Fig. 2: Simultaneous imaging of GCaMP6f and mRGECO reveals the distinct dynamics of cytosolic and mitochondrial Ca²⁺ during action potential firing.

a Left, Somatic mRGECO fluorescence changes from cortical pyramidal neurons during a 50-Hz, 4-sec action potential train in the presence (control) and absence of extracellular Ca²⁺ (Ca²⁺-free). Right, Summary data shows a significant reduction in evoked mitochondrial Ca²⁺ uptake in the absence of extracellular Ca²⁺ (control: n = 5, N = 2; Ca²⁺-free: n = 5, N = 2; paired t test, p = 0.005). b Maximum intensity projection showing cortical neurons from an acute brain slice co-expressing GCaMP6f and mRGECO. c Left, Images depict GCaMP6f and mRGECO fluorescence from a patch-clamped pyramidal neuron during the application of a 50 Hz, 4-sec action potential train. The time (t) relative to stimulus onset is depicted. Right, Kymograph showing the change in GCaMP6f and mRGECO fluorescence along the somatodendritic axis. The ROIs used for producing the kymograph are labelled (a–c). d Sample trace showing that cytosolic Ca²⁺ (GCaMP6f) in the soma and apical dendrite has a rapid stimulus-evoked rise time in comparison to mitochondrial Ca²⁺ (mRGECO). e Stimulus-evoked changes in cytosolic Ca²⁺ have a significantly shorter time to half peak than mitochondrial Ca²⁺ in the soma (GCAMP: n = 8, N = 5; mRGECO: n = 8, N = 5; paired t test, p = 0.005) but not the apical dendrite (GCAMP: n = 5, N = 3; mRGECO: n = 5, N = 3; paired t test, p = 0.23). f Summary data showing the ratio of evoked changes in mitochondrial Ca²⁺ and cytosolic Ca²⁺ (coupling ratio) at 2-sec post-stimulus onset. The coupling ratio is low and not significantly different between the soma and apical dendrite (soma: n = 5, N = 3; dendrite: n = 5, N = 3; paired t test, p = 0.126). g Left, Sample Ca²⁺ traces measured from the soma depict the rapid (seconds) recovery of post-train cytosolic Ca²⁺ and the long-lasting (minutes) recovery of mitochondrial Ca²⁺ to pre-stimulus baseline. The dashed lines depict the fitted monoexponential decay functions. Right, The decay time constant (τ) acquired during the Ca²⁺ recovery period is significantly longer in the mitochondria relative to the cytosol (GCAMP: n = 5, N = 3; mRGECO: n = 5, N = 3; paired t test, p = 0.037). 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.