Simultaneous single neuron recording of O2 consumption, [Ca 2+]i and mitochondrial membrane potential in glutamate toxicity

In order to determine the sequence of cellular processes in glutamate toxicity, we simultaneously recorded O₂ consumption, cytosolic Ca²⁺ concentration ([Ca²⁺]_i), and mitochondrial membrane potential (mδψ) in single cortical neurons. Oxygen consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca²⁺]_i and mδψ were monitored with Fluo-4 and TMRE⁺, respectively, using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca²⁺]_i followed seconds afterwards by an increase in O₂ consumption which reached a maximum level within 1-5 min. A modest increase in mδψ occurred during this time period, and then, shortly before maximal O₂ consumption was reached, the mδψ, as indicated by TMRE⁺ fluorescence, dissipated. Maximal O₂ consumption lasted up to 5 min and then declined together with mδψ and ATP levels, while [Ca²⁺]_i further increased. mδψ and [Ca²⁺]_i returned to baseline levels when neurons were treated with an NMDA receptor antagonist shortly after the [Ca²⁺]_i increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O₂ consumption, [Ca²⁺]_i, and mδψ. The data obtained using this new method are consistent with a model where Ca ²⁺ influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation.