OP (post-photoreceptoral) peak amplitudes elicited by step-wise increases in stimulus intensity.

<p>(A) Representative traces corresponding to responses elicited by a stimulus intensity of 1.36 log cds/m2; (B) OP peak amplitudes measured in control and IUGR offspring at 2 and 14 months of age as a function of stimulus intensity, and (C) Bar graphs depicting comparisons of peak amplitudes of OPs elicited by the maximal stimulus intensity. *P<0.05 compared to controls at the same age. A.u., arbitrary units; AUC, area under the curve.</p

Dark-adapted b/a ratios as a function of age in control and IUGR offspring.

<p>No differences were seen between groups at either 2 or 14 months of age. However, the b/a ratio increased in the IUGR group between 2 and 14 months (P<0.01).</p

Minimum recorded implicit times (IT) in young (2 month) and aged (14 month) offspring.

<p>Photopic a-wave implicit times are not shown because of the lack of complete data sets in each group; notably, 14 month IUGR offspring had only one rat achieve criterion responses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061861#pone-0061861-g003" target="_blank">Figure 3B</a>).</p

Dark-adapted ERG a-wave (photoreceptoral) and b-wave (post-photoreceptoral) elicited by step-wise increases in stimulus strength.

<p>(A) Representative traces corresponding to responses elicited by a stimulus intensity of 1.36 log cds/m2; (B) a-wave, and (C) b-wave amplitudes measured in control and IUGR offspring at 2 and 14 months of age. Adjacent bar graphs depict corresponding peak amplitudes. **P<0.01 compared to controls at the same age.</p

Absence of anatomical changes in the IUGR rat retina at 14 months of age.

<p>(A) Rhodopsin staining of rod photoreceptor outer segments. (B) m-Opsin staining of cone outer segments. (C) m-Opsin (r<i>ed</i>) and s-Opsin (<i>green</i>) staining of cone cell membranes. (D) PKCalpha staining of rod bipolar cells (and a subset of amacrine cells) (<i>green</i>) and bassoon staining of rod and cone ribbon synapses (<i>red</i>). (E) GFAP labelling of astrocytes and Müller cell processes. Nuclei stained with DAPI (<i>blue</i>). Scale bar, 20 µm for panels A–D, 50 µm for panel E. (F) TH staining (<i>green</i>) of low density dopaminergic A18 amacrine cells showing both cell bodies of typical size (7–11 µm, <i>arrows</i>), as well as a very small percentage with large cell bodies (>15 µm, <i>asterisk</i>). (G) ChAT staining (r<i>ed</i>) of type I and II “starburst” amacrine cells; non-displaced, located at proximal border of INL; and displaced, located in GCL. Scale bar 50 µm for panels F, G.</p

Light-adapted ERG a-wave (photoreceptoral) and b-wave (post-photoreceptoral) elicited by step-wise increases in stimulus intensities.

<p>(A) Representative traces corresponding to responses elicited by a stimulus intensity of 1.36 log cds/m²; (B) a-wave, and (C) b-wave amplitudes measured in control and IUGR offspring at 2 and 14 months of age. A-wave data (B) depict responses of each rat tested rather than means because the majority of responses were below the threshold criteria of 10 µV (shaded area). *P<0.05, *P<0.01 compared to controls at the same age.</p

Localization of AE3fl and AE3c in mouse retina and retinal changes in <i>Slc4a3</i>^−/− mice.

<p>(a–d) Retina cross sections of 4 months <i>Slc4a3</i>^+/+ mice (a, c), showing staining of the outer plexiform layer and Müller cells somas (arrows) and processes (arrowheads), and horizontal cells (diamond), using C-terminus antibody (a); somas in the ganglion cell layer were also immunolabeled. N-terminus antibody (specific to AE3c isoforms) labeled horizontal cell somas and dendrites (c). Specific staining was absent in 4 months <i>Slc4a3</i>^−/− littermates (b, d) scale bar = 25 µm. GFAP was restricted to inner limiting membrane in <i>Slc4a3</i>^+/+ mice (e), while it stained radial (arrows) and tangential (stars) processes and was elevated in the inner limiting membrane (arrowheads, a and b) in age-matched <i>Slc4a3</i>^−/− mice (f); scale bar = 20 µm. (g) Western blot analysis of protein samples (50 µg) prepared from whole retinal lysates of wild-type <i>Slc4a3</i>^+/+, and null <i>Slc4a3</i>^−/− mice, resolved by 10% SDS-PAGE (<i>top panel</i>); α-tubulin served as a loading marker (<i>bottom panel</i>). (h) Summary of GFAP expression normalized to α-tubulin. Values are expressed relative to <i>Slc4a3</i>^+/+ protein levels. Brackets represent number of retinas analyzed. *Indicates statistically significant difference (<i>P</i><0.05), compared to <i>Slc4a3</i>^+/+ wild type mice. Immunostaining of <i>Slc4a3</i>^+/+ (i) and null <i>Slc4a3</i>^−/− mice (j) retinas examined by confocal microscopy using double-labeling for Bassoon (OPL, red) and PKC-α (rod bipolar cells, green). Arrows indicate sprouting of processes in the OPL of <i>Slc4a3</i>^−/− mice; scale bar = 20 µm.</p

