Pupillary reflex and behavioral masking responses to light as functional measures of retinal degeneration in mice.

BACKGROUND: Pre-clinical testing of retinal pathology and treatment efficacy depends on reliable and valid measures of retinal function. The electroretinogram (ERG) and tests of visual acuity are the ideal standard, but can be unmeasurable while useful vision remains. Non-image-forming responses to light such as the pupillary light reflex (PLR) are attractive surrogates. However, it is not clear how accurately such responses reflect changes in visual capability in specific disease models. The purpose of this study was to test whether measures of non-visual responses to light correlate with previously determined visual function in two photoreceptor degenerations. METHODS: The sensitivity of masking behavior (light induced changes in running wheel activity) and the PLR were measured in 3-month-old wild-type mice (WT) with intact inner retinal circuitry, Pde6b-rd1/rd1 mice (rd1) with early and rapid loss of rods and cones, and Prph2-Rd2/Rd2 mice (Rd2) with a slower progressive loss of rods and cones. RESULTS: In rd1 mice, negative masking had increased sensitivity, positive masking was absent, and the sensitivity of the PLR was severely reduced. In Rd2 mice, positive masking identified useful vision at higher light levels, but there was a limited decrease in the irradiance sensitivity of negative masking and the PLR, and the amplitude of change for both underestimated the reduction in irradiance sensitivity of image-forming vision. CONCLUSIONS: Together these data show that in a given disease, two responses to light can be affected in opposite ways, and that for a given response to light, the change in the response does not accurately represent the degree of pathology. However, the extent of the deficit in the PLR means that even a limited rescue of rod/cone function might be measured by increased PLR amplitude. In addition, positive masking has the potential to measure effective treatment in both models by restoring responses or shifting thresholds to lower irradiances.

The Efemp1R345W Macular Dystrophy Mutation Causes Amplified Circadian and Photophobic Responses to Light in Mice.

Purpose: The R345W mutation in EFEMP1 causes malattia leventinese, an autosomal dominant eye disease with pathogenesis similar to an early-onset age-related macular degeneration. In mice, Efemp1R345W does not cause detectable degeneration but small subretinal deposits do accumulate. The purpose of this study was to determine whether there were abnormal responses to light at this presymptomatic stage in Efemp1R345W mice. Methods: Responses to light were assessed by visual water task, circadian phase shifting, and negative masking behavior. The mechanism of abnormal responses was investigated by anterior eye exam, electroretinogram, melanopsin cell quantification, and multielectrode recording of retinal ganglion cell activity. Results: Visual acuity was not different in Efemp1R345W mice. However, amplitudes of circadian phase shifting (P = 0.016) and negative masking (P < 0.0001) were increased in Efemp1R345W mice. This phenotype was not explained by anterior eye defects or amplified outer retina responses. Instead, we identified increased melanopsin-generated responses to light in the ganglion cell layer of the retina (P < 0.01). Conclusions: Efemp1R345W increases the sensitivity to light of behavioral responses driven by detection of irradiance. An amplified response to light in melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) is consistent with this phenotype. The major concern with this effect of the malattia leventinese mutation is the potential for abnormal regulation of physiology by light to negatively affect health.

Assessing the quality of the ecological component of English Environmental Statements.

Environmental Impact Assessment (EIA) is a key tool to help ensure sustainable built development in more than 200 countries worldwide. Ecology is frequently a component of EIA and early reviews of Ecological Impact Assessment (EcIA) chapters identified scope for improvement at almost every stage of the EcIA process, regardless of country. However, there have been no reviews of UK EcIA chapters since 2000, despite important changes in biodiversity and planning legislation, policy and guidance. In addition, no UK EcIA chapter reviews have attempted to assign a grade or score to EcIA chapters (as has been done for reviews of US, Finnish and Indian EcIA chapters). Furthermore, no EcIA chapter reviews have attempted to use a scoring system to identify which variables determine EcIA chapter information content, beyond straightforward comparisons of EcIA chapters before and after the introduction of guidelines. A variant of the Biodiversity Assessment Index (BAI) was used to assign scores between zero and one to EcIA chapters based on a series of 47 questions drawn from EU legislation and professional guidance. 112 EcIA chapters for proposed developments that were subsequently granted planning permission in England were assessed. The mean BAI score was less than 0.5, indicating the presence of considerable information gaps in the majority of EcIA chapters. Of 13 predictor variables identified as having the potential to affect EcIA chapter quality, 10 were identified as significantly related to the BAI scores. A backward stepwise Generalized Linear Model identified the use of professional guidance, the ecological consultancy type and the length of the EcIA chapter as having the greatest combined explanatory power. As a result, several recommendations are made to help improve future EcIA chapter content, including formal EcIA chapter review, publicising the professional guidance to consultants, the provision of training and the introduction of an accreditation scheme for consultants involved in EcIA This approach could be replicated in other countries that conduct EIA. Context-dependent EcIA chapter review criteria (as in this paper) would help to identify targeted recommendations for improvement. Alternatively, a global set of review criteria could highlight areas of best practice that could then be exported to other countries.

