QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.123182v1 322/2/443    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Deshpande, L. S. Articles by DeLorenzo, R. J. Search for Related Content PubMed PubMed Citation Articles by Deshpande, L. S. Articles by DeLorenzo, R. J.

NEUROPHARMACOLOGY

Activation of a Novel Injury-Induced Calcium-Permeable Channel That Plays a Key Role in Causing Extended Neuronal Depolarization and Initiating Neuronal Death in Excitotoxic Neuronal Injury

Departments of Neurology (L.S.D., S.S., R.J.D.), Pharmacology and Toxicology, (R.J.D.), and Biochemistry (R.J.D.), Virginia Commonwealth University, Richmond, Virginia; and Department of Neurosurgery, Washington University, School of Medicine, St. Louis, Missouri (D.D.L.)

Received March 21, 2007; accepted May 2, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Protracted elevation in intracellular calcium caused by the activation of the N-methyl-D-aspartate receptor is the main cause of glutamate excitotoxic injury in stroke. However, upon excitotoxic injury, despite the presence of calcium entry antagonists, calcium unexpectedly continues to enter the neuron, causing extended neuronal depolarization and culminating in neuronal death. This phenomenon is known as the calcium paradox of neuronal death in stroke, and it represents a major problem in developing effective therapies for the treatment of stroke. To investigate this calcium paradox and to determine the source of this unexpected calcium entry after neuronal injury, we evaluated whether glutamate excitotoxicity activates an injury-induced calcium-permeable channel responsible for conducting a calcium current that underlies neuronal death. We used a combination of whole-cell and single-channel patch-clamp recordings, fluorescent calcium imaging, and neuronal cell death assays in a well characterized primary hippocampal neuronal culture model of glutamate excitotoxicity/stroke. Here, we report activation of a novel calcium-permeable channel upon excitotoxic glutamate injury that carries calcium current even in the presence of calcium entry inhibitors. Blocking this injury-induced calcium-permeable channel for a significant time period after the initial injury is still effective in preventing calcium entry, extended neuronal depolarization, and delayed neuronal death, thereby accounting for the calcium paradox. This injury-induced calcium-permeable channel represents a major source for the initial calcium entry following stroke, and it offers a new target for extending the therapeutic window for preventing neuronal death after the initial excitotoxic (stroke) injury.

Conventional Ca²⁺ entry antagonists prevent neuronal death and END when administered before and during the injury phase of glutamate excitotoxicity (Coulter et al., 1992; Limbrick et al., 2001), but after excitotoxic insult has occurred, these Ca²⁺ entry antagonists are no longer effective in blocking the Ca²⁺ entry and in reducing the elevated [Ca²⁺]_i (the Ca²⁺ plateau). Thus, traditional Ca²⁺ entry antagonists do not prevent END (Limbrick et al., 2003), block neuronal death (Ikonomidou and Turski, 2002), or improve the outcome after the excitotoxic injury in stroke (Horn and Limburg, 2000). These observations have lead to the Ca²⁺ paradox of neuronal death in stroke, and they refer to the unexpected finding that conventional Ca²⁺ entry antagonists do not prevent Ca²⁺ entry or END after glutamate excitotoxicity (Lee et al., 1999; Horn and Limburg, 2001; Ikonomidou and Turski, 2002). Explaining the cause of the Ca²⁺ paradox of neuronal death in stroke is one of the important problems in neuroscience research, and it underlies the failure of many of the previous clinical trials for potential neuroprotective agents in stroke and brain injury (Lee et al., 1999; Horn and Limburg, 2001; Ikonomidou and Turski, 2002; Wahlgren and Ahmed, 2004). Our previous attempts at elucidating the Ca²⁺ paradox identified that an influx of extracellular Ca²⁺ was underlying the genesis of END. However, that study could not identify the source or nature of this postinjury Ca²⁺ entry (Limbrick et al., 2003). Therefore, it is important to explain this Ca²⁺ paradox and to understand the continued Ca²⁺ entry after injury despite the use of known Ca²⁺ entry inhibitors to develop novel and effective stroke and brain injury therapeutic agents.

In this study, using a well characterized in vitro hippocampal neuronal culture model of glutamate excitotoxicity/stroke, we investigated the cause of the Ca²⁺ entry after injury that is resistant to known Ca²⁺ entry inhibitors. Experiments were directed at evaluating the development of an injury induced Ca²⁺ current that underlies END and cell death that is not blocked by conventional Ca²⁺ entry inhibitors. Studies were also directed at demonstrating that this novel injury-induced Ca²⁺ current accounts for the Ca²⁺ paradox, because traditional Ca²⁺ entry inhibitors, including blockers for L-, N-, P/Q-, and T-type Ca²⁺ channels, NMDA/AMPA/KA channels, stretch-activated channels, and other injury-induced cation channels, such as the TRPM-7 and acid-sensing channels, did not block this Ca²⁺ entry. Furthermore, our results also demonstrate that there is a therapeutic window of opportunity of at least 1 h to block this current, decrease the elevated [Ca²⁺]_i, reverse END, and prevent neuronal death. The development of a novel injury-induced Ca²⁺-permeable channel provides a molecular basis for the postinjury Ca²⁺ entry current responsible for producing END (Limbrick et al., 2003), and it explains why many of the therapeutic trials using conventional strategies to inhibit Ca²⁺ entry have not been effective in treating stroke. Activation of this injury-induced Ca²⁺-permeable channel represents an early step in the cascade leading to excitotoxic neuronal death, and it offers potential insight into developing novel therapeutic interventions to prevent brain injury from stroke.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. All the reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise noted. -Methyl-4-carboxyphenyl glycine (mCPG), SKF-96365, and tetrodotoxin (TTX) were obtained from Tocris Cookson Inc. (Ballwin, MO), Calbiochem (San Diego, CA), and Alomone Labs (Jerusalem, Israel), respectively. 4,4'-Diiso-thiocyanatostilbene-2,2'-disulfonic acid (DIDS), N-nitro-L-arginine methyl ester (L-NAME), and amiloride hydrochloride (AMILO) were purchased from Sigma-Aldrich. Minimal essential medium, L-glutamine, trypsin, penicillin-streptomycin, fetal bovine serum, and horse serum used in the tissue culture preparation were obtained from Invitrogen (Carlsbad, CA).

