Eyeless razor clam Sinonovacula constricta discriminates light spectra through opsins to guide Ca2+ and cAMP signaling pathways.

Phototransduction is based on opsins that drive distinct types of Gα cascades. Although nonvisual photosensitivity has long been known in marine bivalves, the underlying molecular basis and phototransduction mechanism are poorly understood. Here, we introduced the eyeless razor clam Sinonovacula constricta as a model to clarify this issue. First, we showed that S. constricta was highly diverse in opsin family members, with a significant expansion in xenopsins. Second, the expression of putative S. constricta opsins was highly temporal-spatio specific, indicating their potential roles in S. constricta development and its peripheral photosensitivity. Third, by cloning four S. constricta opsins with relatively higher expression (Sc_opsin1, 5, 7, and 12), we found that they exhibited different expression levels in response to different light environments. Moreover, we demonstrated that these opsins (excluding Sc_opsin7) couple with Gαq and Gαi cascades to mediate the light-dependent Ca2+ (Sc_opsin1 and 5) and cAMP (Sc_opsin12) signaling pathways. The results indicated that Sc_opsin1 and 5 belonged to Gq-opsins, Sc_opsin12 belonged to Gi-opsins, while Sc_opsin7 might act as a photo-isomerase. Furthermore, we found that the phototransduction function of S. constricta Gq-opsins was dependent on the lysine at the seventh transmembrane domain, and greatly influenced by the external light spectra in a complementary way. Thus, a synergistic photosensitive system mediated by opsins might exist in S. constricta to rapidly respond to the transient or subtle changes of the external light environment. Collectively, our findings provide valuable insights into the evolution of opsins in marine bivalves and their potential functions in nonvisual photosensitivity.

An Expanding Role for Nonvisual Opsins in Extraocular Light Sensing Physiology.

We live on a planet that is bathed in daily and seasonal sunlight cycles. In this context, terrestrial life forms have evolved mechanisms that directly harness light energy (plants) or decode light information for adaptive advantage. In animals, the main light sensors are a family of G protein-coupled receptors called opsins. Opsin function is best described for the visual sense. However, most animals also use opsins for extraocular light sensing for seasonal behavior and camouflage. While it has long been believed that mammals do not have an extraocular light sensing capacity, recent evidence suggests otherwise. Notably, encephalopsin (OPN3) and neuropsin (OPN5) are both known to mediate extraocular light sensing in mice. Examples of this mediation include photoentrainment of circadian clocks in skin (by OPN5) and acute light-dependent regulation of metabolic pathways (by OPN3 and OPN5). This review summarizes current findings in the expanding field of extraocular photoreception and their relevance for human physiology.

Non-visual Opsins and Novel Photo-Detectors in the Vertebrate Inner Retina Mediate Light Responses Within the Blue Spectrum Region.

In recent decades, a number of novel non-visual opsin photopigments belonging to the family of G protein- coupled receptors, likely involved in a number of non-image-forming processes, have been identified and characterized in cells of the inner retina of vertebrates. It is now known that the vertebrate retina is composed of visual photoreceptor cones and rods responsible for diurnal/color and nocturnal/black and white vision, and cells like the intrinsically photosensitive retinal ganglion cells (ipRGCs) and photosensitive horizontal cells in the inner retina, both detecting blue light and expressing the photopigment melanopsin (Opn4). Remarkably, these non-visual photopigments can continue to operate even in the absence of vision under retinal degeneration. Moreover, inner retinal neurons and Müller glial cells have been shown to express other photopigments such as the photoisomerase retinal G protein-coupled receptor (RGR), encephalopsin (Opn3), and neuropsin (Opn5), all able to detect blue/violet light and implicated in chromophore recycling, retinal clock synchronization, neuron-to-glia communication, and other activities. The discovery of these new photopigments in the inner retina of vertebrates is strong evidence of novel light-regulated activities. This review focuses on the features, localization, photocascade, and putative functions of these novel non-visual opsins in an attempt to shed light on their role in the inner retina of vertebrates and in the physiology of the whole organism.

