Timely Questions Emerging in Chronobiology: The Circadian Clock Keeps on Ticking.

Chronobiology investigations have revealed much about cellular and physiological clockworks but we are far from having a complete mechanistic understanding of the physiological and ecological implications. Here we present some unresolved questions in circadian biology research as posed by the editorial staff and guest contributors to the Journal of Circadian Rhythms. This collection of ideas is not meant to be comprehensive but does reveal the breadth of our observations on emerging trends in chronobiology and circadian biology. It is amazing what could be achieved with various expected innovations in technologies, techniques, and mathematical tools that are being developed. We fully expect strengthening mechanistic work will be linked to health care and environmental understandings of circadian function. Now that most clock genes are known, linking these to physiological, metabolic, and developmental traits requires investigations from the single molecule to the terrestrial ecological scales. Real answers are expected for these questions over the next decade. Where are the circadian clocks at a cellular level? How are clocks coupled cellularly to generate organism level outcomes? How do communities of circadian organisms rhythmically interact with each other? In what way does the natural genetic variation in populations sculpt community behaviors? How will methods development for circadian research be used in disparate academic and commercial endeavors? These and other questions make it a very exciting time to be working as a chronobiologist.

Reflections on Several Landmark Advances in Circadian Biology.

Circadian Biology intersects with diverse scientific domains, intricately woven into the fabric of organismal physiology and behavior. The rhythmic orchestration of life by the circadian clock serves as a focal point for researchers across disciplines. This retrospective examination delves into several of the scientific milestones that have fundamentally shaped our contemporary understanding of circadian rhythms. From deciphering the complexities of clock genes at a cellular level to exploring the nuances of coupled oscillators in whole organism responses to stimuli. The field has undergone significant evolution lately guided by genetics approaches. Our exploration here considers key moments in the circadian-research landscape, elucidating the trajectory of this discipline with a keen eye on scientific advancements and paradigm shifts.

Characterization of neural damage and neuroinflammation in Pax6 small-eye mice.

Aniridia is a panocular condition characterized by a partial or complete loss of the iris. It manifests various developmental deficits in both the anterior and posterior segments of the eye, leading to a progressive vision loss. The homeobox gene PAX6 plays an important role in ocular development and mutations of PAX6 have been the main causative factors for aniridia. In this study, we assessed how Pax6-haploinsufficiency affects retinal morphology and vision of Pax6Sey mice using in vivo and ex vivo metrics. We used mice of C57BL/6 and 129S1/Svlmj genetic backgrounds to examine the variable severity of symptoms as reflected in human aniridia patients. Elevated intraocular pressure (IOP) was observed in Pax6Sey mice starting from post-natal day 20 (P20). Correspondingly, visual acuity showed a steady age-dependent decline in Pax6Sey mice, though these phenotypes were less severe in the 129S1/Svlmj mice. Local retinal damage with layer disorganization was assessed at P30 and P80 in the Pax6Sey mice. Interestingly, we also observed a greater number of activated Iba1+ microglia and GFAP + astrocytes in the Pax6Sey mice than in littermate controls, suggesting a possible neuroinflammatory response to Pax6 deficiencies.

Intrinsically photosensitive retinal ganglion cells in glaucoma.

Glaucoma is a group of eye diseases afflicting more than 70 million people worldwide. It is characterized by damage to retinal ganglion cells (RGCs) that ultimately leads to the death of the cells and vision loss. The diversity of RGC types has been appreciated for decades, and studies, including ours, have shown that RGCs degenerate and die in a type-specific manner in rodent models of glaucoma. The type-specific loss of RGCs results in differential damage to visual and non-visual functions. One type of RGC, the intrinsically photosensitive retinal ganglion cell (ipRGC), expressing the photopigment melanopsin, serves a broad array of non-visual responses to light. Since its discovery, six subtypes of ipRGC have been described, each contributing to various image-forming and non-image-forming functions such as circadian photoentrainment, the pupillary light reflex, the photic control of mood and sleep, and visual contrast sensitivity. We recently demonstrated a link between type-specific ipRGC survival and behavioral deficits in a mouse model of chronic ocular hypertension. This review focuses on the type-specific ipRGC degeneration and associated behavioral changes in animal models and glaucoma patients. A better understanding of how glaucomatous insult impacts the ipRGC-based circuits will have broad impacts on improving the treatment of glaucoma-associated non-visual disorders.

