Identification of the glycosylation sites of Opsin3 and its glycosylation modification function / 生物工程学报

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

The Antidepressant Effect of Light Therapy from Retinal Projections / 神经科学通报·英文版

Observations from clinical trials have frequently demonstrated that light therapy can be an effective therapy for seasonal and non-seasonal major depression. Despite the fact that light therapy is known to have several advantages over antidepressant drugs like a low cost, minimal side-effects, and fast onset of therapeutic effect, the mechanism underlying light therapy remains unclear. So far, it is known that light therapy modulates mood states and cognitive functions, involving circadian and non-circadian pathways from retinas into brain. In this review, we discuss the therapeutic effect of light on major depression and its relationship to direct retinal projections in the brain. We finally emphasize the function of the retino-raphe projection in modulating serotonin activity, which probably underlies the antidepressant effect of light therapy for depression.

Changes of S-opsin expression in guinea pigs with flickering light-induced and form-deprived myopia / 中国实验动物学报

Objective To observe the changes of S-opsin expression in guinea pigs with flickering light-induced and form-deprived myopia,and to investigate the causes.Methods Thirty-six two-week-old healthy guinea pigs were randomly assigned to three groups:Flickering light-induced myopia group(FLM group,n=13),form-deprived myopia group(FDM group,n=12) and control group(n=11).For the FLM group,the cages were equipped with astroboscope(0.5 Hz),and LEDs were used as the light source.The right eyes of the guinea pigs in FDM group wore translucent goggles which did not interfere with the normal activity of their eyelids.No special treatment was given to the guinea pigs in the normal groups.All measurements were performed prior to and then after 6 weeks of treatment.The first measurement day was recorded as 0 week.Biological parameters,such as the refraction,axial length(AL) and corneal radius of curvature(CRC),were measured and fundus photography is performed before and after 6 weeks of the treatment.The expression of S-opsin was observed and analyzed by immunofluorescence technique and image analysis system.Results Before the treatment,no significant difference was found in three biometric measurements including refraction,AL and CRC between the groups at 0 week(P>0.05).After the treatment for 6 weeks,significant differences were found in changes of both the biometric measurements between the FLM and control groups,and between the FDM and control groups(P0.05).Expression of S-opsin differed in the FLM and FDM groups.For the mean gray values of green channel,compared with the control group respectively,significant differences were found in both the FLM and FDM group(P<0.001).The mean gray value of green channel of the FLM group was higher,however the mean gray value of green channel of the FDM group was lower.Conclusions Both guinea pig models of flickering light-induced and form-deprived myopia can be established successfully.S-opsin is increased in the flickering light-induced myopia model and decreased in the form-deprived myopia model,indicating that the mechanisms of formation of these two experimental myopia models may be different.

Establishment of a transgenic zebrafish line with tdTomato expression in the ultraviolet-sensitive cone photoreceptors / 中国实验动物学报

Objective To better understand the mechanisms of cone opsin transport , we set to generate a trans-genic zebrafish line with red fluorescence proteins expressing in the cone photoreceptors .Methods We used sws1 promot-er to drive the expression of a chimerical protein , in which the last 44 amino acids of rhodopsin of Xenopus laevis were fused to the C-terminus of tdTomato to restrict its localization to the outer segment of photoreceptors .Results We successfully i-solated such a transgenic zebrafish line and confirmed the localization of tdTomato by immunostaining analysis .Conclu-sions This transgenic zebrafish line will help us to better understand the transport mechanisms of cone opsins , especially the transport in live photoreceptors .

