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.

Three different seasonally expressed opsins are present in the brain of the Eared Dove, an opportunist breeder.

Birds living at high latitudes perceive the photoperiod through deep-brain photoreceptors (DBP) located in deep-brain neurons. During long photoperiods the information transmitted by these photoreceptors increases the activity of the hypothalamic-pituitary-gonadal (HPG) axis, leading to gonadal development. The presence of photopigments such as VA-Opsin, Opn4, Opn5 and Opn2 in brain areas implicated in reproductive behaviors has been firmly established in several avian species with seasonal breeding, whereas their existence in opportunistic breeding birds remains unconfirmed. The Eared Dove is an urban and peri-urban dove that breeds throughout the year. Males of this species do not exhibit the typical gonadal regression/recrudescence cycle, thus posing the question of what occurs upstream of the HPG axis. We addressed this issue by first studying the presence of diverse opsins located in DBP in the brains of Eared Dove males and whether these photopigments changed their expression throughout the year. We carried out an immunohistochemistry analysis on three different opsins: Opn2 (rhodopsin), Opn3 and Opn5. Our results demonstrate the discrete neuroanatomical distribution of these opsins in the brain of Eared Dove males and strongly indicate different seasonal expressions. In the anterior region of the hypothalamus, Opn2-positive cells were detected throughout the year. By contrast, Opn5 was found to be strongly and seasonally expressed during winter in the anterior and the hypothalamic region. Opn3 was also found to be significantly and seasonally expressed during winter in the hypothalamic region. We thus demonstrate for the first time that males of the Eared Dove, have three different deep-brain opsin-expressing photoreceptors with differential location/distribution in the anterior and hypothalamic region and differential seasonality. The persistence of Opn2 and the strong seasonal expression of nonvisual photopigments Opn3 and Opn5 in two areas of the avian brain, which are associated with reproduction, could be the primary distinction between seasonal and opportunistic breeders.

Arylalkylamine N-acetyltransferase (AANAT): Blue light induction, nuclear translocation, and potential role in the survival of chicken retina neuronal cells.

In vertebrates, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) is the time-keeping and key regulatory enzyme in melatonin (Mel) biosynthesis. AANAT is present in the pineal gland, retina, and other regions where it is controlled by light, cyclic adenosine monophosphate (cAMP) levels, and the molecular clock. AANAT converts serotonin to N-acetyl serotonin (NAS) and the last enzyme in the pathway, hydroxy-o-methyltransferase (HIOMT), forms Mel by NAS methylation. We have previously shown that AANAT is expressed in chicken retinal ganglion cells (RGCs) during daytime at the level of mRNA and enzyme activity. Here we investigated the presence of AANAT protein and mRNA throughout development in the chicken embryonic retina as well as AANAT expression, phosphorylation, and its sub-cellular localization in primary cultures of retinal neurons from E10 embryonic retinas exposed to blue light (BL) and controls kept in the dark (D). From embryonic days 7-10 (E7-10) AANAT mRNA and protein were visualized mainly concentrated in the forming ganglion cell layer (GCL), while from E17 through postnatal days, expression was detectable all through the different retinal cell layers. At postnatal day 10 (PN10) when animals were subjected to a 12:12 h LD cycle, AANAT was mainly expressed in the GCL and inner nuclear layer cells at noon (Zeitgeber Time (ZT 6)) and in the photoreceptor cell layer at night (ZT 21). Primary cultures of retinal neurons exhibited an induction of AANAT protein when cells were exposed to BL for 1 h as compared with D controls. After BL exposure, AANAT showed a significant change in intracellular localization from the cytoplasm to the nucleus in the BL condition, remaining in the nucleus 1-2 h in the D after BL stimulation. BL induction of nuclear AANAT was substantially inhibited when cultures were treated with the protein synthesis inhibitor cycloheximide (CHD). Furthermore, the phosphorylated form of the enzyme (pAANAT) increased after BL in nuclear fractions obtained from primary cultures as compared with D controls. Finally, the knockdown of AANAT by sh-RNA in primary cultures affected cell viability regardless of the light condition. AANAT knockdown also affected the redox balance, sh-AANAT treated cultures showing higher levels of reactive oxygen species (ROS) than in the sh-control. Our results support the idea that AANAT is a BL-sensing enzyme in the inner retina of diurnal vertebrates, undergoing phosphorylation and nuclear importation in response to BL stimulation. Moreover, it can be inferred that AANAT plays a novel role in nuclear function, cell viability, and, likely, through redox balance regulation.

