Fig. 6: Mitochondrial Ca²⁺ uptake clamps cytosolic Ca²⁺ magnitude and reduces the slow afterhyperpolarization duration elicited by high-frequency action potential firing.

a Representative traces of cytosolic Ca²⁺ in the soma measured with Fluo5N (Upper), and membrane potential (Vm) (Lower), from patch-clamped pyramidal neurons during low and high-frequency action potential trains. Upper inset depicts a pyramidal neuron filled with Fluo5N and the morphology marker Alexa594 (A594). Lower inset shows a single action potential from neurons in control and Ru360 conditions. Ru360 dialysis results in substantially larger and more prolonged cytosolic Ca²⁺ responses selectively during high-frequency action potential trains. The train-evoked slow afterhyperpolarization (sAHP) increases in duration with high-frequency trains and is significantly prolonged relative to control when the MCU is blocked by Ru360. b Images depict the change in cytosolic Ca²⁺ (Fluo5N ΔF/F_o) in pyramidal neurons before, during, and after a 50 Hz, 4-sec train of action potentials in the presence (control) and absence of mitochondrial Ca²⁺ buffering (Ru360). The somatodendritic Ca²⁺ responses were substantially larger and more prolonged in the absence of mitochondrial Ca²⁺ buffering. c Left, Ru360 significantly increased the peak cytosolic Ca²⁺ response evoked during high but not low frequency action potential firing (5 Hz train, control: n = 9, N= 5; Ru360: n = 7, N = 4; unpaired t test with Welch’s correction, p = 0.24; 10 Hz train, control: n = 8, N = 4; Ru360: n = 7, N = 4; Mann-Whitney test, p = 0.12; 20 Hz train, control: n = 8, N = 5; Ru360: n = 6, N = 4; unpaired t test with Welch’s correction, p = 0.057; 50 Hz train, control: n = 8, N = 4; Ru360: n = 7, N = 4; Mann-Whitney test, p = 0.04). Middle, Ru360 treatment results in a significantly greater time to 50% recovery from peak Ca²⁺ response at high frequency firing rates (5 Hz train, control: n = 8, N = 5; Ru360: n = 5, N = 4; unpaired t test with Welch’s correction, p = 0.35; 10 Hz train, control: n = 7, N = 4; Ru360: n = 7, N = 4; Mann-Whitney test, p = 0.23; 20 Hz train, control: n = 8, N = 5; Ru360: n = 6, N = 4; Mann-Whitney test, p = 0.0047; 50 Hz train, control: n = 8, N = 4; Ru360: n = 7, N = 4; Mann-Whitney test, p = 0.002). Right, Compared to controls, Ru360 significantly increased the time to 50% recovery from the sAHP following high frequency action potential firing (5 Hz train, control: n = 6, N = 4; Ru360: n = 6, N = 4; Mann-Whitney test, p = 0.94; 10 Hz train, control: n = 6, N = 6; Ru360: n = 6, N = 4; Mann-Whitney test, p = 0.23; 20 Hz train, control: n = 6, N = 5; Ru360: n = 6, N = 4; unpaired t test with Welch’s correction, p = 0.12; 50 Hz train, control: n = 8, N = 6; Ru360: n = 6, N = 4; unpaired t test, p = 0.017). Summary data presented as the mean ± SEM. *P < 0.05, **P < 0.01. 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.