The distribution and degeneration pattern of the cone opsins in rd11 mice / 中华实验眼科杂志

Background The retinal degeneration 11 (rd11) mouse is a newly discovered naturally occurring recessive animal model with lysophosphatidylcholine acyltransferase 1 (Lpcatl) mutation.Previous studies showed that the photoreceptor cells are characterized by typical rod-cone degeneration pattern in rd1 1 mice,while cone degeneration pattern in rd11 mice is unclcar.Objective Using immunofluorescence staining techniques with retinal wholemount,we aim to clarify the degeneration patterns of cone-function related M-opsin or S-opsin in different ages of rd1 1 mice.Methods A total of thirty rd1 1 and C57BL/6J mice at postnatal (P) day 14,28,42 (five in each age group) were sacrificed and retinal wholemounts were prepared.Immunohistochemistry was performed to identify the expression of M-opsin or S-opsin in retinal wholemounts,which were photographed with a fluorescent microscope.Cone opsins were compared between rd1 1 retinas and age-matched normal C57BL/6J retinas by manually counting the opsin positive cone cells in different quadrants of the retinas.Results The number of M-opsin or S-opsin positive fluorescent dots in each quadrant was similar at all ages of normal C57BL/6J retina.M-opsin positive fluorescent dots in dorsal/temporal,ventral/temporal,dorsal/nasal and ventral/nasal quadrants of rdl 1 retina at P28 were (414±32),(300± 8),(324 ± 22) and (250± 20)/0.037 mm2,which were lower than the age-matched normal C57BL/6J mice (t =4.114,15.225,7.505,17.990,all at P＜0.05).At the same time the S-opsin positive fluorescent dots in P28 rd11 were (8 ±4),(175 ± 16),(74 ± 13) and (315 ±20)/0.037 mm2,with significant decrease in comparison with those in the age-matched normal C57BL/6J mice (t =8.555,17.076,21.637,13.498,all at P＜0.05).With the development of retinal degeneration in rd11 mice,the M-opsin degeneration spread from central to ventral,nasal and then to temporal and dorsal peripheral retina;and the S-opsin loss started from dorsal/temporal to ventral/nasal retina.Conclusions Most of the M-opsin and S-opsins,especially the S-opsins in rd11 mice,degenerate in 6 weeks.Retinal wholemount and cone opsin immunofluorescent staining provide a useful tool to show the cone degeneration pattern and to evaluate the therapeutic efficiency in ongoing gene therapy study.

The genetics of New World monkey visual pigments

To have color vision, having at least two cone photopigment types with different spectral sensitivities present in distinct photoreceptors is necessary together with the neural circuitry necessary to extract color information. Visual pigments are highly conserved molecules, but differences can be found among vertebrate groups. Primates have a variety of cone photopigments (i.e., opsins) that are expressed by polymorphic genes. This article examines the diversity of cone photopigments in New World monkeys and their behavioral relevance...

Influence of long-time illumination of monochromatic light on density of cones and opsin expression in guinea pig / 中华实验眼科杂志

Background The visual system of animal have to optimally adjust in various environmental conditions in order to obtain stable and effective visual funetion.However,the color vision system of animals which encounter uncertainty of spectral signals should be plastic.Whether the densities of various cones and expression of opsins change with long-time spectral deprivation is unclear.Objective This study was to investigate the changes of cone density as well as the expression of corresponding opsin and mRNA following the long-term illumination of monochromatic light.Methods Thirty 3-day-old guinea pigs were randomized into 3 groups and exposed tO the 530 nm green light,400 nm purple light and white light for consecutive 8 weeks respectively.The flat-mounted retinal sample was prepared and divided into dorsal zone,ventral zone and mixed zone anatomically according to the distribution of difierent light-sensitive cone.The changes in density of cone cells sensitivited to different colored light were detected by single-1abel or double-label immunocytochemistry.The levels of opsin and its mRNA were determined using Western-blot and real-time PCR respectively.Results The density of green-sensitivity cones was significantly different in the dorsal zone of retina among green light group,purple light group and white light group (F=234.28,P<0.01).Compared with white light group,the density of green-sensitive cones in dorsal retina of green light group was obviously higher but that of purple light group wag evidently lower(q=389.68,P<0.01；q=67.11,P<0.01).No significant difference was found in the density of purple-sensitive cones in the ventral zone of retina among green light group,purple light group and white light group(F=3.14,P>0.05).The density of coexpression of the mixed cone cells was increased in green light group(q=157.55,P<0.01)but decreased in purple light roup (q=254.85,P<0.01)in comparison with white light group.The expression levels of green-opsin and green-opsin mRNA in green light group was significantly elevated(q=184.45,P<0.01；q=4.71,P<0.05),but those of purple light group were evidently declined(q=5.87,P<0.05；q=346.66,P<0.01)in comparison with white light group.There was no statistically significant differences were found in the expression of purple-opsin and its mRNA among all the groups(F=1.24,P>0.05；F=3.27,P>0.05).Conclusion After the exposure of long-time monochromatic light illumination,monochromatic cones density and its opsin in guinea pig occur the corresponding alteration to gain good spatial vision as a compensatory reaction.These outcomes imply that there is some plasticity during the development of color vision.The increase of green-sensitive cones might be from the differentiation of coexpression cones in transition region.