Apoptosis in retinas of <i>Slc4a3</i>^−/− mice.

<p>(A) Absence of apoptotic nuclei in retinas of 4 and 6-month-old <i>Slc4a3</i>^+/+ mice. Nuclei (blue) are stained with DAPI dye. Apoptotic nuclei are shown (green) in retinas of age-matched <i>Slc4a3</i>^−/− mice littermates, with predominant apoptotic nuclei found at 6-months of age. (B) Model demonstrating functional significance of AE3fl expression in Müller glial cells, and functional significance of AE3c in horizontal cells. In Müller cells, cytoplasmic carbonic anhydrase (CAII) can trap CO₂ release by the photoreceptors intracellularly by converting to HCO₃⁻ and H⁺. AE3fl located in Müller cells end feet facilitate the removal intro the vitreous or the blood of the HCO₃⁻ and H⁺, in a process that is maximized by the CAXIV. In horizontal cells, AE3c may contribute to pH_i homeostasis by exchanging intracellularly produced HCO₃⁻ with Cl⁻. CAII and CAXIV are also involved in tight regulation of horizontal cells pH_i, increasing the effectiveness of the bicarbonate buffer system.</p

Histological analysis, funduscopy, and immunohistochemistry examination of the <i>Slc4a3</i>^−/− mouse retina.

<p>Retina cross-sections (20 µm-thick) from <i>Slc4a3</i>^+/+ (a) and <i>Slc4a3</i>^−/− (b) mice stained with hematoxylin-eosin, scale bar = 50 µm. Electron micrographs of <i>Slc4a3</i>^+/+ (c) and <i>Slc4a3</i>^−/− (d) mouse retinas, scale bar = 5 µm; OS = outer segment; IS = inner segment; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cells layer. Fundus photos in 8-month-old <i>Slc4a3</i>^+/+ wild type (e) and <i>Slc4a3</i>^−/− littermate mouse (f). Confocal microscopic pictures of retina flat mounts stained with GFAP (g, h), scale bar = 50 µm. Confocal microscopic reconstructions (<i>z</i>-stack of 50 µm depth) of fluorescein-filled blood vessels in retina flat mounts showing several loops indicative of shunting between venule and arteriole (arrows) in 8 month old <i>Slc4a3</i>^−/− (k) not seen in WT (i); at higher magnification, tortuous vessels are obvious in <i>Slc4a3</i>^−/− (j) compared with straight trajectories in <i>Slc4a3</i>^+/+(l), scale bar = 100 µm. Confocal fluorescence image of six-month old <i>Slc4a3</i>^−/− mouse retina (m), stained with anti-glial fibrilar acidic protein (GFAP) antibody, at the level of the optic nerve head (anterior lamina cribosa), scale bar = 50 µm.</p

Localization of carbonic anhydrases II and XIV in <i>Slc4a3</i>^−/− mouse retina.

<p>Frozen vertical sections of adult wild type <i>Slc4a3</i>^+/+ and <i>Slc4a3</i>^−/− null mouse littermates retina were mounted in the same slide and labeled with goat anti-CAII ((A), 1∶100 dilution) or goat anti-CAXIV ((B), 1∶100 dilution) antibodies. Immunofluorescence signals were visualized by Alexa fluor 594-conjugated anti-goat IgG antibody (red, 1∶100 dilution). Sections were mounted in a DAPI media to identify nuclei, and images collected with a Zeiss LSM 510 laser-scanning confocal microscope with ×40/1.3 oil immersion objective (Neofluar oil). Merged images display CAII and CAXIV labeling overlapping nuclei staining. Scale bar = 20 µm. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cells layer.</p