Ocular and extraocular roles of neuropsin in vertebrates.

The ability to detect and adapt to different levels of ambient light is critical for animal survival. Light detection is the basis of vision, but light also regulates eye development and drives several non-image-forming functions, including synchronizing circadian rhythms to the daily light/dark cycle, restricting pupils in response to changes in light intensity, and modulating mood in response to light. Until the early 2000s, these functions were thought to be solely mediated by ocular photoreceptors. However, neuropsin (OPN5), a UV-sensitive opsin, has been receiving growing attention, as new methods have revealed previously unappreciated functions of OPN5. In fact, OPN5-mediated extraocular and deep-brain photoreception have recently been described for the first time in mammals. This review aims to synthesize current knowledge of the properties and functions of OPN5 across vertebrates.

[Biophysical and Biochemical Research of Animal Rhodopsins].

Opsins (also called animal rhodopsins) are universal photoreceptive proteins that provide the molecular basis of visual and nonvisual photoreception in animals, including humans. Opsins consist of seven helical α-transmembrane domains and use retinal, a derivative of vitamin A, as a chromophore. In many opsins, light absorption triggers photo-isomerization from 11-cis retinal to all-trans retinal, resulting in activation via dynamic structural changes in the protein moiety. Activated opsins stimulate cognate trimeric G proteins to induce signal transduction cascades in cells. Recently, molecular and physiological analyses of diverse opsins have progressively advanced. This review introduces the molecular basis and physiological functions of opsins. Based on the functions of opsins, I will discuss the potential of opsins as target molecules to treat and prevent visual and nonvisual diseases such as sleep disorder and depression.

TMT-Opsins differentially modulate medaka brain function in a context-dependent manner.

Vertebrate behavior is strongly influenced by light. Light receptors, encoded by functional opsin proteins, are present inside the vertebrate brain and peripheral tissues. This expression feature is present from fishes to human and appears to be particularly prominent in diurnal vertebrates. Despite their conserved widespread occurrence, the nonvisual functions of opsins are still largely enigmatic. This is even more apparent when considering the high number of opsins. Teleosts possess around 40 opsin genes, present from young developmental stages to adulthood. Many of these opsins have been shown to function as light receptors. This raises the question of whether this large number might mainly reflect functional redundancy or rather maximally enables teleosts to optimally use the complex light information present under water. We focus on tmt-opsin1b and tmt-opsin2, c-opsins with ancestral-type sequence features, conserved across several vertebrate phyla, expressed with partly similar expression in non-rod, non-cone, non-retinal-ganglion-cell brain tissues and with a similar spectral sensitivity. The characterization of the single mutants revealed age- and light-dependent behavioral changes, as well as an impact on the levels of the preprohormone sst1b and the voltage-gated sodium channel subunit scn12aa. The amount of daytime rest is affected independently of the eyes, pineal organ, and circadian clock in tmt-opsin1b mutants. We further focused on daytime behavior and the molecular changes in tmt-opsin1b/2 double mutants, and found that-despite their similar expression and spectral features-these opsins interact in part nonadditively. Specifically, double mutants complement molecular and behavioral phenotypes observed in single mutants in a partly age-dependent fashion. Our work provides a starting point to disentangle the highly complex interactions of vertebrate nonvisual opsins, suggesting that tmt-opsin-expressing cells together with other visual and nonvisual opsins provide detailed light information to the organism for behavioral fine-tuning. This work also provides a stepping stone to unravel how vertebrate species with conserved opsins, but living in different ecological niches, respond to similar light cues and how human-generated artificial light might impact on behavioral processes in natural environments.

Cardiac optogenetics: a decade of enlightenment.

