Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents

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Experimental Neurology 183 (2003) 573–580

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Neuroprotection against NMDA excitotoxicity by group I metabotropic glutamate receptors is associated with reduction of NMDA stimulated currents Morten Blaabjerg,a Liwei Fang,b Jens Zimmer,a and Andrius Baskysb,c,* a Anatomy and Neurobiology, University of Southern Denmark-Odense, Denmark Long Beach VA Medical Center, 06/116A, 5901 E, Seventh Street, Long Beach, CA 90822, USA c Department of Psychiatry and Human Behavior, University of California, Irvine, CA 92668, USA b

Received 12 December 2002; revised 13 March 2003; accepted 26 March 2003

Abstract The neurotransmitter glutamate can have both excitotoxic and protective effects on neurons. The excitotoxic effects have been intensively studied, whereas the protective effects, including the involvement of metabotropic glutamate receptors (mGluRs), remain unclear. In the present study, we tested the protective effects of the group-I-mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) on organotypic hippocampal slice cultures exposed to excitotoxic concentrations of N-methyl-D-aspartate (NMDA). Effects of DHPG on electrophysiological responses induced by NMDA receptor activation were also recorded. Experiments were performed on organotypic hippocampal slice cultures derived from 7-day-old rats, with cellular uptake of propidium iodide as a marker for neuronal cell death. Slice cultures pretreated with DHPG (10 or 100 ␮M) for 2 h prior to exposure to 50 ␮M NMDA for 30 min displayed reduced propidium iodide uptake, compared to cultures exposed to NMDA only. The neuroprotective effect was confirmed by Hoechst 33342 staining, where the appearance of pycnotic nuclei after NMDA treatment was prevented by the DHPG pretreatment. Using caspase-3 activity to monitor the presence of apoptosis, failed to demonstrate this type of cell death in CA1 after NMDA application. The protective effect of DHPG was abolished by the mGluR1 selective antagonist (S)-(⫹)-␣-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385; 5 or 10 ␮M), whereas the mGluR5-selective antagonist 2-methyl-6-phenylethynylpyridine (MPEP; 1 ␮M) had no effect. Voltage-clamping of CA1 pyramidal cells in cultures treated with 10 ␮M DHPG for 2 h showed a significant depression of NMDA-induced inward currents compared to untreated controls. We conclude that neuroprotection induced by activation of group-I-mGluRs involve mGluR1 and is associated with decreased NMDA-stimulated currents. © 2003 Elsevier Science (USA). All rights reserved. Keywords: mGluR; N-methyl-D-aspartate; Propidium iodide; Neurodegeneration; LY367385; MPEP

Introduction Glutamate is the major excitatory neurotransmitter in the brain but is also a potent excitotoxin involved in neurodegenerative disorders such as ischemia and Alzheimer’s disease. Pretreatment with moderate levels of glutamate agonists can, however, protect neurons from damage caused by subsequent exposure to excitotoxic concentrations of gluta* Corresponding author. Fax: ⫹1-562-826-5969. E-mail address: [email protected] or [email protected] (A. Baskys).

mate. Recent evidence suggests that activation of metabotropic glutamate receptors (mGluRs) is crucial for this neuroprotection (Adamchik and Baskys, 2000; Chiamulera et al., 1992; Koh et al., 1991; Mount et al., 1993; Opitz and Reymann, 1993; Siliprandi et al., 1992), particularly against ischemic nerve cell death (Kalda and Zharkovsky, 1999; Maiese et al., 1996; Pizzi et al., 1996b; Sagara and Schubert, 1998; Schro¨der et al., 1999). Since mGluR-mediated neuroprotection requires protein kinase C (PKC) activation (Kalda and Zharkovsky, 1999; Kalda et al., 2000; Koh et al., 1991; Pizzi et al., 1996b; Sagara and Schubert, 1998), group-I-mGluRs (mGluR1 and

