Ca²⁺ imaging of photoreceptor terminals.

**<p>p<0.01;</p>*<p>p<0.05; n.s., not significant. Significance levels were determined by Kruskal-Wallis ANOVA.</p

Comparison of the sprouting phenotype in the wild-type, ΔEx14–17, and I756T mutant retinae.

<p><b>A–C:</b> Immunocytochemical triple staining of Calbindin (red), PKCα (green), and VGluT1 (blue) on P28 old wild-type (A), ΔEx14–17 (B), I756T (C) outer retinae shows sprouting of ON-bipolar cell dendrites as well as horizontal cell processes into the ONL of both <i>Cacna1f</i> mutants. The VGluT1 labeling shows the existence of presynaptic contacts with the sprouting elements. <b>D:</b> Comparison of the severity of horizontal cell sprouting in the wild-type, ΔEx14–17, I756T mutant outer retina at P6, P14, P28, two months, and eight months. Asterisks indicate the onset of noticeable sprouting in the <i>Cacna1f</i> mutants. In the I756T mutant retina, noticeable horizontal cell sprouting started earlier (P14) than in the ΔEx14–17 mutant retina (P28), but declined at eight months, when sprouting still continued in the ΔEx14–17 mutant retina. ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bar in A for A–C<b>:</b> 10 µm; in D: 20 µm.</p

Measurement of macular pigment optical density using HMP.

<p>A) Determination of the macular pigment optical density (MPOD) from the photometric matches. The modulation sensitivity (1/threshold) curves for central and peripheral fixation are plotted against modulation ratio on a log-log-scale. The curve for central fixation is shifted upward for better visualization (black arrow). The minimum of the modulation sensitivity curves is determined by fitting a theoretical function to the data. Because the CIE luminances <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110521#pone.0110521-Stockman1" target="_blank">[38]</a> of both lights are identical, the position of the flicker minimum on the ordinate is zero for an observer with a luminous sensitivity identical to the CIE standard observer's. A shift of the minimum of the modulation sensitivity curve to the left indicates a higher sensitivity for the 460 nm light, a shift to the right indicates a lower sensitivity. The MPOD (expressed in optical density units at 460 nm) is calculated by subtracting the minimum flicker point for peripheral fixation from that obtained for central fixation. B) Modulation ratio-sensitivity curves together with the fitted models for all 24 observers. The curves measured under central (C) and peripheral (P) absorption were shifted vertically, so that the position on the Y-axis at x = −1.5 for both curves of each observer is equal. For facility of inspection, the curves for the individual observers were ordered in a way that MPOD increases from left to right and from bottom to top.</p

Age-dependent loss of CACNA1F immunoreactivity in the I756T mutant mouse.

<p>Immunocytochemical labeling of CACNA1F in wild-type and I756T mutant outer plexiform layer (OPL) at P6, P14, P28, two, and eight months. Scale bar: 10 µm.</p

Correlation between macular pigment optical densities measured by different methods.

<p>Significant correlations are reported with the Pearson R correlation coefficient and the p-values (without Bonferroni correction) in brackets. The correlations between HMP and MPR as well as HMP and HFP are the primary outcome measures (<b>bold</b>). MPOD measured with HMP correlates significantly with MPOD-MPR, and also with MPOD-HFP at two locations; however, only the correlation with HFP at 0.25° remains significant after Bonferroni correction for testing at four locations.</p><p>*The significances of the correlations with HFP have to be corrected for testing at four locations. The positive correlations with HFP at 0.25° and at 0.5° remain significant even after Bonferroni correction.</p><p>Correlation between macular pigment optical densities measured by different methods.</p

Photoreceptor degeneration in <i>Cacna1f</i> mutant mice.

<p><b>A:</b> Quantification of the percentage of TUNEL positive cells in the ONL from wild-type, ΔEx14–17, and I756T mutants at P28, 2 and 8 months. Values are means ± SD. (*p<0.05; **p<0.01; ***p<0.001, ANOVA). <b>B:</b> Immunocytochemical staining of GFAP on P28 old wild-type, ΔEx14–17, and I756T mutant retina shows more pronounced Müller cell reactivity in the I756T mutant retina. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 µm.</p

Modeling the modulation sensitivity functions.

<p>A) A revision of the Pokorny, Smith and Lutze model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110521#pone.0110521-Pokorny1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110521#pone.0110521-Pokorny2" target="_blank">[17]</a> was fit to the modulation sensitivity threshold data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110521#pone-0110521-g002" target="_blank">Figure 2b</a> for original data and model fits). The derivation of the model is described in detail in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110521#pone.0110521.s004" target="_blank">Appendix S1</a>. In contrast to the original HMP function, the shape of the new function varies with changes in the horizontal position of the equiluminant point. A prediction from the model is that the difference between the heights of the asymptotes of the modulation sensitivity function (ΔS) is equal to the horizontal shift of the minimum flicker point (h). B) The difference in the asymptotes for this theoretical relationship is evident in our data.</p

Age-dependent ONL thickness in <i>Cacna1f</i> mutant mice.

<p><b>A:</b> Labeling of nuclei with DAPI (blue) and of cone photoreceptor outer segments and terminals (arrowheads) with peanut agglutinin (green) on retinal cryostat sections of wild-type, ΔEx14–17, and I756T mutant mice at 8 months. <b>B:</b> Quantification of the number of cell rows in the outer nuclear layer (ONL) from wild-type, ΔEx14–17, and I756T mutants at P28, 2 and 8 months. Values in are means ± SD. (*p<0.05; **p<0.01; ***p<0.001, ANOVA). POS, photoreceptor outer segments; OPL, outer plexiform layer. Scale bar: 10 µm.</p

Agreement between MPOD measured with heterochromatic modulation photometry and with heterochromatic flicker photometry.

<p>a) The relationship between MPOD measured with HMP and with HFP (the latter measured with a 0.5° diameter ring). A significant correlation is found with HFP measurements at 0.25° and 0.5° retinal eccentricities, but not at 1.0° and 1.75°. b) The Bland-Altman plot shows that MPOD_HMP values were systematically smaller than MPOD_HFP at 0.5°: mean difference −0.22 with 95%-confidence interval of [−.28, −.15].</p

Photometric matches for central (bottom) and peripheral fixation (top) and the resultant MPOD values.

<p>Under the assumption that only macular pigment is responsible for differences in luminous sensitivity, the MPOD is calculated as the difference between the HMP minimum points.</p