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Sample records for photoreceptor cell patterning

  1. Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision

    PubMed Central

    Ecker, Jennifer L.; Dumitrescu, Olivia N.; Wong, Kwoon Y.; Alam, Nazia M.; Chen, Shih-Kuo; LeGates, Tara; Renna, Jordan M.; Prusky, Glen T.; Berson, David M.; Hattar, Samer

    2010-01-01

    Using the photopigment melanopsin, intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light to drive circadian clock resetting and pupillary constriction. We now report that ipRGCs are more abundant and diverse than previously appreciated, project more widely within the brain, and can support spatial visual perception. A Cre-based melanopsin reporter mouse line revealed at least five subtypes of ipRGCs with distinct morphological and physiological characteristics. Collectively, these cells project beyond the known brain targets of ipRGCs to heavily innervate the superior colliculus and dorsal lateral geniculate nucleus, retinotopically-organized nuclei mediating object localization and discrimination. Mice lacking classical rod-cone photoreception, and thus entirely dependent on melanopsin for light detection, were able to discriminate grating stimuli from equiluminant gray, and had measurable visual acuity. Thus, non-classical retinal photoreception occurs within diverse cell types, and influences circuits and functions encompassing luminance as well as spatial information. PMID:20624591

  2. Programming Retinal Stem Cells into Cone Photoreceptors

    DTIC Science & Technology

    2015-12-01

    to program human stem cells directly into cones. Using RNA -seq, we identified several genes that are upregulated in advance of the earliest...reverse vision loss. 15. SUBJECT TERMS Cone photoreceptor, retina, retinal stem cell, Otx2, Onecut1, Blimp1, RNA -seq., transcription factors, and...1 Keywords: 1. Cone photoreceptor 2. Retina 3. Retinal stem cell 4. Otx2 5. Onecut1 6. Blimp1 7. RNA -seq. 8. Transcription factors 9

  3. Evolution of eyes and photoreceptor cell types.

    PubMed

    Arendt, Detlev

    2003-01-01

    The evolution of the eye is a matter of debate ever since Darwin's Origin of Species. While morphological comparisons of eye anatomy and photoreceptor cell types led to the view that animal eyes evolved multiple times independently, the molecular conservation of the pax6 eye-specifying cascade has indicated the contrary - that animal eyes evolved from a common, simple precursor, the proto-eye. Morphological and molecular comparative approaches are combined here in a novel Evo-Devo approach, the molecular comparison of cell types ("comparative molecular cell biology"). In the eye, the various types of photoreceptor cells, as well as pigment and lens cells, each require distinct combinations of specifying transcription factors that control their particular differentiation programmes, such as opsin expression in photoreceptors, specific neurotransmitter metabolism, or axonal outgrowth. Comparing the molecular combinatorial codes of cell types of animal extant eyes, their evolutionary histories can be reconstructed. This is exemplified here on the evolution of ciliary and rhabdomeric photoreceptor cells in bilaterian eyes and on the evolution of cell type diversity in the vertebrate retina. I propose that the retinal ganglion, amacrine and horizontal cells are evolutionary sister cell types that evolved from a common rhabdomeric photoreceptor cell precursor.

  4. Avian photoreceptor patterns represent a disordered hyperuniform solution to a multiscale packing problem

    NASA Astrophysics Data System (ADS)

    Jiao, Yang; Lau, Timothy; Hatzikirou, Haralampos; Meyer-Hermann, Michael; Corbo, Joseph C.; Torquato, Salvatore

    2014-02-01

    Optimal spatial sampling of light rigorously requires that identical photoreceptors be arranged in perfectly regular arrays in two dimensions. Examples of such perfect arrays in nature include the compound eyes of insects and the nearly crystalline photoreceptor patterns of some fish and reptiles. Birds are highly visual animals with five different cone photoreceptor subtypes, yet their photoreceptor patterns are not perfectly regular. By analyzing the chicken cone photoreceptor system consisting of five different cell types using a variety of sensitive microstructural descriptors, we find that the disordered photoreceptor patterns are "hyperuniform" (exhibiting vanishing infinite-wavelength density fluctuations), a property that had heretofore been identified in a unique subset of physical systems, but had never been observed in any living organism. Remarkably, the patterns of both the total population and the individual cell types are simultaneously hyperuniform. We term such patterns "multihyperuniform" because multiple distinct subsets of the overall point pattern are themselves hyperuniform. We have devised a unique multiscale cell packing model in two dimensions that suggests that photoreceptor types interact with both short- and long-ranged repulsive forces and that the resultant competition between the types gives rise to the aforementioned singular spatial features characterizing the system, including multihyperuniformity. These findings suggest that a disordered hyperuniform pattern may represent the most uniform sampling arrangement attainable in the avian system, given intrinsic packing constraints within the photoreceptor epithelium. In addition, they show how fundamental physical constraints can change the course of a biological optimization process. Our results suggest that multihyperuniform disordered structures have implications for the design of materials with novel physical properties and therefore may represent a fruitful area for future

  5. FGF Signaling Regulates Rod Photoreceptor Cell Maintenance and Regeneration in Zebrafish

    PubMed Central

    Qin, Zhao; Kidd, Ambrose R.; Thomas, Jennifer L.; Poss, Kenneth D.; Hyde, David R.; Raymond, Pamela A.; Thummel, Ryan

    2011-01-01

    Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgf-dependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways. PMID:21945172

  6. Changing sensitivity to cell death during development of retinal photoreceptors.

    PubMed

    Chiarini, Luciana B; Leal-Ferreira, Mona Lisa; de Freitas, Fabíola G; Linden, Rafael

    2003-12-15

    Photoreceptor cell death occurs during both normal and pathological retinal development. We tested for selective induction and blockade of cell death in either retinal photoreceptors or their precursors. Organotypical retinal explants from rats at postnatal days 3-11 were treated in vitro for 24 hr with thapsigargin, okadaic acid, etoposide, anisomycin, or forskolin. Explant sections were examined for cell death, and identification of either photoreceptors or proliferating/immediate postmitotic cells followed imunohistochemistry for either rhodopsin or bromodeoxyuridine and proliferating cell nuclear antigen, respectively. Photoreceptor cell death was selectively induced by either thapsigargin or okadaic acid, whereas death of proliferating/immediate postmitotic cells was induced by etoposide. Prelabeling of proliferating precursors allowed direct demonstration of changing sensitivity of photoreceptors to various chemicals. Degeneration of both photoreceptors and proliferating/immediate postmitotic cells depended on protein synthesis. Increase of intracellular cyclic AMP blocked degeneration of postmitotic, but not of proliferating, photoreceptor precursors. The selective induction and blockade of cell death show that developing photoreceptors undergo progressive changes in mechanisms of programmed cell death associated with phenotypic differentiation.

  7. Glutathione Peroxidase 4 Is Required for Maturation of Photoreceptor Cells*

    PubMed Central

    Ueta, Takashi; Inoue, Tatsuya; Furukawa, Takahisa; Tamaki, Yasuhiro; Nakagawa, Yasuhito; Imai, Hirotaka; Yanagi, Yasuo

    2012-01-01

    Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells. PMID:22207760

  8. Photoreceptor cell death and rescue in retinal detachment and degenerations

    PubMed Central

    Murakami, Yusuke; Notomi, Shoji; Hisatomi, Toshio; Nakazawa, Toru; Ishibashi, Tatsuro; Miller, Joan W.; Vavvas, Demetrios G.

    2013-01-01

    Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss. PMID:23994436

  9. Choline Accumulation by Photoreceptor Cells of the Rabbit Retina

    NASA Astrophysics Data System (ADS)

    Masland, Richard H.; Mills, John W.

    1980-03-01

    Photoreceptor cells of the rabbit retina accumulate choline from the extracellular environment by an overall process that has a high affinity for choline. These cells do not synthesize acetylcholine; instead, the choline taken up is incorporated into phosphorylcholine and eventually phospholipid. A mechanism for efficient choline accumulation is presumably concomitant to the photoreceptor cell's synthesis of large amounts of membrane for outer segment membrane renewal. Its existence in the photoreceptor cell supports previous evidence that high-affinity choline uptake is not confined to neurons that release acetylcholine, but may be present wherever large amounts of choline are required.

  10. Somatostatin protects photoreceptor cells against high glucose–induced apoptosis

    PubMed Central

    Mazzeo, Aurora; Cazzoni, Daniele; Beltramo, Elena; Hernández, Cristina; Porta, Massimo; Simó, Rafael; Valverde, Ángela M.

    2016-01-01

    Purpose Many cellular and molecular studies in experimental animals and early retinal function tests in patients with diabetic retinopathy (DR) have shown that retinal neurodegeneration is an early event in the pathogenesis of the disease. Somatostatin (SST) is one of the most important neuroprotective factors synthesized by the retina: SST levels are decreased in parallel to retinal neurodegeneration in early stages of DR. In this study, we characterized the induction of apoptosis (programmed cell death) in a 661W photoreceptor-like cell line cultured under high glucose (HG) conditions and the effect of SST. Methods A 661W photoreceptor-like cell line and retinal explants from 10-week-old male C57BL/6 mice were cultured under HG conditions and treated with SST. Results Hyperglycemia significantly reduced the cellular viability by increasing the percentage of apoptotic cells, and this effect was ameliorated by SST (p˂0.05). Activation of caspase-8 by hyperglycemia was found in the 661W cells and retinal explants and decreased in the presence of SST (p˂0.05). Moreover, we detected activation of calpain-2 associated with hyperglycemia-induced cell death, as well as increased protein tyrosine phosphatase 1B (PTP1B) protein levels; both had a pattern of cleavage that was absent in the presence of SST (p˂0.05). Treatment of the 661W cells and retinal explants with SST for 24 h increased the phosphorylation of type 1 insulin-like growth factor receptor (IGF-IR; tyrosine 1165/1166) and protein kinase B (Akt; serine 473), suggesting this survival signaling is activated in the neuroretina by SST (p˂0.05). Conclusions This study has provided new mechanistic insights first into the involvement of calpain-2 and PTP1B in the loss of cell survival and increased caspase-8-dependent apoptosis induced by hyperglycemia in photoreceptor cells and second, on the protective effect of SST against apoptosis by the enhancement of IGF-IR-mediated Akt phosphorylation. PMID:28050125

  11. Programming Retinal Stem Cells into Cone Photoreceptors

    DTIC Science & Technology

    2015-12-01

    this grant, we sought to investigate the mechanisms that regulate the earliest events in cone photoreceptor development and to exploit this knowledge ...identified 236 genes that were differentially expressed (P < 0.01, false discovery rate < 0.25) between DMSO and DAPT conditions at times that preceded

  12. Multipotent stem cells isolated from the adult mouse retina are capable of producing functional photoreceptor cells.

    PubMed

    Li, Tianqing; Lewallen, Michelle; Chen, Shuyi; Yu, Wei; Zhang, Nian; Xie, Ting

    2013-06-01

    Various stem cell types have been tested for their potential application in treating photoreceptor degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Only embryonic stem cells (ESCs) have so far been shown to generate functional photoreceptor cells restoring light response of photoreceptor-deficient mice, but there is still some concern of tumor formation. In this study, we have successfully cultured Nestin(+)Sox2(+)Pax6(+) multipotent retinal stem cells (RSCs) from the adult mouse retina, which are capable of producing functional photoreceptor cells that restore the light response of photoreceptor-deficient rd1 mutant mice following transplantation. After they have been expanded for over 35 passages in the presence of FGF and EGF, the cultured RSCs still maintain stable proliferation and differentiation potential. Under proper differentiation conditions, they can differentiate into all the major retinal cell types found in the adult retina. More importantly, they can efficiently differentiate into photoreceptor cells under optimized differentiation conditions. Following transplantation into the subretinal space of slowly degenerating rd7 mutant eyes, RSC-derived photoreceptor cells integrate into the retina, morphologically resembling endogenous photoreceptors and forming synapases with resident retinal neurons. When transplanted into eyes of photoreceptor-deficient rd1 mutant mice, a RP model, RSC-derived photoreceptors can partially restore light response, indicating that those RSC-derived photoreceptors are functional. Finally, there is no evidence for tumor formation in the photoreceptor-transplanted eyes. Therefore, this study has demonstrated that RSCs isolated from the adult retina have the potential of producing functional photoreceptor cells that can potentially restore lost vision caused by loss of photoreceptor cells in RP and AMD.

  13. Cell Non-autonomous Function of Ceramidase in Photoreceptor Homeostasis

    PubMed Central

    Acharya, Jairaj K.; Dasgupta, Ujjaini; Rawat, Satinder S.; Yuan, Changqing; Sanxaridis, Parthena D.; Yonamine, Ikuko; Karim, Pusha; Nagashima, Kunio; Brodsky, Michael H.; Tsunoda, Susan; Acharya, Usha

    2008-01-01

    SUMMARY Neutral Ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila Ceramidase. We show that secreted Ceramidase functions in a cell non-autonomous manner to maintain photoreceptor homeostasis. In the absence of Ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of Rhodopsin, and do not respond to light stimulus. Consistent with a cell non-autonomous function, our studies show that over expression of Ceramidase in a tissue distant from the photoreceptors can suppress photoreceptor degeneration in an Arrestin mutant and facilitate membrane turnover in a Rhodopsin null mutant. Furthermore, our results show that secreted CDase is internalized and localizes to endosomes. Our findings are the first to establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function. PMID:18184565

  14. Understanding Cone Photoreceptor Cell Death in Achromatopsia.

    PubMed

    Carvalho, Livia S; Vandenberghe, Luk H

    2016-01-01

    Colour vision is only achieved in the presence of healthy and functional cone photoreceptors found in the retina. It is an essential component of human vision and usually the first complaint patients undergoing vision degeneration have is the loss of daylight colour vision. Therefore, an understanding of the biology and basic mechanisms behind cone death under the degenerative state of retinal dystrophies and how the activation of the apoptotic pathway is triggered will provide valuable knowledge. It will also have broader applications for a spectrum of visual disorders and will be critical for future advances in translational research.

  15. Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish

    PubMed Central

    Sotolongo-Lopez, Mailin; Alvarez-Delfin, Karen; Saade, Carole J.; Vera, Daniel L.; Fadool, James M.

    2016-01-01

    The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7) regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub) mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation across species

  16. Photoreceptor Cells Produce Inflammatory Mediators That Contribute to Endothelial Cell Death in Diabetes

    PubMed Central

    Tonade, Deoye; Liu, Haitao; Kern, Timothy S.

