Figure 1.
General morphology of the retinas of young apoE4 and apoE3 mice.
Representative retinal sections of apoE3 and apoE4 mice stained with hematoxylin and eosin are depicted on the left. Quantification of the thickness of the outer and inner nuclear layers (ONL and INL), which respectively, contain photoreceptors and bipolar cells, and of the outer and inner synaptic plexiform layers (OPL and IPL) of the apoE3 and apoE4 mice is shown on the right. The results (mean ± SEM, n = 10) of the apoE4 mice are presented relative to the apoE3 mice whose values were set as 100%.
Figure 2.
Immunostaining of distinct neuronal populations of the retina of apoE3 and apoE4 mice.
Retinal sections were stained with the following neuron-specific markers: Recoverin (photoreceptors), CHX10 (pan bipolar cells), PKCα (rod bipolar), DAPI (ganglion cells), Calbindin (Horizontal cells), and PAX6 (amacrine cells) as described in Materials and Methods. Representative sections are presented in the upper panel. Retinal borders are marked with white dashed lines, and the specific cells are marked with arrows. Scale bar = 50 µm. The results (mean ± SEM, n = 10) are shown in the lower panel and are presented relative to the apoE3 mice whose values were set as 100%.
Figure 3.
The effects of apoE4 on retinal nerve terminals.
(A) Immunohistohemistry of retinal sections that were stained by the pan presynaptic marker synaptophysin and for the glutamatergic, GABAergic and cholinergic presynaptic vesicular transporters VGluT1, VGaT and VAChT, respectively, as described in Materials and Methods. Representative sections are depicted on the upper panel. Scale bar = 80 µm for Synaptophysin and 50 µm for VGluT1, VGaT and VAChT. Quantification of the VGluT1, VGaT and VAChT results (mean ± SEM, n = 10) is shown in the lower panel, and is represented relative to the apoE3 mice whose values were set as 100%. (B) Immunoblots of synaptophysin (Syp), VGluT1, and VGaT of retinal homogenates of apoE3 and apoE4. Representative blots are depicted on the left panels, and quantification of these results (mean ± SEM, n = 10) relative to the apoE3 mice is depicted on the right panel. (C) The ratio of VGluT1/VGaT of each mouse in both immunohistochemistry and western blots. (*P<0.03, **P<0.001). (D) Immunoblots of the post-synaptic markers PSD-95 and Gephyrin, of retinal homogenates of apoE3 and apoE4. Representative blots are depicted on the left panels, and quantification of these results and the ratio of PSD95/Gephyrin of each mouse (mean ± SEM, n = 10) relative to the apoE3 mice is depicted on the right panel.
Figure 4.
The effects of apoE4 on retinal apoE.
(A) Immunohistochmistry. Representative images of retinal sections of both apoE3 and apoE4 stained for cell nuclei (DAPI - blue), GS (green), apoE (red), and the merged image. Scale bar = 50 µm. (B) Quantification of the GS, apoE and their co-localization. Results presented (mean ± SEM, n = 10) are relative to the apoE3 mice whose values were set as 100%. (C) ApoE immunoblot assays. Representative immunoblots are shown in the upper panel, whereas quantification of the results relative to the apoE3 mice (mean± SEM, n = 11) is presented in the lower panel. The immunoblot assays were performed as described in Materials and Methods. *P<0.0001.
Figure 5.
The effects of the apoE genotype on retinal function.
Dark-adapted ERG responses of apoE3 and apoE4 mice were recorded in 13 steps (0.00003–25 cd*s/m2). (A) Representative ERG plots for apoE3 and apoE4 in response to a light flash. The peaks of a- and b-waves of apoE3 and apoE4 retinas are marked with black and red (+) signs, respectively. (B) Intensity response curves of the 6 highest light stimulation intensities (0.1, 0.3, 1, 3, 10, and 25 cd*s/m2) are presented. Results shown represent the mean ± SEM of the amplitudes (µV) and implicit times (ms) of a- and b-waves as a function of stimulus intensity. (n = 10; *P<0.05).