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mGluR5) are likely to be involved, because these receptors are coupled to the IP3/DAG signaling pathway leading to release of Ca2⫹ from intracellular stores and activation of PKC respectively (for review see Conn and Pin, 1997). The role of these receptors, however remain poorly understood and controversial. There is evidence for both neurotoxic and neuroprotective actions of both group-I-mGluR agonists and antagonists (for review see Nicoletti et al., 1999). For example, there are reports that the mGluR1 and -5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG), facilitates NMDA receptor function (Awad et al., 2000; Bandrowski et al., 2001; Fitzjohn et al., 1996; Koga et al., 1996; Orlando et al., 2001; Pisani et al., 2001; Skeberdis et al., 2001) just as the nonselective mGluR agonist 1S,3R-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) or the group-I-mGluR-selective agonist DHPG potentiated NMDA-induced toxicity in spinal cord neurons or hippocampal slice cultures (Blaabjerg et al., 2001; Faden, 1997; Young et al., 1998) and neuronal degeneration by oxygen and glucose deprivation (PellegriniGiampietro et al., 1999) There is, however, at least one report that coapplication of NMDA and ACPD in acute hippocampal slice preparations induced neuroprotection (Pizzi et al., 1996a). Other evidence that group-I-mGluRs are neuroprotective comes from studies of nitric oxide (NO) or ischemia-induced cell death (Lin and Maiese, 2001a, 2001b; Maiese et al., 1995, 2000; Vincent et al., 1997), decrease in apoptotic markers (Allen et al., 2000; Anneser et al., 1998; Maiese and Vincent, 1999; Vincent and Maiese, 2000), and reduction of NMDA toxicity (Adamchik and Baskys, 2000; Colwell and Levine, 1999; Colwell et al., 1996). Bruno et al. (2001) reported that the group-I-mGluR agonist DHPG was protective when repeatedly coapplied with NMDA despite an increase in NMDA-receptor-mediated currents. In the present study, we investigated whether selective activation of group-I-mGluRs induced neuroprotection and to what extent mGluR1 and mGluR5 are involved. The effect of group-I-mGluR activation on NMDA-stimulated currents was also investigated.

Materials and methods Animals Organotypic hippocampal slice cultures were prepared and grown by the interface method (Stoppini et al., 1991) as previously described (Baskys and Adamchik, 2001). Briefly, 7-day-old Wistar rat pups (Charles River, Raleigh, NC) were anesthetized with halothane and rapidly decapitated, and the brains placed into an ice-cold stabilization medium [50% minimal essential medium with no bicarbonate or glutamate, 50% calcium and magnesium-free Hanks’ balanced salt solution, 7.5 mM D-glucose, and 20 mM N-2hydroxyethyl piperazine-N⬘-2-ethanosulfonic acid (HEPES),

pH 7.15]. The middorsal segments of the two hippocampi were dissected out and cut into 400-␮m transverse slices. The slices were separated, and excess tissue was removed and placed on 30-mm Millicel-CM 0.4-␮m-thick inserts (Millipore, Bedford, MA, USA). The inserts were transferred to a 6-well culture plate (Falcon, Becton Dickinson Labware, NJ, USA) on top of 1 ml of culture medium (50% MEM, 25% horse serum, 25% Earl’s balanced salt solution, D-glucose, HEPES, 5000 units/ml penicillin G, and 5 ␮l/ml streptomycin sulfate, pH 7.15). Slices were grown for a minimum of 2 weeks at 36.5°C in 100% humidity, with 95% air and 5% CO2 and fed twice weekly via 50% medium exchange. All experiments were performed according to the standards described in Animal Welfare Regulations and the Guide for the Care and Use of Laboratory Animals. Experimental protocol for propidium iodide (PI) experiments To investigate the potential neuroprotective effects of the group-I-mGluR agonist DHPG it was applied to the culture medium for 2 h prior to a 30-min exposure to 50 ␮M N-methyl-D-aspartate (NMDA), followed by change to normal control medium. In the experiments in which the competitive mGluR1 antagonist (S)-(⫹)-␣-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) or the noncompetitive mGluR5 antagonist 2-methyl-6-phenylethynylpyridine (MPEP) was used, these compounds were applied 30 min before and during the pretreatment period to ensure maximal receptor saturation. The same time schedule was used in experiments where antagonists were applied without DHPG exposure. Cell death, measured as cellular uptake of PI (3,8-diamino-5-[3-(diethylethylamino)propyl]6-phenyl phenanthridinium diiodide; Molecular Probes, Eugene, OR) was recorded by laser scanning confocal microscopy (PCM2000, Nikon). Images of the cultures were obtained before the experiments to determine the baseline cellular uptake of PI and 24 and 48 h after exposure to NMDA. All data were normalized to data obtained after the slices had been kept for 48 h at an ambient temperature of 4°C (except for cultures used for Hoechst 33342 and caspase-3 staining). Measurements of the PI uptake in slices exposed to 4°C was assumed to reflect the maximal cell death level in that same slice and were used for two purposes. First, the high level of PI uptake allowed the visualization of all hippocampal subfields and exclusion of damaged slices from analysis. Second, the PI uptake normalized to the maximal PI uptake in the same slice allowed to quantify and compare cell death levels in individual slices regardless of their size or thickness. Since all comparisons of cell death were made in relation to cell death induced by NMDA alone, data were expressed as a percentage of the NMDA-induced cell death (taken as 100%) in that particular experiment. In order to gage the basal level of cell death, one well with five to six cultures in each experimental run was exposed to culture medium only. The basal cell death