Differential effects of experimental glaucoma on intrinsically photosensitive retinal ganglion cells in mice.

Glaucoma is a group of eye diseases characterized by retinal ganglion cell (RGC) loss and optic nerve damage. Studies, including this study, support that RGCs degenerate and die in a type-specific manner following the disease insult. Here we specifically examined one RGC type, the intrinsically photosensitive retinal ganglion cell (ipRGC), and its associated functional deficits in a mouse model of experimental glaucoma. We induced chronic ocular hypertension (OHT) by laser photocoagulation and then characterized the survival of ipRGC subtypes. We found that ipRGCs suffer significant loss, similar to the general RGC population, but ipRGC subtypes are differentially affected following chronic OHT. M4 ipRGCs, which are involved in pattern vision, are susceptible to chronic OHT. Correspondingly, mice with chronic OHT experience reduced contrast sensitivity and visual acuity. By comparison, M1 ipRGCs, which project to the suprachiasmatic nuclei to regulate circadian rhythmicity, exhibit almost no cell loss following chronic OHT. Accordingly, we observed that circadian re-entrainment and circadian rhythmicity are largely not disrupted in OHT mice. Our study demonstrates the link between subtype-specific ipRGC survival and behavioral deficits in glaucomatous mice. These findings provide insight into glaucoma-induced visual behavioral deficits and their underlying mechanisms.

Opsins outside the eye and the skin: a more complex scenario than originally thought for a classical light sensor.

Since the discovery of melanopsin as a retinal non-visual photopigment, opsins have been described in several organs and cells. This distribution is strikingly different from the classical localization of photopigments in light-exposed tissues such as the eyes and the skin. More than 10 years ago, a new paradigm in the field was created as opsins were shown, to detect not only light, but also thermal energy in Drosophila. In agreement with these findings, thermal detection by opsins was also reported in mammalian cells. Considering the presence of opsins in tissues not reached by light, an intriguing question has emerged: What is the role of a classical light-sensor, and more recently appreciated thermo-sensor, in these tissues? To tackle this question, we address in this review the most recent studies in the field, with emphasis in mammals. We provide the present view about the role of opsins in peripheral tissues, aiming to integrate the current knowledge of the presence and function of opsins in organs that are not directly affected by light.

Clinical implications of the melanopsin-based non-image-forming visual system.

Since the discovery of the non-image-forming visual system, tremendous research efforts have been dedicated to understanding its mechanisms and functional roles. Original functions associated with the melanopsin system include the photoentrainment of circadian sleep-wake cycles and the pupillary light reflex. Recent findings, however, suggest a much broader involvement of this system in an array of physiologic responses to light. This newfound insight into the underlying function of the non-image-forming system has revealed the many connections to human pathology and attendant disease states, including seasonal affective disorder, migraine, glaucoma, inherited mitochondrial optic neuropathy, and sleep dysregulation of aging. In this review, the authors discuss in detail the clinical implications of the melanopsin system.

Lack of Melanopsin Is Associated with Extreme Weight Loss in Mice upon Dietary Challenge.

