MW-opsin restores fast and sensitive light responses

Recent studies have established that vertebrate rhodopsin, found in rod outer segments, may be used ectopically to control G-coupled signaling in cultured cells, RGCs and ON bipolar cells, but runs down with repeated stimulation and deactivates slowly30,31,21,22, raising concern that it may not support vision of natural scenes because of movement of the observer and surrounding objects or saturation and possibly photo-bleaching of the chromophore under photopic lighting conditions. We wondered if another vertebrate opsin would have faster kinetics while maintaining similar light-sensitivity to rhodopsin. We turned to a mammalian cone opsin, MW-opsin, whose activation of the tetrameric GIRK1(F137S) G protein-coupled inward-rectifier potassium channel in HEK293 cells we found to decay 8-fold more rapidly and recover more completely than rhodopsin (Fig. 1a–c).

Fig. 1 Expression and function of MW-opsin in HEK293 cells and RGCs of rd1 mouse retina. a, b Representative traces of activation of homotetramer GIRK(F137S) channels by photo-stimulation of rhodopsin (a) or MW-opsin (b) measured in whole cell patch in 50 mM [K+] ext at V H = −80 mV in response to low intensity (1 mW cm−2) pulses of light at 535 nm (for MW-opsin) or 500 nm (for rhodopsin). c Decay of photo-response (Tau OFF) for rhodopsin (blue) and MW-opsin (green). Values are mean + SEM; n = 6 (rhodopsin), 8 (MW-opsin) cells. d Viral DNA expression cassette. MW-opsin with YFP (green) under control of hSyn-1 promoter. e Schematic of a degenerated rd1 mouse retina with targeted RGCs highlighted (green). ONL outer nuclear layer, IPL inner plexiform layer. Photoreceptor degeneration denoted in light gray and red cross. f, g En face view of flat mount (f) and transverse slice (g) confocal images of MW-opsin expression of rd1 mouse retina 4 weeks after intravitreal injection of AAV2/2-hSyn-MW-opsin-YFP. Images of YFP fused to C-terminal end of MW-opsin (green) show pan-retinal distribution (f) in RGC layer in relation to DAPI staining of nuclei (d, blue). Scales 60 μm (f) and 20 μm (g). h, i MEA recordings from representative uninjected control (e) and MW-opsin expressing (f) rd1 mouse retina. (Top) Raster plot with spikes for each RGC (e: n = 92 cells; g: n = 68 cells). (Bottom) Peristimulus time histogram (PSTH). Light stimulation protocol: 4 pulses of light of 100 ms duration (λ = 535 nm, enlarged green bars) separated by 60 s dark intervals. j Normalized Light response Index (LRI) for rd1 retina without (gray) and with MW-opsin expression (green) (gray: N = 6 retinas, n = 295 cells; green: N = 8 retinas, n = 323 cells). LRI for 1st and 5th light flash without (N = 3 retinas, n = 106 cells) and with (N = 6 retinas, n = 257 cells) 9-cis retinal. Light intensity 2 mW cm−2. Wavelength: λ = 535 nm (MW-opsin), Values are mean + SEM. Cells are sorted units. Statistical significance assessed using Mann–Whitney U test (*p ≤ 0.01) Full size image

We tested MW-opsin in the retina of the rd1 mouse, which has a mutation in the PDE-6-β gene, resulting in progressive loss of rod and cone photoreceptor cells. MW-opsin under control of the human synapsin promoter (hSyn-1), with a yellow fluorescent (YFP) C-terminal tag for tracking expression, was packaged in AAV2/2(4YF) and injected intravitreally at postnatal day 45–60 (Fig. 1d, e). Retinas isolated 4–8 weeks later showed expression to be pan-retinal, with a transfection rate of 45 ± 19% (SD) and localized to the soma and dendritic layers of ON-RGCs and OFF-RGCs (Fig. 1f, g and Supplementary Fig. 1), consistent with previous studies32 and similar to expression of rhodopsin under identical parameters (Supplementary Fig. 2). Retinas were mounted on a multi-electrode array (MEA), with the RGC layer in contact with the electrodes, to test for light-evoked activity. Due to complete photoreceptor degeneration in animals ≥3-months-old33, no light-evoked response was detected in the retina of control rd1 littermates (Fig. 1h), with the exception of a few RGCs which displayed slow responses characteristic of intrinsically photosensitive RGCs34. In contrast, retinas from MW-opsin rd1 animals displayed robust light-evoked increases in firing rate, consisting of a large fast, transient component and a small (~30% in size) slow component (Fig. 2a, Supplementary Fig. 3). Responses across retina were normalized using the Light Response Index (LRI = (peak firing rate in the light—average firing rate in dark)/peak firing rate in the light + average firing rate in dark)) adopted from Tochitsky and colleagues11 and our earlier work20. The light responses ran down with repeated bouts of light stimulation, as expected following removal of the retinal pigment epithelium, a source of 11-cis-retinal. The run down was reduced by the addition of 9-cis-retinal (a stable analog of 11-cis-retinal) to the recording solution (Fig. 1j).