Comparison of drusen and modifying genes in autosomal dominant radial drusen and age-related macular degeneration.

BACKGROUND: Autosomal dominant radial drusen (ADRD), also termed Malattia Leventinese and Doyne honeycomb retinal dystrophy, causes early-onset vision loss because of mutation in EFEMP1. Drusen in an exceedingly rare ADRD human donor eye was compared with eyes affected with age-related macular degeneration (AMD). This study also elucidated whether variations in high-risk AMD genotypes modify phenotypic severity of ADRD. METHODS: Morphologic and histochemical analyses of drusen in one ADRD donor and seven AMD donors. Evaluation of complement factor H (CFH) and ARMS2/HTRA1 alleles in a cohort of 25 subjects with ADRD. RESULTS: Autosomal dominant radial drusen had unique onion skin-like lamination but otherwise shared many compositional features with hard, nodular drusen and/or diffuse soft drusen with basal deposits. Autosomal dominant radial drusen also possessed collagen type IV, an extracellular matrix protein that is absent in age-related drusen. Antibodies directed against the membrane attack complex showed robust labeling of ADRD. Vitronectin and amyloid P were present in drusen of both types. High-risk alleles in the CFH and ARMS2/HTRA1 genes were not associated with increasing ADRD severity. CONCLUSION: Drusen from ADRD and AMD exhibit overlap of some major constituents, but ADRD exhibit distinct alterations in the extracellular matrix that are absent in AMD.

Photophobia and abnormally sustained pupil responses in a mouse model of bradyopsia.

PURPOSE: Mutations in the RGS9 gene cause the visual disorder bradyopsia, which includes difficulty adapting to changes in light and photophobia. The purpose of this study was to determine whether lack of Rgs9 also caused photophobia-like behavior in Rgs9 knockout (Rgs9-/-) mice and to identify useful diagnostic measures of Rgs9 dysfunction. METHODS: We measured two behavioral responses to light and the pupillary light reflex to determine the form and basis of photophobia in Rgs9-/- mice. RESULTS: Rgs9-/- mice spent less time than wild-type mice in both dim and bright light. The mice also showed increased sensitivity to light in negative masking behavior, with a half maximal response at 0.08 lux (0.01 µW·cm(-2)) in Rgs9-/- mice compared to 5.0 lux (0.85 µW·cm(-2)) in wild-type mice. These behaviors were not due to increased anxiety or increased pupil size causing more light to enter the eye. Rather, constriction of the pupil showed that Rgs9-/- mice had an abnormally sustained response to light across multiple irradiance measurement pathways. CONCLUSIONS: Rgs9-/- mice recapitulate a photophobia phenotype of bradyopsia, and the pupil light reflex identifies a simple means to screen for irradiance measurement abnormalities in bradyopsia and potentially other genetic disorders involving photophobia.

RNA interference-based therapy for spinocerebellar ataxia type 7 retinal degeneration.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disease characterized by loss of motor coordination and retinal degeneration with no current therapies in the clinic. The causative mutation is an expanded CAG repeat in the ataxin-7 gene whose mutant protein product causes cerebellar and brainstem degeneration and retinal cone-rod dystrophy. Here, we reduced the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina by RNA interference and evaluated retinal function 23 weeks post injection. We observed a preservation of normal retinal function and no adverse toxicity with ≥50% reduction of mutant and wildtype ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 retinal therapy.

