NMDA and Non-NMDA Receptor-Mediated Excitotoxicity Are Potentiated in Cultured Striatal Neurons by Prior Chronic Depolarization

Experimental Neurology 159, 283–296 (1999) Article ID exnr.1999.7135, available online at http://www.idealibrary.com on

NMDA and Non-NMDA Receptor-Mediated Excitotoxicity Are Potentiated in Cultured Striatal Neurons by Prior Chronic Depolarization Quan Chen,* D. James Surmeier,† and Anton Reiner* *Department of Anatomy and Neurobiology, University of Tennessee–Memphis, Memphis, Tennessee 38163; and †Department of Physiology/NUIN, Searle 5-474, Northwestern University Medical School, 320 E. Superior Street, Chicago, Illinois 60611 Received December 31, 1998

tor subtypes in excitotoxicity in striatum. The excitatory input from cortex and/or thalamus to striatum appears to promote the maturation of glutamate receptors on striatal neurons, but the mechanisms by which it does so have been uncertain. To explore the possibility that the excitatory input to striatum might influence glutamate receptor maturation on striatal neurons, at least in part, by its depolarizing effect on striatal neurons, we examined the influence of chronic KCl depolarization on the development of glutamate receptor-mediated excitotoxic vulnerability and glutamate receptors in cultured striatal neurons. Dissociated striatal neurons from E17 rat embryos were cultured for 2 weeks in Barrett’s medium containing either low (3 mM) or high (25 mM) KCl. The vulnerability of these neurons to NMDA receptor agonists (NMDA and quinolinic acid), non-NMDA receptor agonists (AMPA and KA), and a metabotropic glutamate receptor agonist (trans-ACPD) was examined by monitoring cell loss 24 h after a 1-h agonist exposure. We found that high-KCl rearing potentiated the cell loss observed with 500 ␮M NMDA or 250 ␮M KA and yielded cell loss with 250 ␮M AMPA that was not evident under low KCl rearing. In contrast, neither QA up to 5 mM nor trans-ACPD had a significant toxic effect in either KCl group. ELISA revealed that chronic high KCl doubled the abundance of NMDA NR2A/B, AMPA GluR2/3, and KA GluR5-7 receptor subunits on cultured striatal neurons and more than doubled AMPA GluR1 and GluR4 subunits, but had no effect on NMDA NR1 subunit levels. These receptor changes may contribute to the potentiation of NMDA and non-NMDA receptor-mediated excitotoxicity shown by these neurons following chronic high-KCl rearing. Our studies suggest that membrane depolarization produced by corticostriatal and/or thalamostriatal innervation may be required for maturation of glutamate receptors on striatal neurons, and such maturation may be important for expression of NMDA and non-NMDA receptormediated excitotoxicity by striatal neurons. Striatal cultures raised under chronically depolarized conditions may, thus, provide a more appropriate culture model to study the role of NMDA or non-NMDA recep-

r 1999 Academic

Press

Key Words: basal ganglia; striatum; excitotoxicity; chronic depolarization; non-NMDA receptors; NMDA receptors; neurodegenerative disease.

INTRODUCTION

The striatal part of the basal ganglia receives prominent glutamatergic inputs from the cortex and thalamus (46, 62), and it is the major site of neurodegeneration in Huntington’s disease and a prominent site of neurodegeneration in global ischemia (5, 21, 33, 96). As a consequence, a number of studies have focused on the possibility that glutamate receptor-mediated excitotoxicity might underlie striatal cell loss under these conditions. Consistent with this possibility, intrastriatal injection of glutamate, N-methyl-D-aspartate (NMDA)-receptor selective agonists, or non-NMDA receptor selective agonists have been shown to produce striatal neuron death resembling that in HD or ischemia (5, 6, 16, 33, 36, 37, 97). Because of the potential for an indirect action of the excitotoxins in such in vivo studies, a number of authors have turned to in vitro preparations to elucidate the precise postsynaptic receptor mechanisms by which striatal neurons are affected by glutamatergic excitotoxins. Several studies on cultured striatal neurons have reported that NMDA receptor agonists kill striatal neurons (40, 63). This result is similar to that obtained for cultured neurons from a variety of nonstriatal brain regions, including neocortex (22, 23, 39, 49, 61, 78). Nonetheless, other investigators have found that NMDA agonists have minimal toxic effect on pure cultured striatal neurons (41, 123). The differences in observed NMDA vulnerability between studies of cultured cortical neurons (which found vulnerability) and those studies on cultured striatal neurons that did not find NMDA vulnerability may stem from differences between cortex and striatum in glutamate receptor and glutamate receptor subunit localization (3, 13, 48, 55,

