Mitochondrial and plasma membrane potential of cultured cerebellar neurons during glutamate-induced necrosis, apoptosis, and tolerance

Abstract

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 delta psi(m) or delta psi(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 delta psi(m) that was closely associated with a loss of delta psi(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 delta psi(m) as confirmed using the delta psi(p)-sensitive anionic probe DiBAC2(3). This hyperpolarization of delta psi(m) was closely associated with a significant increase in neuronal glucose uptake, NADPH availability, and ATP levels. Statistical analysis of the changes in delta psi(m) or delta psi(p) at a single-cell level revealed two major correlations; those neurons displaying a more pronounced depolarization of delta psi(p) during the initial phase of glutamate excitation entered apoptosis more rapidly, and neurons that displayed a more pronounced hyperpolarization of delta psi(m) after glutamate excitation survived longer. Indeed, those neurons that were tolerant to transient glutamate excitation (18%) showed the most significant increases in delta psi(m). Our results indicate that a hyperpolarization of delta psi(m) is associated with increased glucose uptake, NADPH availability, and survival responses during excitotoxic injury.

Figure 1.

Potential models and fitting parameters for MATLAB-based analysis. The figure depicts the assumed functional behavior of ΔΨ_m (solid line) and ΔΨ_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 ΔΨ_m only (i) and showing hyperpolarization of ΔΨ_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.

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 ΔΨ_m and ΔΨ_p were established for these control experiments (all experiments were performed 3 times in different cultures). F, Cerebellar granule neurons were loaded with the ΔΨ_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 ΔΨ_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 ΔΨ_p (A) for neurons exposed to oligomycin (2 μg/ml) and FCCP (2 μm). Oligo, Oligomycin.

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.

Figure 4.

Prolonged glutamate excitation induces a rapid loss of ΔΨ_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 ΔΨ_m (i) and traces that show a partial recovery of ΔΨ_m, followed by a secondary collapse of ΔΨ_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 ΔΨ_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 ΔΨ_p or a biphasic (ii) collapse of ΔΨ_p (traces are representative of those obtained from 3 separate experiments). E, Representative traces for modeled changes in ΔΨ_p (C) for neurons undergoing a rapid monophasic (i) collapse of ΔΨ_p or a biphasic (ii) collapse of ΔΨ_p when continuously exposed to glutamate/glycine (100 μm/10 μm).

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 ΔΨ_p (dashed lines) and ΔΨ_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.

Figure 6.

Transient glutamate excitation results in a hyperpolarization ΔΨ_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 ΔΨ_m downstream of excitation (i) and traces that show a collapse of ΔΨ_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 ΔΨ_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 ΔΨ_p for neurons transiently exposed to glutamate.

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 ΔΨ_p (dashed lines) and ΔΨ_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 ΔΨ_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 ΔΨ_m against onset of injury after glutamate excitation (n = 5 populations of neurons).

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.