Fig. 2 Light response in isolated rd1 mouse retina with MW-Opsin in RGCs. a (Top) Average response to light flash of RGC population expressing MW-opsin (green) or rhodopsin (blue) in rd1 mouse retina. (Bottom) Raster plot of average response of rd1 mouse retina RGCs to 5 flashes of 100 ms duration light at 535 nm for MW-opsin (n = 59 cells) and 510 nm for rhodopsin (n = 27 cells) expressing in RGCs. b, c Light sensitivity for MW-opsin (N = 6 retinas) and rhodopsin (N = 4 retinas) in RGCs of rd1 mouse retina. Peak firing rate normalized to maximum at highest intensity. d, e Time-course of light response. Population average traces with time from light onset to max excitation (time to peak: 355 + 21 ms), exponential fits for excitatory phase (Tau ON: 112 + 25 ms) and decay (Tau OFF: 260 + 31 ms) and full width at half max (FWHM: 183 + 85 ms) (e) for MW-opsin (d; e, green; N = 5 retinas, nc = 104 channels), rhodopsin (e, blue; N = 7 retinas, nc = 134 channels) and wt (e, white; N = 3 retina, nc = 97 channels). f, g Dependence of MW-opsin light response on flash duration. f Representative retina light response (n = 117 cells): population average firing rate (top) and raster plot of unit responses (bottom). g Normalized peak responses for different stimulation durations (2 × 100 mW cm−2, N = 2 retinas, nc = 63 channels). Light intensity 2 × 10−1 mW cm−2 unless specified, Wavelength: λ = 535 nm (MW-opsin) or 510 nm (rhodopsin). N = number of retinas, nc = # of channels, n = number of cells/units. Cells are sorted units. Values are mean + SEM. Statistical significance assessed using Mann–Whitney U test (*p ≤ 0.05) Full size image

MW-opsin rd1 mouse retinas were highly sensitive to light, to a degree similar to rhodopsin (Fig. 2b, c), in the range of indoor light intensity, and ~1000-fold higher than channelrhodopsin13,18 and halorhodopsin16,17 (Supplementary Fig. 4). While similar in sensitivity to rhodopsin (Supplementary Fig. 5a) MW-opsin ran down less in response to repeated flashes (Supplementary Fig. 5b) and had faster kinetics: ~5-fold faster rise, ~3-fold shorter time to peak and ~7-fold faster decay following a light pulse (Fig. 2d, e). The time constant of rise, the time to peak, and the time constant of decay changed modestly with decreasing light intensities, maintaining the advantage in speed over most of the intensity range (Supplementary Fig. 5c–e)21,22. The rise and decay kinetics of the response in RGCs of rd1 retina expressing MW-opsin resembled those of the RGC transient ON-response seen in wt retina, except that the former had a longer latency (Fig. 2e and Supplementary Fig. 5f, g). The fast response kinetics and sensitivity of MW-opsin suggested that it would respond to brief flashes of light. Indeed, illumination pulses as short as 25 ms, triggered responses that were ~50% of the maximal peak response (Fig. 2f, g), similar to what is seen in wt retina35.

We examined contrast sensitivity in the excised retina and in primary visual cortex in vivo by measuring responses to full-field gray scale steps. In the excised rd1 retina expressing MW-opsin, RGC activity changed in response to changes in brightness of as little as 25% (Supplementary Fig. 6a, b), approaching but not equivalent to the contrast sensitivity of the wild type retina (Supplementary Fig. 6c). In complementary in vivo experiments on rd1 animals expressing MW-opsin in RGCs, we measured single unit responses and visually evoked potentials across the layers of primary visual cortex in awake, free running animals (Supplementary Fig. 7) and observed similar contrast sensitivity using a standard computer monitor (Supplementary Fig. 8). The cortical responses followed flash frequencies up to at least 4 Hz (Supplementary Fig. 9). The sensitivity and kinetics of the light responses imparted by MW-opsin in RGCs suggested that it may support visually guided behavior.