The effects of varied antigen retrieval methods on immunohistochemistry of functional proteins in retinal paraffin section / 眼科研究

Background Antigen retrieval method is the key of improving the successful rate of immunohistochemical assay in paraffin sections.To study an available method of antigen retrieval is a goal to achieve both good immunochemistry result and preserving retinal proteins.ObjectiveThe aim of present study is to investigate tyrosin digestion,high-temperature heat pressure,water bath heating and microwave repair in antigen retrieval for retinal tissues.MethodsRetinal tissue was isolated and obtained from clean Chinchilla rabbits.Four hundreds retinal paraffin sections were prepared.Four kinds of antigen retrieval methods for retinal tissue including tyrosin digestion,high-temperature heat pressure,water bath heating and microwave repair were used respectively.The depigmentated retinal paraffin section without antigen retrieval was used as control.The positive rates of expression of CRALBP,Rhodopsin and opsin proteins were evaluated and compared among four kinds of antigen retrieval methods by immunochemistry.ResultsCRALBP,Rhodopsin and opsin protein was positively expressed in cytoplasm of retinal pigment epithelial cells and the outer segment of photoreceptor respectively.No significant difference was found in the positive expression rates of these three proteins among four kinds of antigen retrieval methods (P＞0.05),but the differences in tissue integrity and background staining were statistically significant (P＜0.01).The structural damage of retina included loss and pucker of scalera,crack of nucleus and abnormal background stain in high-temperature heat pressure method,water bath heating method and microwave retrieval method.However,stable CRALBP,Rhodopsin and opsin protein expression and strain effectiveness,clear background without unspecific staining and integrated tissue were seen in tyrosin digestion method.ConclusionDuring the clinical pathology analysis of retinal tissue,the application of tyrosin digestion in antigen retrieval could obtain a better effectiveness.

Estudo eletrorretinográfico de visão cromática / Electroretinographic study of chromatic vision

OBJETIVO: O objetivo deste estudo é descrever o traçado eletrorretinográfico no gambá sul-americano (Didelphis aurita) obtido com estímulo cromático de comprimento de onda seletivo. O eletrorretinograma é o registro das variações de voltagem nas células retinianas, desencadeadas por estímulo luminoso. O eletrorretinograma representa a atividade elétrica combinada de diferentes células, e sofre variações dependendo da fisiologia retiniana e do método de exame. MÉTODOS: Foram registrados os eletrorretinogramas de seis animais em adaptação ao escuro utilizando filtros cromáticos Kodak Wratten®, e registrada a sensibilidade espectral para comprimentos de onda específicos nas faixas de cores do azul, verde, amarelo, laranja e vermelho. RESULTADOS: Os resultados eletrorretinográficos mais consistentes foram obtidos quando o animal foi estimulado por faixas espectrais seletivas, ao invés de luz branca; e são consistentes com a curva de absorbância das opsinas descritas em fotorreceptores de marsupiais. Estudos prévios sugeriram a tricromacia dos marsupiais por microespectrofotometria de opsinas e imuno-histoquímica de retina. Esse fundamento morfológico não tinha demonstração fisiológica eletrorretinográfica, até este estudo. CONCLUSÃO: O gambá sul-americano tem se mostrado interessante como animal experimental no estudo comparativo da fisiologia visual em mamíferos, especialmente no estudo filogenético da visão cromática. Os marsupiais apresentam um modelo retiniano que superpõe os sistemas fotópico e escotópico; e o gênero Didelphis conserva características encontradas em fósseis do período pleoceno. Portanto, o sistema visual de um marsupial resgata características dos primórdios da evolução dos mamíferos, até o desenvolvimento dos padrões retinianos modernos.

PURPOSE: To describe the electroretinogram of the South-American opossum (Didelphis aurita) obtained by chromatic stimulus of specific wavelengths. The electroretinogram records voltage variations of retinal cells triggered by light stimulation. The electroretinogram represents the combination of electric activity of many different cells and varies according to retinal physiology and examination methods. METHODS: We recorded the electroretinogram of six animals in dark adaptation using chromatic Kodak Wratten® filters, and recorded the spectral sensitivity to specific wavelengths in the spectrum of blue, green, yellow, orange and red light bands. RESULTS: The most consistent electrorretinographic results were obtained when the animals were stimulated by selective spectral bands instead of white light. These results are consistent with the absorbance curve of the opsins described in marsupial photoreceptors. Previous studies using microspectrophotometry of opsins and retinal immunohistochemistry suggested marsupial trichromacy. This morphologic knowledge has not before been physiologically demonstrated by electroretinographic methods. CONCLUSION: The South-American opossum has proven to be an interesting experimental animal for comparative visual physiology studies among other mammals, especially studies on phylogenetic of chromatic vision. The opossum represents a retinal model that superimposes both the photopic and scotopic systems; and the Didelphis genus shows few changes when compared to the fossils of the Pleocene period. Therefore the marsupial's visual system retrieves characteristics from ancient mammal evolution to the retinal patterns found in modern mammals.

Establishment of transgenic mice harboring mouse rod opsin promoter and enhanced green fluorescent protein fusion gene / 第二军医大学学报

Objective: To clone mouse rod opsin promoter (ROP) and establish transgenic mice harboring mouse rod opsin promoter and enhanced green fluorescent protein(mROP-EGFP) fusion gene. Methods: Mouse ROP was cloned from C57BL/6 mouse genomic DNA by polymerase chain reaction (PCR). Expression vector of mROP-EGFP fusion gene were constructed by recombination DNA technique. It was identified by restriction endonucleases digestion and confirmed by DNA sequencing. After Not I restriction endonuclease digestion, the coding elements were microinjected into male pronuclei of mice zygotes to generate transgenic mice. The pups were evaluated by PCR at genomic DNA level and mated with normal mouse. Expression of GFP in retina of transgenic mice was detected by fluorescent microscope. Results: 2. 1 kb mouse rod opsin promoter fragment was amplified from mice genome DNA. Expression vector pmROP-EGFP was constructed successfully. Following microinjection of coding sequence of pmROP-EGFP, 3 pups were verified to integrate the mROP-EGFP fusion gene in their genomic DNA by PCR assay, named C57-TgN (mROP-EGFP )SMMU21, C57-TgN (mROP-EGFP)SM-MU26 and C57-TgN(mROP-EGFP) SMMU27. They could express GFP in retina. Conclusion: 2. 1 kb mouse rod opsin promoter is cloned and expression vector pmROP-EGFP is constructed. mROP-EGFP fusion gene transgenic mice are established, which harboring mROP-EGFP gene and expressing GFP in their retina. This is valuable for studying the development of brain and retina, pathogenesis of retina disorder and retina transplanting.