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The Journal of Neuroscience, August 1, 2007, 27(31):8238-8249; doi:10.1523/JNEUROSCI.1984-07.2007

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Neurobiology of Disease
Mitochondrial and Plasma Membrane Potential of Cultured Cerebellar Neurons during Glutamate-Induced Necrosis, Apoptosis, and Tolerance

Manus W. Ward,^1 * Heinrich J. Huber,^1,2 * Petronela Weisová,¹ Heiko Düssmann,¹ David G. Nicholls,³ and Jochen H. M. Prehn¹

¹Department of Physiology and Medical Physics and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin 2, Ireland, ²Siemens Medical Division, Siemens Ireland, Dublin 2, Ireland, and ³Buck Institute for Age Research, Mitochondrial Physiology, Novato, California 94945

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
A failure of mitochondrial bioenergetics has been shown to be closely associated with the onset of apoptotic and necrotic neuronal injury. Here, we developed an automated computational model that interprets the single-cell fluorescence for tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either _m or _p, thus allowing for the characterization of responses for populations of single cells and subsequent statistical analysis. Necrotic injury triggered by prolonged glutamate excitation resulted in a rapid monophasic or biphasic loss of _m that was closely associated with a loss of _p and a rapid decrease in neuronal NADPH and ATP levels. Delayed apoptotic injury, induced by transient glutamate excitation, resulted in a small, reversible decrease in TMRM fluorescence, followed by a sustained hyperpolarization of _m as confirmed using the _p-sensitive anionic probe DiBAC₂(3). This hyperpolarization of _m was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in _m or _p at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of _p during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of _m after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in _m. Our results indicate that a hyperpolarization of _m is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.

Key words: excitotoxicity; mitochondria; modeling; plasma and mitochondrial membrane potential; bioenergetics; necrosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glutamate excitotoxicity is an important contributor to neuronal loss associated with ischemic, traumatic, and seizure-induced brain injury (Choi, 1994). Prolonged glutamate receptor overactivation results in a rapid necrotic neuronal injury that is known to be highly dependent on excessive Ca²⁺ uptake leading to a loss of mitochondrial bioenergetics, ionic homeostasis, and cellular integrity (Choi, 1987; Tymianski et al., 1993a; Budd and Nicholls, 1996b; White and Reynolds, 1996; Stout et al., 1998; Vergun et al., 1999; Ward et al., 2000). Transient glutamate receptor activation, in contrast, can trigger a delayed apoptotic neuronal injury characterized by nuclear condensation, cell shrinkage, and a delayed collapse of mitochondrial bioenergetics and Ca²⁺ homeostasis hours after the initial excitation (Ankarcrona et al., 1995; Budd et al., 2000; Luetjens et al., 2000; Ward et al., 2000, 2006). The sequestration of Ca²⁺ within the mitochondrial matrix has been reported to play an integral role in excitotoxic injury (Budd and Nicholls, 1996b; Wang and Thayer, 1996; White and Reynolds, 1997; Stout et al., 1998; Brocard et al., 2001; Ward et al., 2005), resulting in an early impairment of mitochondrial ADP phosphorylation (Kushnareva et al., 2005).

The mitochondrial membrane potential (_m) is the central parameter controlling the accumulation of Ca²⁺ within the mitochondrial matrix, respiration, and ATP synthesis (Nicholls and Ferguson, 1992; Nicholls and Budd, 2000; Nicholls, 2002). The fluorescent membrane-permeant cationic probe tetramethylrhodamine methyl ester (TMRM) has become one of the most frequently used probes in the analysis of _m in intact cells because of its minimal phototoxicity, low photobleaching, and the ability to use it in both a quenched (aggregated probe) and nonquenched (no aggregation of probe) mode (Ehrenberg et al., 1988; Nicholls and Ward, 2000; Buckman et al., 2001; Gerencser and Adam-Vizi, 2005). Previously, we have successfully used TMRM in the quenched mode to monitor mitochondrial function during glutamate-induced injury in cerebellar granule neurons (Ward et al., 2000) identifying a sensitivity of TMRM to changes in _m and the plasma membrane potential (_p). Indeed, the relative contribution of key changes in _m and _p after glutamate excitation have previously been difficult to define (Ankarcrona et al., 1995; Khodorov et al., 1996; White and Reynolds, 1996; Kiedrowski, 1998; Prehn, 1998; Stout et al., 1998; Keelan et al., 1999; Nicholls and Ward, 2000). In this study, we set out to investigate the complex changes that occur at the _m and _p level in relation to changes in neuronal metabolism and define how alterations in these parameters correlate with neuronal injury and survival after glutamate excitation. We have expanded on a computational model for interpreting whole-cell TMRM fluorescence (Ward et al., 2000; Nicholls, 2006) allowing for the rapid, automated, and impartial assessment of changes in _m and _p at a single-cell level and subsequent statistical analysis. From this, we have identified significant alterations in _m and _p that are paralleled with key modifications to neuronal metabolism that directly relate to neuronal outcome (necrosis, apoptosis, and tolerance) after glutamate excitation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials. Fetal calf serum and minimal essential medium were from Invitrogen (Paisley, Strathclyde, UK). Glutamate, glycine, and all other reagents were from Sigma (Poole, Dorset, UK). Fluo-4 AM, DiBAC₂(3), and TMRM were purchased from Invitrogen (Bio Sciences, Dun Laoghaire, Ireland).

