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Originally published In Press as doi:10.1074/jbc.M503090200 on June 24, 2005 Originally published In Press as doi:10.1074/jbc.M503090200 on June 2, 2005

J. Biol. Chem., Vol. 280, Issue 32, 28894-28902, August 12, 2005
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Excitotoxic Injury to Mitochondria Isolated from Cultured Neurons*

Yulia E. Kushnareva{ddagger}, Sandra E. Wiley§, Manus W. Ward, Alexander Y. Andreyev||, and Anne N. Murphy**

From the MitoKor, San Diego, California 92121

Received for publication, March 21, 2005 , and in revised form, June 2, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by ~10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 ± 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 ± 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 ± 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca2+-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutamate excitotoxicity underlies neuronal loss in ischemic and traumatic brain injury (1) and also likely contributes to dysfunction in chronic forms of neurodegeneration. In particular, the evidence for involvement of excitotoxicity in Huntington disease has been significantly strengthened by recent studies (for a review see Ref. 2). It is well established that glutamate-induced neuronal death depends on entry of extracellular Ca2+ as a result of activation of the NMDA1 subtype of glutamate receptors (3, 4). The description of ensuing events in various types of cultured neurons is extensive (5) and includes an initial transient increase in cytoplasmic Ca2+, followed by a loss in ionic homeostasis (also termed delayed Ca2+ deregulation) (4). The initial increase in cytoplasmic Ca2+ is associated with mitochondrial Ca2+ loading and slight mitochondrial depolarization followed by profound depolarization concurrent with the loss of ionic homeostasis. Other early events may include the release of apoptogenic cytochrome c and generation of reactive oxygen species and nitric oxide (5–7). As well, excitotoxic death in cultured neurons is preceded by a significant decline in cellular ATP (8, 9).

Recent imaging studies of cultured neurons have demonstrated that mitochondrial Ca2+ loading is in large part responsible for induction of cell death following exposure to glutamate (9–12), yet the nature of the Ca2+-induced mitochondrial injury remains controversial. An alternative approach to the elucidation of these mechanisms has been the study of mitochondria isolated from the brains of rodents exposed to excitotoxic stimuli. An inherent problem in such studies with brain mitochondria lies in the heterogeneity of these preparations that contain the organelles from both neuronal and glial cells (13, 14). Furthermore, frequently used isolation methods employing gradient centrifugation enrich the preparations in mitochondria from cell bodies, yet the excitotoxic events occur mostly within nerve terminals that are lost in the preparation as synaptosomal fractions. The use of digitonin to avoid the loss of synaptosomal mitochondria (15, 16) has been questioned recently (17). Therefore, we have taken the approach of isolating mitochondria from relatively pure neuronal cultures exposed to glutamate to allow these problems to be overcome.

We investigated the early bioenergetic consequences of exposure to glutamate by isolating mitochondria from primary neuronal cultures exposed to glutamate for 10 min, prior to a commitment to neuronal death. By doing so, we avoid studying potential secondary effects of the cell death process that may affect mitochondrial function. Estimation of the extent of glutamate-induced Ca2+ loading enabled us to test the effects of Ca2+ at concentrations that mimic the glutamate stimulus. Additionally, Ca2+ uptake capacities and susceptibility of mitochondria to the permeability transition were assessed. Our novel finding, which has not been revealed by other current approaches, is that mitochondria of neurons exposed to high levels of glutamate sustain significant Ca2+-induced injury to oxidative phosphorylation, and this injury occurs prior to any commitment to cell death.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Cortical Neurons—Primary cultures of cortical neurons were prepared from embryonic day 18 Sprague-Dawley rats. The cerebral cortices were collected and triturated gently (3–4 times) in ice-cold Hibernate E medium (Brain Bits, Southern Illinois University, School of Medicine) plus 1x B27 supplement (Invitrogen), 100 units/ml penicillin, and 100 µg/ml streptomycin. After the tissue settled, the Hibernate E medium was aspirated, and the tissue was triturated for 1 min in 0.1% trypsin in a Ca2+/Mg2+-free phosphate-buffered saline solution supplemented with glucose (1.5 mM), after which trypsin was inactivated by addition of soybean trypsin inhibitor (0.1 mg/ml). The mixture was transferred into Hibernate E medium containing 20 units/ml DNase (Promega) in 0.2x reaction buffer (Promega), and the cells were centrifuged at 200 x g for 1.5 min. The supernatant was quickly aspirated, and the cells were resuspended in 10 ml of Neurobasal (E) medium (Invitrogen) plus glutamate (0.4 µg/ml), 0.5 mM L-glutamine, penicillin, and streptomycin (100 units/ml and 100 µg/ml, respectively), 1x B27 supplement, and 5 mM sodium pyruvate. Once in suspension, the cells were diluted into 30 ml of the same medium without pyruvate (initial plating medium), and the number of viable cells was determined by trypan blue exclusion. Cells were plated on poly-D-lysine-coated 10-cm plates at a concentration of 750,000 cells per ml in a volume of 10 ml per plate and kept at 37 °C in a 5% CO2 incubator. For measurements of cellular ATP and cytoplasmic Ca2+, cells were plated on Biocoat poly-D-lysine-coated black 96-well plates (BD Biosciences) at 50,000 cells per well. After 4–6 days in vitro, the initial plating medium was diluted with an equal volume of maintenance medium of the same composition lacking glutamate and L-glutamine and supplemented with 1% GlutaMAX-1 (Invitrogen). Cultures were fed every 3–4 days with fresh medium. All experiments were performed with cultures that were 13–15 days in vitro. These cultures were 91–95% neuronal, as estimated by immunocytochemical staining according to the manufacturer's protocols with anti-neuronal nuclei (Chemicon, mAB377) or anti-neurofilament 200 kDa (Calbiochem, IF06L).

