Demonstrating a multi-primary high dynamic range display system for vision experiments.

We describe the design, construction, calibration, and characterization of a multi-primary high dynamic range (MPHDR) display system for use in vision research. The MPHDR display is the first system to our knowledge to allowfor spatially controllable, high dynamic range stimulus generation using multiple primaries.We demonstrate the high luminance, high dynamic range, and wide color gamut output of the MPHDR display. During characterization, the MPHDR display achieved a maximum luminance of 3200 cd=m2, a maximum contrast range of 3; 240; 000 V 1, and an expanded color gamut tailored to dedicated vision research tasks that spans beyond traditional sRGB displays. We discuss how the MPHDR display could be optimized for psychophysical experiments with photoreceptor isolating stimuli achieved through the method of silent substitution. We present an example case of a range of metameric pairs of melanopsin isolating stimuli across different luminance levels, from an available melanopsin contrast of117%at 75 cd=m2 to a melanopsin contrast of23%at 2000 cd=m2.

Large scale expression and purification of mouse melanopsin-L in the baculovirus expression system.

Melanopsin is the mammalian photopigment that primarily mediates non-visual photoregulated physiology. So far, this photopigment is poorly characterized with respect to structure and function. Here, we report large-scale production and purification of the intact long isoform of mouse melanopsin (melanopsin-L) using the baculovirus/insect cell expression system. Exploiting the baculoviral GP67 signal peptide, we obtained expression levels that varied between 10-30pmol/10(6)cells, equivalent to 2-5mg/L. This could be further enhanced using DMSO as a chemical chaperone. LC-MS analysis confirmed that full-length melanopsin-L was expressed and demonstrated that the majority of the expressed protein was N-glycosylated at Asn(30) and Asn(34). Other posttranslational modifications were not yet detected. Purification was achieved exploiting a C-terminal deca-histag, realizing a purification factor of several hundred-fold. The final recovery of purified melanopsin-L averaged 2.5% of the starting material. This was mainly due to low extraction yields, probably since most of the protein was present as the apoprotein. The spectral data we obtained agree with an absorbance maximum in the 460-500nm wavelength region and a significant red-shift upon illumination. This is the first report on expression and purification of full length melanopsin-L at a scale that can easily be further amplified.

Thermal recovery of iodopsin from photobleaching intermediates.

The chloride effect on the photobleaching process of iodopsin, a chicken red-sensitive cone visual pigment, was studied in detail by time-resolved low-temperature spectroscopy at -40 degrees C to -10 degrees C. Decay-associated difference spectra obtained by kinetic analysis using the singular value decomposition method were composed of spectra of BL-iodopsin, lumiiodopsin, metaiodopsin I, metaiodopsin II and metaiodopsin III, essentially identical to those at room temperature. In each conversion step however, iodopsin was partially regenerated, which is not observed in the bleaching process for other visual pigments or iodopsin at room temperature. Moreover, iodopsin was slowly regenerated from the bleached species. The reverse reactions were completely suppressed by substitution of lyotropic NO(3)(-) for Cl(-), suggesting that Cl(-) binding to iodopsin interferes with light-induced cis-trans isomerization of the chromophore. It is likely that the water molecule hydrating Cl(-) forms the additional hydrogen bond(s), by which the protein conformational change necessary to release this steric hindrance becomes enthalpic. As progress of the bleaching process is a consequence of protein conformational change, it is suppressed at low temperatures, resulting in thermal back-isomerization.

Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch.

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.

Isolation and characterization of melanopsin and pinopsin expression within photoreceptive sites of reptiles.

Non-mammalian vertebrates have multiple extraocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection. In this study, we report the full-length cDNA cloning of ruin lizard melanopsin and pinopsin. The high level of identity with opsins in both the transmembrane regions, where the chromophore binding site is located, and the intracellular loops, where the G-proteins interact, suggests that both melanopsin and pinopsin should be able to generate a stable photopigment, capable of triggering a transduction cascade mediated by G-proteins. Phylogenetic analysis showed that both opsins are located on the expected branches of the corresponding sequences of ortholog proteins. Subsequently, using RT-PCR and RPA analysis, we verified the expression of ruin lizard melanopsin and pinopsin in directly photosensitive organs, such as the lateral eye, brain, pineal gland and parietal eye. Melanopsin expression was detected in the lateral eye and all major regions of the brain. However, different from the situation in Xenopus and chicken, melanopsin is not expressed in the ruin lizard pineal. Pinopsin mRNA expression was only detected in the pineal complex. As a result of their phylogenetic position and ecology, reptiles provide the circadian field with some of the most interesting models for understanding the evolution of the vertebrate circadian timing system and its response to light. This characterization of melanopsin and pinopsin expression in the ruin lizard will be important for future studies aimed at understanding the molecular basis of circadian light detection in reptiles.

Molecular cloning, localization and circadian expression of chicken melanopsin (Opn4): differential regulation of expression in pineal and retinal cell types.

The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde-based photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

Melanopsin forms a functional short-wavelength photopigment.

