Behavioral Assays of Brightness Discrimination in Mice

13 Hankins M.W.

Peirson S.N.

Foster R.G. Melanopsin: an exciting photopigment. 14 Lucas R.J.

Freedman M.S.

Muñoz M.

Garcia-Fernández J.M.

Foster R.G. Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors. 1) in the rod-specific cyclic guanosine monophosphate (cGMP) phosphodiesterase, which abolishes rod phototransduction and leads to rod and subsequent cone degeneration. They carry an additional diphtheria toxin-based transgene targeting surviving cones for cytoxic lesion (cl). At the ages employed here, rd/rd cl mice are essentially rodless and coneless [ 14 Lucas R.J.

Freedman M.S.

Muñoz M.

Garcia-Fernández J.M.

Foster R.G. Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors. Figure 1 Melanopsin-Dependent Brightness Discrimination in Mice Show full caption (A) Schematic of swim maze viewed from above. Visual targets (arrays of 64 blue, green, and red LEDs Figure S1 ) appear at the end of two lanes created by a dividing wall. An escape platform (shaded box) could be associated with a visual cue and the animal's ability to learn this association quantified by the frequency with which it chose the correct lane when first passing the end of the dividing wall (choice point). (B) The performance of rd/rd cl mice under training to swim toward a lit (106 melanopic cd/m2) versus dark target, was not significantly better than chance over the first 8 days of testing (filled circles; p > 0.05; two-tailed one sample t test; 6–8 trials per day) but improved over repeated training to be significantly better than chance over days 22–30 (open circles, p < 0.001). Performance with the light occluded (triangles) is shown for comparison. Data are percentage of correct choices over 48 trials for each of seven mice. (C) Immunohistochemical analysis of retinal whole mounts from these rd/rd cl mice revealed a number of remodeled cones immunoreactive for S-opsin (green) in the ventral retina ( Figure S1 for further data and methods). An equivalent image from a wild-type (WT) retina is shown for comparison. Scale bars represent 200 μm. (D) Maze navigation was not dependent upon these surviving S-cones because, although this ability was retained when a “green” stimulus (peak emission 517 nm; half peak bandwidth 30 nm) replaced the white light, their performance was no better than chance under a “blue” light providing an equivalent excitation of S-opsin (see Figure S1 for spectral radiance). (E) rd/rd cl mice could also be trained to identify the escape platform with the brighter of two lit targets, with the percentage of correct choice over 6 days (8 trails per day; n = 4 mice) related to their difference in radiance. Mice performed significantly better than chance (one sample t test; ∗∗p < 0.01, ∗p < 0.05) when asked to distinguish a moderately lit target (104 melanopic cd/m2) from darkness (difference in irradiance = ∞) or targets 105 or 13 (but not 2) times brighter. (F) The frequency with which Opn1mwR and Opn1mwR;Opn4−/− mice previously trained to associate the escape platform with a brighter target chose a “green” lane in preference to a “red” lane is plotted as a function of the green target's radiance. Data show mean ± SEM; n = 4 for Opn1mwR and 5 for Opn1mwR;Opn4−/− mice; fitted with sigmoidal curves; curves differed in the predicted radiance for a 50% green choice between genotypes (F statistic; p < 0.0001) indicating a melanopsin influence on the spectral sensitivity of brightness discrimination. One prediction of the hypothesis that intrinsically photoreceptive retinal ganglion cells (ipRGCs) contribute to assessing brightness is that this aspect of visual discrimination should survive complete loss of outer retinal photoreceptors. ipRGCs remain functional and can support a variety of accessory visual responses in mice lacking rods and cones []. To determine whether brightness discrimination also survives under such conditions, we used a murine model of advanced retinal degeneration (rd/rd cl) []. These mice are homozygous for a loss-of-function mutation (rd) in the rod-specific cyclic guanosine monophosphate (cGMP) phosphodiesterase, which abolishes rod phototransduction and leads to rod and subsequent cone degeneration. They carry an additional diphtheria toxin-based transgene targeting surviving cones for cytoxic lesion (cl). At the ages employed here, rd/rd cl mice are essentially rodless and coneless []. We tested their ability to make visual discriminations in a trapezoid Y water maze in which the mouse was trained to swim toward a lit target (in preference to a dark target in the adjoining lane) to reach an escape platform ( Figure 1 A ). Mice with an intact visual system learned to perform this task rapidly and with few errors (≥94% correct over the first 4 days of testing for each of four mice). By contrast, rd/rd cl animals initially appeared confused, choosing the lit lane no more often than expected by chance over 8 days of training ( Figure 1 B). However, over 21 days of repeated training, there was a gradual improvement in performance. At the end of this period, these animals showed significant preference for the lit target ( Figure 1 B).

15 Thyagarajan S.

van Wyk M.

Lehmann K.

Löwel S.

Feng G.

Wässle H. Visual function in mice with photoreceptor degeneration and transgenic expression of channelrhodopsin 2 in ganglion cells. 14 Lucas R.J.

Freedman M.S.

Muñoz M.

Garcia-Fernández J.M.

Foster R.G. Regulation of the mammalian pineal by non-rod, non-cone, ocular photoreceptors. 16 Allen A.E.

Brown T.M.

Lucas R.J. A distinct contribution of short-wavelength-sensitive cones to light-evoked activity in the mouse pretectal olivary nucleus. 17 Kojima D.

Mori S.

Torii M.

Wada A.

Morishita R.

