Fig. 7: Myelin attenuates internode mt-Ca²⁺ responses.

a Example 2P image of an Atto594-filled mt-GcaMP6f⁺ PV⁺ interneuron. Dashed box indicates the first branch point of the axon, with a characteristic large angle (≥90°). Similar results were obtained in 17 mice. b Example control PV⁺ axon with mt-GCaMP6f-labelled mitochondria. Mt-Ca²⁺ responses are stronger in MBP⁻ mitochondria (mito 1 and 2) compared to those in MBP⁺ segment (Mito 3 and 4). Dashed lines indicate timepoints corresponding to the heatmap, yellow arrowhead indicates branch point. Scale bars indicate (top to bottom) 100% ΔF/F, 50 mV, 0.5 nA and 0.5 s. c Schematic overview of sampled axonal regions. In d, for cuprizone, axonal segments in between branch points were selected. Similar results were obtained in 17 mice. d Left, example Ca²⁺ responses of mitochondria in MBP⁺, MBP^– or putatively demyelinated segments. Right, stronger mitochondrial Ca²⁺ responses in MBP⁻ segments compared to MBP⁺ axon and increased Ca²⁺ responses after demyelination (Kruskal–Wallis test, P < 0.0001; Dunn’s post hoc test MBP^– vs., MBP⁺ ***P < 0.0001; MBP⁻ vs. cuprizone, *P = 0.0195; MBP⁺ vs. cuprizone, **P = 0.0039; MBP⁺, n = 101 mitochondria of 12 cells from 7 mice; MBP^–, n = 27 mitochondria of 10 cells from 6 mice; cuprizone, n = 36 mitochondria of 10 cells from 5 mice). Scale bars indicate (top to bottom) 25% ΔF/F, 50 mV, 1 nA and 0.5 s. e Top: Example confocal image of the AIS, myelinated internode and first branch point of a PV⁺ axon Bottom: time lapse of a two-photon cytosolic (cyt-)GCaMP6f recording of the same axon in response to ~100 APs. Notice only a slight Ca²⁺ response at the MBP edge but practically none in the rest of the internode. Scale bars indicate (top to bottom) 100% ΔF/F, 50 mV, 0.5 nA and 0.5 s. f Cyt-Ca²⁺ responses are stronger in unmyelinated and demyelinated axonal segments (Kruskal–Wallis test, P < 0.0001; Dunn’s post hoc test MBP^– vs MBP⁺, P < 0.0001, MBP⁺ vs cuprizone, P = 0.0004, MBP^– vs cuprizone, P = 0.4351; MBP⁺, n = 18 segments of 6 cells from 3 mice; MBP^–, n = 24 segments of 8 cells from 3 mice; cuprizone, n = 28 segments of 6 cells from 3 mice). Solid bars in d and f represent the mean, error bars indicate SEM. Individual data points in d represent mitochondria, individual data points in f represent segments. Source data are provided as a Source data file.