Mitochondrial Ca²⁺ buffering regulates synaptic transmission between retinal amacrine cells

The diverse functions of retinal amacrine cells are reliant on the physiological properties of their synapses. Here we examine the role of mitochondria as Ca²⁺ buffering organelles in synaptic transmission between GABAergic amacrine cells. We used the protonophore p-trifluoromethoxy-phenylhydrazone (FCCP) to dissipate the membrane potential across the inner mitochondrial membrane that normally sustains the activity of the mitochondrial Ca²⁺ uniporter. Measurements of cytosolic Ca²⁺ levels reveal that prolonged depolarization-induced Ca²⁺ elevations measured at the cell body are altered by inhibition of mitochondrial Ca²⁺ uptake. Furthermore, an analysis of the ratio of Ca²⁺ efflux on the plasma membrane Na-Ca exchanger to influx through Ca²⁺ channels during voltage steps indicates that mitochondria can also buffer Ca²⁺ loads induced by relatively brief stimuli. Importantly, we also demonstrate that mitochondrial Ca²⁺ uptake operates at rest to help maintain low cytosolic Ca²⁺ levels. This aspect of mitochondrial Ca²⁺ buffering suggests that in amacrine cells, the normal function of Ca²⁺-dependent mechanisms would be contingent upon ongoing mitochondrial Ca²⁺ uptake. To test the role of mitochondrial Ca²⁺ buffering at amacrine cell synapses, we record from amacrine cells receiving GABAergic synaptic input. The Ca²⁺ elevations produced by inhibition of mitochondrial Ca²⁺ uptake are localized and sufficient in magnitude to stimulate exocytosis, indicating that mitochondria help to maintain low levels of exocytosis at rest. However, we found that inhibition of mitochondrial Ca²⁺ uptake during evoked synaptic transmission results in a reduction in the charge transferred at the synapse. Recordings from isolated amacrine cells reveal that this is most likely due to the increase in the inactivation of presynaptic Ca²⁺ channels observed in the absence of mitochondrial Ca²⁺ buffering. These results demonstrate that mitochondrial Ca²⁺ buffering plays a critical role in the function of amacrine cell synapses.