ERG responses in <i>Gnat1^−/−;Cnga3^−/−;Opn4^−/−</i> (TKO) mice.

<p><b>A</b>, Dark adapted flash ERG traces from a representative TKO mouse and representative traces from two <i>rd/rd cl</i> mice; arrow depicts flash onset; scale bar  = 50 ms (x-axis), 25 µV (y-axis); numbers to left are stimulus irradiance in log cd/m². <b>B</b>, Mean (±SEM; n = 5) a- and b-wave amplitudes for flash ERG in TKO mice. <b>C</b>, Representative light-adapted ERG traces in wild type (WT) and TKO mice (Scale bar =   = 50 ms (x-axis), 25 µV (y-axis)). <b>D</b>, b-wave amplitude (mean±SEM) at the brightest flash (3.5 log₁₀ cd/m²) in wild-type (n = 6), TKO (n = 4) <i>Gnat1^−/−</i> mice (n = 5) compared by one-way ANOVA (p<0.001) and Bonferroni's post test. <b>E</b>, Estimated threshold irradiance (box shows median±upper lower quartiles, whiskers range of data) for a reliable ERG response in TKO (n = 5), <i>Gnat1^−/−</i> (n = 3) and wild type mice (n = 6) compared with one-way ANOVA (p<0.0001) and bonferroni post test. *** p<0.001; ** p<0.01; ns p>0.05.</p

Light-driven neural activation in the visual cortex of TKO mice.

<p>Multiple immunostaining for c-fos (green) and SMI-32 (red) reveals a clear pattern of activation in response to light (<b>A</b> and <b>C</b>), relative to darkness (<b>B</b> and <b>D</b>). As shown at low magnification in <b>A</b>, light-driven c-fos induction was found in retrosplenial (RSD), primary (V1) and secondary (V2M/L) divisions of visual cortex. The V1 region from <b>A</b> and <b>B</b> is shown at higher magnification in <b>C</b> and <b>D</b> respectively. Light-driven neural activation, as visualized by c-fos positive nuclei, was seen throughout the different layers of primary visual cortex (I-IV). Scale bars: <b>A</b>–<b>B</b> 500 µm, <b>C</b>–<b>D</b> 200 µm.</p

Impact of a PLC-β antagonist on the TKO ERG.

<p>Effects of the intravitreal injection of the PLC-β inhibitor U73122 on the b-wave amplitude (mean±SEM; <b>A</b>) and the average response waveform following vehicle or 1.0 mM U73122 in TKO mice (<b>B</b>). The same data plotted to show paired amplitudes of b-wave and a-wave before and after 1.0 mM U73122 for each individual (<b>C&D</b>). Effects of 1.0 mM U73122 intravitreal injection on wild type b-wave amplitude (<b>E</b>) and the average response waveform (<b>F</b>)<b>.</b> Sample size for TKO n = 4–6 for Vehicle, 0.1 mM and 0.5 mM U73122, and n = 10 for 1.0 mM U73122; for WT n = 4. Drug concentrations given in mM are for the injected preparation, final tissue concentration will be around 10× lower. Data in <b>A</b> analysed by one-way ANOVA and bonferroni post tests. Data in <b>C, D</b> and <b>E</b> analysed by paired two-tailed t-tests. * p<0.05; **p<0.p01. Scale bars in <b>B</b> and <b>F</b> = 50 ms (x-axis), 25 µV (y-axis).</p

Spectral sensitivity of the TKO flash ERG.

