Role of a novel photopigment,melanopsin, in behavioral adaptation to light

Adaptation to changes in the ambient light is of critical importance to life. In mammals, three principal photoadaptation mechanisms depend on ocular photoreception and exhibit spectral sensitivity suggestive of the opsin class of photopigment(s). These include rapid adaptation of the visual system to the ambient light by pupil constriction, direct modulation of neuroendocrine function and entrainment of the circadian clock to the day:night cycle. Surprisingly, these processes can largely function independent of classical rod/cone photoreceptors, suggesting a novel opsin-based signaling mechanism. They appear to involve a recently discovered network of intrinsically photosensitive retinal ganglion cells that make direct or indirect axonal connections to brain centers regulating photoadaptive behaviors. The discovery of a novel opsin, melanopsin, in these cells has offered an exciting entry point to explore, at the molecular level, how mammals adapt to their light environment. There is now genetic proof of a principal role for melanopsin in all three major photoadaptation processes.     

The absence of melanopsin alters retinal clock function and dopamine regulation by light

The retinal circadian clock is crucial for optimal regulation of retinal physiology and function, yet its cellular location in mammals is still controversial. We used laser microdissection to investigate the circadian profiles and phase relations of clock gene expression and Period gene induction by light in the isolated outer (rods/cones) and inner (inner nuclear and ganglion cell layers) regions in wild-type and melanopsin-knockout (Opn ₄ ^?/? ) mouse retinas. In the wild-type mouse, all clock genes are rhythmically expressed in the photoreceptor layer but not in the inner retina. For clock genes that are rhythmic in both retinal compartments, the circadian profiles are out of phase. These results are consistent with the view that photoreceptors are a potential site of circadian rhythm generation. In mice lacking melanopsin, we found an unexpected loss of clock gene rhythms and of the photic induction of Per1-Per2 mRNAs only in the outer retina. Since melanopsin ganglion cells are known to provide a feed-back signalling pathway for photic information to dopaminergic cells, we further examined dopamine (DA) synthesis in Opn ₄ ^?/? mice. The lack of melanopsin prevented the light-dependent increase of tyrosine hydroxylase (TH) mRNA and of DA and, in constant darkness, led to comparatively high levels of both components. These results suggest that melanopsin is required for molecular clock function and DA regulation in the retina, and that Period gene induction by light is mediated by a melanopsin-dependent, DA-driven signal acting on retinal photoreceptors.     

Vertebrate circadian rhythms: Retinal and extraretinal photoreception

Summary ERRs Both the pineal and the SCN are elements of the vertebrate multioscillator system although the relative importance of these 2 areas probably varies between, and possibly within, the different vertebrate classes. Extraretinal photoreception is a universal feature of submammalian vertebrates, and possibly of neonatal mammals, but is absent in adult mammals. Although the pineal systems of sumammalian vertebrates are photosensitive, the pineal system has been directly implicated as an extraocular site for the perception of entraining light cycles only in amphibians. In all other submammalian vertebrates extraretinal entrainment can occur in the absence of the pineal system although it is certainly conceivable that the pineal system may act as an alternate route of photoreception. These extraretinal-extrapineal receptors are located within the brain but the exact location(s) of these receptors within the brain is unknown. The hypothalamus would be likely area for this extraretinal photoreception, however, for several reasons: 1. Neurophysiological studies have identified light sensitive neurons in the frog's hypothalamus⁴³. 2. The avian hypothalamus is a site of photoperiodic photoreception^100–103. 3. The only other light sensitive structures known in vertebrates—the pineal system and the lateral eyes—are all derived embryologically from the hypothalamus. 4. The hypothalamus appears to be the site of a circadian clock and there may be advantages in having the photoreceptors and the clock anatomically close to one another. These considerations, of course, do not exclude the possibility that other brain areas may be involved as well. The reason behind the loss of extraretinal photoreception in mammals is uncertain. The shift to exclusive retinal photoreception in mammals may have been dictated by the extensive reorganization that occurred during the evolution of the mammalian brain. Or, perhaps, the increased size of the mammalian skull and overlying tissue made direct photoreception difficult and necessitated a shift to retinal photoreception. The persistence of extraretinal photoreceptors in submammalian vertebrates, however, underscores their importance in the sensory repertoire of vertebrates.     

Intercellular junctions of hyperplastic retinal pigment epithelium

In rats with retinopathies induced by excess fluorescent light or injections of urethane, the retinal pigment epithelium (RPE) undergoes focal hyperplasia. Neither intravascularly injected horseradish peroxidase or lanthanum nitrate penetrated the sensory retina at these hyperplastic sites. Electron microscopy revealed that this was due to the persistence of intact tight junctions among a single layer of hyperplastic cells facing the sensory retina. These junctions prevented intraocularly injected microperoxidase from passing as well. Cells within the hyperplastic foci were connected only by adherent junctions that presented no permeability barrier.     

Sinuslichtreizung der rezeptiven Felder einzelner Retinaneurone

Summary The action potentials of optic nerve fibres in cats were stereotactically recorded (light pentobarbital anaesthesia, Curarin-immobilization). The activation of retinal neurons stimulated by light spots within the receptive field centre was a non-linear function of the sinusoidal intensity modulation of the stimuli.     

Cone mosaic in a teleost retina: No difference between light and dark adapted states

Summary The mosaic arrangement of retinal cones in the eye of the African cichlid fish,Haplochromis burtoni, is the same in both light and dark adapted states. This is in contrast to Kunz' claim² that the retinal mosaic pattern changes from a square to a row type during dark adaptation, in the guppy (Poecelia reticulata). Kunz' histological procedure may account for this difference in results.Acknowledgments. This work was supported by grants from the Whitehall Foundation and NIH EY02284. I thank L. Shelton for expert histological assistance.     

Light evoked release of radioactivity from rabbit retinas preloaded with (3H)-GABA

Summary Light flashes evoke an increased release of radioactivity in vitro from rabbit retinas preloaded with (³H)-GABA in vivo. Constant light does not affect the release. No light evoked release can be demonstrated from the glia. Pentobarbitone and AOAA depress the evoked release. The results are consistent with GABA being a retinal neurotransmitter, most likely in a class of amacrines.This work was supported by grants from the Swedish Medical Research Council (project 04X2321), The Faculty of Medicine, University of Lund, Royal Physiographic Society.     

Light and temperature affect retinyl ester hydrolase activity and visual pigment composition

Summary Dim light, in combination with high water temperature, resulted in a significant increase in the retinyl ester hydrolase activity in the goldfish retina. This rise in enzyme activity may relate to a selective increase in the availability of retinal chromophores thereby favoring the formation of rhodopsin under these light and temperature conditions.     

Regulation of the retinal interphotoreceptor matrix Na by the retinal pigment epithelium during the light response

We examined the rabbit retinal pigment epithelium (RPE) for Na transport properties which would allow it to buffer undesirable changes in Na concentrations in the interphotoreceptor matrix (IPM) during light and dark cycles. The RPE is selectivity permeable to sodium. Open and short circuit transport studies with RPE indicate a circulating (choroid to retina and back) Na current which does not compromise the electrical integrity of the blood brain barrier but together with the Na permeselectivity is of sufficient magnitude to buffer both upwards and downwards movements of IPM [Na] during light or dark responses.     

Photoreceptor damage following exposure to excess riboflavin

Flavins generate oxidants during metabolism and when exposed to light. Here we report that the photoreceptor layer of retinas from black-eyed rats is reduced in size by a dietary regime containing excess riboflavin. The effect of excess riboflavin was dose-dependent and was manifested by a decrease in photoreceptor length. This decrease was due in part to a reduction in the thickness of the outer nuclear layer, a structure formed from stacked photoreceptor nuclei. These changes were accompanied by an increase in photoreceptor outer segment autofluorescence following illumination at 328 nm, a wavelength that corresponds to the excitation maxima of oxidized lipopigments of the retinal pigment epithelium.     

Light and temperature affect retinyl ester hydrolase activity and visual pigment composition

Dim light, in combination with high water temperature, resulted in a significant increase in the retinyl ester hydrolase activity in the goldfish retina. This rise in enzyme activity may relate to a selective increase in the availability of retinal chromophores thereby favoring the formation of rhodopsin under these light and temperature conditions.     

Periostin in vitreoretinal diseases

Proliferative vitreoretinal diseases such as diabetic retinopathy, proliferative vitreoretinopathy (PVR), and age-related macular degeneration are a leading cause of decreased vision and blindness in developed countries. In these diseases, retinal fibro(vascular) membrane (FVM) formation above and beneath the retina plays an important role. Gene expression profiling of human FVMs revealed significant upregulation of periostin. Subsequent analyses demonstrated increased periostin expression in the vitreous of patients with both proliferative diabetic retinopathy and PVR. Immunohistochemical analysis showed co-localization of periostin with α-SMA and M2 macrophage markers in FVMs. In vitro, periostin blockade inhibited migration and adhesion induced by PVR vitreous and transforming growth factor-β2 (TGF-β2). In vivo, a novel single-stranded RNAi agent targeting periostin showed the inhibitory effect on experimental retinal and choroidal FVM formation without affecting the viability of retinal cells. These results indicated that periostin is a pivotal molecule for FVM formation and a promising therapeutic target for these proliferative vitreoretinal diseases.     

Visual pigment: G-protein-coupled receptor for light signals

The visual pigment present in photoreceptor cells is a prototypical G-protein-coupled receptor (GPCR) that receives a light signal from the outer environment using a light-absorbing chromophore, 11-cis-retinal. Through cis-trans isomerization of the chromophore, light energy is transduced into chemical free energy, which is in turn utilized for conformational changes in the protein to activate the retinal G-protein. In combination with site-directed mutagenesis, various spectroscopic and biochemical studies identified functional residues responsible for chromophore binding, color regulation, intramolecular signal transduction and G-protein coupling. Extensive studies reveal that these residues are localized into specific domains of visual pigments, suggesting a highly manipulated molecular architecture in visual pigments. In addition to the recent findings on dysfunctional mutations in patients with retinitis pigmentosa or congenital night blindness, the mechanism of intramolecular signal transduction in visual pigments and their evolutionary relationship are discussed. Received 20 July 1998; received after revision 9 September 1998; accepted 23 September 1998     

Specific isomerization of rhodopsin-bound 11-cis-retinal to all-trans-retinal under thermal denaturation

The natural ligand of the retinal photoreceptor rhodopsin, 11-cis-retinal, is isomerized to its all-trans configuration as a consequence of light absorption in the first step of the visual phototransduction process. Here we show, by means of difference spectroscopy and high-performance liquid chromatography analysis, that thermal denaturation of rhodopsin induces the same type of isomerization. This effect is likely due to thermally induced conformational rearrangements of amino acid residues in the retinal-binding pocket – possibly implying helical movements – and highlights the tight coupling between 11-cis-retinal and opsin. This effect could have implications in the instability and functional changes seen for certain mutations in rhodopsin associated with retinal disease, and in the stability of the different conformers induced by mutations in other G protein-coupled receptors.Received 25 March 2003; received after revision 6 August 2003; accepted 9 September 2003     

Intercellular junctions of hyperplastic retinal pigment epithelium

Summary In rats with retinopathies induced by excess fluorescent light or injections of urethane, the retinal pigment epithelium (RPE) undergoes focal hyperplasia. Neither intravascularly injected horseradish peroxidase or lanthanum nitrate penetrated the sensory retina at these hyperplastic sites. Electron microscopy revealed that this was due to the persistence of intact, tight junctions among a single layer of hyperplastic cells facing the sensory retina. These junctions prevented intraocularly injected microperoxidase from passing as well. Cells within the hyperplastic foci were connected only by adherent junctions that presented no permeability barrier.Supported by a grant from the National Eye Institute to Dr R. Bellhorn, whose support is greatly appreciated, and an unrestricted grant and a Research Manpower Award from Research to Prevent Blindness, Inc.     

Structural and functional relationships between photoreceptor tetraspanins and other superfamily members

The two primary photoreceptor-specific tetraspanins are retinal degeneration slow (RDS) and rod outer segment membrane protein-1 (ROM-1). These proteins associate together to form different complexes necessary for the proper structure of the photoreceptor outer segment rim region. Mutations in RDS cause blinding retinal degenerative disease in both rods and cones by mechanisms that remain unknown. Tetraspanins are implicated in a variety of cellular processes and exert their function via the formation of tetraspanin-enriched microdomains. This review focuses on correlations between RDS and other members of the tetraspanin superfamily, particularly emphasizing protein structure, complex assembly, and post-translational modifications, with the goal of furthering our understanding of the structural and functional role of RDS and ROM-1 in outer segment morphogenesis and maintenance, and our understanding of the pathogenesis associated with RDS and ROM-1 mutations.     

Functional diversity of melanopsins and their global expression in the teleost retina

Melanopsin (OPN4) is an opsin photopigment that, in mammals, confers photosensitivity to retinal ganglion cells and regulates circadian entrainment and pupil constriction. In non-mammalian species, two forms of opn4 exist, and are classified into mammalian-like (m) and non-mammalian-like (x) clades. However, far less is understood of the function of this photopigment family. Here we identify in zebrafish five melanopsins (opn4m-1, opn4m-2, opn4m-3, opn4x-1 and opn4x-2), each encoding a full-length opsin G protein. All five genes are expressed in the adult retina in a largely non-overlapping pattern, as revealed by RNA in situ hybridisation and immunocytochemistry, with at least one melanopsin form present in all neuronal cell types, including cone photoreceptors. This raises the possibility that the teleost retina is globally light sensitive. Electrophysiological and spectrophotometric studies demonstrate that all five zebrafish melanopsins encode a functional photopigment with peak spectral sensitivities that range from 470 to 484 nm, with opn4m-1 and opn4m-3 displaying invertebrate-like bistability, where the retinal chromophore interchanges between cis- and trans-isomers in a light-dependent manner and remains within the opsin binding pocket. In contrast, opn4m-2, opn4x-1 and opn4x-2 are monostable and function more like classical vertebrate-like photopigments, where the chromophore is converted from 11-cis to all-trans retinal upon absorption of a photon, hydrolysed and exits from the binding pocket of the opsin. It is thought that all melanopsins exhibit an invertebrate-like bistability biochemistry. Our novel findings, however, reveal the presence of both invertebrate-like and vertebrate-like forms of melanopsin in the teleost retina, and indicate that photopigment bistability is not a universal property of the melanopsin family. The functional diversity of these teleost melanopsins, together with their widespread expression pattern within the retina, suggests that melanopsins confer global photosensitivity to the teleost retina and might allow for direct “fine-tuning” of retinal circuitry and physiology in the dynamic light environments found in aquatic habitats.     

Intraocular 6-hydroxydopamine prevents the persistent estrus induced by continuous light

Following the intraocular injection of 6-hydroxydopamine, which can destroy the retinal dopaminergic neurons, female rats showed a normal estrous cycle in LD 12:12 but not a persistent estrus in continuous light.     

Effect of alpha-tocopherol (vitamin E) on the utilization of vitamin A and induction of cytochrome P 450 in rats receiving DDT]

During detoxication processes of DDT, there is, in the Rat, interference between vitamins A and E (D.1.alpha-tocopherol acetate). Vitamin E spares retinal and has a slighter but significant influence on P 450 cytochrome induction. These results are discussed.