MW-opsin restores innate light avoidance

Sighted mice innately avoid illuminated areas, a survival mechanism to evade capture36 that is lost following photoreceptor degeneration in rd1 mice14,23. To determine if this behavior could be restored, rd1 mice expressing MW-opsin were tested in a behavior box consisting of adjoining light and dark compartments (Fig. 3a). The fraction of time spent in each compartment was recorded and compared to untreated rd1 and wt mice (Fig. 3b, Supplementary Fig. 10a–c, Supplementary Table 1). The light compartment was illuminated with low intensity white light, equivalent to indoor office lighting (100 μW cm−2). Untreated rd1 mice spent ~45% of the time in the dark compartment, reflecting a slight location bias in favor of the release compartment (see Supplementary Methods) (Fig. 3b). In contrast, rd1 mice expressing either rhodopsin or MW-opsin showed a strong preference for the dark compartment (~70%), similar to normally sighted wt animals (80%) (Fig. 3b and Supplementary Fig. 10a). When white light was replaced with blue (460 ± 22 nm) or green (535 ± 25 nm) light and the intensity that was reduced to a lower end of the isolated retina intensity-response curves for MW-opsin and rhodopsin (1 μW cm−2; Fig. 2b) both MW-opsin and rhodopsin expressing animals showed green light avoidance (Fig. 3c, left and Supplementary Fig. 10b), but only rhodopsin animals showed blue light-avoidance (Fig. 3c, right and Supplementary Fig. 10c), consistent with their absorption spectra37.

Fig. 3 Light avoidance and learned visually guided behavior in rd1 mice expressing MW-opsin or rhodopsin in RGCs. a Schematic of light/dark box for light avoidance test. b, c Respectively for b, c-left and c-right, proportion of time spent in the dark compartment (proportion of avoidance) for rd1 control (gray; n = 5,8,5 mice), rd1 expressing rhodopsin in RGCs (blue; n = 6,8,5 mice) or MW-opsin (green; n = 11,10,4), and wt mice (white; n = 5 mice) when illuminated with either (b) white light (100 μW cm−2), (c) 1 μW cm−2 blue light (470 nm) (right) or green light (535 nm) (left). d Schematic of freezing response fear conditioning experiment. e Quantification of fear response for discrimination of temporally patterned stimulation. Time freezing above baseline is shown for when illumination transitions from static to 2 Hz frequency stimulation (100 μW cm−2) was paired or unpaired with a electric shock for control rd1, rhodopsin, MW-opsin, and wt mice (n = 4,6,12,10 paired, n = 7,8,8,8 unpaired). f Schematic of pattern discrimination experiment. Mice habituated at day 1, then exposed to electric shock in association with specific pattern of light projected to tablets and paired randomly in either chamber (conditioning days 2 and 3). On day 4 recall tested (time spent in each chamber), in absence of shock with light patterns reversed to avoid location bias (See Supplementary Fig. 11d). g–i Learned pattern discrimination. Time spent avoiding pattern paired with shock. g Horizontal vs. vertical parallel bars. Discrimination of parallel static (h) or moving (i) bars at distances of 1 vs. 6 cm. Respectively for g, h and i: rd1 control (n = 8,5,16), rd1 rhodopsin (blue; n = 8,6 mice), rd1 MW-opsin (n = 17,11,6) and wt (n = 5,6,9). (25 μW cm−2). (Note, proportion of success for these experiments shown in Supplementary Fig. 11). Light intensity = 25–100 μW cm−2; Wavelength: = 535 nm (MW-opsin), 510 nm (rhodopsin) or white light (MW-opsin). n = number of mice. Values are mean + SEM. Statistical significance assessed using Student’s two-tailed t-test with Bonferroni correction: *p < 0.05 Full size image

MW-opsin supports temporal light pattern discrimination

We used a visually cued fear-conditioning paradigm to test the ability of animals to differentiate flashing from constant light. We used a single compartment behavioral apparatus with a low intensity (100 μW cm−2) LED light that switched between constant and flashing (2 Hz) light. For each animal, either constant or flashing light was paired with a mild foot shock for 2 days (Fig. 3d) and freezing time was measured on day 3 in response to light cues in absence of foot shock11,22,38. Freezing time in this “paired group” was compared to that of an “unpaired group,” in which training shocks were randomized (i.e., not paired consistently with a visual cue). Freezing times in untreated rd1 mice did not differ between paired and unpaired conditions, consistent with the inability to see the visual cues (Fig. 3e, gray). In contrast, rd1 mice expressing MW-opsin froze more in the paired condition, as observed in wt animals (Fig. 3e, green and white). Strikingly, rd1 mice expressing rhodopsin did not differ between paired and unpaired conditions (Fig. 3e, blue). This suggests that, unlike blind mice expressing MW-cone opsin, rhodopsin mice cannot discriminate light flashing at 2-Hz from constant light, consistent with the slow light response kinetics observed in MEA (Fig. 2e)