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56, 58, 70, 71, 79, 80–82, 87, 116). The basis of the discrepancy in vulnerability to NMDA receptor agonists in studies of cultured striatal neurons, however, is unclear. In a prior study, we examined striatal neuron survival following exposure to specific agonists and antagonists for each of the four known types of glutamate receptors (NMDA, ␣-amino-3-hydro-5-methyl-4-isoxazole proprionic acid (AMPA), kainate, and metabotropic). We found that the destruction of striatal neurons by glutamate was mediated by non-NMDA receptors, but not by NMDA and metabotropic glutamate receptors. These results raised two important, somewhat opposing possibilities. First, they raised the possibility that death of striatal neurons in degenerative diseases of the basal ganglia may occur via excitotoxicity mediated primarily by non-NMDA receptors. This interpretation, however, assumes that the cultured striatal neurons in our prior in vitro study possessed the characteristics of striatal neurons in vivo. It is possible that they did not. For example, a second and alternative possibility is that the glutamate receptors expressed by pure striatal neurons in culture may not reflect those expressed in vivo, presumably because of the absence of the normal trophic influences and inputs that striatal neurons experience during in vivo development. As a consequence, inferences from such in vitro findings about the in vivo vulnerability of striatal neurons might be problematic. Resolving this concern was necessary before the pertinence of our prior culture study to the relative roles of different glutamate receptors in degenerative diseases of the basal ganglia could be accurately gauged. The fact that afferent innervation is known to influence the developmental profile of target neurons (30, 49) is consistent with the notion that pure cultured striatal neurons, which had been used in most previous in vitro studies of striatal excitotoxicity, may not mature in exactly the same way as striatal neurons do under in vivo conditions. Such a possibility is especially great considering the previous findings that corticostriatal innervation is important for the expression of glutamate receptor-mediated excitotoxicity in the striatum (11, 41, 75, 123). The corticostriatal (and/or thalamostriatal) innervation might regulate the expression of glutamate receptors and receptor-mediated excitotoxicity either by its release of tropic factors or by its depolarizing effect on striatal neurons or by both. Studies of cultured cerebellar granule cells have shown that depolarization can indeed be critical for the expression of NMDA toxicity (29, 115). Such depolarizationdependent NMDA toxicity appears to be due to an up-regulation of specific NMDA receptor subunits (9). The effect of membrane depolarization on the glutamate receptor expression and receptor-mediated excitotoxicity of striatal neurons in vitro has not been studied

previously. We therefore examined the effects of chronic depolarization with high concentration KCl on glutamate receptor-mediated excitotoxicity in striatal cultures. Since non-NMDA receptors as well as NMDA receptors can directly gate Ca2⫹ entry, and metabotropic glutamate receptors can mobilize Ca2⫹ from intracellular stores and potentiate the ability of ionotropic glutamate receptors to allow Ca2⫹ entry (44, 73, 79, 98), all four major types of glutamate receptors (NMDA, AMPA, kainate, and metabotropic) could, in principle, have a Ca2⫹-dependent role in excitotoxicity. We thus examined the effects of high KCl rearing on the toxicity of agonists for each of these four types of glutamate receptors. We also examined the effects of chronic KCl depolarization on the levels of the specific receptor subunits in our cultures. Such studies provided information about the importance of specific subunits for the expression of glutamate receptormediated excitotoxicity by striatal neurons. METHODS

Pure Striatal Tissue Cultures Cultures of dissociated striatal neurons were prepared from E17 embryos obtained from timed-pregnancy Sprague–Dawley rats. The culture methods used have been previously described (18). Briefly, the dissected dorsal striata of each embryo were minced in cold, calcium- and magnesium-free Hanks’ balanced salt solution (pH 7.4) and gently triturated with a series of fire polished Pasteur pipettes. Dissociated striatal neurons then were plated on sterile 35-mmdiameter culture dishes at the density of 1.5 million cells per dish. The culture dishes had been stamped with a 26 ⫻ 26 grid (square area 9 ⫻ 104 µm2 ) and pretreated with polyornithine (40 µg/ml) for 60 min. Dissociated striatal neurons were initially incubated in glutamate-free Barrett’s medium supplemented with 5% fetal bovine serum, insulin (5 µg/ml), 10 mM Hepes, streptomycin (50 µg/ml), and penicillin (50 U/ml), containing 145 mM Na⫹, 3 mM K⫹, 2 mM Ca2⫹, and 1.5 mM Mg2⫹. On the second day after cell plating, striatal cultures were divided into two sets. One set of striatal cultures continued to be maintained in the low (3 mM) KCl containing supplemented Barrett’s medium in which they had been maintained until that point, while the other set was switched to a modified supplemented Barrett’s medium containing high (25 mM) KCl. Culture media were changed at 3- to 4-day intervals thereafter, with Barrett’s medium supplemented with 5% mixed horse and fetal bovine serum, insulin (5 µg/ml), and 10 mM Hepes. On day 6, 30 µl cystosine arabinoside (5 µM) was added to control glial cell proliferation. Cultures were incubated in humid, 5% CO2 atmosphere at 37°C for 14 days in vitro before being used for the study of excitotoxic vulnerability.

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The low-KCl cultures were maintained in low KCl containing supplemented Barrett’s medium, and the high-KCl cultures were maintained in high KCl containing supplemented Barrett’s medium until the assaying of excitotoxic vulnerability. Striatal neurons maintained for 14 days in vitro were used, since cultured striatal neurons by this age have extensive processes and well-developed synaptic contacts from other striatal neurons, possess a profile of striatal neuron types resembling that in vivo, and express functional glutamate receptors (41, 42, 84, 110, 121, 122). Glutamate Receptor Agonist Exposure The effect of various glutamate receptor agonists was assessed by 1-h exposure of: (i) NMDA; (ii) quinolinic acid (QA); (iii) kainic acid (KA); (iv) AMPA; or (v) trans-1-aminocyclopentyl-1,3-dicarboxylic acid (transACPD). Each of these was dissolved in supplemented Barrett’s medium, and the pH and osmolarity of each agonist solution were adjusted to 7.3 and 300 mOsm, respectively, before being applied to the cultures. A set of sister cultures were used in the study of each agonist or set of agonists, and control dishes from each set were exposed to an equal volume of supplemented Barrett’s medium without glutamate agonist. After a 1 h incubation in agonist or control medium, the exposure was terminated by washing each culture dish three times with culture medium. To minimize mechanical cell damage stemming from the washing procedures, we first removed 1 ml from the 2-ml solution in each culture dish, and then added 3 ml supplemented Barrett’s medium, after which an additional 3-ml solution was removed. This ‘‘3 ml in and 3 ml out’’ procedure was repeated at least three times. Finally, 1 ml supplemented Barrett’s medium was added to each dish to bring the total volume of the bathing medium in each dish to 2 ml. After washing, the cultures were returned to the incubator for an additional 23 h. Control dishes received an equal number of solution changed as the dishes exposed to glutamate agonist. The specificity of agonist effects was examined using selective glutamate receptor antagonists added to the agonist solutions. Glutamate agonist solutions containing (⫾)2-amino-5-phosphonopentanoic acid (APV), 1-(4-aminophenyl)-4-menthyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYK152466), or 2-amino-3phosphonopropanoate (AP3) were prepared as described above. Our standard culture medium contained 1.5 mM [Mg2⫹]. Therefore, when we studied NMDA receptormediated toxicity, the striatal cultures were depolarized with 40 mM KCl to relieve any possible voltagedependent Mg2⫹ block of NMDA receptors. Assessment of Neuronal Loss Neuronal cell loss was quantitatively assessed using phase contrast microscopy at 200⫻ magnification. Stria-

