Common morphological features and marker expression in the Trpm1 KO and rd1 mouse retinas. (A–C) Distribution of RBC terminals in the WT, Trpm1 KO, and mGluR6 KO mouse retinas. (A) RBCs and SACs were immunolabeled with PKCα (green) and ChAT (magenta: IPL sublaminar maker), respectively. Scale bar: 20 µm. (B) Intensity profiles of RBC labeling from A relative to the location of the ChAT bands (magenta dotted lines). Solid lines and shaded areas represent the mean ± SD, respectively. (C) Ending point of RBC terminal distribution in the IPL was significantly shifted toward the ON-ChAT band (magenta dotted lines) in the Trpm1 KO mouse retinas compared with the WT and mGluR6 KO mouse retinas (starting point: WT: 114.6 ± 7.9%, n = 6 sections from six mice; Trpm1 KO: 111.8 ± 9.3%, n = 5 sections from five mice; mGluR6 KO: 123.3 ± 18.8%, n = 3 sections from three mice; P = 0.24, ending point: WT: 217.3 ± 6.6%, n = 6 sections from six mice; Trpm1 KO: 194.6 ± 12.1%, n = 6 sections from six mice; mGluR6 KO: 215.1 ± 12.5%, n = 3 sections from three mice; P = 0.004). (D–F) Distribution of RBC and type 6 BC terminals in the WT and Trpm1 KO mouse retinas. (D) RBCs, type 2 and 6 BCs, and SACs were immunolabeled with PKCα (green), Syt2 (cyan), and ChAT (magenta), respectively. Scale bar: 20 µm. (E) Intensity profiles of PKCα and Syt2 labeling from D relative to the location of the ChAT bands (magenta dotted lines). (F) The ending point of RBC terminal distribution (GCL side) was significantly closer to the ON-ChAT band (magenta dotted lines) in the Trpm1 KO mouse retinas compared with the WT mouse retinas (starting point: WT: 116.3 ± 4.2%, n = 3 sections from three mice; Trpm1 KO: 113.3 ± 9.5%, n = 3 sections from three mice; P = 0.65, ending point: WT: 222.1 ± 3.8%, n = 3 sections from three mice; Trpm1 KO: 195.4 ± 6.7%, n = 3 sections from three mice; P = 0.004, unpaired t test). In contrast, the distribution of type 6 BC terminals was similar between WT and Trpm1 KO mouse retinas (starting point: WT: 110.3 ± 7.1%, n = 3 sections from three mice; Trpm1 KO: 112.9 ± 4.1%, n = 3 sections from three mice; P = 0.62, ending point: WT: 182.5 ± 7.8%, n = 3 sections from three mice; Trpm1 KO: 182.9 ± 8.6%, n = 3 sections from three mice; P = 0.96, unpaired t test). (G–K) Altered distribution and reduced volume of RBC terminals in the rd1 mouse retinas. (G–I) Distribution of RBC terminals was shifted toward the INL in the rd1 mouse retinas. Similar analyses are shown in A–C (starting point: WT: 133.7 ± 7.3%, n = 5 sections from five mice; rd1: 118.5 ± 3.4%, n = 5 sections from five mice; P = 0.003, ending point: WT: 238.9 ± 13.2%, n = 5 sections from five mice; rd1: 217.7 ± 9.1%, n = 5 sections from five mice; P = 0.02). (J and K) Volume of RBC terminals in the rd1 mouse retinas was smaller than that in the WT mouse retinas. (J) Synaptic terminals immunolabeled with PKCα (green) and VGluT1 (magenta) in the WT (top) and rd1 (bottom) mouse retinas. Scale bar: 5 µm. (K) RBC terminal volumes were significantly smaller in the rd1 mouse retinas than in the WT mouse retinas (WT: 5.7 ± 5.7 µm3, n = 107 terminals, 3 retinas, 3 mice; rd1: 3.4 ± 2.7 µm3, n = 86 terminals, 3 retinas, 3 mice; P = 7.00 × 10−4). (L–O) Marker expression in the OPL. (L) OPL localization of mGluR6 (green), CtBP2 (magenta), and DAPI (blue) was similar between WT (top) and Trpm1 KO (bottom) mouse retinas. Scale bar: 5 µm. (M) Cone pedicles remained intact in the Trpm1 KO mouse retinas. Cone arrestin (yellow), mGluR6 (magenta), GluK1 (cyan), and DAPI (blue). Scale bar: 5 µm. WT (top) and the Trpm1 KO (bottom) mouse retinas. (N) TRPM1 puncta were moderately reduced in the mGluR6 KO mouse retinas and severely diminished in the rd1 mouse retinas. RBC dendrites showed intact structure in the mGluR6 KO mouse retina but retracted in the rd1 mouse retinas. WT (left), rd1 (center), mGluR6 KO (right) mouse retinas. Scale bar: 10 µm. Insets show an enlarged view of individual RBC dendritic trees. Scale bar: 5 µm. (O) Cone pedicles showed degeneration in the rd1 mouse retinas. Cone arrestin (yellow), mGluR6 (magenta), GluK1 (cyan), and DAPI (blue). Scale bar: 5 µm. WT (top) and rd1 (bottom) mouse retinas. Data are presented as the mean ± SD. Error bars indicate SD. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. SACs, starburst ACs.
Figure 3.

