Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca2+]i increases and NMDA receptor mRNA levels

Brain Research 849 Ž1999. 97–108 www.elsevier.comrlocaterbres

Research report

Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated wCa2qxi increases and NMDA receptor mRNA levels Chialin Cheng a , Daniel M. Fass b , Ian J. Reynolds a

a, )

Department of Pharmacology, UniÕersity of Pittsburgh School of Medicine, E1354 Biomedical Science Tower, Pittsburgh, PA 15261, USA b Department of Neuroscience, UniÕersity of Pittsburgh, Pittsburgh, PA 15261, USA Accepted 17 August 1999

Abstract We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 mM glutamater10 mM glycine at 5 days in vitro ŽDIV., but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca2q, respectively, to measure changes in wCa2q x i . Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar wCa2q x i changes with exposure to 3 or 100 mM glutamate for 5 min, whereas magfura-2 reported larger wCa2q x i increases with 5-min exposure to 100 mM glutamate than with exposure to 3 mM glutamate, 100 mM kainate or 50 mM Kq from 10 DIV onward. This suggests that the magnitude of the wCa2q x i changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca2q. We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated wCa2q x i responses and NMDA receptor subunit mRNA levels Žespecially NR2B. are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: NMDA receptor; Excitotoxicity; Fura-2; Magfura-2; Calcium; mRNA

1. Introduction Excessive amounts of the excitatory neurotransmitter glutamate can be neurotoxic w26,34x. Since glutamate-induced toxicity, or excitotoxicity, is implicated in a wide variety of neurological disorders Že.g., see Ref. w2,8,9,15,43x., there is great interest in elucidating its underlying mechanisms. Using rodent neuronal cortical cell cultures as an in vitro model of excitotoxicity, it has been demonstrated that only a brief Žfew minutes. exposure to a high concentration of glutamate is required to produce neuronal damage that is manifested approximately 24 h later w10x. This type of excitotoxicity involves the activation of a subtype of glutamate receptors, the Nmethyl-D-aspartate ŽNMDA. receptor.

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It is widely believed that overactivation of NMDA receptors causes a large influx of Ca2q resulting in an increase in the intracellular free Ca2q concentration ŽwCa2q x i ., and this increase is central to excitotoxicity w7x. Studies using 45 Ca2q to measure net Ca2q movement support this idea w14,18x. Toxic stimulation with NMDA or glutamate cause greater accumulation of 45 Ca2q within the cell than non-toxic stimulation with other glutamate receptor agonists or cell depolarization. However, data from several studies using the Ca2q indicator dyes fura-2, indo-1 or fluo-3 to measure wCa2q x i do not indicate a correlation between the excitotoxic potential of glutamate receptor agonists and agonist-stimulated increases in wCa2q x i w27,38,40x. Specifically, it has been shown that brief activation of voltage-gated Ca2q channels, AMPArkainate receptors, or NMDA receptors can elicit apparently similar changes in wCa2q x i , yet only stimulation of the NMDA receptor is toxic to neurons w27,38,40x. There are at least two possible explanations that can reconcile the 45 Ca2q

