Fig. 4: Measurement of action potential evoked NAD(P)H dynamics in single pyramidal neurons in situ. | Communications Biology

Fig. 4: Measurement of action potential evoked NAD(P)H dynamics in single pyramidal neurons in situ.

From: Mitochondrial Ca2+ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing

Fig. 4

a Left, Illustration depicts the generation of fluorescent NADH by the TCA cycle and its oxidation to non-fluorescent NAD+ by the electron transport chain (ETC). NADH autofluorescence can be measured using two-photon excitation at 750 nm. b Images of a cortical pyramidal neuron showing the punctate pattern of intracellular NAD(P)H autofluorescence and mitochondria labelled with mRGECO. c Group time series data showing the enlargement of somatic NAD(P)H transients with progressively higher action potential firing frequencies in a cortical pyramidal neuron. Labels during the 50 Hz train highlight the pre-stimulus phase (a), the stimulus-evoked transient decrease (b), and the long-lasting elevation of NAD(P)H autofluorescence (c). Images from these time points are depicted in panel D. d NAD(P)H autofluorescence in a pyramidal neuron soma during the baseline (a), immediately following a 50 Hz, 4-sec action potential train (b), and tens of seconds post-stimulus (c). The region of interest (ROI) used for analysis is depicted. e Relative to unstimulated neurons (n = 6, N = 4) train-evoked evoked NAD(P)H dips (min) (Left) and transient peaks (Right) significantly increase in magnitude with higher action potential firing frequency (NAD(P)H min data, One-way ANOVA and Dunnett’s multiple comparisons test: 1 Hz train, n = 5, N = 2, p = 0.976; 5 Hz train, n = 6, N = 4, p = 0.274; 10 Hz, n = 7, N = 3, p = 0.05; 20 Hz train, n = 5, N = 2, p = 0.028; 50 Hz train, n = 7, N = 4, p = 0.99; NAD(P)H peak stats, One-way ANOVA and Dunnett’s multiple comparisons test: 1 Hz train, n = 5, N = 2, p = 0.99; 5 Hz train, n = 6, N = 4, p > 0.999; 10 Hz, n = 7, N = 3, p = 0.99; 20 Hz train, n = 5, N = 2, p = 0.263; 50 Hz train, n = 7, N = 4, p = 0.0097. f Prior exposure of neurons to 5 min of 750 nm excitation light had no significant effect on NAD(P)H dips or latent peaks triggered by a 50 Hz train of action potentials (NAD(P)H min data, control: n = 7, N = 4; pre-illumination: n = 6, N = 4; unpaired t test, p = 0.92; NAD(P)H peak data, control: n = 7, N = 4; preillumination: n = 6, N = 4; unpaired t test, p = 0.72). 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.

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