Arrows. Scale bar = 50 mm. The results (mean 6 SEM, n = 10) are shown in the lower panel and are presented relative to the apoE3 mice whose values were set as 100 . doi:10.1371/journal.pone.0064949.gApoE4 Induces Retinal ImpairmentsApoE4 Induces Retinal ImpairmentsFigure 3. The effects of apoE4 on retinal nerve terminals. (A) Immunohistohemistry of retinal sections that were MedChemExpress Fruquintinib 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 mm for Synaptophysin and 50 mm for VGluT1, VGaT and VAChT. Quantification of the VGluT1, VGaT and VAChT results (mean 6 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 6 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 16985061 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 6 SEM, n = 10) relative to the apoE3 mice is depicted on the right panel. doi:10.1371/journal.pone.0064949.gtochemistry and western blot, were lower in the apoE4 than in the apoE3 mice (Figure 4B,C; P,0.001), as previously seen in the brain [42]. The quantification of the Muller cells marker, GS, revealed that the levels of GS were lower in apoE4 than in apoE3 retinas (Figure 4B). The effect of apoE4 on retinal function was measured using ERG. In the dark adapted mice, no significant differences weredetected between apoE4 and apoE3 at the first seven luminance intensity levels (0.00003?.03 cd*s/m2). In contrast, the a- and bwave amplitudes of apoE4 mice were significantly lower than those of apoE3 mice at the higher light intensities (Figure 5B; upper panel; P,0.05). The ITs of both a- and b-waves were, however, not affected by mouse genotypes (Figure 5B; lower panel). UnlikeFigure 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 mm. (B) Quantification of the GS, apoE and their colocalization. Results presented (mean 6 SEM, n = 10) are relative to the apoE3 mice whose values were set as 100 . (C) ApoE 1113-59-3 site immunoblot assays. Representative immunoblots are shown in the upper panel, whereas quantification of the results relative to the apoE3 mice (mean6 SEM, n = 11) is presented in the lower panel. The immunoblot assays were performed as described in Materials and Methods. *P,0.0001. doi:10.1371/journal.pone.0064949.gApoE4 Induces Retinal ImpairmentsFigure 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?5 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 wit.Arrows. Scale bar = 50 mm. The results (mean 6 SEM, n = 10) are shown in the lower panel and are presented relative to the apoE3 mice whose values were set as 100 . doi:10.1371/journal.pone.0064949.gApoE4 Induces Retinal ImpairmentsApoE4 Induces Retinal ImpairmentsFigure 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 mm for Synaptophysin and 50 mm for VGluT1, VGaT and VAChT. Quantification of the VGluT1, VGaT and VAChT results (mean 6 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 6 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 16985061 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 6 SEM, n = 10) relative to the apoE3 mice is depicted on the right panel. doi:10.1371/journal.pone.0064949.gtochemistry and western blot, were lower in the apoE4 than in the apoE3 mice (Figure 4B,C; P,0.001), as previously seen in the brain [42]. The quantification of the Muller cells marker, GS, revealed that the levels of GS were lower in apoE4 than in apoE3 retinas (Figure 4B). The effect of apoE4 on retinal function was measured using ERG. In the dark adapted mice, no significant differences weredetected between apoE4 and apoE3 at the first seven luminance intensity levels (0.00003?.03 cd*s/m2). In contrast, the a- and bwave amplitudes of apoE4 mice were significantly lower than those of apoE3 mice at the higher light intensities (Figure 5B; upper panel; P,0.05). The ITs of both a- and b-waves were, however, not affected by mouse genotypes (Figure 5B; lower panel). UnlikeFigure 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 mm. (B) Quantification of the GS, apoE and their colocalization. Results presented (mean 6 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 (mean6 SEM, n = 11) is presented in the lower panel. The immunoblot assays were performed as described in Materials and Methods. *P,0.0001. doi:10.1371/journal.pone.0064949.gApoE4 Induces Retinal ImpairmentsFigure 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?5 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 wit.