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 9 5 - 2

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and fluorescent dye data. First, the high affinity Ca2q indicators used to measure wCa2q x i may become saturated during intense glutamate stimulation w6,21,44x, and thus fail to indicate that the wCa2q xi response to NMDA is indeed larger than from the other stimuli. Second, wCa2q x i changes may not be the only determinant of the excitotoxic potential of a stimulus. Changes in the intracellular concentration of other ions such as Mg 2q w5,19x could also play a role in triggering toxicity. We previously found that the fluorescent indicator magfura-2 is a much better indicator of the excitotoxic potential of a stimulus than dyes such as fura-2 w5x. That is, a normally toxic stimulation with glutamate elicits a much bigger change in the magfura-2 ratio signal than non-toxic stimulation. Glutamate-stimulated changes in magfura-2 signal can be inhibited by NMDA receptor antagonists. While fura-2 has been used to measure wCa2q xi exclusively, magfura-2 has been used to measure either wCa2q x i or wMg 2q x i changes w17,25,31,33,41x. This is because magfura-2 has an affinity for Ca2q in the tens of micromolar and an affinity for Mg 2q in the low millimolar range w23,39x. Therefore, under conditions in which micromolar wCa2q x i and perhaps also low millimolar wMg 2q x i changes occur such as during intense glutamate stimulation, magfura-2 is better suited than fura-2 for testing the hypothesis that the size of the wCa2q x irwMg 2q x i response correlates with the toxicity of a given glutamate stimulus. Several laboratories have demonstrated increased vulnerability to excitotoxic stimuli in older cultured neurons w10,16,36,50x. We wanted to examine whether this increased vulnerability is related to increases in the magnitude of glutamate-stimulated wCa2q x i increases in the older cultured neurons. Our hypothesis is that the increase in vulnerability to excitotoxicity over time in culture correlates with the increase in glutamate-stimulated increases in magfura-2, but not fura-2 ratio signal, reflecting the ability of the former dye to report large wCa2q x i increases more accurately. Several NMDA receptor subunits have been cloned including the NR1 subunit with eight possible splice variants, and four NR2 ŽA–D. subunits w22,24,29,30x. The coexpression of NR1 with one or more of the NR2 subunits generates functional receptors with properties resembling native NMDA receptors. It has been shown that transfection of NMDA receptor subunits into non-neuronal cells produces cytotoxicity which can be blocked with NMDA receptor antagonists w1,4,12x. Also, treating cortical cell cultures with antisense oligonucleotides to NR1 can inhibit NMDA-mediated toxicity w48x. The expression of the NMDA receptor subunits increases over time in culture w28,51x. Therefore, increases in the expression of NMDA receptor subunits over time in culture may underlie increases in glutamate-stimulated wCa2q x i changes and increased vulnerability to excitotoxicity. Although there have been studies on increases in glutamate-stimulated wCa2q x i responses, NMDA receptor sub-

unit expression, and vulnerability to excitotoxicity in neurons over time in culture w3,10,16,36,47,50,51x, these parameters have not been studied simultaneously using the same culture preparation. Differences in cell preparation methods between studies have made it difficult to assess whether the time course of these events are correlated. Therefore, in this study, we examined the glutamatestimulated wCa2q x i changes, expression of NMDA receptor subunits, and the development of vulnerability to excitotoxicity in embryonic rat forebrain neurons over time in culture using a single culture system.

2. Materials and methods 2.1. Materials The lactate dehydrogenase ŽLDH. assay kit, Tox-7, was purchased from Sigma ŽSt. Louis, MO., the BCA protein assay kit from Pierce ŽRockford, IL., the fluorescent dyes fura-2 acetoxymethyl ester ŽAM. and magfura-2 AM from Molecular Probes ŽEugene, OR., and the w a-32 PxUTP Žspecific activity, 3000 Cirmmol. from New England Nuclear ŽBoston, MA.. Plasmids used in synthesizing riboprobes for NMDA receptor NR1 and NR2A-C subunit mRNAs were kind gifts from Drs. J. Zhong and P.B. Molinoff ŽUniversity of Pennsylvania, Philadelphia, PA.. All other chemicals and reagents are from commercial sources. 2.2. Cell culture Primary cultures of day 17 embryonic Sprague–Dawley rat forebrains were prepared as previously described w49x. Briefly, the isolated forebrains were treated with 0.1% trypsin in Ca2q-, Mg 2q-free medium Žcontaining minimum essential medium ŽMEM., amino acids and phenol red. for 30 min. Dissociated cells were plated onto 31 mm poly-Dlysine coated glass coverslips in Dulbecco’s modified Eagle’s medium Žsupplemented with 10% fetal bovine serum, 100 mgrml streptomycin and 100 unitsrml penicillin. at a density of 4.5 = 10 5 cellsrcoverslip. Twenty-four hours after plating, the medium was changed to Dulbecco’s modified Eagle’s medium supplemented with 10% horse serum and antibiotics Žmaintenance medium., and the coverslips were inverted to suppress non-neuronal cell proliferation. The resulting cell cultures were neuronally enriched. The cells were maintained in culture in a humidified 95% airr5% CO 2 incubator at 378C until the day of experiment. In the first day or so after the cells were plated, the cells adhered to the coverslip and neuronal extensions were evident by 2 days in vitro ŽDIV. ŽFig. 1.. Over time in culture, the neuronal processes grow and extend such that by 14 DIV, there was a full meshwork of neuronal processes surrounding the neurons that is main-

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Fig. 1. Representative phase-contrast microphotographs of neurons over days in culture Žspecific day marked in the upper left corner of each picture.. Scale bar s 50 mm.

tained until after 21 DIV. By 28 DIV, the meshwork have started to disintegrate. Also, while most of the neurons appear to be phase bright under the microscope, a few of the neurons contained vacuoles within the cell body. Thus we did not use neuronal cultures older than 28 DIV in these experiments. Cultures maintained for more than 20 days were supplemented with 1 ml maintenance medium in each well 15 days after plating. All efforts were made to minimize animal suffering, to reduce the number of animals used, and the animal experimental protocols were in

strict accordance with the National Institutes of Health ‘Guide for the Care and Use of Laboratory Animals’ and were approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. 2.3. LDH release assay On the specified culture day, the maintenance medium on the cell culture was replaced with MEM followed by HEPES buffered saline solution ŽHBSS. containing 137 mM NaCl, 5 mM KCl, 900 mM MgCl 2 , 1.4 mM CaCl 2 , 3

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mM NaHCO 3 , 600 mM Na 2 HPO4 , 600 mM KH 2 PO4 , 20 mM HEPES and 5.6 mM glucose ŽpH adjusted to 7.4 with NaOH.. The coverslips were inverted again so that the neurons were facing upward before the cells were treated for 5 min at room temperature with the appropriate drugs in HBSS. After the exposure, the HBSS was removed and replaced with MEM, and the cells were placed back into the incubator for 22–24 h. The next day, LDH in the culture medium was measured using a LDH assay kit ŽTox-7.. Briefly, the medium samples were mixed with the Tox-7 reaction mixture Žcontaining NADq, diaphorase, and tetrazolium dye INT., and the absorbance of the product was read at 490 nm after a 10-min incubation at room temperature. The absorbance was normalized to the protein content of each well Žprotein estimation was performed using the BCA protein assay.. 2.4. Microfluorimetric recordings Measurements of wCa2q x i in cultured rat forebrain neurons were made using ratiometric fluorescence recording

techniques as previously described w5,20,42x. Briefly, on the day of the recordings, the maintenance medium in the culture wells was replaced with HBSS and the coverslips were inverted so that the neurons were facing upward. The cells were dye-loaded by incubation with either 5 mM fura-2 AM or magfura-2 AM in HBSS containing 5 mgrml bovine serum albumin for approximately 45 or 15 min, respectively. After loading, the cells were rinsed and placed in HBSS for 10 min before the coverslip was mounted into a chamber for recording. For the duration of the recordings, the cells were continuously superfused at a rate of 20 mlrmin. Neurons were illuminated using light from a 150 W Hg–Xe lamp that was alternately filtered through 340 and 380 nm narrow band-pass filters and directed to a Nikon Diaphot microscope using a liquid light guide and quartz optics. Fluorescence output from individual neurons passed through a 510-nm narrow band-pass filter, and light collection was limited to the cell body by means of a rectangular diaphragm placed in the light path. Background fluorescence was recorded at the end of the experiment by moving the coverslip to a cell-free area, and this value was then subtracted from the neuronal signal.

Fig. 2. Representative phase-contrast microphotographs of neurons 24 h after various stimulations. Rat forebrain neurons at 5 Ži., 14 Žii. or 21 Žiii. days in vitro ŽDIV. were exposed for 5 min to HBSS ŽA., 100 mM glutamater10 mM glycine ŽB., or 100 mM H 2 O 2 ŽC.. Scale bar s 100 mm.

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Since the results of this study and others w21,44x indicate that the fura-2 signal can be saturated with Ca2q during strong glutamate stimulations, conversion of the fura-2 data to Ca2q concentration under these conditions could lead to underestimation of the actual wCa2q x i . Also, the contribution of glutamate-stimulated wMg 2q x i changes to the magfura-2 signal cannot be determined at this time to allow for correction of Mg 2q binding in the calibration of the dye w44x. Therefore, we have elected to report the data as ratio of the fura-2 or magfura-2 fluorescence signals at 340 and 380 nm. 2.5. RNA collection Cultures were prepared for RNA isolation by removal of maintenance media, rinsing with HBSS and coverslip inversion Žresulting in neurons facing up.. RNA was isolated by guanidinium thiocyanate–phenol–chloroform extraction w11x. Each RNA sample consists of contents from two cell culture wells with 45 mg of yeast RNA added as a carrier. Briefly, HBSS was removed from the first well and the cells were mechanically disrupted in 0.5 ml homogenization buffer Ž4 M guanidinium thiocyanate, 25 mM sodium citrate at pH 7.0, 0.5% Sarcosyl, and 0.1 M 2-mercaptoethanol.. HBSS was removed from the second well and cells were mechanically disrupted in the homogenization buffer transferred over from the first well. Then the cellrbuffer mixture was collected into a microfuge tube and subjected to phenolrchloroform extraction. RNA

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was precipitated with isopropanol and sodium acetate at y208C. 2.6. RNase protection assay mRNA levels were analyzed by RNase protection assay as described by Takimoto et al. w45x. Samples were subjected to overnight solution hybridization at 508C with 5 = 10 5 c.p.m. of 32 P-labelled RNA probes to the NMDA receptor subunits NR1 and NR2A-C and b-actin. The antisense RNA probes Ždetailed description of the NMDA receptor subunit probes is presented in Ref. w51x. were made by in vitro transcription of the following templates: NR1 — plasmid pGEM-NR1, linearized with EcoRI, transcribed with SP6 polymerase; NR2A — plasmid pSP72-NR2A, linearized with BamHI, transcribed with T7 polymerase; NR2B — plasmid pGEM-NR2B, linearized with EcoRI, transcribed with SP6 polymerase; NR2C — plasmid pSP72-NR2C, linearized with HindIII, transcribed with T7 polymerase; b-actin — plasmid pTRI-b-actin125-rat, transcribed with T7 polymerase. The length of the RNA probes were sufficiently different to allow simultaneous assay of NR1 with NR2C, and NR2A with NR2B in the same RNA sample. For presentation, the air-dried gels were exposed to X-ray film with an intensifying screen for approximately 15 h at y808C. For quantitation, the gels were exposed to phosphor screens for 1 h, and the density of bands corresponding to target mRNAs was measured by analysis in a phosphorimager ŽMolecular Dynamics, Sunnyvale, CA..

Fig. 3. Sensitivity of neurons to toxicity over time in culture. Forebrain neurons at the indicated days in culture were treated for 5 min with 100 mM glutamater10 mM glycine Žsolid bars. or 100 mM H 2 O 2 Žstippled bars., and then viability was assessed 22–24 h later by measuring LDH release. Data were normalized to the control treatment of HBSS Ždashed horizontal line. for each respective day. Each bar represents the mean" S.E.M. of at least seven experiments performed in triplicate. Statistical analyses were performed using the one-way ANOVA with Bonferroni’s post hoc test. Significant differences vs. HBSS controls are indicated by UU Ž p - 0.01..

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2.7. Data analysis

3. Results

Statistical significance was tested using one- or two-way ANOVA and post hoc analyses were performed using Bonferroni’s or Fisher’s LSD multiple comparison tests as calculated by Prism 2.01 ŽGraphPad Software, San Diego, CA. or NCSS 2000 ŽNCSS Statistical Software, Kaysville, UT., respectively.

3.1. Susceptibility to glutamate-induced toxicity The ability of various stimuli to produce toxicity in neurons maintained 5, 14 and 21 DIV was assessed by measuring LDH release in the culture media and confirmed by morphological inspection ŽFig. 2.. The amount

Fig. 4. Profile of glutamate-stimulated fura-2 and magfura-2 ratio signals at various days in culture. Average traces of ratiometric measurements from neurons at 2, 14, or 28 DIV loaded with either fura-2 ŽA. or magfura-2 ŽB. and stimulated for 5 min with 100 mM glutamater10 mM glycine and followed for 25 min post-stimulation. Each trace is the mean of the fura-2 or magfura-2 ratio signal of 10–22 neurons from at least four culture batches. For simplicity, only a few representative error bars Žindicating S.E.M.. of each trace are included.