Hippocampal Neuronal Cultures. All animal use procedures were in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by Virginia Commonwealth University's Institutional Animal Care and Use Committee. Cultured hippocampal neurons were prepared as described previously from 2-day postnatal Sprague-Dawley rats (Harlan, Frederick, MD) with slight modifications (Sombati et al., 1991; Coulter et al., 1992; Limbrick et al., 2001). Cultures were fed thrice weekly with neuronal feed, maintained at 37°C in a 5% CO₂, 95% air atmosphere, and used after 14 days in vitro.

Electrophysiology. Whole-cell patch-clamp analyses were performed as described using an Axopatch 200A amplifier or an Axo-patch 1D amplifier (Molecular Devices, Sunnyvale, CA) in voltage-clamp mode (Hamill et al., 1981; Coulter et al., 1992). Membrane potential was sequentially stepped from–90 to +60 mV from a steady holding potential of–60 mV. Voltage steps were 50 ms in duration and applied at a frequency of 0.2 Hz. Current responses were sampled at 20 kHz and low-pass filtered at 1 kHz using a four-pole Bessel filter (Frequency Devices, Haverhill, MA). The recording solution contained 145 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM CaCl₂, and 1 mM MgCl₂, pH 7.3 (290 ± 10 mOsM). The pipette solution contained 140 mM K⁺ gluconate, 10 mM HEPES, 1.1 mM EGTA, and 1 mM MgCl₂, pH 7.2 (290 ± 10 mOsM). Depending upon the experiments, various inhibitors were included in the recording solution. In establishing the whole-cell configuration, gigaseal formation was verified, pipette capacitance was canceled, and gentle suction was applied. Cells that required >3 applications of suction for whole-cell access were discarded. Once whole-cell access was established, whole cell capacitance was canceled. Series resistance was generally 4 to 9 M, but it was reduced by 75 to 80% using the compensation circuit of the amplifier. Series resistance error was generally between 3 and 5 mV (but always <9 mV).

Cell-attached single-channel recordings were performed as described previously (Hamill et al., 1981). Fire-polished Slygard (Dow Corning, Midland, MI)-coated borosilicate glass pipettes had a resistance of 7 to 10 m when filled with recording solution containing 145 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 10 mM glucose, 2 mM CaCl₂, and 1 mM MgCl₂, pH 7.3 (290 ± 10 mOsM). Depending upon the experiment, various inhibitors were included in the pipette solution. Neurons were bath perfused with a "high-K⁺" solution in which the extracellular KCl concentration was raised from 2.5 to 40 mM to clamp the resting membrane potential near 0 mV. The patch was voltage-clamped at various voltages by applying a voltage of opposite sign to the patch pipette, and the recording was started. Current amplification was accomplished with an Axopatch 200A amplifier and recorded on a VHS tape via a Neurocorder (Neurodata, NY) using pClamp9 via Digidata 1322A (Molecular Devices). Data were sampled at 10 kHz and filtered at 2 kHz.

Excitotoxic Glutamate Exposure. Excitotoxic injury was induced as described previously (Choi et al., 1987; Michaels and Rothman, 1990; Sombati et al., 1991; Coulter et al., 1992; Dubinsky, 1993; Limbrick et al., 1995). Bath application of glutamate was performed by gravity-feed perfusion at a rate of 1 ml/min, and solution changes were controlled through a six-valve perfusion system (Warner Instruments, Hamden, CT). Glutamate (500 µM) was dissolved in recording solution and applied with 10 µM glycine for 10 min. Glutamate washout was performed with control recording solution (2 mM CaCl₂) or Ca²⁺-free recording solution (0 mM CaCl₂; omitting CaCl₂ but contained no Ca²⁺ chelator), or high-Ca²⁺ recording solution (10 mM CaCl₂; equimolar replacement of NaCl), or Na⁺-free recording solution [equimolar substitution of N-methyl-D-glucamine (NMDG) chloride for NaCl].

Calcium Microfluorometry. Fura-2 acetoxymethyl ester was loaded in the neurons and then transferred to a heated stage (37°C) of an Olympus IX-70 inverted microscope (Olympus, Tokyo, Japan) coupled to an ultrahigh-speed fluorescence imaging system (Olympus/PerkinElmer Life and Analytical Sciences, Boston, MA) (Dubinsky, 1993; Limbrick et al., 1995). Ratio images were acquired by using alternating excitation wavelengths (340/380 nm) with a filter wheel (Sutter Instrument Company, Novato, CA) and Fura filter cube at 510/540-nm emissions with a dichroic mirror at 400 nm. Image pairs were captured and digitized every 15s, and the images at each wavelength were averaged over four frames and corrected for background fluorescence by imaging a nonindicator-loaded field.

Cell Death Assay. Neuronal death was characterized using the Vybrant apoptosis assay kit 3 (Invitrogen) (Raza et al., 2001). Cells were treated with fluorescein isothiocyanate Annexin-V (5 µl/100 µl of total volume) and 1 µl of 100 µg/ml propidium iodide (PI) solution. After 15 min, 400 µl of 1x Annexin binding buffer was added, and cells were visualized. Fraction of neuronal death was calculated as fraction dead = (dead_treat–dead_control)/dead_glutamate.

Data Analysis. Averaging the final 17 ms of each step to minimize any effect of 60-Hz noise generated steady-state current-voltage (I-V) relationships. Permeability ratios were calculated using the Goldman-Hodgkin-Katz voltage equation. Statistical differences in the magnitude of current responses or END potentials or cell death were tested using a one-way analysis of variance followed by post hoc Tukey's test. Single-channel analyses were accomplished using Clampfit 9 (Molecular Devices). Statistical tests were run using SigmaStat 2.0, and graphs were generated with SigmaPlot 8.0 (both from SPSS Inc., Chicago, IL).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Excitotoxic Glutamate Injury Produces a Persistent Inward Excitotoxic Injury Current (I_EIC). To investigate the molecular basis of END, whole-cell recordings were conducted on control and glutamate-injured neurons in the presence of a mixture of Ca²⁺ entry inhibitors that were demonstrated to inhibit voltage-gated Ca²⁺ channels (5 µM nifedipine), NMDA channel (10 µM MK-801), AMPA/KA channels (10 µM CNQX), the TRP channel family, and stretch-activated ion channels (10 µM GdCl₃) (Sattler et al., 1998; Aarts et al., 2003). Recordings in the presence this Ca²⁺ entry inhibitor cocktail have been shown by our laboratory and others (Sattler et al., 1998; Aarts et al., 2003) to inhibit the standard mechanisms of Ca²⁺ entry and to provide the ability to investigate the presence or absence of Ca²⁺ entry following excitotoxic injury.

