Validating the cAMP biosensor.

<p>A HEK293 cell line expressing the Glosensor™ cAMP biosensor under a tetracycline inducible promoter (FLP-IN™ system; Invitrogen) was generated. The reporter was validated by determining the effect of known doses of forskolin on levels of cAMP, determined by ELISA (<b>a</b>) and Glosensor bioluminescence (<b>b</b>). Data show mean for 2 (ELISA) and 3 (luminescence) separate experiments each of which contained samples in triplicate. Fits show sigmoidal dose response curves of the form y = a + b/1+10^(c-x) where a = bottom, b = top-bottom and c =  LogEC50, and yield EC50 values of 51 and 21 µM for ELISA and luminescence assays respectively. (<b>c</b>) A comparison of cAMP concentration and RLU for each forskolin concentration was used to infer the relationship between these parameters. This could be fit by a first order polynomial (R² value of 0.999), suggesting that, under these conditions, cAMP concentration can therefore be estimated from RLU as follows: [cAMP] µM  = 4.785 + (0.0003971a) + (0.000005261â2) where a  =  RLU/µl.</p

OptoXR sequence alignment.

<p>A number of structural variants on a published OptoXR chimera comprising elements of the human β2AR and human rhodopsin sequences were generated in an attempt to increase response amplitude/reproducibility. An amino acid alignment of these variants and, for comparison the human β2AR and rhodopsin (Genbank NM_000539.3, in red and NM_000024, in black), and JellyOp (Genbank AB435549, in blue) sequences are shown. Structural boundaries are based on bovine rhodopsin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030774#pone.0030774-Palczewski1" target="_blank">[14]</a> with putative cytoplasmic regions shaded in dark grey. The lysine residue in TM7, which forms a Schiff-base linkage with retinaldehyde chromophore, is highlighted in green. Note that the terminal 9 amino acids of rod opsin are included as an epitope tag (1D4) in all receptors used in this study (light grey shading). In addition to the published OptoXR in which the entire cytoplasmic surface of rod opsin is replaced by that of the β2AR (Rh1B2AR 1-t), variants in which either 1^st or 1^st and 2^nd intracellular loops from rod opsin were retained (Rh1B2AR 2-t and Rh1B2AR C3,t) in the hope of improving chimera stability were generated. Other variants on Rh1B2AR 1-t employed site directed mutagenesis of phosphorylation sites (highlighted in red) important for arrestin binding and receptor inactivation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030774#pone.0030774-Seibold1" target="_blank">[18]</a> (Rh1B2AR 1-t phos mutant) or a fusion of the human Gαs subunit at the C-terminal tail in purple (Rh1B2AR 1-t::Gαs).</p

Real time analysis of a standard OptoXR response.

<p>Light induced changes in cAMP biosensor (Glosensor) luminescence in HEK293 cells transiently transfected with Rh1B2AR 1-t. Data for cells incubated with 9 <i>cis</i> retinaldehyde (black) or all <i>trans</i> retinaldehyde (grey) are shown; yellow arrows depict presentation of light flash. Data points show mean ± SEM n = 7. Inset shows immunocytochemical staining for the 1D4 epitope (in red, alexa 555 secondary antibody) of HEK293 cells expressing Rh1B2AR 1-t. DAPI shown in blue, scale bar  = 10 µm.</p

JellyOp driven light responses.

<p>(<b>a</b>) Light induced changes in cAMP biosensor luminescence (depicted _ormalized to baseline before light exposure) in HEK293 cells transiently transfected with JellyOp (n = 5). (<b>b,c</b>) The peak increase in luminescence following the first light flash was significantly greater for 9-<i>cis</i> pretreated cells expressing JellyOp than either the Rh1B2AR 1-t (1-t) or Rh1B2AR 2-t (2-t) chimera. It was also significantly greater for all-<i>trans</i> pretreated cells expressing JellyOp than Rh1B2AR 1-t. (<b>d,e</b>) Sustained responses were further enhanced, with luminescence over 5 minutes of light exposure (flashes once per minute) reaching a plateau significantly higher in JellyOp than either Rh1B2AR chimera. (<b>f</b>) Tracking changes in luminescence with higher temporal resolution (reading every 30s) in HEK293 cells stably transfected with JellyOp revealed high magnitude responses that could be sustained over 15 minutes of repeated stimulation (n = 4). Inset shows immunocytochemical staining for 1D4 epitope (red) in JellyOp expressing HEK293 cells. Nuclei stained blue with DAPI; scale bar 10 µm. (<b>g</b>) Cells stably expressing JellyOp show markedly repressed responses to a light flash when treated with 100 µM MDL2330A (adenylate cyclase inhibitor; dashed line), n = 1 (<b>h</b>) cAMP biosensor luminescence responses in HEK293 cells transiently transfected with JellyOp (continuous line) are abolished in the JellyOp lysine mutant (dashed line), n = 1 (<b>i</b>) Irradiance response curves for cAMP reporter activity in cells stably expressing JellyOp and induced with 10s white light (LED) pulses. RLU values are _ormalized to the peak response (n = 3). <b>A</b>-<b>I</b> show cells pre-incubated with either 9-<i>cis</i> (black) or all-<i>trans</i> (grey) retinaldehyde; mean ± SEM; yellow arrows depict timing of light flash. Data in <b>b</b>-<b>e</b> were analysed using one-way ANOVA with Dunnett's post hoc comparisons to JellyOp as control group, *p<0.05, **p<0.01; n≥4.</p

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 Stimulation Protocols.

