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

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

Responses of cells sensitive to the melanopsin-step over a range of irradiances.

<p>A: PSTHs for firing rate across melanopsin-isolating steps (71% contrast) over a range of backgrounds (across both increasing and decreasing arms of the ramp; n = 24 responsive cells mean±SEM). See 5Cii for significance values. B: Scatter plot displaying mean onset vs offset latencies for each cell as a function of background irradiance. Ci: Mean change in firing rate associated with the melanopsin-isolating step (firing rate during step—firing rate over previous 10 s) with increasing irradiance during the upwards (dark blue) and downwards (light blue) phases of the ramping protocol. Ci and ii Significant responses were recorded for steps against backgrounds ≥ 12.1 log melanopsin photons/cm²/s (ND2) on the ramp up and 13.1 log melanopsin photons/cm²/s (ND1) on the ramp down (2-way RM ANOVA step vs baseline firing rage x irradiance; main effects of irradiance (p<0.05), step vs baseline (p<0.001) and interaction (p<0.001); Bonferroni post-hoc comparisons p<0.01 for ND2 and above for upward ramp and ND1 for downward). D: Firing rate (mean±SEM; n = 54) of dLGN stepping cells from protocol 1 to a bright blue 10 sec step from dark (log 14.1 melanopsin photons/cm²/s; purple bar). Note sustained activity after the termination of the step, considered to be a feature of the melanopsin light response. E: There was no significant change in time averaged firing rate of cells responsive to the melanopsin step as a function of ramp progression (mean±SEM n = 24).</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

Retinal responses in <i>rd/rd cl</i> mice.

<p>A: Trial bin count examples (5 second bins of time) of three cells responding to a 20s 71% contrast step (background log 12.8 to step log 13.56 melanopsin photons/cm2/s) in a <i>rd/rd cl</i> retina. Note the variety in the latency of response offset in examples 1 and 2, and the poor response reproducibility that is typical of <i>rd/rd cl</i> mice in example 3. Colour bar to the right of the plot (FR Hz) denotes the firing rate of cells in this and subsequent Trial bin count figures. B: Averaged plots for firing rate over time of melanopsin-step responsive cells (mean±SEM) to a 20s step (Bi n = 15) and a 60s step (Bii n = 17). Both step durations elicit a significant change in firing to the baseline rate (**** P<0.0001). C: A plot of mean onset and offset latencies for each individual responsive cell over multiple stimulus repeats reveals the extremely sluggish nature of light responses in <i>rd/rd cl</i> mice.</p

A subset of dLGN cells track the irradiance ramp.

<p>A: Trial Bin Count plots from a representative irradiance tracking unit to repeated 130 sec epochs across 3 presentations of the irradiance ramps. Note increases in firing rate in line with the intensity of ND wedge (Black = ND4, white = ND0). B: firing rate (mean±SEM) of 33 units that tracked irradiance (across 3 repeats of the ramp) track irradiance cleanly upwards of ND2 (log 12.1 background melanopsin photons/cm²/s). C: Histological localisation of units responding to the melanopsin-isolating steps in protocol 1 or 2 (n = 78; black dots) superimposed upon images from the appropriate elements of a mouse brain atlas showing the boundaries of the dLGN, ventral LGN (vLGN) and neighbouring nuclei (IGL; intergeniculate leaflet).</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-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