Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.     

The distribution of melanopsin (OPN4) protein in the human brain

Until now, melanopsin (OPN4) – a specialized photopigment being responsive especially to blue light wavelengths – has not been found in the human brain at protein level outside the retina. More specifically, OPN4 has only been found in about 2% of retinal ganglion cells (i.e. in intrinsically photosensitive retinal ganglion cells), and in a subtype of retinal cone-cells. Given that Allen Institute for Brain Science has described a wide distribution of OPN4 mRNA in two human brains, we aimed to investigate whether OPN4 is present in the human brain also at protein level. Western blotting and immunohistochemistry, as well as immunoelectron microscopy, were used to analyse the existence and distribution of OPN4 protein in 18 investigated areas of the human brain in samples obtained in forensic autopsies from 10 male subjects (54 ± 3.5 years). OPN4 protein expression was found in all subjects, and, furthermore, in 5 out of 10 subjects in all investigated brain areas localized in membranous compartments and cytoplasmic vesicles of neurons. To our opinion, the wide distribution of OPN4 in central areas of the human brain evokes a question whether ambient light has important straight targets in the human brain outside the retinohypothalamic tract (RHT). Further studies are, however, needed to investigate the putative physiological phototransductive actions of inborn OPN4 protein outside the RHT in the human brain.     

Expression and comparative characterization of Gq-coupled invertebrate visual pigments and melanopsin

A non-visual pigment melanopsin, which is localized in photosensitive retinal ganglion cells and is involved in the circadian photoentrainment and pupillary responses in mammals, is phylogenetically close to the visual pigments of invertebrates, such as insects and cephalopods. Recent studies suggested that melanopsin is a bistable pigment and drives a Gq-mediated signal transduction cascade, like the invertebrate visual pigments. Because detailed electrophysiological properties are somewhat different between the visual cells and the photosensitive ganglion cells, we here expressed and purified the invertebrate visual pigment and melanopsin to comparatively investigate their Gq-activation abilities. We successfully expressed and purified UV and blue light-sensitive visual pigments of the honeybee as well as the amphioxus melanopsin. Although the purified UV-sensitive pigment and the melanopsin lost their bistable nature during purification, reconstitution of the pigments in lipid vesicles resulted in return of the bistable nature. The light-dependent Gq-activation abilities among these reconstituted pigments are similar, suggesting that the electrophysiological differences do not depend on the Gq-activation step but rather on the other signal transduction steps and/or on cell properties. Our findings are also important in that this is the first report describes a heterologous large-scale expression of the Gq-coupled invertebrate visual pigments in cultured cells.     

Contribution of human melanopsin retinal ganglion cells to steady-state pupil responses

The recent discovery of melanopsin-containing retinal ganglion cells (mRGCs) has led to a fundamental reassessment of non-image forming processing, such as circadian photoentrainment and the pupillary light reflex. In the conventional view of retinal physiology, rods and cones were assumed to be the only photoreceptors in the eye and were, therefore, considered responsible for non-image processing. However, signals from mRGCs contribute to this non-image forming processing along with cone-mediated luminance signals; although both signals contribute, it is unclear how these signals are summed. We designed and built a novel multi-primary stimulation system to stimulate mRGCs independently of other photoreceptors using a silent-substitution technique within a bright steady background. The system allows direct measurements of pupillary functions for mRGCs and cones. We observed a significant change in steady-state pupil diameter when we varied the excitation of mRGC alone, with no change in luminance and colour. Furthermore, the change in pupil diameter induced by mRGCs was larger than that induced by a variation in luminance alone: that is, for a bright steady background, the mRGC signals contribute to the pupillary pathway by a factor of three times more than the L- and M-cone signals.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

哺乳动物昼夜节律调节的神经基础——昼夜光感受器

哺乳动物昼夜光感受器为一组具有直接感光功能的特殊视网膜神经节细胞 ,其基本感光色素为黑视素 .昼夜光感受器具有直接、广谱和稳定感受昼夜光变化的功能特点 .昼夜光感受器的功能是通过导引作用 ,使下丘脑视交叉上核内的昼夜节律活动与外界明 暗周期变化同步     

