Robust activity-dependent mitochondrial calcium dynamics at the AIS is dispensable for action potential generation

Mitochondria are diverse and multifaceted intracellular organelles regulating oxidative energy supply, lipid metabolism and calcium (Ca ²⁺ ) signaling. In neurons the spatial sequestration of cytoplasmic Ca ²⁺ by mitochondria plays a critical role in determining activity-dependent spine plasticity, shaping the presynaptic transmitter release characteristics and contributing to sustained action potential firing. Here, we tested the hypothesis that mitochondria at the axon initial segment (AIS) affect the microdomain cytoplasmic Ca ²⁺ transients, thereby regulating Ca ²⁺ -dependent voltage-gated ion channels at the plasma membrane and initiation of action potentials. Using 3D electron microscopy (EM) reconstructions and virally injecting genetically encoded fluorescence indicators we visualized the ultrastructure and distribution of mitochondria selectively in thick-tufted layer 5 pyramidal neurons. We found that most mitochondria were stably clustered to the proximal AIS, while few were observed at distal sites. Simultaneous two-photon imaging of action potential-dependent cytoplasmic and mitochondrial Ca ²⁺ , combined with electrophysiological recordings showed the AIS mitochondria exhibit powerful activity-dependent cytosolic Ca ²⁺ uptake. However, while intracellular application of the mitochondrial Ca ²⁺ uniporter inhibitor Ru360 fully blocked mitochondrial Ca ²⁺ import, it did not affect action potential input-output function, action potential dynamics nor the ability to produce high-frequency burst output. Together, the results indicate that AIS mitochondria are dispensable for temporal and rate encoding, suggesting that mt-Ca ²⁺ buffering at the AIS may be involved in non-electrical roles, including AIS maintenance or axonal transport.