Recently, melanopsin has emerged as the leading candidate for the elusive photopigment of the mammalian circadian system. This novel opsin-like protein is expressed in retinal ganglion cells that form the retinohypothalamic tract, a neuronal connection between the retina and the suprachiasmatic nucleus. These hypothalamic structures contain the circadian pacemaker, which generates daily rhythms in physiology and behavior. In mammals, proper synchronization of these rhythms to the environmental light-dark cycle requires retinal input. Surprisingly, rod and cone photoreceptors are not required. Instead, the melanopsin-containing ganglion cells are intrinsically sensitive to light, perhaps responding via a melanopsin-based signaling pathway. To test this hypothesis, we have characterized melanopsin following heterologous expression in COS cells. We found that melanopsin absorbed maximally at 424 nm after reconstitution with 11-cis-retinal. Furthermore, melanopsin activated the photoreceptor G-protein, transducin, in a light-dependent manner. In agreement with the measured absorbance spectrum, melanopsin was most efficiently excited by blue light (420-440 nm). In contrast, published action spectra suggest that the photopigment underlying the intrinsic light sensitivity of SCN-projecting RGCs has an absorption maximum near 484 nm. In summary, our experiments constitute the first direct demonstration that melanopsin forms a photopigment capable of activating a G-protein, but its spectral properties are not consistent with the action spectrum for circadian entrainment.

Teleost multiple tissue (tmt) opsin: a candidate photopigment regulating the peripheral clocks of zebrafish?

Isolated organs and cell lines from zebrafish exhibit circadian oscillations in clock gene expression that can be entrained to a 24-h light/dark cycle. The mechanism underlying this cellular photosensitivity is unknown. We report the identification of a novel opsin family, tmt-opsin, that has a genomic structure characteristic of vertebrate photopigments, an amino acid identity equivalent to the known photopigment opsins, and the essential residues required for photopigment function. Significantly, tmt-opsin is expressed in a wide variety of neural and non-neural tissues, including a zebrafish embryonic cell line that exhibits a light entrainable clock. Collectively the data suggest that tmt-opsin is a strong candidate for the photic regulation of zebrafish peripheral clocks.

New fluorescent probes for visual proteins. Part II. 5-(Oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoate.

A new dual-fluorescent compound, 5-(oxo)penta-2,4-dienyl-p-(N,N-dimethylamino)benzoate (1), a derivative of dimethylaminobenzoic acid, has been synthesised and studied photophysically. This compound continues the series of potential fluorescent probes for visual and proton-pumping opsin proteins. The photophysical behaviour of this molecule, including charge-transfer interaction in the ground state and dual-fluorescence emission, is similar to that of the previously studied analogue cis-3-(oxo)propenyl-p-(N,N-dimethylamino)benzoate (cis-2). The presence of several theoretically calculated conformers of compound 2 was suggested to be responsible for the observed strongly red-shifted absorption and excitation wavelength dependence. These photophysical anomalies were also observed for molecule 1, though the models put forward to explain them in the cases of 1 and 2 are rather different. Based on theoretical calculations and experimental results, we propose that some of the stable conformers might be connected with either a charge-transfer complex or mesomeric interactions in the ground state. Upon changing the electronic nature of the oxo-pentadienyl acceptor moiety, e.g. protonation, chemical or biochemical reaction, the charge-transfer absorption disappears, which leads to a dramatic increase in the fluorescence quantum yield.

Zebrafish melanopsin: isolation, tissue localisation and phylogenetic position.

Photoreception is best understood in retinal rods and cones, but it is not confined to these cells. In non-mammals, intrinsically photosensitive cells have been identified within several structures including the pineal, hypothalamus and skin. More recently novel light sensitive cells have been identified in the inner/basal retina of both teleosts and rodents. Melanopsin has been proposed as the photopigment mediating many of these non-rod, non-cone responses to light. However, much about the melanopsin gene family remains to be clarified including their potential role as photopigments, and taxonomic distribution. We have isolated the first orthologue of melanopsin from a teleost fish and show expression of this gene in a sub-set of retinal horizontal cells (type B). Zebrafish melanopsin, and orthologues of this gene, differ markedly from the vertebrate photopigment opsins. The putative counterion is not a glutamate but a tyrosine, the putative G-protein binding domain in the third cytoplasmic loop is not conserved, and they show low levels of amino acid identity (approximately 27%) to both the known photopigment opsins and to other members of the melanopsin family. Mouse melanopsin is only 58% identical to Xenopus, and 68% identical to zebrafish. By contrast, the photosensory opsin families show approximately 75% conservation. On the basis of their structure, genomic organisation, discrete evolutionary lineage, and their co-expression with other opsins, the melanopins are not obvious photosensory opsins. They might represent a separate branch of photopigment evolution in the vertebrates or they may have a non-direct photosensory function, perhaps as a photoisomerase, in non-rod, non-cone light detection.

Assays for activation of opsin by all-trans-retinal.

The data collected with the techniques discussed in this chapter suggest significant differences between the active conformation(s) of the opsin/atr complex, which are reversibly formed in the dark, and the active conformation (R*) of the meta-II photoproduct. First, there is good evidence for noncovalent opsin/atr complexes with considerable activity (although covalent binding of atr is found in mutant opsins. Even more intriguing, all-trans-retinal in an amount that saturates the activity of the opsin/atr complex toward Gt does not measurably inhibit the access of 11-cis-retinal to the light-sensitive binding site during regeneration (Fig. 2C). On the other hand, forced protonation at or near Glu-134 appears to be an integral mechanism for both the meta-II and the opsin-like activities (Fig. 4). Thus, it is not inconceivable that these two activities of the receptor arise from two fundamentally different conformations, one meta-II-like and one opsin-like. They would be similar with respect to the Gt (or RK) protein-protein interaction but different in their mode of retinal-protein interaction.

A novel rod-like opsin isolated from the extra-retinal photoreceptors of teleost fish.

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra-retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod-opsins from the retina of these species. By PCR, we have determined that the novel rod-like opsin is not expressed in the salmon retina, and the retinal rod-opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod-like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod-like opsins, their extra-retinal sites of expression, and phylogenetic position we have termed these novel opsins 'extra-retinal rod-like opsins' (ERrod-like opsins). We speculate that the differences between retinal rod-opsins and ERrod-like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.

Cloning and expression of a Xenopus short wavelength cone pigment.

The short wavelength visual pigment from Xenopus responsible for vision in the blue/violet portion of the spectrum was characterized by sequence spectroscopic analysis. The amino acid sequence was deduced by sequencing clones isolated by reverse transcription PCR, from retinal cDNA and genomic libraries. The gene contains 5 exons spanning 8.4 kb of genomic DNA and produces an mRNA of 2.4 kb in length. The deduced amino acid sequence predicts a protein of 347 amino acids with 76-78% identity to other short wavelength opsins. The mRNA encoding the Xenopus violet pigment was detected using in situ hybridization in cones, comprising a few percent of the total photoreceptors in the adult retina. The Xenopus violet opsin cDNA, modified to contain an epitope from the carboxyl terminus of bovine rhodopsin, was expressed in COS1 cells by transient transfection and analysed by UV-visible absorption spectroscopy. The protein expressed in COS1 cells migrated at 34 kD and was glycosylated at a single site in the amino terminus, exhibiting a diffuse pattern on SDS PAGE similar to bovine rhodopsin expressed in COS1 cells. Following incubation with 11-cis retinal, a light-sensitive pigment was formed with the lambdamax=425+/-2 nm. A Schiff base linkage between retinal and the violet opsin was demonstrated by acid denaturation. Xenopus violet opsin was sensitive to hydroxylamine in the dark, reacting with a half-time of 5 min at room temperature. This is the first group S pigment for amphibians. The pigment was expressed and purified from COS1 cells in a form that has permitted for the first time determination of the extinction coefficient, reactivity to hydroxylamine and presence of a Schiff base.

Regeneration of ultraviolet pigments of vertebrates.

We report here the regeneration of the visual pigments of mouse, rat, goldfish and pigeon, which have wavelengths of maximal absorption at 359 nm, 358 nm, 359 nm, and 393 nm, respectively. The construction and functional assays of the ultraviolet or near-ultraviolet pigments from a wide range of vertebrate species will allow us to study the molecular bases of ultraviolet vision for the first time.

The pineal organ as a folded retina: immunocytochemical localization of opsins.

The most simple pineal complex (the pineal and parapineal organs of lampreys), consists of saccular evaginations of the diencephalic roof, and has a retina-like structure containing photoreceptor cells and secondary neurons. In more differentiated vertebrates, the successive folding of the pineal wall multiplies the cells and reduces the lumen of the organ, but the pattern of the histological organization remains similar to that of lampreys; therefore, we consider the histological structure of the pineal organ of higher vertebrates as a 'folded retina'. The cell membrane of several pineal photoreceptor outer-segments of vertebrates immunoreact with anti-retinal opsin antibodies supporting the view of retina-like organization of the pineal. Some other pineal outer segments do not react with retinal anti-opsin antibodies, a result suggesting the presence of special pineal photopigments in different types of pinealocytes that obviously developed during evolution. The chicken pinopsin, detected in the last years, may represent one of these unknown photopigments. Using antibodies against chicken pinopsin, we compared the immunoreactivity of different photoreceptors of the pineal organs from cyclostomes to birds at the light and electron microscopic levels. We found pinopsin immunoreaction on all pinealocytes of birds and on the rhodopsin-negative large reptilian pinealocytes. As the pinopsin has an absorption maximum at 470 nm, these avian and reptilian immunoreactive pinealocytes can be regarded as green-blue light-sensitive photoreceptors. Only a weak immunoreaction was observed on the frog and fish pinealocytes and no reaction was seen in cyclostomes and in the frontal organ of reptiles. Some photoreceptors of the retina of various species also reacted the pinopsin antibodies, therefore, pinopsin must have certain sequential similarity to individual retinal opsins of some vertebrates.