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 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.
Department of Physiology and Medical Physics and RCSI Neuroscience Research Centre, Royal College of Surgeons in Ireland, Dublin 2, Ireland.
Manus W
MW
Ward
Heinrich J
HJ
Huber
Petronela
P
Weisová
Heiko
H
Düssmann
David G
DG
Nicholls
Jochen H M
JH
Prehn
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D004361:Drug Tolerance / Q000502:physiology; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000502:physiology*; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D018698:Glutamic Acid