Article Figures & Data
Figures
- Fig. 1.
Nav1.2 was found throughout the unmyelinated zone of adult RGC axons, whereas Nav1.6 was localized specifically to axonal initial segments. A, Double-labeling of rat retina with PAN (red) and anti-Nav1.6 (green) revealed no colocalization in fascicles of unmyelinated RGC axons (f). Nav1.6 immunoreactivity was selectively observed in RGC somata and in short segments of axons, representing the distal portion of putative initial segments (arrows indicate 2 examples). B, An image taken from a region of the peripheral retina that is devoid of axon fascicles. Bright PAN-stained processes (red) coincide with Nav1.6 immunoreactivity (green). The arrows indicate a bright segment of PAN staining seemingly connected by a more dimly stained process to an adjacent RGC soma. C, Examples of PAN-stained RGCs, illustrating the relationship of the brightly stained axonal segment to the cell body.Arrows indicate the start of the brightly stained segment, asterisks indicate the cell body, andarrowheads indicate the more dimly stained proximal portion of the axon. In these examples, the distance from the soma to the brightly stained segment was ∼15–20 μm. D, Double-labeling with PAN (red) and anti-Nav1.2 (green) reveals Nav1.2 immunoreactivity throughout RGC axons. This image was taken from an intermediate zone of the retina with a higher density of axon fascicles. Scale bars: A, B,D, 50 μm; C, 20 μm. All images were obtained from flat mounts of intact retinas. Images inA, B, and D are projections of two consecutive optical sections, whereas images inC are projections of a series of optical sections spanning 6–7 μm from near the vitreal surface to the RGC layer.
- Fig. 2.
Nav1.6, ankyrin-G (AnkG), and neurofascin (nf) colocalize at initial segments of adult RGC axons. A, Flat mount of adult rat retina stained for PAN (green) and ankyrin-G (red) shows clusters of ankyrin-G immunoreactivity colocalized with intense PAN-positive regions representing RGC initial segments. Little ankyrin-G immunofluorescence was observed in RGC axon bundles.B, Cryosections of rat retina revealed colocalization between Nav1.6 (green) and ankyrin-G (red) at RGC initial segments. C, Brightly PAN-positive regions of RGC axons (green) colocalized with neurofascin immunoreactivity (red) in cryosections, also confirming these regions as initial segments. Scale bar, 20 μm. Individual optical sections are shown in A andC; the image in B is a projection of sections spanning 1.5 μm.
- Fig. 3.
Nav1.2 immunoreactivity was detected throughout unmyelinated RGC axons, including the initial segment.A, Nav1.2 immunostaining in a flat mount of adult rat retina. B, Ankyrin-G immunostaining in the same field of view shown in A. C, Superposition of Nav1.2 and Ankyrin-G immunofluorescence. Ankyrin-G immunoreactivity (red) was used to mark initial segments (arrow, B). Anti-Nav1.2 immunostaining (green) was present in all parts of the RGC axon and colocalized with ankyrin-G in the initial segment (arrow, A). The cell body is not visible in this single optical section.
- Fig. 4.
Nav1.2 immunofluorescence colocalized with Nav1.6 immunostaining at initial segments. Cryosections of P35 rat retina double-labeled with polyclonal anti-Nav1.2 (green) and monoclonal anti-Nav1.6 (red) further demonstrated the presence of Nav1.2 at initial segments. A, Nav1.2 staining was located in the Nav1.6-enriched initial segment (arrow) and in the flanking region of the RGC axon. B, Brightly Nav1.6-labeled initial segment (arrow) was also dimly stained with anti-Nav1.2, which also stained axon regions without detectable Nav1.6 immunoreactivity. Scale bar, 10 μm. Images show single confocal optical sections.
- Fig. 5.
Nav1.6 appears at RGC initial segments during postnatal development. Flat mounts of rat retina were labeled with anti-Nav1.6 (green) and anti-ankyrin-G (AnkG; red) at different stages of postnatal development. No colocalization was detected at P2. Nav1.6 immunoreactivity was present in a subset of initial segments by P9 and in 80% of initial segments by P14, with variable degrees of intensity. Brightest Nav1.6 immunofluorescence was observed in adult retina, in which >90% of initial segments contained Nav1.6. Scale bar, 40 μm. Images are single optical sections, except the image at P14, which spans 4 μm.
- Fig. 6.
Early initial segments during retinal development were brightly labeled with anti-Nav1.2. The presence of Nav1.2 is maintained at RGC initial segments throughout development. Flat mounts of rat retina were immunostained for Nav1.2 and ankyrin-G (AnkG) at different postnatal ages. Nav1.2 staining was detected at ankyrin-G-defined initial segments at all ages, as well as throughout the unmyelinated RGC fibers. Scale bar, 40 μm. Images are single optical sections.
- Fig. 7.
Hypomyelination of RGC axons does not disrupt Nav1.6 targeting at RGC initial segments in the retina of Shiverer mice. A, Cryosections from adult Shiverer retina were stained with anti-Nav1.6 (green) and PAN (red,left) or anti-ankyrin-G (AnkG;red, right). Images are single confocal sections (left) or projections of optical sections spanning 3 μm (right). The staining pattern in adult Shiverer retina was indistinguishable from that in wild-type mice (data not shown) or rat retina (Figs. 1, 2). B, In the retina of P7 Shiverer mice, anti-Nav1.2 (green) stains all brightly PAN-labeled axonal regions (red, left), in which pronounced anti-Nav1.6 labeling was observed in the adult (A). Similarly, Nav1.2 immunoreactivity (green, right) colocalized with ankyrin-G-positive initial segments (red, right) in P7 Shiverer retina. Images on the left are of single optical sections, and images on the right are projections of sections spanning 2.5 μm. Scale bar, 20 μm.
Tables
- Table 1.
Percentage of total initial segments labeled by isoform-specific sodium-channel antibodies
Age Nav1.2-positive initial segments (percentage of total) Nav1.6-positive initial segments (percentage of total) P2 95.7 (176/184) 2.6 (7/274) P9 99.4 (172/173) 40.9 (99/242) P14 97.3 (181/186) 80 (132/165) Adult 92 (92/100) 95.3 (242/254) Initial segments were defined by ankyrin-G immunostaining.
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