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tal neurons were counted in the same defined fields before agonist or control exposure and 24 h after exposure onset. In each culture dish, the neurons in at least eight randomly selected squares were counted, typically totaling 80–100 neurons in each dish. Only intact neurons were counted. Counts made at 24 h after exposure onset were expressed as a percentage of neurons present prior to exposure. The effect of glutamate agonists was evident as cell loss (following death, disintegration, and detachment of affected neurons). Trypan blue exclusion assessment confirmed our criteria for identifying remaining neurons as being alive, as in our prior study (18). Statistical analysis of the results was carried out using a two-way repeated measures ANOVA with a priori planned comparisons. Enzyme-Linked Immunosorbent Assay (ELISA) For assessment of glutamate receptor abundance on cultured neurons, ELISA was used. Striatal cultures were prepared and maintained as described above, reared in either low or high KCl. At 14DIV, striatal cultures were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) (PB) for 30 min. Following this fixation, the cultured neurons were treated with 5% normal horse serum for 1 h to reduce nonspecific binding of antibody and then incubated with primary antibody specific for individual ionotropic glutamate receptor subunits or combinations of individual subunits. The antisera used, their target antigens, the antisera concentrations used, and the sources of the antisera were: (i) a mouse monoclonal antibody against the NR1 subunit of the NMDA receptor, 1:700 (PharMingen, San Diego, CA); (ii) a rabbit polyclonal antibody recognizing an epitope common to both the NR2A and NR2B NMDA receptor subunits, 1:200 (Chemicon, Temecula, CA); (iii) a rabbit polyclonal antibody recognizing the GluR1 AMPA receptor subunit, 1:200 (Chemicon); (iv) a rabbit polyclonal antibody recognizing an epitope common to the GluR2 and GluR3 AMPA receptor subunits, 1:200 (Chemicon) (v) a rabbit polyclonal antibody recognizing the GluR4 AMPA receptor subunit, 1:350 (Chemicon); and (vi) a mouse monoclonal antibody recognizing an epitope common to the GluR5, GluR6, and GluR7 KA receptor subunits, 1:1000 (PharMingen). These antisera have been shown to be specific for their target antigens and to be effective in detecting their target antigens in rat striatum in vivo (17, 19, 20). Separate cultures were incubated with different antisera. Antisera were diluted with Triton X-100/0.2% sodium azide/PB. After a 2-h incubation in primary antibody, the striatal cultures were washed three times with PB and then incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (for the NR2A/2B, GluR1, GluR2/3, and GluR4 primary antisera), AP-conjugated goat anti-mouse IgG (for the NR1 antibody), or AP-conjugated goat anti-mouse

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IgG⫹IgM (for the GluR5/6/7 antibody). All secondary antisera were diluted 1:5000 with Triton X-100/0.2% sodium azide/PB and cultures were incubated for 1 h at room temperature. After incubation in the AP-linked secondary antiserum, striatal cultures were washed and incubated with p-nitrophenylophosphate (1.0 mg/ml in 10% diethanolamine buffer, pH 9.8, containing 0.01% MgCl and 0.2% sodium azide). The reaction was stopped 20 min later by adding an equal amount of 5% EDTA solution. The final reaction solution was read at a wavelength of 410 nM with a MR 700 spectrophotometer. Pure glial cultures were used as a control for the measurement of nonspecific background labeling with each of the antibodies used. Previous studies suggest that NR2C and NR2D subunits are not significantly expressed in the striatum, and for this reason we did not examine their levels (107, 119). RESULTS

The Effects of Activation of NMDA Receptors We found that NMDA produced significant neuronal cell loss in high-KCl-reared striatal cultures (Fig. 1). As shown in Fig. 1B, 500 µM NMDA killed about 50% of the striatal neurons in high-KCl-reared striatal cultures. Increasing the concentration of NMDA to 1 mM appeared to cause slightly more cell loss (about 60% of striatal neurons were killed). While low KCl rearing also yielded a significant toxic effect with 500 µM NMDA (about 30% neuron loss), this effect was significantly less than in the high-KCl-reared cultures (Fig. 1). Thus, high KCl treatment significantly potentiated NMDA-induced cell loss, from 25% loss in the low KCl to 47% loss in the high KCl (P ⬍ 0.01) (normalized to the no-excitotoxin control), as shown more clearly in Fig. 1C. The specificity of NMDA toxicity was confirmed by antagonist studies, in which 500 µM NMDA-induced cell death was completely blocked by the competitive NMDA receptor antagonist APV (500 µM) (Fig. 2). Finally, consistent with the results of our prior studies, QA up to 5 mM showed no significant toxicity in cultures of low-KCl-reared striatal neurons, and this was still the case in striatal neurons grown in high KCl (P ⬎ 0.05) (Fig. 1). The Effects of Activation of Non-NMDA or Metabotropic Glutamate Receptors We confirmed our prior finding (18) that KA was slightly toxic for striatal neurons grown in low KCl (Fig. 3). We additionally found that KA-induced (250 µM) cell death appeared to be perhaps slightly potentiated when the neurons were reared in high KCl, but this seeming potentiation did not reach statistical significant (Fig. 3C). With 250 µM AMPA we confirmed our prior observation of the apparent absence of AMPA