    2016-01-01

    Purpose Recent studies suggest that photoreceptor cells regulate local inflammation in the retina in diabetes. The purpose of this study was to determine if photoreceptor cells themselves produce inflammatory proteins in diabetes and if soluble factors released by photoreceptors in elevated glucose induce inflammatory changes in nearby cells. Methods Laser capture microdissection was used to isolate the outer retina (photoreceptors) from the inner retina in nondiabetic and diabetic mice. Diabetes-induced changes in the expression of inflammatory targets were assessed by reverse transcription polymerase chain reaction and immunohistochemistry. Cell culture experiments were carried out to determine if photoreceptors in vitro and ex vivo release soluble mediators that can stimulate nearby cells. Photoreceptor contribution to leukocyte-mediated endothelial cell death was tested using coculture models. Results Messenger ribonucleic acid and protein expression levels for inflammatory proteins intercellular adhesion molecule 1 (ICAM1), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) were increased in photoreceptors cells in diabetes. In vitro and ex vivo studies show that photoreceptor cells in elevated glucose release mediators that can induce tumor necrosis factor-α in leukocytes and endothelial cells, but not in glia. The soluble mediators released by photoreceptor cells in elevated glucose are regulated by transforming growth factor β-activated kinase 1 and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) signaling. In contrast to enhanced leukocyte-mediated killing of endothelial cells by leukocytes from wild-type diabetic mice, leukocytes from diabetic mice lacking photoreceptor cells (opsin−/−) did not kill endothelial cells. Conclusions These data indicate that photoreceptor cells are a source of inflammatory proteins in diabetes, and their release of soluble mediators can contribute to the death of retinal capillaries

  17. The Role of Mislocalized Phototransduction in Photoreceptor Cell Death of Retinitis Pigmentosa

    PubMed Central

    Nakao, Takeshi; Tsujikawa, Motokazu; Notomi, Shoji; Ikeda, Yasuhiro; Nishida, Kohji

    2012-01-01

    Most of inherited retinal diseases such as retinitis pigmentosa (RP) cause photoreceptor cell death resulting in blindness. RP is a large family of diseases in which the photoreceptor cell death can be caused by a number of pathways. Among them, light exposure has been reported to induce photoreceptor cell death. However, the detailed mechanism by which photoreceptor cell death is caused by light exposure is unclear. In this study, we have shown that even a mild light exposure can induce ectopic phototransduction and result in the acceleration of rod photoreceptor cell death in some vertebrate models. In ovl, a zebrafish model of outer segment deficiency, photoreceptor cell death is associated with light exposure. The ovl larvae show ectopic accumulation of rhodopsin and knockdown of ectopic rhodopsin and transducin rescue rod photoreceptor cell death. However, knockdown of phosphodiesterase, the enzyme that mediates the next step of phototransduction, does not. So, ectopic phototransduction activated by light exposure, which leads to rod photoreceptor cell death, is through the action of transducin. Furthermore, we have demonstrated that forced activation of adenylyl cyclase in the inner segment leads to rod photoreceptor cell death. For further confirmation, we have also generated a transgenic fish which possesses a human rhodopsin mutation, Q344X. This fish and rd10 model mice show photoreceptor cell death caused by adenylyl cyclase. In short, our study indicates that in some RP, adenylyl cyclase is involved in photoreceptor cell death pathway; its inhibition is potentially a logical approach for a novel RP therapy. PMID:22485131

  18. Photoreceptor Cells Influence Retinal Vascular Degeneration in Mouse Models of Retinal Degeneration and Diabetes

    PubMed Central

    Liu, Haitao; Tang, Jie; Du, Yunpeng; Saadane, Aicha; Tonade, Deoye; Samuels, Ivy; Veenstra, Alex; Palczewski, Krzysztof; Kern, Timothy S.

    2016-01-01

    Purpose Loss of photoreceptor cells is associated with retinal vascular degeneration in retinitis pigmentosa, whereas the presence of photoreceptor cells is implicated in vascular degeneration in diabetic retinopathy. To investigate how both the absence and presence of photoreceptors could damage the retinal vasculature, we compared two mouse models of photoreceptor degeneration (opsin−/− and RhoP23H/P23H ) and control C57Bl/5J mice, each with and without diabetes. Methods Retinal thickness, superoxide, expression of inflammatory proteins, ERG and optokinetic responses, leukocyte cytotoxicity, and capillary degeneration were evaluated at 1 to 10 months of age using published methods. Results Retinal photoreceptor cells degenerated completely in the opsin mutants by 2 to 4 months of age, and visual function subsided correspondingly. Retinal capillary degeneration was substantial while photoreceptors were still present, but slowed after the photoreceptors degenerated. Diabetes did not further exacerbate capillary degeneration in these models of photoreceptor degeneration, but did cause capillary degeneration in wild-type animals. Photoreceptor cells, however, did not degenerate in wild-type diabetic mice, presumably because the stress responses in these cells were less than in the opsin mutants. Retinal superoxide and leukocyte damage to retinal endothelium contributed to the degeneration of retinal capillaries in diabetes, and leukocyte-mediated damage was increased in both opsin mutants during photoreceptor cell degeneration. Conclusions Photoreceptor cells affect the integrity of the retinal microvasculature. Deterioration of retinal capillaries in opsin mutants was appreciable while photoreceptor cells were present and stressed, but was less after photoreceptors degenerated. This finding proves relevant to diabetes, where persistent stress in photoreceptors likewise contributes to capillary degeneration. PMID:27548901

  19. Gene expression analysis of embryonic photoreceptor precursor cells using BAC-Crx-EGFP transgenic mouse.

    PubMed

    Muranishi, Yuki; Sato, Shigeru; Inoue, Tatsuya; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

    2010-02-12

    Crx is a transcription factor which is predominantly expressed in developing and mature photoreceptor cells in the retina, and plays a crucial role in the terminal differentiation of both rods and cones. Crx is one of the earliest-expressed genes specifically in photoreceptor precursors, allowing us to trace photoreceptor precursor cells from embryonic stages to adult stage by visualizing Crx-expressing cells. In the current study, we generated a transgenic mouse line which expresses enhanced green fluorescence protein (EGFP) in the retina driven by the Crx promoter using bacterial artificial chromosome (BAC) transgenesis. EGFP-positive cells were observed in the presumptive photoreceptor layer in the retina at embryonic day 15.5 (E15.5), and continued to be expressed in developing and mature photoreceptor cells up to adult stage. We sorted EGFP-positive photoreceptor precursors at E17.5 using fluorescence-activated cell sorter (FACS), and subsequently performed microarray analysis of the FACS-sorted cells. We observed various photoreceptor genes, especially cone genes, are enriched in the EGFP-positive cells, indicating that embryonic cone photoreceptor precursors are enriched. In addition, we found that most of the EGFP-positive cells were post-mitotic cells. Thus, the transgenic line we established can serve as a useful tool to study both developing and mature photoreceptor cells, including embryonic cone precursors whose analysis has been difficult.

  20. The mouse Crx 5'-upstream transgene sequence directs cell-specific and developmentally regulated expression in retinal photoreceptor cells.

    PubMed

    Furukawa, Akiko; Koike, Chieko; Lippincott, Pia; Cepko, Constance L; Furukawa, Takahisa

    2002-03-01

    Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression of Crx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.

  1. Measurement of Photon Statistics with Live Photoreceptor Cells

    NASA Astrophysics Data System (ADS)

    Sim, Nigel; Cheng, Mei Fun; Bessarab, Dmitri; Jones, C. Michael; Krivitsky, Leonid A.

    2012-09-01

    We analyzed the electrophysiological response of an isolated rod photoreceptor of Xenopus laevis under stimulation by coherent and pseudothermal light sources. Using the suction-electrode technique for single cell recordings and a fiber optics setup for light delivery allowed measurements of the major statistical characteristics of the rod response. The results indicate differences in average responses of rod cells to coherent and pseudothermal light of the same intensity and also differences in signal-to-noise ratios and second-order intensity correlation functions. These findings should be relevant for interdisciplinary studies seeking applications of quantum optics in biology.

  2. Pineal Photoreceptor Cells Are Required for Maintaining the Circadian Rhythms of Behavioral Visual Sensitivity in Zebrafish

    PubMed Central

    Li, Xinle; Montgomery, Jake; Cheng, Wesley; Noh, Jung Hyun; Hyde, David R.; Li, Lei

    2012-01-01

    In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry)] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity. PMID:22815753

  3. Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina.

    PubMed

    Berta, Ágnes I; Boesze-Battaglia, Kathleen; Genini, Sem; Goldstein, Orly; O'Brien, Paul J; Szél, Ágoston; Acland, Gregory M; Beltran, William A; Aguirre, Gustavo D

    2011-01-01

    A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.

  4. Photoreceptor Cell Death, Proliferation and Formation of Hybrid Rod/S-Cone Photoreceptors in the Degenerating STK38L Mutant Retina

    PubMed Central

    Berta, Ágnes I.; Boesze-Battaglia, Kathleen; Genini, Sem; Goldstein, Orly; O'Brien, Paul J.; Szél, Ágoston; Acland, Gregory M.; Beltran, William A.; Aguirre, Gustavo D.

    2011-01-01

    A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7–14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene. PMID:21980341

  5. The Degeneration and Apoptosis Patterns of Cone Photoreceptors in rd11 Mice

    PubMed Central

    Li, Xia; Dai, Xufeng; Han, Juanjuan; Qi, Yan; Liu, Yan; Chang, Bo

    2017-01-01

    The retinal degeneration 11 (rd11) mouse is a new animal model with rapid photoreceptor degeneration. The long-term efficacy of gene therapy has a direct relationship with the onset of photoreceptor degeneration or apoptosis, whereas the degeneration or apoptosis patterns of photoreceptors are still unclear in rd11 mice. The distribution patterns of cone function-related L- and S-opsin were examined by immunofluorescence staining, and the apoptosis was performed by TUNEL assay in rd11 mice. The expression pattern of L-opsin or S-opsin in rd11 retina at postnatal day (P) 14 was similar to the pattern observed in wildtype retina. With increasing age, the expression of L-opsin and S-opsin, especially S-opsin, decreased significantly in rd11 mice. The degeneration of L-opsin began around the optic nerve and expanded to the periphery of the retina, from the ventral/nasal to dorsal/temporal retina, whereas the expression of S-opsin gradually decreased from the dorsal/temporal to ventral/nasal retina. Apoptotic signal appeared at P14 and was strongest at P28 of rd11 mice. The key genes associated with apoptosis confirmed those changes. These indicated that the degeneration and apoptosis of cone photoreceptors began at P14 of rd11 mice, which was a key point for gene therapy. PMID:28168050

  6. bcl-2 Overexpression Reduces Apoptotic Photoreceptor Cell Death in Three Different Retinal Degenerations

    NASA Astrophysics Data System (ADS)

    Chen, Jeannie; Flannery, John G.; Lavail, Matthew M.; Steinberg, Roy H.; Xu, Jun; Simon, Melvin I.

    1996-07-01

    Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

  7. In vitro transdifferentiation of human peripheral blood mononuclear cells to photoreceptor-like cells.

    PubMed

    Komuta, Yukari; Ishii, Toshiyuki; Kaneda, Makoto; Ueda, Yasuji; Miyamoto, Kiyoko; Toyoda, Masashi; Umezawa, Akihiro; Seko, Yuko

    2016-06-15

    Direct reprogramming is a promising, simple and low-cost approach to generate target cells from somatic cells without using induced pluripotent stem cells. Recently, peripheral blood mononuclear cells (PBMCs) have attracted considerable attention as a somatic cell source for reprogramming. As a cell source, PBMCs have an advantage over dermal fibroblasts with respect to the ease of collecting tissues. Based on our studies involving generation of photosensitive photoreceptor cells from human iris cells and human dermal fibroblasts by transduction of photoreceptor-related transcription factors via retrovirus vectors, we transduced these transcription factors into PBMCs via Sendai virus vectors. We found that retinal disease-related genes were efficiently detected in CRX-transduced cells, most of which are crucial to photoreceptor functions. In functional studies, a light-induced inward current was detected in some CRX-transduced cells. Moreover, by modification of the culture conditions including additional transduction of RAX1 and NEUROD1, we found a greater variety of retinal disease-related genes than that observed in CRX-transduced PBMCs. These data suggest that CRX acts as a master control gene for reprogramming PBMCs into photoreceptor-like cells and that our induced photoreceptor-like cells might contribute to individualized drug screening and disease modeling of inherited retinal degeneration.

  8. Dendritic stratification differs among retinal OFF bipolar cell types in the absence of rod photoreceptors

    PubMed Central

    Puller, Christian; Arbogast, Patrick; Keeley, Patrick W.; Reese, Benjamin E.; Haverkamp, Silke

    2017-01-01

    Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the ‘cone-full’ Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells’ dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL. PMID:28257490

  9. Subretinal transplantation of MACS purified photoreceptor precursor cells into the adult mouse retina.

    PubMed

    Eberle, Dominic; Santos-Ferreira, Tiago; Grahl, Sandra; Ader, Marius

    2014-02-22

    Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to

  10. Optogenetic control of cell function using engineered photoreceptors.

    PubMed

    Pathak, Gopal P; Vrana, Justin D; Tucker, Chandra L

    2013-02-01

    Over the past decades, there has been growing recognition that light can provide a powerful stimulus for biological interrogation. Light-actuated tools allow manipulation of molecular events with ultra-fine spatial and fast temporal resolution, as light can be rapidly delivered and focused with sub-micrometre precision within cells. While light-actuated chemicals such as photolabile 'caged' compounds have been in existence for decades, the use of genetically encoded natural photoreceptors for optical control of biological processes has recently emerged as a powerful new approach with several advantages over traditional methods. Here, we review recent advances using light to control basic cellular functions and discuss the engineering challenges that lie ahead for improving and expanding the ever-growing optogenetic toolkit.

  11. Direct observation of light focusing by single photoreceptor cell nuclei.

    PubMed

    Błaszczak, Zuzanna; Kreysing, Moritz; Guck, Jochen

    2014-05-05

    The vertebrate retina is inverted with respect to its optical function, which requires light to pass through the entire tissue prior to detection. The last significant barrier for photons to overcome is the outer nuclear layer formed by photoreceptor cell (PRC) nuclei. Here we experimentally characterise the optical properties of PRC nuclei using bright-field defocusing microscopy to capture near-field intensity distributions behind individual nuclei. We find that some nuclei efficiently focus incident light confirming earlier predictions based on comparative studies of chromatin organisation in nocturnal and diurnal mammals. The emergence of light focusing during the development of mouse nuclei highlights the acquired nature of the observed lens-like behaviour. Optical characterisation of these nuclei is an important first step towards an improved understanding of how light transmission through the retina is influenced by its constituents.

  12. Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice

    PubMed Central

    Barnea-Cramer, Alona O.; Wang, Wei; Lu, Shi-Jiang; Singh, Mandeep S.; Luo, Chenmei; Huo, Hongguang; McClements, Michelle E.; Barnard, Alun R.; MacLaren, Robert E.; Lanza, Robert

    2016-01-01

    Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease. PMID:27405580

  13. Brief report: self-organizing neuroepithelium from human pluripotent stem cells facilitates derivation of photoreceptors.

    PubMed

    Boucherie, Cédric; Mukherjee, Sayandip; Henckaerts, Els; Thrasher, Adrian J; Sowden, Jane C; Ali, Robin R

    2013-02-01

    Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling.