Measurement of Viability of Cortical Neurons Treated with Glutamate—Cell viability was measured in neurons cultured in 96-well plates using a cytotoxicity detection kit (lactate dehydrogenase, LDH) according to the manufacturer's recommendations (Roche Applied Science). Data are expressed as percent cell death based upon measurement of LDH activity that was cell-associated versus that which was released to the medium. Cells were treated in HEPES-buffered salt solution (HBSS, containing 137 mM NaCl, 5 mM KCl, 10 mM NaHCO3, 20 mM HEPES, 5.5 mM glucose, 0.6 mM KH2PO4, 1.4 mM CaCl2, 0.9 mM MgSO4) with 100 µM glutamate plus 10 µM glycine with or without pretreatment with 10 µM of the NMDA receptor antagonist MK-801. Control cultures were exposed to HBSS without glutamate or glycine. Following a 10-min incubation at 37 °C, an equal volume of maintenance medium was added (supplemented with glutamate plus glycine and/or MK-801 to maintain constant concentration of these effectors). The cells were then placed back in the incubator for 24 h prior to measurement of viability. Alternatively, at the addition of the maintenance medium, 2x (20 µM) MK-801 was included to block NMDA receptor activity.

Measurement of Neuronal Ca2+—Cytoplasmic Ca2+ was measured in neurons cultured in the 96-well plates using magfluo-4, a cell permeant low affinity Ca2+ dye (Molecular Probes). A stock solution of magfluo-4 (1 mg/ml) was prepared on the day of the experiment in Me2SO and then diluted in HBSS to a final concentration of 4 µM. The culture medium was carefully aspirated, and cells were loaded with the dye (100 µl of a 4 µM solution per well) for 25 min in the incubator. The loading buffer was then replaced with dye-free HBSS, and the plates were assayed in a fluorescent plate reader (FLIPR, Molecular Devices) at 488 (excitation) and 525 nm (emission).

Measurement of Neuronal ATP—The level of neuronal ATP was determined by using a CellTiter-GloTM luminescent assay kit (Promega). Cells cultured in 96-well plates were treated with glutamate for varying lengths of time (from 10 to 40 min). Control cells were exposed to HBSS without glutamate and glycine for 10 min. At the end of the treatment, CellTiter-GloTM reagent was added, and the plates were placed on a shaker for 5 min. In some experiments, the glutamate-containing medium was aspirated, and cells were incubated in Ca2+-free HBSS (containing 100 µM EGTA) for 3–6 min prior to the addition of CellTiter-GloTM reagent. Luminescence was measured in a PolarStar plate reader (BMG). ATP levels are expressed as fmol/cell based upon an ATP standard curve, and data are corrected for background luminescent signal. Each set of data was collected from multiple replicate wells (n = 12) and plotted as mean ± S.E.

Preparation of Cerebellar Granule Cells—Granule cells were prepared as described previously (18) from 7-day-old postnatal Wistar rats. Cells were plated on poly-D-lysine-coated 10-cm plates at a concentration of 1 x 106 cells per ml in a volume of ~9 ml per plate. Cells were cultured in minimal essential medium containing Earle's salts (Invitrogen) plus 10% (v/v) fetal calf serum (HyClone), 25 mM KCl, 30 mM glucose, 2 mM glutamine, penicillin, and streptomycin (100 units/ml and 100 µg/ml, respectively). After 24 h, 10 mM cytosine arabinose was added to inhibit non-neuronal cell proliferation. Cells were maintained at 37 °C in a 5% CO2 incubator and were used after 7–8 days in vitro.

Isolation of Mitochondria from Control and Glutamate-treated Neuronal Cultures—A method for isolation of functional mitochondria from primary neuronal cultures has been described previously (19). We used a modified protocol for rapid preparation of neuronal mitochondria suitable for assessment of bioenergetic parameters and the Ca2+ content. The culture medium was aspirated, and cells (cortical neurons or cerebellar granule cells) were exposed to 100 µM glutamate and 10 µM glycine in HBSS. Control cells were exposed to HBSS without glutamate and glycine. After glutamate addition, cells were returned to the incubator for 10 min. Next, the plates were placed on ice and washed twice with ice-cold Ca2+-free and Mg2+-free phosphate-buffered saline solution (Invitrogen) supplemented with 2 mM EGTA. Cells were rapidly scraped in 1 ml (per plate) of ice-cold mitochondrial isolation buffer containing 210 mM mannitol, 70 mM sucrose, 10 mM HEPES-KOH, pH 7.4, 2 mM EGTA, and 0.1% fatty acid-free bovine serum albumin and were then homogenized using a Dounce homogenizer (10 passes with a loose pestle and 10 passes with a tight pestle). Cell homogenates were centrifuged for 10 min at 1060 x g at 4 °C, and the supernatant was collected. The pellet was resuspended in the isolation buffer and centrifuged at 1060 x g for 5 min. The first and second supernatants were pooled together and centrifuged at 14,600 x g for 10 min at 4 °C. The mitochondrial pellet was resuspended in the isolation buffer lacking EGTA and albumin and centrifuged at 14,600 x g for 10 min. All isolation steps were performed on ice. The protein concentration was measured using the BCA protein assay kit (Pierce). Typically, 10–15 plates of cells were used for one mitochondrial preparation. Note that mitochondria were isolated in the absence of digitonin that is commonly used for disruption of the cholesterol-rich synaptosomal membranes to release mitochondria entrapped in synaptosomes (15). Our mitochondrial preparations from cultured neurons appear to be relatively free of synaptosomes as evidenced by the lack of an effect of digitonin on the maximal rate of respiration in the presence of succinate and rotenone (data not shown). Succinate is a mitochondrial substrate that is poorly permeable to the plasma membrane.

Measurements of Mitochondrial Respiration, Membrane Potential, and Ca2+ in Isolated Neuronal Mitochondria—Simultaneous measurements of mitochondrial respiration (O2 consumption), Ca2+ fluxes, membrane potential, and optical density of the mitochondrial suspension were conducted in a custom-constructed thermostatically controlled chamber (B. Krasnikov, Burke Medical Research Institute, NY). Membrane potential ({Delta}{psi}) was monitored by distribution of tetraphenylphosphonium (2 µM) with a tetraphenylphosphonium-selective electrode (20). Extramitochondrial Ca2+ was monitored with a Selectophore I membrane (Fluka)-based Ca2+-selective electrode. A Clark-type electrode (Diamond General) was used to measure oxygen consumption. In addition, measurements of optical density at 660 nm by means of a light-emitting diode and photodetector permitted changes in mitochondrial volume to be followed. This parameter was also used to confirm consistency of protein concentrations between experimental runs. Digital data acquisition using a DynaRes 16 board and WorkBench software (Strawberry Tree) permitted simultaneous recording of all four parameters as well as plots of their first derivatives in real time. The first derivatives were used for quantification of respiration and Ca2+ uptake rates.

