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. 2020 Sep 17;9:e62184. doi: 10.7554/eLife.62184

Figure 4. Rod synapses lack invaginating HC and RBC neurites in the absence of Cav1.4 Ca2+ signals.

(A), confocal micrographs of the ONL and OPL of WT, G369i KI and Cav1.4 KO retinas immunolabeled for calbindin and PKCα. The schematic below illustrates neurite sprouting in each genotype. (B), quantification of the area in the ONL occupied by HCs (calbindin) or RBCs (PKCα). One-way ANOVA with Fishers least significant difference post-hoc test. ****p<0.0001, **p<0.01, *p<0.05. n = 6 mice for each genotype. (C), confocal micrographs of the OPL of WT and the ONL and OPL of G369i KI retinas immunolabeled for Ribeye and Calbindin (upper panels) or PKCα (lower panels). (D), confocal micrographs of the ONL of G369i KI retinas that were immunolabeled for CtBP2 and Cav1.4 (left), PSD95 (middle) or Bassoon (right). (E), TEM image of a rod terminal in a WT retina. (F,G), serial block-face scanning electron microscopy images (F’,G’) and 3D reconstructions (F’’–G’’’) of rod terminals in a G369i KI retina. Rod terminals located within the OPL (F) and ONL (G). The yellow star F’’ indicates a neurite from HC2 sprouting into the ONL. Insets in F’’ are a rotated view. G’’’ is a side view of G’’. Arrows depict anchored ribbons. Arrowheads in G’’’ depict additional ribbon. Asterisks in G’ indicate rod somas. Scale bars, 10 μm in A, 2 μm in C-D, 1 μm in E,F’,G’. Rib, ribbon. RBC, rod bipolar cell. HC, horizontal cell.

Figure 4—source data 1. The image archive contains all images used in quantitative analyses in Figure 4B.
The data were exported as 8-bit ‘tif’ files (800 × 600 pixels). Values obtained for individual data points (and outlier analysis) for summary graphs in are contained in ‘.xlsx’ files.

Figure 4.

Figure 4—figure supplement 1. Multiple ribbons are found in rod terminals of G369i KI mice.

Figure 4—figure supplement 1.

(A) Electron micrographs of rod terminals with multiple ribbons (left) and a club-shaped ribbon (right). Arrows depict separate ribbons. (B) Number of total and floating ribbons in four rod terminals of G369i KI mice. Filled circles represent the means. Bars represent the SEM. (C) Additional SBFSEM reconstruction of a G369i KI spherule at the OPL/ONL border. Panels are labeled as in Figure 4F–G.

Figure 4—figure supplement 2. Nearest-neighbor analysis of Ribeye- and mGluR6-labeled structures.

Figure 4—figure supplement 2.

(A) Deconvolved confocal images from Figure 2B-D. Arrows indicate arc-shaped ribbons in apposition to mGluR6/TRPM1. (B) Cumulative frequency of distances between Ribeye and mGluR6. (C) Violin plot of Ribeye-mGluR6 distances. Solid lines represent the medians. Dashed lines represent the quartiles. p-value determined by unpaired t-test. WT, n = 864. G369i KI, n = 1390.
Figure 4—figure supplement 2—source data 1. The image archive contains all images used in quantitative analyses in Figure 4—figure supplement 2B; Figure 4—figure supplement 2C.
The data were exported as 8-bit ‘tif’ files (1024 × 1024 pixels). Values for individual data points (and outlier analysis) for summary graphs in Figure 4—figure supplement 2B are Figure 4—figure supplement 2C contained in excel files.