  14. Reprogramming Müller glia via in vivo cell fusion regenerates murine photoreceptors

    PubMed Central

    Simonte, Giacoma; Di Vicino, Umberto; Romo, Neus; Pinilla, Isabel; Nicolás, Marta

    2016-01-01

    Vision impairments and blindness caused by retinitis pigmentosa result from severe neurodegeneration that leads to a loss of photoreceptors, the specialized light-sensitive neurons that enable vision. Although the mammalian nervous system is unable to replace neurons lost due to degeneration, therapeutic approaches to reprogram resident glial cells to replace retinal neurons have been proposed. Here, we demonstrate that retinal Müller glia can be reprogrammed in vivo into retinal precursors that then differentiate into photoreceptors. We transplanted hematopoietic stem and progenitor cells (HSPCs) into retinas affected by photoreceptor degeneration and observed spontaneous cell fusion events between Müller glia and the transplanted cells. Activation of Wnt signaling in the transplanted HSPCs enhanced survival and proliferation of Müller-HSPC hybrids as well as their reprogramming into intermediate photoreceptor precursors. This suggests that Wnt signaling drives the reprogrammed cells toward a photoreceptor progenitor fate. Finally, Müller-HSPC hybrids differentiated into photoreceptors. Transplantation of HSPCs with activated Wnt functionally rescued the retinal degeneration phenotype in rd10 mice, a model for inherited retinitis pigmentosa. Together, these results suggest that photoreceptors can be generated by reprogramming Müller glia and that this approach may have potential as a strategy for reversing retinal degeneration. PMID:27427986

  15. Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

    PubMed

    Kianianmomeni, Arash; Hallmann, Armin

    2015-02-01

    Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study

  16. Anatomy of the Hesse photoreceptor cell axonal system in the central nervous system of amphioxus.

    PubMed

    Castro, Antonio; Becerra, Manuela; Manso, María Jesús; Sherwood, Nancy M; Anadón, Ramón

    2006-01-01

    The present study reports the organization of the Hesse cell axonal system in the central nervous system of the amphioxus, with the use of a polyclonal antiserum raised against lamprey gonadotropin-releasing hormone-I (GnRH-I). In the spinal cord, the rhabdomeric photoreceptor cells of the bicellular organs were well labeled with this antibody. These cells sent smooth, straight, lateral processes that bent and became beaded as they passed ventrally and crossed to the contralateral side of the cord. There, the processes of several cells aggregated to give rise to a longitudinal fiber bundle. Beaded collaterals of these processes were directed to ventral neuropil and did not appear to contact giant Rohde cell axons. The crossed projections of the Hesse photoreceptors are compared with those of vertebrate retinal ganglion cells. Other antisera raised against GnRH weakly labeled rhabdomeric photoreceptors located dorsally in the brain, the Joseph cells. The finding that GnRH antibodies label amphioxus photoreceptor cells and axons is not definitive proof that the photoreceptors contain GnRH. Regardless of whether the antibody recognizes amphioxus GnRH, which has not yet been identified by structure, the antibody has revealed the processes of the Hesse photoreceptor cells.

  17. Expression pattern in retinal photoreceptors of POMGnT1, a protein involved in muscle-eye-brain disease

    PubMed Central

    Uribe, Mary Luz; Haro, Carmen; Campello, Laura; Cruces, Jesús; Martín-Nieto, José

    2016-01-01

    Purpose The POMGNT1 gene, encoding protein O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, is associated with muscle-eye-brain disease (MEB) and other dystroglycanopathies. This gene’s lack of function or expression causes hypoglycosylation of α-dystroglycan (α-DG) in the muscle and the central nervous system, including the brain and the retina. The ocular symptoms of patients with MEB include retinal degeneration and detachment, glaucoma, and abnormal electroretinogram. Nevertheless, the POMGnT1 expression pattern in the healthy mammalian retina has not yet been investigated. In this work, we address the expression of the POMGNT1 gene in the healthy retina of a variety of mammals and characterize the distribution pattern of this gene in the adult mouse retina and the 661W photoreceptor cell line. Methods Using reverse transcription (RT)–PCR and immunoblotting, we studied POMGNT1 expression at the mRNA and protein levels in various mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of its protein product in mouse retinal sections and in 661W cultured cells. The intranuclear distribution of POMT1 and POMT2, the two enzymes preceding POMGnT1 in the α-DG O-mannosyl glycosylation pathway, was also analyzed. Results POMGNT1 mRNA and its encoded protein were expressed in the neural retina of all mammals studied. POMGnT1 was located in the cytoplasmic fraction in the mouse retina and concentrated in the myoid portion of the photoreceptor inner segments, where the protein colocalized with GM130, a Golgi complex marker. The presence of POMGnT1 in the Golgi complex was also evident in 661W cells. However, and in contrast to retinal tissue, POMGnT1 additionally accumulated in the nucleus of the 661W photoreceptors. Colocalization was found within this organelle between POMGnT1 and POMT1/2, the latter associated with euchromatic regions of the nucleus. Conclusions

  18. Macrophage- and RIP3-dependent inflammasome activation exacerbates retinal detachment-induced photoreceptor cell death

    PubMed Central

    Kataoka, K; Matsumoto, H; Kaneko, H; Notomi, S; Takeuchi, K; Sweigard, J H; Atik, A; Murakami, Y; Connor, K M; Terasaki, H; Miller, J W; Vavvas, D G

    2015-01-01

    Detachment of photoreceptors from the retinal pigment epithelium is seen in various retinal disorders, resulting in photoreceptor death and subsequent vision loss. Cell death results in the release of endogenous molecules that activate molecular platforms containing caspase-1, termed inflammasomes. Inflammasome activation in retinal diseases has been reported in some cases to be protective and in others to be detrimental, causing neuronal cell death. Moreover, the cellular source of inflammasomes in retinal disorders is not clear. Here, we demonstrate that patients with photoreceptor injury by retinal detachment (RD) have increased levels of cleaved IL-1β, an end product of inflammasome activation. In an animal model of RD, photoreceptor cell death led to activation of endogenous inflammasomes, and this activation was diminished by Rip3 deletion. The major source of Il1b expression was found to be infiltrating macrophages in the subretinal space, rather than dying photoreceptors. Inflammasome inhibition attenuated photoreceptor death after RD. Our data implicate the infiltrating macrophages as a source of damaging inflammasomes after photoreceptor detachment in a RIP3-dependent manner and suggest a novel therapeutic target for treatment of retinal diseases. PMID:25906154

  19. Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival

    PubMed Central

    Rice, Dennis S.; Calandria, Jorgelina M.; Gordon, William C.; Jun, Bokkyoo; Zhou, Yongdong; Gelfman, Claire M.; Li, Songhua; Jin, Minghao; Knott, Eric J.; Chang, Bo; Abuin, Alex; Issa, Tawfik; Potter, David; Platt, Kenneth A.; Bazan, Nicolas G.

    2015-01-01

    The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity. PMID:25736573

  20. Adalimumab Reduces Photoreceptor Cell Death in A Mouse Model of Retinal Degeneration

    PubMed Central

    Martínez-Fernández de la Cámara, Cristina; Hernández-Pinto, Alberto M.; Olivares-González, Lorena; Cuevas-Martín, Carmen; Sánchez-Aragó, María; Hervás, David; Salom, David; Cuezva, José M.; de la Rosa, Enrique J.; Millán, José M; Rodrigo, Regina

    2015-01-01

    Growing evidence suggests that inflammation is involved in the progression of retinitis pigmentosa (RP) both in patients and in animal models. The aim of this study was to investigate the effect of Adalimumab, a monoclonal anti-TNFα antibody, on retinal degeneration in a murine model of human autosomal recessive RP, the rd10 mice at postnatal day (P) 18. In our housing conditions, rd10 retinas were seriously damaged at P18. Adalimumab reduced photoreceptor cell death, as determined by scoring the number of TUNEL-positive cells. In addition, nuclear poly (ADP) ribose (PAR) content, an indirect measure of PAR polymerase (PARP) activity, was also reduced after treatment. The blockade of TNFα ameliorated reactive gliosis, as visualized by decreased GFAP and IBA1 immunolabelling (Müller cell and microglial markers, respectively) and decreased up-regulation of TNFα gene expression. Adalimumab also improved antioxidant response by restoring total antioxidant capacity and superoxide dismutase activity. Finally, we observed that Adalimumab normalized energetic and metabolic pattern in rd10 mouse retinas. Our study suggests that the TNFα blockade could be a successful therapeutic approach to increase photoreceptor survival during the progression of RP. Further studies are needed to characterize its effect along the progression of the disease. PMID:26170250

  1. Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration

    PubMed Central

    Chen, Yu; Palczewska, Grazyna; Masuho, Ikuo; Gao, Songqi; Jin, Hui; Dong, Zhiqian; Gieser, Linn; Brooks, Matthew J.; Kiser, Philip D.; Kern, Timothy S.; Martemyanov, Kirill A.; Swaroop, Anand; Palczewski, Krzysztof

    2016-01-01

    Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration–approved drugs that act on different G protein (guanine nucleotide–binding protein)–coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders. PMID:27460988

  2. Ectopic photoreceptors and cone bipolar cells in the developing and mature retina.

    PubMed

    Günhan, Emine; van der List, Deborah; Chalupa, Leo M

    2003-02-15

    An antibody against recoverin, the calcium-binding protein, labels photoreceptors, cone bipolar cells, and a subpopulation of cells in the ganglion cell layer. In the present study, we sought to establish the origin and identity of the cells expressing recoverin in the ganglion cell layer of the rat retina. By double labeling with rhodopsin, we demonstrate that early in development some of the recoverin-positive cells in the ganglion cell layer are photoreceptors. During the first postnatal week, these rhodopsin-positive cells are eliminated from the ganglion cell layer, but such neurons remain in the inner nuclear layer well into the first postnatal month. Another contingent of recoverin-positive cells, with morphological features equivalent to those of bipolar cells, is present in the postnatal retina, and approximately 50% of these neurons survive to maturity. The incidence of such cells in the ganglion cell layer was not affected by early transection of the optic nerve, a manipulation that causes rapid loss of retinal ganglion cells. These recoverin-positive cells were not double-labeled by cell-specific markers expressed by photoreceptors, rod bipolar cells, or horizontal and amacrine cells. Based on their staining with recoverin and salient morphological features, these ectopic profiles in the ganglion cell layer are most likely cone bipolar cells. Collectively, the results provide evidence for photoreceptors in the ganglion cell and inner nuclear layers of the developing retina, and a more permanent subpopulation of cone bipolar cells displaced to the ganglion cell layer.

  3. Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

    PubMed Central

    Cho, Kyoung-in; Yu, Minzhong; Hao, Ying; Qiu, Sunny; Pillai, Indulekha C. L.; Peachey, Neal S.; Ferreira, Paulo A.

    2013-01-01

    Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial

  4. Xenopus Bsx links daily cell cycle rhythms and pineal photoreceptor fate.

    PubMed

    D'Autilia, Silvia; Broccoli, Vania; Barsacchi, Giuseppina; Andreazzoli, Massimiliano

    2010-04-06

    In the developing central nervous system, the cell cycle clock plays a crucial role in determining cell fate specification. A second clock, the circadian oscillator, generates daily rhythms of cell cycle progression. Although these two clocks interact, the mechanisms linking circadian cell cycle progression and cell fate determination are still poorly understood. A convenient system to address this issue is the pineal organ of lower vertebrates, which contains only two neuronal types, photoreceptors and projection neurons. In particular, photoreceptors constitute the core of the pineal circadian system, being able to transduce daily light inputs into the rhythmical production of melatonin. However, the genetic program leading to photoreceptor fate largely remains to be deciphered. Here, we report a previously undescribed function for the homeobox gene Bsx in controlling pineal proliferation and photoreceptor fate in Xenopus. We show that Xenopus Bsx (Xbsx) is expressed rhythmically in postmitotic photoreceptor precursors, reaching a peak during the night, with a cycle that is complementary to the daily rhythms of S-phase entry displayed by pineal cells. Xbsx knockdown results in increased night levels of pineal proliferation, whereas activation of a GR-Xbsx protein flattens the daily rhythms of S-phase entry to the lowest level. Furthermore, evidence is presented that Xbsx is necessary and sufficient to promote a photoreceptor fate. Altogether, these data indicate that Xbsx plays a dual role in contributing to shape the profile of the circadian cell cycle progression and in the specification of pineal photoreceptors, thus acting as a unique link between these two events.

  5. Effects of Ranibizumab and Aflibercept on Human Müller Cells and Photoreceptors under Stress Conditions

    PubMed Central

    Shen, Weiyong; Yau, Belinda; Lee, So-Ra; Zhu, Ling; Yam, Michelle; Gillies, Mark C.

    2017-01-01

    Anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of retinal vascular diseases. However, constitutive VEGF also acts as a trophic factor on retinal non-vascular cells. We have studied the effects of aflibercept and ranibizumab on human Müller cells and photoreceptors exposed to starvation media containing various concentrations of glucose, with or without CoCl2-induced hypoxia. Cell survival was assessed by calcein-AM cell viability assays. Expression of heat shock proteins (Hsp) and redox proteins thioredoxin 1 and 2 (TRX1, TRX2) was studied by Western blots. The production of neurotrophic factors in Müller cells and interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors was measured by enzyme-linked immunosorbent assays. Aflibercept and ranibizumab did not affect the viability of both types of cells. Neither aflibercept nor ranibizumab affected the production of neurotrophic factors or expression of Hsp60 and Hsp90 in Müller cells. However, aflibercept but not ranibizumab affected the expression of Hsp60, Hsp9, TRX1 and TRX2 in photoreceptors. Aflibercept and ranibizumab both inhibited the production of IRBP in photoreceptors, aflibercept more so than ranibizumab. Our data indicates that the potential influence of aflibercept and ranibizumab on photoreceptors should be specifically monitored in clinical studies. PMID:28257068

  6. Nrf2 protects photoreceptor cells from photo-oxidative stress induced by blue light.

    PubMed

    Chen, Wan-Ju; Wu, Caiying; Xu, Zhenhua; Kuse, Yoshiki; Hara, Hideaki; Duh, Elia J

    2017-01-01

    Oxidative stress plays a key role in age-related macular degeneration and hereditary retinal degenerations. Light damage in rodents has been used extensively to model oxidative stress-induced photoreceptor degeneration, and photo-oxidative injury from blue light is particularly damaging to photoreceptors. The endogenous factors protecting photoreceptors from oxidative stress, including photo-oxidative stress, are continuing to be elucidated. In this study, we evaluated the effect of blue light exposure on photoreceptors and its relationship to Nrf2 using cultured murine photoreceptor (661W) cells. 661W cells were exposed to blue light at 2500 lux. Exposure to blue light for 6-24 h resulted in a significant increase in intracellular reactive oxygen species (ROS) and death of 661W cells in a time-dependent fashion. Blue light exposure resulted in activation of Nrf2, as indicated by an increase in nuclear translocation of Nrf2. This was associated with a significant induction of expression of Nrf2 as well as an array of Nrf2 target genes, including antioxidant genes, as indicated by quantitative reverse transcription PCR (qRT-PCR). In order to determine the functional role of Nrf2, siRNA-mediated knockdown studies were performed. Nrf2-knockdown in 661W cells resulted in significant exacerbation of blue light-induced reactive oxygen species levels as well as cell death. Taken together, these findings indicate that Nrf2 is an important endogenous protective factor against oxidative stress in photoreceptor cells. This suggests that drugs targeting Nrf2 could be considered as a neuroprotective strategy for photoreceptors in AMD and other retinal conditions.