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of LDH release produced by the various stimuli were normalized to the amounts produced by the control stimulation on each DIV since LDH release with HBSS exposure alone increases slightly over time in culture Ž0.75 " 0.07 at 5 DIV, 1.19 " 0.10 at 14 DIV, 1.53 " 0.14 at 21 DIV in absorbance= 100rmg protein.. The neuronal cultures were susceptible to H 2 O 2-induced cell death at all of the cultures days examined ŽFigs. 2 and 3.. In contrast, glutamate-induced toxicity increases over time in culture. At 5 DIV, there is essentially no glutamate-induced LDH release, whereas at 14 DIV, the LDH release was approximately double that of control, and by 21 DIV, the LDH release was greater than double that of control. The LDH release caused by 5-min exposure to glutamate and glycine in these cultures was 80%–90% of the release triggered by overnight exposure to the agonists Ždata not shown.. Thus, cultured neurons can be killed by H 2 O 2 at any age, whereas the vulnerability to excitotoxicity develops over time in culture. 3.2. Glutamate-stimulated [Ca 2 q ]i changes We also examined glutamate-stimulated changes in the fura-2 and magfura-2 ratio in neurons over time in culture. Exposure to 100 mM glutamate produced a fura-2 ratio change that increased from 2 to 14 or 28 DIV ŽFig. 4A.. Glutamate stimulates a rise in the fura-2 signal that leads to a plateau. Although 100 mM glutamate stimulated similar fura-2 ratio changes between 14 and 28 DIV, there was more variability and less recovery of the signal post-stimu-

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lation at 28 DIV. The post-stimulation return to baseline in the fura-2 ratio traces was erratic, especially at later culture dates. In contrast, 100 mM glutamate did not stimulate an appreciable change in the magfura-2 ratio at 2 DIV ŽFig. 4B.. At 14 and 28 DIV, glutamate stimulated an increasing magfura-2 ratio change over the duration of the 5-min stimulation without a plateau. Upon the removal of glutamate, the magfura-2 ratio change reversed such that by 25 min post-stimulation, the magfura-2 signal at all the culture days examined had almost returned to baseline. The plateau and erratic recovery kinetics in the fura-2 ratio signal suggest that this high affinity Ca2q indicator may have been saturated by large wCa2q x i increases stimulated by 100 mM glutamate. In contrast, the lack of a plateau and smooth recovery kinetics in the magfura-2 ratio signal suggest that this low affinity Ca2q dye accurately measured these large wCa2q x i increases, although it is less sensitive to small wCa2q x i changes near resting values Že.g., at 2 DIV.. Alternatively, magfura-2 may have been detecting a combination of wCa2q x i and wMg 2q x i changes, whereas fura-2 may have been only detecting wCa2q x i changes. Next, we compared the fura-2 and magfura-2 ratio changes produced by non-toxic and toxic stimulations in neurons over time in culture. Exposure for 5 min with 100 mM glutamate is normally toxic ŽFigs. 2 and 3., whereas 3 mM glutamate, 50 mM Kq Žcell depolarization. or 100 mM kainate are not w38,44x. Both 3 and 100 mM glutamate stimulated maximal increases in the fura-2 and magfura-2 ratio by 10 DIV ŽFigs. 5 and 6.. The two glutamate stimulations produced similar changes in the fura-2 ratio at

Fig. 5. Magnitude of glutamate-stimulated fura-2 ratio changes in neurons over time in culture. These results are the summary of fura-2 ratio increases in neurons at various days in culture in response to 5-min stimulation with 3 mM glutamate Žstippled bars. or 100 mM glutamate Žsolid bars., both with 10 mM glycine. The D fura-2 ratio was obtained by subtracting the mean of the basal fura-2 ratio Žaverage of 10 data points. from the peak fura-2 ratio during the glutamate stimulation. Each bar represents the mean " S.E.M. of 7–15 neurons from at least four culture batches. Statistical analyses were performed using the two-way ANOVA with Fisher’s LSD multiple comparison test. Over time in culture, there was a progressively larger fura-2 ratio increase between 2 and 10 DIV Ž p - 0.001..

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Fig. 6. Differences in agonist-stimulated magfura-2 changes in neurons over time in culture. Summary of magfura-2 ratio increases in neurons at various days in culture in response to 5 min stimulation with 50 mM Kq Žstriped bars., 100 mM kainate Žopen bars., 3 mM glutamate Žstippled bars. or 100 mM glutamate Žsolid bars., both with 10 mM glycine. Each bar represents the mean " S.E.M. of 3–26 neurons from at least three culture batches. The D magfura-2 ratio was obtained by subtracting the mean of the basal magfura-2 ratio Žaverage of 10 data points. from the peak magfura-2 ratio during the stimulation. Statistical analyses were performed using the two-way ANOVA with Fisher’s LSD multiple comparison test. Significant difference Ž p - 0.001. between glutamate and high Kq ŽU . or kainate Ž§. are indicated. Over time in culture, the magfura-2 ratio changes due to 100 mM glutamate were similar between 2 and 5 DIV, but were significantly larger at all later culture days examined Ž p - 0.001.. In contrast, magfura-2 ratio changes due to stimulation with 3 mM glutamate were only significantly larger at 28 DIV Ž p - 0.001.. Cell depolarization or kainate stimulated similar magfura-2 ratio changes at all the days examined.

all the days examined. In contrast, from 10 DIV onward, the magfura-2 ratio increase during exposure to 3 mM glutamate appear to be smaller than during exposure to 100 mM glutamate. Moreover, 100 mM glutamate stimulated significantly larger magfura-2 ratio changes than kainate Ž100 mM. or cell depolarization Ž50 mM Kq. by 10 DIV ŽFig. 6B.. The shape of the magfura-2 transient due to cell depolarization or kainate were also different from the that due to glutamate. While the glutamatestimulated magfura-2 ratio steadily increases during the entire stimulation, there was an initial peak in the cell depolarization- or kainate-stimulated magfura-2 ratio change, and then the signals declined towards baseline even before the removal of the stimulus Ždata not shown.. These results suggest that magfura-2 was better at predicting the excitotoxic potential of a stimulus than fura-2. Moreover, the differences in the magfura-2 signal produced by toxic and non-toxic stimuli developed over the time frame in which these cultured cells became vulnerable to glutamate-induced toxicity. 3.3. Expression of NMDA receptor subunit mRNAs One possible explanation for the increases in the glutamate-stimulated magfura-2 ratio change could be increased expression of NMDA receptor channels over time in cul-

ture w51x. To test this idea in our culture system, we measured the expression of NMDA receptor subunit mRNAs over time in culture using RNA probes to NR1 and NR2A-C and the RNase protection assay. The expression of NR1 and NR2A mRNA increases over time, although there was a slight lag in the NR2A mRNA increase before 10 DIV ŽFig. 7.. NR2B mRNA increases dramatically during the first 10 days, but then plateaus in subsequent days. NR2C mRNA was not detected at any of the days examined. b-actin mRNA was also measured to confirm that the samples were fully loaded into each lane. The expression of the b-actin mRNA increased over time consistent with the increase in surface area of the neurons due to neuronal process extensions and also an increase in glial growth. Therefore, the b-actin mRNA level was not used to normalize the NMDA subunit mRNA levels. However, the presence of expected levels of b-actin mRNA in each lane suggests that loading of the samples was consistent. By 28 DIV, the cell culture exhibited a number of degenerating patches of neurons and debris which could account for the lack of a further increase in the NR2A mRNA and the decreased mRNA levels of all other mRNA examined including b-actin. The increases in the NMDA receptor subunit mRNAs are consistent with the increases in the glutamate-stimulated magfura-2 ratio changes over time in culture. Intriguingly, the emergence of vulnerabil-

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Fig. 7. NMDA receptor subunit mRNA levels over time in culture. ŽA. Representative autoradiograms from a RNase protection assay measuring mRNA levels of the NMDA receptor subunits NR1, NR2A and NR2B, and b-actin. Lanes contain 50 mg yeast RNA ŽYeast. as a control for non-specific signals or total RNA from neuronal cultures isolated on the indicated days. ŽB. Summary of the NMDA receptor subunit mRNA levels over time in culture. Each point represents the mean " S.E.M. from three culture batches.

ity to excitotoxicity and the large wCa2q xi increases coincides with the appearance of the NR2B mRNA.

4. Discussion In this study we examined the relationship between the emergence of vulnerability to excitotoxicity, glutamate-

stimulated increases in the fura-2 and magfura-2 ratio, and the expression of NMDA receptor subunit mRNAs in cultured rat embryonic forebrain neurons over time in vitro. It was important to study these parameters concurrently in the same culture system to rule out differences in the cell culture methods as an explanation for variability in the time course of changes of these three parameters reported in the literature w3,10,16,36,47,50,51x.