For respiration measurements, mitochondria were incubated at 25 °C in a basal saline medium (125 mM KCl, 5 mM HEPES/KOH, pH 7.4, and 2 mM phosphate) supplemented with 2 mM MgCl2 and either complex I-linked substrates (a mixture of 5 mM glutamate and 5 mM malate) or a complex II substrate (5 mM succinate in the presence of 2 µM rotenone). Protein concentrations were 1–1.5 mg/ml for cortical mitochondria and 0.7–1.3 mg/ml for cerebellar mitochondria. State 3 (phosphorylating) respiration was initiated by addition of 200 µM ADP, after which state 4 (resting) respiration was induced by addition of 100 µM atractyloside, an inhibitor of the ADP/ATP antiporter. Finally, to measure uncoupler-stimulated respiration (state 3u), sequential additions of the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20–50 nM) were made until the respiration rate reached maximum. The acceptor control ratio was determined as the ratio of state 3 to state 4 respiration. The experiments were completed within 2–3 h after the isolation procedure.

Mitochondrial Ca2+ content was determined in the same basal medium (above) supplemented with glutamate and malate. Protein concentrations were in the same range as for the respiration measurements. After 2–3 min of incubation to allow for uptake of contaminating Ca2+ from the medium, mitochondrial Ca2+ was released into the medium by addition of FCCP (0.5 µM) followed 5 min later by the addition of the pore-forming peptide alamethicin (40 µg/ml) to ensure complete Ca2+ release. In some experiments, A23187 [GenBank] (1.2 µM), a Ca2+ ionophore, was also used to release sequestered Ca2+. The amount of Ca2+ released from mitochondria was calculated based upon calibration of the Ca2+ electrode performed at the end of each experimental day by additions of known concentrations of CaCl2 to the medium. EGTA (1 mM) was then added to chelate free Ca2+ in the medium. According to the Nernst equation, the voltage changes on the electrode are linearly related to the logarithm of Ca2+ concentration values in the medium. The concentration of contaminating Ca2+ in the medium was also experimentally determined. It was defined as an independent variable in the regression analysis shown in Equation 1,

(Eq. 1)
where a and b are regression parameters expressed in relative units, and [Ca2+]0 is the concentration of contaminating Ca2+.

R2 values for all calibration curves were no less than 0.995. The best fit parameters were used to calculate Ca2+ concentrations from the voltage changes in the experimental runs. The mitochondrial Ca2+ load was determined as the difference in Ca2+ concentration after addition of the Ca2+-releasing agent (FCCP or alamethicin) minus the contaminating Ca2+ in the medium and was normalized to mitochondrial protein concentration.

Mitochondrial Ca2+ uptake capacity was measured in the same medium as that used for mitochondrial Ca2+ content determination. Energized mitochondria were pulsed with successive additions of Ca2+ (30–50 µM) every 2–3 min. The increasing Ca2+ load caused a successive decline in the membrane potential and Ca2+ uptake rates. Maximal Ca2+ uptake capacity was defined as an amount of Ca2+ (per mg of protein) required to decrease the Ca2+ uptake rate by >90%. Note that because of the duration of the experimental run (25–40 min), most of these measurements were performed in an open chamber to avoid anoxia.

Data are expressed as mean ± S.E., and n indicates the number of independent experiments. Each mitochondrial preparation was obtained from a separate neuronal harvest (or neuronal preparation).

Statistical Analysis—One-way analysis of variance followed by post hoc Tukey test was performed using SigmaStat. Where differences are noted, analysis of variance detected significant variance at p < 0.001, and pairwise comparisons are indicated in the figure legends.

Materials—Unless indicated otherwise, all reagents were obtained from Sigma.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Measurements of Cytoplasmic Ca2+ in Cortical Cultures— Mitochondrial Ca2+ accumulation has been well established in different excitotoxicity models (21–24). An intact cell study utilizing a dye with low affinity to Ca2+ has demonstrated that a significant fraction of accumulated Ca2+ is retained within the mitochondria during brief glutamate exposures until FCCP is added to induce Ca2+ efflux (25). Based on this approach, we performed measurements of cytoplasmic Ca2+ in situ with our cortical cultures to demonstrate that the conditions of glutamate treatment used in our model produces results consistent with literature data. Fig. 1 depicts results of a representative experiment in which cortical neurons were challenged with various concentrations of glutamate (in the presence of 10 µM glycine) for 10 min followed by chelation of extracellular Ca2+ with EGTA and subsequent addition of FCCP. The responses to glutamate at concentrations between 30 and 300 µM were similar, and the pattern is typified by an initial peak in cytoplasmic Ca2+ that is followed relatively rapidly in these cultures by a more sustained and profound rise in cytoplasmic Ca2+. The response to 100 µM glutamate appeared to be maximal with regard to cytoplasmic Ca2+ and FCCP-induced release of Ca2+, as noted previously by others (25); therefore, we chose to probe in greater depth the bioenergetic changes occurring at this 10-min time point of glutamate exposure.

Effect of Glutamate Treatment on Cellular ATP—As shown in Fig. 2, 10 min of glutamate exposure results in a significant drop in the ATP level (Fig. 2A). Longer exposures to glutamate (up to 40 min) did not further decrease the content of ATP. Partial ATP depletion could be a direct consequence of acute activation of ATP-requiring efforts by the cell to re-establish ionic homeostasis during glutamate stimulation, and/or the ATP loss could result from impairment of cellular ATP production. To test whether ATP could be readily recovered if the glutamate and Ca2+ challenge were halted, cells were exposed to glutamate for 10 min followed by incubation of the cells in a glutamate- and Ca2+-free buffer. Under these conditions, cytoplasmic Ca2+ fairly rapidly returns to a steady state level (see Fig. 1 described above); however, ATP did not recover within this time frame. This result suggests that the compromise in cellular ATP levels reflects a compromise in the ability of the cell to produce ATP at a normal rate.