FIG. 1. The effects of NMDA or QA exposure on striatal neuron survival in low-KCl- and high-KCl-reared striatal cultures. Striatal cultures grown in either low KCl (A) or high KCl (B) were exposed to 5 mM QA, 500 µM NMDA, or 1000 µM NMDA for 1 h. Neuron survival at 24 h after exposure onset was expressed as a percentage of neurons counted before exposure. The NMDA significantly killed striatal neurons grown in both low (3 mM) and high (25 mM) KCl (P ⬍ 0.01). However, QA had no statistically significant toxic effect in either condition. (C) Directly compares the data results shown in A and B for the 5 mM QA and 500 µM NMDA exposures, in this case with the neuronal survival normalized to that observed in the no-excitotoxin control. (C) More clearly shows that chronic high-KCl treatment significantly potentiated NMDA toxicity (P ⬍ 0.05), but had no evident effect on QA toxicity. The data presented for each condition in A–C are the mean ⫾SEM for six dishes. Asterisks in A and B indicate a significant difference from the non-drug-treated control, while in C the asterisks indicate a significant difference from the low-KCltreated condition.

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receptor-mediated toxicity in pure striatal cultures grown in low KCl (18). In contrast to the nontoxic effect of AMPA in low-KCl cultures, the same concentration of AMPA (250 µM) was found to be toxic in high-KCl cultures. A concentration of 250 µM AMPA killed about 26.7% of the neurons in the cultures raised in high KCl, after correction for the cell loss in the nonexcitotoxin control group (P ⬍ 0.01) (Fig. 3C). Exposure to higher concentrations of AMPA was not carried out, since AMPA in high concentrations is likely to activate KA receptors (51, 106). The specificity of KA- and AMPAinduced cell death was confirmed by antagonist studies. As shown in Fig. 4, the non-NMDA receptor antagonist GYKI52466 (100 µM) reversed the toxic effects of KA (250 µM) and AMPA (250 µM) in high-KCl cultures. Finally, the metabotropic glutamate receptor agonist trans-ACPD (500 µM) was not toxic to striatal neurons grown in either low or high KCl (Fig. 3). Effects of High KCl on Ionotropic Glutamate Receptor Subunit Levels Previous electrophysiological and pharmacological studies have demonstrated that cultured striatal neurons at 14DIV express functional NMDA, non-NMDA, and metabotropic glutamate receptors (41, 84, 121). Because of the effect of chronic KCl depolarization on

FIG. 2. The specificity of NMDA-induced excitotoxicity in cultures chronically treated with high KCl. Striatal cultures chronically maintained in high KCl were exposed to 500 µM NMDA or 500 µM NMDA plus 500 µM APV for 1 h. Neuron survival at 24 h after exposure onset was expressed as a percentage of neurons counted before exposure. APV completely blocked the NMDA-induced neuron loss observed in the NMDA-treated condition (P ⬍ 0.05). The data presented for each condition are the mean ⫾ SEM for six dishes. Asterisks indicate a significant difference between the NMDA plus APV condition and the NMDA alone condition.

FIG. 3. The effects of trans-ACPD, AMPA, or KA exposure on neuron survival in low-KCl- and high-KCl-reared striatal cultures. Striatal cultures grown in either low KCl (A) or high KCl (B) were exposed to 500 µM trans-ACPD, 250 µM AMPA, or 250 µM KA for 1 h. Neuron survival at 24 h after exposure onset was expressed as a percentage of neurons counted before exposure. AMPA significantly killed striatal neurons grown in high KCl (25 mM) (P ⬍ 0.01), but not in low KCl (3 mM). The KA significantly killed striatal neurons grown in both the low and the high KCl (P ⬍ 0.01). The trans-ACPD had no toxic effect on striatal neurons grown in either high or low KCl. (C) Directly compares the data results shown in A and B for the 250 µM AMPA or 250 µM KA exposures, in this case with the neuronal survival normalized to that observed in the no-excitotoxin control. (C) More clearly shows that chronic high-KCl treatment yielded significant AMPA toxicity compared to the low-KCl condition (P ⬍ 0.01), but did not significantly potentiate KA toxicity. The data presented for each condition in A–C are the mean ⫾ SEM for six dishes. Asterisks in A and B indicate a significant difference from the non-drug-treated control, while in C the asterisks indicate a significant difference from the low-KCl-treated condition.