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level measured in 8 wells (n ⫽ 5– 6 slices each) after 24 h of culturing was 0.96 ⫾ 0.10 % in relation to the maximal level of cell death in all areas of the hippocampus. This low level of basal cell death was ignored and not considered in cell death calculations. PI images were analyzed off-line by measuring the pixel intensity, using ScionImage analysis software (available at http://www.scioncorp.com). The areas of interest (CA1, CA3 pyramidal cell layers) were outlined and superimposed on the images obtained at baseline, 24 h and 48 h. DHPG, MPEP and LY367385 were obtained from Tocris Cookson Inc. (Ellisville, MO) and NMDA from Sigma (St. Louis, MO). The drugs were dissolved in sterile distilled water, aliquoted, and stored at ⫺20°C until use. Processing of tissue for immunohistochemistry To investigate whether the lesion induced by NMDA treatment resulted in apoptotic or necrotic cell death, we performed double stainings of Hoechst 33342 and activated caspase-3. Cultures were treated with NMDA as described above and fixed after 24 and 48 h in 4% paraformaldehyde for 1 h followed by cryoprotection in 20% sucrose in phosphate buffer for 24 h. Cultures were then frozen on CO2 ice and cryostat-sectioned in 20-␮m thick sections and kept at ⫺20°C until processed for immunohistochemistry. Immunohistochemistry After thawing to room temperature, sections were rinsed in 0.15 M Tris-buffered saline (TBS) with 1% Triton-X for 3 ⫻ 15 min and incubated in 5% goat serum for 30 min. Sections were then incubated with anti-caspase-3 active (1:5000; R&D Systems, Abingdon, UK) at 4°C for 48 h followed by 3 ⫻ 15 min rinse in TBS with 1% Triton-X. To avoid unspecific binding of the secondary antibody, sections were again incubated in 5% goat-serum followed by incubation with anti-rabbit/CY3 (1:100) and Hoechst 33342 (2 ␮g/ml) for 2 h. After a final 3 ⫻ 15 min rinse in TBS, sections were washed with distilled water air-dried and cover-slipped in Flouromount (BDH). As a positive control for the caspase-3 staining we included sections from cultures treated with colchicine (1 ␮M for 48 h) known to induce apoptosis in the dentate granule cells (Kim et al., 2002; Kristensen et al., 2003). Digital images of the stained sections where obtained by using a fluorescence microscope (Olympus, Vanox-T) equipped with a digital camera (Sensys KAF 1400 G2: Photometric, Tucson, AZ). Electrophysiology Cultured hippocampal slices treated with DHPG (10 ␮M for 2 h) or untreated age-matched controls were transferred to a submersion-type chamber with the attached membrane for recording and superfused with ASCF (in micromolar