The inner clock—Blue light sets the human rhythm

Visible light synchronizes the human biological clock in the suprachiasmatic nuclei of the hypothalamus to the solar 24‐hour cycle. Short wavelengths, perceived as blue color, are the strongest synchronizing agent for the circadian system that keeps most biological and psychological rhythms internally synchronized. Circadian rhythm is important for optimum function of organisms and circadian sleep–wake disruptions or chronic misalignment often may lead to psychiatric and neurodegenerative illness. The beneficial effect on circadian synchronization, sleep quality, mood, and cognitive performance depends not only on the light spectral composition but also on the timing of exposure and its intensity. Exposure to blue light during the day is important to suppress melatonin secretion, the hormone that is produced by the pineal gland and plays crucial role in circadian rhythm entrainment. While the exposure to blue is important for keeping organism's wellbeing, alertness, and cognitive performance during the day, chronic exposure to low‐intensity blue light directly before bedtime, may have serious implications on sleep quality, circadian phase and cycle durations. This rises inevitably the need for solutions to improve wellbeing, alertness, and cognitive performance in today's modern society where exposure to blue light emitting devices is ever increasing.      

The electroretinogram as a method for studying circadian rhythms in the mammalian retina

Circadian clocks are thought to regulate retinal physiology in anticipation of the large variation in environmental irradiance associated with the earth’s rotation upon its axis. In this review we discuss some of the rhythmic events that occur in the mammalian retina, and their consequences for retinal physiology. We also review methods of tracing retinal rhythmicity in vivo and highlight the electroretinogram (ERG) as a useful technique in this field. Principally, we discuss how this technique can be used as a quick and noninvasive way of assessing physiological changes that occur in the retina over the course of the day. We highlight some important recent findings facilitated by this approach and discuss its strengths and limitations.     

Immunohistochemical detection of opsins in turbellarians

Using the antiserum against the C-terminus of opsin of Dugesia japonica, immunohistochemistry revealed that opsins were actually localized in the photoreceptive organs of several turbellarians. The antiserum produced positive reactions in eyes of the Polycladida Notoplana koreana as well as those of the Tricladida Dugesia dorotocephala, Bdellocephala brunnea, Phagocata kawakatsui, Phagocata vivida and Polycelis auriclata, but not to those of Convoluta naikaiensis, Stenostomum sp. and Macrostomum tuba. This suggests that these opsin proteins of triclad and polyclad species have functional similarity in the C-terminus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Divergent photic thresholds in the non-image-forming visual system: entrainment, masking and pupillary light reflex

Light is the principal cue that entrains the circadian timing system, but the threshold of entrainment and the relative contributions of the retinal photoreceptors—rods, cones and intrinsically photosensitive retinal ganglion cells—are not known. We measured thresholds of entrainment of wheel-running rhythms at three wavelengths, and compared these to thresholds of two other non-image-forming visual system functions: masking and the pupillary light reflex (PLR). At the entrainment threshold, the relative spectral sensitivity and absolute photon flux suggest that this threshold is determined by rods. Dim light that entrained mice failed to elicit either masking or PLR; in general, circadian entrainment is more sensitive by 1–2 log units than other measures of the non-image-forming visual system. Importantly, the results indicate that dim light can entrain circadian rhythms even when it fails to produce more easily measurable acute responses to light such as phase shifting and melatonin suppression. Photosensitivity to one response, therefore, cannot be generalized to other non-image-forming functions. These results also impact practical problems in selecting appropriate lighting in laboratory animal husbandry.     

Molecular characterization of crustacean visual pigments and the evolution of pancrustacean opsins