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striatal neuron vulnerability to various glutamate receptor agonists, we evaluated the effects of high KCl rearing on glutamate receptor subunit levels in our striatal cultures by using antibodies to specific ionotropic glutamate receptor subunits. The levels of the NR1 and NR2A/2B NMDA subunits; the GluR1, GluR2/3, and GluR4 AMPA receptor subunits; and the GluR5/6/7 KA receptor subunits in both high- and low-KCl cultures were determined by ELISA. As summarized in Table 1, striatal neurons grown under high- and low-KCl conditions showed detectable levels of the NR1 and NR2A/2B subunits. Our studies further showed that NR2A/2B immunoreactivity in high-KCl cultures was significantly increased compared to that in low-KCl cultures (199.5% of low-KCl cultures) (P ⬍ 0.01). We did not observe any significant change in the NR1 subunit levels with high-KCl rearing. While GluR2/3 AMPA receptor subunits were also detectable in low-KCl cultures, their levels were markedly and significantly increased in the high-KCl group (to 223.9% of the low-KCl group) (P ⬍ 0.01) (Table 1). In contrast, the GluR1 and GluR4 AMPA receptor subunits were either very low (GluR1) or undetectable (GluR4) in low KCl (i.e., not significantly different from that in glial cell control cultures), but were detectable and significantly greater than in the glial cell culture controls (P ⬍ 0.05) in the high-KCl cultures. Nonethe-

TABLE 1 Effects of Chronic KCl Depolarization on the Expression of Ionotropic Glutamate Receptor Subunits by Cultured Striatal Neurons

Receptor subunits assayed by ELISA

Abundance in 3 mM KCl

Abundance in 25 mM KCl

Receptor abundance at 25 mM as percentage of that at 3 mM KCl

NR1 NR2A/2B GluR1 GluR2/3 GluR4 GluR5/6/7

88.1 4.39 1.0 6.05 ⬃0.0 10.80

71.8 8.75 10.55 13.58 1.90 21.29

81.5 199.5* ⬎250* 223.9* ⬎250* 197.2*

Note. Effects of KCl depolarization on the relative levels of various ionotropic glutamate receptor subunits in striatal cultures. Glutamate receptor subunit levels were measured by ELISA in 2-week-old striatal cultures grown in the chronic presence of either low (3 mM) or high KCl (25 mM). The relative abundance of each subunit or set of subunits detected is expressed as a ratio to the signal intensity for the GluR1 receptor raised in low KCl. Note that the GluR4 subunits were undetectable under the 3 mM condition (i.e., not significantly different from that in the glial control group). Because of the low levels of the GluR1 and GluR4 subunits in the low-KCl condition, the increases in these subunits stemming from high-KCl rearing are extremely great and are indicated only as greater than 250%. The data shown represent the mean ⫾ SEM of 16 wells for each subunit per KCl condition. Asterisks indicate significant differences between the high- and low-KCl groups.

less, the GluR4 ELISA signal, at least, was low even in the high-KCl cultures, when compared with the expression level of the other subunits. The GluR5/6/7 KA receptor subunit signal was prominent in both highand low-KCl cultures, and their expression level appeared to be higher than that for AMPA receptor subunits. The GluR5/6/7 KA receptor subunits were, nonetheless, significantly increased by high KCl to 197.2% of the low-KCl group (P ⬍ 0.01) (Table 1). DISCUSSION

FIG. 4. The specificity of AMPA-induced and KA-induced excitotoxicity in cultures chronically treated with high KCl. Striatal cultures chronically maintained in high KCl were exposed to 250 µM AMPA or 250 µM KA in the presence or absence of GYKI52466 for 1 h. Neuron survival at 24 h after exposure onset was expressed as a percentage of neurons counted before exposure. GYKI52466 blocked both AMPA-induced and KA-induced neuron loss compared to the non-AMPA/non-KA-treated control condition (P ⬍ 0.01). The data presented for each condition are the mean ⫾ SEM for six dishes. Asterisks indicate a significant difference between the blocked condition and the non-blocked condition for the same agonist.

We have found that high-KCl rearing potentiates the excitotoxic vulnerability of cultured striatal neurons to all three types of ionotropic glutamate receptors, and this effect seems to be related to depolarization-induced upregulation of specific ionotropic glutamate receptor subunits. Our findings are discussed in further detail below. NMDA Receptors and Striatal Excitotoxicity Quinolinic acid. In our prior studies of excitotoxic vulnerability in striatal cultures maintained 14DIV, we found no evidence for QA toxicity over a wide range of concentrations and exposure durations, even when the block of NMDA receptors by Mg2⫹ was relieved (18, 32, 99). These prior studies of ours were consistent with

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published reports that the activation of NMDA receptors with QA has minimal toxic effect in pure striatal cultures (41, 123). The present results confirm these prior findings and further show that rearing the cultures under chronic depolarization does not induce the expression of QA toxicity in pure striatal cultures. The lack of a toxic effect of QA in striatal cultures has been surprising in light of the previous findings that direct intrastriatal infusion of QA in intact animals causes extensive striatal neuron death (5, 6, 36–38, 100). Prior electrophysiological and pharmacological studies have shown that functional NMDA receptors with the ability to elevate intracellular [Ca2⫹] are expressed in striatal neurons grown under low-KCl conditions (41, 65, 84). Our present ELISA studies confirm that NR1 NMDA receptor subunits, the key functional NMDA receptor subunit, and NR2A/2B subunits are expressed in our striatal cultures, and our toxicity studies here show that such cultures do show vulnerability to NMDA, irrespective of the KCl-rearing condition. Our current studies further show that the enhanced levels of NR2A/B produced by high-KCl rearing (which seem associated with a potentiation of vulnerability to NMDA) still do not yield QA toxicity in pure striatal cultures. Thus, the lack of QA toxicity is unlikely to be due to a failure to express functional NMDA receptors. Possible explanations for the discrepancy between in vivo and in vitro results with QA are: (i) QA does not directly kill striatal neurons in vivo, but relies on some other features of striatal organization to produce its effects; and/or (ii) QA does directly kill striatal neurons in vivo and would kill striatal neurons in vitro if they were allowed to mature under conditions that simulate some requisite set of in vivo conditions. With respect to the first possibility, the striatum receives an extensive glutamatergic input from the cortex (46, 62, 124), and available evidence indicates that the integrity of this input is necessary for the expression of QA toxicity in vivo (16, 74, 75, 100). The cortical input to the striatum may promote QA toxicity by: (i) QA stimulated release of endogeneous glutamate from corticostriatal terminals; or (ii) QA-mediated damage to glial cells that normally take up the glutamate released by corticostriatal terminals. With respect to the possibility that QA stimulates glutamate release, introduction of QA has been shown to cause overrelease of glutamate in striatum and cortex, presumably by presynaptic mechanisms (27, 34). With respect to the possibility that QA kills striatal glial cells, glial cell destruction in striatum is known to result in damage to striatal neurons (69, 95), glial cells appear to possess NMDA receptors (1, 54, 109), and there is some evidence that they are transiently damaged by QA (12). The present studies appear to rule out the possibility that the chronic depolarization of striatal neurons produced by the cortical input in vivo is the basis of the

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occurrence of QA toxicity in vivo. It is possible, however, that the glutamatergic input in vivo exerts effects on NMDA receptor maturation by mechanisms additional to chronic depolarization. For example, it is possible that growth factors released by cortical terminals might be involved in such a process, based on the finding that BDNF induces NR2A expression in cultured rat cortical neurons (104). It is also possible that QA effects may be limited to particular types of NMDA receptors (e.g., receptors with unique subunit configuration) that mature beyond the time window we examined and/or critically depend upon some unknown environmental factors that exist in vivo, but were absent in our cultures (14, 31, 80, 93, 103). NMDA. In contrast to QA, we found that NMDA (which is a more potent NMDA receptor agonist than QA) (31) did cause excitotoxic destruction of striatal neurons raised in low KCl, and this loss was greatly increased in the high-KCl-reared cultures. This potentiation with high KCl is consistent with that observed in previous studies of cultured cerebellar granule cells KCl (9, 29, 115). Our ELISA data show that high-KCl treatment markedly upregulated NR2A/2B subunits in our striatal cultures, but had no significant influence on NR1 subunit expression. Since coexpression of NR2 subunits with NR1 subunits potentiates NMDA receptor activity (76, 85), it is likely that chronic KCl depolarization potentiated NMDA toxicity through its regulation of NMDA subunit expression. A similar effect has been reported for cultured cerebellar granule cells, in which 25 mM KCl-induced chronic depolarization was observed to selectively increase NR2A mRNA levels, but have no effects on NR1 or NR2B mRNA levels (9). Since incubation with antisense oligonucleotides targeted to NR2A mRNAs blocked NMDA toxicity in cerebellar granule cells, it was concluded that upregulation of NR2A was critical for the expression of NMDA toxicity (9). By contrast, since NR2B subunits are considerably more abundant than NR2A subunits in adult rat striatum (107), it seems likely that the chronic high KCl preferentially upregulated NR2B subunits in our cultured striatal neurons. Glutamatergic input and development of NMDA excitotoxicity. Striatal neurons in vivo fluctuate between a resting hyperpolarized state and periods of 5–20 mV depolarization above this resting state (28, 126). The neurons frequently fire bursts of spikes from the depolarized ‘‘up’’ state, and both the ‘‘up’’ state and the spike bursts depend on the integrity of the corticostriatal (125, 127). Prominent and prolonged spontaneous synaptic currents are also observed in striatal neurons cocultured with cortical cells, while such synaptic activity does not occur in pure striatal cultures (123). In vitro studies suggest the possible importance of this input for development of NMDA vulnerability, since NMDA toxicity is prominent in striatal neurons cocul-

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tured with cortical cells, while it is otherwise absent or minimal (41, 123). Corticostriatal innervation might influence the development of glutamate receptormediated excitotoxicity through its chronic depolarizing effect on striatal neurons. Chronic depolarization has long been known to influence the survival, growth, differentiation, and neurotransmitter expression of neurons during development, including striatal cholinergic neurons (30, 43, 49, 57, 60, 68, 118). Incubation in 25 mM KCl, as confirmed by previous intracellular recording studies, produces a sustained depolarizing effect (about 25 mV) that is maintained unless a normal K⫹ concentration is restored (86). Thus, the KCl-induced chronic depolarization in our striatal cultures may mimic the bioelectric effects of corticostriatal innervation on striatal neurons in vivo, although the depolarization magnitude and pattern produced by chronic high KCl is likely to differ from that in vivo. Our studies together with previously published findings thus provide evidence suggesting that the chronic depolarization produced by corticostriatal and/or thalamostriatal innervation in vivo may potentiate NMDA toxicity in the striatum by selective upregulation of NR2 subunits. As noted above, depolarization is also found to affect NMDA receptor subunit expression and receptor-mediated toxicity in cerebellar granule cells, which like striatal cells receive glutamatergic afferent innervation, from mossy fibers in the case of the cerebellar granule cells (102). Non-NMDA Receptors and Striatal Excitotoxicity Kainic acid. Our prior studies showed that glutamate-induced cell loss in pure striatal cultures raised in low KCl was largely attributable to activation of non-NMDA-type glutamate receptors and was most prominent with KA as the agonist (18). The present data and those of Mesco et al. (77) are consistent with our prior findings. In addition, the present studies show that the excitotoxic destruction of neurons by KA in pure striatal cultures may be slightly enhanced in striatal cultures reared with chronic depolarization. The high toxic potency of KA that we observed is consistent with the electrophysiological finding that KA is the only glutamatergic agonist that produces a sustained inward current (113, 114). Such sustained activation of glutamate receptors with KA is accompanied in cultured striatal neurons by a large increase in intracellular Ca2⫹ (84). Note that although KA has only low affinity for AMPA receptors, it produces a sustained activation of these receptors (101, 105). In contrast, AMPA produces rapidly desensitizing responses at AMPA receptors, which might explain why AMPA had no excitotoxic effect in our low-KCl-reared cultures. This raises the possibility that sustained AMPA receptor activation might have contributed to our KAinduced striatal neuron death. Consistent with this