"Disruption of the molecular clock severely affects lipid metabolism in a hepatocellular carcinoma cell model".

Involved in triglyceride (TG) and glycerophospholipid metabolism, the liver plays a crucial physiological role in the human body both as a major metabolic integrator and a central hub for lipid and energy homeostasis. Metabolic disorders can be caused by various factors that promote abnormal lipid accumulation in storage organelles called lipid droplets (LDs), as in hepatic steatosis, a metabolic syndrome manifestation that can progress to a hepatocellular carcinoma, the most common primary liver malignancy worldwide. Modern life involves conditions that disrupt the biological clock, causing metabolic disorders and higher cancer risk. A circadian clock is present in the liver and in immortalized cell lines and temporally regulates physiological processes by driving transcriptional and metabolic rhythms. Here we investigated metabolic rhythms in HepG2 cells, a human hepatocellular carcinoma-derived cell line, and the link between these rhythms and the circadian clock in control (Bmal1-wildtype) and Bmal1-disrupted (B-D) cells having their molecular clock impaired. Rhythms in the expression of lipid-synthesizing enzymes ChoKα, Pcyt2, and Lipin1, in the metabolism of particular glycerophospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine, and in the phosphatidylcholine/phosphatidylethanolamine ratio and TG and LD content were observed in Bmal1-wildtype cells. By contrast, in the B-D model, the whole hepatic metabolism was severely altered with a significant reduction in the TG and LD content as well as in ChoKα and other related lipid enzymes. Together, our results suggest a very strong crosstalk between the molecular clock and lipid metabolism, which exhibits an exacerbated pathological condition in B-D cells.

Glial cell response to constant low light exposure in rat retina.

To study the macroglia and microglia and the immune role in long-time light exposure in rat eyes, we performed glial cell characterization along the time-course of retinal degeneration induced by chronic exposure to low-intensity light. Animals were exposed to light for periods of 2, 4, 6, or 8 days, and the retinal glial response was evaluated by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. Retinal cells presented an increased expression of the macroglia marker GFAP, as well as increased mRNA levels of microglia markers Iba1 and CD68 after 6 days. Also, at this time-point, we found a higher number of Iba1-positive cells in the outer nuclear layer area; moreover, these cells showed the characteristic activated-microglia morphology. The expression levels of immune mediators TNF, IL-6, and chemokines CX3CR1 and CCL2 were also significantly increased after 6 days. All the events of glial activation occurred after 5-6 days of constant light exposure, when the number of photoreceptor cells has already decreased significantly. Herein, we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that occurs independently of glial-derived immune responses.

The Intrinsic Blue Light Responses of Avian Müller Glial Cells Imply Calcium Release from Internal Stores.

The retina of vertebrates is responsible for capturing light through visual (cones and rods) and non-visual photoreceptors (intrinsically photosensitive retinal ganglion cells and horizontal cells) triggering a number of essential activities associated to image- and non-image forming functions (photic entrainment of daily rhythms, pupillary light reflexes, pineal melatonin inhibition, among others). Although the retina contains diverse types of neuronal based-photoreceptors cells, originally classified as ciliary- or rhabdomeric-like types, in recent years, it has been shown that the major glial cell type of the retina, the Müller glial cells (MC), express blue photopigments as Opn3 (encephalopsin) and Opn5 (neuropsin) and display light responses associated to intracellular Ca2 + mobilization. These findings strongly propose MC as novel retinal photodetectors (Rios et al., 2019). Herein, we further investigated the intrinsic light responses of primary cultures of MC from embryonic chicken retinas specially focused on Ca2 + mobilization by fluorescence imaging and the identity of the internal Ca2 + stores responsible for blue light responses. Results clearly demonstrated that light responses were specific to blue light of long time exposure, and that the main Ca2 + reservoir to trigger downstream responses came from intracellular stores localized in the endoplasmic reticulum These observations bring more complexity to the intrinsic photosensitivity of retinal cells, particularly with regard to the detection of light in the blue range of visible spectra, and add novel functions to glial cells cooperating with other photoreceptors to detect and integrate ambient light in the retinal circuit and participate in cell to cell communication.Summary statement:Non-neuronal cells in the vertebrate retina, Muller glial cells, express non-canonical photopigments and sense blue light causing calcium release from intracellular stores strongly suggesting a novel intrinsic photosensitivity and new regulatory events mediating light-driven processes with yet unknown physiological implications.

