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Physiological function of S-cone system is not enhanced in rd7 mice.

The rd7mouse is a mutant mouse with a relatively late development of retinal degeneration. Earlier studies have shown that rd7 mice have a distinctive pattern of retinal dysplasia with an increased number of cone cells, particularly those with S (short wavelength)-opsin immunoreactivity. These alterations of the rd7 retina are caused by a mutation in the photoreceptor cell-specific nuclear receptor gene, Nr2e3, which is involved in the signaling pathway regulating photoreceptor cell differentiation, cell maintenance, and cell-cell interactions. The purpose of this study was to determine the physiological properties of the rd7 retina using electroretinographic (ERG) techniques. We found that the maximal a-wave amplitude of the ERG in rd7 mice was already reduced to half of the congenic controls at 6 weeks of age with normal phototransduction sensitivity. The photopic ERGs of rd7 mice were not supernormal, and the amplitudes of the S-cone ERGs were not significantly different from those recorded in controls. These results suggested that even though the number of cones expressing S-opsin is increased, the physiological function of the S-cone system is not enhanced in rd7 mice.

Animals↗

Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme.

To develop an allele independent ribozyme for the treatment of autosomal dominant retinitis pigmentosa (ADRP) associated with mutations in the rhodopsin (RHO) gene, a ribozyme targeting dog, mouse, human but not rat rhodopsin (RHO) mRNA was designed and tested in vitro. Activity of this ribozyme was tested in tissue culture by co-transfection of HEK 293 cells with plasmids expressing opsin mRNA and ribozyme, followed by quantitative RT-PCR to evaluate the level of RHO mRNA. For experiments in vivo, Rz525 driven by the mouse opsin proximal promoter was inserted in plasmids with AAV 2 terminal repeats (TR) and packaged in AAV serotype 5 capsids. AAV-Rz525 was injected subretinally into the right eyes of P23H rat pups. Left eyes were injected with virus expressing GFP from the identical promoter. Animals were analyzed at 4, 8 and 12 weeks post-injection by full field scotopic electroretinography (ERG). After 12 weeks, animals were sacrificed and retinas were dissected, fixed and sectioned. Rz525 had high catalytic activity in vitro and led to a 50% reduction of RHO mRNA in cells. AAV-Rz525 injection into P23H transgenic rats led to significant preservation (about 50%) of scotopic ERG a- and b-wave amplitudes. Histological analysis showed an increased number of ONL nuclei in the central and superior retina of treated eyes relative to control eyes. RT-PCR analysis revealed 46% reduction of transgenic (mouse) RHO mRNA in right eyes relative to left eyes and no change in rat RHO mRNA. AAV5 delivery of Rz525 resulted in a partial rescue of the light response and structural preservation of photoreceptors in transgenic rats. This ribozyme may be a useful component of an RNA replacement gene therapy for ADRP.

Alleles↗

Connecting the navigational clock to sun compass input in monarch butterfly brain.

Migratory monarch butterflies (Danaus plexippus) use a time-compensated sun compass to navigate to their overwintering grounds in Mexico. Although polarized light is one of the celestial cues used for orientation, the spectral content (color) of that light has not been fully explored. We cloned the cDNAs of three visual pigment-encoding opsins (ultraviolet [UV], blue, and long wavelength) and found that all three are expressed uniformly in main retina. The photoreceptors of the polarization-specialized dorsal rim area, on the other hand, are monochromatic for the UV opsin. Behavioral studies support the importance of polarized UV light for flight orientation. Next, we used clock protein expression patterns to identify the location of a circadian clock in the dorsolateral protocerebrum of butterfly brain. To provide a link between the clock and the sun compass, we identified a CRYPTOCHROME-staining neural pathway that likely connects the circadian clock to polarized light input entering brain.

Animal Migration↗

Modeling color percepts of dichromats.