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concentrations): 129 NaCl, 3 KCl, 1.25 NaH2PO4 ⫻ H2O, 2 MgCl2 ⫻ 6H2O, 10 D-glucose, 26 NaHCO3, and 2 CaCl2, pH 7.5, heated to 30°C, osmolarity ⫽ 300 mOsm) supplied at a rate of 1.8 –2 ml/min. Recording microelectrodes were prepared from borosilicate glass (WPI Inc; Sarasota, FL) by a Kopf Instruments micropipette puller (Tujunga, CA) (resistance ranging from 4 to 6 M⍀) and filled with solution of the following composition (in micromolar concentrations): K⫹ gluconate 142.5, potassium methylsulfate 20, NaCl 8, Hepes 10, EGTA 0.1, MgATP 2, and GTP 0.2. The pH of the internal solution was adjusted to 7.2 with KOH and the osmolarity was adjusted to 300 mOsm with H2O and sucrose. After making a high-resistance seal the whole-cell configuration was established by rupturing the membrane under the patch pipette. Recordings were done using a Axopatch 1D preamplifier (Axon Instruments; Foster City, CA) and filtered at 5 kHz. Pipette and cell capacitance transients were compensated with the appropriate capacitance controls on the amplifier and series resistance compensation was applied to a level of 80 –90%. Cells included in analysis were voltage-clamped between ⫺53 and ⫺77 mV and had action potential amplitudes ⬎70 mV and stable input resistance for at least 10 min prior to the NMDA application. Statistics Statistical significance was assessed in GraphPad Instat (GraphPad Software, San Diego, CA), using one-way ANOVA with Bonferonni correction for comparison between groups of interest. In voltage clamp studies Student’s t-test was used for comparison. Differences were considered significant at P ⬍ 0.05.

Results Neuroprotection induced by selective activation of group-I-mGluRs To investigate the potential neuroprotective effects of group-I-mGluR activation, cultures were pretreated with DHPG (1, 10, or 100 ␮M) for 2 h followed by exposure to a toxic 50 ␮M concentration of NMDA for 30 min. It has been shown earlier that at this concentration NMDA causes damage to approximately 30% of all neurons in an organotypic hippocampal culture (Adamchik and Baskys, 2000). After 24 h cultures exposed to NMDA showed an increased PI uptake in hippocampal subfields (Fig. 1B and M). When pretreated with 1 ␮M DHPG for 2 h prior to the NMDA exposure, cultures displayed no reduction in PI uptake compared to cultures exposed to NMDA only (Fig. 1M). Pretreatment with DHPG at 10 or 100 ␮M did, however, induce significant concentration-dependent reduction in PI uptake in CA1 and CA3 pyramidal cell areas (Fig. 1C–D and M). To verify that reduced PI uptake in DHPG ⫹ NMDA-

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Fig. 1. DHPG-induced neuroprotection against NMDA excitotoxicity. (A–D) PI uptake after 24 h in organotypic hippocampal slice cultures exposed to NMDA (50 ␮M for 30 min) compared to slices pretreated with DHPG before the NMDA exposure. (E–H) High magnification (100⫻) of CA1 pyramidal cells after similar treatment. I and J correspond to E and F but in caspase-3 staining. As a positive control for the caspase-3 staining, sections from cultures treated with colchicine (1 ␮M for 48 h) were included (K and L). Untreated age-matched control cultures displayed a small baseline level of PI uptake (A), normal nuclear morphology (E), and no caspase-3 activity (I). Cultures treated with NMDA had increased PI uptake in hippocampal subfields (B) with nuclei appearing pycnotic and densely stained (F; arrow) but without caspase-3 activity (J). In contrast nuclei in cultures treated with colchicine showed fragmentation (K; arrowhead) and increased caspase-3 activation (L; arrowhead). Pretreatment with DHPG for 2 h before the NMDA exposure reduced the NMDA-induced PI uptake (C and D) as well as the number of pycnotic nuclei (G and H). Graph (M) shows quantification of PI uptake in CA1 (black columns) and CA3 subfields (gray columns) after 24 h with NMDA set to 100%. Pretreatment for 2 h with DHPG at 1 ␮M had no effect on NMDA-induced PI uptake, whereas pretreatment with DHPG at 10 and 100 ␮M significantly reduced PI uptake in both CA1 and CA3 in a concentration dependent manner. Data are shown as mean ⫾ SEM, ***P ⬍ 0.001 (compared to NMDA in CA1 alone). ##P ⬍ 0.01, and ###P ⬍ 0.001 (compared to NMDA in CA3 alone) with n ⫽ 21– 42 cultures per group.