Metabolic disorders have been established as major risk factors for ocular complications and poor vision. However, little is known about the inverse possibility that ocular disease may cause metabolic dysfunction. To test this hypothesis, we assessed the metabolic consequences of a robust dietary challenge in several mouse models suffering from retinal mutations. To this end, mice null for melanopsin (Opn4-/-), the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs), were subjected to five weeks of a ketogenic diet. These mice lost significantly more weight than wild-type controls or mice lacking rod and cone photoreceptors (Pde6brd1/rd1). Although ipRGCs are critical for proper circadian entrainment, and circadian misalignment has been implicated in metabolic pathology, we observed no differences in entrainment between Opn4-/- and control mice. Additionally, we observed no differences in any tested metabolic parameter between these mouse strains. Further studies are required to establish the mechanism giving rise to this dramatic phenotype observed in melanopsin-null mice. We conclude that the causality between ocular disease and metabolic disorders merits further investigation due to the popularity of diets that rely on the induction of a ketogenic state. Our study is a first step toward understanding retinal pathology as a potential cause of metabolic dysfunction.

Measuring and using light in the melanopsin age.

Light is a potent stimulus for regulating circadian, hormonal, and behavioral systems. In addition, light therapy is effective for certain affective disorders, sleep problems, and circadian rhythm disruption. These biological and behavioral effects of light are influenced by a distinct photoreceptor in the eye, melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), in addition to conventional rods and cones. We summarize the neurophysiology of this newly described sensory pathway and consider implications for the measurement, production, and application of light. A new light-measurement strategy taking account of the complex photoreceptive inputs to these non-visual responses is proposed for use by researchers, and simple suggestions for artificial/architectural lighting are provided for regulatory authorities, lighting manufacturers, designers, and engineers.

The role of intrinsically photosensitive retinal ganglion cells in nonimage-forming responses to light.

Light exerts many effects on behavior and physiology. These effects can be characterized as either image-forming or nonimage-forming (NIF) visual processes. Image-forming vision refers to the process of detecting objects and organisms in the environment and distinguishing their physical characteristics, such as size, shape, and direction of motion. NIF vision, in contrast, refers to effects of light that are independent of fine spatiotemporal vision. NIF effects are many and varied, ranging from modulation of basal physiology, such as heart rate and body temperature, to changes in higher functions, such as mood and cognitive performance. In mammals, many NIF effects of light are dependent upon the inner retinal photopigment melanopsin and the cells in which melanopsin is expressed, the intrinsically photosensitive retinal ganglion cells (ipRGCs). The ipRGCs project broadly throughout the brain. Many of these projections terminate in areas known to mediate NIF effects, while others terminate in regions whose link to photoreception remains to be established. Additionally, the presence of ipRGC projections to areas of the brain with no known link to photoreception suggests the existence of additional ipRGC-mediated NIF effects. This review summarizes the known NIF effects of light and the role of melanopsin and ipRGCs in driving these effects, with an eye toward stimulating further investigation of the many and varied effects of light on physiology and behavior.

Light enhances learned fear.

The ability to learn, remember, and respond to emotional events is a powerful survival strategy. However, dysregulated behavioral and physiological responses to these memories are maladaptive. To fully understand learned fear and the pathologies that arise during response malfunction we must reveal the environmental variables that influence learned fear responses. Light, a ubiquitous environmental feature, modulates cognition and anxiety. We hypothesized that light modulates responses to learned fear. Using tone-cued fear conditioning, we found that light enhances behavioral responses to learned fear in C57BL/6J mice. Mice in light freeze more in response to a conditioned cue than mice in darkness. The absence of significant freezing during a 2-wk habituation period and during intertrial intervals indicated that light specifically modulates freezing to the learned acoustic cue rather than the context of the experimental chamber. Repeating our assay in two photoreceptor mutant models, Pde6b(rd1/rd1) and Opn4(-/-) mice, revealed that light-dependent enhancement of conditioned fear is driven primarily by the rods and/or cones. By repeating our protocol with an altered lighting regimen, we found that lighting conditions acutely modulate responses when altered between conditioning and testing. This is manifested either as an enhancement of freezing when light is added during testing or as a depression of freezing when light is removed during testing. Acute enhancement, but not depression, requires both rod/cone- and melanopsin-dependent photoreception. Our results demonstrate a modulation by light of behavioral responses to learned fear.

