Rods and cones mediate visual perception over 9 log units of light intensities, with both photoreceptor types contributing to a middle 3-log unit range that comprises most night-time conditions. Rod ...function in this mesopic range has been difficult to isolate and study in vivo because of the paucity of mutants that abolish cone signaling without causing photoreceptor degeneration. Here we describe a novel Gnat2 knockout mouse line (Gnat2-/-) ideal for dissecting rod and cone function. In this line, loss of Gnat2 expression abolished cone phototransduction, yet there was no loss of cones, disruption of the photoreceptor mosaic, nor change in general retinal morphology up to at least 9 months of age. Retinal microglia and Müller glia, which are highly sensitive to neuronal pathophysiology, were distributed normally with morphologies indistinguishable between Gnat2-/- and wildtype adult mice. ERG recordings demonstrated complete loss of cone-driven a-waves in Gnat2-/- mice; comparison to WT controls revealed that rods of both strains continue to function at light intensities exceeding 104 photoisomerizations rod−1 s−1. We conclude that the Gnat2-/- mouse is a preferred model for functional studies of rod pathways in the retina when degeneration could be an experimental confound.
•New genetic knockout of cone G-protein alpha subunit abolishes cone function.•Loss of Gnat2 does not alter rod signaling nor cause retinal degeneration.•In vivo, rods saturate at >104 photoisomerizations rod−1 s−1.•Gnat2-/- mouse model is ideal for dissecting rod and cone contributions to vision.
In the eye, the function of same-type photoreceptors must be regionally adjusted to process a highly asymmetrical natural visual world. Here, we show that UV cones in the larval zebrafish area ...temporalis are specifically tuned for UV-bright prey capture in their upper frontal visual field, which may use the signal from a single cone at a time. For this, UV-photon detection probability is regionally boosted more than 10-fold. Next, in vivo two-photon imaging, transcriptomics, and computational modeling reveal that these cones use an elevated baseline of synaptic calcium to facilitate the encoding of bright objects, which in turn results from expressional tuning of phototransduction genes. Moreover, the light-driven synaptic calcium signal is regionally slowed by interactions with horizontal cells and later accentuated at the level of glutamate release driving retinal networks. These regional differences tally with variations between peripheral and foveal cones in primates and hint at a common mechanistic origin.
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•Larval zebrafish prey detection uses specialized single UV cones in the acute zone•High-gain UV cones in this region boost detection of UV-bright contrasts•Cellular and molecular mechanisms of this tuning tally with those of the primate fovea•Further optimization occurs at the level of UV cone glutamate release
Yoshimatsu et al. show that larval zebrafish rely on single UV cones at a time to support visual prey capture. For this, zebrafish combine molecular, cellular, and circuit tuning to regionally boost detectability of prey in their acute zone. The mechanisms of this specialization tally with those of the primate fovea.
Leber congenital amaurosis (LCA) is the most common cause of inherited retinal degeneration in children. LCA patients with RPE65 mutations show accelerated cone photoreceptor dysfunction and death, ...resulting in early visual impairment. It is therefore crucial to develop a robust therapy that not only compensates for lost RPE65 function but also protects photoreceptors from further degeneration. Here, we show that in vivo correction of an Rpe65 mutation by adenine base editor (ABE) prolongs the survival of cones in an LCA mouse model. In vitro screening of ABEs and sgRNAs enables the identification of a variant that enhances in vivo correction efficiency. Subretinal delivery of ABE and sgRNA corrects up to 40% of Rpe65 transcripts, restores cone-mediated visual function, and preserves cones in LCA mice. Single-cell RNA-seq reveals upregulation of genes associated with cone phototransduction and survival. Our findings demonstrate base editing as a potential gene therapy that confers long-lasting retinal protection.