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.

Circadian Regulation and Clock-Controlled Mechanisms of Glycerophospholipid Metabolism from Neuronal Cells and Tissues to Fibroblasts.

Along evolution, living organisms developed a precise timekeeping system, circadian clocks, to adapt life to the 24-h light/dark cycle and temporally regulate physiology and behavior. The transcriptional molecular circadian clock and metabolic/redox oscillator conforming these clocks are present in organs, tissues, and even in individual cells, where they exert circadian control over cellular metabolism. Disruption of the molecular clock may cause metabolic disorders and higher cancer risk. The synthesis and degradation of glycerophospholipids (GPLs) is one of the most highly regulated metabolisms across the 24-h cycle in terms of total lipid content and enzyme expression and activity in the nervous system and individual cells. Lipids play a plethora of roles (membrane biogenesis, energy sourcing, signaling, and the regulation of protein-chromatin interaction, among others), making control of their metabolism a vital checkpoint in the cellular organization of physiology. An increasing body of evidence clearly demonstrates an orchestrated and sequential series of events occurring in GPL metabolism across the 24-h day in diverse retinal cell layers, immortalized fibroblasts, and glioma cells. Moreover, the clock gene Per1 and other circadian-related genes are tightly involved in the regulation of GPL synthesis in quiescent cells. However, under proliferation, the metabolic oscillator continues to control GPL metabolism of brain cancer cells even after molecular circadian clock disruption, reflecting the crucial role of the temporal metabolism organization in cell preservation. The aim of this review is to examine the control exerted by circadian clocks over GPL metabolism, their synthesizing enzyme expression and activities in normal and tumorous cells of the nervous system and in immortalized fibroblasts.

Adjusting the Molecular Clock: The Importance of Circadian Rhythms in the Development of Glioblastomas and Its Intervention as a Therapeutic Strategy.

Gliomas are solid tumors of the central nervous system (CNS) that originated from different glial cells. The World Health Organization (WHO) classifies these tumors into four groups (I-IV) with increasing malignancy. Glioblastoma (GBM) is the most common and aggressive type of brain tumor classified as grade IV. GBMs are resistant to conventional therapies with poor prognosis after diagnosis even when the Stupp protocol that combines surgery and radiochemotherapy is applied. Nowadays, few novel therapeutic strategies have been used to improve GBM treatment, looking for higher efficiency and lower side effects, but with relatively modest results. The circadian timing system temporally organizes the physiology and behavior of most organisms and daily regulates several cellular processes in organs, tissues, and even in individual cells, including tumor cells. The potentiality of the function of the circadian clock on cancer cells modulation as a new target for novel treatments with a chronobiological basis offers a different challenge that needs to be considered in further detail. The present review will discuss state of the art regarding GBM biology, the role of the circadian clock in tumor progression, and new chrono-chemotherapeutic strategies applied for GBM treatment.

Temporal regulation of tumor growth in nocturnal mammals: In vivo studies and chemotherapeutical potential.

Tumors of the nervous system including glioblastoma multiforme (GBM) are the most frequent and aggressive form of brain tumors; however, little is known about the impact of the circadian timing system on the formation, growth, and treatment of these tumors. We investigated day/night differences in tumor growth after injection of A530 glioma cells isolated from malignant peripheral nerve sheath tumor (MPNSTs) of NPcis (Trp53+/- ; Nf1+/- ) mice. Synchronized A530 cell cultures expressing typical glial markers were injected at the beginning of the day or night into the sciatic nerve zone of C57BL/6 mice subject to a 12:12 hours light/dark (LD) cycle or after being released to constant darkness (DD). Tumors generated in animals injected early at night in the LD cycle or in DD showed higher growth rates than in animals injected diurnally. No differences were found when animals were injected at the same time with cultures synchronized 12 hours apart. Similar experiments performed with B16 melanoma cells showed higher tumor growth rates in animals injected at the beginning of the night compared to those injected in the daytime. A higher tumor growth rate than that in controls was observed when mice were injected with knocked-down clock gene Bmal1 cells. Finally, when we compared day/night administration of different doses of the proteasome inhibitor Bortezomib (0.5-1.5 mg/kg) in tumor-bearing animals, we found that low-dose chemotherapy displayed higher efficacy when administered at night. Results suggest the existence of a precise temporal control of tumor growth and of drug efficacy in which the host state and susceptibility are critical.

