FST results with two colors under dark-adapted conditions and on a range of white and chromatic backgrounds.

<p>(A,B) FST-TVI with blue (A) and red (B) stimuli on white backgrounds in BCM patients and normals. Gray region defines the expected desensitization of the rod system. Rectangles define the data further explored in panels C-F. (C-F) Comparison of thresholds with chromatic and white backgrounds; blue stimuli on white (BonW) or yellow (BonY), and red stimuli on white (RonW) or on blue (RonB) backgrounds are shown. Different panels show the effectiveness of the background for the scotopic (C,D), S-cone (E) and photopic (F) systems. Lines with unity slope are fit to the data, and offset between the lines is shown in log units. (G,H) Sensitivity loss and predicted photoreceptor mediation using a pair of RonW and RonB FST thresholds. Both individual results and group averages (Avg) are shown. Error bars, when visible, are ±1SD.</p

Spectral sensitivity functions in normal subjects and BCM patients recorded at 14° superior field.

<p>(A) Sensitivities (mean±1 SD) to six spectrally distinct stimuli in normal subjects (n = 3) under dark-adapted (left), and on 1 (middle) and 10 cd.m^-2 (right) white backgrounds. (B) Sensitivities to the spectrally distinct stimuli in BCM patients for the same three adaptation conditions as in Panel A. Results from P8 are shown at the correct ordinate location; results from remaining patients have been adjusted by 1 log increments for visibility. Theoretical functions describing rod (green), S cone (blue), L/M cone (orange) sensitivities are shown after vertical shifts to fit relevant normal and BCM data in Panels A and B. (C) Comparison of individual normal and BCM sensitivities at 500 nm. (D,E) Comparison of individual normal and BCM sensitivities at 440, 500 and 560 nm. Symbols in Panels C, D, and E are painted by colors derived from the fit of theoretical functions to the spectral data. N = normal, B = BCM.</p

Nystagmus and foveal function in BCM.

<p>(A) Fixation locations in a normal subject and 3 BCM patients. For each subject, 10 s long epochs of eye movement data during fixation to a large visible red target (Target I) are shown in spatial (left) and spatio-temporal (right) coordinates. Spatial distribution of fixation clouds are shown on infrared SLO images of each macula with standard circles centered on the anatomical foveal depression. Spatio-temporal distribution of eye movements are shown on chart records for X and Y directions; up is nasal retina for X and superior retina for Y. All results are presented as equivalent right eyes for comparability. Horizontal dashed lines on the chart records depict the location of the anatomical fovea. (B) Fixation location and instability in BCM patients as a function of the bright red standard target (I), a green target (II) scotopically-matched to the standard target but expected to show greater visibility to S-cones, and a dim red target (III). N.S., not significant; *, P<0.05. (C) Distribution of fixation locations with the standard target in all patients. I = inferior, N = nasal, S = superior, and T = temporal retina. (D) Fixation location and instability as a function of best-corrected visual acuity. (E) Test pattern used with microperimetric stimuli to evaluate visual function under chromatic adaptation displayed on a normal near-infrared reflectance image. Stimulus locations are divided into 5 groups; f, foveal region, s, superior, i, inferior, t, temporal and n, nasal retina. (F,G) Sensitivities to blue stimuli on yellow background (BonY) and red stimuli on cyan background (RonC) in individual BCM patients (bars left to right; P2, P3, P4, P6, P8, P9, P10, P15, P16, P17, P18, P20, P25, P26, P28, and P29) compared to normal results (symbols; mean ±1sd) at the five regions shown in panel E. BCM results plotted below the zero line in Panel G represent those cases where the brightest available stimulus was not seen.</p

Visual fields of BCM patients evaluated with kinetic and static perimetry.