Preparation of primary cerebellar granule neurons. Cerebellar granule neurons were prepared as described previously (Ward et al., 2000) with minor modifications. Cerebella from 7-d-old Wistar rats of both sexes were dissected and pooled. The tissue was placed in 20 ml of filter sterilized PBS supplemented with 0.25 mg/ml trypsin and incubated at 37°C for 20 min. Trypsinization was terminated by the addition of an equal volume of filter-sterilized PBS supplemented with 0.05 mg/ml soybean trypsin inhibitor, 3 mM MgSO₄, and 30 U/ml DNase I. The neurons were then triturated, and the resulting neurons were resuspended in supplemented culture medium. Cells were then plated on poly-L-lysine-coated glass coverslips, glass Willco (Amsterdam, The Netherlands) dishes, 6-well plates, and 24-well plates at 1 x 10⁶ cells per milliliter and maintained at 37°C in a humidified atmosphere of 5% CO₂/95% air.

Confocal microscopy. Cerebellar granule neurons on Willco dishes were loaded with TMRM (30 nM) only or coloaded with Fluo-4 AM (3 µM) for 30 min at 37°C (in the dark) in experimental buffer [containing (in mM) 120 NaCl, 3.5 KCl, 0.4 KH₂PO₄, 20 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid N-[Tris(hydroxymethyl)methyl]-2-aminoethanosulfonic acid (Tes), 5 NaHCO₃, 1.2 Na₂SO₄, 1.2 CaCl₂, 1.2 MgCl₂, and 15 glucose, pH 7.4]. The Willco dishes with cells were washed in fresh medium after loading before being mounted in a nonperfusion (37°C) holder and placed on the stage of an LSM 510 Meta Zeiss (Oberkochen, Germany) confocal microscope. No MgCl₂ was present in buffers for experiments that involved the addition of glutamate. For glutamate-induced apoptosis, neurons were exposed to glutamate and glycine (100 and 10 µM) for 5 min, and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801; 10 µM; Sigma) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonoamide (NBQX; 10 µM; Sigma) were added to block glutamate receptor activation. In the model of glutamate-induced necrosis, MK-801 and NBQX were not added. Fluo-4 AM was excited at 488 nm with an argon laser (1%), and the emission was collected through a 505–550 nm barrier filter; TMRM was excited at 543 nm with a helium neon laser (3%), and the emission was collected through a 560 nm long-pass filter. Images were collected at 15 s intervals during glutamate excitation and every 5 min during the rest of the experiment, and the resulting fluorescent images were processed using MetaMorph Software version 7.1, release 3 (Molecular Devices, Berkshire, UK).

DiBAC₂(3) is a fluorescent probe that has been successfully used to characterize changes in _p (Freedman and Novak, 1989). Here, cerebellar granule neurons were incubated with an experimental buffer containing 1 µM DiBAC₂(3) for 30 min at 37°C. A concentration of 1 µM DiBAC₂(3) was also present in the experimental buffer. DiBAC₂(3) is a bis-barbituric acid oxonol compound that partitions into the membrane as a function of membrane potential. Hyperpolarization causes extrusion of the dye and decreased fluorescence, whereas depolarization causes enhanced fluorescence. Fluorescence was monitored with an LSM 510 (Zeiss) confocal microscope. The probe was excited at 488 nm with an argon laser (1%), and the emission was collected through a 530–600 nm barrier filter. Images were collected at 15 s intervals during additions [glutamate, oligomycin, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)] and every 2 min throughout the rest of the experiment. The resulting fluorescent images were processed using MetaMorph software.

Computational modeling. We have used MATLAB software (version 7.0; Mathworks, Cambridge, UK) for the creation of an automated computational model for the analysis of single-cell fluorescence responses for monovalent cationic probes [TMRM, tetramethylrhodamine ethyl ester (TMRE), rhodamine-123] in populations of cells providing rapid predictive output on changes in _p and _m for large data sets. Previous models have been restricted by the manual fitting of parameters, limited flexibility within the parameters, and low throughput of data sets. This model provides automated output for large data sets with minimal parameter fitting.

This model, as in previous models (Ward et al., 2000; Nicholls, 2006), was based on the Nernstian equilibration of a generic membrane-permeant monovalent cation c⁺ (TMRM, TMRE, rhodamine-123). The equilibration concentration for free c⁺ in the extracellular medium, cytosol, and mitochondrial matrix at 37°C results in the relationships described below:

where _p and _m are the plasma and mitochondrial membrane potentials and the divisor 61.5 is the value (in millivolts) for RT/F log(2) at 37°C. Re-equilibration of the cytosolic and extracellular compartment attributable to changes in the potentials of _p and _m are taken into account by assuming a first-order flux between the extracellular medium and the cytosol:

where k_kinetic is a constant proportional to the permeability of the c⁺ probe across the plasma membrane. The equilibration effects between the mitochondria and the cytosol are assumed as immediate. From this, we obtain the whole-cell fluorescence f_[cell] as the sum of the cytosolic and mitochondrial matrix signals by taking into consideration the distinct behavior attributable to quenching of the fluorescence signal within the mitochondria with the quench threshold concentration c⁺_[quench]:

Here, q is the quantum yield of the probe with V_[cyto] and V_[mito] denoting the cytosolic and mitochondrial volume.

To provide a more accurate model, we re-evaluated the mitochondrial volume (V_[mito]) within the neurons as well as the rate constant k_kinetic for TMRM (see Fig. 2) and implemented the model into MATLAB. A Newton routine for fitting the membrane potentials to the experimental fluorescent traces was implemented to avoid the need and bias of manual parameter fitting. The mitochondrial volume within the neurons (6.2 ± 0.8% SEM) was determined by using the Zeiss LSM 510 confocal microscope to create high-resolution z-stacks (80 x 0.2 µm steps, 0.8 µm optical slice) of TMRM-loaded neurons (see Fig. 2A). The rate constant k_kinetic was determined as 0.008 s^–1 by remodeling the redistribution of TMRM after the induction of mitochondrial depolarization by the protonophore FCCP in the presence of oligomycin in both the quenched and unquenched mode (see Fig. 2B,D). Because of variation in both the kinetic constant and the mitochondrial volume between neurons, we included them into the set of parameters for the subsequent fitting routine with an allowed variation of 0.008 ± 0.001 s^–1 and 6.2 ± 1.7%, respectively.