The electromechanical function of the heart involves complex, coordinated activity over time and space. Life-threatening cardiac arrhythmias arise from asynchrony in these space-time events; therefore, therapies for prevention and treatment require fundamental understanding and the ability to visualize, perturb and control cardiac activity. Optogenetics combines optical and molecular biology (genetic) approaches for light-enabled sensing and actuation of electrical activity with unprecedented spatiotemporal resolution and parallelism. The year 2020 marks a decade of developments in cardiac optogenetics since this technology was adopted from neuroscience and applied to the heart. In this Review, we appraise a decade of advances that define near-term (immediate) translation based on all-optical electrophysiology, including high-throughput screening, cardiotoxicity testing and personalized medicine assays, and long-term (aspirational) prospects for clinical translation of cardiac optogenetics, including new optical therapies for rhythm control. The main translational opportunities and challenges for optogenetics to be fully embraced in cardiology are also discussed.

Minimally invasive neural stimulation with a novel ultra-sensitive step function opsin: implications and future directions.

Optogenetics has become a popular tool to probe the link between neural circuits and behavior, since the technique was first introduced in 2005. Recently, Gong et al. (Gong X, Mendoza-Halliday D, Ting JT, Kaiser T, Sun X, Bastos AM, Wimmer RD, Guo B, Chen Q, Zhou Y, Pruner M, Wu CWH, Park D, Deisseroth K, Barak B, Boyden ES, Miller EK, Halassa MM, Fu Z, Bi G, Desimone R, Feng G. Neuron 107: 38-51, 2020) developed an ultra-sensitive step-function opsin capable of activating any region of the mouse brain and cortical areas in macaques with external illumination, thus aiming toward minimally invasive light delivery. In this article, we highlight and discuss the new opsin's potential in nonhuman primate research.

Visual system diversity in coral reef fishes.

Coral reefs are one of the most species rich and colourful habitats on earth and for many coral reef teleosts, vision is central to their survival and reproduction. The diversity of reef fish visual systems arises from variations in ocular and retinal anatomy, neural processing and, perhaps most easily revealed by, the peak spectral absorbance of visual pigments. This review examines the interplay between retinal morphology and light environment across a number of reef fish species, but mainly focusses on visual adaptations at the molecular level (i.e. visual pigment structure). Generally, visual pigments tend to match the overall light environment or micro-habitat, with fish inhabiting greener, inshore waters possessing longer wavelength-shifted visual pigments than open water blue-shifted species. In marine fishes, particularly those that live on the reef, most species have between two (likely dichromatic) to four (possible tetrachromatic) cone spectral sensitivities and a single rod for crepuscular vision; however, most are trichromatic with three spectral sensitivities. In addition to variation in spectral sensitivity number, spectral placement of the absorbance maximum (λmax) also has a surprising degree of variability. Variation in ocular and retinal anatomy is also observed at several levels in reef fishes but is best represented by differences in arrangement, density and distribution of neural cell types across the retina (i.e. retinal topography). Here, we focus on the seven reef fish families most comprehensively studied to date to examine and compare how behaviour, environment, activity period, ontogeny and phylogeny might interact to generate the exceptional diversity in visual system design that we observe.

Functional evolution of vertebrate sensory receptors.

Sensory receptors enable animals to perceive their external world, and functional properties of receptors evolve to detect the specific cues relevant for an organism's survival. Changes in sensory receptor function or tuning can directly impact an organism's behavior. Functional tests of receptors from multiple species and the generation of chimeric receptors between orthologs with different properties allow for the dissection of the molecular basis of receptor function and identification of the key residues that impart functional changes in different species. Knowledge of these functionally important sites facilitates investigation into questions regarding the role of epistasis and the extent of convergence, as well as the timing of sensory shifts relative to other phenotypic changes. However, as receptors can also play roles in non-sensory tissues, and receptor responses can be modulated by numerous other factors including varying expression levels, alternative splicing, and morphological features of the sensory cell, behavioral validation can be instrumental in confirming that responses observed in heterologous systems play a sensory role. Expression profiling of sensory cells and comparative genomics approaches can shed light on cell-type specific modifications and identify other proteins that may affect receptor function and can provide insight into the correlated evolution of complex suites of traits. Here we review the evolutionary history and diversity of functional responses of the major classes of sensory receptors in vertebrates, including opsins, chemosensory receptors, and ion channels involved in temperature-sensing, mechanosensation and electroreception.

