Fig. 3: The coupling between activity and mitochondrial Ca²⁺ loading in pyramidal neurons is dependent on action potential firing frequency and varies between brain regions.

a Upper, Images depict the peak fluorescence change of mRGECO from a current-clamped cortical pyramidal neuron delivered action potential trains of progressively increasing frequencies. Stimuli of frequencies >5 Hz produced large and protracted elevations of mitochondrial Ca²⁺. All trains were comprised of 200 action potentials elicited by 200 current pulses (+2 nA, 5 ms). Lower, Traces depict changes in mRGECO fluorescence measured from the pyramidal neuron soma. b Left, Group data depicting the peak somatic fluorescence change of mRGECO and the low affinity sensor, mLaRGECO, in relation to action potential train frequency. Peak responses at each frequency were compared to the nominal responses elicited during 1-Hz trains (mRGECO data: n = 9, N = 4; Friedman’s ANOVA and Dunn’s multiple comparisons test: 5 Hz train, p > 0.99; 10 Hz train, p = 0.029; 20 Hz train, p = 0.002; 50 Hz train, p = 0.0006; mLaRGECO data: n = 8, N = 3; Friedman’s ANOVA and Dunn’s multiple comparisons test: 5 Hz train, p > 0.99; 10 Hz train, p > 0.99; 20 Hz train, p = 0.224; 30 Hz train, p = 0.01; 50 Hz train, p = 0.003). Right, Summary data showing the significantly greater time to recovery between low and high-frequency train-evoked fluorescence changes for mRGECO and mLaRGECO expressing neurons. For mRGECO experiments, statistical comparisons were made between responses at 5 Hz with all other frequencies (n = 9, N = 4; Friedman’s ANOVA with Dunn’s multiple comparison test: 10 Hz train, p > 0.99; 20 Hz train, p = 0.0042; 50 Hz train, p = 0.0021). For mLaRGECO experiments, statistical comparisons were made between the response to the 10 Hz train and all other frequencies (n = 8, N = 3; Friedman test with Dunn’s multiple comparisons test; 20 Hz train, p > 0.99; 30 Hz train, p = 0.0995; 50 Hz train, p = 0.0002). c Patch-clamped and dye (A488) filled mRGECO expressing hippocampal CA1 pyramidal neuron. d Representative traces from a CA1 pyramidal neuron showing somatic mRGECO fluorescence changes in response to action potential firing frequencies greater than 1 Hz. e A CA1 pyramidal neuron showing progressive mitochondrial Ca²⁺ accumulation (Upper) when stimulated to fire action potential bursts applied at theta frequency (50 Hz bursts at 5 Hz) (Lower). f Left, Relationship between the train-evoked peak mRGECO responses and spike firing frequency in cortical (data set repeated from panel B) and hippocampal pyramidal neurons. The hippocampal data set included cells from the CA1 and CA3 subregions. For each neuron, the peak responses at each train frequency were normalized to the largest evoked response out of the series of action potential trains. Normalized peak responses are significantly greater in hippocampal vs cortical neurons during low frequency firing (cortex: n = 9, N = 4; hippocampus: n = 8, N = 5; Mann-Whitney test for all; 1 Hz train, p = 0.001; 5 Hz train, p = 0.0003; 10 Hz train, p = 0.0345; 20 Hz train, p = 0.672; 50 Hz train, p = 0.015). Right, The time to recovery from peak mRGECO response was significantly longer in neurons of the hippocampus relative to the cortex during the 5 and 10 Hz action potential trains (5 Hz train, cortex: n = 9, N = 4; hippocampus: n = 9, N = 5; Mann-Whitney test, p = 0.0142; 10 Hz train, cortex: n = 9, N = 4; hippocampus: n = 9, N = 5; Mann-Whitney test, p = 0.0244; 20 Hz train, cortex: n = 9, N = 4; hippocampus: n = 7, N = 5; unpaired t test, p = 0.611; 50 Hz train, cortex: n = 8, N = 4; hippocampus: n = 8, N = 5; unpaired t test, p = 0.71). Summary data presented as the mean ± SEM. * P < 0.05, **P < 0.01, *** 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.