Experimental design.

<p>Both retinas from each mouse were studied, the left treated ones, and their right contralateral to the lesion, which were used as control.</p><p>Experimental design.</p

Intraocular pressure values.

<p><b>A</b>: Graph showing the temporal course of IOP from 24h till 14 weeks after laser photocoagulation (LP). In the left eye (LE) there is a significant increase of the IOP at 24h, 48h and 5 days compared to the contralateral right eye (RE) and to prelaser (PRE) values. IOP values are back to normal from 1 week to 14 weeks, the latest time analyzed. In <b>B</b> are shown the IOP values during the first week post-LP measured at shorter intervals. One hour after LP, IOP has already increased significantly in the left eye. n = number of animals analyzed.</p

Correlation analysis.

<p>Correlation between the number of Brn3a⁺RGCs vs OHSt⁺RGCs (<b>A</b>) and mRGCs vs OHSt-RGCs (<b>B</b>) for each analyzed retina (open circles) at 2 and 4 weeks after OHT induction. The r² value for each regression line is shown at the bottom right of each graph.</p

Sectorial loss of RGCs after OHT.

<p>Distribution of traced- (A-F), Brn3a⁺ (A'-F') and melanopsin⁺ (A'',F'') RGCs at 2 (<b>A-C''</b>) and 4 weeks (<b>D-F''</b>) after the induction of ocular hypertension (OHT). Maps labelled with the same letter are from the same retina (<b>A-A''</b>, <b>B-B''</b> and so on). <b>A-F'</b>: isodensity maps. <b>A''-F''</b>: neighbour maps. Colour scale for isodensity maps in A goes from purple (0 RGCs/mm²) to red (≥4,800 RGCs/mm²), and for neighbour maps in A'' goes from purple (1–2 neighbours in a radius of 0.165 mm) to red (≥11 neighbours in the same radius). At the bottom of each map is shown the number of RGCs or mRGCs counted in its respective retina. S: superior, V: ventral, N: nasal, T: temporal. Bar scale in A: 500 μm.</p

Primary antibodies used in this work.

<p>Primary antibodies used in this work.</p

Cone distribution in the mouse retina.

<p>Isodensity maps showing the topography of cones in pigmented and albino mice. For each population and strain two left and two right retinas are shown. Notice that because L-opsin⁺ and S-opsin⁺ cones were double immunodetected the first and third, and the second and fourth map in each row are from the same retinas. All-cones maps were generated from another set of retinas in which both α-opsin antibodies were visualized using secondary antibodies coupled with the same fluorophore, hence all cones were taken into account irrespectively of their opsin expression. Below each map is shown the number of cones quantified in its corresponding retina. The colour scale density code is placed in the middle of the figure. LR: left retina, RR: right retina. S: superior, N: nasal, I: inferior, T: temporal. Bar: 1 mm.</p

Opsin immunodetection in pigmented (C57/BL6) and albino (Swiss) mouse strains.

<p>Flat mounted left retinas from a pigmented (<b>A,B</b>) and an albino (<b>C,D</b>) mouse where the L- and the S- opsin have been double immunodetected. S: superior, N: nasal, I: inferior, T: temporal. Bar: 1 mm.</p

How to sample the mouse retina to accurately infer each cone population.

<p>Schematic drawings of a right retina showing the minimum number of sampling areas of 0.05² needed to manually quantify and infer the total cone population in both strains. In each drawing are shown the cone density regions wherefrom the samples should be taken. The inference of the total cone population can be calculated following the mathematical formula at the bottom of the figure, where the retinal area should be previously measured in mm², % of area low is 25, % of area medium is 30–90, % of area high is 30–45 (depending on the cone population, see drawings), sampling area is 0.05 mm² and is the mean number of cones per density area (i.e. average of cones in the samples taken within each density region). S: superior, N: nasal, I: inferior, T: temporal.</p

Total number of cones in the mouse retina.

<p>Total number of cones quantified in individual retinas and their mean number ± standard deviation (SD). <b>L-opsin:</b> number of cones that express the L-opsin (L-opsin⁺cones: genuine-L and dual cones); <b>S-opsin:</b> number of cones that express the S-opsin (S-opsin⁺cones: genuine-S and dual cones). <b>Both opsins:</b> quantification of all-cones after double immunodetection of S- and L-opsins with the same fluorophore (all-cones: genuine-L, genuine-S, and dual cones). Last rows show the retinal area and the mean cone density across the retina. In addition are shown the maximum (max) and minimum (min) densities found. These values were extracted from the intense scrutiny carried out during the sampling method analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102392#pone-0102392-g006" target="_blank">figure 6</a>), were samples of 0.05 mm² were independently quantified. LR: left retina, RR: right retina. There are significantly more L-opsin⁺cones and all-cones in the pigmented than in the albino strain (Ttest, <b>^§</b>p = 0.006, *p≤0.001, respectively) while for the S-opsin⁺cones is the reverse (<b>^#</b>p = 0.01).</p

Opsin expression in pigmented and albino mice.

<p>Magnifications from pigmented (<b>A</b>) and albino (<b>B</b>) mice flat mounted retinas showing L-opsin⁺cones, S-opsin⁺cones and their merged image. The rightmost column shows all-cones (both opsins developed with the same fluorophore). In the retinal drawings on the left, is shown the area where the magnifications were taken from. S-opsin⁺cones are sparse in the superior retina of the pigmented strain and abundant in the albino one. In both strains most genuine S-cones are found in the inferior retina. In the pigmented strain majority of genuine L-cones lay in the superior retina, while in the albino mouse majority of L-cones are dual. And so dual cones are found across the retina in the albino strain and mostly restricted to the ventral retina in the pigmented one. S: superior, N: nasal, I: inferior, T: temporal. Bar: 100 µm.</p