Fukada Y. UV-sensitive photoreceptor protein OPN5 in humans and mice. Residual visual discrimination in rd/rd mice has previously been correlated with surviving cone photoreceptors, even at very late stages of degeneration []. Based upon previous analyses [], we were confident that the rd/rd cl mice used in our maze experiments would lack cones expressing M-opsin but concerned that a few S-opsin-expressing cones might survive (these predictions were born out in subsequent immunohistochemical analyses; Figure 1 C; see also Figure S1 available online). We therefore set out to test the possibility that surviving S-cones allowed brightness discrimination in this cohort of rd/rd cl mice. To this end, we adjusted the spectral composition and intensity of the light-emitting diode (LED) array to produce stimuli enriched for either short or longer wavelengths that should appear equally bright (isoluminant) for S-opsin (calculated according to the method in []). Under these circumstances, the longer wavelength stimulus was estimated to provide ∼30× greater excitation of melanopsin. We found that these rd/rd cl mice, previously trained to choose a lit over a dark target, were able to navigate the maze when the lit target was replaced with the longer but not the shorter wavelength light ( Figure 1 D). This finding excludes surviving S-cones, any other UV sensitive pigment [], or some nonvisual output of the array (e.g., heat) as explanations for their maze navigation ability.

2 or 104 melanopic cd/m2) in preference to a dark target. Over the last 5 days of this training period, the mice swam toward the lit target more often than expected by chance (65% ± 2% correct, mean ± SEM; p < 0.01 one sample t test), confirming that this moderate target radiance was within the melanopsin sensitivity range. The dark target was then replaced with a target of equivalent spectral composition, but 100× higher radiance. When the escape platform was associated with this brighter target, the rd/rd cl mice readily learned to swim toward it. This ability was maintained when the difference in target radiance was reduced to ×13 ( These experiments confirm that rd/rd cl mice can detect a visual signal and employ it for purposes of spatial navigation. To determine whether they could distinguish quantitative differences in brightness, we trained a new cohort of four rd/rd cl mice over 17 days to swim toward a lit target of moderate radiance (64 red+green+blue LED triplets each with radiance 13 W/sr/mor 10melanopic cd/m) in preference to a dark target. Over the last 5 days of this training period, the mice swam toward the lit target more often than expected by chance (65% ± 2% correct, mean ± SEM; p < 0.01 one sample t test), confirming that this moderate target radiance was within the melanopsin sensitivity range. The dark target was then replaced with a target of equivalent spectral composition, but 100× higher radiance. When the escape platform was associated with this brighter target, the rd/rd cl mice readily learned to swim toward it. This ability was maintained when the difference in target radiance was reduced to ×13 ( Figure 1 E).

2 each) in preference to the null lane, which had a “red” target (64 red LEDs; 953 W/sr/m2 each). For this purpose, we used Opn1mwR mice that carry a knockin of the human red cone pigment (L-opsin) at the mouse M-cone opsin locus, causing cones that ordinarily would express M-opsin to instead express the human pigment [ 18 Smallwood P.M.

Olveczky B.P.

Williams G.L.

Jacobs G.H.

Reese B.E.

Meister M.

Nathans J. Genetically engineered mice with an additional class of cone photoreceptors: implications for the evolution of color vision. 19 Lall G.S.

Revell V.L.

Momiji H.

Al Enezi J.

Altimus C.M.

Güler A.D.

Aguilar C.

Cameron M.A.

Allender S.

Hankins M.W.

Lucas R.J. Distinct contributions of rod, cone, and melanopsin photoreceptors to encoding irradiance. 20 Jacobs G.H.

Neitz J.

Deegan 2nd, J.F. Retinal receptors in rodents maximally sensitive to ultraviolet light. The performance of rd/rd cl mice in the water maze is consistent with the hypothesis that ipRGCs contribute to brightness discrimination. However, given the possibility of compensatory reorganization following this aggressive retinal degeneration, we were particularly interested to determine whether melanopsin also contributes to visual discrimination in animals with an intact visual system. To this end, we set out to determine whether melanopsin influences the spectral sensitivity of brightness perception in mice. Using the same swim maze paradigm employed for the rd/rd cl experiments, we initially trained mice to associate the escape platform with the appearance of a “green” target (64 green LEDs; 300 W/sr/meach) in preference to the null lane, which had a “red” target (64 red LEDs; 953 W/sr/meach). For this purpose, we used Opn1mwR mice that carry a knockin of the human red cone pigment (L-opsin) at the mouse M-cone opsin locus, causing cones that ordinarily would express M-opsin to instead express the human pigment []. Whereas the mouse M-opsin has a rather similar spectral sensitivity to melanopsin, L-opsin is shifted to longer wavelengths []. During the training phase, although the radiance of the red target was greater than that of the green, the reduced sensitivity of all photopigments (including the introduced L-opsin) at the longer wavelengths meant that the green target was calculated to appear “brighter” irrespective of whether the mice were basing their decision on the activity of cones, rods, or melanopsin. Accordingly, both Opn1mwR mice and Opn1mwR mice lacking melanopsin (Opn1mwR;Opn4−/−) rapidly learnt this task. Because mouse S-opsin is very insensitive to either red or green wavelengths [], we felt it most unlikely that the mice were using color to discriminate between the two lanes. Nevertheless, to confirm that their choice was based on assessments of brightness, we replaced the green light with a (4×) dimmer red light. Mice of both genotypes reliably swam toward the higher radiance panel without any further training (mean >80% correct over 4–8 trials for each genotype).