Parameter fitting was implemented using the built-in Newton-based root-finding algorithm FITCURVE of MATLAB, allowing for up to 50,000 iterations. To pose biological restrictions on the fits, we assumed a functional behavior for the time course of (_p) and (_m) as given in Figure 1 with parameters subject to fitting within a given range (supplemental Table 1, available at www.jneurosci.org as supplemental material) (Ward et al., 2000; Dussmann et al., 2003; Nicholls, 2006). For the apoptotic model (Fig. 1A), we assumed an initial depolarization (_p,initial) of the plasma membrane being present between the onset of glutamate and the addition of MK-801, returning to a higher value (_p,relax) [–83 mV (Nicholls, 2006)] rapidly after the addition of the antagonist. Moreover, to render the change of experimental fluorescence, we allowed _m to vary to a distinct value (^Hyp_m,max) at the time of glutamate addition and then to exponentially relax (constant k_relax) to a certain rate (^Hyp_{m, relax}). During the collapse of _m and the onset of apoptosis, we assumed a variant (ii) of our model allowing for exponential decay of both potentials (k_m,DCD, k_p,DCD) to a final, remnant value (_p,final, _m,final) after the onset of injury (t_DCD).

View larger version (27K): [in this window] [in a new window]   Figure 1. Potential models and fitting parameters for MATLAB-based analysis. The figure depicts the assumed functional behavior of m (solid line) and p (dashed line) that is used for fitting the TMRM fluorescent responses as taken from biological assumptions described in Materials and Methods. Parameters subject to fitting (potential values, decay constants) are indicated by arrows. A, Potential model for the apoptotic cells showing hyperpolarizion of m only (i) and showing hyperpolarization of m with a secondary collapse (ii). B, Necrotic model; monophasic response. C, Necrotic model; biphasic response. a.u., Arbitrary units; hyp, hyperpolarizion; max, maximum; dep init, initial depolarization; dep final, final depolarization; inc, increase; dec, decay; Glut, glutamate; DCD, delayed calcium deregulation.

 
For the monophasic necrotic model (Fig. 1B), we assumed a permanent initial depolarization of the plasma (_p,final) and mitochondrial membrane (_m,final) with the decay constants k_{p, decay}, k_{m, decay}, respectively. For the biphasic necrotic model (Fig. 1C), we allowed for a change of the mitochondrial membrane (_m,_relax^Hyp/Dep, k_m,inc/dec) and a decay of the plasma potential (_p,relax, k_p,_decay) after onset of continuous glutamate lasting and before the decay of both potentials, at the time point of injury (t_DCD).

For computational modeling of the DiBAC₂(3) responses, we assumed the measured fluorescence F_initial, F_actual(t) proportional to the cytosolic concentration of the dye with the Nernstian equation:

We obtained the following:

Epi-fluorescence NADPH microscopy. Seven- to 8-d-old cerebellar granule neurons on Willco dishes were loaded with TMRM (30 nM) for 30 min at 37°C (in the dark), in experimental buffer. The Willco dishes with cells were washed in fresh medium after loading before being mounted in a nonperfusion (37°C) holder and placed on the stage of a Zeiss Axiovert 200M microscope. During experiments, cells were treated as described above (see Confocal microscopy). NADPH auto fluorescence as well as TMRM fluorescence was observed using an Axiovert 200M inverted microscope equipped with a 63x, 1.4 numerical aperture oil-immersion objective (Zeiss), dichroic beam splitters, and filter wheels in the excitation and emission light path containing the appropriate filter sets (NADPH: excitation, 375 ± 25 nm; emission, 448 ± 32 nm; TMRM: excitation, 530 ± 21 nm; emission, 592 ± 22 nm; filters and dichroic mirrors were made by Semrock, Rochester, NY). Emission and bright-field images were recorded using a back-illuminated, cooled EM CCD camera (Ixon BV 887-DCS; Andor, Belfast, UK). The imaging setup was controlled by MetaMorph software.

ATP luciferase assays. Cerebellar granule neurons were maintained on poly-D-lysine (5 µg/ml)-coated 24-well plates for 7–8 d before use. The culturing medium was replaced with an experimental buffer (in mM: 120 NaCl, 3.5 KCl, 0.4 KH₂PO₄, 20 TES, 5 NaHCO₃, 1.2 Na₂SO₄, 1.2 CaCl₂, and 15 glucose, pH 7.4), and the neurons were excited with glutamate/glycine for 5 min or continuously (100 and 10 µM) and lysed at the times indicated. In separate experiments, cerebellar granule neurons were exposed to combinations of oligomycin (2 µg/ml), FCCP (2 µM), and MK-801 (10 µM) and maintained for 30 min before being lysed. The neurons were lysed using a hypotonic lysis buffer (Tris-acetate buffer, pH 7.75). Fifty microliters of the sample and 50 µl of the luciferin–luciferase reaction kit (ENLITEN ATP Assay System Bioluminescence Detection kit; Promega, Southampton, UK) for ATP were reacted to quantify ATP content. The amount of ATP was determined by a concentration standard curve, and ATP content values were normalized according to the protein concentration for each sample (moles ATP/µg protein).