Control neurons exhibited a membrane potential of –56.7 ± 1.3 mV and mean input resistance of 240.7 ± 32.5 M (n = 10; Table 1). Whole-cell voltage-clamp recordings from control neurons in the presence of cocktail of Ca²⁺ entry inhibitors showed no steady-state inward currents (Fig. 1A). Outward currents consistent with activation of voltage-dependent K⁺ channels were observed in control neurons at voltages positive to–40 mV, and they were sensitive to K⁺ channel blockers (4-aminopyridine and charybdotoxin). In contrast, excitotoxic glutamate injured neurons in the presence of Ca²⁺ entry inhibitors mixture manifested END (Sombati et al., 1991; Coulter et al., 1992) and exhibited a membrane potential of –14.0 ± 1.6 mV and an input resistance of 26.6 ± 3.4 M that were statistically different from control neurons (p < 0.001 for both; Table 1). Neurons in END revealed a persistent I_EIC (Fig. 1A). We contend that this persistent inward current represents the basis of the Ca²⁺ paradox. The peak inward current for the END conditions in the presence of Ca²⁺ entry inhibitors was–3934.6 ± 635.5 pA (n = 12). Steady-state outward currents in excitotoxic glutamate-injured neurons were identical in magnitude to those observed in control neurons. The net I-V relationships (Aarts et al., 2003) for glutamate-injured neurons (total EIC minus control; Fig. 1B) were studied in the remaining experiments. This net inward current had a reversal potential (E_rev) of approximately +40 mV.

View larger version (29K): [in this window] [in a new window]   Fig. 1. Induction of an inward current, IEIC, following glutamate excitotoxicity. Current traces (A) and I-V relationship (B) from a representative control (n = 10) or END (n = 12) neuron. Note the large inward current, IEIC, observed in END neuron that is not blocked by standard Ca2+ entry inhibitors. C, representative trace showing induction of single-channel activity in cell-attached patches and I-V relationship from excitotoxic glutamate-injured neurons. Glutamate-injured neurons demonstrated robust single-channel activity in the presence of cocktail of Ca2+ entry inhibitors. No single-channel events were observed in control neurons (n = 6). D, excitotoxic glutamate-induced channel had a slope conductance of 48.5 ± 4.2 pS. I-V curves (E) and single-channel activity (F) for omission of [Ca2+]e or treatment with 100 µM Gd3+ (n = 7). These treatments abolished IEIC channel activity in the excitotoxic glutamate-injured neurons.

Cell-attached single-channel recordings from glutamate-injured neurons demonstrated distinct openings and closings of an ion channel that existed in the presence of Ca²⁺ entry inhibitors and corresponded to the appearance of I_EIC during END (Fig. 1C). This channel exhibited an essentially linear I-V relationship with a unitary conductance of 49.5 ± 5.2 pS (Fig. 1D) (n = 7). Conversely, control neurons demonstrated no channel activity in the presence of Ca²⁺ entry inhibitors.

We recently showed that END could be reversed by application of 100 µM GdCl₃ or removal of [Ca²⁺]_e after glutamate excitotoxicity (Limbrick et al., 2003). To investigate whether activation of I_EIC and the observed channel were responsible for mediating END, we recorded whole-cell and single-channel currents under these conditions (Limbrick et al., 2003). Application of 100 µMGd³⁺ or removal of [Ca²⁺]_e in the postglutamate END period abolished the inward current and restored the I-V profile identical to that of a control neuron (Fig. 1, E and F). In addition, Gd³⁺-treated neurons (n = 14) and neurons recorded in absence of [Ca²⁺]_e (n = 11) had a mean membrane potential of –52.4 ± 3.9 and –49.6 ± 3.4 mV, respectively. The mean input resistance for these two conditions was 246.3 ± 41.3 and 257.4 ± 39.7 M, respectively. These parameters were not significantly different from control neurons (p = 0.075 and 0.745, respectively; Table 1). Both the inward current and channel activity were not voltage-dependent over the range of voltages studied. Furthermore, the current was not inhibited by cocktail of Ca²⁺ entry inhibitors, ruling out the involvement of voltage-dependent Ca²⁺ channels. Together, these results indicate that excitotoxic glutamate exposure produces END and activates a novel channel activity that carries I_EIC in injured neurons.

Calcium Ions Are the Primary Permeant Ions for I_EIC and the I_EIC Channel. To confirm the ionic basis of END, I_EIC, and the channel-mediating I_EIC, major cations were sequentially replaced in the recording solution. Equimolar substitution of NaCl with NMDG failed to reverse END (Fig. 2A), they did not affect the magnitude of I_EIC (Fig. 2B), and they had no effect on channel activity (Fig. 2C). In addition, removal of Na⁺ did not affect the membrane potential (–9.4 ± 2.0 mV) or input resistance (38.3 ± 5.2 M), compared with END neurons (p = 0.101 and 0.093, respectively; n = 16; Table 1). These results indicate that Na⁺ influx did not significantly contribute to I_EIC.

View larger version (29K): [in this window] [in a new window]   Fig. 2. IEIC is highly selective for Ca2+. A, representative current-clamp traces show that [Ca2+]e but not [Na+]e was required for the maintenance of END. B, I-V curves for omission of [Ca2+]e or [Na+]e. Omission of Ca2+ (n = 11) but not Na+ (n = 12) after glutamate excitotoxicity abolished IEIC (n = 10). C, excitotoxic glutamate injury-induced ion channel is Ca2+-selective. Representative single-channel traces demonstrate no channel events in the absence of Ca2+ in the pipette (n = 6), whereas omission of Na+ maintained channel activity (n = 6). D, direct measurements of [Ca2+]i, expressed as Fura-2 ratios, demonstrate that removal of [Ca2+]e (n = 8) but not [Na+]e (n = 7) reversed elevated [Ca2+]i (n = 9) following glutamate excitotoxicity. E, I-V curves for changes in [Ca2+]e. Lowering [Ca2+]e decreased the magnitude of IEIC (n = 9). Conversely, raising [Ca2+]e increased the magnitude of IEIC (n = 12). F, varying [Ca2+]e caused shifts in the net Erev according to Nernstian predictions.