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  		FIG. 1.
Glutamate-induced changes in cytoplasmic and mitochondrial Ca2+ levels in cortical neurons. Cytoplasmic Ca2+ was monitored using magfluo-4 as described under "Experimental Procedures." FCCP-releasable Ca2+ represents the mitochondrial pool. The arrows indicate timing of addition of glutamate plus 10 µM glycine (Glu), 10 mM EGTA, and 4 µM FCCP. Concentrations of glutamate were 316 (a), 100 (b), 31.6 (c), 10 (d), 3.2 (e), and 1 µM(f). Control cells (g) did not receive glutamate or glycine. The data are representative of six independent experiments. Data points are means ± S.E. of the fluorescent signal obtained from six replicate wells.

 
Effect of Glutamate Treatment on Neuronal Viability—We next tested the effects of glutamate exposure on the viability of the cortical cultures. By using LDH release as a method of assessing plasma membrane permeability, we found that 24 h of continuous exposure of these cultures to 100 µM glutamate resulted in a significant loss in viability that was inhibited by 10 µM of the NMDA receptor antagonist, MK-801 (Fig. 3). Even though ionic homeostasis appears to be lost and mitochondrial Ca2+ loading is maximal after 10 min of glutamate exposure (Fig. 1), if this treatment is halted by addition of MK-801 for the ensuing 24 h, the injury is no longer sufficient to induce LDH release substantially above control levels (Fig. 3), at least at this time point. Therefore, cellular events leading up to the 10 min time point may be critical to initiation of the cell death pathway, but these events have not resulted in commitment of the cells to acute cell death.

Quantitative Estimation of Ca2+ in Mitochondria from Control and Glutamate-treated Neurons—The ability of mitochondria in glutamate-treated cells to release accumulated Ca2+ in response to FCCP argues against pervasive induction of a permeability transition in the mitochondrial population, as this would prevent retention of mitochondrial Ca2+. As shown on Fig. 4A, trace b, addition of FCCP to mitochondria from glutamate-treated cells caused a rapid release of a significant amount of Ca2+ presumably via reversal of the Ca2+ uniporter as a result of depolarization of the membrane (26). This FCCP-induced release is relatively slow and was still progressing at a significant rate 5 min after the uncoupler addition (Fig. 4A, trace c; note that the y scale is logarithmic). Therefore, we used a pore-forming peptide alamethicin to quickly attain complete release (Fig. 4A, trace c). Quantitative estimation of mitochondrial Ca2+ (performed as described under "Experimental Procedures") revealed that the total Ca2+ released in response to FCCP and alamethicin reached 167 ± 19 nmol/mg protein (n = 8). In the 5-min time frame, FCCP-released about half of the total Ca2+ (86 ± 14 nmol/mg protein, n = 8). More importantly, the amount of Ca2+ released from mitochondria isolated from untreated cells was very low (1.9 ± 0.8 nmol/mg protein, n = 5; Fig. 4A, trace b). Note that the amount of contaminating Ca2+ taken up into mitochondria from the incubation medium (2.5 ± 1.6 nmol/mg protein, n = 13) was subtracted from the amount of Ca2+ released into the medium. We do not exclude the possibility that a fraction of damaged mitochondria could be lost during preparation, but there was no apparent difference in the yields of mitochondria isolated from control and glutamate-treated cells. As well, we cannot exclude the possibility that a subfraction of mitochondria may have undergone the permeability transition and released their accumulated Ca2+ prior to isolation. Both of these possibilities would result in an underestimation of the quantities of retained Ca2+. Although it is possible that mitochondrial Ca2+ could be lost during isolation, the standard conditions for preparation of mitochondria, such as low temperature, the presence of EGTA, and the absence of Na+ in the isolation buffers, minimize changes in mitochondrial Ca2+ during the isolation procedure. This statement is supported by direct Ca2+ measurements in Ca2+-loaded brain mitochondria after prolonged incubation under conditions that mimic our isolation procedure (27).



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  		FIG. 2.
ATP level in control and glutamate-challenged cortical neurons. A, neurons were treated with glutamate for 10, 20, 30, and 40 min as indicated. B, after treatment of the cells with glutamate for 10 min, the medium was removed, and the cells were incubated in a Ca2+-free medium without glutamate for 3 or 6 min as indicated. Data are representative of three independent experiments. Each data set is mean ± S.E. of the signal obtained from 12 replicate wells. All time points are statistically different from their respective controls (p < 0.01). Other details are as specified under "Experimental Procedures."

 



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  		FIG. 3.
Cortical neuron viability following glutamate exposure. Neurons were treated and viability was measured using LDH release as specified under "Experimental Procedures." For Glu, 10 min, cells were exposed to glutamate for 10 min, followed by dilution with maintenance media containing MK-801, and Glu, 24 h indicates the continuous presence of glutamate. Where indicated, MK-801 was present from time 0. The data presented are means ± S.E. (n = 6). *, significantly different from other groups (p < 0.01).

 



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  		FIG. 4.
Effect of glutamate treatment on Ca2+ content in mitochondria from cortical neurons. A, trace a, electrode calibration by sequential additions of CaCl2; trace b, control mitochondria; trace c, mitochondria from glutamate-treated neurons. The arrows indicate the timing of additions of mitochondria (Mito), FCCP (0.5 µM), and alamethicin (Alm) (40 µg/ml). Data are representative of multiple (n = 7) independent experiments. Inset shows electrode calibration presented in semilogarithmic scale. B, lack of contribution of contaminating endoplasmic reticulum into Ca2+ content of mitochondria from glutamate-treated neurons. Trace a, mitochondria from glutamate-treated neurons, trace b, mitochondria from neurons treated with glutamate following pretreatment with 10 µM cyclopiazonic acid, an inhibitor of Ca2+-ATPase of endoplasmic reticulum. Experimental conditions and quantification of the amount of Ca2+ in the incubation medium are specified under "Experimental Procedures."

 
Because alamethicin is nonselective, and endoplasmic reticulum is a persistent contaminant of mitochondrial preparations, we performed additional experiments to confirm that the released Ca2+ was primarily of mitochondrial origin. Pretreatment of intact cortical neurons for 15 min prior to glutamate addition with 10 µM cyclopiazonic acid, an endoplasmic reticulum Ca2+-ATPase inhibitor, did not affect Ca2+ release in response to FCCP and alamethicin (Fig. 4B, traces a versus b). This observation suggests that the Ca2+ released by alamethicin is primarily mitochondrial.



