Mitochondria are increasingly implicated in neurodegenerative mechanisms from several perspectives, including intracellular calcium regulation, free radical production, energy utilization, and release of apoptotic factors. Initially our group demonstrated that chelation of intracellular calcium by membrane-permeant calcium chelators was neuroprotective both in in vitro neuronal cultures and in vivo, significantly diminishing ischaemic stroke volume (Tymianski et al. 1993). All in vitro and in vivo experiments were performed according to local and national guidelines, including humane killing of animals. We then showed that an 8 min ischaemic (hypoxia/hypoglycaemia) insult to an organotypic hippocampal slice culture, promoted glutamate-mediated generation of free radicals with concomitant elevation of intracellular calcium (Perez Velazquez et al. 1997). In this same model, the mitochondrial complex I inhibitor, rotenone, the mitochondrial permeability transition blocker, cyclosporin A (CsA), and a blocker of NAD⁺, nicotinamide, decreased ischaemia-induced free radical generation and increased mitochondrial calcium (Frantseva et al. 2001). Interestingly, CsA did not diminish the increase in the cytoplasmic calcium, but did reduce the increased mitochondrial calcium, suggesting that mitochondrial calcium could be the most important mediator of neurodegenerative processes.

Synaptic transmission could be an early and sensitive target for cerebral ischaemia. Membrane-permeant calcium chelators diminish synaptic transmission in the in vitro hippocampal CA1 region (Ouanounou et al. 1996). Brief hypoxia diminished stratum radiatum-evoked synaptic transmission, which was resistant to intracellular calcium chelation by a membrane-permeant calcium chelator (Ouanounou et al. 1999). Preliminary experiments in acutely prepared rat hippocampal slices have shown that during and following 8 min of hypoxia/ hypoglycaemia, stratum radiatum evoked synaptic transmission in the CA1 subfield is depressed along with an increase in mitochondrial calcium which persists for up to 1 h following the ischaemic insult. We can now specifically load stratum radiatum presynaptic terminals with calcium fluorescent and mitochondrial dyes. Further experiments delineating the role of pre- and postsynaptic mitochondria in hypoxia/hypoglycaemia induced depression of synaptic transmission and the role of membrane-permeant calcium chelators will be discussed. We hypothesize that the neuroprotective actions of intracellular calcium chelation could be by limiting rises in intramitochondrial calcium and that the maintenance of ‘healthy’ mitochondria will be neuroprotective against ischaemic insults.

This work was supported by CIHR and the Alzheimer’s Association.

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