Postnatal development and functional adaptations of the melanopsin photoreceptive system in the albino mouse retina.

PURPOSE: To study the melanopsin system of the albino CD1 mouse retina during postnatal development. METHODS: Pups were kept under different ambient conditions: light/dark (LD) cycles, constant light (LL), constant darkness (DD), LL followed by LD, and DD followed by LL. Using immunohistochemistry, melanopsin-expressing cells were classified as M1 or M2 according to the location of their somata and dendritic processes and were counted. RESULTS: Under LD cycles an increase in the number of immunoreactive cells was observed within the first week of postnatal development. When mice were maintained in DD, the increase in the number of immunopositive cells detected was significantly higher than that in LD. On the contrary, when mice were exposed to LL within the same period, no increase was detected. To determine whether the effect of LL during the early postnatal period was reversible, the authors studied animals born in LL and subsequently maintained under LD cycles. After 3 days in LD, these animals showed a significant increase in melanopsin cell number. However, after 1 month in LD, the number was similar to that of the LD controls. Surprisingly, when mice born in DD were exposed to LL, no decrease was detected, though the immunostaining was of low intensity. CONCLUSIONS: The amount of melanopsin protein per cell varies, depending on ambient light conditions. Periods of darkness or, more likely, the sequence of light and dark periods occurring under the daily cycles might be necessary for the normal development of the melanopsin system.

Morphology and mosaics of melanopsin-expressing retinal ganglion cell types in mice.

Melanopsin is the photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). Melanopsin immunoreactivity reveals two dendritic plexuses within the inner plexiform layer (IPL) and morphologically heterogeneous retinal ganglion cells. Using enhanced immunohistochemistry, we provide a fuller description of murine cell types expressing melanopsin, their contribution to the plexuses of melanopsin dendrites, and mosaics formed by each type. M1 cells, corresponding to the originally described ganglion-cell photoreceptors, occupy the ganglion cell or inner nuclear layers. Their large, sparsely branched arbors (mean diameter 275 microm) monostratify at the outer limit of the OFF sublayer. M2 cells also have large, monostratified dendritic arbors (mean diameter 310 microm), but ramify in the inner third of the IPL, within the ON sublayer. There are approximately 900 M1 cells and 800 M2 cells per retina; each type comprises roughly 1-2% of all ganglion cells. The cell bodies of M1 cells are slightly smaller than those of M2 cells (mean diameters: 13 microm for M1, 15 microm for M2). Dendritic field overlap is extensive within each type (coverage factors approximately 3.8 for M1 and 4.6 for M2 cells). Rare bistratified cells deploy terminal dendrites within both melanopsin-immunoreactive plexuses. Because these are too sparsely distributed to permit complete retinal tiling, they lack a key feature of true ganglion cell types and may be anomalous hybrids of the M1 and M2 types. Finally, we observed weak melanopsin immunoreactivity in other ganglion cells, mostly with large somata, that may constitute one or more additional types of melanopsin-expressing cells.

Light modulates the melanophore response to alpha-MSH in Xenopus laevis: an analysis of the signal transduction crosstalk mechanisms involved.

