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 ⌬⌿ 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 DiBAC 2 (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.

Dynamics of Intracellular Oxygen in PC12 Cells upon Stimulation of Neurotransmission

“…6B) very difficult to resolve and interpret. However, the difficulties in defining absolute changes ⌬⌿ m and ⌬⌿ p can be partially resolved when utilizing mathematical models that interpret changes in TMRM fluorescence (36,37). From this we could establish that a 30 -40-mV depolarization of ⌬⌿ p was coupled with a 5-10-mV depolarization of ⌬⌿ m (Fig.…”

“…TMRM has been widely used to monitor changes in ⌬⌿ m in models of injury (36,37,45,46,59). However, TMRM is also sensitive to alterations in ⌬⌿ p ; therefore care has to be taken in the interpretation of TMRM fluorescent signals.…”

“…⌬⌿ m and ⌬⌿ p were calculated using the models provided by Ward et al (36) and Nicholls (37). Fitting parameters for cerebellar granule neurons were adapted.…”

“…Single Cell Characterization of ⌬⌿ m and ⌬⌿ p Relative to O 2 Consumption Following K ϩ Stimulation-The potentiometric probe TMRM has been used extensively as a tool for the single cell characterization of ⌬⌿ m (36,37,(43)(44)(45)(46) in vitro. Here we have utilized a nonquenching concentration of TMRM (20 nM) to monitor mitochondrial bioenergetics in d PC12 cells relative to changes in i O 2 .…”

AMP kinase–mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis

“…We next quantified the levels of pyknotic nuclei after NMDA excitation in neurons from puma-, bid-, or bim-deficient mice. Bonfoco et al, 1995;Ward et al, 2000Ward et al, , 2007. Conversely, if the ATP demand is less severe and the capacity of cells to generate ATP is sufficient, neurons restore their ionic gradients, and only a transient decrease in mitochondrial membrane potential ( m ) or cellular ATP levels can be detected.…”