MITOCHONDRIA TO THE RESCUE IN TURTLE NEURONS

When neurons are deprived of oxygen, NMDA receptors become activated, allowing an influx of calcium ions into the neuron. As the mitochondria can't efficiently produce ATP in the absence of oxygen (anoxia), ATP-fuelled pumps can't pump out the ions and calcium accumulation eventually leads to cell death. However, in anoxia-tolerant animals, such as the western painted turtle, calcium flow through the NMDA receptors is rapidly reduced during anoxic periods and neuronal death is avoided. So how do they do it? It's a question that intrigues Leslie Buck, from the University of Toronto, Canada, and over the last few years, Buck and his team have found that a small increase in intracellular calcium (below the levels that induce cell death) is necessary to reduce the NMDA receptor's activity. Buck suspected that mitochondria might be the source of this calcium and that the mitochondrial permeability transition pore (mPTP) was involved; to find out, he recruited postgraduate student Peter Hawrysh to the project (p. 4375).To test the mPTP's involvement in regulating NMDA receptor activity, the duo surgically removed small flaps of cerebral cortex from their turtles. ‘It's a very nice system because it functions like an intact brain, all the connections in that particular area are present’, explains Buck. ‘We'd put this piece of tissue in a recording chamber where we could control the flow of the saline into the chamber. We could then gas that saline flow with nitrogen or oxygen and CO2 to give us what we thought was a fairly good representation of a transition from a normoxic to an anoxic situation. We could then put recording electrodes into the tissue and record various ion channel activities.’ To begin with they added NMDA during normoxia to measure NMDA currents, and when the tissue was made anoxic, the level of NMDA receptor activity decreased as expected. When the duo blocked the mPTP with cyclosporine A during anoxia, the NMDA receptor activity returned to normoxic levels and when the pair added atractyloside (which activates the mPTP) during normoxic conditions, they saw NMDA receptor activity was decreased. By using dyes that reacted to calcium, Buck and Hawrysh could also show that activation of the mPTP corresponded to an increase in intracellular calcium.Next, the duo moved on to working out how the mPTP was activated. ‘We measured membrane potential with rhodamine, which is a mitochondrial membrane potential-sensitive indicator, and we saw it change and that connected up with changes in the NMDA receptor activity’, recalls Buck. In addition, when they pharmacologically activated potassium channels to depolarise the membrane, they also saw mPTP activation.However, the duo found that membrane depolarisation was carefully controlled: ‘It doesn't fully depolarise’, explains Buck. ‘If we use an uncoupling compound like FCCP, we get a much larger decrease in membrane potential, which likely represents the full depolarised state. Under normoxic conditions it's [the membrane's] polarised and now we have a new intermediate state during the anoxic phase.’ To maintain this intermediate state, the duo found that the F1/F0 ATP synthase, which usually uses a gradient of hydrogen ions to produce ATP, switches instead to using ATP to pump hydrogen ions across the mitochondrial membrane to maintain this intermediate potential. On the surface, this might seem to use up valuable ATP when reserves are running low, but by carefully controlling the activation of the mPTP channel, and thus facilitating a small intracellular change in calcium, it seems that the turtles have found a way to prevent unregulated calcium entry via the NMDA receptor.

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