Translational thresholds in a core circadian clock model.

Organisms have evolved in a daily cyclic environment, developing circadian cell-autonomous clocks that temporally organize a wide range of biological processes. Translation is a highly regulated process mainly associated with the activity of microRNAs (miRNAs) at the translation initiation step that impacts on the molecular circadian clock dynamics. Recently, a molecular titration mechanism was proposed to explain the interactions between some miRNAs and their target mRNAs; new evidence also indicates that regulation by miRNA is a nonlinear process such that there is a threshold level of target mRNA below which protein production is drastically repressed. These observations led us to use a theoretical model of the circadian molecular clock to study the effect of miRNA-mediated translational thresholds on the molecular clock dynamics. We model the translational threshold by introducing a phenomenological Hill equation for the kinetics of PER translation and show how the parameters associated with translation kinetics affect the period, amplitude, and time delays between clock mRNA and clock protein expression. We show that our results are useful for analyzing experiments related to the translational regulation of negative elements of transcriptional-translational feedback loops. We also provide new elements for thinking about the translational threshold as a mechanism that favors the emergence of circadian rhythmicity, the tuning of the period-delay relationship and the cell capacity to control the protein oscillation amplitude with almost negligible changes in the mRNA amplitudes.

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.

Differential regulation of feeding rhythms through a multiple-photoreceptor system in an avian model of blindness.

All organisms have evolved photodetection systems to synchronize their physiology and behavior with the external light-dark (LD) cycles. In nonmammalian vertebrates, the retina, the pineal organ, and the deep brain can be photoreceptive. Inner retinal photoreceptors transmit photic information to the brain and regulate diverse nonvisual tasks. We previously reported that even after preventing extraretinal photoreception, blind GUCY1* chickens lacking functional visual photoreceptors could perceive light that modulates physiology and behavior. Here we investigated the contribution of different photoreceptive system components (retinal/pineal and deep brain photoreceptors) to the photic entrainment of feeding rhythms. Wild-type (WT) and GUCY1* birds with head occlusion to avoid extraocular light detection synchronized their feeding rhythms to a LD cycle with light >12 lux, whereas at lower intensities blind birds free-ran with a period of >24 h. When released to constant light, both WT and blind chickens became arrhythmic; however, after head occlusion, GUCY1* birds free-ran with a 24.5-h period. In enucleated birds, brain illumination synchronized feeding rhythms, but in pinealectomized birds only responses to high-intensity light (≥800 lux) were observed, revealing functional deep brain photoreceptors. In chickens, a multiple photoreceptive system, including retinal and extraretinal photoreceptors, differentially contributes to the synchronization of circadian feeding behavior.

Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina.

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

Differential responses of the mammalian retinal ganglion cell line RGC-5 to physiological stimuli and trophic factors.

The rat retinal ganglion cell (RGC) line RGC-5 constitutes a widely used model for studying physiological processes in retinal cells. In this paper we investigated the expression of clock and immediately early genes, and calcium mediated responses to physiological stimuli in differentiated and mitotically active RGC-5 cells. To this end, we attempted to differentiate the RGC-5 cells with a variety of effectors classically used to induce morphological differentiation. No sign of morphological differentiation was observed after 24 h of treatment with BDNF (80 ng/mL), NGF (100 ng/mL) and retinoic acid (20 ng/mL), among others. Only staurosporine (SSP) was able to promote neurite outgrowth at concentrations ranging from 53.5 to 214 nM. However, apoptotic nuclei were seen at 24 h of treatment using DNA staining, and a few cells remained at 72 h post-treatment. Concentrations of SSP lower than 214 nM were partially effective in inducing cell differentiation. Dividing RGC-5 cells express the RGC marker Thy-1 and different clock genes such as Per1, Clock and Bmal1. When characterizing the responsiveness of proliferative RGC-5 cells we found that in most of them, brief pulses of 50% FBS induced c-Fos and PER1 expression. Subsets of RGC-5 cells displayed significant changes in intracellular Ca2+ levels by ATP (100 microM) but not by glutamate (100-200 microM) stimulation. On the basis of cell morphology, size and complexity and effector responsiveness it was possible to distinguish different subpopulations within the cell line. The results demonstrate that only SSP is effective in promoting RGC-5 morphological differentiation, though the treatment provoked cell death. Proliferative cells expressing the RGC marker Thy-1 and a number of clock genes, responded differentially to diverse physiological stimuli showing a rapid c-Fos and PER1 induction by FBS stimulation, and an increase in intracellular Ca2+ by ATP.

Inner retinal circadian clocks and non-visual photoreceptors: novel players in the circadian system.

Daily and annual changes in ambient illumination serve as specific stimuli that associate light with time and regulate the physiology of the organism through the eye. The eye acts as a dual sense organ linking light and vision, and detecting light that provides specific stimuli for non-classical photoreceptors located in the inner retina. These photoreceptors convey information to the master circadian pacemaker, the hypothalamic suprachiasmatic nuclei (SCN). Responsible for sensing the light that regulates several non-visual functions (i.e. behavior, pupil reflex, sleep, and pineal melatonin production), the retina plays a key role in the temporal symphony orchestra playing the musical score of life: it is intrinsically rhythmic in its physiological and metabolic activities. We discuss here recent evidence in support of the hypothesis that retinal oscillators distributed over different cell populations may act as clocks, inducing changes in the visual and circadian system according to the time of the day. Significant progress has recently been made in identifying photoreceptors/photopigments localized in retinal ganglion cells (RGCs) that set circadian rhythms and modulate non-visual functions. Autonomous retinal and brain oscillators could have a more complex organization than previously recognized, involving a network of "RGC clock/SCN clock cross-talk". The convergence of oscillatory and photoreceptive capacities of retinal cells could deeply impact on the circadian system, which in turn may be severely impaired in different retinal pathologies. The aim of this review is to discuss the state of the art on inner retinal cell involvement in the light and temporal regulation of health and disease.

A nonmammalian vertebrate model of blindness reveals functional photoreceptors in the inner retina.

In mammals, photoreceptors located in the inner retina convey photic information to the brain, regulating diverse non-image-forming tasks such as pupillary light reflexes and photic synchronization (entrainment) of daily activity rhythms. In nonmammalian vertebrates, the retina, deep brain photoreceptors, and pineal organ may be photoreceptive. Here we investigated light perception in the absence of functional cone and rod photoreceptors using GUCY1* chickens, birds carrying a null mutation that causes blindness at hatch. They showed light responses in both the pupillary light reflex and the entrainment of feeding rhythms to a 12:12 h light-dark cycle. Light responses persisted even when the extraretinal photoperception was abolished, but they were lost after enucleation; this strongly indicates the essential role played by the inner retina. A sensitivity spectrum study for the pupillary reflex that combined pupil responses to different monochromatic lights of various intensities demonstrated that a single opsin/vitamin A-based photopigment peaking at 484 nm drives photic responses; the best fit (lowest sum of squares, R(2)=0.9622) was attained with an opsin:vitamin A2 template. The results are the first characterization of functional inner retinal photoreceptors participating in the regulation of non-image-forming activities in nonmammalian vertebrates.