MW-opsin restores spatial pattern discrimination

We asked if MW-opsin in RGCs would enable rd1 mice to detect spatial light patterns. We used a behavioral chamber with two adjoining compartments (Fig. 3f), each with a low-intensity LCD tablet (iPad) mounted on a wall that displayed a pair of parallel lines: in one, the lines were oriented vertically (||) and in the other horizontally (=). For MW-opsin the wavelength was centered at 535 nm (520–560) and for rhodopsin at 497 nm (480–520). On day 1, mice were habituated to the compartments with the visual displays off. During a 2-day training period, an aversive foot shock was paired with either the vertical or horizontal lines (Supplementary Fig. 10d). On day 4 the locations of the stimuli were switched to avoid location bias and conditioned avoidance was tested. We found that rd1 animals expressing MW-opsin showed avoidance of the aversive visual cue at a level significantly higher than did untreated rd1 controls, and similar to wt mice (Fig. 3g and Supplementary Fig. 10e). Rd1 animals expressing rhodopsin behaved like the blind untreated rd1 controls. These results indicate that MW-opsin restores the ability to recognize spatial light patterns, but rhodopsin does not.

We next asked if mice could discriminate differences between lines of identical orientation but different spacing, a visual task adopted from tests of visual acuity in humans and animals39,40. Parallel vertical lines were separated by distances of 1 or 6 cm. As above, an aversive foot shock was paired with one of the stimuli during the training period on days 2 and 3, and recall was tested on day 4. We found that rd1 mice expressing MW-opsin are able to distinguish between the two patterns with a performance that is similar to that of wt mice, whereas rhodopsin expressing animals are similar to untreated rd1 mice (Fig. 3h. Supplementary Figs. 10f and 12). MW-opsin also supported line differentiation when the parallel lines were in motion (1 cm/s) (Fig. 3i and Supplementary Fig. 10g).

Light adaptation of MW-opsin response and visual function

A fundamental characteristic of vision is the ability to distinguish objects across a wide range of ambient light intensities41,42. We wondered whether some aspect of adaptation would operate in the rd1 retina expressing MW-opsin. Isolated retinas were kept in complete darkness for 15 min (dark-adapted) and then tested in a series of brief (100 ms) flashes of green light (535 + 25 nm) at 60-s intervals and over a range of intensities. Retinas were then adapted for 5 min to a moderate indoor light level (light-adapted; white light at 100 μW cm−2) and retested. We first examined the kinetics of the light responses. The light response decayed rapidly, as shown above, displaying similar response kinetics for both the light and dark-adapted retina (Fig. 4a Supplementary Fig. 12a, b). The intensity-response curve showed a high light sensitivity in the dark-adapted retina (responding at ~0.5 μW cm−2) and lower sensitivity after adaptation to moderate light (responding to ~200 μW cm−2) (Fig. 4b, c and Supplementary Fig. 12a, b). This adaptation represented a shift by 2–3 orders of magnitude on the intensity axis (321 + 89, N = 3) (Fig. 4b, c).

Fig. 4 Light adaptation in RGC activity and visually guided behavior by MW-opsin. a–c MEA recordings in isolated retina of RGC light response mediated by MW-opsin in RGCs of rd1 mouse retina show sensitivity difference with retina adapted to dark versus light. a Light response decay (Tau OFF) as a function of flash intensity in dark versus light adapted condition (N = 3 retinas, nc = 88 channels). b Example intensity-response curve for representative retina first dark adapted (filled symbols) then light adapted (open symbols) (n = 56 cells). White light adaptation. ChR2 minimum value from Bi et al.13 and Sengupta et al.18. c Average (error bars are SEM) normalized Light response Index (LRI) at 3 flash intensities in same retina, first dark adapted and then light adapted (N = 3 retinas, nc = 88 channels). d–f Behavior shows light adaptation in visually guided tasks. d Schematic of adaptation to dark or light prior to testing of innate avoidance behavior or learned pattern discrimination behavior. e Proportion of time spent in the dark compartment (proportion of avoidance) under 100 μW cm−2 (bright light) or 1 μW cm−2 (dim indoor light) following 1 h. of adaptation to dark (N = 11 mice) or adaptation to light (white light; 1 mW cm−2/535 nm spectral component; ~50 μW cm−2; N = 11,13 mice). f Learned pattern discrimination of parallel bars spaced at distances of 1 versus 6 cm displayed at low (0.25 μW cm−2) or indoor (10 μW cm−2) light levels following 1 h. of adaptation to dark (N = 11 and 8 mice for each display) or light (white light; 1 mW cm−2/535 nm spectral component; 50 μW cm−2; N = 10 and 7 mice for each display). Performance was also reported in cohorts experiencing 4 and 8 h. of light adaptation (N = 7 mice). Dotted line denotes average performance of untreated rd1 control mice and performance (gray N = 5 mice) reproduced from Fig. 3h for reference and comparison. Wavelength: λ = 535 nm. N = # of animals, n = # of retina, nc = number of channels. Cells are identified as sorted units. Values are mean + SEM. Statistical significance assessed using Mann-Whitney U test (*p < 0.01). Student’s two-tailed t-test with Bonferroni correction: *p < 0.05 Full size image

We asked if MW-opsin would provide visually useful light adaptation in the behaving animal, first in the context of light avoidance behavior. Rd1 mice expressing MW-opsin were either dark adapted or light adapted to bright indoor illumination for 1 h (white light, 1 mW cm−2/535 nm light component, 50 μW cm−2) (Fig. 4d). They were then tested immediately in the two-chamber light-dark box for light avoidance at either 1 μW cm−2 (dim) or 100 μW cm−2 (bright). The light adapted MW-opsin expressing rd1 mice showed stronger light avoidance with the brighter test light, whereas the dim test light produced a high level of light avoidance in the dark-adapted animals (Fig. 4e and Supplementary Fig. 12c). Pattern recognition was also influenced by light adaptation. Rd1 mice expressing MW-opsin were trained by pairing mild foot shock with a display of parallel lines at one of two spacing, similar to that described above (Fig. 3h and Supplementary Fig. 10d). They were then dark-adapted (1 h) or light-adapted (1, 4 or 8 h) before testing. We found that the dark-adapted animals were able to discriminate between the line patterns whether they were presented at the low (0.25 μW cm−2) or moderate (10 μW cm−2) indoor intensity (Fig. 4f and Supplementary Fig. 12d), but that light-adapted animals only succeeded with the brighter test line patterns and were identical in performance between the groups that were light adapted for 1, 4, and 8 h (Fig. 4f and Supplementary Fig. 12e). The results show that spatial pattern recognition mediated by MW-opsin is adaptive over a range of natural light intensities.

MW-opsin restores novel object exploration

Our experiments above show that MW-opsin enables pattern recognition across a wide range of light intensities using illuminated displays. We wondered how it would operate in a natural environment, where ambient, incidental light illuminates three-dimensional objects. To address this, we employed an open field arena that is commonly used to test novel object recognition and exploratory behavior43,44. Mice naturally avoid open spaces and maintain proximity to walls of their environment. Exploratory excursions from these places of safety can be motivated by novel stimuli. Although mice employ multiple sensory modalities during exploration, vision has been shown to be critical for spatial navigation45. Our arena consisted of a cube containing two distinct novel objects. The mouse was placed against the arena wall, far enough from the objects, which themselves were far enough apart, so that the chance of an accidental encounter was low whether the animal walked along the wall or explored the other object. We filmed rd1 untreated, rd1-sham injected, rd1 expressing rhodopsin or rd1 expressing MW-opsin mice, as well as wt animals. Their movements were tracked for 10 min the first time that they were placed into the arena (Fig. 5a–d). We found that wt animals travel 1.6-fold farther and moved at an average velocity 1.59-fold faster than blind rd1 animals, consistent with the known visual component of exploratory behavior. Strikingly, like wt animals, rd1 animals expressing MW-opsin traveled farther (by 1.42-fold) and faster (by 1.41-fold) than their untreated rd1 littermates (Fig. 5e, f), suggesting that MW-opsin supports normal novel object exploration. To analyze this further, we focused on aspects of exploratory behavior that most likely depend on vision at a distance; the latency to exploration of the novel objects and the velocity and distance traveled on the excursions to the objects. Sham injected and rhodopsin expressing rd1 mice performed similarly to untreated rd1 animals, but MW-opsin mice reached the first and second objects in 5.12-fold and 4.25-fold shorter times, respectively (Fig. 5g, h), moved at velocities that were 2.2-fold and 1.89-fold faster to the first and second objects, respectively (Fig. 5i, j), and took shorter pathways that were 0.60-fold and 0.55-fold the distance to the first and second objects, respectively (Fig. 5k, l), as compared to untreated rd1 mice. In each of these measures, MW-opsin expressing rd1 mice reached levels that were similar to those of wt animals (Fig. 5e–l). These results suggest that MW-opsin in RGCs provides previously blind animals with naturalistic vision of objects under ambient light.