Functional integrity of visual coding following advanced photoreceptor degeneration.

Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises hope for vision restoration treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons. The success of these approaches is dependent on the capacity of surviving circuits of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodeling. To determine whether retinally degenerate animals possess this capacity, we generated a transgenic mouse model expressing the optogenetic actuator ReaChR in ON bipolar cells (second-order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR-derived responses with photoreceptor-driven responses in wild-type (WT) mice at the level of retinal ganglion cells and the visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to the WT. ReaChR rd1 responses had high trial-to-trial reproducibility and showed sensitivity normalization to code contrast across background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variations in response polarity, contrast sensitivity, and temporal frequency tuning as the WT. Units from the WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system.

Optogenetics for visual restoration: From proof of principle to translational challenges.

Degenerative retinal disorders are a diverse family of diseases commonly leading to irreversible photoreceptor death, while leaving the inner retina relatively intact. Over recent years, innovative gene replacement therapies aiming to halt the progression of certain inherited retinal disorders have made their way into clinics. By rendering surviving retinal neurons light sensitive optogenetic gene therapy now offers a feasible treatment option that can restore lost vision, even in late disease stages and widely independent of the underlying cause of degeneration. Since proof-of-concept almost fifteen years ago, this field has rapidly evolved and a detailed first report on a treated patient has recently been published. In this article, we provide a review of optogenetic approaches for vision restoration. We discuss the currently available optogenetic tools and their relative advantages and disadvantages. Possible cellular targets will be discussed and we will address the question how retinal remodelling may affect the choice of the target and to what extent it may limit the outcomes of optogenetic vision restoration. Finally, we will analyse the evidence for and against optogenetic tool mediated toxicity and will discuss the challenges associated with clinical translation of this promising therapeutic concept.

A systematic comparison of optogenetic approaches to visual restoration.

During inherited retinal degenerations (IRDs), vision is lost due to photoreceptor cell death; however, a range of optogenetic tools have been shown to restore light responses in animal models. Restored response characteristics vary between tools and the neuronal cell population to which they are delivered: the interplay between these is complex, but targeting upstream neurons (such as retinal bipolar cells) may provide functional benefit by retaining intraretinal signal processing. In this study, our aim was to compare two optogenetic tools: mammalian melanopsin (hOPN4) and microbial red-shifted channelrhodopsin (ReaChR) expressed within two subpopulations of surviving cells in a degenerate retina. Intravitreal adeno-associated viral vectors and mouse models utilising the Cre/lox system restricted expression to populations dominated by bipolar cells or retinal ganglion cells and was compared with non-targeted delivery using the chicken beta actin (CBA) promoter. In summary, we found bipolar-targeted optogenetic tools produced faster kinetics and flatter intensity-response relationships compared with non-targeted or retinal-ganglion-cell-targeted hOPN4. Hence, optogenetic tools of both mammalian and microbial origins show advantages when targeted to bipolar cells. This demonstrates the advantage of bipolar-cell-targeted optogenetics for vision restoration in IRDs. We therefore developed a bipolar-cell-specific gene delivery system employing a compressed promoter with the potential for clinical translation.

Zfhx3 modulates retinal sensitivity and circadian responses to light.

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3Sci/+ ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3Sci/+ mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3Sci/+ retina show an increased sensitivity of ipRGC light responses.

Chimeric human opsins as optogenetic light sensitisers.

Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild-type LWS opsin and LWS opsin/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry (ICC) demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.

ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.

PURPOSE: Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. METHODS: Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. RESULTS: A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. CONCLUSION: Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies.

The functional characteristics of optogenetic gene therapy for vision restoration.

Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.

Expression and Localization of Kcne2 in the Vertebrate Retina.

