Wenqin Hu, Cuiping Tian, Tun Li, Mingpo Yang, Han Hou & Yousheng Shu

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1 Distinct contributions of Na v 1.6 and Na v 1.2 in action potential initiation and backpropagation Wenqin Hu, Cuiping Tian, Tun Li, Mingpo Yang, Han Hou & Yousheng Shu Supplementary figure and legend Supplementary Figure 1 Peak distribution of Na v 1.6 at the distal AIS. (a c) Double stainings for AnkG (red) and Na v 1.6 (green) in thick sections (100 μm). Note the peak distribution (arrows) of Na v 1.6 at the distal AIS. Arrow heads indicate the root of the axon hillock. Scale bar: 10 μm. Images were projections of confocal z-stacks. (d) Plot of the normalized fluorescence intensity as a function of distance from the soma (mean ± s.e.m., n = 7). 1

2 Supplementary Figure 2 Dependence of the activation curve slope on the amplitude of Na + current. (a) The slope of the activation curve for Na + currents in control (open, mean ± s.e.m., n = 8), with TTX (gray, n = 8) and in low-na + ACSF (black, n = 9). No statistical difference was found between the slope with TTX and that in low-na + ACSF (P = 0.92). **, P < (b) A plot of the slope as a function of peak Na + current revealed the dependence of activation slope on current amplitude. Data was obtained from the experiments shown in a (color coded). Solid line, single exponential fit. Supplementary Figure 3 Activation and inactivation curves for Na + channels implemented in the simulations. Green traces, Na v 1.2 (nasoma). Blue traces, Na v 1.6 (naaxon). Experimental data is shown for comparison (black symbols, data from somatic patches; red symbols, data from patches obtained from axon regions distal to the AIS, > 70 μm from the soma). 2

3 Supplementary Figure 4 Contribution of the proximal AIS Na v 1.2 channels in action potential backpropagation to the soma (simulations). (a) With the presence of Na v 1.2 channels at the proximal AIS, action potential backpropagation was dependent of the V m levels. (b) Removal of Na v 1.2 channels from the proximal AIS caused a decrease in the amplitude of the spikelets and a complete failure of the action potential backpropagation. Action potentials were evoked through current injection at the axon (see Supplementary Fig. 8). 3

4 Supplementary Figure 5 Differential contributions of perisomatic and distal AIS Na + channels to somatic action potentials. (a) Top, schematic graph showing whole-cell recording while puffing TTX locally at the soma. Bottom, action potentials evoked before (black) and after TTX application (red, early; blue, late). Arrow indicates the thresholds of action potentials. (b) Phase plots of the action potentials shown in a. Notice that the high-threshold component (somatodendritic potential) of the action potential was dramatically reduced after the TTX application, while the low-threshold component (AIS potential) was largely preserved. Inset: the rising phase was expanded for clarity. (c) Same as in a except that TTX puffing was made at the distal portion of the AIS. Note that the voltage threshold was increased and the rising phase was less steep after the TTX application. (d) Phase plots of the action potentials shown in c. The AIS potential was eliminated by TTX, while the somatodendritic potential was preserved. Inset: the rising phase was expanded for clarity. 4

5 Supplementary Figure 6 Light staining of AnkG in the axon bleb. (a1 a3) Double stainings for AnkG (red) and Na v 1.6 (green) in prefrontal cortical slices (thickness of 300 μm). Arrows indicate the root of the axon hillock. (b1 b3) Higher magnification of the area in the box shown in left panels. Note that the fluorescence intensity of Na v 1.6 in the bleb membrane is similar to that in the nearby axon trunk. The proximal AIS is not stained for Na v 1.6. Scale bar: 10 μm. 5

6 Supplementary Figure 7 Non-uniform distribution of Na v 1.6 channels at the AIS of layer 5 pyramidal neurons in somatosensory cortex. (a1 a3) Double stainings for AnkG (red) and Na v 1.6 (green). Note that the distal portion of the AIS was heavily stained for Na v 1.6. Arrow heads indicate the root of the axon hillock. (b1 b3) Another example shows that both thick and thin axons exhibit a proximal-distal gradient of immunoreactivity for Na v 1.6 at the AIS. Scale bar: 10 μm. 6

7 Supplementary Figure 8 Schematic diagram of the simulation on action potential backpropagation. Action potential was evoked by stimulating the axon with current injection. Action potential backpropagation failure occurred when the somatic V m was set equal to or lower (more negative) than the failure threshold. 7