Axes of visual adaptation in the ecologically diverse family Cichlidae.

The family Cichlidae contains approximately 2000 species that live in diverse freshwater habitats including murky lakes, turbid rivers, and clear lakes from both the Old and New Worlds. Their visual systems are similarly diverse and have evolved specific sensitivities that differ along several axes of variation. Variation in cornea and lens transmission affect which wavelengths reach the retina. Variation in photoreceptor number and distribution affect brightness sensitivity, spectral sensitivity and resolution. Probably their most dynamic characteristic is the variation in visual pigment peak sensitivities. Visual pigments can be altered through changes in chromophore, opsin sequence and opsin expression. Opsin expression varies by altering which of the seven available cone opsins in their genomes are turned on. These opsins can even be coexpressed to produce seemingly infinitely tunable cone sensitivities. Both chromophore and opsin expression can vary on either rapid (hours or days), slower (seasonal or ontogenetic) or evolutionary timescales. Such visual system shifts have enabled cichlids to adapt to different habitats and foraging styles. Through both short term plasticity and longer evolutionary adaptations, cichlids have proven to be ecologically successful and an excellent model for studying organismal adaptation.

Vision in sharks and rays: Opsin diversity and colour vision.

The visual sense of elasmobranch fishes is poorly studied compared to their bony cousins, the teleosts. Nevertheless, the elasmobranch eye features numerous specialisations that have no doubt facilitated the diversification and evolutionary success of this fascinating taxon. In this review, I highlight recent discoveries on the nature and phylogenetic distribution of visual pigments in sharks and rays. Whereas most rays appear to be cone dichromats, all sharks studied to date are cone monochromats and, as a group, have likely abandoned colour vision on multiple occasions. This situation in sharks mirrors that seen in other large marine predators, the pinnipeds and cetaceans, which leads us to reassess the costs and benefits of multiple cone pigments and wavelength discrimination in the marine environment.

Origin and adaptation of green-sensitive (RH2) pigments in vertebrates.

One of the critical times for the survival of animals is twilight where the most abundant visible lights are between 400 and 550 nanometres (nm). Green-sensitive RH2 pigments help nonmammalian vertebrate species to better discriminate wavelengths in this blue-green region. Here, evaluation of the wavelengths of maximal absorption (λmax s) of genetically engineered RH2 pigments representing 13 critical stages of vertebrate evolution revealed that the RH2 pigment of the most recent common ancestor of vertebrates had a λmax of 503 nm, while the 12 ancestral pigments exhibited an expanded range in λmax s between 474 and 524 nm, and present-day RH2 pigments have further expanded the range to ~ 450-530 nm. During vertebrate evolution, eight out of the 16 significant λmax shifts (or |Δλmax | ≥ 10 nm) of RH2 pigments identified were fully explained by the repeated mutations E122Q (twice), Q122E (thrice) and M207L (twice), and A292S (once). Our data indicated that the highly variable λmax s of teleost RH2 pigments arose from gene duplications followed by accelerated amino acid substitution.

The BioLuminescent-OptoGenetic in vivo response to coelenterazine is proportional, sensitive, and specific in neocortex.