Glucose uptake assay. Cerebellar granule neurons were maintained on poly-D-lysine (5 µg/ml)-coated 96-well plates for 7–8 d before use. The culturing medium was then replaced with an experimental buffer (in mM: 120 NaCl, 3.5 KCl, 0.4 KH₂PO₄, 20 TES, 5 NaHCO₃, 1.2 Na₂SO₄, 1.2 CaCl₂, and 1 glucose, pH 7.4) minus MgCl₂. The cerebellar granule neurons were treated with glutamate/glycine (100 and 10 µM) for 10 min (37°C) or exposed to sham conditions (experimental buffer). The excitation buffer was replaced with buffer minus glucose and containing 100 µM 2-deoxyglucose and ³H-2-deoxyglucose. ³H-2-deoxyglucose is taken up by neurons through the glucose transporters; however, it is not fully metabolized by the neuron and is retained within the cell. The neurons were maintained for 20 min in the experimental buffer containing the ³H-2-deoxyglucose; the buffer was then removed, and the neurons were washed twice with cold buffer minus glucose before the addition of 100 µl of scintillation fluid. The plates were left for 20 min on ice before reading on a Matrix-96 beta counter (Packard, Grove, IL). All conditions were repeated six times per experiment, and the same experiment was performed in at least three different cultures.

Statistics. Data are given as means ± SEM. For statistical comparison, one-way ANOVA followed by Tukey's test were used. p values <0.05 were considered to be statistically significant. Data for _{m, p} and the onset of neuronal injury were plotted using Origin 7 software (OriginLab, Northampton, MA) a linear regression was preformed on the data, and the R² value was calculated for each data set.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization and computational modeling of whole-cell TMRM fluorescence in cerebellar granule neurons
Cellular TMRM fluorescence is not only highly dependent on the changes in both _p and _m but is also directly related to the total mitochondrial volume within the cell (Ward et al., 2000; Dussmann et al., 2003; Nicholls, 2006). Therefore, a stringent determination of the mitochondrial volume was performed in cerebellar granule neurons. High-resolution z-stacks with 0.2 µm steps were taken of three fields of neurons, and the cellular volume (296.9 ± 32.9 µm³) and the mitochondrial volume (17.6 ± 1.5 µm³) were determined for each neuron (Fig. 2A). The mitochondrial volume (6.2 ± 0.8%) was found to be larger than that described previously (Ward et al., 2000; Nicholls, 2006)

View larger version (40K): [in this window] [in a new window]   Figure 2. Characterization of TMRM fluorescence in cerebellar granule neurons with high-resolution single-cell confocal microscopy. A, Cerebellar granule neurons were loaded with 30 nM TMRM, and the mitochondrial volume (17.6 ± 1.5 µm3; 6.2 ± 0.8%; n = 9) was determined by measuring the volume of individual mitochondria within the neurons after the acquisition of high-resolution z-stacks with an LSM 510 confocal microscope. B, D, Neurons loaded with 100 nM TMRM (B) and 30 nM TMRM (D) were exposed to FCCP (2 µM) in the presence of oligomycin (2 µg/ml) and monitored over a 60 min period. C, E, Using MATLAB software, the traces in B and D were modeled, and the changes in both m and p were established for these control experiments (all experiments were performed 3 times in different cultures). F, Cerebellar granule neurons were loaded with the p-sensitive probe DiBAC2(3) (1 µM) and exposed to oligomycin (2 µg/ml), followed by FCCP (2 µM). A rapid increase in fluorescence (depolarization of p) was associated with the addition of FCCP (traces are representative of those obtained from 3 separate experiments). G, Representative traces for modeled changes in p (A) for neurons exposed to oligomycin (2 µg/ml) and FCCP (2 µM). Oligo, Oligomycin.

 
To determine the rate constant (k_kinetic) for the redistribution of TMRM, several control experiments were performed (Fig. 2B,C). The protonophore FCCP (2 µM) was added to neurons loaded with TMRM in the presence of oligomycin (2 µg/ml) to uncouple _m, and the fluorescent signals were monitored for neurons in both the quenched (Fig. 2B) and nonquenched (Fig. 2D) mode. From the redistribution of the probe after the addition of FCCP, we established a kinetic constant for the redistribution of TMRM of 0.008 s^–1.

The MATLAB-based computational model was used to interpret the changes in TMRM fluorescence in response to the addition of FCCP/oligomycin and provide output on changes in _m and _p. Surprisingly, in the modeled traces (Fig. 2C,E) the expected rapid collapse in _m after the addition of FCCP was also closely paralleled with a rapid collapse of _p. To validate these findings, we used the _p-sensitive fluorescent probe DiBAC₂(3) to monitor changes in _p after the addition of oligomycin and FCCP (Fig. 2F,G). A rapid and prolonged increase in DiBAC₂(3) fluorescence after the addition of FCCP that reflected a substantial depolarization of _p (Fig. 2F,G) was observed.

In addition to monitoring TMRM and DiBAC₂(3) fluorescent responses, changes in Ca²⁺ dynamics (Fluo-4) and neuronal ATP levels were monitored within the neurons after the addition of FCCP in the presence of oligomycin (Fig. 3). A rapid collapse of Ca²⁺ homeostasis was identified within the neurons after exposure to the protonophore FCCP (Fig. 3A). The collapse in Ca²⁺ homeostasis (Fig. 3A) after the addition of FCCP was completely blocked by the NMDA antagonist MK-801 (Fig. 3B). The addition of oligomycin alone had no significant impact on neuronal ATP levels (Fig. 3); however, oligomycin plus FCCP resulted in a significant decrease (*p < 0.01) in ATP levels that was blocked by the presence of the NMDA antagonist MK-801 (Fig. 3C). These results suggest that any Ca²⁺ entering the neuron is the direct result of NMDA receptor overactivation attributable to glutamate release from the presynaptic terminals. Because the ATP synthase inhibitor oligomycin was present throughout (Fig. 3), energy restrictions alone could not account for the release of glutamate. This is intriguing in that it suggests presynaptic, polarized mitochondria play a key role in the regulation of glutamate within the synapse.