Expression of Non-visual Opsins Opn3 and Opn5 in the Developing Inner Retinal Cells of Birds. Light-Responses in Müller Glial Cells.

The avian retina is composed of different types of photoreceptors responsible for image and non-image forming tasks: the visual photoreceptor cells (cones and rods), the melanopsin-expressing intrinsically photoresponsive retinal ganglion cells (ipRGCs) and horizontal cells. Furthermore, the non-visual opsins Opn3 (encephalopsin/panaopsin) and Opn5 (neuropsin) have been shown to be expressed in the vertebrate inner retina, responding to blue (BL) and UV light, respectively. Here we investigated the expression and localization of Opn3 and Opn5 in the developing chick retina at different embryonic days (E) as well as in primary cultures of retinal Müller glial cells (MCs). Opn3 and Opn5 mRNAs and proteins appeared as early as E10 although traces of Opn3- and Opn5-like proteins were seen earlier by E7 in the forming RGC layer and in glial cells extending throughout the developing nuclear layer. Later on, at postnatal days 1-10 (PN1-10) a significant expression of Opn3 was observed in inner retinal cells and processes in plexiform layers, together with expression of the glial markers glutamine synthetase (GS) and the glial fibrillary acidic protein (GFAP). Opn3 and Opn5 were found to be expressed in primary MC cultures prepared at E8 and kept for 2 weeks. In addition, significant effects of BL exposure on Opn3 expression and subcellular localization were observed in MCs as BL significantly increased its levels and modified its nuclear location when compared with dark controls, through a mechanism dependent on protein synthesis. More importantly, a subpopulation of MCs responded to brief BL pulses by increasing intracellular Ca2+ levels; whereas light-responses were completely abolished with the retinal bleacher hydroxylamine pretreatment. Taken together, our findings show that these two opsins are expressed in inner retinal cells and MCs of the chicken retina at early developmental phases and remain expressed in the mature retina at PN days. In addition, the novel photic responses seen in MCs may suggest another important role for the glia in retinal physiology.

Proliferative Glioblastoma Cancer Cells Exhibit Persisting Temporal Control of Metabolism and Display Differential Temporal Drug Susceptibility in Chemotherapy.

Even in immortalized cell lines, circadian clocks regulate physiological processes in a time-dependent manner, driving transcriptional and metabolic rhythms, the latter being able to persist without transcription. Circadian rhythm disruptions in modern life (shiftwork, jetlag, etc.) may lead to higher cancer risk. Here, we investigated whether the human glioblastoma T98G cells maintained quiescent or under proliferation keep a functional clock and whether cells display differential time responses to bortezomib chemotherapy. In arrested cultures, mRNAs for clock (Per1, Rev-erbα) and glycerophospholipid (GPL)-synthesizing enzyme genes, 32P-GPL labeling, and enzyme activities exhibited circadian rhythmicity; oscillations were also found in the redox state/peroxiredoxin oxidation. In proliferating cells, rhythms of gene expression were lost or their periodicity shortened whereas the redox and GPL metabolisms continued to fluctuate with a similar periodicity as under arrest. Cell viability significantly changed over time after bortezomib treatment; however, this rhythmicity and the redox cycles were altered after Bmal1 knock-down, indicating cross-talk between the transcriptional and the metabolic oscillators. An intrinsic metabolic clock continues to function in proliferating cells, controlling diverse metabolisms and highlighting differential states of tumor suitability for more efficient, time-dependent chemotherapy when the redox state is high and GPL metabolism low.

Identification and characterization of key circadian clock genes of tobacco hairy roots: putative regulatory role in xenobiotic metabolism.