  7. DICER1 is essential for survival of postmitotic rod photoreceptor cells in mice

    PubMed Central

    Sundermeier, Thomas R.; Zhang, Ning; Vinberg, Frans; Mustafi, Debarshi; Kohno, Hideo; Golczak, Marcin; Bai, Xiaodong; Maeda, Akiko; Kefalov, Vladimir J.; Palczewski, Krzysztof

    2014-01-01

    Photoreceptor cell death is the proximal cause of blindness in many retinal degenerative disorders; hence, understanding the gene regulatory networks that promote photoreceptor survival is at the forefront of efforts to combat blindness. Down-regulation of the microRNA (miRNA)-processing enzyme DICER1 in the retinal pigmented epithelium has been implicated in geographic atrophy, an advanced form of age-related macular degeneration (AMD). However, little is known about the function of DICER1 in mature rod photoreceptor cells, another retinal cell type that is severely affected in AMD. Using a conditional-knockout (cKO) mouse model, we report that loss of DICER1 in mature postmitotic rods leads to robust retinal degeneration accompanied by loss of visual function. At 14 wk of age, cKO mice exhibit a 90% reduction in photoreceptor nuclei and a 97% reduction in visual chromophore compared with those in control littermates. Before degeneration, cKO mice do not exhibit significant defects in either phototransduction or the visual cycle, suggesting that miRNAs play a primary role in rod photoreceptor survival. Using comparative small RNA sequencing analysis, we identified rod photoreceptor miRNAs of the miR-22, miR-26, miR-30, miR-92, miR-124, and let-7 families as potential factors involved in regulating the survival of rods.—Sundermeier, T. R., Zhang, N., Vinberg, F., Mustafi, D., Kohno, H., Golczak, M., Bai, X., Maeda, A., Kefalov, V. J., Palczewski, K. DICER1 is essential for survival of postmitotic rod photoreceptor cells in mice. PMID:24812086

  8. Green tea extract attenuates MNU-induced photoreceptor cell apoptosis via suppression of heme oxygenase-1.

    PubMed

    Emoto, Yuko; Yoshizawa, Katsuhiko; Kinoshita, Yuichi; Yuki, Michiko; Yuri, Takashi; Tsubura, Airo

    2016-01-01

    The effects of green tea extract (GTE) on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis were examined, and the possible mechanisms of action of GTE were assessed. Alterations in the retinal morphological architecture were determined by hematoxylin-eosin staining, vimentin immunoreactivity, and photoreceptor cell apoptosis (TUNEL labeling). Expression of oxidant marker, heme oxygenase (HO)-1, mRNA levels in outer nuclear cells was assessed by laser capture microdissection (LCM). Sprague-Dawley rats were given 40 mg/kg MNU at 7 weeks of age in the absence and presence of 250 mg/kg GTE treatment (once daily from 3 days prior to MNU for a maximum 10 days). Although photoreceptor cell degeneration began 24 hr after MNU, the morphological effects of GTE at the time point were not definitive. However, GTE lowered TUNEL labeling and HO-1 mRNA expression. At 7 days after MNU, photoreceptor damage was attenuated by GTE treatment. Therefore, the ability of GTE to reduce MNU-induced photoreceptor cell apoptosis may be due to its antioxidant properties.

  9. Cell Type-Specific Epigenomic Analysis Reveals a Uniquely Closed Chromatin Architecture in Mouse Rod Photoreceptors

    PubMed Central

    Hughes, Andrew E. O.; Enright, Jennifer M.; Myers, Connie A.; Shen, Susan Q.; Corbo, Joseph C.

    2017-01-01

    Rod photoreceptors are specialized neurons that mediate vision in dim light and are the predominant photoreceptor type in nocturnal mammals. The rods of nocturnal mammals are unique among vertebrate cell types in having an ‘inverted’ nuclear architecture, with a dense mass of heterochromatin in the center of the nucleus rather than dispersed clumps at the periphery. To test if this unique nuclear architecture is correlated with a unique epigenomic landscape, we performed ATAC-seq on mouse rods and their most closely related cell type, cone photoreceptors. We find that thousands of loci are selectively closed in rods relative to cones as well as >60 additional cell types. Furthermore, we find that the open chromatin profile of photoreceptors lacking the rod master regulator Nrl is nearly indistinguishable from that of native cones, indicating that Nrl is required for selective chromatin closure in rods. Finally, we identified distinct enrichments of transcription factor binding sites in rods and cones, revealing key differences in the cis-regulatory grammar of these cell types. Taken together, these data provide insight into the development and maintenance of photoreceptor identity, and highlight rods as an attractive system for studying the relationship between nuclear organization and local changes in gene regulation. PMID:28256534

  10. Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

    PubMed Central

    Viringipurampeer, I A; Shan, X; Gregory-Evans, K; Zhang, J P; Mohammadi, Z; Gregory-Evans, C Y

    2014-01-01

    Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6cw59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6cw59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6cw59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment. PMID:24413151

  11. Photoreceptor Cells With Profound Structural Deficits Can Support Useful Vision in Mice

    PubMed Central

    Thompson, Stewart; Blodi, Frederick R.; Lee, Swan; Welder, Chris R.; Mullins, Robert F.; Tucker, Budd A.; Stasheff, Steven F.; Stone, Edwin M.

    2014-01-01

    Purpose. In animal models of degenerative photoreceptor disease, there has been some success in restoring photoreception by transplanting stem cell–derived photoreceptor cells into the subretinal space. However, only a small proportion of transplanted cells develop extended outer segments, considered critical for photoreceptor cell function. The purpose of this study was to determine whether photoreceptor cells that lack a fully formed outer segment could usefully contribute to vision. Methods. Retinal and visual function was tested in wild-type and Rds mice at 90 days of age (RdsP90). Photoreceptor cells of mice homozygous for the Rds mutation in peripherin 2 never develop a fully formed outer segment. The electroretinogram and multielectrode recording of retinal ganglion cells were used to test retinal responses to light. Three distinct visual behaviors were used to assess visual capabilities: the optokinetic tracking response, the discrimination-based visual water task, and a measure of the effect of vision on wheel running. Results. RdsP90 mice had reduced but measurable electroretinogram responses to light, and exhibited light-evoked responses in multiple types of retinal ganglion cells, the output neurons of the retina. In optokinetic and discrimination-based tests, acuity was measurable but reduced, most notably when contrast was decreased. The wheel running test showed that RdsP90 mice needed 3 log units brighter luminance than wild type to support useful vision (10 cd/m2). Conclusions. Photoreceptors that lack fully formed outer segments can support useful vision. This challenges the idea that normal cellular structure needs to be completely reproduced for transplanted cells to contribute to useful vision. PMID:24569582

  12. Hypoxia-induced metabolic stress in retinal pigment epithelial cells is sufficient to induce photoreceptor degeneration

    PubMed Central

    Kurihara, Toshihide; Westenskow, Peter D; Gantner, Marin L; Usui, Yoshihiko; Schultz, Andrew; Bravo, Stephen; Aguilar, Edith; Wittgrove, Carli; Friedlander, Mollie SH; Paris, Liliana P; Chew, Emily; Siuzdak, Gary; Friedlander, Martin

    2016-01-01

    Photoreceptors are the most numerous and metabolically demanding cells in the retina. Their primary nutrient source is the choriocapillaris, and both the choriocapillaris and photoreceptors require trophic and functional support from retinal pigment epithelium (RPE) cells. Defects in RPE, photoreceptors, and the choriocapillaris are characteristic of age-related macular degeneration (AMD), a common vision-threatening disease. RPE dysfunction or death is a primary event in AMD, but the combination(s) of cellular stresses that affect the function and survival of RPE are incompletely understood. Here, using mouse models in which hypoxia can be genetically triggered in RPE, we show that hypoxia-induced metabolic stress alone leads to photoreceptor atrophy. Glucose and lipid metabolism are radically altered in hypoxic RPE cells; these changes impact nutrient availability for the sensory retina and promote progressive photoreceptor degeneration. Understanding the molecular pathways that control these responses may provide important clues about AMD pathogenesis and inform future therapies. DOI: http://dx.doi.org/10.7554/eLife.14319.001 PMID:26978795

  13. Serial sectioning for examination of photoreceptor cell architecture by focused ion beam technology.

    PubMed

    Mustafi, Debarshi; Avishai, Amir; Avishai, Nanthawan; Engel, Andreas; Heuer, Arthur; Palczewski, Krzysztof

    2011-05-15

    Structurally deciphering complex neural networks requires technology with sufficient resolution to allow visualization of single cells and their intimate surrounding connections. Scanning electron microscopy (SEM), coupled with serial ion ablation (SIA) technology, presents a new avenue to study these networks. SIA allows ion ablation to remove nanometer sections of tissue for SEM imaging, resulting in serial section data collection for three-dimensional reconstruction. Here we highlight a method for preparing retinal tissues for imaging of photoreceptors by SIA-SEM technology. We show that this technique can be used to visualize whole rod photoreceptors and the internal disc elements from wild-type (wt) mice. The distance parameters of the discs and photoreceptors are in good agreement with previous work with other methods. Moreover, we show that large planes of retinal tissue can be imaged at high resolution to display the packing of normal rods. Finally, SIA-SEM imaging of retinal tissue from a mouse model (Nrl⁻/⁻) with phenotypic changes akin to the human disease enhanced S-cone syndrome (ESCS) revealed a structural profile of overall photoreceptor ultrastructure and internal elements that accompany this disease. Overall, this work presents a new method to study photoreceptor cells at high structural resolution that has a broad applicability to the visual neuroscience field.

  14. Development and plasticity of mitochondria and electrical properties of the cell membrane in blowfly photoreceptors.

    PubMed

    Rudolf, Jerneja; Meglič, Andrej; Zupančič, Gregor; Belušič, Gregor

    2014-07-01

    Blowfly photoreceptors are highly energy demanding sensory systems. Their information processing efficiency is enabled by the high temporal resolution of the cell membrane, requiring heavy metabolic support by the mitochondria. We studied the developmental changes of the mitochondrial apparatus and electrical properties of the photoreceptor membrane in the white eyed Calliphora vicina Chalky. Using in vivo microspectrophotometry and Western blot analysis, we found an age-dependent increase in the concentration of mitochondrial pigments. The maximal change occurred during the first week. The age-related changes were smaller in dark-bred than in light-bred flies. The mitochondrial pigment content increased after the switch from dark to light rearing and decreased after the switch from light to dark rearing. The electrical parameters of the photoreceptors were investigated with intracellular recordings. The resting membrane resistance and time constant decreased significantly after eclosion. The decrease was again most significant during the first week of adult life, paralleled with changes in the Na/K pump-dependent hyperpolarizing afterpotential. We conclude that the photoreceptor mitochondria exhibit remarkable ontogenetic and phenotypic plasticity, because the quantity of mitochondrial pigments tightly follows the development of the cell membrane as well as the energy demands of the photoreceptors under different rearing conditions.

  15. Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

    NASA Astrophysics Data System (ADS)

    Unachukwu, Uchenna John; Warren, Alice; Li, Ze; Mishra, Shawn; Zhou, Jing; Sauane, Moira; Lim, Hyungsik; Vazquez, Maribel; Redenti, Stephen

    2016-03-01

    To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α – CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment.

  16. Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

    PubMed Central

    Unachukwu, Uchenna John; Warren, Alice; Li, Ze; Mishra, Shawn; Zhou, Jing; Sauane, Moira; Lim, Hyungsik; Vazquez, Maribel; Redenti, Stephen

    2016-01-01

    To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α – CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment. PMID:26935401

  17. Transcriptome Dynamics of Developing Photoreceptors in Three-Dimensional Retina Cultures Recapitulates Temporal Sequence of Human Cone and Rod Differentiation Revealing Cell Surface Markers and Gene Networks.

    PubMed

    Kaewkhaw, Rossukon; Kaya, Koray Dogan; Brooks, Matthew; Homma, Kohei; Zou, Jizhong; Chaitankar, Vijender; Rao, Mahendra; Swaroop, Anand

    2015-12-01

    The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.

  18. Kinetics of Inhibitory Feedback from Horizontal Cells to Photoreceptors: Implications for an Ephaptic Mechanism

    PubMed Central

    Warren, Ted J.; Van Hook, Matthew J.; Tranchina, Daniel

    2016-01-01

    Inhibitory feedback from horizontal cells (HCs) to cones generates center-surround receptive fields and color opponency in the retina. Mechanisms of HC feedback remain unsettled, but one hypothesis proposes that an ephaptic mechanism may alter the extracellular electrical field surrounding photoreceptor synaptic terminals, thereby altering Ca2+ channel activity and photoreceptor output. An ephaptic voltage change produced by current flowing through open channels in the HC membrane should occur with no delay. To test for this mechanism, we measured kinetics of inhibitory feedback currents in Ambystoma tigrinum cones and rods evoked by hyperpolarizing steps applied to synaptically coupled HCs. Hyperpolarizing HCs stimulated inward feedback currents in cones that averaged 8–9 pA and exhibited a biexponential time course with time constants averaging 14–17 ms and 120–220 ms. Measurement of feedback-current kinetics was limited by three factors: (1) HC voltage-clamp speed, (2) cone voltage-clamp speed, and (3) kinetics of Ca2+ channel activation or deactivation in the photoreceptor terminal. These factors totaled ∼4–5 ms in cones meaning that the true fast time constants for HC-to-cone feedback currents were 9–13 ms, slower than expected for ephaptic voltage changes. We also compared speed of feedback to feedforward glutamate release measured at the same cone/HC synapses and found a latency for feedback of 11–14 ms. Inhibitory feedback from HCs to rods was also significantly slower than either measurement kinetics or feedforward release. The finding that inhibitory feedback from HCs to photoreceptors involves a significant delay indicates that it is not due to previously proposed ephaptic mechanisms. SIGNIFICANCE STATEMENT Lateral inhibitory feedback from horizontal cells (HCs) to photoreceptors creates center-surround receptive fields and color-opponent interactions. Although underlying mechanisms remain unsettled, a longstanding hypothesis proposes that

  19. The Hippo Pathway Controls a Switch between Retinal Progenitor Cell Proliferation and Photoreceptor Cell Differentiation in Zebrafish

    PubMed Central

    Asaoka, Yoichi; Hata, Shoji; Namae, Misako; Furutani-Seiki, Makoto; Nishina, Hiroshi

    2014-01-01

    The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap’s TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1

  20. Drosophila N-cadherin mediates an attractive interaction between photoreceptor axons and their targets

    PubMed Central

    Prakash, Saurabh; Caldwell, Jason C; Eberl, Daniel F; Clandinin, Thomas R

    2008-01-01

    Classical cadherins have been proposed to mediate interactions between pre- and postsynaptic cells that are necessary for synapse formation. We provide the first direct, genetic evidence in favor of this model by examining the role of N-cadherin in controlling the pattern of synaptic connections made by photoreceptor axons in Drosophila. N-cadherin is required in both individual photoreceptors and their target neurons for photoreceptor axon extension. Cell-by-cell reconstruction of wild-type photoreceptor axons extending within mosaic patches of mutant target cells shows that N-cadherin mediates attractive interactions between photoreceptors and their targets. This interaction is not limited to those cells that will become the synaptic partners of photoreceptors. Multiple N-cadherin isoforms are produced, but single isoforms can substitute for endogenous N-cadherin activity. We propose that N-cadherin mediates a homophilic, attractive interaction between photoreceptor growth cones and their targets that precedes synaptic partner choice. PMID:15735641

  1. Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina

    PubMed Central

    Kuse, Yoshiki; Tsuruma, Kazuhiro; Sugitani, Sou; Izawa, Hiroshi; Ohno, Yuta; Shimazawa, Masamitsu; Hara, Hideaki

    2016-01-01

    Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU+ cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin+ photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway. PMID:27030285

  2. Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol

    PubMed Central

    Wang, Wen-Der; Hsu, Hwei-Jan; Li, Yi-Fang; Wu, Chang-Yi

    2017-01-01

    Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf), and PBZ-treated embryos (2–72 hpf) were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of gnat1 (rod photoreceptor marker), gnat2 (cone photoreceptor marker), aldehyde dehydrogenases (encoding key enzymes for RA synthesis), and phospho-histone H3 (an M-phase marker) in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division. PMID:28085063

  3. Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

    PubMed

    Gonzalez-Cordero, Anai; West, Emma L; Pearson, Rachael A; Duran, Yanai; Carvalho, Livia S; Chu, Colin J; Naeem, Arifa; Blackford, Samuel J I; Georgiadis, Anastasios; Lakowski, Jorn; Hubank, Mike; Smith, Alexander J; Bainbridge, James W B; Sowden, Jane C; Ali, Robin R

    2013-08-01

    Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

  4. A new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent, cobalt chloride.

    PubMed

    Hara, Akira; Niwa, Masayuki; Aoki, Hitomi; Kumada, Masako; Kunisada, Takahiro; Oyama, Takeru; Yamamoto, Tetsuya; Kozawa, Osamu; Mori, Hideki

    2006-09-13

    Retinal photoreceptor cell degeneration was induced by cobalt chloride, a chemical hypoxia-mimicking agent in rodents. Time course and dose-response of photoreceptor cell degeneration in mouse retina after intravitreal injection of cobalt chloride were examined by conventional histological analysis by hematoxylin and eosin staining and in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method with the use of paraffin-embedded sections. The dose-response of photoreceptor cell degeneration in rat retina was also examined. Photoreceptor cells progressively degenerated with time and under dose-response relationship. The suitable dose of cobalt chloride for the selective photoreceptor cell degeneration in mice is 10-12 nmol intravitreal injection at the volume of 2 microl. The retinal morphology of the mice 2 weeks after the 10-12 nmol intravitreal injection was similar to that of retinal degeneration in the mutant rd mouse. Retinal damage of total retinal layers was induced by an excessive dose of cobalt chloride. The progression of retinal damage after cobalt chloride injection, measured morphologically, was completed at 1 week. However, nuclear DNA fragmentation, mainly detected at outer nuclear layer by TUNEL, peaked at 48 h after 12 nmol cobalt chloride injection. Thus, the selective photoreceptor cell degeneration induced by cobalt chloride follows DNA fragmentation at outer nuclear layer. The photoreceptor cell degeneration is established optionally by cobalt chloride without use of the retinal degeneration mutant animals. Thus, we have described the development of a new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent.