treated cultures was indeed associated with reduced neuronal cell death, cryostat sectioned slices was stained with Hoechst 33342. This revealed small pycnotic nuclei in CA1 pyramidal cells at 24 and 48 h (Fig. 1F; images at 48 h are not shown) after the NMDA treatment compared to untreated controls (Fig. 1E). In DHPG ⫹ NMDA-treated cultures we observed less pycnotic CA1 pyramidal cells (Fig. 1G and H) when compared to cultures exposed to NMDA only (Fig. 1F). Since presence of pycnotic cells following NMDA treatment could be interpreted as an indicator of necrotic cell death, we measured caspase-3 activity in control and NMDA-treated cultures. (Increases in caspase-3 activity has been associated with apoptotic cell death; Kim et al., 2002; Kristensen et al., 2003.) We found no increase in caspase-3 activity following 24 or 48 h after NMDA

treatment (Fig. 1J; image of 48 h not shown) compared to untreated controls (Fig. 1I). As a control for the caspase-3 staining we included sections from cultures treated with colchicine (1 ␮M for 48 h) known to induce apoptosis in the dentate granule cells. In Hoechst staining dentate granule cells from these cultures had fragmented nuclei (Fig. 1K) with increased caspase-3 activity (Fig. 1L), suggesting that the staining protocol was appropriate and that the NMDA treatment did not induce caspase-3 activity. To investigate whether the timing of agonist application was crucial for neuroprotection we also performed experiments with 2 h DHPG treatment immediately after the NMDA exposure. Using this protocol, DHPG did, however, not induce neuroprotection (DHPG 10 ␮M ⫽ 91.1 ⫾ 28.1%; DHPG 100 ␮M ⫽ 85.6 ⫾ 10.0%).

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Fig. 2. Effect of group I antagonist on DHPG-induced neuroprotection. Graphs showing quantification of PI uptake in cultures coexposed to the mGluR1 antagonist LY367385 (A) or the mGluR5 antagonist MPEP (B) during the 2-h pretreatment with DHPG 10 ␮M. Antagonists were applied 30 min before and during DHPG treatment to ensure receptor saturation. Blocking the mGluR1 receptor with 10 ␮M LY367385 during the DHPG pretreatment attenuated neuroprotection (A), whereas blocking the mGluR5 receptor with 1 ␮M MPEP was ineffective (B). Data are shown as mean ⫹ SEM, *P ⬍ 0.05, **P ⬍ 0.01 with n ⫽ 6 –12.

Effects of group-I-mGluR antagonists on DHPG-induced neuroprotection We examined the contribution of mGluR1 and mGluR5 receptors to the DHPG-induced neuroprotection by application of selective antagonists during the 2 h DHPG pretreatment. An mGluR1 selective antagonist LY367385 (5 and 10 ␮M) abolished neuroprotection in a concentration-dependent manner (Fig. 2A), whereas the mGluR5-selective antagonist MPEP (1 ␮M) had no significant effect (Fig. 2B).

0.05) in all four neurons. The mean values of the NMDAstimulated inward current were significantly (P ⬍ 0.05, two-sided t test) different between the two groups at 5, 6, and 7 min after the start of NMDA application (Fig. 3). Thus, the peak value of the NMDA-stimulated current in neurons from the control cultures was 42.5 ⫾ 2.5 pA (n ⫽ 4 neurons from four cultures) at 6 min after the onset of NMDA application (Fig. 3). In contrast, in the group of cultures treated with DHPG (10 ␮M, 2 h) NMDA-stimulated pA (n ⫽ 3 neurons from three cultures).