Photoreceptor cells with profound structural deficits can support useful vision in mice.

PURPOSE: In animal models of degenerative photoreceptor disease, there has been some success in restoring photoreception by transplanting stem cell-derived photoreceptor cells into the subretinal space. However, only a small proportion of transplanted cells develop extended outer segments, considered critical for photoreceptor cell function. The purpose of this study was to determine whether photoreceptor cells that lack a fully formed outer segment could usefully contribute to vision. METHODS: Retinal and visual function was tested in wild-type and Rds mice at 90 days of age (Rds(P90)). Photoreceptor cells of mice homozygous for the Rds mutation in peripherin 2 never develop a fully formed outer segment. The electroretinogram and multielectrode recording of retinal ganglion cells were used to test retinal responses to light. Three distinct visual behaviors were used to assess visual capabilities: the optokinetic tracking response, the discrimination-based visual water task, and a measure of the effect of vision on wheel running. RESULTS: Rds(P90) mice had reduced but measurable electroretinogram responses to light, and exhibited light-evoked responses in multiple types of retinal ganglion cells, the output neurons of the retina. In optokinetic and discrimination-based tests, acuity was measurable but reduced, most notably when contrast was decreased. The wheel running test showed that Rds(P90) mice needed 3 log units brighter luminance than wild type to support useful vision (10 cd/m(2)). CONCLUSIONS: Photoreceptors that lack fully formed outer segments can support useful vision. This challenges the idea that normal cellular structure needs to be completely reproduced for transplanted cells to contribute to useful vision.

Ecological mitigation measures in English Environmental Impact Assessment.

Built development is one of the main drivers of biodiversity loss in the UK. Major built developments usually require an Environmental Impact Assessment (EIA) to be conducted, which frequently includes an Ecological Impact Assessment (EcIA) chapter. By identifying the flaws in EcIA mitigation measure proposals and their implementation in completed developments, it may be possible to develop measures to reduce biodiversity loss and help meet the UK's EU obligation to halt biodiversity loss by 2020. A review of 112 English EcIAs from 2000 onwards was conducted to provide a broad-scale overview of the information provision and detail of ecological mitigation measures. Audits of seven EIA development case study sites provided finer-scale detail of mitigation measure implementation, and the effectiveness of their grassland and marginal habitat creation and management measures was assessed using standard NVC methodology. Despite higher than expected levels of mitigation measure implementation in completed developments, EcIA mitigation proposal information and detail has seen little improvement since a 1997 review, and the effectiveness of the habitat mitigation measures studied was poor. This suggests that measures to improve ecological mitigation measures are best targeted at ecological consultants. A recommendation for EcIA-specific training of Competent Authorities is also made.

Ultraviolet light provides a major input to non-image-forming light detection in mice.

The change in irradiance at dawn and dusk provides the primary cue for the entrainment of the mammalian circadian pacemaker. Irradiance detection has been ascribed largely to melanopsin-based phototransduction [1-5]. Here we examine the role of ultraviolet-sensitive (UVS) cones in the modulation of circadian behavior, sleep, and suprachiasmatic nucleus (SCN) electrical activity. UV light exposure leads to phase-shifting responses comparable to those of white light. Moreover, UV light exposure induces sleep in wild-type and melanopsin-deficient (Opn4(-/-)) mice with equal efficacy. Electrical recordings from the SCN of wild-type mice show that UV light elicits irradiance-dependent sustained responses that are similar to those induced by white light, with characteristic fast transient components occurring at the light transitions. These responses are retained in Opn4(-/-) mice and preserved under saturating photopic conditions. The sensitivity of phase-shifting responses to UV light is unaffected by the loss of rods but is severely attenuated by the additional loss of cones. Our data show that UVS cones play an important role in circadian and sleep regulation in mice.

Different inner retinal pathways mediate rod-cone input in irradiance detection for the pupillary light reflex and regulation of behavioral state in mice.

