Figure S3.

Nodal and Internodal Architecture of Thick-Tufted L5 Axons, Related to Figure 1

(A), Low magnification photomicrograph of a biocytin-stained cell (cell #6). Nodes were identified by an increased staining intensity and/or branch point, indicated by the position of arrows (red). (B), Immuofluorescence staining for biocytin-streptavidin (green) and the nodal/AIS marker βIV spectrin (magenta). Nodes of Ranvier, identified by the overlap of spectrin and biocytin, are indicated with arrows (red). (C), High magnification of the AIS and two nodes (2^nd and 6^th) from the axon shown in (B). (D), Comparison of internode length as a function of internode number for the biocytin-stained axons (cells #2–6) and the immunofluorescence identified L5 axons (n = 8) revealed a similarity in the sequence of internode lengths (two-way ANOVA, group-internode interaction (p > 0.890). The first 2–3 internodes are short, with collaterals emerging from the nodes of Ranvier. With increasing distance from the soma, internodes are progressively longer and lack collaterals. Data represent mean ± SEM (E), Internode length (L) scaled linearly with axon core diameter d, based on all immunofluorescence-identified internodes, (n = 42; r² = 0.653). (F), Example of a L5 neuron filled with HRP and recovered for EM analysis. EM images are shown for the two indicated locations. (G), The number of myelin lamellae was constant with distance from the soma (red line, linear regression slope ≈0, F test p > 0.836; R² = 0.00191, n = 25 internodes from 6 axons, represented by different symbols).