bands are produced differing in 21 base pairs. The
large band is NR1b, and the smaller band is NR1a. For analysis of NR2
the products of the PCR reaction were digested with one of three
different restriction enzymes. The digestions refer to the different
NR2 subunit digested by the different enzymes: a, NR2a;
b, NR2b, c, NR2c; , no enzyme. Undigested
product is observed for all digestions as one enzyme digests only one
subtype and all three subtypes are present in each reaction.
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Oligonucleotide Knockdown and Neurotoxicity--
In order to
determine if different NMDA subunit subtype composition influences the
susceptibility of CGNs to glutamate toxicity in our model, we tested
the effect of oligonucleotides specific for each of the five subunit
subtypes on NAM toxicity and compared this to the effects of the
oligonucleotides on CGNs exposed to AM (Fig.
6). The oligonucleotides had no effect on
CGN survival in the presence of AM. The anti-NR1b oligonucleotide
decreased NAM toxicity the most. The anti-NR2a oligonucleotide
decreased NAM toxicity whereas anti-NR2c enhanced toxicity, suggesting
that the two subunits have antagonistic roles in terms of sensitivity to NAM toxicity. These results suggest that the changes in some of the
NMDA receptor subunit types (NR1b and NR2a) we observed in NAM-treated
cultures can be directly related to their increased sensitivity to NAM
toxicity. As NAM toxicity is related to either glutamate or NMDA,
changes in the subunits caused by NAM may regulate sensitivity to
glutamate.

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Fig. 6.
Oligonucleotide knockdown of NR1 or NR2
subtypes. CGN cultures were treated with either AM
(open bars) or NAM (black
bars) for 4 days. Additional cultures treated in this way
were also co-treated with one of five antisense oligonucleotides at 100 nM. After 4 days of treatment, the survival of the cultures
was determined using an MTT assay. Shown are the mean and S.E. for four
experiments with three determinations each.
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Death Signal from NMDA Receptors--
We investigated the effect
of NAM in terms of inducing cell death through the NMDA receptor.
Nordihydroguaiaretic acid (NDGA) is known to inhibit toxicity mediated
through NMDA receptors. Treatment with NDGA but not indomethacin
blocked the toxicity of NAM to CGNs (Fig.
7A). These results suggest
that NAM might modify NMDA receptors in a way that initiates synthesis
of arachidonic acid when high glutamate is present. To investigate this
further, we measured the levels of lipoxygenase in CGNs grown under
various conditions. Increased lipoxygenase mRNA was detected using
RT-PCR in cultures of CGNs exposed to NAM or co-cultured. The greatest effect was seen with NAM. NAM produced without serum had a weaker effect (Fig. 7B). These results suggest that regulation of
the arachidonic acid synthesis pathway is involved in the toxicity induced by NAM.

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Fig. 7.
Role of arachidonic acid in NAM
toxicity. A, CGN cultures were treated with NAM at 50%
of total culture volume. The cultures were co-treated either with
iodomethacin ( ) or NDGA ( ) at various concentrations. After 4 days of treatment, the survival of the cultures was determined using an
MTT assay. B, quantitative RT-PCR for 5-lipoxygenase was
carried out on CGN cultures gown under conditions of no-treatment
(control), co-cultured with astrocytes (co-cultured), or treated with
conditioned media. The conditioned medium was either AM or NAM with
serum or serum-free NAM (SF-NAM). The results were
quantitated densitometrically and shown in C. Values were
compared with control as a percentage increase. Shown are the mean and
S.E. for four experiments.
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DISCUSSION |
In this article, we have investigated two model systems in
parallel. The first, co-culture of astrocytes and CGNs, represents a
complete model of the effect of astrocytes on neurons in terms of their
interaction to modulate effects of glutamate. The second involves use
of conditioned medium from astrocytes, which have themselves been
pre-treated with conditioned medium from neurons. The effects of this
conditioned medium (NAM) show similarities to that of co-culture in
that NAM sensitizes CGNs to the toxicity of glutamate. CGNs withdrawn
from co-culture with astrocytes are also more sensitive to glutamate
and NMDA toxicity. With this system we were then able to dissect
changes in the composition of NMDA receptors that underlie this change
in sensitivity to glutamate toxicity.