PURPOSE: Detection of light in the eye contributes both to spatial awareness (form vision) and to responses that acclimate an animal to gross changes in light (irradiance detection). This dual role means that eye disease that disrupts form vision can also adversely affect physiology and behavioral state. The purpose of this study was to investigate how inner retinal circuitry mediating rod-cone photoreceptor input contributes to functionally distinct irradiance responses and whether that might account for phenotypic diversity in retinal disease. METHODS: The sensitivity of the pupillary light reflex and negative masking (activity suppression by light) was measured in wild-type mice with intact inner retinal circuitry, Nob4 mice that lack ON-bipolar cell function, and rd1 mice that lack rods and cones and, therefore, have no input to ON or OFF bipolar cells. RESULTS: An expected increase in sensitivity to negative masking with loss of photoreceptor input in rd1 was duplicated in Nob4 mice. In contrast, sensitivity of the pupillary light reflex was more severely reduced in rd1 than in Nob4 mice. CONCLUSIONS: Absence of ON-bipolar cell-mediated rod-cone input can fully explain the phenotype of outer retina degeneration for negative masking but not for the pupillary light reflex. Therefore, inner retinal pathways mediating rod-cone input are different for negative masking and the pupillary light reflex.

Light aversion in mice depends on nonimage-forming irradiance detection.

Detection of light in the eye underlies image-forming vision, but also regulates adaptive responses in physiology and behavior. Typically these adaptive responses do not involve image-forming vision, but depend on a relatively absolute measure of brightness (nonimage-forming irradiance detection). The goal of this study was to further understand how image-forming vision and nonimage-forming irradiance detection contribute to the effects of light on behavior. Three light dependent behaviors were assessed in wild-type, Rpe65-/- and rd1 mice. In Rpe65-/- mice, nonimage-forming irradiance detection is severely attenuated, but rod based visual acuity is relatively preserved. In rd1 mice visual acuity is nonrecordable, but nonimage-forming responses are less severely attenuated than Rpe65-/-. Positive masking, an image-forming vision dependent increase in wheel running, was absent in rd1 and restricted to higher irradiances in Rpe65-/-. Negative masking, a suppression of wheel running sensitivity with nonimage-forming irradiance detection input, was increased in rd1, but reduced in Rpe65-/- mice. By contrast, light aversion, an avoidance of brightly lit areas, was abolished in both Rpe65-/- and rd1. This shows that image-forming vision is not sufficient for light aversion, suggesting nonimage-forming irradiance detection motivates this behavior. Further, the differing effects of disease suggest that negative masking and light aversion are distinct responses with specialized nonimage-forming irradiance detection pathways.

Reduced frequency of known mutations in a cohort of LHON patients from India.

BACKGROUND: Three mitochondrial mutations account for 95% of Leber's hereditary optic neuropathy (LHON) in the European population: G3640A, G11778A and T14484C. The purpose of the study was to investigate the frequency of these mitochondrial DNA mutations in LHON patients from a South Indian population. METHODS: LHON was diagnosed by inheritance pattern, ophthalmologic examination, and by exclusion of non-LHON forms of optic neuropathy. Ninety unrelated LHON patients and 20 at-risk family members (5 with LHON and 15 without LHON) underwent molecular screening for the mitochondrial DNA mutations G3640A, G11778A and T14484C by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR). Positive results were confirmed with bi-directional sequencing. RESULTS: The G11778A mutation was detected in 8 of 90 (8.9%) LHON families. The T14484 mutation was detected in 3 of 90 (3.3%) LHON families. No instances of the G3460A mutation were detected. Other variants were incidentally detected by the DNA sequencing assay. CONCLUSIONS: Three mitochondrial mutations (G3640A, G11778A and T14484C) account for the vast majority of LHON cases in Europe. However, these mutations were detected in only 11 (12%) of 90 LHON families from Southern India in our study. These results suggest that a different set of LHON-causing mutations is present in the South Indian population than in the European population. Further study of subjects with LHON from India may lead to the discovery of novel disease-causing mutations and/or genes.

Effects of hereditary retinal degeneration due to a CEP290 mutation on the feline pupillary light reflex.

