(a) Changes in mitoGCaMP6m and Fura-2 fluorescence elicited by a train of two APs in a representative L5 pyramidal cell. Left, The image obtained by merging two optical sections through part of a L5 neuron at excitation wavelengths of 760 and 960 nm, eliciting the Fura-2 and mitoGCaMP6m fluorescence, respectively. Right, Somatic mitoGCaMP6m (red) and Fura-2 (green) ΔF/F transients elicited by a train of two APs. MitoGCaMP6m trace (red) is an ensemble average of 16 consecutive sweeps. (b) Small-amplitude mitoGCaMP6m transients decay rapidly. Left, Localization of the mitoGCaMP6m labeled mitochondria in a representative L5 neuron. The image was obtained by merging the fourteen, 1 µm thick optical sections at excitation wavelengths of 760 and 960 nm, eliciting the Fura-2 and mitoGCaMP6m fluorescence, respectively. The yellow color indicates colocalization of the Fura-2 (green) and mitoGCaMP6m (red) fluorescence, revealing the position of the mitochondria. The rectangle indicates the region of interest from which the optical sweeps were obtained. Right, somatic mitoGCaMP6m (red) and Fura-2 (green) transients elicited by 5, 20, and 50 APs in a cell shown on the left. Black lines are the best single or double exponential fits of the decay. Notice that after the relatively small [Ca2+]m elevations evoked by five or 20 APs, the Ca2+ clearance is rapid and follows the single exponential time course (τ ~ 0.7 s). The decay of larger transients evoked by 50 APs is biexponential (τ1 of ~0.7 s and τ2 of ~28 s). (c) Decay time course of mitochondrial Ca2+ transients depends on their amplitude. Each dot represents a decay time constant of the mitoGCaMP6m transient (τ1 for monoexponential, τ1 and τ2 for bi-exponential decays) obtained in recordings from 42 neurons and plotted against its peak amplitude. Continuous lines are mean τ1 (n = 55, black) and τ2 (n = 23, red); dashed lines represent standard deviation from the mean. Arrow represents the amplitude of the smallest mitoGCaMP6m transient (ΔF/F = 44 %), which decayed biexponentially. (d) The amplitude of spike-evoked mitoGCaMP6m fluorescence transients varies between different neuronal processes. Left, Localization of the mitoGCaMP6m labeled mitochondria in a representative L5 pyramidal neuron. The rectangles indicate the regions within the apical dendrite (cyan), basal dendrite (magenta), and axon initial segment (red) from which fluorescence measurements were obtained. Right, Mitochondrial and cytosolic (green) Ca2+ transients elicited by a train of 5 APs at 50 Hz in different neuronal compartments. (e) Mean peak ΔF/F of the mitoGCaMP6m transients as a function of the number of action potentials in soma, apical, and basal dendrites, and axon initial segment. Shown are mean values ± SE (n = 5–29).