Effects of DHPG on NMDA receptor/channel function To examine whether DHPG pretreatment had any effect on the NMDA receptor/channel function we performed whole-cell recordings of the NMDA-stimulated current in CA1 pyramidal cells in organotypic slice cultures treated with DHPG (10 ␮M, 2 h) and untreated control cultures. To measure the NMDA-stimulated current, neurons were voltage-clamped (Vhold of control neurons ⫽ ⫺73, ⫺72, ⫺56, ⫺53 mV; DHPG-treated neurons ⫽ ⫺77, ⫺76, ⫺66 mV) and the holding current (Ihold) values were recorded each minute after application of NMDA (1 ␮M, 5 min) for 10 min. The average Ihold prior to NMDA application was 115 ⫾ 42 pA (210, 160, 60, 30 pA) and 113 ⫾ 49 pA (210, 50, 80 pA) in the control and DHPG-treated cultures respectively. The average membrane input resistance (RN) prior to NMDA application was 872 ⫾ 322 and 904 ⫾ 335 M⍀ in the control and DHPG-treated groups, which was similar to RN values reported in cultured hippocampal cells (Mynlieff, 1999). In the control but not the DHPG-treated cultures, NMDA produced a robust inward current recorded as an increase in the negative Ihold (Fig. 3), which was associated with a decreased RN(490 ⫾ 107 M⍀, not significant, P ⬎

Fig. 3. NMDA stimulated inward current in CA1 pyramidal cells from hippocampal slice cultures. In control cultures (solid circles), NMDA induced a clear inward current measured as holding current (Ihold). This current was strongly suppressed in cultures treated with DHPG (10 ␮M, 2 h, open circles). The difference between the DHPG-treated and control cultures reached significance after 5, 6, and 7 min. Data are shown as mean ⫾ SEM, *P ⬍ 0.05, **P ⬍ 0.01 using Student’s t-test with n ⫽ 3– 4 neurons from individual cultures per group.

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Discussion The results of the present study show that selective activation of group-I-mGluRs protect neurons against a subsequent excitotoxic stimulation of NMDA receptors in a concentration-dependent manner in hippocampal CA1 and CA3 subfields. This finding is consistent with previous results in organotypic hippocampal slice cultures, where a similar ACPD pretreatment induced neuroprotection (Adamchik and Baskys, 2000), confirming the important role of these receptors. DHPG-induced neuroprotection could be reversed by inhibition of the mGluR1 receptor with LY367385, but not by the mGluR5 receptor antagonist MPEP. Earlier studies with LY367385 show a lack of a discernible effect on NMDA receptors, which, taken together, suggest that the neuroprotective effects of DHPG were likely due to inhibition of group-I-mGluRs. Interestingly, the neuroprotective pretreatment with DHPG also significantly decreased the NMDA-stimulated inward currents in voltage-clamped CA1 pyramidal cells, suggesting that activation of group-I-mGluRs could lead to inhibition of NMDA-receptor function and although the nature of the current recorded in the voltage-clamp experiments was not investigated in this study, it most likely represents the NMDA receptor/channel mediated inward current (Collingridge and Lester 1989). Several possible mechanisms could underlie this inhibition. Moderate increases in [Ca2⫹] cause inactivation of NMDA receptors (Krupp et al., 1998; Tong et al., 1995) and activation of group-I-mGluRs may bring about such an increase in [Ca2⫹] (for review see Baskys, 1994). Other possible mechanisms may involve modifications of NMDA receptors that significantly reduce agonist binding (Bi et al., 1998), changes in NMDA receptor pentameric structure following mGluR stimulation (Nicoletti et al., 1999), or downregulation of NMDA receptors (Yu et al., 1997). Consistent with this, it has recently been shown that DHPG treatment results in internalization of NMDA and AMPA receptors in primary hippocampal neurons (Snyder et al., 2001), which could explain neuroprotection. That the mechanism by which DHPG induce neuroprotection is due to an increase in [Ca2⫹]i is supported by electrophysiological evidence showing that LY367385 blocks the DHPG-induced increase in [Ca2⫹]i and the direct depolarization in CA1 pyramidal cells, whereas MPEP blocks Ca2⫹ activated K⫹ currents and potentiation of NMDA receptors (Mannaioni et al., 2001). Taken together these data are consistent with the results of the present study and show that DHPG-mediated neuroprotection against NMDA toxicity occurs via the mGluR1 receptor and that neuroprotective DHPG treatment can greatly reduce NMDA-stimulated currents. A similar observation has been made in cerebellar granule cells, in which treatment with trans-ACPD or (R,S)-DHPG significantly reduces Ca2⫹ influx induced by application of NMDA or glutamate (Pizzi et al., 1996b) and in mouse cortical cultures where DHPG reduced NMDA whole-cell