To further establish that Ca²⁺ influx was responsible for END and that it was the major ion permeating the I_EIC channel, we measured [Ca²⁺]_i using Fura-2 imaging. Substituting NaCl with NMDG had no effect on [Ca²⁺]_i. Conversely, removing [Ca²⁺]_e reversed the elevated [Ca²⁺]_i observed after glutamate excitotoxicity (Fig. 2D).

The Ca²⁺ dependence of I_EIC was then evaluated by investigating the effects of variable [Ca²⁺]_e on magnitude of I_EIC. The net peak inward currents reduced from–3932.3 ± 635.5 to–1119.7 ± 327.4 pA at –90 mV during 2 and 0.5 mM [Ca²⁺]_e (Fig. 2E), and it also caused a significant shift in the net E_rev from +40.5 ± 2.2 to –20.6 ± 2.0 mV, respectively. This value agreed with the theoretical E_rev value of approximately –22 mV determined by the Nernst equation for 0.5 mM [Ca²⁺]_e, and it resulted in the predicted linear relationship of E_rev versus log₁₀ [Ca²⁺_]e (Fig. 2F). The _^membrane potential for neurons in 0.5 mM CaCl₂ was –25.9 ± 4.6 mV, and the input resistance was 59.1 ± 6.9 M. These values represent significant changes from those measured in 2 mM CaCl₂ for END (p = 0.013 and < 0.001, respectively; n = 9; Table 1).

Conversely, increasing [Ca²⁺]_e from 2 to 10 mM resulted in an increased net peak inward current of –5590.8 ± 457.8 pA (Fig. 2E), and it resulted in a membrane potential of –1.5 ± 2.4 mV and an input resistance of 22.9 ± 1.7 M. Increasing [Ca²⁺]_e to 10 mM caused a shift in the net E_rev from +40.5 ± 2.2 to +50.1 ± 4.2 mV. The observed shift of E_rev with 10 mM CaCl₂ was slightly less than the predicted E_rev (approximately +62 mV), and it may be due to saturation of E_rev when plotted against log₁₀ [Ca²⁺]_e (Fig. 2F). This observation is consistent with the concept of permeant ion rectification, where saturation of I_EIC occurs with respect to [Ca²⁺] (Hille, 2001). Thus, I_EIC followed Nernstian predictions in response to changes in [Ca²⁺]_e.

Taken together, the following evidence supports the conclusion that the injury induced Ca²⁺-permeable channel is responsible for carrying the I_EIC that mediates END: 1) activation of the ion channel in the postglutamate END period coincides with the appearance of I_EIC; 2) END, I_EIC, and the Ca²⁺-permeable channel demonstrate identical ionic selectivity and are abolished in the absence of [Ca²⁺]_e; 3) END, I_EIC, and the Ca²⁺-permeable channel all manifest identical insensitivity to blockade by Ca²⁺ entry inhibitors mixture; and 4) they all are inhibited by higher concentrations of Gd³⁺. These data provide the first direct evidence of a novel Ca²⁺-permeable channel that can clarify the Ca²⁺ paradox and explain the persistent entry of Ca²⁺ despite the use of Ca²⁺ entry inhibitors.

Traditional Routes of Ca²⁺ Entry or Internal Stores Do Not Mediate I_EIC. We used extensive pharmacological studies to establish that the I_EIC-Ca²⁺-permeable channel represented a new route of Ca²⁺ entry. END neurons are characterized by membrane potentials of approximately –15 to –20 mV. Activation of voltage-gated Ca²⁺ channels (VGCCs) is expected to occur at these potentials. Thus, the observed injury-mediated channel activity could be due to the activation of the voltage-gated Ca²⁺ channels. To investigate contribution of VGCCs to END, a combination of effective concentrations of L-type VGCC antagonist (5 µM nifedipine), N-type VGCC antagonist (1 µM -conotoxin GVIA), P/Q-type VGCC antagonist (100 nM -conotoxin MVIIC), or T-type VGCC antagonist (1 mM ethosuximide), along with other components of Ca²⁺ entry inhibitor mixture (such as MK-801 and CNQX), was used to wash out glutamate. Because alterations in VGCC gating could also occur during or after excitotoxic glutamate exposure, we included 200 µM CoCl₂ in the inhibitor combination. This combination had no effect on END membrane potentials, suggesting that the VGCCs are not mediating the injury-induced ion channel activity (Fig. 3A). Most of the cation channels, including the stretch-activated channels and other Ca²⁺ channels are completely or maximally blocked at GdCl₃ concentrations of 10 µM (Caldwell et al., 1998). However, this concentration did not inhibit END or decrease I_EIC or single-channel activity after glutamate excitotoxicity. Thus, although use of 10 µM GdCl₃ inhibited these other channels under our conditions, it had no effect on END. In fact, END and related physiological processes were abolished only when the Gd³⁺ concentration was raised to 100 µM. This is a very large difference in concentration, and it provides a major distinction. This dose-dependent effect of Gd³⁺ clearly differentiates the Ca²⁺ channel observed in our study from the traditional GdCl₃-sensitive Ca²⁺ channels previously identified. In addition, voltagegated Ca²⁺ channels undergo inactivation rapidly after the onset of depolarization. But, we observed a persistent channel activity for a prolonged period in the depolarized END phase. Moreover, VGCCs are characterized by low single-channel conductance. Taken together, we can conclude that VGCCs are not the mediators of the glutamate injury induced Ca²⁺-permeable channel.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Evidence that IEIC accounts for the Ca2+ paradox of excitotoxic neuronal death. A, mean membrane potentials for control (CTRL), END, and END + test agents conditions: Ca2+ entry inhibitors (CEIs): MK-801, CNQX, nifedipine, and 10 µMGd3+ along with -conotoxin and ethosuximide), mCPG, TTX, bepridil (BEP), DIDS, SKF-96365, ATP, L-NAME, AMILO, 10 Gd3+, and 5 Zn2+ (n = 6). Treatment with these agents after glutamate excitotoxicity did not abolish END. B, treatment after glutamate injury with 0 Ca2+, 100 Gd3+, and 500 Zn2+ but not 0 Na+ (n = 5) inhibited END. C, treatment with 100 µMGd3+ was neuroprotective and prevented neuronal cell death. Conversely, inhibition of TRPM-7 (L-NAME and 10 µMGd3+) or ASICs (AMILO) had no effect on fraction of dead cell count. D and E, blockade of the IEIC-Ca2+-permeable channel at different times after glutamate excitotoxicity demonstrates an the extension of the therapeutic window for preventing cell death and the postinjury [Ca2+]i plateau. Glutamate-injured neurons were treated with 100 µMGd3+ (D) or 0-Ca2+ solutions (E) starting at 0 h and up to 4 h after glutamate excitotoxicity. The percentage of neurons undergoing cell death or returning to basal [Ca2+]i ratios was measured (n = 6). Data are expressed as mean ± S.E.M. For A and B, asterisks denote differences from control and from glutamate (GLU) for C, D, and E (*, p < 0.05). Color scheme: black, control; white, END/glutamate; and gray, inhibitors.

