Table 1.
Antibodies.
Figure 1.
Synaptic proteins investigated in the CaV1.4(α1F)-KO.
In photoreceptor synapses, proteins are divided in several compartments: the ribbon associated proteins are shown in red, the active zone associated proteins in blue, and vesicle associated proteins in green. Additional proteins are important for synaptic functions and some of these proteins are presented in grey.
Figure 2.
Distribution of photoreceptor terminals at different ages in the CaV1.4(α1F)-KO.
Photoreceptor terminals are visualized with complexin 4 (Cplx4). A–J: Vertical sections from P9-WT (A), P9-KO (B), P11-KO (C), P13-KO (D), P15-KO (E), P18-KO (F), P23-KO (G), P25-KO (H), P30-KO (I) and P30-WT (J) mouse retinae. At all ages, ectopic terminals are distinguishable as indicated with white arrowheads. Scale bar = 5 µm.
Figure 3.
Some isolated terminals are new terminals in CaV1.4(α1F)-KO retinae.
(A–F) Vertical sections from P15 (A, B), P25 (C, D) and P30 (E–F) mouse CaV1.4(α1F)-KO retinae. (A–F) Confocal micrographs of retinae co-immunolabeled for complexin 4 (Cplx4) and C-terminal binding protein 2 (CtBP2). CtBP2 is presented alone in grayscale in the top row, the CtBP2 (magenta) and Cplx4 (green) signals are presented merged in the bottom row. Potentially quickly retracting isolated terminals that are Cplx4-positive/CtBP2-positive are indicated by white arrows, new or degenerating terminals that are Cplx4-positive/CtBP2-negative are indicated by grey arrows (see text for explanation). Scale bar = 5 µm.
Figure 4.
Association of Ribeye and Bassoon at different ages.
(A–H) Vertical sections from P13 WT (A) and CaV1.4(α1F)-KO (B–D), and P30 WT (E) and CaV1.4(α1F)-KO (F–H) retinae. Confocal micrographs of retinae co-immunolabeled for Bassoon (Bsn - magenta) and CtPB2 (green) are merged in A–B (P13) and E–F (P30); in addition each staining is presented separately in grayscale: C and G depict Bassoon staining; D and H show CtBP2 distribution. White arrowheads indicate the terminals where CtBP2 and Bassoon are associated, grey arrowheads indicate isolated Bassoon in the ONL, and grey arrows indicate isolated CtBP2 in the ONL. Scale bar = 5 µm. I–K: Electron micrographs of cone pedicles in WT and CaV1.4(α1F)-KO at different ages. (I) Cone pedicle containing three presynaptic ribbons in a P13 WT mouse. (J) Cone pedicle containing two presynaptic ribbons in a P13 CaV1.4(α1F)-KO. (K) Synaptic terminal containing two spherical presynaptic ribbons (white arrowheads) in an adult CaV1.4(α1F)-KO. Scale bar = 0.5 µm.
Figure 5.
Expression of additional presynaptic proteins.
(A–L) Vertical sections from P13-WT (A, E and I), P13-KO (B, F, and J), P30-KO (C, G and K) and P30-WT (D, H and L). The immunoreactivity of Veli3 is presented alone in the top row in grayscale (A–D). In the second row Veli3 (magenta) and glycogen phosphorylase (Glypho, green) signals are presented merged (E–H). PSD-95 immunoreactivity is shown in panels (I–L). Scale bar = 5 µm.
Figure 6.
Expression of vesicular proteins.
(A–I) Vertical sections from P13-KO (A, D and G), P25-KO (B, E and H), and P25-WT (C, F and I). Confocal micrographs of retinae immunolabeled for vGluT 1 (A–C), VAMP2. (D–F) and synaptophysin (G–I). Scale bar = 5 µm.
Figure 7.
Mature cones in CaV1.4(α1F)-KO.
(A–E) Vertical sections from P46 CaV1.4(α1F)-KO (A, C, E) and P46-WT (B,D). A: S-opsin labeling shows the morphology of cones in CaV1.4(α1F)-KO. Arrowheads indicate the various cone branches and synapse-like terminals. The expression of two cone markers, PNA (B–C) and mCar (D–E) is presented in B–E. Arrowheads indicate the immunopositive outer segments of cones in both WT and CaV1.4(α1F)-KO. (F–K) Glycogen phosphorylase (glypho) immunoreactivity shows the overall cone population in the dorsal (F–H) and ventral (I–K) retina of WT at 6 months (F, I) and of CaV1.4(α1F)-KO at 6 months (G, J) and 10 months(H, K). Scale bars = 10 µm.
Figure 8.
Cones in CaV1.4(α1F)-KO at different ages.
(A–F) Vertical sections from WT (A, F) and CaV1.4(α1F)-KO (B–E) at different ages. S-opsin labeling shows the morphology of cones in CaV1.4(α1F)-KO at P13 (B), P15 (C), P18 (D) and P23 (E), and in WT at P13 (A) and P23 (F). Arrowheads indicate the various cone branches and synapse-like terminals. Scale bar = 5 µm.
Figure 9.
Cones in CaV1.4(α1F)-KO establish new synapses.
(A–D) Vertical sections from CaV1.4(α1F)-KO at different ages (A–C) and WT (D). S-opsin (green) and calbindin (magenta) labeling show the morphology of cones in CaV1.4(α1F)-KO and sprouting of horizontal cells at P13 (A), P15 (B), P18 (C–D). Arrowheads indicate the fasciculation or apposition of HC neurites onto cones and the arrow points to a cone sprout. (E–H) Vertical sections from CaV1.4(α1F)-KO at different ages (E–G) and WT (H). S-opsin (green) and complexin 3 (magenta) immunoreactivity shows progressive appearance of Cplx3 in new cone synapses (white arrowheads). Grey arrowheads indicate the Cplx3-positve cone pedicles. (I–L) Vertical sections from CaV1.4(α1F)-KO at different ages (I–K) and WT (L). S-opsin (green) and CtPB2 (magenta) expression shows progressive appearance of ribbons in new cone synapses (white arrowheads). Scale bar = 5 µm.
Figure 10.
Summary of the elements affected in the photoreceptor terminals in the CaV1.4(α1F)-KO.
Several protein–protein interactions are affected such as the association of Ribeye, Piccolo and Bassoon. The investigated proteins in the model are depicted with a red outline. The proteins with an abnormal expression and/or distribution are shown in grey. Synaptic ribbons form a complex with several other proteins such as CAST, RIM2, Rab3a, and Munc13. In the CaV1.4(α1F)-KO, RIM2 expression is compromised. The vesicle related machinery (VAMP2, synaptophysin and complexin 3 and 4) is not affected in the CaV1.4(α1F)-KO. Other presynaptic proteins such PMCA, PSD-95 and Veli3 are also affected in the CaV1.4(α1F)-KO. Furthermore, β-Dystroglycan, a protein necessary for the invagination of bipolar cells is absent from the photoreceptor synapses in the CaV1.4(α1F)-KO.