currents (Yu et al., 1997). Moreover, it has been suggested that functional activity and the number of functional NMDA receptors are regulated by the strength of the glutamatergic input (Cebers et al., 2001). Group-I-mGluRs have been shown to increase glutamate release (Rodriguez-Moreno et al., 1998; Ye and Sontheimer, 1999) from both neurons and astrocytes, and as a result, increased concentration of ambient glutamate may decrease NMDA receptor-mediated effects. It should be noted that neuroprotection could only be found if the activation of group-I-mGluRs was prior to the NMDA exposure and no neuroprotection was observed if DHPG was applied for 2 h immediately after the insult. The conflicting results on the role of glutamate in neuroprotection and neurodegeneration, discussed in the introduction, may be due to the timing of agonist application. Coapplication of a group-I-mGluR agonist and NMDA may potentiate NMDA responses through phosphorylation of the NMDA receptors relieving the Mg2⫹ block and increasing Ca2⫹ influx through the associated channel to a toxic level (Bruno et al., 1995). Pretreatment with the group-I-mGluR agonist, however, would increase [Ca2⫹]i causing inhibition of NMDA receptors or resulting in their internalization, so that a subsequent exposure to NMDA would yield a decreased response. This idea is supported by our earlier finding that coapplication of NMDA with ACPD potentiated NMDA toxicity and that this potentiation was reversed by a 30-min pretreatment with ACPD before the coapplication (Blaabjerg et al., 2001). Another mechanism of mGluR-induced neuroprotection could be mGluR receptor desensitization. Due to a high intracellular concentration of glutamate, initial disintegration of neurons after the insult would result in a massive glutamate release that would activate extrasynaptic mGluRs, thus increasing Ca2⫹ influx and cell death. mGluRs, however, undergo rapid PKCmediated receptor desensitization upon agonist binding (Herrero et al., 1994; Gereau and Heinemann, 1998). It is not unlikely that, if mGluRs are activated prior to exposure to NMDA, the receptors may be desensitized and, therefore, respond weakly to the glutamate released by a subsequent toxic NMDA exposure, resulting in neuroprotection. Several studies have shown that activation of group-ImGluR appears to reduce apoptotic cell death (Allen et al., 2000; Kalda and Zharkovsky, 1999; Vincent and Maiese, 2000; Vincent et al., 1997) while potentiating necrotic cell death (Allen et al., 2000). Interestingly we observed pycnotic densely stained nuclei, with no caspase-3 activation, after NMDA treatment, corresponding to necrotic cell death. One study on mouse hippocampal slice cultures report NMDA-induced apoptosis independent of caspase-3 activation by measuring the intrahistonic DNA fragmentation (Djebaili et al., 2002). We cannot rule out that the same mechanism could be present in our model; however, we found no nuclear fragmentation 24 or 48 h after the NMDA exposure.

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In summary, this study strongly supports the role of mGluR1 in neuroprotection against NMDA-induced cell death. It also points to changes in NMDA receptor/channel function as a putative mechanism of such protection.

Acknowledgments This study was supported by the Codan Foundation, the Fuhrmann Foundation (M.B.), the FP5 EU Grant QLK3CT-2001-00407 (J.Z.), and Mental Illness Research and Education Clinical Center (MIRECC) (A.B.). The authors acknowledge the editorial services of Jennifer Kahle from BPS International as well as technical assistance from Dorte Bramsen, University of Southern Denmark, Odense.

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