OBJECTIVE: To understand how progressive rod cone degeneration due to a mutation in CEP290 affects the pupillary light reflex (PLR) in domestic cats. ANIMALS STUDIED: Domestic cats identified as either normal wildtype (WT; n = 6), or homozygous for the rdAc mutation in CEP290 and having early stage retinal degeneration (stage 2, S2; n = 4), or advanced retinal degeneration (S4; n = 6). METHODS: The effect of light on pupil size was measured over a series of 10-s pulses of white and chromatic light in cats lightly sedated with medetomidine. RESULTS: In WT cats, the PLR was characterized by a pronounced initial constriction that rapidly re-dilated during the stimulus (pupil escape), to a stable or sustained constriction. There was then a marked constriction at stimulus offset. Each component of the PLR was retained in affected cats, but with progressively reduced irradiance sensitivity from early to advanced retinal disease. CONCLUSIONS: The PLR of cats had multiple phases, with a remarkably high-amplitude 'paradoxical' off-constriction even in the absence of retinal disease. In rdAc cats, reduced irradiance sensitivity was consistent with progressive loss of rod and cone function. Based on previously characterized retinal pathology, this suggests the visual streak of the retina has a proportionally large contribution to PLR input. These findings support the hypothesis that the efficacy of planned therapeutic trials can be determined by careful evaluation of the PLR in cats.

Mutations that are a common cause of Leber congenital amaurosis in northern America are rare in southern India.

PURPOSE: To test patients from southern India for the presence of mutations that most commonly cause Leber congenital amaurosis (LCA) in northern America. METHODS: A review of the literature identified 177 unique LCA causing mutations in eight different genes: aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), crumbs homolog 1 (CRB1), cone-rod homeobox (CRX), guanylate cyclase 2D (GUCY2D), nephronophthisis 6 (NPHP6), retinol dehydrogenase 12 (RDH12), retinal pigment epithelium-specific protein 65 kDa (RPE65), and retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1). Allele-specific ligation assay and bidirectional sequencing were used to test 38 unrelated LCA patients from southern India for 104 of these mutations, which contribute to more than 30% of the LCA cases in a northern American population. RESULTS: Only one participant was found to harbor one of the 104 mutations in the allele-specific assay (homozygous RPE65 Tyr368His). A mutation that was not part of the assay (homozygous RPE65 Tyr143Asp) was incidentally detected in a second patient when an equivocal signal from one allele on the assay was followed up with automated DNA sequencing. CONCLUSIONS: Mutations that contribute to 30% of the LCA cases in northern America were detected in only 2.6% of LCA cases in our cohort from southern India. There were no instances of IVS26 c.2991+1655 A>G in NPHP6, the most commonly detected mutation in LCA. These data suggest that LCA in India is caused primarily by a different set of mutations in the same genes associated with disease in northern America, or by mutations in other genes that have not yet been discovered. Therefore, mutation-specific assays developed for European and northern American cohorts may not be suited for testing LCA patients from India or other ethnically distinct populations.

The effects of rod and cone loss on the photic regulation of locomotor activity and heart rate.

Behavioral responses to light indirectly affect cardiovascular output, but in anesthetized rodents a direct effect of light on heart rate has also been described. Both the basis for this response and the contribution of rods, cones and melanopsin-based photosensitive retinal ganglion cells (pRGCs) remains unknown. To understand how light acutely regulates heart rate we studied responses to light in mice lacking all rod and cone photoreceptors (rd/rd cl ) along with wild-type controls. Our initial experiments delivered light to anesthetized mice at Zeitgeber time (ZT)16 (4 h after lights off, mid-activity phase) and produced an increase in heart rate in wild-type mice, but not in rd/rd cl animals. By contrast, parallel experiments in freely-moving mice demonstrated that light exposure at this time suppressed heart rate and activity in both genotypes. Because of the effects of anesthesia, all subsequent studies were conducted in freely-moving animals. The effects of light were also assessed at ZT6 (mid-rest phase). At this timepoint, wild-type mice showed an irradiance-dependent increase in heart rate and activity. By contrast, rd/rd cl mice failed to show any modulation of heart rate or activity, even at very high irradiances. Increases in heart rate preceded increases in locomotor activity and remained elevated when locomotor activity ceased, suggesting that these two responses are at least partially uncoupled. Collectively, our results show an acute and phase-dependent effect of light on cardiovascular output in mice. Surprisingly, this irradiance detection response is dependent upon rod and cone photoreceptors, with no apparent contribution from melanopsin pRGCs.

Divergent phenotypes of vision and accessory visual function in mice with visual cycle dysfunction (Rpe65 rd12) or retinal degeneration (rd/rd).