The bivalent metal cation zinc (Zn²⁺) is known to regulate a number of ligand-gated, voltage-gated, and nonselective cation channels, several of which gate Ca²⁺ entry into the neurons (Christine and Choi, 1990; Chen et al., 1997). In particular, Zn²⁺ blocks NMDA and AMPA receptors and most types of VGCCs. However, Zn²⁺ at a lower 5 µM concentration, a concentration that was also greater than the dissociation constant of Zn²⁺ at the NMDA receptor Zn²⁺ site (Christine and Choi, 1990), had no effect on the postglutamate membrane potential (Fig. 3A). Conversely, inclusion of 500 µM ZnCl₂ following removal of glutamate allowed a rapid and complete repolarization to resting membrane potential (Fig. 3B). Thus, the ability of ZnCl₂ to reverse END was independent of its effects on NMDA receptors.

Other mechanisms of ionic entry were also tested. Inhibiting the forward and reverse mode of Na⁺/Ca²⁺ exchanger (50 µM bepridil or [Na⁺]_o removal), chloride channels (100 µM DIDS), and stretch receptors channels (10 µM GdCl₃) also had no effect on diminishing the END potentials after glutamate excitotoxicity (Fig. 3A). In addition, the strict ionic selectivity, the differential permeability of I_EIC, and the fact that changes in [Ca²⁺]_i could be measured throughout our experiments demonstrated that I_EIC was not the result of a nonspecific ion leak or membrane disruption following glutamate excitotoxicity.

Studies were also done to evaluate the contribution of internal Ca²⁺ stores that are known to play a major role in [Ca²⁺] homeostasis (Duchen, 2000). Blocking store-operated Ca²⁺ channels (10 µM SKF-96365), ryanodine receptors (20 µM dantrolene), or inositol trisphosphate receptors (2 mg/ml heparin) or intracellular Ca²⁺ release inhibitor (1 µM thap-sigargin) (data not shown) had no effect on reducing END. Compensating for mitochondrial injury by addition of an ATP-regenerating system (4 mM ATP and 22 mM phospho-creatine) or by inhibiting mitochondrial Ca²⁺ release (10 µM rhodamine; data not shown) also had no effect on reducing END (Fig. 3A). Indeed, mitochondria maintain their resting level for Ca²⁺ for about 45 min after glutamate excitotoxicity, even in the face of rising cytosolic Ca²⁺ levels (Bano et al., 2005). Moreover, mitochondrial respiration is retained for a relatively long time in cerebellar neurons undergoing excitotoxicity (Jekabsons and Nicholls, 2004), and the final mitochondrial Ca²⁺ deregulation and the permeability transition were downstream rather than upstream of the secondary Ca²⁺ overload following glutamate excitotoxicity (Bano et al., 2005). Although intracellular Ca²⁺ stores and other cation conductances may play role in ischemia-induced [Ca²⁺]_i accumulation, our results demonstrate that I_EIC is responsible for majority of the early Ca²⁺ influx after glutamate excitotoxicity.

Neuroprotection with Gadolinium: Evidence That I_EIC Accounts for the Ca²⁺ Paradox. To test whether activation of I_EIC could explain the Ca²⁺ paradox, we investigated whether blocking this channel could prevent neuronal death after glutamate excitotoxicity. Treatment with 100 µMGd³⁺ produced a significant reduction in the number of Annexin-positive cells (Figs. 3C and 4), and it also conferred neuroprotection when administered 1 h after excitotoxic injury (Fig. 3D). Furthermore, with Gd³⁺ intervention, up to 50% neuroprotection was observed even out to 2 h after glutamate excitotoxicity (Fig. 3D).

View larger version (116K): [in this window] [in a new window]   Fig. 4. Effect of Ca2+ removal and 100 µM GdCl3 treatment on [Ca2+]i dynamics and neuronal death after excitotoxic glutamate injury. The right side panels show fluorescent Fura-2 Ca2+ images from the same field of neurons before glutamate exposure (A), during END (B), and upon removal of [Ca2+]e (C). As depicted in the fluorescent scale bar, blue/green color indicates a low 340/380 ratio that corresponds to low [Ca2+]i. Conversely, red indicates a high 340/380 ratio that corresponds to elevated [Ca2+]i. As shown in the fluorescent images, control (A) is characterized by predominantly bluish green neurons. Upon glutamate exposure and during END, the majority of the neurons show bright red color in the cytoplasm, which corresponds to elevated [Ca2+]i. When [Ca2+]e is removed after excitotoxic injury, IEIC is blocked, which results in restoration of elevated [Ca2+]i, now characterized by greenish color in the majority of neurons (C). The right panels show fluorescent images from a cell death assay using the Annexin and PI stains. Annexin-fluorescein isothiocyanate identifies apoptotic cells by binding to exposed phosphatidyl-serine and emitting a green fluorescence. PI is impermeant to live cells and apoptotic cells, but it stains dead cells with red fluorescence, binding tightly to the nucleic acids in the cell. Viable neurons are the phase-bright neurons. The top panel on right side shows a fluorescent image from a control plate stained with Annexin-PI. There are only a few neurons emitting green or red fluorescence, indicating low cell death. END (B) is characterized by a preponderance of neurons emitting green/red fluorescence, showing significant cell death. However, treatment with 100 mM GdCl3 (C) after glutamate injury blocks END and attenuates neuronal death as characterized by only a few neurons emitting green fluorescence, demonstrating significant neuroprotection.