Investigations of opsin evolution outside of vertebrate systems have long been focused on insect visual pigments, whereas other groups have received little attention. Furthermore, few studies have explicitly investigated the selective influences across all the currently characterized arthropod opsins. In this study, we contribute to the knowledge of crustacean opsins by sequencing 1 opsin gene each from 6 previously uncharacterized crustacean species (Euphausia superba, Homarus gammarus, Archaeomysis grebnitzkii, Holmesimysis costata, Mysis diluviana, and Neomysis americana). Visual pigment spectral absorbances were measured using microspectrophotometry for species not previously characterized (A. grebnitzkii=496 nm, H. costata=512 nm, M. diluviana=501 nm, and N. americana=520 nm). These novel crustacean opsin sequences were included in a phylogenetic analysis with previously characterized arthropod opsin sequences to determine the evolutionary placement relative to the well-established insect spectral clades (long-/middle-/short-wavelength sensitive). Phylogenetic analyses indicate these novel crustacean opsins form a monophyletic clade with previously characterized crayfish opsin sequences and form a sister group to insect middle-/long-wavelength-sensitive opsins. The reconstructed opsin phylogeny and the corresponding spectral data for each sequence were used to investigate selective influences within arthropod, and mainly "pancrustacean," opsin evolution using standard dN/dS ratio methods and more sensitive techniques investigating the amino acid property changes resulting from nonsynonymous replacements in a historical (i.e., phylogenetic) context. Although the conservative dN/dS methods did not detect any selection, 4 amino acid properties (coil tendencies, compressibility, power to be at the middle of an alpha-helix, and refractive index) were found to be influenced by destabilizing positive selection. Ten amino acid sites relating to these properties were found to face the binding pocket, within 4 A of the chromophore and thus have the potential to affect spectral tuning.     

Estimating photoreceptor excitations from spectral outputs of a personal light exposure measurement device

The intrinsic circadian clock requires photoentrainment to synchronize the 24-hour solar day. Therefore, light stimulation is an important component of chronobiological research. Currently, the chronobiological research field overwhelmingly uses photopic illuminance that is based on the luminous efficiency function, V(λ), to quantify light levels. However, recent discovery of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are activated by self-contained melanopsin photopigment and also by inputs from rods and cones, makes light specification using a one-dimensional unit inadequate. Since the current understanding of how different photoreceptor inputs contribute to the circadian system through ipRGCs is limited, it is recommended to specify light in terms of the excitations of five photoreceptors (S-, M-, L-cones, rods and ipRGCs; Lucas et?al., 2014 Lucas RJ, Peirson SN, Berson DM, et al. (2014). Measuring and using light in the melanopsin age. Trends Neurosci. 37:1–9[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]). In the current study, we assessed whether the spectral outputs from a commercially available spectral watch (i.e. Actiwatch Spectrum) could be used to estimate photoreceptor excitations. Based on the color sensor spectral sensitivity functions from a previously published work, as well as from our measurements, we computed spectral outputs in the long-wavelength range (R), middle-wavelength range (G), short-wavelength range (B) and broadband range (W) under 52 CIE illuminants (25 daylight illuminants, 27 fluorescent lights). We also computed the photoreceptor excitations for each illuminant using human photoreceptor spectral sensitivity functions. Linear regression analyses indicated that the Actiwatch spectral outputs could predict photoreceptor excitations reliably, under the assumption of linear responses of the Actiwatch color sensors. In addition, R, G, B outputs could classify illuminant types (fluorescent versus daylight illuminants) satisfactorily. However, the assessment of actual Actiwatch recording under several testing light sources showed that the spectral outputs were subject to great non-linearity, leading to less accurate estimation of photoreceptor excitations. Based on our analyses, we recommend that each spectral watch should be calibrated to measure spectral sensitivity functions and linearization characteristics for each sensor to have an accurate estimation of photoreceptor excitations. The method we provided to estimate photoreceptor excitations from the outputs of spectral watches could be used for chronobiological studies that can tolerate an error in the range of 0.2–0.5 log units. Our method can be easily expanded to incorporate linearization functions to have more accurate estimations.     

The expression of L-type voltage-gated calcium channels in retinal photoreceptors is under circadian control

Photoreceptors are non-spiking neurons, and their synapses mediate the continuous release of neurotransmitters under the control of L-type voltage-gated calcium channels (VGCCs). Photoreceptors express endogenous circadian oscillators that play important roles in regulating photoreceptor physiology and function. Here, we report that the L-type VGCCs in chick cone photoreceptors are under circadian control. The L-type VGCC currents are greater when measured during the subjective night than during the subjective day. Using antibodies against the VGCCalpha1C and VGCCalpha1D subunits, we found that the immunofluorescence intensities of both VGCCalpha1C and VGCCalpha1D in photoreceptors are higher during the subjective night. However, the mRNA levels of VGCCalpha1D, but not VGCCalpha1C, are rhythmic. Nocturnal increases in L-type VGCCs are blocked by manumycin A, PD98059, and KN93, which suggest that the circadian output pathway includes Ras, Erk, and calcium-calmodulin dependent kinase II. In summary, four independent lines of evidence show that the L-VGCCs in cone photoreceptors are under circadian control.     