Common morphological features and marker expression in the Trpm1 KO and rd1 mouse retinas. (A–C) Distribution of RBC terminals in the WT, Trpm1 KO, and mGluR6 KO mouse retinas. (A) RBCs and SACs were immunolabeled with PKCα (green) and ChAT (magenta: IPL sublaminar maker), respectively. Scale bar: 20 µm. (B) Intensity profiles of RBC labeling from A relative to the location of the ChAT bands (magenta dotted lines). Solid lines and shaded areas represent the mean ± SD, respectively. (C) Ending point of RBC terminal distribution in the IPL was significantly shifted toward the ON-ChAT band (magenta dotted lines) in the Trpm1 KO mouse retinas compared with the WT and mGluR6 KO mouse retinas (starting point: WT: 114.6 ± 7.9%, n = 6 sections from six mice; Trpm1 KO: 111.8 ± 9.3%, n = 5 sections from five mice; mGluR6 KO: 123.3 ± 18.8%, n = 3 sections from three mice; P = 0.24, ending point: WT: 217.3 ± 6.6%, n = 6 sections from six mice; Trpm1 KO: 194.6 ± 12.1%, n = 6 sections from six mice; mGluR6 KO: 215.1 ± 12.5%, n = 3 sections from three mice; P = 0.004). (D–F) Distribution of RBC and type 6 BC terminals in the WT and Trpm1 KO mouse retinas. (D) RBCs, type 2 and 6 BCs, and SACs were immunolabeled with PKCα (green), Syt2 (cyan), and ChAT (magenta), respectively. Scale bar: 20 µm. (E) Intensity profiles of PKCα and Syt2 labeling from D relative to the location of the ChAT bands (magenta dotted lines). (F) The ending point of RBC terminal distribution (GCL side) was significantly closer to the ON-ChAT band (magenta dotted lines) in the Trpm1 KO mouse retinas compared with the WT mouse retinas (starting point: WT: 116.3 ± 4.2%, n = 3 sections from three mice; Trpm1 KO: 113.3 ± 9.5%, n = 3 sections from three mice; P = 0.65, ending point: WT: 222.1 ± 3.8%, n = 3 sections from three mice; Trpm1 KO: 195.4 ± 6.7%, n = 3 sections from three mice; P = 0.004, unpaired t test). In contrast, the distribution of type 6 BC terminals was similar between WT and Trpm1 KO mouse retinas (starting point: WT: 110.3 ± 7.1%, n = 3 sections from three mice; Trpm1 KO: 112.9 ± 4.1%, n = 3 sections from three mice; P = 0.62, ending point: WT: 182.5 ± 7.8%, n = 3 sections from three mice; Trpm1 KO: 182.9 ± 8.6%, n = 3 sections from three mice; P = 0.96, unpaired t test). (G–K) Altered distribution and reduced volume of RBC terminals in the rd1 mouse retinas. (G–I) Distribution of RBC terminals was shifted toward the INL in the rd1 mouse retinas. Similar analyses are shown in A–C (starting point: WT: 133.7 ± 7.3%, n = 5 sections from five mice; rd1: 118.5 ± 3.4%, n = 5 sections from five mice; P = 0.003, ending point: WT: 238.9 ± 13.2%, n = 5 sections from five mice; rd1: 217.7 ± 9.1%, n = 5 sections from five mice; P = 0.02). (J and K) Volume of RBC terminals in the rd1 mouse retinas was smaller than that in the WT mouse retinas. (J) Synaptic terminals immunolabeled with PKCα (green) and VGluT1 (magenta) in the WT (top) and rd1 (bottom) mouse retinas. Scale bar: 5 µm. (K) RBC terminal volumes were significantly smaller in the rd1 mouse retinas than in the WT mouse retinas (WT: 5.7 ± 5.7 µm3, n = 107 terminals, 3 retinas, 3 mice; rd1: 3.4 ± 2.7 µm3, n = 86 terminals, 3 retinas, 3 mice; P = 7.00 × 10−4). (L–O) Marker expression in the OPL. (L) OPL localization of mGluR6 (green), CtBP2 (magenta), and DAPI (blue) was similar between WT (top) and Trpm1 KO (bottom) mouse retinas. Scale bar: 5 µm. (M) Cone pedicles remained intact in the Trpm1 KO mouse retinas. Cone arrestin (yellow), mGluR6 (magenta), GluK1 (cyan), and DAPI (blue). Scale bar: 5 µm. WT (top) and the Trpm1 KO (bottom) mouse retinas. (N) TRPM1 puncta were moderately reduced in the mGluR6 KO mouse retinas and severely diminished in the rd1 mouse retinas. RBC dendrites showed intact structure in the mGluR6 KO mouse retina but retracted in the rd1 mouse retinas. WT (left), rd1 (center), mGluR6 KO (right) mouse retinas. Scale bar: 10 µm. Insets show an enlarged view of individual RBC dendritic trees. Scale bar: 5 µm. (O) Cone pedicles showed degeneration in the rd1 mouse retinas. Cone arrestin (yellow), mGluR6 (magenta), GluK1 (cyan), and DAPI (blue). Scale bar: 5 µm. WT (top) and rd1 (bottom) mouse retinas. Data are presented as the mean ± SD. Error bars indicate SD. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001. SACs, starburst ACs.

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