Fig 2: Distribution of NaV1.1 at the AIS of interneurons in L2–L6. (A) Double staining of AnkG (red) and NaV1.1 (green) showed that positive stainings of NaV1.1 were only found in the thin AIS of a putative interneuron (boxed area). Note that the orientation and thickness of the AIS were different from those of putative PCs. (B) High magnification of the boxed area shown in A. NaV1.1 immunosignals were more concentrated at the proximal AIS. (C–E) Other examples of NaV1.1 stainings at the AIS of interneurons. The distribution pattern and the relative fluorescence intensity varied across interneurons. Note that the two AIS in E showed different fluorescence intensity. (F–G) Triple staining of PV (green), AnkG (blue), and NaV1.1 (red). (F) NaV1.1 immunosignals accumulated at the AIS of the PV-positive neuron, especially at the very proximal AIS region. (G) In a putative interneuron (PV-negative), NaV1.1 signals could be also found at the AIS, but distributed evenly. Note the axon trunk beyond the AIS was also stained. Pia locates to the top in (A–E), and to the left in (F,G). Thickness of the Z-stacks is 3.5 µm in (A), 3 µm in (B,D,E), 1 µm in (C), 5 µm in (F), 8 µm in (G). Scale bars represent 20 µm in (A), 10 µm in (B–G).

Fig 3: Co-localization of FGF14 and Nav1.6 in mouse cortex using 1% formaldehyde and 0.5% MeOH fixation. (A–D) The gray and red channels represent FGF14 immunoreactivity visualized with an Alexa 568-conjugated secondary antibody, the green channel represents PanNav (Sigma-Aldrich, rabbit anti PanNav, catalog number S6936) in (A), Nav1.1 (Alomone Labs) in (B), Nav1.2 (Alomone Labs) in (C), and Nav1.6 (Alomone Labs) in (D) visualized with an Alexa 488-conjugated secondary antibody and the blue represents Topro3 nuclear staining in the cortex. Right panels represent overlaid images (third column from the left) and high magnification of boxed ROI from the merged images. Scale bars represent 20 µm.

Fig 4: Polarized distribution of NaV1.1 and NaV1.6 at the AIS of PV neurons.(A) Triple staining using antibodies for PV (blue), AnkG (red), and NaV1.2 (green) revealed the absence of NaV1.2 at the AIS of PV neuron (arrowheads). Note that neighboring PV-negative AIS (presumably from PCs, asterisks) show strong immunosignals for NaV1.2. (B) Triple staining for PV, AnkG (green), and NaV1.6 (red). Note that distal regions of AIS were heavily stained for NaV1.6 (arrowheads). Neighboring axons (asterisks) also showed strong immunosignals. (C) Triple staining for PV, NaV1.6, and NaV1.1 shows polarized distribution of these subtypes at the AIS. (D) Plots of the averaged fluorescence intensity (± s.e.m., see Materials and Methods) as a function of distance from soma at the AIS. Data were obtained from triple-staining experiments similar to (C). Images are projections of confocal z stacks. Scale bars represent 10 µm. Error bars represent s.e.m.

Fig 5: NaV1.2 but not NaV1.1 channels accumulate at the nodes of Ranvier in adult human cortical tissue. (A) Double staining of AnkG (red) and NaV1.1 (green) in the white matter indicating the absence of NaV1.1 at the nodes. (B) Double labeling of AnkG and NaV1.2 (green). Note that NaV1.2 positive puncta scattered in the white matter and colocalized with segmental AnkG-positive axon processes, indicating these puncta were nodes immunoreactive to NaV1.2. There were also AnkG-positive nodes without NaV1.2 expressed. (C) High-magnification images of the box areas shown in (B). These individual nodes (C1–C5) expressed NaV1.2 but showed diversification in their length, shape and labeling pattern. (D) Group data of the percentage of nodes with different Na+ channel subtypes. The thickness of Z-stacks is 2 µm in (A), 6 µm in (B,C2), 3 µm in (C1,C3,C4), 1 µm in (C5). Scale bars represent 10 µm in (A,B), and 2 µm in (C).