CALCIUM-INDUCED ACTIVATION OF THE MITOCHONDRIAL PERMEABILITY TRANSITION IN HIPPOCAMPAL-NEURONS

The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mP T in hippocampal neurons, cultures were loaded with the mitochondrial dye JC-l and observed with confocal and conventional microscopy, After pretreatment with 4Br-A23187 and subsequent calcium addition, the ini tially rodlike mitochondria increased in diameter until mitochondria b ecame rounded in appearance. Morphological changes reversed when calci um was removed by EGTA, When neurons were loaded with both fura-2-AM a nd rhodamine 123, calcium loading produced an increase in cytosolic ca lcium, mitochondrial depolarization, and similar alterations in mitoch ondrial morphology, Smaller calcium challenges produced calcium cyclin g, delaying morphological changes until after secondary depolarization and calcium release to the cytosol, In neurons exposed to glutamate, confocal observation of JC-l fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substi tuted for calcium, or following pretreatment with the mPT inhibitor, c yclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. I n addition, the timing of changes suggested that induction of the perm eability transition in situ represents a sequence of multiple events t hat may reflect the multiple open conformations of the mPT pore. (C) 1 998 Wiley-Liss, Inc.