<p>A: LED spectral power densities and <i>in vivo</i> photoreceptor spectral sensitivity (normalised). The output of blue and yellow LEDs was adjusted to produce equivalent effects on rods (black line). By contrast, the blue LED, always appeared brighter for melanopsin (green line). B: Protocol 1. Stimuli (30 or 20s melanopsin-isolating steps in dLGN and retina, respectively) presentations of the blue LED were interleaved with 210 or 180 sec of the (dLGN and retina, respectively) yellow to produce a ‘step’ visible only to melanopsin. The magnitude of this melanopsin step could be varied by mixing blue and yellow in the step (increasing the yellow and decreasing the blue elicited decreasing levels of contrast). C: Protocol 2. Starting at ND4, irradiance slowly ramped up (0.5 ND per 200 seconds) before remaining at a steady state for 10 seconds. At each 0.5ND, a blue melanopsin-isolating step (71%) is given for 30 seconds (total time per 0.5ND cycle = 4 minutes). This process was repeated until reaching ND0, at which point light-levels instead slowly ramped down and the process was repeated. D: The effective change in photon flux for melanopsin (green) and rods (black) across a full repeat of Protocol 2. Settings of ND filter at the point of each melanopsin isolating step are provided above. Di and ii: The starting position of the ramp (ND4 or ND0) was varied across experiments.</p

Responses to melanopsin-steps in the dLGN.

<p>A: Example cell responses (Ai and ii) to 30s melanopsin-isolating steps (blue shading) at ND0 (background = 14.3 log melanopsin photons/cm²/s). Top panels: trial bin count data for multiple repeats of the 71% contrast step (data normalised to mean firing rate of each repeat). Lower panels: PSTHs displaying firing rate over time for 71, 60, 51 and 38% contrast steps (mean±SEM of 14–16 repeats). Bin size in both plots is 5 seconds. B: Average PSTHs (mean±SEM) for all step-responsive cells across a range of melanopsin contrasts. A small but significant change in firing rate can be seen for contrasts ≥32% (two way ANOVA comparing step firing rate vs baseline firing rate (p<0.001), contrast (p>0.05), interaction (p<0.001) with Bonferroni post hoc tests displaying significance level; ** p<0.01; ***p<0.001. C: Mean onset and offset latencies for individual step-responsive units over a range of contrasts. Ci: Scatter plot displaying each single cell response onset vs response offset and ii: mean latencies across all step responsive units.</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

Online calibration and rod control verify silent substitution paradigm.

<p>Online calibration of the rod-isoluminant settings (A) and tests of rod contrast sensitivity (B) were performed both <i>in vitro</i> and <i>in vivo</i>. Displayed are representative data from dLGN. A. A 100 ms blue ‘flash’ (transition from yellow to blue LED) was presented at ND3 to provide conditions preferable to rods. Raster plots and associated PSTHs for an example melanopsin-stepping cell over a range of settings for the blue LED. In the middle (outlined by black dotted box) was the setting at which there was no change in firing, taken as the point of rod isoluminance, while decreasing (plots to left) or increasing (plots to right) the blue LED produced measurable responses in line with the appearance of negative or positive contrast for rods. Numbers above in the grey panels are estimated Michelson contrast for rods and melanopsin calculated according to known pigment absorption nomograms [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123424#pone.0123424.ref021" target="_blank">21</a>]. B: We determined the ability of fast flicker and extended step stimuli to reveal responses to low contrast rod-isolating stimuli (yellow step on low blue background; values above are estimated Michelson contrast). Rod responses were apparent for estimated rod contrasts ≥15% under both a 4Hz flicker (Bi; raster above and PSTH of mean firing rate) and 30sec step (Bii; mean±SEM firing rate) in this representative cell. Firing rates for both A and B match scale bar (bottom left 5 spikes/s).</p

Responses to melanopsin-isolating steps and gradual irradiance ramps in retina.

<p>A: Trial bin count examples of two cells responding to a 71% contrast step (blue bar at 0 to 20 seconds) presented during protocol 1 (background log 12.75 melanopsin photons/cm²/s to step log 13.5 photons/cm²/s) before (i) and during (ii) synaptic blockade. Before and after graphs are scaled to same axis to show changes in baseline activity upon synaptic blockade. B: Example raster plots and PSTH to a 50ms rod favouring yellow flash from dark (flash intensity log 11.45 rod photons/cm²/s) under normal conditions (aCSF) and under synaptic blockade (5 minutes after application of L-AP4 + NBQX) in a cell that was identified to respond to our melanopsin-isolating stimulus. C: Averaged plots for firing rate over time of consistent (n = 31) melanopsin-step responsive cells (mean±SEM) before (left) and during (right) synaptic blockade. D: Mean response onset and offset latencies for individual melanopsin-step responsive cells in the presence (purple symbols) and absence (green) of synaptic blockade. E: Under protocol 2, retinal cells responding to a melanopsin-step (n = 22/ 314 cellls; 71% contrast) do so over a range of background irradiances on both the upward and downward phases of the ramp (Repeated measures 2-way ANOVA, step vs baseline firing rate x irradiance; main effects of irradiance (p<0.001), step vs baseline (p<0.001) and interaction (p<0.05); Bonferroni post-hoc comparisons marked on figure as ** and *** p<0.01 for ND2 upward ramp and ND1.5 for downward; n = 22 cells). F: Retinal melanopsin-step responsive cells also tracked the gradual change in irradiance during protocol 2, revealed as a change in firing rate (mean± SEM) as a function of ramp progression.</p