<p>(A) Light-adapted (LA) vertical sensitivity profiles from a normal subject and a BCM patient using achromatic (black line) and 600-nm (orange line) stimuli on a 10 cd.m^-2 white background, or 440-nm (blue line) stimuli on a yellow background (YB). (B) S-cone sensitivity profiles (filled circles) of the BCM patients using a 440-nm stimulus on YB compared to normal limits (gray = ±2SD). (C) LA white vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). Blue line is the S-cone sensitivities from Panel C shifted according to the difference in effectiveness between the white and 440-nm stimuli. (D) LA 600-nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (E) Sensitivity differences between LA white and LA 600-nm stimuli are shown for the BCM patients (filled circles) and normal (unfilled circles). Predicted differences for rod (green dashes) and L/M cone (orange dashes) mediation are shown. (F) Dark-adapted (DA) vertical sensitivity profiles from a normal subject and a BCM patient using 500-nm (green line) and 650-nm (red line) stimuli. Above the results it is shown whether there is rod (R) or mixed (M) mediation, as determined by the differences between sensitivities to the stimuli. (G) DA 500-nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (H) DA 650 nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (I) Sensitivity differences between DA 500- and DA 650-nm stimuli are consistent with rod mediation (gray) at all locations except for the normal results with 650 nm at fixation. S, superior; I, inferior. (J) DA 650-nm sensitivities at fixation in normal and BCM. Normal 650-nm sensitivities are mediated by the L/M cones (C) whereas BCM sensitivities are mediated by the rods (R). Error bars are ±1SD.</p

OCT abnormalities in <i>rd16;Nrl^−/−</i> mice.

<p>(A) Upper panels: Representative OCT scans vertically across ∼2 mm of retina (centered at the ONH, optic nerve head) in a WT mouse and in two <i>rd16;Nrl^−/−</i> mice of different ages. Lower panels: Magnified parts of the superior region of the retinal sections with overlaid longitudinal reflectivity profiles (LRPs) to demonstrate the reflective abnormalities in the outer retinal region in <i>rd16;Nrl^−/−</i> mice (b and c) compared with C57BL6 WT (a). (B) Upper two panels: Vertical OCT sections quantified for ONL+ thickness in two age groups of <i>rd16;Nrl^−/−</i> mice. Regions of outer retina with pseudorosettes were excluded in the measurement. ONL+ profiles in the older (P83–89, n = 12 eyes) age group were thinner than those in younger (P31–41, n = 35 eyes) mice; gray bands in the P83–89 plot represent mean±2 SD for ONL+ thickness of the P31–41 mice. For reference, insets at lower right of the upper two plots show original raw data before suppression of pseudorosette regions. Third panel from top: Means of ONL+ data across the vertical meridian in two age groups (error bar: ± SD; P31–41, open circles; P83–89, filled triangles). Lowest panel: Histograms showing average ONL+ fraction across vertical meridian of two age groups (*represents <i>p</i><0.001). (C) Histological sections of <i>rd16;Nrl^−/−</i> retina at 4 different ages from P21 to P80, compared with a WT retinal section. Histograms show ONL fraction (based on the earlier age group) in <i>rd16;Nrl^−/−</i> mice from peripheral retina (n = 6 eyes in each of the two age groups, *represents <i>p</i> = 0.01).</p

Structure and function in the <i>rd16;Nrl^−/−</i> mouse retina.

<p>(A) ERG b-wave amplitudes of responses to UV- and M-cone stimuli as a function of age in <i>rd16;Nrl^−/−</i> mice from P34 to P83 (n = 95) with comparisons to data from previously recorded signals in <i>Nrl^−/−</i> (squares <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Cheng1" target="_blank">[11]</a>; square with cross <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Mears1" target="_blank">[19]</a>), and <i>rd16</i> (crosses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092928#pone.0092928-Cideciyan3" target="_blank">[7]</a>) mice. Upper: Cone b-wave responses to ultraviolet (UV, 360 nm peak) stimuli in the <i>rd16;Nrl^−/−</i> mice are severely reduced compared with those of <i>Nrl^−/−</i> mice at comparable ages. Amplitudes in <i>rd16</i> mice are low compared to the other mice. It is also notable that ERGs of the <i>Nrl^−/−</i> mice remain relatively stable throughout this age range, while ERGs of the <i>rd16; Nrl^−/−</i> and <i>rd16</i> mice decline in amplitude with age. Lower: Responses to green (510 nm) stimuli are substantially lower in amplitude than those from UV-cone stimuli. Again, <i>Nrl^−/−</i> mice have the largest amplitudes and do not decline with increasing age within this time period. The <i>rd16;Nrl^−/−</i> waveforms are lower in amplitude and there is a reduction with age. Only limited data were available for <i>rd16</i> mice and these fell within the range of <i>rd16;Nrl^−/−</i> amplitudes. Waveforms for representative <i>rd16;Nrl^−/−</i> mice at various ages (grey-filled circles) are illustrated in the panels at right. Grey lines: linear regression fit to log-converted data (dashes) and 95% prediction intervals (solid). Squares with cross at earliest age in graphs: <i>Nrl^−/−</i> data from Mears et al., 2001 (B) Photoreceptor structure (ONL+) as a function of the combined UV- and M-cone ERG b-wave amplitudes. ONL+ remains similar to the value at P31 (youngest age <i>rd16;Nrl^−/−</i> we studied) across various degrees of ERG amplitude reduction. Horizontal dashed line is the reference level for the lower limit of retinal structure thickness at P31 (−2SD from the mean at this age); photoreceptor structure above this lower limit indicates no difference compared to the data of P31 (error bars, +2SD from mean).</p