View larger version (23K): [in this window] [in a new window]   Figure 3. Protonophore addition induces a rapid collapse of Ca2+ homeostasis that is blocked with MK-801. A, B, Neurons were loaded with TMRM (30 nM) and Fluo-4 (3 µM) for 30 min at 37°C and exposed to FCCP (2 µM) in the presence of oligomycin (2 µg/ml) with (A) or without (B) MK-801 (10 µM). A rapid increase in cytosolic Ca2+ accompanied the FCCP addition in neurons that did not have MK-801 present (experiments were repeated 3 times for each condition, with similar results). C, Neurons were treated with a combination of FCCP (2 µM), oligomycin (2 µg/ml), and MK-801 (10 µM) for 30 min, and ATP levels were measured moles ATP/µg protein. All data were normalized to control values for comparison between experiments (experiments were performed in triplicate in 3 separate cultures;**p < 0.001; *p < 0.01 difference from control). Error bars indicate SEM. Oligo, Oligomycin; Arb units, arbitrary units.

 
Characterization of _m and _p during necrotic cell injury induced by prolonged glutamate excitation in cerebellar granule neurons
We have previously characterized a model of excitotoxic necrosis in cerebellar granule neurons induced by prolonged glutamate excitation (Castilho et al., 1998, 1999; Ward et al., 2000). In agreement with previous studies, we identified two major subgroups of neurons after prolonged glutamate excitation: (1) neurons that underwent a rapid collapse of _m (5–20 min) (Fig. 4A,B) with monophasic loss (33.1%) of TMRM fluorescence, rapid loss of Ca²⁺ homeostasis, and neuronal swelling; and (2) neurons that underwent a delayed neuronal injury (15 min to 4 h) (Fig. 4A,B, ii) with a biphasic drop (63.4%) in TMRM fluorescence, a delayed loss of Ca²⁺ homeostasis, neuronal swelling, and early membrane lysis (Ward et al., 2000; Ward et al., 2005).

View larger version (60K): [in this window] [in a new window]   Figure 4. Prolonged glutamate excitation induces a rapid loss of m and early necrotic injury in cerebellar granule neurons. Cerebellar granule neurons plated on Willco dishes were loaded with 30 nM TMRM and continuously exposed to glutamate/glycine (100 µM/10 µM). TMRM fluorescence was then monitored over time, and images were taken at 1 min intervals. A, Differential interference contrast (DIC) and TMRM fluorescent images were chosen at selected time points (0, 30, and 120 min during glutamate excitation) from a representative experiment. B, Representative traces for whole-cell TMRM fluorescence in neurons during prolonged glutamate excitation. Two major subgroups were identified: traces that show a rapid collapse of m (i) and traces that show a partial recovery of m, followed by a secondary collapse of m (ii). C, Whole-cell TMRM fluorescence in control neurons after 30 min and neurons treated with glutamate for 30 min. p < 0.001, difference between TMRM fluorescence for control neurons and neurons after 30 min continuous glutamate excitation (control, n = 39; necrotic, n = 63). D, Cerebellar granule neurons were loaded with the p-sensitive probe DiBAC2(3) (1 µM) and continuously exposed to glutamate/glycine (100 µM/10 µM). Neurons undergo a rapid monophasic (i) collapse of p or a biphasic (ii) collapse of p (traces are representative of those obtained from 3 separate experiments). E, Representative traces for modeled changes in p (C) for neurons undergoing a rapid monophasic (i) collapse of p or a biphasic (ii) collapse of p when continuously exposed to glutamate/glycine (100 µM/10 µM).

 
From the data obtained (Fig. 4), we used the MATLAB-based computational model (Fig. 1B,C) for the high throughput assessment of the TMRM fluorescence responses to define potential changes in _m and _p. Representative traces from neurons exposed to glutamate are shown from separate (Fig. 5A,C,E) experiments and modeled output traces for _m and _p determined for each (Fig. 5B,D,F): (1) neurons that entered a rapid necrotic cell death with a monophasic decrease in _m and _p after glutamate excitation (Fig. 5A) and (2) neurons that entered a delayed necrotic injury (Fig. 5C,E) with a biphasic decrease in _m and _p. The rate at which neurons entered the second phase of injury varied considerably (15 min to 4 h) between neurons and cultures; however, all neurons entered a necrotic-like injury with a rapid swelling of the neuron and loss of cellular integrity shortly after the collapse of _m.

View larger version (29K): [in this window] [in a new window]   Figure 5. Computational modeling of TMRM traces for neurons during prolonged glutamate excitation. Cerebellar granule neurons plated on Willco dishes were loaded with 30 nM TMRM and continuously exposed to glutamate/glycine (100 µM/10 µM). TMRM fluorescence was then monitored over time, and images were taken at 2 min intervals. A, Representative TMRM fluorescent trace (solid line) and fitted trace (dashed line) for a neuron that had a rapid monophasic response during glutamate induced necrosis. C, E, Representative TMRM fluorescent traces and fitted traces for neurons that have a biphasic response during glutamate-induced necrosis. B, D, F, Modeled changes in both p (dashed lines) and m (solid line) for the fitted traces in figures A, C, and E, respectively. TMRM (A, C, E) traces are representative of traces obtained from eight separate experiments from different cultures. a.u., Arbitrary units.

 
To validate that the modeled _p responses above (Fig. 5B,D,F) were accurate, we directly monitored _p with the plasma membrane-sensitive probe DiBAC₂(3) (Fig. 4D). Again, two major groups of neurons were identified: neurons that underwent a rapid collapse of _p (Fig. 4D, i) and neurons that underwent a secondary loss of _p (Fig. 4D, ii). When the DiBAC₂(3) responses were modeled using the MATLAB software and the predictive changes in _p were calculated (Fig. 4E), the values for _p were found to be similar to those predicted form the modeled TMRM data (Fig. 5B,D,F). Therefore, it would appear that MATLAB-based computational models can accurately determine changes in both _m and _p from TMRM responses alone.