BioLuminescent (BL) light production can modulate neural activity and behavior through co-expressed OptoGenetic (OG) elements, an approach termed "BL-OG." Yet, the relationship between BL-OG effects and bioluminescent photon emission has not been characterized in vivo. Further, the degree to which BL-OG effects strictly depend on optogenetic mechanisms driven by bioluminescent photons is unknown. Crucial to every neuromodulation method is whether the activator shows a dynamic concentration range driving robust, selective, and nontoxic effects. We systematically tested the effects of four key components of the BL-OG mechanism (luciferin, oxidized luciferin, luciferin vehicle, and bioluminescence), and compared these against effects induced by the Luminopsin-3 (LMO3) BL-OG molecule, a fusion of slow burn Gaussia luciferase (sbGLuc) and Volvox ChannelRhodopsin-1 (VChR1). We performed combined bioluminescence imaging and electrophysiological recordings while injecting specific doses of Coelenterazine (substrate for sbGluc), Coelenteramide (CTM, the oxidized product of CTZ), or CTZ vehicle. CTZ robustly drove activity in mice expressing LMO3, with photon production proportional to firing rate. In contrast, low and moderate doses of CTZ, CTM, or vehicle did not modulate activity in mice that did not express LMO3. We also failed to find bioluminescence effects on neural activity in mice expressing an optogenetically nonsensitive LMO3 variant. We observed weak responses to the highest dose of CTZ in control mice, but these effects were significantly smaller than those observed in the LMO3 group. These results show that in neocortex in vivo, there is a large CTZ range wherein BL-OG effects are specific to its active chemogenetic mechanism.

Defining parameters of specificity for bioluminescent optogenetic activation of neurons using in vitro multi electrode arrays (MEA).

In Bioluminescent Optogenetics (BL-OG) a biological, rather than a physical, light source is used to activate light-sensing opsins, such as channelrhodopsins or pumps. This is commonly achieved by utilizing a luminopsin (LMO), a fusion protein of a light-emitting luciferase tethered to a light-sensing opsin. Light of the wavelength matching the activation peak of the opsin is emitted by the luciferase upon application of its small molecule luciferin, resulting in activation of the fused opsin and subsequent effects on membrane potential. Using optimized protocols for culturing, transforming, and testing primary neurons in multi electrode arrays, we systematically defined parameters under which changes in neuronal activity are specific to bioluminescent activation of opsins, rather than due to off-target effects of either the luciferin or its solvent on neurons directly, or on opsins directly. We further tested if there is a direct effect of bioluminescence on neurons. Critical for assuring specific BL-OG effects are testing the concentration and formulation of the luciferin against proper controls, including testing effects of vehicle on LMO expressing and of luciferin on nonLMO expressing targets.

Bioluminescence-driven optogenetic activation of transplanted neural precursor cells improves motor deficits in a Parkinson's disease mouse model.

The need to develop efficient therapies for neurodegenerative diseases is urgent, especially given the increasing percentages of the population living longer, with increasing chances of being afflicted with conditions like Parkinson's disease (PD). A promising curative approach toward PD and other neurodegenerative diseases is the transplantation of stem cells to halt and potentially reverse neuronal degeneration. However, stem cell therapy does not consistently lead to improvement for patients. Using remote stimulation to optogenetically activate transplanted cells, we attempted to improve behavioral outcomes of stem cell transplantation. We generated a neuronal precursor cell line expressing luminopsin 3 (LMO3), a luciferase-channelrhodopsin fusion protein, which responds to the luciferase substrate coelenterazine (CTZ) with emission of blue light that in turn activates the opsin. Neuronal precursor cells were injected bilaterally into the striatum of homozygous aphakia mice, which carry a spontaneous mutation leading to lack of dopaminergic neurons and symptoms of PD. Following transplantation, the cells were stimulated over a period of 10 days by intraventricular injections of CTZ. Mice receiving CTZ demonstrated significantly improved motor skills in a rotarod test compared to mice receiving vehicle. Thus, bioluminescent optogenetic stimulation of transplanted neuronal precursor cells shows promising effects in improving locomotor behavior in the aphakia PD mouse model and encourages further studies to elucidate the mechanisms and long-term outcomes of these beneficial effects.