  5. The translocation of signaling molecules in dark adapting mammalian rod photoreceptor cells is dependent on the cytoskeleton.

    PubMed

    Reidel, Boris; Goldmann, Tobias; Giessl, Andreas; Wolfrum, Uwe

    2008-10-01

    In vertebrate rod photoreceptor cells, arrestin and the visual G-protein transducin move between the inner segment and outer segment in response to changes in light. This stimulus dependent translocation of signalling molecules is assumed to participate in long term light adaptation of photoreceptors. So far the cellular basis for the transport mechanisms underlying these intracellular movements remains largely elusive. Here we investigated the dependency of these movements on actin filaments and the microtubule cytoskeleton of photoreceptor cells. Co-cultures of mouse retina and retinal pigment epithelium were incubated with drugs stabilizing and destabilizing the cytoskeleton. The actin and microtubule cytoskeleton and the light dependent distribution of signaling molecules were subsequently analyzed by light and electron microscopy. The application of cytoskeletal drugs differentially affected the cytoskeleton in photoreceptor compartments. During dark adaptation the depolymerization of microtubules as well as actin filaments disrupted the translocation of arrestin and transducin in rod photoreceptor cells. During light adaptation only the delivery of arrestin within the outer segment was impaired after destabilization of microtubules. Movements of transducin and arrestin required intact cytoskeletal elements in dark adapting cells. However, diffusion might be sufficient for the fast molecular movements observed as cells adapt to light. These findings indicate that different molecular translocation mechanisms are responsible for the dark and light associated translocations of arrestin and transducin in rod photoreceptor cells.

  6. Transcription factor NF-Y is involved in differentiation of R7 photoreceptor cell in Drosophila.

    PubMed

    Yoshioka, Yasuhide; Ly, Luong Linh; Yamaguchi, Masamitsu

    2012-01-15

    The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in eye discs with GMR-GAL4 and UAS-dNF-YAIR resulted in a rough eye phenotype and monitoring of differentiation of photoreceptor cells by LacZ expression in seven up-LacZ and deadpan-lacZ enhancer trap lines revealed associated loss of R7 photoreceptor signals. In line with differentiation of R7 being regulated by the sevenless (sev) gene and the MAPK cascade, the rough eye phenotype and loss of R7 signals in dNF-YA-knockdown flies were rescued by expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade. The sev gene promoter contains two dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YA antibody and S2 cells, the sev gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates from transgenic flies by polymerase chain reaction, indicating that dNF-Y is necessary for appropriate sev expression and involved in R7 photoreceptor cell development.

  7. Müller Cell Reactivity in Response to Photoreceptor Degeneration in Rats with Defective Polycystin-2

    PubMed Central

    Vogler, Stefanie; Pannicke, Thomas; Hollborn, Margrit; Grosche, Antje; Busch, Stephanie; Hoffmann, Sigrid; Wiedemann, Peter; Reichenbach, Andreas; Hammes, Hans-Peter; Bringmann, Andreas

    2013-01-01

    Background Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2. Methods Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution. Findings Müller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators. Interpretation Cellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as

  8. Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina.

    PubMed

    Zhou, Liang; Wang, Wei; Liu, Yongqing; Fernandez de Castro, Juan; Ezashi, Toshihiko; Telugu, Bhanu Prakash V L; Roberts, R Michael; Kaplan, Henry J; Dean, Douglas C

    2011-06-01

    Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.

  9. Cobalamin C Deficiency Shows a Rapidly Progressing Maculopathy With Severe Photoreceptor and Ganglion Cell Loss

    PubMed Central

    Bonafede, Lucas; Ficicioglu, Can H.; Serrano, Leona; Han, Grace; Morgan, Jessica I. W.; Mills, Monte D.; Forbes, Brian J.; Davidson, Stefanie L.; Binenbaum, Gil; Kaplan, Paige B.; Nichols, Charles W.; Verloo, Patrick; Leroy, Bart P.; Maguire, Albert M.; Aleman, Tomas S.

    2015-01-01

    Purpose To describe in detail the retinal structure and function of a group of patients with cobalamin C (cblC) disease. Methods Patients (n = 11, age 4 months to 15 years) with cblC disease (9/11, early onset) diagnosed by newborn screening underwent complete ophthalmic examinations, fundus photography, near-infrared reflectance imaging, and spectral-domain optical coherence tomography (SD-OCT). Electroretinograms (ERGs) were performed in a subset of patients. Results Patients carried homozygous or compound heterozygote mutations in the methylmalonic aciduria and homocystinuria type C (MMACHC) gene. Late-onset patients had a normal exam. All early-onset patients showed a maculopathy; older subjects had a retina-wide degeneration (n = 4; >7 years of age). In general, retinal changes were first observed before 1 year of age and progressed within months to a well-established maculopathy. Pseudocolobomas were documented in three patients. Measurable visual acuities ranged from 20/200 to 20/540. Nystagmus was present in 8/11 patients; 5/6 patients had normal ERGs; 1/6 had reduced rod-mediated responses. Spectral-domain OCT showed macular thinning, with severe ganglion cell layer (GCL) and outer nuclear layer (ONL) loss. Inner retinal thickening was observed in areas of total GCL/ONL loss. A normal lamination pattern in the peripapillary nasal retina was often seen despite severe central and/or retina-wide disease. Conclusions Patients with early-onset cblC and MMACHC mutations showed an early-onset, unusually fast-progressing maculopathy with severe central ONL and GCL loss. An abnormally thickened inner retina supports a remodeling response to both photoreceptor and ganglion cell degeneration and/or an interference with normal development in early-onset cblC. PMID:26658511

  10. Rod photoreceptor-specific gene expression in human retinoblastoma cells.

    PubMed Central

    Di Polo, A; Farber, D B

    1995-01-01

    Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7732024

  11. Retinal bipolar cells: temporal filtering of signals from cone photoreceptors.

    PubMed

    Burkhardt, Dwight A; Fahey, Patrick K; Sikora, Michael A

    2007-01-01

    The temporal dynamics of the response of neurons in the outer retina were investigated by intracellular recording from cones, bipolar, and horizontal cells in the intact, light-adapted retina of the tiger salamander (Ambystoma tigrinum), with special emphasis on comparing the two major classes of bipolars cells, the ON depolarizing bipolars (Bd) and the OFF hyperpolarizing bipolars (Bh). Transfer functions were computed from impulse responses evoked by a brief light flash on a steady background of 20 cd/m(2). Phase delays ranged from about 89 ms for cones to 170 ms for Bd cells, yielding delays relative to that of cones of about 49 ms for Bh cells and 81 ms for Bd cells. The difference between Bd and Bh cells, which may be due to a delay introduced by the second messenger G-protein pathway unique to Bd cells, was further quantified by latency measurements and responses to white noise. The amplitude transfer functions of the outer retinal neurons varied with light adaptation in qualitative agreement with results for other vertebrates and human vision. The transfer functions at 20 cd/m(2) were predominantly low pass with 10-fold attenuation at about 13, 14, 9.1, and 7.7 Hz for cones, horizontal, Bh, and Bd cells, respectively. The transfer function from the cone voltage to the bipolar voltage response, as computed from the above measurements, was low pass and approximated by a cascade of three low pass RC filters ("leaky integrators"). These results for cone-->bipolar transmission are surprisingly similar to recent results for rod-->bipolar transmission in salamander slice preparations. These and other findings suggest that the rate of vesicle replenishment rather than the rate of release may be a common factor shaping synaptic signal transmission from rods and cones to bipolar cells.

  12. Structure and function of the interphotoreceptor matrix surrounding retinal photoreceptor cells.

    PubMed

    Ishikawa, Makoto; Sawada, Yu; Yoshitomi, Takeshi

    2015-04-01

    The interphotoreceptor matrix (IPM) is a highly organized structure with interconnected domains surrounding cone and rod photoreceptor cells and extends throughout the subretinal space. Based on known roles of the extracellular matrix in other tissues, the IPM is thought to have several prominent functions including serving as a receptor for growth factors, regulating retinoid transport, participating in cytoskeletal organization in surrounding cells, and regulation of oxygen and nutrient transport. In addition, a number of studies suggest that the IPM also may play a significant role in the etiology of retinal degenerative disorders. In this review, we describe the present knowledge concerning the structure and function of the IPM under physiological and pathological conditions.

  13. Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.

    PubMed

    Mazzoni, Francesca; Safa, Hussein; Finnemann, Silvia C

    2014-09-01

    RPE cells are the most actively phagocytic cells in the human body. In the eye, RPE cells face rod and cone photoreceptor outer segments at all times but contribute to shedding and clearance phagocytosis of distal outer segment tips only once a day. Analysis of RPE phagocytosis in situ has succeeded in identifying key players of the RPE phagocytic mechanism. Phagocytic processes comprise three distinct phases, recognition/binding, internalization, and digestion, each of which is regulated separately by phagocytes. Studies of phagocytosis by RPE cells in culture allow specifically analyzing and manipulating these distinct phases to identify their molecular mechanisms. Here, we compare similarities and differences of primary, immortalized, and stem cell-derived RPE cells in culture to RPE cells in situ with respect to phagocytic function. We discuss in particular potential pitfalls of RPE cell culture phagocytosis assays. Finally, we point out considerations for phagocytosis assay development for future studies.

  14. Novel Methodology for Creating Macaque Retinas with Sortable Photoreceptors and Ganglion Cells

    PubMed Central

    Choudhury, Shreyasi; Strang, Christianne E.; Alexander, John J.; Scalabrino, Miranda L.; Lynch Hill, Julie; Kasuga, Daniel T.; Witherspoon, C. Douglas; Boye, Sanford L.; Gamlin, Paul D.; Boye, Shannon E.

    2016-01-01

    Purpose: The ability to generate macaque retinas with sortable cell populations would be of great benefit to both basic and translational studies of the primate retina. The purpose of our study was therefore to develop methods to achieve this goal by selectively labeling, in life, photoreceptors (PRs) and retinal ganglion cells (RGCs) with separate fluorescent markers. Methods: Labeling of macaque (Macaca fascicularis) PRs and RGCs was accomplished by subretinal delivery of AAV5-hGRK1-GFP, and retrograde transport of micro-ruby™ from the lateral geniculate nucleus, respectively. Retinas were anatomically separated into different regions. Dissociation conditions were optimized, and cells from each region underwent fluorescent activated cell sorting (FACS). Expression of retinal cell type- specific genes was assessed by quantitative real-time PCR to characterize isolated cell populations. Results: We show that macaque PRs and RGCs can be simultaneously labeled in-life and enriched populations isolated by FACS. Recovery from different retinal regions indicated efficient isolation/enrichment for PRs and RGCs, with the macula being particularly amendable to this technique. Conclusions: The methods and materials presented here allow for the identification of novel reagents designed to target RGCs and/or photoreceptors in a species that is phylogenetically and anatomically similar to human. These techniques will enable screening of intravitreally-delivered AAV capsid libraries for variants with increased tropism for PRs and/or RGCs and the evaluation of vector tropism and/or cellular promoter activity of gene therapy vectors in a clinically relevant species. PMID:27990105

  15. Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

    PubMed

    González-Sastre, Alejandro; Molina, Ma Dolores; Saló, Emili

    2012-01-01

    Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.

  16. In vitro expanded stem cells from the developing retina fail to generate photoreceptors but differentiate into myelinating oligodendrocytes.

    PubMed

    Czekaj, Magdalena; Haas, Jochen; Gebhardt, Marlen; Müller-Reichert, Thomas; Humphries, Peter; Farrar, Jane; Bartsch, Udo; Ader, Marius

    2012-01-01

    Cell transplantation to treat retinal degenerative diseases represents an option for the replacement of lost photoreceptor cells. In vitro expandable cells isolated from the developing mammalian retina have been suggested as a potential source for the generation of high numbers of donor photoreceptors. In this study we used standardized culture conditions based on the presence of the mitogens FGF-2 and EGF to generate high numbers of cells in vitro from the developing mouse retina. These presumptive 'retinal stem cells' ('RSCs') can be propagated as monolayer cultures over multiple passages, express markers of undifferentiated neural cells, and generate neuronal and glial cell types upon withdrawal of mitogens in vitro or following transplantation into the adult mouse retina. The proportion of neuronal differentiation can be significantly increased by stepwise removal of mitogens and inhibition of the notch signaling pathway. However, 'RSCs', by contrast to their primary counterparts in vivo, i.e. retinal progenitor cells, loose the expression of retina-specific progenitor markers like Rax and Chx10 after passaging and fail to differentiate into photoreceptors both in vitro or after intraretinal transplantation. Notably, 'RSCs' can be induced to differentiate into myelinating oligodendrocytes, a cell type not generated by primary retinal progenitor cells. Based on these findings we conclude that 'RSCs' expanded in high concentrations of FGF-2 and EGF loose their retinal identity and acquire features of in vitro expandable neural stem-like cells making them an inappropriate cell source for strategies aimed at replacing photoreceptor cells in the degenerated retina.

  17. Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9.

    PubMed

    Lv, Ji-Neng; Zhou, Gao-Hui; Chen, Xuejiao; Chen, Hui; Wu, Kun-Chao; Xiang, Lue; Lei, Xin-Lan; Zhang, Xiao; Wu, Rong-Han; Jin, Zi-Bing

    2017-02-20

    Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.