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  		FIG. 5.
Ca2+ uptake capacity measured in mitochondria from cortical neurons. Sequential Ca2+ pulses (8 additions of 30 µM Ca2+ and 6 additions of 50 µM Ca2+) caused gradual decreases in the Ca2+ uptake rates (traces a and b) and membrane potential decline ({Delta}{psi}, trace c). In this experiment, total Ca2+ added to suppress Ca2+ uptake was 479 nmol per mg of protein. No changes in optical density (trace d) at the increasing Ca2+ load were detected. The arrows indicate the timing of addition of mitochondria (Mito) and Ca2+. Data are representative of five independent experiments. Inset, lack of Ca2+-induced permeability transition. Maximal Ca2+ accumulation (Ca (1) through Ca(n) was followed by addition of the pore-forming agent alamethicin (Alm). Note that alamethicin, but not Ca2+, induces hallmarks of permeability transition, swelling and release of previously retained Ca2+. Other experimental details are specified under "Experimental Procedures."

 
Determination of Ca2+ Uptake Capacity in Mitochondria from Cortical Neurons—As discussed above, the results of Fig. 1 imply that the mitochondrial Ca2+ load is saturated under our standard conditions (100 µM glutamate and 10 min of treatment). At a low level of glutamate, the mitochondrial Ca2+ load appears to be limited by Ca2+ availability as indicated by a near basal steady state level of cytosolic Ca2+ (Fig. 1, trace e). In contrast, one might presume that under the standard conditions, the mitochondrial Ca2+ load represents the Ca2+ uptake capacity of the mitochondria. To determine whether the uptake capacity of the mitochondria has indeed been saturated by glutamate exposure, we quantitatively determined the maximal Ca2+ uptake capacity of mitochondria isolated from control cells that were not exposed to glutamate. Pulses of Ca2+ were added to mitochondria from control neurons until virtually complete inhibition of Ca2+ uptake was achieved. Repeated Ca2+ additions caused gradual decreases in the membrane potential and Ca2+ uptake rates (Fig. 5) to a point at which Ca2+-induced respiratory inhibition prevented further establishment of membrane potential (respiratory tracing not shown). The maximal Ca2+ uptake capacity in cortical mitochondria was 363 ± 35 nmol/mg protein (n = 7), a value that is significantly higher than the glutamate-induced Ca2+ load (167 ± 19 nmol/mg protein). Most interestingly, even at the maximal Ca2+ concentrations, mitochondria retained all the accumulated Ca2+ and did not swell (Fig. 5). The addition of the pore-forming peptide alamethicin permitted detection of swelling under these conditions. These observations rule out induction of the permeability transition by Ca2+ in this experiment. It should be noted that the measurements were performed in the absence of added Mg2+ and adenine nucleotides, which are known to inhibit the classical permeability transition (28, 29), and to enhance significantly the Ca2+ uptake capacity of brain mitochondria (16, 30, 31). Therefore, the mitochondrial Ca2+ uptake capacity may be even higher in intact neurons, as the cytoplasm contains millimolar concentrations of Mg2+ and adenine nucleotides. However, factors other than the intrinsic mitochondrial Ca2+ uptake capacity may limit Ca2+ accumulation in mitochondria during acute glutamate treatment (see "Discussion").

Effect of Glutamate Treatment on Mitochondrial Respiration and Membrane Potential—Mitochondria isolated from untreated cortical neurons preserved functional integrity as evidenced by the ability to establish membrane potential (Fig. 6A) and by an acceptor control ratio of 4.8 ± 0.3 (n = 16) in the presence of complex I-linked substrates (Fig. 6B). Exposure of the cells to 100 µM glutamate for 10 min did not compromise the ability of subsequently isolated mitochondria to generate membrane potential (Fig. 6A). During the isolation procedure, mitochondrial Ca2+ transport processes that were taking place following glutamate exposure are inhibited by the low temperature and by permeabilization in medium containing EGTA. Therefore, these conditions are unlike those in the intact cell in which mitochondrial Ca2+ cycling is ongoing. Our observation is in agreement with evidence that mitochondrial depolarization during acute glutamate treatment is reversible upon glutamate removal (5).

Most importantly, glutamate treatment markedly reduced (on average, by 44%) ADP-stimulated respiration (state 3) and decreased the amplitude of ADP-induced depolarization (Fig. 6, A and B). Because state 3 respiration is dependent upon both electron transport and ADP phosphorylation, the observed inhibition could result from injury to either of the two processes. Uncoupler-stimulated respiration (state 3u) is an indicator of the maximal rate of electron transport. As shown in Fig. 6, A and B, state 3u respiration was reduced by ~10%. Under resting conditions, respiration rates (state 4) remained relatively unchanged. The acceptor control ratio in mitochondria from glutamate-treated cells dropped to 2.7 ± 0.2 (n = 15). Therefore, the most significant effect of glutamate treatment on mitochondria from cortical neurons was inhibition of ADP phosphorylation, whereas the effect on the maximal rate of electron transport was less pronounced, and there was no evidence of significant respiratory uncoupling.

Deleterious effects of elevated Ca2+ on neural mitochondria, such as induction of the permeability transition (16, 31–33) or inhibition of respiration (34–37), have been described in the literature. However, the pathophysiological relevance of the amounts of Ca2+ used in many studies remains questionable. We examined the effects of adding exogenous Ca2+ to control mitochondria at a concentration close to the quantity we measured for glutamate-induced Ca2+ loading (Fig. 6B). In these experiments, mitochondria from untreated cortical neurons were exposed to a bolus of 150 µM Ca2+ (100–125 nmol/mg). After sequestration of the added Ca2+ was complete, respiration was measured under the same conditions as in Fig. 6A. As shown on Fig. 6B, which summarizes the data on respiration from multiple experiments, the patterns of respiratory changes induced by 150 µM Ca2+ and glutamate treatment were similar, although the added Ca2+ caused a more dramatic inhibition of state 3 respiration. The data demonstrate that in this model Ca2+ per se mimics the mitochondrial bioenergetic effects of acute glutamate treatment.