Melanopsin is a visual pigment that is expressed in a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs). It is involved in regulating non-image forming visual behaviors, ...such as circadian photoentrainment and the pupillary light reflex, while also playing a role in many aspects of image-forming vision, such as contrast sensitivity. Melanopsin was initially discovered in the melanophores of the skin of the frog Xenopus, and subsequently found in a subset of ganglion cells in rat, mouse and primate retinas. ipRGCs were initially thought to be a single retinal ganglion cell population, and melanopsin was thought to activate a single, invertebrate-like Gq/transient receptor potential canonical (TRPC)-based phototransduction cascade within these cells. However, in the 20 years since the discovery of melanopsin, our knowledge of this visual pigment and ipRGCs has expanded dramatically. Six ipRGC subtypes have now been identified in the mouse, each with unique morphological, physiological and functional properties. Multiple subtypes have also been identified in other species, suggesting that this cell type diversity is a general feature of the ipRGC system. This diversity has led to a renewed interest in melanopsin phototransduction that may not follow the canonical Gq/TRPC cascade in the mouse or in the plethora of other organisms that express the melanopsin photopigment. In this Review, we discuss recent findings and discoveries that have challenged the prevailing view of melanopsin phototransduction as a single pathway that influences solely non-image forming functions.
Human color vision is achieved by mixing neural signals from cone photoreceptors sensitive to different wavelengths of light. The spatial arrangement and proportion of these spectral types in the ...retina set fundamental limits on color perception, and abnormal or missing types are responsible for color vision loss. Imaging provides the most direct and quantitative means to study these photoreceptor properties at the cellular scale in the living human retina, but remains challenging. Current methods rely on retinal densitometry to distinguish cone types, a prohibitively slow process. Here, we show that photostimulation-induced optical phase changes occur in cone cells and carry substantial information about spectral type, enabling cones to be differentiated with unprecedented accuracy and efficiency. Moreover, these phase dynamics arise from physiological activity occurring on dramatically different timescales (from milliseconds to seconds) inside the cone outer segment, thus exposing the phototransduction cascade and subsequent downstream effects. We captured these dynamics in cones of subjectswith normal color vision and a deuteranope, and at different macular locations by: (i) marrying adaptive optics to phase-sensitive optical coherence tomography to avoid optical blurring of the eye, (ii) acquiring images at high speed that samples phase dynamics at up to 3 KHz, and (iii) localizing phase changes to the cone outer segment, where photoactivation occurs. Our method should have broad appeal for color vision applications in which the underlying neural processing of photoreceptors is sought and for investigations of retinal diseases that affect cone function.
The visual phototransduction cascade begins with a cis–trans photoisomerization of a retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. Visual opsins release ...their all-trans-retinal chromophore following photoactivation, which necessitates the existence of pathways that produce 11-cis-retinal for continued formation of visual pigments and sustained vision. Proteins in the retinal pigment epithelium (RPE), a cell layer adjacent to the photoreceptor outer segments, form the well-established “dark” regeneration pathway known as the classical visual cycle. This pathway is sufficient to maintain continuous rod function and support cone photoreceptors as well although its throughput has to be augmented by additional mechanism(s) to maintain pigment levels in the face of high rates of photon capture. Recent studies indicate that the classical visual cycle works together with light-dependent processes in both the RPE and neural retina to ensure adequate 11-cis-retinal production under natural illuminances that can span ten orders of magnitude. Further elucidation of the interplay between these complementary systems is fundamental to understanding how cone-mediated vision is sustained in vivo. Here, we describe recent advances in understanding how 11-cis-retinal is synthesized via light-dependent mechanisms.
Melanopsin is expressed in distinct types of intrinsically photosensitive retinal ganglion cells (ipRGCs), which drive behaviors from circadian photoentrainment to contrast detection. A major ...unanswered question is how the same photopigment, melanopsin, influences such vastly different functions. Here we show that melanopsin’s role in contrast detection begins in the retina, via direct effects on M4 ipRGC (ON alpha RGC) signaling. This influence persists across an unexpectedly wide range of environmental light levels ranging from starlight to sunlight, which considerably expands the functional reach of melanopsin on visual processing. Moreover, melanopsin increases the excitability of M4 ipRGCs via closure of potassium leak channels, a previously unidentified target of the melanopsin phototransduction cascade. Strikingly, this mechanism is selective for image-forming circuits, as M1 ipRGCs (involved in non-image forming behaviors), exhibit a melanopsin-mediated decrease in excitability. Thus, melanopsin signaling is repurposed by ipRGC subtypes to shape distinct visual behaviors.