The circadian clock is an endogenous system that allows organisms to daily adapt and optimize their physiology and metabolism. We studied the key circadian clock gene (CCG) orthologs in Nicotiana tabacum seedlings and in hairy root cultures (HRC). Putative genes involved in the metabolism of xenobiotic compounds (MXC) were selected and their expression profiles were also analyzed. Seedlings and HRC displayed similar diurnal variations in the expression profiles for the CCG examined under control conditions (CC). MXC-related genes also showed daily fluctuations with specific peaks of expression. However, when HRC were under phenol treatment (PT), the expression patterns of the clock and MXC-related genes were significantly affected. In 2-week-old HRC, PT downregulated the expression of NtLHY, NtTOC1, and NtPRR9 while NtFKF1 and NtGI genes were upregulated by phenol. In 3-week-old HRC, PT also downregulated the expression of all CCG analyzed and NtTOC1 was the most affected. Following PT, the expression of the MXC-related genes was upregulated or displayed an anti-phasic expression profile compared to the expression under CC. Our studies thus provide a glimpse of the circadian expression of clock genes in tobacco and the use of HRC as a convenient system to study plant responses to xenobiotic stresses.

Non-Visual Photopigments Effects of Constant Light-Emitting Diode Light Exposure on the Inner Retina of Wistar Rats.

The retina is part of the central nervous system specially adapted to capture light photons and transmit this information to the brain through photosensitive retinal cells involved in visual and non-visual activities. However, excessive light exposure may accelerate genetic retinal diseases or induce photoreceptor cell (PRC) death, finally leading to retinal degeneration (RD). Light pollution (LP) caused by the characteristic use of artificial light in modern day life may accelerate degenerative diseases or promote RD and circadian desynchrony. We have developed a working model to study RD mechanisms in a low light environment using light-emitting diode (LED) sources, at constant or long exposure times under LP conditions. The mechanism of PRC death is still not fully understood. Our main goal is to study the biochemical mechanisms of RD. We have previously demonstrated that constant light (LL) exposure to white LED produces a significant reduction in the outer nuclear layer (ONL) by classical PRC death after 7 days of LL exposure. The PRCs showed TUNEL-positive labeling and a caspase-3-independent mechanism of cell death. Here, we investigate whether constant LED exposure affects the inner-retinal organization and structure, cell survival and the expression of photopigments; in particular we look into whether constant LED exposure causes the death of retinal ganglion cells (RGCs), of intrinsically photosensitive RGCs (ipRGCs), or of other inner-retinal cells. Wistar rats exposed to 200 lx of LED for 2 to 8 days (LL 2 and LL 8) were processed for histological and protein. The results show no differences in the number of nucleus or TUNEL positive RGCs nor inner structural damage in any of LL groups studied, indicating that LL exposure affects ONL but does not produce RGC death. However, the photopigments melanopsin (OPN4) and neuropsin (OPN5) expressed in the inner retina were seen to modify their localization and expression during LL exposure. Our findings suggest that constant light during several days produces retinal remodeling and ONL cell death as well as significant changes in opsin expression in the inner nuclear layer.

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR).

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as "visual cycle" involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.

Horizontal cells expressing melanopsin x are novel photoreceptors in the avian inner retina.

In the vertebrate retina, three types of photoreceptors-visual photoreceptor cones and rods and the intrinsically photosensitive retinal ganglion cells (ipRGCs)-converged through evolution to detect light and regulate image- and nonimage-forming activities such as photic entrainment of circadian rhythms, pupillary light reflexes, etc. ipRGCs express the nonvisual photopigment melanopsin (OPN4), encoded by two genes: the Xenopus (Opn4x) and mammalian (Opn4m) orthologs. In the chicken retina, both OPN4 proteins are found in ipRGCs, and Opn4x is also present in retinal horizontal cells (HCs), which connect with visual photoreceptors. Here we investigate the intrinsic photosensitivity and functioning of HCs from primary cultures of embryonic retinas at day 15 by using calcium fluorescent fluo4 imaging, pharmacological inhibitory treatments, and Opn4x knockdown. Results show that HCs are avian photoreceptors with a retinal-based OPN4X photopigment conferring intrinsic photosensitivity. Light responses in HCs appear to be driven through an ancient type of phototransduction cascade similar to that in rhabdomeric photoreceptors involving a G-protein q, the activation of phospholipase C, calcium mobilization, and the release of the inhibitory neurotransmitter GABA. Based on their intrinsic photosensitivity, HCs may have a key dual function in the retina of vertebrates, potentially regulating nonvisual tasks together with their sister cells, ipRGCs, and with visual photoreceptors, modulating lateral interactions and retinal processing.