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  		FIG. 6.
Glutamate-induced bioenergetic changes in mitochondria from neurons. A, respiration (traces a and b) and membrane potential ({Delta}{psi}, traces c and d) measured in mitochondria from control (traces a and c) and glutamate-treated (traces b and d) cortical neurons. Glutamate and malate were present as complex I-linked respiratory substrates. The arrows indicate the timing of additions of mitochondria (Mito), ADP, atractyloside (Atr), and FCCP titration (six sequential additions of 50 nM FCCP). Oxygen consumption data are given in nmol of O2 per min per mg of protein. Data are representative of multiple (n > 10) independent experiments. Other details are as specified under "Experimental Procedures." B, summary of effects of glutamate treatment and added Ca2+ (150 µM) on respiration measured in mitochondria from cortical neurons. For Ca2+ loading, mitochondria were incubated with 150 µM CaCl2 (100–125 nmol/mg) for 2–3 min to allow for Ca2+ accumulation, and respiration was measured in the presence of complex I-linked substrates as shown in A. Data are means ± S.E.; n indicates the number of independent experiments. *, statistically different from state 3 in control (p < 0.01). **, statistically different from the respective state 3 in the treatment group (p < 0.01). Other experimental details are specified under "Experimental Procedures." C, effect of glutamate treatment on respiration measured in mitochondria from cortical neurons in the presence of complex II substrate. Trace a, control mitochondria; trace b, mitochondria from glutamate-treated neurons. Data are representative of three independent experiments. Other details are as specified under "Experimental Procedures."

 



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  		FIG. 7.
Effect of Ca2+ depletion by A23178 on state 3 respiration measured in mitochondria from glutamate-treated cortical neurons. A, induction of Ca2+ release by A23187 [GenBank] (1.2 µM). Ruthenium Red (0.2 µM) was added to prevent Ca2+ re-uptake via the Ca2+ uniporter. Released Ca2+ was then chelated by EGTA (500 µM). The arrows indicate the timing of additions of mitochondria (Mito), Ruthenium Red (RR), A23187 [GenBank] , and EGTA. B, restoration of glutamate-inhibited state 3 respiration in Ca2+-depleted mitochondria. After removal of the accumulated Ca2+ by the combination of A23187 [GenBank] plus EGTA, respiration was measured in the presence of complex I-linked substrates as shown on Fig. 1. Data are means ± S.E. from three independent experiments. *, statistically different from state 3 in glutamate (p < 0.01). Other experimental details are specified under "Experimental Procedures."

 
Because the extent of Ca2+-mediated injury may depend on the type of respiratory substrate (31, 33, 35, 36, 38, 39), we also assessed the effects of glutamate treatment in the presence of the complex II substrate succinate. Mitochondria energized with succinate showed a similar pattern of inhibition of respiration as in the presence of complex I-linked substrates (Fig. 6, C versus A). In the experiment shown in Fig. 6C, succinate-supported state 3 and state 3u respiration were inhibited by 47 and 19%, respectively.

Reversibility of Glutamate-induced Inhibition of ADP Phosphorylation—Next, we addressed the question of whether glutamate-induced partial inhibition of state 3 respiration can be reversed upon removal of the accumulated Ca2+. Addition of the Ca2+ ionophore A23187 [GenBank] to mitochondria from glutamate-treated cells induced rapid release of the endogenous Ca2+ that was subsequently chelated by EGTA (Fig. 7A). Ruthenium Red, the inhibitor of the Ca2+ uniporter, was present in these experiments to prevent Ca2+ re-uptake during A23187 [GenBank] -induced Ca2+ release. Respiration measurements showed that Ca2+ depletion partially restored glutamate-inhibited state 3 respiration (Fig. 7B). This observation is consistent with previous studies with Ca2+-challenged heart (40) and brain (34) mitochondria, demonstrating that Ca2+-induced inhibition of oxidative phosphorylation can be at least partially relieved by treatment with A23187 [GenBank] and EGTA or after Ca2+ release via the Ca2+/Na+ exchanger.

Measurements of Respiration, Ca2+ Content, and Ca2+ Uptake Capacity in Mitochondria from Cerebellar Granule Cells— Little is known about the differences in mitochondria from different neuronal types, although such information is highly desirable in an effort to understand further the varying susceptibility of different neurons to injury in acute and chronic forms of neurodegeneration. Isolated mitochondria from different brain regions have been studied with regard to susceptibility to the permeability transition (41), susceptibility to dysfunction induced by traumatic brain injury (42), ischemic injury (27, 43), and various toxicants (44) with the caveat that these are studies of mixed populations of mitochondria from varied cell types. The current approach allows more appropriate comparison of bioenergetic function between enriched neuronal cultures. A series of experiments was performed to compare the glutamate-induced changes in mitochondria from cortical neurons with those in mitochondria isolated from cerebellar granule cells. Under control conditions, the rates of respiration of mitochondria from the two neuronal cell types were comparable (Fig. 6B and Fig. 8).



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  		FIG. 8.
Effect of glutamate treatment on respiration measured in mitochondria from cerebellar granule cells. Respiration was measured in the presence of complex I-linked respiratory substrates as described for Fig. 6, and data are expressed in nmol of O2 per min per mg of protein. Data are means ± S.E. from six independent experiments. *, statistically different from control (p < 0.05). Other experimental details are specified under "Experimental Procedures."

 
With regard to glutamate-induced mitochondrial injury, Atlante et al. (45) have reported progressive inhibition of succinate-supported state 3 respiration measured in homogenates prepared from cerebellar granule cell cultures at different times after glutamate exposure. In agreement with this study, glutamate treatment for 10 min induced 20–30% inhibition of both state 3 and state 3u respiration in the presence of complex I-linked substrates in cerebellar mitochondria (Fig. 8). ADP-stimulated respiration was therefore less inhibited than in cortical mitochondria (Fig. 8 versus Fig. 6). The observation of partial inhibition of ADP-stimulated respiration does not contradict a previous conclusion that cerebellar mitochondria in situ remain capable of producing ATP throughout excitotoxic glutamate exposure (Refs. 5 and 46; see "Discussion").