Purpose: To characterize the retinal expression and localization of Kcne2, an ancillary (ß) ion-channel subunit with an important role in fine-tuning cellular excitability. Methods: We analyzed available single-cell transcriptome data from tens of thousands of murine retinal cells for cell-type-specific expression of Kcne2 using state-of-the-art bioinformatics techniques. This evidence at the transcriptome level was complemented with a comprehensive immunohistochemical characterization of mouse retina (C57BL/6, ages 8-12 weeks) employing co-labeling techniques and cell-type-specific antibody markers. We furthermore examined how conserved the Kcne2 localization pattern in the retina was across species by performing immunostaining on zebrafish, cowbird, sheep, mice, and macaque. Results: Kcne2 is distinctly expressed in cone photoreceptors and rod bipolar cells. At a subcellular level, the bulk of Kcne2 immunoreactivity can be observed in the outer plexiform layer. Here, it localizes into cone pedicles and likely the postsynaptic membrane of the rod bipolar cells. Thus, the vast majority of Kcne2 immunoreactivity is observed in a thin band in the outer plexiform layer. In addition to this, faint Kcne2 immunoreactivity can also be observed in cone inner segments and the somata of a small subset of cone ON bipolar cells. Strikingly, the localization of Kcne2 in the outer plexiform layer was preserved among all of the species studied, spanning at least 300 million years of evolution of the vertebrate kingdom. Conclusions: The data we present here suggest an important and specific role for Kcne2 in the highly specialized photoreceptor-bipolar cell synapse.

Defining the impact of melanopsin missense polymorphisms using in vivo functional rescue.

Melanopsin (OPN4) is an opsin photopigment expressed within intrinsically photosensitive retinal ganglion cells (ipRGCs) that mediate non-image forming (NIF) responses to light. Two single-nucleotide polymorphisms (SNPs) in human melanopsin (hOPN4), Pro10Leu and Thr394Ile, have recently been associated with abnormal NIF responses to light, including seasonal affective disorder. It has been suggested these behavioural changes are due to altered melanopsin signalling. However, there is currently no direct evidence to support this. Here we have used ipRGC-specific delivery of hOPN4 wild-type (WT), Pro10Leu or Thr394Ile adeno-associated viruses (AAV) to determine the functional consequences of hOPN4 SNPs on melanopsin-driven light responses and associated behaviours. Immunohistochemistry confirmed hOPN4 AAVs exclusively transduced mouse ipRGCs. Behavioural phenotyping performed before and after AAV injection demonstrated that both hOPN4 Pro10Leu and Thr394Ile could functionally rescue pupillary light responses and circadian photoentrainment in Opn4-/- mice, with no differences in NIF behaviours detected for animals expressing either SNP compared to hOPN4 WT. Multi-electrode array recordings revealed that ipRGCs expressing hOPN4 Thr394Ile exhibit melanopsin-driven light responses with significantly attenuated response amplitude, decreased sensitivity and faster offset kinetics compared to hOPN4 WT. IpRGCs expressing hOpn4 Pro10Leu also showed reduced response amplitude. Collectively these data suggest Thr394Ile and Pro10Leu may be functionally significant SNPs, which result in altered melanopsin signalling. To our knowledge, this study provides the first direct evidence for the effects of hOPN4 polymorphisms on melanopsin-driven light responses and NIF behaviours in vivo, providing further insight into the role of these SNPs in melanopsin function and human physiology.

Functional characterisation of naturally occurring mutations in human melanopsin.

Melanopsin is a blue light-sensitive opsin photopigment involved in a range of non-image forming behaviours, including circadian photoentrainment and the pupil light response. Many naturally occurring genetic variants exist within the human melanopsin gene (OPN4), yet it remains unclear how these variants affect melanopsin protein function and downstream physiological responses to light. Here, we have used bioinformatic analysis and in vitro expression systems to determine the functional phenotypes of missense human OPN4 variants. From 1242 human OPN4 variants collated in the NCBI Short Genetic Variation database (dbSNP), we identified 96 that lead to non-synonymous amino acid substitutions. These 96 missense mutations were screened using sequence alignment and comparative approaches to select 16 potentially deleterious variants for functional characterisation using calcium imaging of melanopsin-driven light responses in HEK293T cells. We identify several previously uncharacterised OPN4 mutations with altered functional properties, including attenuated or abolished light responses, as well as variants demonstrating abnormal response kinetics. These data provide valuable insight into the structure-function relationships of human melanopsin, including several key functional residues of the melanopsin protein. The identification of melanopsin variants with significantly altered function may serve to detect individuals with disrupted melanopsin-based light perception, and potentially highlight those at increased risk of sleep disturbance, circadian dysfunction, and visual abnormalities.

