The doughnut-like profiles of HC process helical coils manifesting on cross-sections from mGluR6-knockout mice were also found in wild-type mice, although less frequently (
Figs. 3A,
3B). At high magnification, electron-dense material was found in the cytoplasm of HCs facing the rod BC dendrite in both genotypes (
Figs. 3C,
3D). At higher magnification, membranes of the HC process and the rod BC dendrite were observed in opposition across the interstitial cleft, and electron-dense fibrous material was observed on the intracellular side of the HC process membrane (
Figs. 3E,
3F). Thus, specific conformations of cytoskeletal molecules may underlie the helical structure in both wild-type and mGluR6-knockout retinas.
To examine mGluR6-associated and age-related differences in these fasciculation patterns, we measured the number and the length of helical coils (defined as at least one complete circle around a rod BC dendrite) in an area (14 by 19 µm) of three wild-type and three mGluR6-knockout mouse retinas at 12 and 90 weeks of age (
Figs. 3G,
3J,
3K). When a single rod BC dendrite had two separate segments with helical encirclement, we counted it as one case.
It was difficult to exactly count all the rod BC dendrites in our mouse samples, because rod BC dendrites, HC processes, and cone pedicle processes were piled up in the narrow interstitial space of five tiers of rod spherules. Therefore, we estimated the density of rod BC dendrites from the mean rod density and the mean divergence as described in
Supplementary Table S1. Based on the numbers of rod BC dendrites in 266 µm
2, percentages of the rod BC dendrites encircled by HC coils were estimated to be 2.4% for wild-type and 23% for mGluR6-knockout mice (three mice for each genotype at mixed 12 and 90 weeks; unpaired, two-tailed Student’s
t-test,
*P = 0.002) (
Fig. 3Hα). Thus, the HC coils around rod BC dendrites were approximately 10 times more frequent in mGluR6-knockout mice than wild-type mice (
Fig. 3Hα). The HC coils occurred slightly more frequently at 90 weeks than at 12 weeks, but the
t-test was not applicable due to the small sampling size (
Fig. 3Hβ). The greatest length of the HC coiling portion was 2 µm in wild-type mice and 3.4 µm in mGluR6-knockout mice. The length distribution of HC coiling portions did not differ between ages (
n = 44 per area at 12 weeks and
n = 52 per area at 90 weeks; Mann–Whitney
U test,
P = 0.32) (
Fig. 3J), but it did differ between genotypes (
n = 17 in three areas of wild-type mice vs.
n = 131 in three areas of mGluR6-knockout mice; Mann–Whitney
U test,
*P = 0.005) (
Fig. 3K).