Fig. 1: Pyramidal neuron action potential firing triggers a long-lasting mitochondrial Ca²⁺ elevation mediated by the MCU.

a mRGECO fluorescence in neurons of the cortex is stable over time (ΔF/F_o) (time 1 [t1] vs time 2 [t2]), apart from infrequent spontaneous fluorescence transients seen in the neuropil (arrow). b Left, mRGECO fluorescence from regions of interest (ROI) in the soma and neuropil, showing the stability of the signal and an occasional spontaneous event. Right, Summary data demonstrates the low rate of spontaneous mitochondrial Ca²⁺ elevations, which did not significantly differ between the neuropil and the soma (soma: n = 5, N = 3; neuropil: n = 5, N = 3; Mann-Whitney test, p = 0.063). c Upper Left, Maximum intensity projection of a patch-clamped pyramidal neuron expressing mRGECO and dialyzed with Alexa488 (A488). A 50-Hz train of depolarizing current pulses (+2 nA, 5 ms each) elicited under current-clamp recording conditions (Lower Left) produced a marked increase in mRGECO fluorescence (ΔF/F_o) in the cell body and apical dendrite that lasted for several minutes (Middle & Right). Kymograph reveals the spatial pattern of mitochondrial Ca²⁺ uptake along the somatodendritic axis after the onset of the action potential train. d Upper, mRGECO fluorescence measured from ROIs in the soma and apical dendrite shows the onset of mitochondrial Ca²⁺ loading shortly after the start of the action potential train. Lower, The somatic mitochondrial Ca²⁺ levels slowly recovered to prestimulus baseline in a monoexponential manner (decay time constant [τ]). e Summary data showing a significantly larger evoked mRGECO fluorescence change in the soma relative to the apical dendrite (soma: n = 7, N = 4; dendrite: n = 7, N = 4; paired t test, p = 0.031). f Left, Illustration depicting the method for blocking the MCU by intracellular dialysis of Ru360 via the internal solution (IS) of the patch pipette. Middle & Right, Action potential evoked mitochondrial Ca²⁺ uptake is significantly reduced by 20 µM Ru360, but not 2 µM Ru360, relative to control (control: n = 7, N = 4; 2 µM Ru360: n = 7, N = 2; 20 µM Ru360: n = 7, N = 3; One-way ANOVA with Dunnett’s multiple comparisons test: 2 µM Ru360, p = 0.62; 20 µM Ru360, p < 0.0001). The Control data set is reproduced from the summary data in panel E. Summary data presented as the mean ± SEM. *P < 0.05, ***P < 0.001. The number of cell replicates is shown in the graphs as well as the ‘n’ value in the text. The number of animal replicates is represented by the ‘N’ value. Source data used for all summary figures are provided in the Supplementary Data 1 file.