Melanin granule (melanosome) dispersion within Xenopus laevis melanophores is evoked either by light or alpha-MSH. We have previously demonstrated that the initial biochemical steps of light and alpha-MSH signaling are distinct, since the increase in cAMP observed in response to alpha-MSH was not seen after light exposure. cAMP concentrations in response to alpha-MSH were significantly lower in cells pre-exposed to light as compared to the levels in dark-adapted melanophores. Here we demonstrate the presence of an adenylyl cyclase (AC) in the Xenopus melanophore, similar to the mammalian type IX which is inhibited by Ca(2+)-calmodulin-activated phosphatase. This finding supports the hypothesis that the cyclase could be negatively modulated by a light-promoted Ca(2+) increase. In fact, the activity of calcineurin PP2B phosphatase was increased by light, which could result in AC IX inhibition, thus decreasing the response to alpha-MSH. St-Ht31, a disrupting agent of protein kinase A (PKA)-anchoring kinase A protein (AKAP) complex totally blocked the melanosome dispersing response to alpha-MSH, but did not impair the photo-response in Xenopus melanophores. Sequence comparison of a melanophore AKAP partial clone with GenBank sequences showed that the anchoring protein was a gravin-like adaptor previously sequenced from Xenopus non-pigmentary tissues. Co-immunoprecipitation of Xenopus AKAP and the catalytic subunit of PKA demonstrated that PKA is associated with AKAP and it is released in the presence of alpha-MSH. We conclude that in X. laevis melanophores, AKAP12 (gravin-like) contains a site for binding the inactive PKA thus compartmentalizing PKA signaling and also possesses binding sites for PKC. Light diminishes alpha-MSH-induced increase of cAMP by increasing calcineurin (PP2B) activity, which in turn inhibits adenylyl cyclase type IX, and/or by activating PKC, which phosphorylates the gravin-like molecule, thus destabilizing its binding to the cell membrane.

A missense variant (P10L) of the melanopsin (OPN4) gene in seasonal affective disorder.

BACKGROUND: Melanopsin, a non-visual photopigment, may play a role in aberrant responses to low winter light levels in Seasonal Affective Disorder (SAD). We hypothesize that functional sequence variation in the melanopsin gene could contribute to increasing the light needed for normal functioning during winter in SAD. METHODS: Associations between alleles, genotypes, and haplotypes of melanopsin in SAD participants (n=130) were performed relative to controls with no history of psychopathology (n=90). RESULTS: SAD participants had a higher frequency of the homozygous minor genotype (T/T) for the missense variant rs2675703 (P10L) than controls, compared to the combined frequencies of C/C and C/T. Individuals with the T/T genotype were 5.6 times more likely to be in the SAD group than the control group, and all 7 (5%) of individuals with the T/T genotype at P10L were in the SAD group. LIMITATIONS: The study examined only one molecular component of the non-visual light input pathway, and recruitment methods for the comparison groups differed. CONCLUSION: These findings support the hypothesis that melanopsin variants may predispose some individuals to SAD. Characterizing the genetic basis for deficits in the non-visual light input pathway has the potential to define mechanisms underlying the pathological response to light in SAD, which may improve treatment.

Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms.

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.

Meeting report: the role of environmental lighting and circadian disruption in cancer and other diseases.

Light, including artificial light, has a range of effects on human physiology and behavior and can therefore alter human physiology when inappropriately timed. One example of potential light-induced disruption is the effect of light on circadian organization, including the production of several hormone rhythms. Changes in light-dark exposure (e.g., by nonday occupation or transmeridian travel) shift the timing of the circadian system such that internal rhythms can become desynchronized from both the external environment and internally with each other, impairing our ability to sleep and wake at the appropriate times and compromising physiologic and metabolic processes. Light can also have direct acute effects on neuroendocrine systems, for example, in suppressing melatonin synthesis or elevating cortisol production that may have untoward long-term consequences. For these reasons, the National Institute of Environmental Health Sciences convened a workshop of a diverse group of scientists to consider how best to conduct research on possible connections between lighting and health. According to the participants in the workshop, there are three broad areas of research effort that need to be addressed. First are the basic biophysical and molecular genetic mechanisms for phototransduction for circadian, neuroendocrine, and neurobehavioral regulation. Second are the possible physiologic consequences of disrupting these circadian regulatory processes such as on hormone production, particularly melatonin, and normal and neoplastic tissue growth dynamics. Third are effects of light-induced physiologic disruption on disease occurrence and prognosis, and how prevention and treatment could be improved by application of this knowledge.