Fura-2-imaging experiments demonstrated that injured neurons restored excitotoxic glutamate induced elevated [Ca²⁺]_i to basal levels for up to 1 h after removal of [Ca²⁺]_e (Figs. 3E and 4). However, beyond this time point, the percentage of neurons that could restore basal [Ca²⁺]_i levels started to decrease despite absence of [Ca²⁺]_e. These results demonstrate that there is a window of opportunity for neuroprotection of up to 1 h after glutamate excitotoxicity, during which it is possible to reverse the increased [Ca²⁺]_i while the I_EIC channel is the major source of elevated [Ca²⁺]_i. In addition, as shown in Fig. 1A, injured neurons revealed I_EIC despite the presence of Ca²⁺ entry inhibitors. Thus, these data provide an explanation for the Ca²⁺ paradox by demonstrating that blocking I_EIC-Ca²⁺-permeable channel was able to prevent Ca²⁺ entry and inhibitor-resistant Ca²⁺ entry and reverse END, and inhibit cell death.

Comparison of I_EIC to Other Injury-Induced Cation Currents. It is important to compare I_EIC to other nonselective cation currents that have been observed in association with different types of neuronal injury. Recently, TRPM-7 channels were shown to carry an anoxia-induced cation current (I_OGD) in cortical neurons (Aarts et al., 2003). Two hours of anoxia induced I_OGD, which was inhibited by 300 µM L-NAME or 10 µM GdCl₃ and increased in magnitude upon [Ca²⁺]_e removal (Aarts et al., 2003). In contrast, induction of I_EIC was rapid, and treatments with L-NAME or 10 µM GdCl₃ during and after glutamate excitotoxicity failed to reverse END (Fig. 3A), inhibit Ca²⁺ influx (data not shown), or prevent neuronal death (Fig. 3C). Moreover, I_EIC decreased in magnitude upon [Ca²⁺]_e removal. Thus, based on the kinetic characteristics and pharmacological comparisons, it seems that TRPM-7 channels are not responsible for mediating I_EIC.

Acidosis from ischemia induces the activation of amiloride-sensitive high Na⁺-low Ca²⁺-permeable cation channels (ASICs) (Xiong et al., 2004). Amiloride (100 µM) or its derivative, bepridil (50 µM), had no significant effect on END (Fig. 3A), they failed to block Ca²⁺ influx (data not shown), and they did not prevent cell death after glutamate excitotoxicity (Fig. 3C). Furthermore, the activation of ASICs was unlikely because our perfusion conditions prevented the development of acidic conditions, demonstrating that ASICs are not responsible for mediating I_EIC.

Excitotoxic injury with NMDA can also induce a postexposure current (I_pe) that was shown to be not selective for Ca²⁺ (P_Ca:P_Na = 7:1) and not altered by removal of [Ca²⁺]_e in acutely isolated hippocampal neurons (Chen et al., 1997). In contrast, steady-state I_EIC has a high Ca²⁺ selectivity (P_Ca: P_Na = 50:1), and it is abolished upon omission of [Ca²⁺]_e, demonstrating that I_pe is not responsible for mediating I_EIC.

Taken together, these results demonstrate that TRPM-7, ASIC, or I_pe are not significantly contributing to the initial Ca²⁺ entry or END during the first hour of the postglutamate treatment paradigm and that I_EIC is a unique Ca²⁺ current that is mediated by a novel Ca²⁺-permeable channel.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study demonstrates that glutamate excitotoxicity activates a previously undetected Ca²⁺-permeable channel in cultured hippocampal neurons that carries the injury-induced current (I_EIC), primarily responsible for the initial sustained increases in [Ca²⁺]_i following neuronal injury that maintains END and ultimately leads to neuronal death. Inhibiting this I_EIC-Ca²⁺-permeable channel within the window of opportunity after glutamate excitotoxicity reversed END, blocked Ca²⁺entry, and prevented delayed neuronal death. To our knowledge, the development of the Ca²⁺ entry inhibitor-resistant I_EIC-Ca²⁺-permeable channel following glutamate excitotoxicity provides the first molecular insight into the Ca²⁺ paradox, and it explains why many of the clinical trials using conventional strategies to inhibit Ca²⁺ entry have not been effective in treating excitotoxic neuronal injury and stroke (Ikonomidou and Turski, 2002; Wahlgren and Ahmed, 2004).

Numerous studies have demonstrated a causal relationship between ischemia induced neuronal death and [Ca²⁺]_i accumulation (Kristián and Siesjö, 1998). Thus, a critical question relates to the source of this Ca²⁺. Whereas intracellular Ca²⁺ stores and other cation conductances may play some role in ischemia induced Ca²⁺ elevations, our data demonstrate that the activation of the I_EIC-Ca²⁺-permeable channel is responsible for the majority of the Ca²⁺ influx during the 1st hour after glutamate excitotoxicity. During this 1-h time window, intervention with Gd³⁺ or removal of [Ca²⁺]_e can restore elevated [Ca²⁺]_i. However, beyond this time point, in addition to I_EIC other irreversible ionic mechanisms are activated, including Ca²⁺ release from mitochondria, from intracellular stores, and from alterations in Ca²⁺ homeostatic mechanisms such that it is no longer possible to lower the elevated [Ca²⁺]_i, despite removal of [Ca²⁺]_e.

The activation, but not the maintenance of END and the I_EIC-Ca²⁺-permeable channel is dependent upon NMDA receptor activation during excitotoxicity (Limbrick et al., 2003), since the presence of the NMDA channel inhibitor MK-801 during glutamate injury prevents cell death and END. In contrast, MK-801 administered after excitotoxic injury did not prevent cell death, inhibit Ca²⁺ entry during END, or inhibit the I_EIC-Ca²⁺-permeable channel. In addition, depolarization induced Ca²⁺ entry alone did not produce END (Coulter et al., 1992). Treatments with high concentrations of potassium chloride or substitution of [Ca²⁺]_e with [Ba²⁺]_e during glutamate excitotoxicity both caused neuronal depolarization, but they did not cause the induction of END (Coulter et al., 1992). These findings indicate that the I_EIC-Ca²⁺-permeable channel is activated by an NMDA/Ca²⁺ mechanism. Since I_EIC could be observed immediately following 10-min glutamate stimulation, it is unlikely that I_EIC represents a newly synthesized channel, since this may be too rapid a time frame for de novo protein synthesis and insertion into the membrane. It is more likely that excitotoxic stimulation would trigger second messenger cascades, leading to post-translational modifications of existing membrane proteins or activation of a dormant channel to activate I_EIC. Indeed, NMDA-dependent changes in protein phosphorylation (Churn et al., 1995; Durkin et al., 1997), protease activity such as the calpains (Minger et al., 1998; Simpkins et al., 2003), and numerous other second messenger effects have been reported during glutamate excitotoxicity. Such alterations in key proteins or enzyme activities could result in the activation of the I_EIC-Ca²⁺-permeable channel. Future studies are planned to investigate these possibilities and to elucidate the molecular basis of the activation of the I_EIC-Ca²⁺-permeable channel using both in vitro and in vivo models of stroke and brain injury.

Protracted Ca²⁺ increases upon excitotoxicity persist well beyond the period of glutamatergic injury (Dubinsky, 1993; Limbrick et al., 1995) and cause diverse pathophysiological changes, including generation of free radicals, neuronal acidity, activation of proteases all of which can trigger neurodegenerative processes (Lipton, 1999). These sustained elevations in [Ca²⁺]_i represent a prolonged imbalance in Ca²⁺ homeostasis, and they correlate with subsequent excitotoxic neuronal death (Dubinsky, 1993; Limbrick et al., 1995). This [Ca²⁺]_i deregulation could result either from a persistent influx of [Ca²⁺]_e and/or from sustained impairment of neuronal Ca²⁺ sequestration/extrusion mechanisms. Indeed, recent discoveries that TRPM-7 (Aarts et al., 2003) and ASICs (Xiong et al., 2004) allow for Ca²⁺ entry upon hypoxia-ischemia and that the plasma membrane Na⁺/Ca²⁺ exchanger undergoes cleavage upon excitotoxicity (Bano et al., 2005) suggests that both these possibilities exist. These observations demonstrate the importance of elucidating the mechanisms underlying the sustained [Ca²⁺]_i elevations following glutamate excitotoxicity.

Earlier attempts to elucidate the basis of the postexcitotoxic injury-induced Ca²⁺ paradox indicated that an influx of extracellular Ca²⁺ was responsible for END (Limbrick et al., 2003). However that study did not identify the source or nature of this postinjury Ca²⁺ entry. The data presented here are a major advance over this earlier work, and they demonstrate that a unique Ca²⁺ current is activated upon excitotoxic injury that is the molecular basis for the influx of extracellular Ca²⁺ responsible for END. This study not only documents the existence of this novel Ca²⁺-permeable channel but also provides a careful pharmacological characterization of the channel and differentiates it from other types of calcium channels reported in the literature. Our findings demonstrate that the I_EIC-Ca²⁺-permeable channel is activated by glutamate excitotoxicity and blocking its activity after the excitotoxic insult prevents [Ca²⁺]_i accumulation and neuronal cell death. These findings suggest that activation of I_EIC-Ca²⁺-permeable channel could represent an early step in the genesis of the injury induced [Ca²⁺]_i plateau. The possible identification of a novel molecular target compliant to pharmacological manipulations opens exciting avenues for the treatment of acute and chronic neurological disorders associated with glutamate excitotoxicity. Elucidation of the I_EIC may provide a new target for a significant extension of the therapeutic window to prevent neuronal death in stroke and offer new hope in the search for novel agents to treat stroke and excitotoxic brain injury.

	Acknowledgements

 
We thank our research colleagues, Drs. Clive Baumgarten, Leslie Satin, William Dewey, David Sun, and Robert Blair, for critical suggestions. We thank Elisa Attkinson for preparation and maintenance of neuronal cultures.

	Footnotes

 
This work was supported by National Institute of Neurological Disorders and Stroke Grants R01NS051505, R01NS052529, and U01 NS058213, the Milton L. Markel Alzheimer's Disease Research Fund, and the Sophie and Nathan Gumenick Neuroscience Research Fund (to R.J.D.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.123182.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; END, extended neuronal depolarization; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; KA, kainic acid; mCPG, -methyl-4-carboxyphenyl glycine; SKF-96365, 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]-ethyl]imidazole; TTX, tetrodotoxin; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; L-NAME, N-nitro-L-arginine methyl ester; AMILO, amiloride hydrochloride; NMDG, N-methyl-D-glucamine; PI, propidium iodide; I-V, current-voltage; I_EIC, excitotoxic injury current; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); VGCC, voltage-gated calcium channels; APV, D(–)-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; NBQX, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide; NOS, nitricoxide synthase; ASIC, acid-sensing ion channel; I_OGD, anoxia-induced cation current; I_pe, postexposure current.

Address correspondence to: Dr. Robert J. DeLorenzo, Virginia Common-wealth University, School of Medicine, P.O. Box 980599, Richmond, VA 23298. E-mail: rdeloren{at}hsc.vcu.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, and Tymianski M (2003) A key role for TRPM7 channels in anoxic neuronal death. Cell 115: 863–877.[CrossRef][Medline]

Bano D, Young KW, Guerin CJ, LeFeuvre R, Rothwell NJ, Naldini L, Rizzuto R, Carafoli E, and Nicotera P (2005) Cleavage of the plasma membrane Na⁺/Ca²⁺ exchanger in excitotoxicity. Cell 120: 275–285.[CrossRef][Medline]

Bonita R, Mendis S, Truelsen T, Bogousslavsky J, Toole J, and Yatsu F (2004) The global stroke initiative. Lancet Neurol 3: 391–393.[CrossRef][Medline]

Caldwell RA, Clemo HF, and Baumgarten CM (1998) Using gadolinium to identify stretch-activated channels: technical considerations. Am J Physiol 275: C619–C621.[Medline]

Chen QX, Perkins KL, Choi DW, and Wong RKS (1997) Secondary activation of a cation conductance is responsible for NMDA toxicity in acutely isolated hippocampal neurons. J Neurosci 17: 4032–4036.[Abstract/Free Full Text]

Choi DW (1995) Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 18: 58–60.[CrossRef][Medline]

Choi DW, Maulucci-Gedde M, and Kriegstein AR (1987) Glutamate neurotoxicity in cortical cell culture. J Neurosci 7: 357–368.[Abstract]

Christine CW and Choi DW (1990) Effect of zinc on NMDA receptor-mediated channel currents in cortical neurons. J Neurosci 10: 108–116.[Abstract]

Churn SB, Limbrick D, Sombati S, and DeLorenzo RJ (1995) Excitotoxic activation of the NMDA receptor results in inhibition of calcium/calmodulin kinase II activity in cultured hippocampal neurons. J Neurosci 15: 3200–3214.[Abstract]

Coulter DA, Sombati S, and DeLorenzo RJ (1992) Electrophysiology of glutamate neurotoxicity in vitro: induction of a calcium-dependent extended neuronal depolarization. J Neurophysiol 68: 362–373.[Abstract/Free Full Text]