PURPOSE: Tests of vision for mice remain limited and the visual phenotype of some retinal disorders in mice remain poorly understood. A novel assay of vision was used to determine how the form and extent of retinal disease affects visual phenotype in mice. METHODS: Retinal histology, the suppression of locomotion by light and visual guidance of locomotion, were assessed in mice with progressive photoreceptor degeneration (rd/rd) or visual cycle dysfunction (Rpe65 rd12). RESULTS: In wild-type mice, there was visual guidance of locomotor activity in dim light and suppression of activity (negative masking) in bright light. In rd/rd mice, vision was sufficient to guide locomotion at postnatal day (P)34 but was lost from P46 onward. In bright light rd/rd mice had enhanced negative masking. Although Rpe65 rd12 mice had no dim light response, with high illumination, vision was sufficient to guide locomotion at all ages tested. CONCLUSIONS: A major concern for gene and cell replacement therapies is the development of visual pathways through which restored retinal function can connect to visual centers of the brain. The residual retinal response to high illumination in Rpe65 rd12 mice translates into useful vision, and visual pathways remain functional--a prerequisite for restoring vision in disorders of the retina. Similarly, useful vision in young rd/rd mice shows that there is visual pathway function before photoreceptor degeneration and suggests the potential for early therapy. Together, these findings recommend observation of masking responses in the assessment of gene and cell replacement therapies.

Negative and positive masking responses to light in retinal degenerate slow (rds/rds) mice during aging.

Bright light suppresses locomotor activity in mice (negative masking) but dim light augments activity (positive masking). Retinal degeneration slow mice (rds/rds) were tested for responses to light at 3 months, 1 and 2 years old. The suppressive effect of light increased between 1 and 2 years, but the positive response to dim lights was severely reduced at 1 year. No such effects occurred in aging wildtypes. The results indicate that enhancement of negative masking depends on the degree of degeneration of the classical photoreceptors, and that residual function in photoreceptors lacking outer segments is initially sufficient for positive masking.

Classical and melanopsin photoreception in irradiance detection: negative masking of locomotor activity by light.

Studies in mice lacking either classical or melanopsin photoreception have been useful in describing the photoreceptor contribution to irradiance detection in accessory visual responses. However, application of these findings to irradiance detection in intact animals is problematical because retinal degeneration or manipulation can induce secondary changes in the retina. Among responses dependent on irradiance detection, the suppression of activity by light (negative masking) has had limited study. To further understand the function of classical and melanopsin photoreceptors we studied irradiance and spectral sensitivity of masking by light, primarily in mice with intact retinae. The sensitivity of negative masking was equivalent for medium ( approximately 500 nm) and short wavelengths ( approximately 365 nm) in three strains of wild-type mice, identifying a marked short-wavelength-sensitive-cone input. At medium wavelengths, spectral sensitivity above 500 nm had closest fit to the nomogram for the medium-wavelength-sensitive-cone, but a combined input of cone and melanopsin photoreceptors in wild-type mice seems likely. Under white light a decompression of the irradiance range of masking in C3H rd/rd cl mice, lacking rods and cones, identified a functional deficiency presumably resulting from the absence of classical photoreceptor input. Together the evidence demonstrates a pronounced and sustained classical photoreceptor input to irradiance detection for negative masking, and suggests one role of classical photoreceptor input is to constrain dynamic range.

The acute light-induction of sleep is mediated by OPN4-based photoreception.

Sleep is regulated by both homeostatic and circadian mechanisms. The latter, termed 'process c', helps synchronize sleep-wake patterns to the appropriate time of the day. However, in the absence of a circadian clock, overall sleep-wake rhythmicity is preserved and remains synchronized to the external light-dark cycle, indicating that there is an additional, clock-independent photic input to sleep. We found that the direct photic regulation of sleep in mice is predominantly mediated by melanopsin (OPN4)-based photoreception of photosensitive retinal ganglion cells (pRGCs). Moreover, OPN4-dependent sleep regulation was correlated with the activation of sleep-promoting neurons in the ventrolateral preoptic area and the superior colliculus. Collectively, our findings describe a previously unknown pathway in sleep regulation and identify the pRGC/OPN4 signaling system as a potentially new pharmacological target for the selective manipulation of sleep and arousal states.