Hyperpolarization of _m in apoptotic neurons after transient glutamate receptor excitation
Transient excitotoxicity results in a delayed neuronal injury that has mitochondrial dysfunction intricately linked with injury onset (Lankiewicz et al., 2000; Luetjens et al., 2000; Ward et al., 2000, 2006). Here, cerebellar granule neurons were stimulated for 5 min with glutamate/glycine, allowed to recover, and monitored over a 24 h period (Ward et al., 2006). After transient glutamate excitation, only 2.9% of the neurons underwent rapid necrotic injury with an early loss of _m (46.2 ± 16.7 min), neuronal swelling, and early membrane lysis. However, the majority of neurons monitored (78.8%) displayed a delayed loss of _m (11.8 ± 0.8 h), cell shrinkage, and condensation of the nucleus within a 24 h period. An additional 18.2% survived this injury and retained a hyperpolarized _m and normal cellular morphology for at least 24 h (Fig. 6A,B).

View larger version (64K): [in this window] [in a new window]   Figure 6. Transient glutamate excitation results in a hyperpolarization m and a late apoptotic injury in cerebellar granule neurons. Cerebellar granule neurons plated on Willco dishes were loaded with 30 nM TMRM and exposed to glutamate/glycine (100 µM/10 µM) for 5 min. TMRM fluorescence was then monitored over time, and images were taken at 5 min intervals. A, Differential interference contrast (DIC) and TMRM fluorescent images were chosen at selected time points (0 min, 2 h, and 14 h after glutamate) from a representative experiment. B, Representative traces for whole-cell TMRM fluorescence in neurons during prolonged glutamate excitation. Two major subgroups are shown: traces that show a secondary collapse of m downstream of excitation (i) and traces that show a collapse of m within 24 h (ii). C, Whole-cell TMRM fluorescence in control neurons after 120 min and neurons 120 min after glutamate excitation. p < 0.01, difference between TMRM fluorescence for control neurons and apoptotic neurons 120 min after transient glutamate excitation; p < 0.001 difference between TMRM fluorescence for apoptotic neurons 120 min after transient excitation compared with neurons that survive for >24 h after glutamate excitation (control, n = 39; apoptotic, n = 134; live, n = 31). D, Cerebellar granule neurons were loaded with the p-sensitive probe DiBAC2(3) (1 µM) and transiently (5 min) exposed to glutamate/glycine (100 µM/10 µM). Fluorescence recovers close to pre-exposure levels (average response dark trace) after the addition of glutamate (traces are representative of those obtained from 3 separate experiments). E, Representative traces for modeled changes in p for neurons transiently exposed to glutamate.

 
The MATLAB-based model was used (Fig. 1A) to interpret the changes in TMRM fluorescence for populations of individual neurons and provide output on changes in _m and _p during and after transient glutamate excitation. In the majority of neurons (97.1%), a transient depolarization of _p was associated with glutamate receptor overactivation with values for _p returning to prestimulation levels within a relatively short (5–15 min) period of time (Fig. 7B,D,F,H). Interestingly, the values for _m after glutamate excitation were found to be significantly higher (p < 0.001) than before glutamate excitation (Fig. 7B,D,F,H). Similar to the previous experiments, the output on the changes in _m and _p deducted from the computational analysis of the TMRM responses were validated with the _p-sensitive probe DiBAC₂(3). An increase in DiBAC₂(3) fluorescence was associated with the initial glutamate excitation (_p depolarization) (Fig. 6D); after inhibition of glutamate receptor overactivation, the fluorescence recovered to prestimulation levels with no hyperpolarization of the _p (lower fluorescence than baseline) identified (Fig. 6D). Interestingly, when the DiBAC₂(3) responses were calculated using MATLAB software and the predictive changes in _p were calculated (Fig. 6E), the values for _p were again found to be similar to those predicted from the modeled TMRM data (Fig. 7B,D,F,G). What is most significant from these results is the fact that the increase in TMRM fluorescence after glutamate excitation (Figs. 6, 7) is almost entirely attributable to a hyperpolarization of _m with little or no contribution from changes in _p (Fig. 6D,E).

View larger version (31K): [in this window] [in a new window]   Figure 7. Computational modeling of TMRM traces for cerebellar granule neurons after transient glutamate excitation. Cerebellar granule neurons plated on Willco dishes were loaded with 30 nM TMRM and exposed to glutamate/glycine (100 µM/10 µM) for 5 min. TMRM fluorescence was then monitored over time, and images were taken at 5 min intervals. A, C, Representative TMRM fluorescent traces (solid line) and fitted traces (dashed lines) for neurons that undergo apoptosis after transient glutamate excitation. E, G, Representative TMRM fluorescent traces and fitted traces for neurons that do not undergo apoptosis after transient glutamate excitation. B, D, F, H, Modeled changes in both p (dashed lines) and m (solid line) for the fitted traces in A, C, E, and F, respectively. TMRM traces (A, C, E, G) are representative of traces obtained from eight separate experiments from different cultures. I, The fitted initial depolarization of p during glutamate excitation is plotted against the onset of injury (collapse of TMRM signal; n = 5 populations of neurons). J, The fitted maximum mitochondrial hyperpolarization of m against onset of injury after glutamate excitation (n = 5 populations of neurons).