Differential roles for cryptochromes in the mammalian retinal clock.

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.

Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy.

Optogenetic strategies to restore vision in patients who are blind from end-stage retinal degenerations aim to render remaining retinal cells light sensitive once photoreceptors are lost. Here, we assessed long-term functional outcomes following subretinal delivery of the human melanopsin gene (OPN4) in the rd1 mouse model of retinal degeneration using an adeno-associated viral vector. Ectopic expression of OPN4 using a ubiquitous promoter resulted in cellular depolarization and ganglion cell action potential firing. Restoration of the pupil light reflex, behavioral light avoidance, and the ability to perform a task requiring basic image recognition were restored up to 13 mo following injection. These data suggest that melanopsin gene therapy via a subretinal route may be a viable and stable therapeutic option for the treatment of end-stage retinal degeneration in humans.

Expression and localisation of two-pore domain (K2P) background leak potassium ion channels in the mouse retina.

Two-pore domain (K2P) potassium channels perform essential roles in neuronal function. These channels produce background leak type potassium currents that act to regulate resting membrane potential and levels of cellular excitability. 15 different K2P channels have been identified in mammals and these channels perform important roles in a wide number of physiological systems. However, to date there is only limited data available concerning the expression and role of K2P channels in the retina. In this study we conduct the first comprehensive study of K2P channel expression in the retina. Our data show that K2P channels are widely expressed in the mouse retina, with variations in expression detected at different times of day and throughout postnatal development. The highest levels of K2P channel expression are observed for Müller cells (TWIK-1, TASK-3, TRAAK, and TREK-2) and retinal ganglion cells (TASK-1, TREK-1, TWIK-1, TWIK-2 and TWIK-3). These data offer new insight into the channels that regulate the resting membrane potential and electrical activity of retinal cells, and suggests that K2P channels are well placed to act as central regulators of visual signalling pathways. The prominent role of K2P channels in neuroprotection offers novel avenues of research into the treatment of common retinal diseases.

Constant Light Desynchronizes Olfactory versus Object and Visuospatial Recognition Memory Performance.

Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light (rLL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes Period1 and Period2 (Per1 and Per2), as well as the immediate-early gene Fos in the SCN, dorsal hippocampus, and olfactory bulb. Under rLL, rhythmic Per1 and Fos expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, Per1 and Fos expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under rLL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals.SIGNIFICANCE STATEMENT A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod).

Modulation of recognition memory performance by light requires both melanopsin and classical photoreceptors.

Acute light exposure exerts various effects on physiology and behaviour. Although the effects of light on brain network activity in humans are well demonstrated, the effects of light on cognitive performance are inconclusive, with the size, as well as direction, of the effect depending on the nature of the task. Similarly, in nocturnal rodents, bright light can either facilitate or disrupt performance depending on the type of task employed. Crucially, it is unclear whether the effects of light on behavioural performance are mediated via the classical image-forming rods and cones or the melanopsin-expressing photosensitive retinal ganglion cells. Here, we investigate the modulatory effects of light on memory performance in mice using the spontaneous object recognition task. Importantly, we examine which photoreceptors are required to mediate the effects of light on memory performance. By using a cross-over design, we show that object recognition memory is disrupted when the test phase is conducted under a bright light (350 lux), regardless of the light level in the sample phase (10 or 350 lux), demonstrating that exposure to a bright light at the time of test, rather than at the time of encoding, impairs performance. Strikingly, the modulatory effect of light on memory performance is completely abolished in both melanopsin-deficient and rodless-coneless mice. Our findings provide direct evidence that melanopsin-driven and rod/cone-driven photoresponses are integrated in order to mediate the effect of light on memory performance.

Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4-/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4-/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

Characterisation of light responses in the retina of mice lacking principle components of rod, cone and melanopsin phototransduction signalling pathways.

