Presynaptic mitochondria in functionally different motor neurons exhibit similar affinities for Ca²⁺ but exert little influence as Ca ²⁺ buffers at nerve firing rates in situ

Mitochondria accumulate within nerve terminals and support synaptic function, most notably through ATP production. They can also sequester Ca ²⁺ during nerve stimulation, but it is unknown whether this limits presynaptic Ca²⁺ levels at physiological nerve firing rates. Similarly, it is unclear whether mitochondrial Ca²⁺ sequestration differs between functionally different nerve terminals. We addressed these questions using a combination of synthetic and genetically encoded Ca ²⁺ indicators to examine cytosolic and mitochondrial Ca²⁺ levels in presynaptic terminals of tonic (MN13-Ib) and phasic (MNSNb/d-Is) motor neurons in Drosophila, which, as we determined, fire during fictive locomotion at ∼42 Hz and ∼8 Hz, respectively. Mitochondrial Ca²⁺ sequestration starts in both terminals at ∼250 nM, exhibits a similar Ca²⁺-uptake affinity (∼410 nM), and does not require Ca ²⁺ release from the endoplasmic reticulum. Nonetheless, mitochondrial Ca²⁺ uptake in type Is terminals is more responsive to low-frequency nerve stimulation and this is due to higher cytosolic Ca²⁺ levels. Since type Ib terminals have a higher mitochondrial density than Is terminals, it seemed possible that greater mitochondrial Ca²⁺ sequestration may be responsible for the lower cytosolic Ca²⁺ levels in Ib terminals. However, genetic and pharmacological manipulations of mitochondrial Ca ²⁺ uptake did not significantly alter nerve-stimulated elevations in cytosolic Ca²⁺ levels in either terminal type within physiologically relevant rates of stimulation. Our findings indicate that presynaptic mitochondria have a similar affinity for Ca²⁺ in functionally different nerve terminals, but do not limit cytosolic Ca²⁺ levels within the range of motor neuron firing rates in situ.