 
Hyperpolarization of _m is a predictor of survival time and tolerance to transient glutamate receptor excitation
Statistical analysis of the MATLAB-based computational model was performed to investigate whether changes in _m and _p after transient glutamate receptor overactivation could predict downstream injury or survival. Two major phases of change were determined during transient glutamate excitation: (1) the extent of the initial depolarization of _p during the glutamate excitation phase and (2) the hyperpolarization of _m after the excitation phase (Figs. 6, 7). When the single-cell data from a number of neuronal populations were characterized, correlations between the onset of injury (collapse of _m within the neuron) and changes in _m and _p could be established. A strong correlation was identified (r² = 0.28; n = 5 neuronal populations) between the extent of the initial depolarization and the onset of neuronal injury in individual population subsets (Fig. 7I), with a large depolarization of _p during the initial glutamate excitation phase associated with a more rapid onset of injury. The second and possibly more interesting correlation defined (r² = 0.23; n = 5 neuronal populations) is that between the extent of hyperpolarization after transient excitation within a population of cells and the onset of injury (Fig. 7J), with those neurons having a more pronounced hyperpolarization of _m during the recovery phase surviving longer. On further analysis of the TMRM fluorescence for those neurons that tolerated the glutamate insult, we identified that those neurons tolerant to transient glutamate excitation had a significantly (p < 0.001) higher TMRM fluorescence (202.8 ± 19.2; n = 31) than neurons that underwent apoptosis (Fig. 6C). These data suggest that the extent of the hyperpolarization of _m (increase in TMRM fluorescence) after transient glutamate excitation is more closely associated with neuronal survival rather than injury.

Enhanced glucose uptake and NADPH availability in response to transient glutamate excitation
Because an increased _m is highly suggestive of alterations in neuronal metabolism, we investigated how glucose uptake, NADPH production, and neuronal ATP levels may be altered in relation to glutamate excitation. Prolonged glutamate excitation (dashed line) resulted in a significant (^#p < 0.01) rapid and continuous decrease in both neuronal NADPH (Fig. 8A,B) and ATP (Fig. 8C) levels within the neurons. However, transient glutamate excitation (solid line) resulted in a transient decrease in both neuronal NADPH and ATP levels that was followed by a significant (*p < 0.01) and sustained increase in both (Fig. 8A,B) NADPH and ATP. This increase in NAHPD and ATP levels was also coupled to a significant increase in glucose uptake (Fig. 8D) as measured with ³H-2-deoxyglucose. These alterations in neuronal metabolism, in particular the NADPH levels that could be determined in real time, closely paralleled the hyperpolarization of _m identified after transient glutamate excitation (Figs. 6B, 7).

View larger version (31K): [in this window] [in a new window]   Figure 8. Glutamate (Glut) induced changes in neuronal NADPH, ATP, and glucose uptake. Cerebellar granule neurons plated on Willco dishes were exposed to glutamate/glycine (100 µM/10 µM) continuously (dashed line) or for 5 min (solid line) before termination of NMDA receptor activation with MK-801 (10 µM). A, B, NADPH autofluorescence was monitored over time (A) and at selected time points (0, 10, and 60 min; B) chosen before, during, and after glutamate excitation for statistical analysis. (At least 5 cells were analyzed per experiment, and the experiment was repeated in three different cultures. #p < 0.01; *p < 0.01, difference from respective control.) C, Cerebellar granule neurons plated in 24-well plates were exposed to glutamate/glycine (100 µM/10 µM) for 5 min or continuously, and their ATP content was measured (moles ATP/µg protein) at the times indicated. Data are represented as percentage of control response (n = 3 experiments in triplicate; #p < 0.01; *p < 0.01, difference from respective control). D, Cerebellar granule neurons plated in 96-well plates were exposed to glutamate/glycine (100 µM/10 µM) for 10 min and washed, and the medium was replaced with buffer containing 3H-2-deoxyglucose for 20 min. The cells were washed twice, and the 3H-2-deoxyglucose was measured in a Matrix-96 beta counter for 20 min. Data are presented as counts per minute (CPM) for control (washed neurons) and glutamate-treated neurons (6 wells were examined per condition per experiment and repeated in 3 separate cultures; *p < 0.01). Error bars indicate SEM.

 

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Computational modeling of _m and _p from TMRM fluorescent traces
Cationic fluorescent probes (TMRM, TMRE, rhodamine-123, JC-1) are an invaluable tool for monitoring mitochondrial function in intact cells during excitotoxic injury (Khodorov et al., 1996; White and Reynolds, 1996; Kiedrowski, 1998; Prehn, 1998; Stout et al., 1998; Ward et al., 2000; Buckman et al., 2001). However, because of the complex nature of these probes and their sensitivity to changes in both _m and _p, the fluorescence responses obtained have often been misinterpreted because of an overestimation of changes in _m by underestimating the significance of changes in _p on the total cellular fluorescence (Nicholls and Ward, 2000; Ward et al., 2000). Here, we have successfully created an automated MATLAB-based computational model that uses the sensitivity of TMRM fluorescence to changes in both _m and _p providing predictive output on changes in _m and _p, removing the need for additional plasma membrane indicators or manual parameter fitting. Using the plasma-sensitive probe DiBAC₂(3) (Ehrenberg et al., 1988; Gonzalez and Maher, 2002), we have validated the sensitivity and accuracy of the computational model created in predicting changes in _p. With this technology, we have been able to evaluate changes that occur at a _m and _p level in models of glutamate-induced necrosis, apoptosis, and tolerance, providing us with novel insights into the bioenergetic/metabolic mechanisms associated with glutamate regulation and glutamate-induced injury and tolerance.