Delorenzo RJ, Sun DA, and Deshpande LS (2005) Cellular mechanisms underlying acquired epilepsy: the calcium hypothesis of the induction and maintenance of epilepsy. Pharmacol Ther 105: 229–266.[CrossRef][Medline]

Dingledine R, Borges K, Bowie D, and Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51: 7–61.[Abstract/Free Full Text]

Dubinsky JM (1993) Intracellular calcium levels during the period of delayed excitotoxicity. J Neurosci 13: 623–631.[Abstract]

Duchen MR (2000) Mitochondria and calcium: from cell signalling to cell death. J Physiol 529: 57–68.[Abstract/Free Full Text]

Durkin JP, Tremblay R, Chakravarthy B, Mealing G, Morley P, Small D, and Song D (1997) Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons. J Neurochem 68: 1400–1412.[Medline]

Glaum SR, Scholz WK, and Miller RJ (1990) Acute- and long-term glutamate-mediated regulation of [Ca²⁺]i in rat hippocampal pyramidal neurons in vitro. J Pharmacol Exp Ther 253: 1293–1302.[Abstract/Free Full Text]

Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391: 85–100.[CrossRef][Medline]

Hewett SJ, Corbett JA, McDaniel ML, and Choi DW (1993) Inhibition of nitric oxide formation does not protect murine cortical cell cultures from N-methyl-D-aspartate neurotoxicity. Brain Res 625: 337–341.[CrossRef][Medline]

Hille B (2001) Ion Channels of Excitable Membranes, Sinauer Associates, Inc., Sunderland, MA.

Horn J and Limburg M (2000) Calcium antagonists for acute ischemic stroke. Cochrane Database Syst Rev: CD001928.

Horn J and Limburg M (2001) Calcium antagonists for ischemic stroke: a systematic review. Stroke 32: 570–576.[Abstract/Free Full Text]

Ikonomidou C and Turski L (2002) Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury? Lancet Neurol 1: 383–386.[CrossRef][Medline]

Jekabsons MB and Nicholls DG (2004) In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate. J Biol Chem 279: 32989–33000.[Abstract/Free Full Text]

Kristián T and Siesjö BK (1998) Calcium in ischemic cell death. Stroke 29: 705–718.[Abstract/Free Full Text]

Lee JM, Zipfel GJ, and Choi DW (1999). The changing landscape of ischaemic brain injury mechanisms. Nature 399: A7–A14.[Medline]

Limbrick DD, Jr., Churn SB, Sombati S, and DeLorenzo RJ (1995) Inability to restore resting intracellular calcium levels as an early indicator of delayed neuronal cell death. Brain Res 690: 145–156.[CrossRef][Medline]

Limbrick DD, Jr., Pal S, and DeLorenzo RJ (2001) Hippocampal neurons exhibit both persistent Ca²⁺ influx and impairment of Ca²⁺ sequestration/extrusion mechanisms following excitotoxic glutamate exposure. Brain Res 894: 56–67.[CrossRef][Medline]

Limbrick DD Jr., Sombati S, and DeLorenzo RJ (2003) Calcium influx constitutes the ionic basis for the maintenance of glutamate-induced extended neuronal depolarization associated with hippocampal neuronal death. Cell Calcium 33: 69–81.[CrossRef][Medline]

Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79: 1431–1568.[Abstract/Free Full Text]

Michaels R and Rothman S (1990) Glutamate neurotoxicity in vitro: antagonist pharmacology and intracellular calcium concentrations. J Neurosci 10: 283–292.[Abstract]

Minger SL, Geddes JW, Holtz ML, Craddock SD, Whiteheart SW, Siman RG, and Pettigrew LC (1998) Glutamate receptor antagonists inhibit calpain-mediated cytoskeletal proteolysis in focal cerebral ischemia. Brain Res 810: 181–199.[CrossRef][Medline]

Raza M, Pal S, Rafiq A, and DeLorenzo RJ (2001) Long-term alteration of calcium homeostatic mechanisms in the pilocarpine model of temporal lobe epilepsy. Brain Res 903: 1–12.[CrossRef][Medline]

Sattler R, Charlton MP, Hafner M, and Tymianski M (1998) Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity. J Neurochem 71: 2349–2364.[Medline]

Siesjö BK and Bengtsson F (1989) Calcium fluxes, calcium antagonists, and calciumrelated pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis. J Cereb Blood Flow Metab 9: 127–140.[Medline]

Simpkins KL, Guttmann RP, Dong Y, Chen Z, Sokol S, Neumar RW, and Lynch DR (2003) Selective activation induced cleavage of the NR2B subunit by calpain. J Neurosci 23: 11322–11331.[Abstract/Free Full Text]

Sombati S, Coulter DA, and DeLorenzo RJ (1991) Neurotoxic activation of glutamate receptors induces an extended neuronal depolarization in cultured hippocampal neurons. Brain Res 566: 316–319.[CrossRef][Medline]

Vigé X, Carreau A, Scatton B, and Nowicki JP (1993) Antagonism by N^G-nitro-L-arginine of L-glutamate-induced neurotoxicity in cultured neonatal rat cortical neurons. Prolonged application enhances neuroprotective efficacy. Neuroscience 55: 893–901.[CrossRef][Medline]

Wahlgren NG and Ahmed N (2004). Neuroprotection in cerebral ischaemia: facts and fancies-the need for new approaches. Cerebrovasc Dis 17 (Suppl 1): 153–166.[Medline]

Xiong ZG, Zhu XM, Chu XP, Minami M, Hey J, Wei WL, MacDonald JF, Wemmie JA, Price MP, Welsh MJ, et al. (2004) Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels. Cell 118: 687–698.[CrossRef][Medline]

Yamauchi M, Omote K, and Ninomiya T (1998) Direct evidence for the role of nitric oxide on the glutamate-induced neuronal death in cultured cortical neurons. Brain Res 780: 253–259.[CrossRef][Medline]

This article has been cited by other articles:

				A. T. E. Hartwick, C. M. Hamilton, and W. H. Baldridge Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells J. Physiol., July 15, 2008; 586(14): 3425 - 3446. [Abstract] [Full Text] [PDF]

				T. A. Vander Jagt, J. A. Connor, and C. W. Shuttleworth Localized Loss of Ca2+ Homeostasis in Neuronal Dendrites Is a Downstream Consequence of Metabolic Compromise during Extended NMDA Exposures J. Neurosci., May 7, 2008; 28(19): 5029 - 5039. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.123182v1 322/2/443    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted