Maturation of vulnerability to excitotoxicity: intracellular mechanisms in cultured postnatal hippocampal neurons.
Neuronal vulnerability to excitotoxicity changes dramatically during postnatal maturation. To study the intracellular mechanisms by which maturation alters vulnerability in single neurons, we developed techniques to maintain hippocampal neurons from postnatal rats in vitro. After establishing their neuronal phenotype with immunohistochemistry and electrophysiology, we determined that these neurons exhibit developmentally regulated vulnerability to excitotoxicity. At 5 days in vitro, NMDA-induced cell death at 24 h increased from 3.6% in 3-day-old rats to >90% in rats older than 21 days. Time-lapse imaging of neuronal morphology following NMDA demonstrated increasingly prevalent and severe injury as a function of postnatal age. Neither high- nor low-affinity calcium dyes demonstrated differences in peak NMDA-induced [Ca(2+)](i) increases between neurons from younger and older animals. However, neurons from older animals were uniformly distinguished from those from younger animals by their subsequent loss of [Ca(2+)](i) homeostasis. Because of the role of mitochondrial Ca(2+) buffering in [Ca(2+)](i) homeostasis, we measured NMDA-induced changes in mitochondrial membrane potential (DeltaPsi) as a function of postnatal age. NMDA markedly dissipated DeltaPsi in neurons from mature rats, but minimally in those from younger rats. These data demonstrate that, in cultures of postnatal hippocampal neurons, (a) vulnerability to excitotoxicity increases as a function of the postnatal age of the animal from which they were harvested, and (b) developmental regulation of vulnerability to NMDA occurs at the level of the mitochondrion.
Marks
J D
JD
Department of Pediatrics, The University of Chicago, 5841 S. Maryland Avenue, Chicago, IL 60637, USA. j-marks1@uchicago.edu
Bindokas
V P
VP
Zhang
X M
XM
D000375:Aging / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn / Q000502:physiology*; D015415:Biomarkers; D002118:Calcium / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004351:Drug Resistance; D004594:Electrophysiology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006706:Homeostasis / Q000502:physiology; D007425:Intracellular Membranes / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D008928:Mitochondria / Q000502:physiology; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000145:classification / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009498:Neurotoxins / Q000494:pharmacology*; D009994:Osmolar Concentration; D010100:Oxygen / Q000494:pharmacology; D017966:Pyramidal Cells / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley
D015415:Biomarkers; D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D016202:N-Methylaspartate; D010100:Oxygen; D002118:Calcium