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•Melanopsin enhances contrast sensitivity of M4 ipRGCs (ON alpha cells)•Melanopsin phototransduction increases M4 ipRGC intrinsic excitability•Melanopsin phototransduction closes leak potassium channels in M4 ipRGCs•Melanopsin modulates M4 ipRGC physiology across a wide range of light intensities
Sonoda et al. identify leak potassium channels as the major target of melanopsin phototransduction in M4 ipRGCs/ON alpha retinal ganglion cells. Melanopsin-dependent closure of these channels enhances cell excitability and contrast sensitivity across a wide range of light intensities.
To test the hypothesis that a simple model having properties consistent with activation and deactivation in the rod approximates the whole time course of the photoresponse.
Routinely, an exponential ...of the form f = α·(1 - exp(-(τ·(t - teff)s-1))), with amplitude α, rate constant τ (often scaled by intensity), irreducible delay teff, and time exponent s-1, is fit to the early period of the flash electroretinogram. Notably, s (an integer) represents the three integrating stages in the rod amplification cascade (rhodopsin isomerization, transducin activation, and cGMP hydrolysis). The time course of the photoresponse to a 0.17 cd·s·m-2 conditioning flash (CF) was determined in 21 healthy eyes by presenting the CF plus a bright probe flash (PF) in tandem, separated by interstimulus intervals (ISIs) of 0.01 to 1.4 seconds, and calculating the proportion of the PF a-wave suppressed by the CF at each ISI. To test if similar kinetics describe deactivation, difference of exponential (DoE) functions with common α and teff parameters, respective rate constants for the initiation (I) and quenching (Q) phases of the response, and specified values of s (sI, sQ), were compared to the photoresponse time course.
As hypothesized, the optimal values of sI and sQ were 3 and 2, respectively. Mean ± SD α was 0.80 ± 0.066, I was 7700 ± 2400 m2·cd-1·s-3, and Q was 1.4 ± 0.47 s-1. Overall, r2 was 0.93.
A method, including a DoE model with just three free parameters (α, I, Q), that robustly captures the magnitude and time-constants of the complete rod response, was produced. Only two steps integrate to quench the rod photoresponse.
Rhodopsin (Rho), a prototypical G-protein-coupled receptor (GPCR) in vertebrate vision, activates the G-protein transducin (GT) by catalyzing GDP-GTP exchange on its α subunit (GαT). To elucidate the ...determinants of GT coupling and activation, we obtained cryo-EM structures of a fully functional, light-activated Rho-GT complex in the presence and absence of a G-protein-stabilizing nanobody. The structures illustrate how GT overcomes its low basal activity by engaging activated Rho in a conformation distinct from other GPCR-G-protein complexes. Moreover, the nanobody-free structures reveal native conformations of G-protein components and capture three distinct conformers showing the GαT helical domain (αHD) contacting the Gβγ subunits. These findings uncover the molecular underpinnings of G-protein activation by visual rhodopsin and shed new light on the role played by Gβγ during receptor-catalyzed nucleotide exchange.
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•Structures of a Rho-GT complex ± nanobody at 3.3 and 3.9 Å resolution, respectively•Extensive Rho-GT contacts enable striking signal-to-noise ratio in phototransduction•Rho-GT ± nanobody structures illustrate perturbations resulting from nanobody binding•The Gβγ-GαHD “latching switch” is essential for fast GPCR-catalyzed GDP-GTP exchange
High-resolution cryo-EM structures of a light-activated, signaling-active rhodopsin-transducin complex (Rho-GT) reveal a previously unknown mechanism involving G-protein βγ subunits and the α subunit helical domain (HD) in receptor-catalyzed activation as well as provide a structural basis for vertebrate visual phototransduction and illustrate the structural perturbations caused by a nanobody in a GPCR-G-protein complex.
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