Effects of "mitochondrial" toxins on _p, Ca²⁺ uptake, and glutamate release
In control experiments (Fig. 3), a combination of the ATP synthase inhibitor oligomycin and the protonophore FCCP were used to collapse _m in neurons, a technique routinely used to nullify mitochondrial function within neurons (Stout et al., 1998; Ward et al., 2005). The addition of oligomycin alone resulted in only minor changes in _m and neuronal ATP levels; however, the addition of FCCP resulted in the expected collapse of _m that was coupled with an unexpected decrease in _p, loss of Ca²⁺ homeostasis, and a significant decrease in neuronal ATP levels. Because the addition of the NMDA receptor antagonist MK-801 blocked these events, it implies that a depolarization of synaptic mitochondria was sufficient to induce glutamate release. Presynaptic mitochondria have a functional role in the regulation of Ca²⁺ (Yang et al., 2003; Brown et al., 2006; Ly and Verstreken, 2006; Mironov and Symonchuk, 2006) within synaptic vesicles; indeed, recent studies have shown that the endoplasmic reticulum (ER) and mitochondria bidirectionally exchange Ca²⁺ within the synapse and that Ca²⁺ released from the ER or mitochondria is sufficient to evoke exocytosis (Mironov and Symonchuk, 2006). If synaptic mitochondria are highly involved in the regulation of synaptic Ca²⁺ (Mironov and Symonchuk, 2006), a mitochondrial depolarization and reversal of the Ca²⁺ uniporter would provide sufficient Ca²⁺ to enable glutamate exocytosis to occur. Yang et al. (2003) also identified that Ca²⁺ released from mitochondria through the mitochondrial Na⁺–Ca²⁺ exchanger is sufficient to induce synaptic glutamate release. Additionally, Budd and Nicholls (1996a) previously characterized a protonophore releasable pool of Ca²⁺ within neurons. Therefore, it appears that the manipulation of _m with mitochondrial toxins may alter Ca²⁺ sequestration within synaptic mitochondria and thereby alter glutamate regulation within the synapse.

Glutamate-induced necrosis
The injury identified during prolonged glutamate excitation in this study is very much in agreement with previous studies including work performed by ourselves (Ankarcrona et al., 1995; White and Reynolds, 1996; Prehn, 1998; Stout et al., 1998; Ward et al., 2000), with prolonged glutamate excitation resulting in a monophasic or biphasic collapse of _m, paralleled with a decrease in _p, rapid neuronal swelling, and early membrane lysis. Prolonged glutamate excitation also resulted in the rapid decay of ATP as a consequence of the increased activity of both the Na⁺/K⁺ and Ca²⁺ ATPases at the plasma membrane.

What is intriguing from the data presented here is the biphasic DiBAC₂(3) responses (Fig. 4E) during glutamate excitation, where the loss of _p is not a single event but appears to be a slowly developing process. So what may induce a slow loss of _p? The preservation of a polarized plasma membrane is dependent on the regulation of ionic homeostasis by Na⁺/K⁺ and Ca²⁺ ATPases, as well as the Na⁺/Ca²⁺ exchanger. During glutamate excitation, a high concentration of Ca²⁺ is retained within the cytosol and mitochondrial matrix (Tymianski et al., 1993a; Budd and Nicholls, 1996b; Stout et al., 1998; Brocard et al., 2001; Ward et al., 2005). This prolonged increase in cytosolic and mitochondrial Ca²⁺ has been shown to increase calpain activity (Siman et al., 1989; Faddis et al., 1997; Lankiewicz et al., 2000), and it has been suggested that this increased calpain activation leads to cleavage of the Na⁺/Ca²⁺ exchanger, resulting in the progressive disruption of cellular homeostasis (Bano et al., 2005). Additionally, the activation of TRP (transient receptor potential) channels, post glutamate excitation, may also play an integral part in the development of this secondary, slow _p depolarization (Aarts et al., 2003).

Glutamate-induced apoptosis and tolerance
In contrast to the necrotic injury induced by prolonged glutamate excitation, transient glutamate excitation resulted in a reversible depolarization of _p that was followed by a prolonged hyperpolarization of _m, with the subsequent collapse of both _m and _p, cell shrinkage and nuclear condensation occurring hours downstream of the initial excitation (Lankiewicz et al., 2000; Luetjens et al., 2000; Ward et al., 2000). Using the MATLAB-based computational model, we were able to detect changes in both _m and _p relative to injury onset for populations of single cells after transient glutamate excitation. A correlation was identified between the extent of the initial depolarization of _p during the glutamate excitation phase and the onset of injury within neurons, with those neurons having a more pronounced depolarization of _p during the excitation phase entering a more rapid cell death. Previous studies have identified that excitotoxic injury is highly dependent on the duration and intensity of glutamate/NMDA receptor overactivation (Choi, 1987; Tymianski et al., 1993b; Sattler et al., 1998). Because of the variability in the initial _p depolarization identified after glutamate excitation in this study, it is evident that there are marked variations in the type and number of glutamate/NMDA receptors present at the plasma membrane between neurons within a "homogenous" culture.

The most significant findings in this study involve the relationship between the hyperpolarization of _m, the changes in neuronal metabolism, and neuronal survival after transient glutamate excitation. A hyperpolarization of _m has previously been described in hippocampal neurons exposed to staurosporine (Poppe et al., 2001) and 30 min oxygen glucose deprivation (Iijima et al., 2003), and also in primary rodent cortical neurons (Perry et al., 2005) after the induction of the regulatory protein Tat (transactivator of transcription) by HIV-1. The increase in _m identified was associated with the onset of apoptotic injury, as is true for the majority of neurons in this study. Here, we could rapidly interpret the TMRM fluorescent responses for a number of populations of single-cell data with the MATLAB-based computational model. From this, a positive correlation was established between _m and neuronal survival, with neurons displaying a more pronounced hyperpolarization of _m surviving longer, a correlation that may have been inadvertently missed in previous studies (Poppe et al., 2001; Iijima et al., 2003