The most significant difference between cortical and cerebellar mitochondria was observed in the Ca2+ uptake capacity. The maximal Ca2+ uptake capacity for cerebellar mitochondria was 686 ± 71 nmol/mg protein (n = 4), almost twice as high as for cortical mitochondria. Despite the higher Ca2+ uptake capacity, glutamate-induced increases in the Ca2+ content were similar to those in cortical mitochondria. The Ca2+ pool released by FCCP within 5 min in cerebellar mitochondria was 96 ± 34 nmol/mg protein (n = 7). The total Ca2+ released in response to FCCP plus alamethicin was 173 ± 64 nmol/mg protein (n = 8). These data suggest that in cerebellar granule cells, similar to cortical neurons, glutamate-induced Ca2+ loading does not saturate the mitochondrial Ca2+ uptake capacity, but the extent of loading represents a smaller proportion of the maximal uptake capacity. As with cortical neuronal mitochondria, the alamethicin-releasable pool of Ca2+ in mitochondria from untreated cells was a small fraction of the pool released following glutamate treatment (2.0 ± 1.4% (n = 4)).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In our cortical neuronal cultures, we identified impairment of ADP phosphorylation as the most dramatic consequence of glutamate exposure to mitochondrial bioenergetics, whereas inhibition of maximal respiratory chain activity was quite modest. The inhibition of oxidative phosphorylation occurred prior to commitment to cell death and therefore did not result from sequelae of the cell death process. The effect of excitotoxin exposure on oxidative phosphorylation was Ca2+-dependent as we found that it could be mimicked by additions in vitro of the relevant concentrations of Ca2+. Moreover, glutamate-induced inhibition of state 3 respiration could be reversed by rapid depletion of the accumulated Ca2+ using ionophore. We found that the cellular ATP level was compromised within the first minutes of glutamate exposure (Fig. 2A), and it was not readily recovered at 6 min after glutamate/Ca2+ removal (Fig. 2B). The lack of recovery at this time point likely reflects the protracted elevation of matrix Ca2+ following an excitotoxic stimulus because of the relatively slow kinetics of the mitochondrial Ca2+ efflux pathway and continued Ca2+ cycling (23, 25, 47, 48). We propose that the early impairment of ADP phosphorylation contributes to cellular ATP depletion, and we further suggest that this inhibition is likely key to both ensuing neuronal dysfunction and/or cell death.

Certainly, there are other potential modes by which mitochondrial Ca2+ sequestration may trigger neuronal dysfunction and/or death. Mitochondrial Ca2+ overload may result in respiratory inhibition and/or the permeability transition that prevents sufficient mitochondrial ATP production to meet the increased energy demand. Alternatively, Ca2+ sequestration may result in increased oxidative stress that compromises multiple components of cell function including Ca2+ extrusion (5). Nicholls and co-workers (9, 11, 49) have reported that the cause of delayed Ca2+ deregulation, at least in cerebellar granule cells, is a mitochondrial event other than insufficient ATP production. It should be noted, however, that the approaches in these elegant studies, including fluorescence imaging of intact cells and in situ respiration of monolayers, are capable of detecting whether ADP phosphorylation can take place, but they are unable to assess whether significant but incomplete inhibition of oxidative phosphorylation has occurred. This limitation is inherent to studies of mitochondrial bioenergetics using intact cells because of the inability to control ADP and substrate provision to the mitochondria. The isolation technique employed here overcomes this limitation. Jekabsons and Nicholls (49) found that glutamate stimulated the respiration of intact cerebellar granule cells to 40–50% of their maximal electron transport chain capacity. We would suggest that this increase, although stimulated by ATP turnover, is not meeting ATP demand in a manner consistent with continued normal cell function and viability. There are potentially important differences, however, between the Ca2+ responses of our cortical cultures and the cerebellar granule cells studied by Jekabsons and Nicholls (49). Foremost, Ca2+ deregulation is much more delayed in a majority of the granule cells compared with our cortical neurons (Refs. 9, 11, and 49 versus Fig. 1). The inhibition of oxidative phosphorylation, the persistent decline in ATP, and the rapidity of the loss in ionic homeostasis in our cortical neurons are most consistent with death involving insufficient ATP generation. In fact, the pattern of cytoplasmic Ca2+ responses to glutamate in cortical neurons appears similar to those in cerebellar granule cells when both respiration and mitochondrial ATP synthesis have been chemically blocked (11). As a result, we propose that mechanisms of injury are common in different neurons, but the events most critical to loss in ionic homeostasis and cell death may vary in different cultures and may depend on multiple factors, including the glycolytic capacity to produce ATP, reactive oxygen species-detoxifying systems, and the proportion of the mitochondria of the cells that sequesters excessive loads of Ca2+.

More importantly, the fact that our cortical cultures do not undergo cell death if the stimulus has been halted at 10 min (Fig. 3), despite the fact that they have undergone Ca2+ deregulation (Fig. 1), clearly implies that additional event(s) critical to the commitment to cell death occur after this profound increase in cytoplasmic Ca2+. In fact, others have found that although antioxidants have no effect on the onset of delayed Ca2+ deregulation, they protect against cell death measured 24 h later (50), suggesting that an oxidative event critical to death occurs after the loss of ionic homeostasis. Furthermore, our data suggest that inhibition of oxidative phosphorylation may be a consequence of glutamate exposure that, if brief, may result in a compromise in normal neuronal function but not necessarily result in neuronal death. This may have profound implications for the chronic dysfunction of neurons in diseases such as Huntington disease, in which expansion of the huntingtin protein may lead to NMDA receptor activation, exaggerated cytoplasmic Ca2+ signaling via inositol 1,4,5-trisphosphate-mediated mechanisms, and thus enhanced mitochondrial Ca2+ loading (2, 51).

Inhibition of respiration and/or ADP phosphorylation by elevated Ca2+ is not a unique feature of neuronal mitochondria. A similar injury was observed in earlier studies with mitochondria from tumor (38, 52) and transformed neural cells,2 brain (34, 35), and heart (40). The precise mechanisms underlying this injury remain to be elucidated. The maximal rate of respiration (FCCP-stimulated respiration) is mildly decreased by glutamate treatment in both cortical and cerebellar mitochondria in our hands. Inhibition of maximal respiration in response to glutamate has been observed in cerebellar granule cells (45, 49) and cortical neurons (53), although Atlante et al. (45) and Almeida et al. (53) did not correct for losses in cell viability and measured respiration at later time points. The effect that we observe is independent of the type of respiratory substrate (complex I versus complex II substrates) and therefore could be explained by a partial loss of cytochrome c (33, 54, 55). ADP phosphorylation (state 3 respiration) was significantly more inhibited than FCCP-stimulated respiration (state 3u respiration) in our cortical cultures, indicating that components other than the respiratory chain are affected, e.g. the ATP synthase and/or availability of adenine nucleotides and phosphate. As one potential mechanism, Ca2+ has been proposed to bind the adenine nucleotides in the mitochondrial matrix causing inhibition of state 3 respiration and ATP synthase activity (34, 56).

