Real-time monitoring of basal and activity-dependent mitochondrial bioenergetics in cultured mouse dopamine neurons.

Mitochondria play an essential role in multiple cellular processes including ATP production, intracellular Ca2+ signaling, regulation of reactive oxygen species and apoptotic signalling. Neuronal functions and synaptic activity are strongly dependent on mitochondrial function for ATP supply and Ca2+ buffering. The identification of gene mutations in Parkinson's disease (PD) leading to impaired mitochondrial function and dopamine (DA) neuron death makes it critical to characterize mitochondrial bioenergetics in DA neurons. In the present study we quantified basal and activity-dependent mitochondrial energy metabolism in primary cultures of postnatal mesencephalic mouse DA neurons, grown on an astrocytes monolayer. Using a Seahorse XF24 analyzer, we measured simultaneously the rate of oxygen consumption (OCR), indicative of mitochondrial respiration and the rate of extracellular acidification (ECAR), reflecting glycolysis, thus providing a physiological readout of energetic metabolism. We first compared OCR and ECAR of mesencephalic DA neuron cultures to that of astrocytes cultured alone. We find that DA neuron cultures show considerably higher basal OCR and higher maximal OCR, measured in the presence of CCCP, an uncoupler agent. Quantification of ECAR revealed that cultured DA neurons also show elevated ECAR compared to astrocytes, but the relative difference was considerably less than for OCR, compatible with the known dependence of astrocytes on glycolysis rather than oxydative phosphorylation to produce ATP and the existence of more efficient mitochondrial energetic metabolism in neurons. To gain insight into the energetic metabolism of DA neurons compared to other types of mesencephalic neurons, we performed parallel experiments in purified DA neuron cultures prepared from TH-GFP transgenic mice. Our findings suggest that cultured DA neurons have higher basal OCR compared to other mesencephalic neurons. Finally, we used veratridine, an agent preventing Na+ channel desensitization and TTX, a Na+ channel blocker, to evaluate the activity-dependence of mitochondrial bioenergetics in cultured DA neurons. Veratridine enhanced basal OCR and inhibited CCCP-stimulated respiration, while TTX had the oppostite effect. These treatments were without effect in astrocyte cultures. Together these data provide a first overview of mitochondrial bioenergetics in DA neurons. Further experiments are now planned to evaluate these parameters in genetic models of PD.