棉铃虫生物钟基因HeDbt的克隆和表达模式分析

【目的】克隆并分析棉铃虫Helicoverpa armigera生物钟基因Double-time (Dbt),明确该基因的昼夜表达模式,探讨其表达水平的影响因子,为研究夜蛾科昆虫复眼中生物钟基因的作用机制奠定基础,为理解外周组织中生物钟基因功能提供参考。【方法】采用RT-PCR和RACE技术从2日龄棉铃虫雌成虫复眼中克隆生物钟基因Dbt,并利用在线网站和软件进行生物信息学分析。采用qPCR技术检测棉铃虫雌、雄成虫不同组织(头、脑、复眼、触角、胸、腹、足和翅)中Dbt的表达水平;检测光周期14L∶10D和持续黑暗(DD)下雌、雄成虫头和复眼中Dbt的昼夜表达模式;在暗期用棉铃虫敏感波段光(UV、蓝光和绿光)照射2日龄成虫6 h,检测复眼中Dbt表达水平的变化;在暗期进行雌、雄成虫交配,检测交配结束及3 h后复眼中Dbt表达水平的变化。【结果】成功克隆到棉铃虫生物钟基因Dbt的cDNA序列,命名为HeDbt(GenBank登录号: KM233159),开放阅读框长1 026 bp,编码314个氨基酸组成的多肽。HeDbt理论推测分子量为39.79 kD,等电点(pI)为9.55,不具有跨膜拓扑结构,包含典型的昆虫DBT蛋白保守区域,其与甜菜夜蛾Spodoptera exigua和柞蚕Antheraea pernyi DBT的同源性较高, 氨基酸序列一致性分别为99%和97%。qPCR结果表明,HeDbt在成虫各组织中均有表达,在头、脑和复眼中表达水平较低,在胸和腹中表达水平较高;在14L∶10D和DD下,头和复眼中HeDbt未呈现明显的昼夜表达节律。暗期光照和交配后,复眼中HeDbt的表达均显著下调,但雌、雄成虫间HeDbt表达水平整体相似。【结论】成功克隆得到棉铃虫生物钟基因HeDbt,其在棉铃虫成虫头和复眼中表达水平较低,且不具有昼夜规律性,但复眼中Dbt的表达受到光照和交配的影响。本研究为进一步探索夜蛾外周组织生物钟基因功能奠定了基础。     

Circadian rhythms in the retina of rats with photoreceptor degeneration

Previous studies have demonstrated that the mammalian retina contains a circadian clock system that controls several retinal functions. In mammals the location of the retinal circadian clock is unknown whereas, in non-mammalian vertebrates, earlier work has demonstrated that photoreceptor cells contain the circadian clock. New experimental evidence has suggested that in mammals the retinal circadian clock may be located outside the photoreceptor cells. In this study we report that circadian rhythms in Aa-nat mRNA (in vivo) and melatonin synthesis (in vitro) are still present in the retina of rats lacking photoreceptors. The circadian pacemaker(s) controlling such rhythms is probably located in kainic acid sensitive neurons in the inner retina since kainic acid injections abolished the rhythmicity. These data are the first direct demonstration that circadian rhythmicity in the mammalian retina can be generated independently from the photoreceptors and the suprachiasmatic nuclei of the hypothalamus.     

The evolution of eyes and visually guided behaviour

The morphology and molecular mechanisms of animal photoreceptor cells and eyes reveal a complex pattern of duplications and co-option of genetic modules, leading to a number of different light-sensitive systems that share many components, in which clear-cut homologies are rare. On the basis of molecular and morphological findings, I discuss the functional requirements for vision and how these have constrained the evolution of eyes. The fact that natural selection on eyes acts through the consequences of visually guided behaviour leads to a concept of task-punctuated evolution, where sensory systems evolve by a sequential acquisition of sensory tasks. I identify four key innovations that, one after the other, paved the way for the evolution of efficient eyes. These innovations are (i) efficient photopigments, (ii) directionality through screening pigment, (iii) photoreceptor membrane folding, and (iv) focusing optics. A corresponding evolutionary sequence is suggested, starting at non-directional monitoring of ambient luminance and leading to comparisons of luminances within a scene, first by a scanning mode and later by parallel spatial channels in imaging eyes.