possibility, AMPA toxicity can be produced in cultured hippocampal neurons by coadministration of cyclothiazide, which blocks AMPA receptor desensitization (72, 88). Our ELISA studies showed that GluR5, GluR6, and/or GluR7 KA receptor subunits were highly abundant on our cultured striatal neurons, which is consistent with their abundance on striatal neurons in vivo (19, 129). Our studies further showed that the levels of these subunits were approximately doubled with chronic KCl depolarization. The slight apparent increased KA vulnerability observed in the high-KCl cultures is consistent with this elevation. In situ hybridization studies show that striatal neurons in vivo express large amounts of GluR6 mRNA and some GluR7 mRNA, but little GluR5 mRNA (10, 128, 129). For this reason and because of the demonstrated association of the GluR6 subunit with excitotoxic vulnerability (83), it seems likely that increases in the GluR6 subunit occurred and played a particularly important role in the high-KCl potentiation of KA toxicity. The increase in AMPA receptor subunits levels obtained with high KCl may also have contributed to the enhanced KA toxicity observed in these studies, for the reasons noted above. Corticostriatal input and KA toxicity. Previous in vivo studies have shown that KA toxicity in the striatum increases progressively with age (16, 75). Such an increase in the susceptibility of striatum to KA has been reported to correlate with the development of the corticostriatal glutamatergic innervation (16). Our studies suggest that the corticostriatal innervation might potentiate KA toxicity through depolarization-induced increases in KA receptor subunit expression. Nonetheless, we found that KA was highly toxic to striatal neurons grown in even low-KCl (nondepolarized) conditions. Since our 14DIV cultures are comparable in age to rats at postnatal day (PND) 10, and since most corticostriatal projections do not reach striatum until PND 21 (16), our low-KCl toxicity and receptor subunit expression studies suggest that functional KA receptors may be expressed by striatal neurons in abundance prior to or in the absence of cortical input. This interpretation is supported by in vivo data showing that KA receptor levels are high in rat striatum already at PND 1. In this light, it is difficult to explain the in vivo finding that striatal neurons became insensitive to KA after being deprived of their corticostriatal innervation during adulthood (75). We, however, cannot exclude the possibility that loss of established corticostriatal innervation results in loss of striatal KA receptors, which may be critical for KA toxicity in vivo. AMPA. As in our prior studies, we found that AMPA receptor-mediated excitotoxicity was not evident in pure striatal cultures reared in low KCl (18). Our ELISA studies verified the presence of GluR2 and/or GluR3 AMPA receptor subunits in our pure striatal

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cultures reared in low KCl. Electrophysiological studies have directly confirmed the presence of functional AMPA receptors in pure cultured striatal neurons such as ours (108). Note, however, that since our antibody recognizes an epitope common to both the GluR2 and GluR3 subunits, it is uncertain whether the observed GluR2/3 signal detected by ELISA was due to GluR2 or GluR3 or both. Since GluR2 or GluR3 alone or combined can constitute functional AMPA receptor channels (52, 53, 101), functional AMPA receptors constituted from GluR2 or GluR3 alone could have been present in our pure striatal cultures. If GluR2 homomers or GluR2–GluR3 heteromers predominate in lowKCl cultures, the nontoxic effects of AMPA observed would be explained, since the GluR2 subunit renders AMPA receptor channels impermeable to Ca2⫹ (15, 52, 59). Activation of AMPA receptors by AMPA itself characteristically elicits a rapidly desensitizing current, and such rapid AMPA desensitization has been suggested to protect cells from overexcitation (105, 113, 130). For this additional reason, it is also not surprising that AMPA was not toxic in our pure striatal cultures raised in low KCl. In these studies, the levels of AMPA subunits may have been so low that desensitization was sufficient to protect them from AMPA receptormediated excitotoxicity. We did, however, find significant AMPA toxicity in striatal cultures reared with chronic depolarization. Our ELISA studies showed that the GluR2/3 subunit level was markedly elevated, and GluR1 and GluR4 subunits became detectable. The induced AMPA toxicity may be the result of depolarization-dependent increases in the levels of GluR1 and GluR4 subunits, which may lead to formation of some functional AMPA receptors without GluR2 and thus receptor channels permeable to Ca2⫹. The levels of the GluR4 subunits in the high-KCl-treated striatal cultures, however, were low compared to the levels of GluR2/3. This is not surprising in light of the in vivo localization of the GluR4 subunit in intact striatum to parvalbuminergic striatal interneurons, which make up fewer than 2% of striatal neurons (19, 111). It seems likely that this neuron type was also rare in our striatal cultures (110). Within intact striatum, the GluR1 subunit is also found in parvalbuminergic interneurons, as well as in the dendrites and dendritic spines of striatal projection neurons, which make up over 90% of striatal neurons (8, 20). The fact that cultured striatal projection neurons (which make up the vast majority of striatal neurons in our cultures) do not develop spines in the absence of cortical input (110) may explain why GluR1 levels were also relatively low in our cultures. Thus, it seems unlikely that enhanced GluR4 expression is the major cause of the AMPA-induced cell death observed in our high-KCl cultures, but enhanced GluR1 expression may have contributed. It also may be that in-