<p><b>A</b>, ERG b-wave amplitude (mean±SEM; n = 7) from TKO mice (expressed as % of maximum response at 458 nm) for 458 nm (blue) and 580 nm (yellow) flash stimuli. <b>B</b>–<b>D</b>, The same data plotted with stimulus irradiance at the two wavelengths normalized according to the spectral sensitivity of rod opsin (<b>B</b>), MWS opsin (<b>C</b>), and melanopsin (<b>D</b>). F test statistic allows the use of a single curve for the two wavelengths (p>0.05) when normalised for rod opsin, but not for MWS opsin (p<0.01), or melanopsin (p<0.0001). <b>E</b>. The relationship between p for the F statistic and the λ_max of the putative pigment used to normalize irradiance across the two wavelengths peaked close to 498 nm, the known spectral sensitivity of mouse rod opsin. <b>F.</b> b-wave amplitude (mean±SEM) for a range of monochromatic stimuli calculated to be isoluminant for rods (filled circles) or MWS-cones (open circles). Lines depict best fit by linear regression analysis, slope significantly different from 0 for the MWS-cone (p<0.05) but not rod (p>0.05) conditions. Note that the 580 nm datapoint contributes to both series.</p

ERG responses in <i>Gnat1^−/−;Gnat2^cpfl3/cpfl3</i> mice.

<p><b>A</b>, Comparison of b-wave amplitude of scotopic ERG in TKO and <i>Gnat1^−/−;Gnat2^cpfl3/cpfl3</i> mice (mean±SEM; n = 5 for each group). <b>B</b>, Representative scotopic ERG traces from TKO and <i>Gnat1^−/−;Gnat2^cpfl3/cpfl3</i> mice recorded in response to white flash stimuli (arrow depicts flash onset; scale bar =  50 ms (x-axis), 25 µV (y-axis); numbers to left are stimulus irradiance in log cd/m²). <b>C</b>, Representative traces for light-adapted ERGs in wildtype and <i>Gnat1^−/−;Gnat2^cpfl3/cpfl3</i> mice (arrow depicts flash onset; scale bar  = 50 ms (x-axis), 25 µV (y-axis)). Immunocytochemistry for Gnat2 using an anti-Gαt2 antibody in WT retina (<b>D-E</b>) revealed strong immunoreactivity in cones (arrows) with a lower level of staining found in rods. In TKO retina (<b>F</b>), staining in cones was absent, while immunoreactivity in rods persisted, even though these mice lack Gnat1, the most likely target of any cross-reactivity for this antibody. Pre-absorption with blocking peptide abolished staining in both rods and cones (<b>G</b>). Similarly, immunoreactivity for the γ subunit of cone transducin was detected in both cones and, to a lesser extent, rods of WT (<b>H</b>; arrow points to cones) and TKO (<b>I</b>; arrows point to rods) retinas. In WT retina there was a strong signal in cone outer segments, which was displaced to inner segments in TKO mice. Confocal settings were identical for images shown in D&F, E&G and H&I to facilitate comparison. Scale bars: <b>D</b>&<b>F</b> 10 µm, all others 20 µm.</p

The melanopic sensitivity function accounts for the spectral sensitivity of tonic LGN firing activity in mice with functional rods/cones.

<p>(<b>A</b>) Example response of an <i>Opn1mw^R</i> ‘sustained’ LGN neuron to the three test stimuli depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053583#pone-0053583-g001" target="_blank">Fig. 1A</a> at a range of irradiances (numbers above traces indicate log light intensity relative to the maximum achievable-3.4 log m-lux). (<b>B</b>) Mean ± SEM response to the three stimuli (and below in overlay) at maximum irradiance (n = 46 cells; each unit's response normalised to the largest change in firing rate across all stimuli). (<b>C–E</b>) Irradiance response relationship for <i>Opn1mw^R</i> LGN sustained firing responses (20–30 s after stimulus onset) to the three stimuli. Sensitivity could be explained by a single linear function (F-test) when irradiances were expressed in melanopic lux (C; 0.432), but not in effective photon flux for L- or S-cones (<b>D</b> & <b>E</b>; P = 0.001 & 0.022 respectively).</p

The melanopic sensitivity function accounts for OPN responses to spectrally modulated stimuli in rodless/coneless mice.