Gnat(-/-), Cnga3(-/-), Opn4(-/-) triple knockout (TKO) mice lack essential components of phototransduction signalling pathways present in rods, cones and photosensitive retinal ganglion cells (pRGCs), and are therefore expected to lack all sensitivity to light. However, a number of studies have shown that light responses persist in these mice. In this study we use multielectrode array (MEA) recordings and light-induced c-fos expression to further characterise the light responses of the TKO retina. Small, but robust electroretinogram type responses are routinely detected during MEA recordings, with properties consistent with rod driven responses. Furthermore, a distinctive pattern of light-induced c-fos expression is evident in the TKO retina, with c-fos expression largely restricted to a small subset of amacrine cells that express disabled-1 (Dab1) but lack expression of glycine transporter-1 (GlyT-1). Collectively these data are consistent with the persistence of a novel light sensing pathway in the TKO retina that originates in rod photoreceptors, potentially a rare subset of rods with distinct functional properties, and which is propagated to an atypical subtype of AII amacrine cells. Furthermore, the minimal responses observed following UV light stimulation suggest only a limited role for the non-visual opsin OPN5 in driving excitatory light responses within the mouse retina.

Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light.

Light plays a critical role in the regulation of numerous aspects of physiology and behaviour, including the entrainment of circadian rhythms and the regulation of sleep. These responses involve melanopsin (OPN4)-expressing photosensitive retinal ganglion cells (pRGCs) in addition to rods and cones. Nocturnal light exposure in rodents has been shown to result in rapid sleep induction, in which melanopsin plays a key role. However, studies have also shown that light exposure can result in elevated corticosterone, a response that is not compatible with sleep. To investigate these contradictory findings and to dissect the relative contribution of pRGCs and rods/cones, we assessed the effects of light of different wavelengths on behaviourally defined sleep. Here, we show that blue light (470 nm) causes behavioural arousal, elevating corticosterone and delaying sleep onset. By contrast, green light (530 nm) produces rapid sleep induction. Compared to wildtype mice, these responses are altered in melanopsin-deficient mice (Opn4-/-), resulting in enhanced sleep in response to blue light but delayed sleep induction in response to green or white light. We go on to show that blue light evokes higher Fos induction in the SCN compared to the sleep-promoting ventrolateral preoptic area (VLPO), whereas green light produced greater responses in the VLPO. Collectively, our data demonstrates that nocturnal light exposure can have either an arousal- or sleep-promoting effect, and that these responses are melanopsin-mediated via different neural pathways with different spectral sensitivities. These findings raise important questions relating to how artificial light may alter behaviour in both the work and domestic setting.

An extended family of novel vertebrate photopigments is widely expressed and displays a diversity of function.

Light affects animal physiology and behavior more than simply through classical visual, image-forming pathways. Nonvisual photoreception regulates numerous biological systems, including circadian entrainment, DNA repair, metabolism, and behavior. However, for the majority of these processes, the photoreceptive molecules involved are unknown. Given the diversity of photophysiological responses, the question arises whether a single photopigment or a greater diversity of proteins within the opsin superfamily detect photic stimuli. Here, a functional genomics approach identified the full complement of photopigments in a highly light-sensitive model vertebrate, the zebrafish (Danio rerio), and characterized their tissue distribution, expression levels, and biochemical properties. The results presented here reveal the presence of 42 distinct genes encoding 10 classical visual photopigments and 32 nonvisual opsins, including 10 novel opsin genes comprising four new pigment classes. Consistent with the presence of light-entrainable circadian oscillators in zebrafish, all adult tissues examined expressed two or more opsins, including several novel opsins. Spectral and electrophysiological analyses of the new opsins demonstrate that they form functional photopigments, each with unique chromophore-binding and wavelength specificities. This study has revealed a remarkable number and diversity of photopigments in zebrafish, the largest number so far discovered for any vertebrate. Found in amphibians, reptiles, birds, and all three mammalian clades, most of these genes are not restricted to teleosts. Therefore, nonvisual light detection is far more complex than initially appreciated, which has significant biological implications in understanding photoreception in vertebrates.