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

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