Spatial and temporal distribution of pseudorosettes in <i>rd16;Nrl^−/−</i> retina.

<p>(A) Histological sections from peripheral retina of two <i>rd16;Nrl^−/−</i> mice at different ages demonstrating the presence of pseudorosettes (arrows). Calibration = 50 μm. (B) Schematic drawing of the mouse retina indicating the coverage of the central OCT raster scans (red circle). ONH is centered in the drawing. Integrated <i>en face</i> image of the central region of a P41 <i>rd16;Nrl^−/−</i> mouse showing how pseudorosettes appear as white dots (B, right panel, red circle). (C) Pseudorosette distribution within the central retinal region in a young (P31) and an older (P83) <i>rd16;Nrl^−/−</i> eyes. Insets (up and right) show average pseudorosettes as density in different sectors of the central retinal region sampled (n = 8 eyes for both age groups). (D) Upper: Histograms comparing number of pseudorosettes in the central retina by OCT at two different ages (P31, n = 10 eyes; P83, n = 8 eyes). Lower: Pseudorosette counts from histological sections of peripheral retina of two different age groups (P21–40, n = 6 eyes; P60–80, n = 6 eyes). Both data sets in <i>rd16;Nrl^−/−</i> mice indicate that the number of rosettes decreases with age (*represents <i>p</i><0.001 and <i>p = </i>0.01 for the upper and lower graphs, respectively). Error bars, ± SD from the mean.</p

Expression of photoreceptor proteins is reduced over time in <i>rd16;Nrl^−/−</i> mice.

<p>Representative cross sections (20X) from central and peripheral retina of P21, P40, P60 and P80 <i>rd16;Nrl^−/−</i> mice were immunostained for the presence of cone transducin alpha (GNAT2) (A) or S- cone opsin (B) and PNA (A,B). Despite maintenance of cone outer segment sheaths (PNA), both GNAT2 and S-opsin expression are markedly reduced by P60 in both central and peripheral retina. INL- inner nuclear layer, ONL- outer nuclear layer, IS/OS- inner segments/outer segments. Calibration = 35 μm.</p

Structure and function in the central retina of <i>CEP290</i>-LCA patients.

<p>(A) Cross-sectional OCT scans along the horizontal meridian through the fovea in a normal subject, a <i>CEP290</i>-LCA patient, and an RP patient. ONL is highlighted in blue. Inset shows location of scan. (B) Relationship of foveal ONL thickness and visual acuity in <i>CEP290</i>-LCA patients. Bar graph represents the average ±1SD foveal ONL thickness of eyes in the different visual acuity ranges (n = 3, for 0.1–1 LogMAR; n = 5, for 1–2 LogMAR; and n = 11, for 2–NLP). Dashed line is lower limit of normal and emphasizes that despite low acuities, foveal ONL is within normal limits. Inset, data from a series of RP patients plotted similarly to show the more expected relationship between structure and visual acuity in retinal degenerations (n = 3, for 0–0.2 LogMAR; n = 20, for 0.2–1 LogMAR; and n = 3 for 1–2 LogMAR). Dashed line is also lower limit of normal. (C) Relationship in <i>CEP290</i>-LCA patients of width of the ONL in the central retina and patient age at time of examination. ONL width was unable to be defined in a 32-year-old <i>CEP290</i>-LCA patient with maculopathy. Solid line is linear regression. Inset, traced central ONL peaks in representative patients of different ages.</p