Fig. 4

EAE and CFA treatments increase M-opsin expression in cone photoreceptors. A Retinal flatmounts were stained for M-opsin and S-opsin by immunofluorescence, 18 days after EAE induction (n = 6) or CFA (n = 3) or PBS (naïve; n = 5) injection. Dorso-ventral gradients of M-opsin and S-opsin could be observed for all groups, with predominant M-opsin staining in the dorsal retina and stronger S-cone signal in the ventral regions. However, in EAE retinae, and to a lower extent in CFA retinae, M-opsin expression appeared relatively higher in ventral cells (Boxes a10 to a12), where S-opsin normally prevails in naive animals (a4–a6). The number of M-opsin-labeled cells was higher in EAE and in CFA ventral retinae than in naive animals. However, this difference was only maintained in the most ventral areas of EAE retinae (a12). B Consistently, in the whole retina, the average number of cells expressing M-opsin was increased after EAE induction or CFA injection relative to naive controls, but more strongly in EAE conditions. C, D Increased expression of M-opsin in the ventral quadrant of EAE retinae could be observed on retinal cross-sections as well. Peanut agglutinin (PNA) was used as a general marker of cone photoreceptor outer segments. E On day 18, by RT-qPCR, the mRNA level of Opn1mw gene, coding for M-opsin, was higher in EAE retinal lysates (n = 7) than in CFA retinal samples (n = 5) and naive animals (n = 6). Moderate but significant changes in the expression of Opn1sw, coding for S-opsin, were found between CFA-treated retinae and other groups. No change was noticed between EAE and naive conditions. Interestingly, the transcript level of Gnat2, coding for cone-specific alpha transducing subunit was elevated in EAE/CFA mice, although that of Gnat1, coding for rod-specific alpha transducin subunit, did not vary between experimental groups. F By Western blotting, M-opsin was upregulated in EAE retinal lysates (n = 5) compared with naive lysates (n = 5). Scale bars: A = 100 µm, C, D = 400 µm on left, 100 µm (Right close-ups). Statistics: one-way ANOVA, Tukey post hoc test, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. OD optic disk