  18. Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9

    PubMed Central

    Lv, Ji-Neng; Zhou, Gao-Hui; Chen, Xuejiao; Chen, Hui; Wu, Kun-Chao; Xiang, Lue; Lei, Xin-Lan; Zhang, Xiao; Wu, Rong-Han; Jin, Zi-Bing

    2017-01-01

    Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa. PMID:28216641

  19. IGF-I maintains calpastatin expression and attenuates apoptosis in several models of photoreceptor cell death.

    PubMed

    Arroba, Ana I; Wallace, Deborah; Mackey, Ashley; de la Rosa, Enrique J; Cotter, Thomas G

    2009-09-01

    Retinitis pigmentosa is a heterogeneous group of inherited retinal dystrophies in which the loss of photoreceptor cells via apoptosis leads to blindness. In this study we have experimentally mimicked this condition by treating 661W cells and wild-type mouse retinal explants with a Ca(2+) ionophore. Ca(2+) overload induced apoptosis, which was correlated with calpain-2 activation, loss of calpastatin, its endogenous inhibitor, as well as the loss of its transcriptional activator, phospho-cAMP response element binding (CREB). All are similar changes to those observed in the rd1 mouse model of retinitis pigmentosa. Insulin like-growth factor-I (IGF-I) attenuated this Ca(2+)-induced apoptosis, as well as decreased the activation of calpain-2 and maintained calpastatin levels through the activation of the Akt-CREB pathway. Similarly, IGF-I decreased photoreceptor apoptosis in rd1 mouse retinal explants in parallel with reduced activation of calpain-2 and increased levels of calpastatin and activation of phospho-CREB. In conclusion, IGF-I seems to protect neural cells following a physiopathological or an experimental increase in intracellular Ca(2+), an observation that may have therapeutic consequences in neurodegenerative diseases such as retinitis pigmentosa.

  20. Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy.

    PubMed

    LaVail, M M; Yasumura, D; Matthes, M T; Drenser, K A; Flannery, J G; Lewin, A S; Hauswirth, W W

    2000-10-10

    Ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapeutic measure for dominantly inherited diseases. We previously demonstrated that two ribozymes targeted to the P23H mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (P) 15. We now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in the degeneration after significant photoreceptor cell loss. Recombinant adeno-associated virus (rAAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. For the study of long-term survival, rAAV was administered by subretinal injection at P15, and the rats were allowed to live up to 8 months of age. For the study of late-stage gene transfer, rAAV was administered at P30 or P45, when 40-45% of the photoreceptors already had degenerated. Eyes were examined functionally by the electroretinogram and structurally by morphometric analysis. When injected at P15, expression of either ribozyme markedly slowed the rate of photoreceptor degeneration for at least 8 months and resulted in significantly greater electroretinogram amplitudes at least up to P180. When injected at P30 or P45, virtually the same number of photoreceptors survived at P130 as when injected at P15. Ribozyme rescue appears to be a potentially effective, long-term therapy for autosomal dominant retinal degeneration and is highly effective even when the gene transfer is done after significant photoreceptor cell loss.

  1. Damage to the photoreceptor cells of the rabbit retina from 56Fe ions: effect of age at exposure, 1

    NASA Technical Reports Server (NTRS)

    Williams, G. R.; Lett, J. T.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    Optic and proximate tissues of New Zealand white (NZW) rabbits at ages (approximately 3.5 years) near the middle of their median lifespan (5-7 years) were given 0.5-3.5 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition at a linear energy transfer (LET infinity) of 220 +/- 31 keV micrometer-1. Dose-dependent losses of retinal photoreceptor cells (rods) occurred until 1-2 years after irradiation, the period of this interim report. Similar cumulative losses of photoreceptor cells were seen during the period 1-2 years post-irradiation for rabbits given comparable exposures when young (6-9 weeks old). Since losses of photoreceptor cells at early times had not been determined previously, the current experiment, which was designed to simulate the responses of mature astronauts, redressed that deficiency.

  2. Mertk gene expression and photoreceptor outer segment phagocytosis by cultured rat bone marrow mesenchymal stem cells

    PubMed Central

    Peng, Rong-mei; Jin, Ying; Sun, Yu-zhao; Sun, Yi-qian; Zhang, Pei

    2017-01-01

    Background Bone marrow mesenchymal stem cells (BM-MSCs) are multipotential stem cells that have been used for a broad spectrum of indications. Several investigations have used BM-MSCs to promote photoreceptor survival and suggested that BM-MSCs are a potential source of cell replacement therapy for some forms of retinal degeneration. Purpose To investigate the expression of the MER proto-oncogene, tyrosine kinase (Mertk), involved in the disruption of RPE phagocytosis and the onset of autosomal recessive retinitis pigmentosa in rat BM-MSCs and to compare phagocytosis of the photoreceptor outer segment (POS) by BM-MSCs and RPE cells in vitro. Methods MSCs were isolated from the bone marrow of Brown Norway rats. Reverse transcription-PCR (RT–PCR) and western blot analyses were used to examine the expression of Mertk. The phagocytized POS was detected with double fluorescent labeling, transmission electron microscopy, and scanning electron microscopy. Results Mertk expression did not differ among the first three passages of BM-MSCs. Mertk gene expression was greater in the BM-MSCs than the RPE cells. Mertk protein expression in the BM-MSCs was similar to that in the RPE cells in the primary passage and was greater than that in the RPE cells in the other two passages. BM-MSCs at the first three passages phagocytized the POS more strongly than the RPE cells. The process of BM-MSC phagocytosis was similar to that of the RPE cells. Conclusions BM-MSCs may be an effective cell source for treating retinal degeneration in terms of phagocytosis of the POS. PMID:28210098

  3. Photoreceptor engineering

    PubMed Central

    Ziegler, Thea; Möglich, Andreas

    2015-01-01

    Sensory photoreceptors not only control diverse adaptive responses in Nature, but as light-regulated actuators they also provide the foundation for optogenetics, the non-invasive and spatiotemporally precise manipulation of cellular events by light. Novel photoreceptors have been engineered that establish control by light over manifold biological processes previously inaccessible to optogenetic intervention. Recently, photoreceptor engineering has witnessed a rapid development, and light-regulated actuators for the perturbation of a plethora of cellular events are now available. Here, we review fundamental principles of photoreceptors and light-regulated allostery. Photoreceptors dichotomize into associating receptors that alter their oligomeric state as part of light-regulated allostery and non-associating receptors that do not. A survey of engineered photoreceptors pinpoints light-regulated association reactions and order-disorder transitions as particularly powerful and versatile design principles. Photochromic photoreceptors that are bidirectionally toggled by two light colors augur enhanced spatiotemporal resolution and use as photoactivatable fluorophores. By identifying desirable traits in engineered photoreceptors, we provide pointers for the design of future, light-regulated actuators. PMID:26137467

  4. Proteoglycan synthesis in flat cell-free cultures of chick embryo retinal neurons and photoreceptors.

    PubMed

    Needham, L K; Adler, R; Hewitt, A T

    1988-04-01

    Extracellular matrix and cell surface proteoglycans are thought to play important roles in neural development and regeneration. Central nervous system proteoglycans have been isolated and characterized from rat and sheep brain and from chick neural retina. An experimental advantage offered by the latter tissue is that it is avascular and can be isolated free of connective tissue and pigment epithelium. Therefore, proteoglycans synthesized by this tissue are derived exclusively from neural cells. However, it has not yet been determined whether neurons and photoreceptors contribute to proteoglycan synthesis or whether these molecules are largely glial in origin. In the present study we have addressed this question using cultures of chick neural retinal cells free of flat, glial-like cells. Proteoglycans synthesized by cultures of retinal neurons, photoreceptors, and undifferentiated, process-free round cells from 8-day embryonic chick neural retina were metabolically labeled in vitro using [35S]sulfate and [3H]glucosamine as precursors. Radiolabeled proteoglycans accumulated in the medium, and could also be extracted from the cell layer by sequential treatments with Triton X-100 and with guanidine HCl. The proteoglycans were isolated by ion-exchange chromatography, and characterized by gel filtration chromatography and by susceptibility to degradation by enzymatic and chemical treatments. Overall, heparan sulfate proteoglycans were the predominant type of proteoglycan synthesized in vitro by the cultured neural retinal cells at this developmental stage. The medium and the Triton extract contained different proportions of both chondroitin sulfate and heparan sulfate proteoglycans, while heparan sulfate was the only proteoglycan recovered from the guanidine extract. These studies demonstrate that heparan sulfate and chondroitin sulfate proteoglycans are actively synthesized by cultures of neural retinal cells free of flat, glial-like cells.

  5. Knockout of Ccr2 alleviates photoreceptor cell death in rodent retina exposed to chronic blue light.

    PubMed

    Hu, Zizhong; Zhang, Yi; Wang, Junling; Mao, Pingan; Lv, Xuehua; Yuan, Songtao; Huang, Zhengru; Ding, Yuzhi; Xie, Ping; Liu, Qinghuai

    2016-11-10

    Age-related macular degeneration (AMD), the leading cause of visual loss after the age of 60 years, is a degenerative retinal disease involving a variety of environmental and hereditary factors. Although it has been implicated that immune system is involved in the disease progression, the exact role that microglia has is still unclear. Here we demonstrated that knockout of Ccr2 gene could alleviate photoreceptor cell death in mice retinas exposed to chronic blue light. In Ccr2(-/-) mice, a damaged microglia recruitment was shown in retina and this could protect the visual function in electroretinogram and alleviate the photoreceptor apoptosis, which thus helped attenuate the blue light-induced retinopathy. We further found an increased co-location of NLRP3, Iba-1, and IL-1β in fluorescence and a concomitant increased protein expression of NLRP3, caspase-1, and IL-1β in western blotting in chronic blue light-induced retinopathy. Moreover, the activation of microglia and their cellular NLRP3 inflammasomes occurred as an earlier step before the structural and functional damage of the mice retinas, which collectively supported that microglial NLRP3 inflammasome might be the key to the chronic blue light-induced retinopathy.

  6. CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    PubMed Central

    Xu, Ying; Wang, Tao

    2016-01-01

    ABSTRACT Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster, the main light-sensing rhodopsin (Rh1; encoded by ninaE) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, we discovered a protein we named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd-null mutant flies. Our data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity. PMID:26598556

  7. Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

    PubMed

    Lakowski, Jorn; Gonzalez-Cordero, Anai; West, Emma L; Han, Ya-Ting; Welby, Emily; Naeem, Arifa; Blackford, Samuel J I; Bainbridge, James W B; Pearson, Rachael A; Ali, Robin R; Sowden, Jane C

    2015-08-01

    Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.

  8. Hypoxia increases the yield of photoreceptors differentiating from mouse embryonic stem cells and improves the modeling of retinogenesis in vitro.

    PubMed

    Garita-Hernández, Marcela; Diaz-Corrales, Francisco; Lukovic, Dunja; González-Guede, Irene; Diez-Lloret, Andrea; Valdés-Sánchez, M Lourdes; Massalini, Simone; Erceg, Slaven; Bhattacharya, Shomi S

    2013-05-01

    Retinitis pigmentosa (RP), a genetically heterogeneous group of diseases together with age-related macular degeneration (AMD), are the leading causes of permanent blindness and are characterized by the progressive dysfunction and death of the light sensing photoreceptors of the retina. Due to the limited regeneration capacity of the mammalian retina, the scientific community has invested significantly in trying to obtain retinal progenitor cells from embryonic stem cells (ESC). These represent an unlimited source of retinal cells, but it has not yet been possible to achieve specific populations, such as photoreceptors, efficiently enough to allow them to be used safely in the future as cell therapy of RP or AMD. In this study, we generated a high yield of photoreceptors from directed differentiation of mouse ESC (mESC) by recapitulating crucial phases of retinal development. We present a new protocol of differentiation, involving hypoxia and taking into account extrinsic and intrinsic cues. These include niche-specific conditions as well as the manipulation of the signaling pathways involved in retinal development. Our results show that hypoxia promotes and improves the differentiation of mESC toward photoreceptors. Different populations of retinal cells are increased in number under the hypoxic conditions applied, such as Crx-positive cells, S-Opsin-positive cells, and double positive cells for Rhodopsin and Recoverin, as shown by immunofluorescence analysis. For the first time, this manuscript reports the high efficiency of differentiation in vivo and the expression of mature rod photoreceptor markers in a large number of differentiated cells, transplanted in the subretinal space of wild-type mice.

  9. Revised lineage of larval photoreceptor cells in Ciona reveals archetypal collaboration between neural tube and neural crest in sensory organ formation.

    PubMed

    Oonuma, Kouhei; Tanaka, Moeko; Nishitsuji, Koki; Kato, Yumiko; Shimai, Kotaro; Kusakabe, Takehiro G

    2016-12-01

    The Ciona intestinalis larva has two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus, that are asymmetrically situated in the brain. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. Precise elucidation of the lineage of the photoreceptor cells will be key to understanding the developmental mechanisms of these cells as well as the evolutionary relationships between the photoreceptor organs of ascidians and vertebrates. Photoreceptor cells of the pigmented ocellus have been thought to develop from anterior animal (a-lineage) blastomeres, whereas the developmental origin of the nonpigmented ocellus has not been determined. Here, we show that the photoreceptor cells of both ocelli develop from the right anterior vegetal hemisphere: those of the pigmented ocellus from the right A9.14 cell and those of the nonpigmented ocellus from the right A9.16 cell. The pigmented ocellus is formed by a combination of two lineages of cells with distinct embryonic origins: the photoreceptor cells originate from a medial portion of the A-lineage neural plate, while the pigment cell originates from the lateral edge of the a-lineage neural plate. In light of the recently proposed close evolutionary relationship between the ocellus pigment cell of ascidians and the cephalic neural crest of vertebrates, the ascidian ocellus may represent a prototypic contribution of the neural crest to a cranial sensory organ.

  10. Mouse retinal adaptive response to proton irradiation: Correlation with DNA repair and photoreceptor cell death

    NASA Astrophysics Data System (ADS)

    Tronov, V. A.; Vinogradova, Yu. V.; Poplinskaya, V. A.; Nekrasova, E. I.; Ostrovsky, M. A.

    2015-01-01

    Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous study revealed non-linear dose-response relationship for cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In present study we used the mouse model for MNU-induced retinal degeneration to evaluate adaptive response of retina to proton irradiation and implication in it of glial Muller cells. The data showed that the recovery of retina after genotoxic agents has been associated with increased efficacy of DNA damage repair and lowered death of retinal photoreceptor cells.

  11. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

    PubMed

    Wiley, Luke A; Burnight, Erin R; DeLuca, Adam P; Anfinson, Kristin R; Cranston, Cathryn M; Kaalberg, Emily E; Penticoff, Jessica A; Affatigato, Louisa M; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-07-29

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

  12. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

    PubMed Central

    Wiley, Luke A.; Burnight, Erin R.; DeLuca, Adam P.; Anfinson, Kristin R.; Cranston, Cathryn M.; Kaalberg, Emily E.; Penticoff, Jessica A.; Affatigato, Louisa M.; Mullins, Robert F.; Stone, Edwin M.; Tucker, Budd A.

    2016-01-01

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

  13. Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors.

    PubMed

    Simón, María V; De Genaro, Pablo; Abrahan, Carolina E; de los Santos, Beatriz; Rotstein, Nora P; Politi, Luis E

    2012-02-01

    Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.