Of particular interest in the current work was the analysis of changes
in NR1 and NR2 subtype. This was our main interest in using the rat
system for this analysis because of the broader knowledge basis
available. In particular it has been suggested that the subunit NR1b is
associated with increased sensitivity to glutamate toxicity (28, 29).
In our experiments, we observed that both co-culture with astrocytes
and treatment with NAM resulted in an increase in NR1b. Anti-NR1b
oligonucleotide inhibited NAM toxicity, implying that this subunit is
involved in the increased toxicity of NAM. As CGNs are protected by
astrocytic clearance of glutamate when co-cultured, this would explain
why the expression of this subunit in co-cultured CGNs does not result
in increased cell death. Despite its role in sensitivity to glutamate,
the NR1b isoform has been shown to be necessary for normal regeneration in certain systems such as the retina (30).
During normal granule cell development, there is a known progression in
the expression of the NR2 subunits subtypes. Early in development, the
predominant subtype is NR2b but as development progresses there is a
switch to NR2a and then an increase in the levels of NR2c (31, 32).
Change in the subunit composition of NMDA receptors alters
electrophysiological responses of CGNs (33, 34). Co-cultured CGNs
demonstrated a NR2 subtype profile more like that of a more mature CGN.
The CGNs in NAM showed an increase in type NR2a, but this seemed to
be associated with increased sensitivity to NAM whereas NR2c appeared
to be protective. Therefore, increased sensitivity to glutamate in the
NAM-treated CGNs might represent the inadequacies of a transition phase
in development.
These findings regarding NMDA subunits are important with relation to
treatment to prevent excitotoxic damage in vivo. The suppression of certain subtypes of NMDA subunits using antisense oligonucleotides might represent a possible effective way to diminish damage by glutamate or analogues in many diseases. Alternatively, targeting astrocytes to alter release of substances that modulate NMDA
receptor composition might be beneficial. However, it would first be
important to determine the precise nature of the molecules released and
the mechanisms governing them.
These findings also continue our investigation into the interaction of
astrocytes and neurons in terms of regulating and preventing glutamate
toxicity. We have previously suggested (17, 18, 21) that astrocytes
release a factor upon stimulation by neurons (i.e. released
into NAM) that causes neurons to change their sensitivity to glutamate.
We have shown in this work that this change in sensitivity is mediated
by changes induced in NMDA receptor composition. We have also observed
that astrocyte conditioned medium altered the level of synaptophysin.
This effect appears to be inhibited if the astrocytes producing the
conditioned medium were first exposed to medium from neurons (see Fig.
4). This was possibly because a factor released by neurons inhibited
this effect of astrocytes via their conditioned medium. This change in
synaptophysin was also not observed in co-culture. The changes in NMDA
receptors do not follow this pattern; therefore, what we observe is the stimulation of production of an astrocytic factor rather than an
inhibition of one. This factor regulating NMDA receptor composition we
tentatively describe as neuron-dependent astrocyte-derived maturation factor (NDADM).
NDADM is itself not toxic, as serum-free NAM does not induce cell death
but increases CGN sensitivity to NMDA. One of the results of the effect
of NDADM or another factor released into NAM is the alteration in the
activity of lipoxygenase. This enzyme is involved in the metabolism of
arachidonic acid. Arachidonic acid is released in response to NMDA
receptor stimulation (35). Low levels of arachidonic acid are
beneficial to neurons but at high levels are toxic as they generate
oxidized metabolites (36). We found that NDGA, which inhibits
lipoxygenase activity, inhibited NAM toxicity, which supports the idea
that NAM toxicity is mediated via arachidonic acid as others have found
(37).
Our findings provide further evidence that regulation of NMDA receptor
subunit expression modulates death mediated through NMDA receptors and
that controlling this expression is important for understanding the
mechanism of excitotoxicity. The continued emergence of astrocytes as a
key regulator of neuronal survival now indicates that, in addition to
protecting directly by clearance of glutamate, they also modulate
neuronal sensitivity to glutamate. The doorway is now open for the
discovery of a new class of modulating factors released by astrocytes,
the control of which might be the key to preventing excitotoxicity.