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We have found that while our cultured forebrain neurons are susceptible to a non-receptor mediated toxic stimulation at all the culture days examined, their sensitivity to glutamate-induced toxicity develops over time. Around the same time that the cells become susceptible to excitotoxicity, there were greater increases in the glutamate-stimulated wCa2q xi responses. The magnitude of the wCa2q x i increases correlates more with excitotoxicity when magfura-2, not fura-2, was used. However, in addition to Ca2q, binding of Mg 2q to magfura-2 produces similar changes in its spectral properties w39x. Since the affinity of magfura-2 for Mg 2q Žlow millimolar range. is within the physiological range of wMg 2q x i whereas its affinity for Ca2q Žtens of micromolar range. is several orders of magnitude above the resting wCa2q x i of approximately 100 nM w23,39x, magfura-2 is likely measuring only wMg 2q x i changes under physiological conditions. On the other hand, if the wCa2q x i changes approach the affinity of magfura-2 for Ca2q, the change in the magfura-2 signal will be due to increases in wCa2q x i . Could the glutamate stimulations used in this study cause micromolar increases in wCa2q x i? Previously, our laboratory and others believed that glutamate stimulated wCa2q x i increases in the submicromolar range w13,37,46x. Thus we attributed the glutamate-stimulated magfura-2 ratio change to increases in wMg 2q x i w5x. Recently, however, there have been indications that the wCa2q x i levels reached during glutamate stimulation may be higher than previously appreciated w6,21,44x. Therefore, the most parsimonious explanation is that magfura-2 is most likely measuring large increases in wCa2q x i during glutamate stimulation. This does not necessarily exclude the possibility that there is also some concurrent increase in wMg 2q x i that contributes to the magfura-2 signal since it has also been shown that glutamate can both increase wMg 2q x i and induce neurotoxicity independent of wCa2q x i changes w19x. Resolution of this issue awaits the development of indicators specific for Mg 2q. However, despite the ambiguity over whether magfura-2 is measuring Ca2q or a combination of Ca2q and Mg 2q, it is clear that it is a better indicator of the excitotoxic potential of a stimulus than fura-2. The ability of glutamate to stimulate a progressively larger increase in wCa2q x i over time in culture could be due to two reasons: an increase in Ca2q influx or a change in intracellular Ca2q buffering. Since the expression of NR1, NR2A, and NR2B increase over time in our culture system, the larger wCa2q x i increase in older cells could be due, in part, to an increase in the number of channels and increased Ca2q influx. This is supported by the finding that glutamate-induced currents increase over time in culture in cortical and hippocampal neurons w32,35x. The increase in the expression of NMDA receptors may also underlie the increase in vulnerability to excitotoxicity. This idea is supported by the fact that treating cortical cell cultures with antisense oligonucleotides directed against NR1 inhibits NMDA receptor-mediated 45 Ca2q influx and

decreased NMDA-mediated toxicity w48x. Interestingly, the time course of the increase in NR2B mRNA most closely follows the increases in the magfura-2 signal and neuronal vulnerability. Mizuta et al. w28x also concluded that glutamate neurotoxicity were mediated mainly by NR1rNR2B receptors in murine cortical cultures. It is tempting to conclude that NR2B expression is the critical determinant of glutamate-triggered injury. However, these results do not preclude the possibility that other factors may also change over time in culture which could also increase vulnerability to excitotoxicity. For example, this study has not established that expression of the NR2B protein is altered over the same time course, or that the protein is actually incorporated into functional receptors. It is also possible that NR2B expression plays a permissive role in the presence of a critical level of expression of one of the other subunits, such as NR2A. Nonetheless, increases in the expression and function of NMDA receptors may be part of the mechanism in which cultured forebrain neurons become vulnerable to excitotoxicity.

Acknowledgements We thank K.D. Rothermund and G.J. Kress for their assistance in preparing the neuronal cell cultures, Drs. J. Zhong and P.B. Molinoff for their generous gift of plasmids containing portions of the cDNA for NR1 and NR2A-C, Dr. K. Takimoto for sharing his expertise on the RNase protection assay, Drs. E. Aizenman and J.D. Sinor for their assistance on the RNase protection assay, and Dr. E.K. Jackson for his assistance on statistics. This work was supported by NIH grants NS34138 ŽI.J.R.., T32GM 08424 ŽC.C.. and HL55312 Žawarded to Edwin S. Levitan which supported D.M.F... I.J.R. is an Established Investigator of the American Heart Association.

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