<p>(<b>A</b>) Spectral profile of stimuli that differ in irradiance but not melanopic illuminance (4-fold difference in total photons between ‘dim’ and ‘bright’), termed ‘melanopsin silent’. (<b>B</b>) Mean (± SEM) firing rate of 131 PON neurons to transitions between the two melanopsin silent stimuli. These transitions evoked no significant change in firing activity (paired t-test, P = 0.944). (<b>C</b>) Spectral profile of stimuli which differ substantially in melanopic illuminance (21-fold between dim and bright) but not total photons (<1% difference), termed ‘melanopsin active’. (<b>D</b>) Mean (± SEM) firing rate of PON neurons to transitions between the two melanopsin active stimuli. Transitions to the melanopsin ‘bright’ condition evoked a significant increase firing activity (paired t-test, P = 0.014, n = 131). Yellow bar in <b>C</b> & <b>D</b> indicates presentation of the ‘bright’ stimulus, with dim stimulus present at all other times.</p

The temporal profile of melanopsin-dependent responses is equivalent under short and long wavelength adaptation.

<p>(<b>A</b>) Mean firing rate (± SEM) around a 10 s step of 470 nm (3.8 log m-lux; 10 repeats; interstimulus interval = 240 s) alternating with a background of 470 nm (blue line) or 630 nm (orange line). Backgrounds were calculated to provide −0.1 log m-lux, but differed in terms of total photons (10.6 and 14.6 log photons/m2/s, respectively). (<b>B</b>) Firing rate over the 10s stimulus was not significantly different between conditions (P = 0.34 two-sample T-test). (<b>C</b>) One-phase exponential decay curves were fitted to responses of each unit following light offset, and no significant difference was found in span, k-constant or plateau between the backgrounds (P = 0.40, P = 0.72 and P = 0.33, respectively, following two-sample T-test). (<b>C&D</b>) Long wavelength flashes (15.3 photons/cm2/s) calculated to produce a negligible change (∼1% increase) in melanopsin excitation drive no change in FR immediately following flash onset, and do not have any cumulative effects on ongoing responses (<b>E&F</b>).</p

The melanopic sensitivity function accounts for the spectral sensitivity of LGN responses in rodless/coneless mice.

<p>(<b>A</b>) Spectral profile of the 3 test stimuli and the melanopic sensitivity function (V^zλ: shaded area). (<b>B</b>) Example response of an <i>rd/rd cl</i> LGN neuron to the three test stimuli presented at a range of different irradiances (numbers above traces indicate log light intensity relative to the maximum achievable: 3.4 log m-lux). (<b>C</b>) Mean ± SEM responses to the three stimuli (and below in overlay) at maximum irradiance (n = 30 cells; each unit's response normalised to the largest change in firing rate across all stimuli). (<b>D–F</b>) Irradiance response relationship for <i>rd/rd cl</i> LGN responses to the three stimuli. A single curve best explains all the data (F-test) when irradiances are expressed in melanopic lux (<b>D</b>; P = 0.641), but not number of photons between 470–480 nm (<b>E</b>; P = 0.04), photopic lux (<b>F</b>; P = 0.001), total photons or total optical power (not shown).</p

The melanopic sensitivity function accounts for the impact of long wavelength conditioning stimuli on pupillary responses in rodless/coneless mice.

<p>(<b>A</b>) Sensitivity of <i>rd/rd cl</i> pupil responses to shortwavelength (<500 nm) flash (100 ms) is not altered by 30 min pretreatment with short (498 nm) or long (644 nm) wavelength conditioning stimuli matched for melanopic illuminance (0.4 m-lux; F-test, P = 0.931). (<b>B</b>) Sensitivity of <i>rd/rd cl</i> pupil responses to <500 nm flicker (1 Hz; ON duration  = 71 ms), alternating with with short (<500 nm) or long (>600 nm) wavelength background illumination can be predicted when time-averaged irradiance is expressed in m-lux (F-test; P = 0.256).</p