This study estimates that the glutamate-induced mitochondrial Ca2+ loading for both cortical and cerebellar neurons is ~170 nmol/mg protein. This amount was determined by releasing the cation into the medium with the pore-forming agent alamethicin. We found that alamethicin was more effective than FCCP at rapidly releasing Ca2+, likely because of the fact that the bulk of Ca2+ in mitochondria is stored in the form of the poorly soluble Ca2+-phosphate complex (16, 57–60). The rate of solubilization of this complex may be slower in response to uncoupler as opposed to when alamethicin-induced pores allow dilution of the matrix contents. As noted earlier, one of the shortcomings of our current approach is that mitochondrial Ca2+ can potentially be lost during the isolation. Although there is evidence against this (27), it must be stated that the actual mitochondrial Ca2+ load may be even higher than our estimates. A second reason for this statement is that nonmitochondrial protein typically contaminates mitochondrial preparations. As judged by Western blot analysis (data not shown), the degree of cytosolic and membranous contaminations of our cortical preparation was comparable with that of brain mitochondria isolated according to a published protocol (15), and therefore, the Ca2+ loads represent reasonable estimates for comparison to isolated mitochondria models.

The glutamate-induced mitochondrial Ca2+ load is significantly below the maximal Ca2+ uptake capacity that we measured in vitro. There are a number of potential explanations for this observation, including that the quantity of available phosphate (the necessary counter-ion in Ca2+ transport) could limit glutamate-induced mitochondrial Ca2+ uptake (61), that the activity of the mitochondrial Na+/Ca2+ exchanger is enhanced in response to cytosolic Na+ (23, 48), or that there is loss of loaded Ca2+ because of the permeability transition or the isolation procedure. Furthermore, mitochondrial Ca2+ uptake may be altered by glutamate-induced generation of reactive oxygen species or nitric oxide (for reviews see Refs. 7 and 21). An equally tenable explanation for the apparent "intermediate" Ca2+ loading in situ is that only a subpopulation of mitochondria may be sequestering Ca2+ in intact neurons, for which there is strong evidence (62, 63). Mitochondria within cells display functional and morphological heterogeneity that, among other factors, reflects their proximity to Ca2+ sources (63–65). One can hypothesize that heterogeneous Ca2+ loading causes more dramatic respiratory injury to a subpopulation of mitochondria that are in close proximity to NMDA receptors. Inhibition of ATP production to a critical subset of mitochondria may produce a localized inability to re-establish ionic homeostasis and membrane potential at the plasma membrane that ultimately perpetuates a collapse of ionic homeostasis.

The difference between the glutamate-induced Ca2+ load and the maximal Ca2+ uptake capacity is more dramatic in mitochondria from cerebellar granule cells. The higher Ca2+ uptake capacity of cerebellar mitochondria (as compared with cortical mitochondria) correlates with the relative resistance of this brain region in excitotoxic and ischemia/reperfusion models (66, 67). Mitochondria isolated from cerebellar tissue are less susceptible to a Ca2+-induced injury than mitochondria isolated from cortex and hippocampus (41) potentially because of higher adenine nucleotide content (38, 41). Therefore, these data lead us to suggest that the proportional load of mitochondrial Ca2+ resulting from glutamate exposure compared with the maximal Ca2+ uptake capacity may determine susceptibility to excitotoxic neuronal death.

Our data do not support that glutamate exposure induces pervasive stimulation of the mitochondrial permeability transition in cortical neurons or cerebellar granule cells. If the permeability pore was open at the 10-min time point that we began our isolation, the EGTA in the buffer would sequester the matrix Ca2+ (68). We observe substantial alamethicin-induced Ca2+ release from the mitochondria, and therefore a significant portion of mitochondria was impermeable to EGTA at the time of isolation. However, we cannot eliminate the possibility that a subpopulation of mitochondria has undergone the permeability transition, which would lead to an underestimation of the glutamate-induced mitochondrial Ca2+ load. However, if the permeability transition occurs in a subpopulation of mitochondria during a 10-min glutamate exposure, this event is insufficient to induce significant neuronal death (Fig. 3). Neuronal mitochondria were also resistant to a permeability transition when they were loaded with Ca2+ in vitro. At Ca2+ concentrations sufficient to saturate Ca2+ uptake capacity, no permeability increases were detected (Fig. 5). Although others have found indications of increased mitochondrial permeability in intact neurons (24, 69–74), the lack of consistent protective effects of permeability transition inhibitors in excitotoxicity models (11, 70, 74–76) renders the literature data controversial. Recently, it has been suggested that the permeability transition pore in brain mitochondria has altered sensitivity to conventional pore inhibitors (77). Regardless, the extent of permeability pore contribution to excitotoxic neuronal death requires further investigation.

In conclusion, these results have revealed that glutamate exposure of cortical neurons results in early inhibition of oxidative phosphorylation, in the absence of profound respiratory inhibition, or changes that might be the downstream result of commitment to cell death. Although the approach taken here has caveats, it revealed a type of Ca2+-induced injury that fluorescence imaging or whole cell respiration experiments are unable to detect. We propose that Ca2+-mediated inhibition of oxidative phosphorylation may have pathological relevance to acute as well as chronic excitotoxic injury.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: Del E. Webb Center for Neuroscience and Aging, The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 92037. Back

§ Present address: Walther Cancer Institute, University of California San Diego, La Jolla, CA 92093. Back

Present address: Royal College of Surgeons in Ireland, Dept of Physiology, 123 St Stephen's Green, Dublin 2, Ireland. Back

|| Present address: Dept. of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093. Back

** To whom correspondence should be addressed: BioEnergetix, LLC, 310 Cole Ranch Rd., Encinitas, CA 92024. Fax: 760-635-0033; E-mail: anmurphy{at}cox.net.

1 The abbreviations used are: NMDA, N-methyl-D-aspartic acid; LDH, lactate dehydrogenase; HBSS, HEPES-buffered salt solution; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Back

2 A. Y. Andreyev, S. E. Wiley, Y. E. Kushnareva, and A. N. Murphy, unpublished observations. Back



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 DISCUSSION
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