  14. Basal bodies exhibit polarized positioning in zebrafish cone photoreceptors

    PubMed Central

    Ramsey, Michelle; Perkins, Brian D.

    2012-01-01

    The asymmetric positioning of basal bodies, and therefore cilia, is often critical for proper cilia function. This planar polarity is critical for motile cilia function but has not been extensively investigated for non-motile cilia or for sensory cilia such as vertebrate photoreceptors. Zebrafish photoreceptors form an organized mosaic ideal for investigating cilia positioning. We report that in the adult retina, the basal bodies of red, green-, and blue-sensitive cone photoreceptors localized asymmetrically on the cell edge nearest to the optic nerve. In contrast, no patterning was seen in the basal bodies of ultraviolet-sensitive cones or in rod photoreceptors. The asymmetric localization of basal bodies was consistent in all regions of the adult retina. Basal body patterning was unaffected in the cones of the XOPS-mCFP transgenic line, which lacks rod photoreceptors. Finally, the adult pattern was not seen in 7 day post fertilization (dpf) larvae as basal bodies were randomly distributed in all the photoreceptor subtypes. These results establish the asymmetrical localization of basal bodies in red-, green-, and blue-sensitive cones in adult zebrafish retinas but not in larvae. This pattern suggests an active cellular mechanism regulated the positioning of basal bodies after the transition to the adult mosaic and that rods do not seem to be necessary for the patterning of cone basal bodies. PMID:23171982

  15. Differentiation potential of human adipose tissue derived stem cells into photoreceptors through explants culture and enzyme methods

    PubMed Central

    Xu, Wei-Wei; Huang, Li; Chong, Kelvin K.L.; Leung, Doreen S.Y.; Li, Benjamin F.L.; Yin, Zheng-Qin; Huang, Yi-Fei; Pang, Chi Pui

    2017-01-01

    AIM To investigate the retinal photoreceptor differentiation potential of human orbital adipose tissue-derived stem cells (ADSCs) generated by enzyme (EN) and explant (EX) culture methods. METHODS We investigated potentials of human orbital ADSCs to differentiate into photoreceptors through EN and EX culture methods. EN and EX orbital ADSCs were obtained from the same donor during rehabilitative orbital decompression, and then were subject to a 3-step induction using Noggin, DKK-1, IGF-1 and b-FGF at different time points for 38d. Stem cell, eye-field and photoreceptor-related gene and protein markers were measured by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescent (IMF) staining. RESULTS Both EX and EN orbital ADSCs expressed CD133, a marker of cell differentiation. Moreover, PAX6 and rhodopsin, markers of the retinal progenitor cells, were detected from EX and EN orbital ADSCs. In EX orbital ADSCs, PAX6 mRNA was detected on the 17th day and then the rhodopsin mRNA was detected on the 24th day. In contrast, the EN orbital ADSCs expressed PAX6 and rhodopsin mRNA on the 31st day. EX orbital ADSCs expressed rhodopsin protein on the 24th day, while EN orbital ADSCs expressed rhodopsin protein on the 31st day. CONCLUSION Orbital ADSCs isolated by direct explants culture show earlier and stronger expressions of markers towards eye field and retinal photoreceptor differentiation than those generated by conventional EN method. PMID:28149772

  16. Cloning, localization and functional properties of a cGMP-gated channel in photoreceptor cells from fish pineal gland.

    PubMed

    Decressac, Sonia; Grechez-Cassiau, Aline; Lenfant, Jacques; Falcón, Jacky; Bois, Patrick

    2002-11-01

    The perception of photic information and its translation into a rhythmic melatonin signal differ considerably among vertebrates. In the fish pineal gland, melatonin biosynthesis is controlled directly by the natural light/dark cycle. There are indications that the mechanisms of phototransduction are similar in the retinal and pineal photoreceptor cells. Here we report the molecular cloning of a novel ionic cyclic guanosine monophosphate (cGMP)-gated channel from trout pineal photoreceptors. The deduced amino acid sequence exhibits a high sequence homology to cyclic nucleotide-gated-3 (CNG) channels from retinal cones. In situ hybridization with sections of trout pineal gland revealed the expression of CNG channel in photoreceptor cells of the pineal organ. Electrophysiological studies by means of patch-clamp technique indicated that the native channel in photoreceptor cells and the expressed channel in a human cell line (HEK 293 cells) have properties similar to those of cone-CNG (cCNG)-3 channels. They are activated by cGMP, insensitive to cyclic adenosine monophosphate (cAMP) and blocked by intracellular Mg2+ ions at positive voltage values. They have a single-channel conductance close to 42 pS in negative voltage range. In transfected HEK cells loaded with the calcium indicator dye Fura 2, direct activation of CNG channels by 8-Br-cGMP increased fluorescence. The signal was blocked by the addition of Mg2+ ions. From these results, it is suggested that the pineal cyclic nucleotide-gated channel is a good candidate for mediating calcium entry into the pineal photoreceptors. It is most probably a key element in the signalling pathways that control the rhythmic production of melatonin.

  17. Connectivity map of bipolar cells and photoreceptors in the mouse retina

    PubMed Central

    Behrens, Christian; Schubert, Timm; Haverkamp, Silke; Euler, Thomas; Berens, Philipp

    2016-01-01

    In the mouse retina, three different types of photoreceptors provide input to 14 bipolar cell (BC) types. Classically, most BC types are thought to contact all cones within their dendritic field; ON-BCs would contact cones exclusively via so-called invaginating synapses, while OFF-BCs would form basal synapses. By mining publically available electron microscopy data, we discovered interesting violations of these rules of outer retinal connectivity: ON-BC type X contacted only ~20% of the cones in its dendritic field and made mostly atypical non-invaginating contacts. Types 5T, 5O and 8 also contacted fewer cones than expected. In addition, we found that rod BCs received input from cones, providing anatomical evidence that rod and cone pathways are interconnected in both directions. This suggests that the organization of the outer plexiform layer is more complex than classically thought. DOI: http://dx.doi.org/10.7554/eLife.20041.001 PMID:27885985

  18. The involvement of ATF4 and S-opsin in retinal photoreceptor cell damage induced by blue LED light

    PubMed Central

    Ooe, Emi; Tsuruma, Kazuhiro; Kuse, Yoshiki; Kobayashi, Saori; Shimazawa, Masamitsu

    2017-01-01

    Purpose Blue light is a high-energy emitting light with a short wavelength in the visible light spectrum. Blue light induces photoreceptor apoptosis and causes age-related macular degeneration or retinitis pigmentosa. In the present study, we investigated the roles of endoplasmic reticulum (ER) stress induced by blue light-emitting diode (LED) light exposure in murine photoreceptor cells. Methods The murine photoreceptor cell line was incubated and exposed to blue LED light (464 nm blue LED light, 450 lx, 3 to 24 h). The expression of the factors involved in the unfolded protein response pathway was examined using quantitative real-time reverse transcription (RT)-PCR and immunoblot analysis. The aggregation of short-wavelength opsin (S-opsin) in the murine photoreceptor cells was observed with immunostaining. The effect of S-opsin knockdown on ATF4 expression in the murine photoreceptor cell line was also investigated. Results Exposure to blue LED light increased the bip, atf4, and grp94 mRNA levels, induced the expression of ATF4 protein, and increased the levels of ubiquitinated proteins. Exposure to blue LED light in combination with ER stress inducers (tunicamycin and dithiothreitol) induced the aggregation of S-opsin. S-opsin mRNA knockdown prevented the induction of ATF4 expression in response to exposure to blue LED light. Conclusions These findings indicate that the aggregation of S-opsin induced by exposure to blue LED light causes ER stress, and ATF4 activation in particular. PMID:28331281

  19. Whole-cell clamp of dissociated photoreceptors from the eye of Lima scabra.

    PubMed

    Nasi, E

    1991-01-01

    Voltage-dependent membrane currents were investigated in enzymatically dissociated photoreceptors of Lima scabra using the whole-cell clamp technique. Depolarizing steps to voltages more positive than -10 mV elicit a transient inward current followed by a delayed, sustained outward current. The outward current is insensitive to replacement of a large fraction of extracellular Cl- with the impermeant anion glucuronate. Superfusion with tetraethylammonium and 4-aminopyridine reversibly abolishes the outward current, and internal perfusion with cesium also suppresses it, indicating that it is mediated by potassium channels. Isolation of the inward current reveals a fast activation kinetics, the peak amplitude occurring as early as 4-5 ms after stimulus onset, and a relatively rapid, though incomplete inactivation. Within the range of voltages examined, spanning up to +90 mV, reversal was not observed. The inward current is not sensitive to tetrodotoxin at concentrations up to 10 microM, and survives replacement of extracellular Na with tetramethylammonium. On the other hand, it is completely eliminated by calcium removal from the perfusing solution, and it is partially blocked by submillimolar concentrations of cadmium, suggesting that it is entirely due to voltage-dependent calcium channels. Analysis of the kinetics and voltage dependence of the isolated calcium current indicates the presence of two components, possibly reflecting the existence of separate populations of channels. Barium and strontium can pass through these channels, though less easily than calcium. Both the activation and the inactivation become significantly more sluggish when these ions serve as the charge carrier. A large fraction of the outward current is activated by preceding calcium influx. Suppression of this calcium-dependent potassium current shows a small residual component resembling the delayed rectifier. In addition, a transient outward current sensitive to 4-aminopyridine (Ia) could

  20. Light-induced degeneration and microglial response in the retina of an epibenthonic pigmented teleost: age-dependent photoreceptor susceptibility to cell death.

    PubMed

    Bejarano-Escobar, Ruth; Blasco, Manuel; Martín-Partido, Gervasio; Francisco-Morcillo, Javier

    2012-11-01

    Constant intense light causes apoptosis of photoreceptors in the retina of albino fish. However, very few studies have been performed on pigmented species. Tench (Tinca tinca) is a teleost inhabiting dimly lit environments that has a predominance of rods within the photoreceptor layer. To test the hypothesis that constant high intensity light can result in retinal damage in such pigmented epibenthonic teleost species, photodegeneration of the retina was investigated in the larvae and in juveniles of tench to assess whether any damage may also be dependent on fish age. We exposed both groups of animals to 5 days of constant darkness, followed by 4 days of constant 20,000 lx light, and then by 6 days of recovery in a 14 h light:10 h dark cycle. The results showed that the retina of the larvae group exhibited abundant photoreceptor cell apoptosis during the time of exposition to intense light, whereas that of juveniles was indifferent to it. Damaged retinas showed a strong TUNEL signal in photoreceptor nuclei, and occasionally a weak cytoplasmic TUNEL signal in Müller glia. Specific labelling of microglial cells with Griffonia simplicifolia lectin (GSL) histochemistry revealed that photoreceptor cell death alerts microglia in the degenerating retina, leading to local proliferation, migration towards the injured outer nuclear layer (ONL), and enhanced phagocytosis of photoreceptor debris. During the first days of intense light treatment, Müller cells phagocytosed dead photoreceptor cells but, once microglial cells became activated, there was a progressive increase in the phagocytic capacity of the microglia.

  1. Retinal signal transmission in Duchenne muscular dystrophy: evidence for dysfunction in the photoreceptor/depolarizing bipolar cell pathway.

    PubMed Central

    Fitzgerald, K M; Cibis, G W; Giambrone, S A; Harris, D J

    1994-01-01

    There have been reports of abnormal retinal neurotransmission determined by electroretinography in boys with Duchenne and Becker muscular dystrophy. Dystrophin may play a role in transmitting signals between photoreceptors and the excitatory synapse of the ON-bipolar cell. These electroretinographic changes appeared to be limited to the rod ON-pathway but we felt there was also similar abnormality in the cone ON-pathway. We used long-duration stimuli to separate ON-(depolarizing bipolar cell) and OFF (hyperpolarizing bipolar cell) contributions to the cone-dominated ERG to better understand how the retina functions in boys with Duchenne muscular dystrophy. We recorded the electroretinograms of 11 boys with Duchenne muscular dystrophy and found abnormal signal transmission at the level of the photoreceptor and ON-bipolar cell in both the rod and cone generated responses. The OFF-bipolar cell that responds to the offset of the stimulus continues to function normally. The results support our hypothesis that retinal dystrophin plays a role in receptor function or controlling ion channels at the level of the photoreceptor and depolarizing bipolar cell. PMID:8200977

  2. Calcium and melatonin production in dissociated trout pineal photoreceptor cells in culture.

    PubMed

    Bégay, V; Bois, P; Collin, J P; Lenfant, J; Falcón, J

    1994-07-01

    Trout pineal cells maintained in primary culture produce melatonin in high amounts during night time and low amounts during daytime. The dark-induced increase in melatonin production was enhanced, in a dose-dependent manner, by elevating extracellular calcium concentration. Low external calcium concentration reduced nocturnal and diurnal melatonin production. Bay K 8644 increased, in a dose-dependent manner, the dark-induced rise in melatonin output, and this effect was antagonized by nifedipine and verapamil. This suggests a role for the dihydropyridine calcium channels in the regulation of the melatonin output. To confirm this, patch-clamp recordings (whole-cell perforated) were run in a 20 mmol/l barium medium at different holding potentials from -80 mV. A voltage-dependent inward current was activated from -30 mV to +40 mV with a maximal amplitude being observed at 0 mV. This current was drastically increased in the presence of Bay K 8644. Nifedipine inhibited the current both in the absence or in the presence of Bay K 8644. Our results are consistent with the idea that extracellular calcium participates in the control of melatonin secretion by photoreceptor cells. It is suggested that activation of the voltage-dependent L-type channel may modulate this secretion.

  3. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists.

  4. Colored lenses suppress blue light-emitting diode light-induced damage in photoreceptor-derived cells

    NASA Astrophysics Data System (ADS)

    Hiromoto, Kaho; Kuse, Yoshiki; Tsuruma, Kazuhiro; Tadokoro, Nobuyuki; Kaneko, Nobuyuki; Shimazawa, Masamitsu; Hara, Hideaki

    2016-03-01

    Blue light-emitting diodes (LEDs) in liquid crystal displays emit high levels of blue light, exposure to which is harmful to the retina. Here, we investigated the protective effects of colored lenses in blue LED light-induced damage to 661W photoreceptor-derived cells. We used eight kinds of colored lenses and one lens that reflects blue light. Moreover, we evaluated the relationship between the protective effects of the lens and the transmittance of lens at 464 nm. Lenses of six colors, except for the SY, PN, and reflective coating lenses, strongly decreased the reduction in cell damage induced by blue LED light exposure. The deep yellow lens showed the most protective effect from all the lenses, but the reflective coating lens and pink lens did not show any effects on photoreceptor-derived cell damage. Moreover, these results were correlated with the lens transmittance of blue LED light (464 nm). These results suggest that lenses of various colors, especially deep yellow lenses, may protect retinal photoreceptor cells from blue LED light in proportion to the transmittance for the wavelength of blue LED and the suppression of reactive oxygen species production and cell damage.

  5. Rb deficiency during Drosophila eye development deregulates EMC, causing defects in the development of photoreceptors and cone cells.