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creased levels of either GluR2 and/or GluR3, which are both common in striatal projection neurons in vivo (7, 19, 20, 108, 111, 112), were responsible for the occurrence of AMPA toxicity in our high-KCl cultures. For example, if GluR3 was preferentially upregulated, this might have resulted in the occurrence of AMPA subunit combinations with Ca2⫹ permeability (e.g., GluR1–3 heteromers or GluR3 homomers) in many neurons. Since the permeability of AMPA receptors to Ca2⫹ is inversely correlated with the relative GluR2 abundance in the receptor composition (45), this might make those striatal neurons rich in GluR1–3 heteromers or GluR3 monomers more vulnerable to destruction by AMPA via Ca2⫹-mediated excitotoxicity (47). Elevated neuronal activity in status epilepticus has indeed been found to cause a selective increase in GluR3 subunit mRNAs, with no effect on GluR2 subunit gene expression (26). Alternatively, the GluR2 subunit may be predominantly increased in cultured striatal neurons by high-KCl rearing. Under these circumstances, it could be that the enhanced excitability of these neurons stemming from the higher GluR2 levels renders them vulnerable. Consistent with this notion, overexpression of the GluR2 subunit has been shown to be toxic for neurons, despite its role in mitigating Ca2⫹ entry through AMPA channels (67). Metabotropic Glutamate Receptors and Striatal Excitotoxicity In our prior study of striatal cultures reared in low KCl, we saw no evidence that the potent agonist trans-ACPD had a toxic effect (18). In the present study, we confirmed our prior finding and further showed that there was also no toxic effect of trans-ACPD in highKCl depolarized cultures. Direct exposure of cortical neurons to 1 mM trans-ACPD for 24 h also has not been found to have a toxic effect, although trans-ACPD does elevate intracellular [Ca2⫹] (64). Additionally, direct introduction of trans-ACPD into the striatum in vivo has also not been found to be toxic for striatal cells (24, 25, 99). Thus, broad-spectrum activation of metabotropic glutamate receptors does not seem to have an obvious direct toxic effect on striatal neurons. Metabotropic glutamate receptors, however, consist of eight major receptor subtypes that have been divided into three functional groups (35). It may be that selective activation of specific subtypes or groups of metabotropic glutamate receptors has a direct excitotoxic effect or modulates the excitotoxic effect of ionotropic glutamate receptor agonists (66, 94, 98, 117). In our prior study of striatal cultures reared in low KCl, however, we also saw no evidence that the metabotropic glutamate receptor antagonist AP3 attenuated glutamate excitotoxicity (18). Similarly, knockout of the mGluR1-type metabotropic receptor does not attenuate ischemic or excitotoxic neuronal damage (35). Prior

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studies have, however, shown that activation of metabotropic glutamate receptors inhibits NMDA-induced responses of striatal neurons, and decreases cell loss caused by QA in striatum (24, 25, 89). It is possible that metabotropic glutamate receptor agonists would modulate glutamate responses of striatal neurons in highKCl-reared cultures, a possibility that we did not explore. Mechanism Underlying Effect of KCl on Receptor Expression Previous studies suggest that depolarization may exert its maturational effects on target cells by elevating cytosolic [Ca2⫹] through the activation of voltagedependent Ca2⫹ channels. Calcium is a well-known trophic factor for neuronal development (9, 118). Voltage-dependent Ca2⫹ channel blockers (e.g., nifedipine, verapamil, and Mg2⫹ ) or low-Ca2⫹ media have been found to block depolarization-induced potentiation of cell survival, neurotransmitter content, and NMDA receptor expression. Calcium agonists (e.g., BAY K8644, SZ-202-791) have been shown to potentiate the positive effects of chronic depolarization on cells (43). Interaction of glutamate with glutamate receptors may also produce membrane depolarization that activates voltage-dependent Ca2⫹ channels, thus causing elevation of intracellular [Ca2⫹] (4). Such findings may in part help explain the QA toxicity in rat striatal cultures observed in one prior study and the relatively high toxic potency of NMDA in low-K⫹-reared mouse striatal cultures reported by another group, since cell culture media in these studies contained high glutamate and aspartate (50 µM each) (40, 63). GENERAL CONCLUSIONS

Our studies suggest that abnormalities in depolarization in vivo could influence the expression of glutamate receptor-mediated excitotoxicity by selective upregulation of some glutamate receptor subunits (i.e., NR2 and GluR3) that are critical for glutamate excitotoxicity. An upregulation of hippocampal AMPA receptor subunit GluR3 expression has been observed in induced-status epilepticus, which is characterized by prolonged intense neuronal activation (26). Similarly, GluR2 subunits on hippocampal CA1 neurons are preferentially downregulated in global ischemia and this downregulation appears to be critical for CA1 vulnerability in global ischemia (2, 90, 91). Hypoxia is also found to induce NR2C subunit expression in cortex and hippocampus (92). Therefore, knowledge of the dynamic changes in glutamate receptor subunit expression under pathological conditions is critical for understanding the vulnerability of striatal neurons to excitatory amino acids. Finally, attempts to use cultures of a specific neuronal population to explore the role of specific

receptor types in the disease-related vulnerability of those neurons in vivo need to consider whether those neurons are reared in a culture environment that promotes expression of the relevant receptor types that resembles that in vivo. ACKNOWLEDGMENTS We thank Lorina Dudkin for her technical assistance and Dr. M. G. Honig for her advice on culture procedures. This research was carried out by Q.C. in partial fulfillment of the requirements for a Ph.D. in the Department of Anatomy and Neurobiology at the University of Tennessee–Memphis. This study was supported by NS-28889 (D.J.S.), NS-19620 and NS-28721 (A.R.), and the University of Tennessee Neuroscience Center of Excellence (Q.C.).

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