Figure 2. Progressive RGS7 loss in the ON-BC neurons of a conditional mouse knockout model (cDKO).
(A) Schematic representation of a breeding strategy for the conditional inactivation of RGS7 on Rgs11−/− background. To induce recombination and RGS7 loss mice were administered tamoxifen for 5 days followed by testing. (B) Immunohistochemical analysis of RGS7 expression and localization in the outer plexiform layer of mouse retinas following tamoxifen administration. Retina sections were stained with RGS7 (green) and mGluR6 (red) antibodies from cDKO:Cre+ mice at 12, 17, 22, 28, and 35 days after the start of tamoxifen administration. Retinas lacking RGS7 (Rgs7−/−) were used as a control. Two independent experiments yielding similar data were conducted. Note progressive loss of RGS7 immunoreactivity from postsynaptic puncta denoted by mGluR6. (C) Analysis of changes in RGS7 expression in total retina lysates by Western blotting. cDKO:Cre+ mice were treated with tamoxifen and retinas were collected at indicated time points. To generate retina lysates containing various RGS7 protein content, varying amounts of RGS11 knockout lysates were spiked into lysates isolated from RGS7 and RGS11 double knockout retinas (DKO). To determine the level of RGS7 reduction, a calibration curve was plotted from densities of varying amounts of RGS7 protein and used to determine the protein content in retina extracts obtained from tamoxifen-treated mice. Retinas from three separate animals were used in these experiments. (D) Quantification of RGS7 protein content at different time points following tamoxifen treatment. Values representing density of RGS7 band (red) and fluorescence intensity in mGluR6-positive puncta determined separately for rods (green) and cones (blue) were normalized to respective values observed before tamoxifen treatment (day 0). Averaged RGS7 percentages from western and IHC experiments are fitted with a single exponential decay function. Error bars are SEM values. (E) Intact synaptic morphology and retina cytoarchitecture at 28 days post-tamoxifen treatment. Retina cross-section from Rgs11−/− and cDKO:Cre+ mice were co-stained with postsynaptic marker mGluR6 and pre-synaptic marker CtBP2 and their direct apposition was noted. Immunostaining with TRPM1 (green) reveals intact morphology of ON-BC, their dendritic branching and distribution of postsynaptic puncta. Figure 2—figure supplement 1: assignment of rod vs cone synapses by immunohistochemistry. Figure 2—figure supplement 2: intact synaptic morphology and retina cytoarchitecture at 28 days post-tamoxifen treatment.
Figure 2—figure supplement 1. Assignment of rod vs cone synapses by immunohistochemistry.
Figure 2—figure supplement 2. Intact synaptic morphology and retina cytoarchitecture at 28 days post-tamoxifen treatment.