Melanopsin and the Non-visual Photochemistry in the Inner Retina of Vertebrates.

Melanopsin (Opn4), a member of the G-protein-coupled receptor family, is a vitamin A-based opsin in the vertebrate retina that has been shown to be involved in the synchronization of circadian rhythms, pupillary light reflexes, melatonin suppression and other light-regulated tasks. In nonmammalian vertebrates there are two Opn4 genes, Opn4m and Opn4x, the mammalian and Xenopus orthologs respectively. Opn4x is only expressed in nonmammalian vertebrates including reptiles, fish and birds, while Opn4m is found in a subset of retinal ganglion cells (RGCs), the intrinsically photosensitive (ip) RGCs of the inner retina of both mammals and nonmammalian vertebrates. All opsins described utilize retinaldehyde as chromophore, photoisomerized from 11-cis- to all-trans-retinal upon light exposure. Visual retinal photoreceptor cones and rods, responsible for day and night vision respectively, recycle retinoids through a process called the visual cycle that involves the retinal pigment epithelium or glial Müller cells. Although Opn4 has been characterized as a bistable photopigment, little is known about the mechanism/s involved in its chromophore regeneration. In this review, we will attempt to shed light on the visual cycle taking place in the inner retina and discuss the state of the art in the nonvisual photochemistry of vertebrates.

Circadian control of the pupillary light responses in an avian model of blindness, the GUCY1* chickens.

PURPOSE: The vertebrate inner retina has a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the nonvisual photopigment melanopsin. The intrinsically photosensitive retinal ganglion cells send light information from the environment to the brain to control, among other parameters, the amount of energy entering the eyes through the pupillary light reflex (PLR). A daily variation in the PLR in both mice and humans has recently been shown, indicating circadian control of this response. In a previous work involving the sensitivity spectra for the PLR, we showed that blind chickens (GUCY1*) display the highest sensitivity to light of 480 nm. The aim of the present study was to evaluate the potential circadian control of PLRs in blind birds under scotopic conditions. METHODS: Circadian PLR was performed on GUCY1* chickens with lights of different wavelengths (white or blue light of 475 nm) under scotopic conditions. RESULTS: We found a significant daily variation in the PLRs of chickens exposed to white or blue light of 475 nm, with increased sensitivity at circadian time 6 during the subjective day. CONCLUSIONS: Our observations clearly point to circadian control of PLRs even in blindness, strongly indicating that both the entry of light into the eyes and its quality are differentially regulated during the day in diurnal animals.

Effects of different per translational kinetics on the dynamics of a core circadian clock model.

Living beings display self-sustained daily rhythms in multiple biological processes, which persist in the absence of external cues since they are generated by endogenous circadian clocks. The period (per) gene is a central player within the core molecular mechanism for keeping circadian time in most animals. Recently, the modulation PER translation has been reported, both in mammals and flies, suggesting that translational regulation of clock components is important for the proper clock gene expression and molecular clock performance. Because translational regulation ultimately implies changes in the kinetics of translation and, therefore, in the circadian clock dynamics, we sought to study how and to what extent the molecular clock dynamics is affected by the kinetics of PER translation. With this objective, we used a minimal mathematical model of the molecular circadian clock to qualitatively characterize the dynamical changes derived from kinetically different PER translational mechanisms. We found that the emergence of self-sustained oscillations with characteristic period, amplitude, and phase lag (time delays) between per mRNA and protein expression depends on the kinetic parameters related to PER translation. Interestingly, under certain conditions, a PER translation mechanism with saturable kinetics introduces longer time delays than a mechanism ruled by a first-order kinetics. In addition, the kinetic laws of PER translation significantly changed the sensitivity of our model to parameters related to the synthesis and degradation of per mRNA and PER degradation. Lastly, we found a set of parameters, with realistic values, for which our model reproduces some experimental results reported recently for Drosophila melanogaster and we present some predictions derived from our analysis.