Protanopes and deuteranopes, despite lacking a chromatic dimension at the receptor level, use the color terms "red" and "green", together with "blue" and "yellow", to describe their color percepts. Color vision models proposed so far fail to account for these findings in dichromats. We confirmed, by the method of hue scaling, the consistent use of these color terms, as well as their dependence on intensity, in subjects shown to have only a single X-chromosomal opsin gene each. We present a model for the processing of photoreceptor signals which, under physiologically plausible assumptions, achieves a trichromat-like representation of dichromatic receptor signals. Key feature of the dichromat model is the processing of the photoreceptor signals in parallel channels with different gains and nonlinearities. In this way, the two-dimensional receptor signals are represented on a manifold in a higher-dimensional space, supporting categorization for efficient image segmentation. Introducing a third cone opsin yields a model that explains normal, trichromat hue scaling.

Base Sequence↗

The nature of the primary photochemical events in rhodopsin and isorhodopsin.

The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B. (b) The presence of a small activation barrier (~0.2 kcal mol-1) in the 9-cis - 9-trans excited-state surface is associated with the location of the counterion as well as the intrinsic photophysical properties of the 9-cis chromophore. The principal difference between the 1 1-cis -c 1 -transphoto reaction surface and the 9-cis - 9-trans photoreaction surface is the lack of effective electrostatic stabilization of distorted 9 = 10 conformations due to incomplete charge polarization. (c) Hydrogen bonding to the imine proton, ifpresent, does not involve the counterion. We conclude that water in the active site, or secondary interactions with the protein (not involving the CTN), are responsible. (d) All photochemical transformations involve one-bond photoisomerizations.This prediction is based on the observation of a very small excited state barrier for the I -- B photoreaction and a negative barrier for the R - B phototransformation, coupled with the theoretical prediction that all two-bond photoisomerizations have significant S, barriers while one-bond photoisomerizations have small to negative S, barriers.(e) Rhodopsin is energetically stabilized relative to isorhodopsin due to both electrostatic interactions and conformational distortion, both favoring stabilization of R. The INDO-PSDCI calculations suggest that rhodopsin chromophore-CTN electrostatic interactions provide an enhanced stabilization of -2 kcal mol-1 relative to I. Conformational distortion of the 9-cis chromophore-lysine system accounts for -3 kcal mol-1. (f) Energy storage in bathorhodopsin is-60% conformational distortion and 40% charge separation. Our model predicts that the majority of the chromophore protein conformational distortion energy involves interaction of the C,3(-CH3)=CI4--C,5=N-lysine moiety with nearby (unknown) protein residues. (g) Strong interactions between the counterion and the chromophore in R and I will generate weak, but potentially observable charge-transfer bands in the near infrared. The key predictions are the presence of an observable charge-transfer transition at 859 nm (1 1,640 cm- 1) in I and an analogous, but slightly weaker band at 897 nm (11,150 cm-1) in R. Both transitions involve the transfer of an electron from the counterion into low-lying l theta* molecular orbitals.

Binding Sites↗

Photopigments and photoentrainment in the Syrian golden hamster.

The Syrian golden hamster (Mesocricetus auratus) is an important model in the study of circadian rhythms. However, as in other mammals, little is known about the photoreceptors that mediate circadian entrainment. Using immunocytochemistry and RNA blot hybridization, we found no evidence for the presence of blue-/UV-sensitive opsin. In contrast, green-sensitive cone opsin was demonstrated in the retina both by immunocytochemistry and reverse-transcription PCR. When used as a probe in RNA blot hybridization, this PCR fragment labelled one transcript (5.8 kb) in hamster retinal RNA. These findings are in accordance with preliminary data from other investigators using electroretinography, which showed one cone-mediated photoreceptive mechanism with a maximum sensitivity of 501 nm, but none at shorter wavelengths. However, we found that non-saturating pulses of ultraviolet radiation (357 nm) caused phase shifts in locomotor behaviour. These results corroborate earlier reports that UV radiation can regulate the photoperiodic response in this animal. Having confirmed these apparently contradictory earlier reports, we discuss the mechanisms that might create a UV-triggered non-visual response in a green cone monochromat. Finally, we propose the use of the Syrian golden hamster as a model for photoreceptor development and function in the absence of S/UV cones.

Amino Acid Sequence↗

Isoprenoid lipid metabolism in the retina: dynamics of squalene and cholesterol incorporation and turnover in frog rod outer segment membranes.

Frogs were injected intravitreally with [3H]acetate, and the formation of [3H]-labeled squalene and cholesterol in the retina and their incorporation into rod outer segment (ROS) membranes were evaluated biochemically over a 60-day time course. ROS [3H]squalene specific activity was maximal by 1-3 days, then declined with a half-time of approximately 20-30 days. In contrast, the specific activity of ROS [3H]cholesterol initially increased to a level substantially less than that of [3H]squalene, and then remained constant. Thus, ROS squalene appears to turn over without obligatory conversion to, or coturnover with, ROS cholesterol. When [3H]acetate was injected into one eye, radiolabel in non-saponifiable lipids of the contralateral retina represented < 1% of those recovered from the ipsilateral retina; hence, systemic contributions to de novo synthesis were obviated. Long-term (> or = 8 hr) in vitro incubations of isolated retinas with [3H]acetate resulted in incorporation of [3H]-labeled sterols and squalene into ROS, at levels comparable to those observed in ROS from companion incubated eyecup preparations and from retinas 8 hr after intravitreal injection of [3H]acetate. These results demonstrate that the in vitro system faithfully reflects the in vivo biosynthetic capacity with respect to isoprenoid lipid metabolism, and suggest that de novo synthesis within the neural retina is responsible for generating most, if not all, of the [3H]squalene and [3H]cholesterol formed under the given conditions. Treatment of retinas in vitro with brefeldin A or energy poisons blocked transport of newly synthesized opsin, but not squalene, to the ROS. Furthermore, frogs maintained at 8 degrees C exhibited marked suppression of incorporation of newly synthesized protein into the ROS, while [3H]squalene incorporation was only minimally reduced, compared with frogs maintained at 22 degrees C. These results are consistent with prior findings that suggest that lipids are transported to the ROS by a mechanism distinct and independent from that employed for intracellular trafficking of opsin and other ROS-destined membrane proteins.

Animals↗

Pinopsin expressed in the retinal photoreceptors of a diurnal gecko.

Retinal cDNAs encoding the putative opsins, dg3 and dg4, were isolated from a diurnal gecko, Phelsuma madagascariensis longinsulae. dg3 mRNA is localized in about 20% of the thin members of type C double cones, and likely encodes an opsin of the ultraviolet-sensitive pigment. Surprisingly, dg4 is very similar to chicken pinopsin, a pineal-specific photoreceptive molecule. An anti-dg4 antiserum recognized a small population of photoreceptor outer segments in the retina and a large number of pinealocytes. Our results suggest that P. m. longinsulae expresses pinopsin in its retina, which usually plays a role as a photoreceptive molecule in the pineal organ.

Amino Acid Sequence↗

Spectral tuning of a circadian photopigment in a subterranean 'blind' mammal (Spalax ehrenbergi).

The atrophied subcutaneous eyes of Spalax ehrenbergi (the blind mole rat) express a long wavelength sensitive (LWS) cone opsin. Our data provide strong evidence that this photopigment is spectrally tuned to enhance photon capture in the red light environment of the eye. Furthermore, novel mechanisms appear partially responsible for this sensory fine-tuning. These data support the hypothesis that the LWS opsin of Spalax acts as a functional photopigment and that it is not a 'residue' of the pre-subterranean visual system. As the eye of Spalax has only one known function, the entrainment of circadian rhythms to environmental light, the LWS photopigment is implicated in this task. These results, together with our recent findings that rod and cone photopigments are not required for murine photoentrainment, suggest that multiple photopigments (classical and novel) mediate the effects of light on the mammalian circadian system.

Amino Acid Sequence↗

Transport to the photoreceptor outer segment by myosin VIIa and kinesin II.

This paper reviews our studies in which we have used mutant mice to investigate the roles of myosin VIIa and kinesin II in the transport of proteins to the photoreceptor outer segment. These studies suggest that both motors participate in moving opsin along the connecting cilium. Given the velocities measured for these motors in vitro, it is predicted that the resulting concentration of opsin in the plasma membrane of the connecting cilium is surprisingly low.

Animals↗

Cone pigment variations in four genera of new world monkeys.

Previous research revealed significant individual variations in opsin genes and cone photopigments in several species of platyrrhine (New World) monkeys and showed that these in turn can yield significant variations in color vision. To extend the understanding of the nature of color vision in New World monkeys, electroretinogram flicker photometry was used to obtain spectral sensitivity measurements from representatives of four platyrrhine genera (Cebus, Leontopithecus, Saguinus, Pithecia). Animals from each genus were found to be polymorphic for middle to long-wavelength (M/L) sensitive cones. The presence of a short-wavelength sensitive photopigment was established as well so these animals conform to the earlier pattern in predicting that all male monkeys are dichromats while, depending on their opsin gene array, individual females can be either dichromatic or trichromatic. Across subjects a total of five different M/L cone pigments were inferred with a subset of three of these present in each species.

Adaptation, Ocular↗

Rayleigh match ranges of red/green color-deficient observers: psychophysical and molecular studies.

Large-field Rayleigh match ranges were measured in 27 red/green color-deficient male observers, using bright, temporally alternating, 3-9 deg annular test fields. The observers' X-linked opsin gene arrays were characterized by molecular genetic techniques, and used to infer the absorption maxima of each observer's L and/or M cone photopigment(s). Measured match ranges decreased rapidly as the inferred separation in pigment absorption maxima increased from 0 to 2-3 nm, and varied irregularly thereafter. Following He & Shevell [(1995) Vision Research, 35, 2579-2588] predicted match ranges were calculated for various pigment separations and assumed values of pigment optical density. The predicted variations in match range encompassed the measured match ranges of most (but not all) of the color-deficient observers. The calculations also showed that differences in pigment optical density, in two cone types containing the same pigment, are sufficient to allow a moderate degree of chromatic discrimination. Such models thus provide a possible account of the fact that some color-deficient observers, with only a single X-linked opsin gene, can make red/green chromatic discriminations.

Adolescent↗

Visual pigments of African cichlid fishes: evidence for ultraviolet vision from microspectrophotometry and DNA sequences.

We have found evidence for ultraviolet visual capabilities in a Lake Malawi cichlid fish, Metriaclima zebra. Microspectrophotometry of single cones revealed a visual pigment with peak sensitivity at 368+/-4 nm. M. zebra also expresses a putative ultraviolet opsin gene whose sequence is closely related to the SWS-1 opsin for other fishes. Several other African cichlids have a functional copy of this UV gene in their genomic DNA, but do not appear to express this gene as adults. These results suggest that ultraviolet vision is important for some cichlid fishes. UV wavelengths should therefore be included in future studies of cichlid vision, behavior and color patterns.

Amino Acid Sequence↗

Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland.

The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian pacemaker entrained to environmental light-dark cycles [G.M. Cahill, Brain Res. 708 (1996) 177-181]. Although pinopsin has been found in the pineal glands of birds and reptiles, the molecular identity responsible for fish pineal photosensitivity remains unclear. This study reports identification of a novel opsin gene expressed in the zebrafish pineal gland. The deduced amino acid sequence is similar to, but not identical (74% identity) with that of canonical rhodopsin in the zebrafish retina. This novel rhodopsin is expressed in the majority of pineal cells but not in retinal cells, and hence named exo-rhodopsin after extra-ocular rhodopsin. This study first shows that two different rhodopsin genes are expressed in an individual animal each within a unique location. A phylogenetic analysis indicated that the exo-rhodopsin gene was produced by a duplication of the rhodopsin gene at an early stage in the ray-finned fish lineage. As expected, the exo-rhodopsin gene was found in the medakafish and European eel genomes, suggesting strongly that exo-rhodopsin is a pineal opsin common to teleosts. Identification of exo-rhodopsin in the zebrafish provides an opportunity for studying the role of pineal photoreceptive molecules by using genetic approaches.

Amino Acid Sequence↗

Cloning and expression of the red visual pigment gene of goat (Capra hircus).

We have cloned and sequenced the red opsin gene, rCh, of goat (Capra hircus). When rCh is expressed in cultured cells and reconstituted with 11-cis retinal, the resulting visual pigment has a wavelength of maximal absorption (lambda max) of 553 nm. This result and the deduced aa sequence of the goat red opsin suggest that the lambda max values of the red pigments of goat and human are based mainly on Y277 and T285. The slightly lower lambda max value of the red pigment in goat than in human (approximately 560 nm) can be explained by a single aa difference between the goat (A180) and human (S180) red pigments.

Animals↗

The Python pit organ: imaging and immunocytochemical analysis of an extremely sensitive natural infrared detector.

The Python infrared-sensitive pit organ is a natural infrared imager that combines high sensitivity, ambient temperature function, microscopic dimensions, and self-repair. We are investigating the spectral sensitivity and signal transduction process in snake infrared-sensitive neurons, neither of which is understood. For example, it is unknown whether infrared receptor neurons function on a thermal or a photic mechanism. We imaged pit organs in living Python molurus and Python regius using infrared-sensitive digital video cameras. Pit organs were significantly more absorptive and/or emissive than surrounding tissues in both 3-5 microns and 8-12 microns wavelength ranges. Pit organs exhibited greater absorption/emissivity in the 8-12 microns range than in the 3-5 microns range. To directly test the relationship between photoreceptors and pit organ infrared-sensitive neurons, we performed immunocytochemistry using antisera directed against retinal photoreceptor opsins. Retinal photoreceptors were labeled with antisera specific for retinal opsins, but these antisera failed to label terminals of infrared-sensitive neurons in the pit organ. Infrared-receptive neurons were also distinguished from retinal photoreceptors on the basis of their calcium-binding protein content. These results indicate that the pit organ absorbs infrared radiation in two major atmospheric transmission windows, one of which (8-12 microns) matches emission of targeted prey, and that infrared receptors are biochemically distinct from retinal photoreceptors. These results also provide the first identification of prospective biochemical components of infrared signal transduction in pit organ receptor neurons.

Animals↗

Evolution of color vision.

Color vision is achieved by comparing the inputs from retinal photoreceptor neurons that differ in their wavelength sensitivity. Recent studies have elucidated the distribution and phylogeny of opsins, the family of light-sensitive molecules involved in this process. Interesting new findings suggest that animals have evolved a strategy to achieve specific sensitivity through the mutually exclusive expression of different opsin genes in photoreceptors.

Animals↗

Developmental patterns of protein expression in photoreceptors implicate distinct environmental versus cell-intrinsic mechanisms.

The present study has examined the spatial and temporal expression patterns of various proteins associated with the structure and function of mature photoreceptor outer segments in the developing ferret's retina using immunocytochemistry and RT-PCR. One set of proteins, including rod opsin, arrestin, and recoverin, was detected progressively in photoreceptors as they became postmitotic, being expressed well before the differentiation of outer segments. A second set of proteins, including beta- and gamma-transducin, cGMP-phosphodiesterase, phosducin, rhodopsin kinase, rod cGMP-gated cation channel protein, and peripherin, displayed a contrasting temporal onset and pattern of spatial emergence. These latter proteins first became detectable either shortly before or coincident with outer segment formation, and were expressed simultaneously in both older and younger photoreceptor cells. A third set, the short wavelength-sensitive (SWS) and medium wavelength-sensitive (MWS) cone opsin proteins, was the last to be detected, but materialized in a spatio-temporal pattern reminiscent of the neurogenetic gradient of the cones. These different spatial and temporal patterns indicate that cellular maturation must play a primary role in regulating the onset of expression of some of these proteins, while extrinsic signals must act to coordinate the expression of other proteins across photoreceptors of different ages.

3',5'-Cyclic-GMP Phosphodiesterases↗