    PubMed

    Popova, Milena K; He, Wei; Korenjak, Michael; Dyson, Nicholas J; Moon, Nam-Sung

    2011-12-15

    Retinoblastoma tumor suppressor protein (pRb) regulates various biological processes during development and tumorigenesis. Although the molecular mechanism by which pRb controls cell cycle progression is well characterized, how pRb promotes cell-type specification and differentiation is less understood. Here, we report that Extra Macrochaetae (EMC), the Drosophila homolog of inhibitor of DNA binding/differentiation (ID), is an important protein contributing to the developmental defects caused by Rb deficiency. An emc allele was identified from a genetic screen designed to identify factors that, when overexpressed, cooperate with mutations in rbf1, which encodes one of the two Rb proteins found in Drosophila. EMC overexpression in an rbf1 hypomorphic mutant background induces cone cell and photoreceptor defects but has negligible effects in the wild-type background. Interestingly, a substantial fraction of the rbf1-null ommatidia normally exhibit similar cone cell and photoreceptor defects in the absence of ectopic EMC expression. Detailed EMC expression analyses revealed that RBF1 suppresses expression of both endogenous and ectopic EMC protein in photoreceptors, thus explaining the synergistic effect between EMC overexpression and rbf1 mutations, and the developmental defect observed in rbf1-null ommatidia. Our findings demonstrate that ID family proteins are an evolutionarily conserved determinant of Rb-deficient cells, and play an important role during development.

  6. Colored lenses suppress blue light-emitting diode light-induced damage in photoreceptor-derived cells.

    PubMed

    Hiromoto, Kaho; Kuse, Yoshiki; Tsuruma, Kazuhiro; Tadokoro, Nobuyuki; Kaneko, Nobuyuki; Shimazawa, Masamitsu; Hara, Hideaki

    2016-03-01

    Blue light-emitting diodes (LEDs) in liquid crystal displays emit high levels of blue light, exposure to which is harmful to the retina. Here, we investigated the protective effects of colored lenses in blue LED light-induced damage to 661W photoreceptor-derived cells. We used eight kinds of colored lenses and one lens that reflects blue light. Moreover, we evaluated the relationship between the protective effects of the lens and the transmittance of lens at 464 nm. Lenses of six colors, except for the SY, PN, and reflective coating lenses, strongly decreased the reduction in cell damage induced by blue LED light exposure. The deep yellow lens showed the most protective effect from all the lenses, but the reflective coating lens and pink lens did not show any effects on photoreceptor-derived cell damage. Moreover, these results were correlated with the lens transmittance of blue LED light (464 nm). These results suggest that lenses of various colors, especially deep yellow lenses, may protect retinal photoreceptor cells from blue LED light in proportion to the transmittance for the wavelength of blue LED and the suppression of reactive oxygen species production and cell damage.

  7. Reprogramming amacrine and photoreceptor progenitors into retinal ganglion cells by replacing Neurod1 with Atoh7.

    PubMed

    Mao, Chai-An; Cho, Jang-Hyeon; Wang, Jing; Gao, Zhiguang; Pan, Ping; Tsai, Wen-Wei; Frishman, Laura J; Klein, William H

    2013-02-01

    The specification of the seven retinal cell types from a common pool of retina progenitor cells (RPCs) involves complex interactions between the intrinsic program and the environment. The proneural basic helix-loop-helix (bHLH) transcriptional regulators are key components for the intrinsic programming of RPCs and are essential for the formation of the diverse retinal cell types. However, the extent to which an RPC can re-adjust its inherent program and the mechanisms through which the expression of a particular bHLH factor influences RPC fate is unclear. Previously, we have shown that Neurod1 inserted into the Atoh7 locus activates the retinal ganglion cell (RGC) program in Atoh7-expressing RPCs but not in Neurod1-expressing RPCs, suggesting that Atoh7-expressing RPCs are not able to adopt the cell fate determined by Neurod1, but rather are pre-programmed to produce RGCs. Here, we show that Neurod1-expressing RPCs, which are destined to produce amacrine and photoreceptor cells, can be re-programmed into RGCs when Atoh7 is inserted into the Neurod1 locus. These results suggest that Atoh7 acts dominantly to convert a RPC subpopulation not destined for an RGC fate to adopt that fate. Thus, Atoh7-expressing and Neurod1-expressing RPCs are intrinsically different in their behavior. Additionally, ChIP-Seq analysis identified an Atoh7-dependent enhancer within the intronic region of Nrxn3. The enhancer recognized and used Atoh7 in the developing retina to regulate expression of Nrxn3, but could be forced to use Neurod1 when placed in a different regulatory context. The results indicate that Atoh7 and Neurod1 activate distinct sets of genes in vivo, despite their common DNA-binding element.

  8. Sodium formate induces autophagy and apoptosis via the JNK signaling pathway of photoreceptor cells.

    PubMed

    Wang, Ying; Xu, Shao-Lin; Xu, Wen-Jing; Yang, Hai-Yan; Hu, Ping; Li, Yu-Xin

    2016-02-01

    Incidents associated with methanol intoxication resulting from the consumption of fake wine occur not infrequently worldwide. Certain individuals are made blind due to methanol poisoning. The present study aimed to investigate the effects of sodium formate exposure on photoreceptor cells (661W cells). The 661W cells were exposed to sodium formate for 6‑24 h and cell viability was determined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetrazolium bromide (MTT) assay. Subsequently, the 661W cells were exposed to 15 or 30 mM sodium formate for 24 h. The level of apoptosis was determined using Hoechst 33342/propidium iodide staining, visualizing the cells under a fluorescence microscope, and annexin V‑fluorescein isothiocyanate staining, using flow cytometric analysis. Intracellular reactive oxygen species (ROS) were measured using 2',7'‑dichlorofluorescein diacetate (DCFH‑DA) staining, followed by flow cytometric analysis. Autophagy of the 661W cells was measured by monodansylcadaverine staining. The activation of phosphorylated c‑Jun N‑terminal kinase (p‑JNK), B‑cell lymphoma (Bcl‑2), Bcl‑2‑associated X protein, cleaved caspase‑3, cleaved caspase‑9 and microtubule‑associated protein 1A/1B‑light chain 3 (LC3) was assessed by western blotting. The effects of Z‑VAD‑fmk (a pan‑caspase inhibitor) and SP600125 (a JNK inhibitor) on the viability of the sodium formate‑induced 661W cells were determined using an MTT assay. Sodium formate treatment induced a decrease in the viability of the 661W cells in a time‑ and a dose‑dependent manner. In addition, sodium formate at concentrations of 15 or 30 mM markedly increased the level of apoptosis and the ROS levels, as measured by DCFH‑DA staining of the 661W cells. Additionally, 661W cells exposed to sodium formate for 24 h exhibited increased levels of p‑JNK, Bax, cleaved caspase‑3, cleaved caspase‑9 and LC3II (the phosphatidylethanolamine‑modified form

  9. Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell‐Derived Self‐Forming Retina

    PubMed Central

    Gonzalez‐Cordero, Anai; West, Emma L.; Han, Ya‐Ting; Welby, Emily; Naeem, Arifa; Blackford, Samuel J. I.; Bainbridge, James W. B.; Pearson, Rachael A.; Ali, Robin R.

    2015-01-01

    Abstract Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell‐derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post‐mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(−) facilitates the isolation of photoreceptor precursors from three‐dimensional self‐forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell‐derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation‐competent cells for therapeutic application. Stem Cells 2015;33:2469—2482 PMID:25982268

  10. The rat retina has five types of ganglion-cell photoreceptors

    PubMed Central

    Reifler, Aaron N.; Chervenak, Andrew P.; Dolikian, Michael E.; Benenati, Brian A.; Meyers, Benjamin S.; Demertzis, Zachary D.; Lynch, Andrew M.; Li, Benjamin Y.; Wachter, Rebecca D.; Abufarha, Fady S.; Dulka, Eden A.; Pack, Weston; Zhao, Xiwu; Wong, Kwoon Y.

    2014-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are inner retinal photoreceptors that mediate non-image-forming visual functions, e.g. pupillary constriction, regulation of pineal melatonin release, and circadian photoentrainment. Five types of ipRGCs were recently discovered in mouse, but whether they exist in other mammals remained unknown. We report that the rat also has five types of ipRGCs, whose morphologies match those of mouse ipRGCs; this is the first demonstration of all five cell types in a non-mouse species. Through immunostaining and λmax measurements, we showed that melanopsin is likely the photopigment of all rat ipRGCs. The various cell types exhibited diverse spontaneous spike rates, with the M1 type spiking the least and M4 spiking the most, just like we had observed for their mouse counterparts. Also similar to mouse, all ipRGCs in rat generated not only sluggish intrinsic photoresponses but also fast, synaptically driven ones. However, we noticed two significant differences between these species. First, whereas we learned previously that all mouse ipRGCs had equally sustained synaptic light responses, rat M1 cells’ synaptic photoresponses were far more transient than those of M2–M5. Since M1 cells provide all input to the circadian clock, this rat-versus-mouse discrepancy could explain the difference in photoentrainment threshold between mouse and other species. Second, rat ipRGCs’ melanopsin-based spiking photoresponses could be classified into three varieties, but only two were discerned for mouse ipRGCs. This correlation of spiking photoresponses with cell types will help researchers classify ipRGCs in multielectrode-array (MEA) spike recordings. PMID:25450063

  11. Protective effects of naringenin eye drops on N-methyl-N-nitrosourea-induced photoreceptor cell death in rats

    PubMed Central

    Lin, Jun-Li; Wang, Yan-Dong; Ma, Yan; Zhong, Chun-Mei; Zhu, Mei-Rong; Chen, Wen-Pei; Lin, Bao-Qin

    2014-01-01

    AIM To investigate the effects of naringenin eye drops on N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell death in rats. METHODS Photoreceptor cell death was induced by single intraperitoneal injection of MNU (60 mg/kg) in rats. Both eyes of all animals were instilled with one drop of vehicle, 0.5% or 1.0% naringenin eye drops three times per day from 7d before to 17d after MNU injection. Effects of naringenin on MNU-induced photoreceptor cell death were evaluated by electrophysiological and histological analysis. RESULTS Flash electroretinography (FERG) and oscillatory potentials (OPs) recordings showed that the vehicle control group had remarkable reduction of amplitudes and prolongation of latency times. FERG and OPs responses were significantly reversed in MNU-induced rats treated with 0.5% or 1.0% naringenin eye drops compared with the vehicle control. The retinal morphological results showed that naringenin dose-dependently preserved the outer nuclear layer, outer retina and total retina. CONCLUSION These results indicate that topical treatment with naringenin eye drops prevented retinal neurons from MNU-induced structural and functional damages. PMID:24967179

  12. Luminescence- and nanoparticle-mediated increase of light absorption by photoreceptor cells: Converting UV light to visible light.

    PubMed

    Li, Lei; Sahi, Sunil K; Peng, Mingying; Lee, Eric B; Ma, Lun; Wojtowicz, Jennifer L; Malin, John H; Chen, Wei

    2016-02-10

    We developed new optic devices - singly-doped luminescence glasses and nanoparticle-coated lenses that convert UV light to visible light - for improvement of visual system functions. Tb(3+) or Eu(3+) singly-doped borate glasses or CdS-quantum dot (CdS-QD) coated lenses efficiently convert UV light to 542 nm or 613 nm wavelength narrow-band green or red light, or wide-spectrum white light, and thereby provide extra visible light to the eye. In zebrafish (wild-type larvae and adult control animals, retinal degeneration mutants, and light-induced photoreceptor cell degeneration models), the use of Tb(3+) or Eu(3+) doped luminescence glass or CdS-QD coated glass lenses provide additional visible light to the rod and cone photoreceptor cells, and thereby improve the visual system functions. The data provide proof-of-concept for the future development of optic devices for improvement of visual system functions in patients who suffer from photoreceptor cell degeneration or related retinal diseases.

  13. Luminescence- and nanoparticle-mediated increase of light absorption by photoreceptor cells: Converting UV light to visible light

    PubMed Central

    Li, Lei; Sahi, Sunil K.; Peng, Mingying; Lee, Eric B.; Ma, Lun; Wojtowicz, Jennifer L.; Malin, John H.; Chen, Wei

    2016-01-01

    We developed new optic devices – singly-doped luminescence glasses and nanoparticle-coated lenses that convert UV light to visible light – for improvement of visual system functions. Tb3+ or Eu3+ singly-doped borate glasses or CdS-quantum dot (CdS-QD) coated lenses efficiently convert UV light to 542 nm or 613 nm wavelength narrow-band green or red light, or wide-spectrum white light, and thereby provide extra visible light to the eye. In zebrafish (wild-type larvae and adult control animals, retinal degeneration mutants, and light-induced photoreceptor cell degeneration models), the use of Tb3+ or Eu3+ doped luminescence glass or CdS-QD coated glass lenses provide additional visible light to the rod and cone photoreceptor cells, and thereby improve the visual system functions. The data provide proof-of-concept for the future development of optic devices for improvement of visual system functions in patients who suffer from photoreceptor cell degeneration or related retinal diseases. PMID:26860393

  14. Different RPGR exon ORF15 mutations in Canids provide insights into photoreceptor cell degeneration.

    PubMed

    Zhang, Qi; Acland, Gregory M; Wu, Wen X; Johnson, Jennifer L; Pearce-Kelling, Sue; Tulloch, Brian; Vervoort, Raf; Wright, Alan F; Aguirre, Gustavo D

    2002-05-01

    The canine disease, X-linked progressive retinal atrophy (XLPRA), is similar to human RP3, an X-linked form of retinitis pigmentosa, and maps to the same region in the X chromosome. Analysis of the physical map of the XLPRA and RP3 intervals shows a high degree of conservation in terms of genes and their order. We have found different mutations in exon ORF15 of the RPGR gene in two distinct mutant dog strains (XLPRA1, XLPRA2). Microdeletions resulting in a premature stop or a frameshift mutation result in very different retinal phenotypes, which are allele-specific and consistent for each mutation. The phenotype associated with the frameshift mutation in XLPRA2 is very severe and manifests during retinal development; the phenotype resulting from the XLPRA1 nonsense mutation is expressed only after normal photoreceptor morphogenesis. Splicing of RPGR mRNA transcripts in retina is complex, and either exon ORF15 or exon 19 can be a terminal exon. The retina-predominant transcript contains ORF15 as a terminal exon, and is expressed in normal and mutant retinas. The frameshift mutation dramatically alters the deduced amino acid sequence, and the protein aggregates in the endoplasmic reticulum of transfected cells. The cellular and molecular results in the two canine RPGR exon ORF15 mutations have implications for understanding the phenotypic variability found in human RP3 families that carry similar mutations.

  15. Lipofuscins prepared by modification of photoreceptor cells via glycation or lipid peroxidation show the similar phototoxicity

    PubMed Central

    Dontsov, Alexander; Koromyslova, Anna; Ostrovsky, Mikhail; Sakina, Natalia

    2016-01-01

    AIM To investigate the effect of two ways of lipofuscin production (lipid peroxidation and glycation) on lipofuscin fluorescence characteristics and phototoxicity and to compare them with the properties of natural lipofuscin. METHODS Model lipofuscins were prepared on the basis of bovine photoreceptor outer segments (POS) with bisretinoid A2E addition. One set of samples was prepared from POS modified by lipid peroxidation, while another set from POS modified by glycation with fructose. Fluorescent properties and kinetics of photoinduced superoxide generation of model lipofuscins and human retinal pigment epithelium (RPE) lipofuscin were compared. The fluorescence spectra of samples were measured at 365 nm excitation wavelength and 380-650 emission wavelength. RESULTS The fluorescence spectra of model lipofuscins are almost the same as the spectrum of natural lipofuscin. Visible light irradiation of both model lipofuscins and natural lipofuscin isolated from RPE cells leads to decrease of a fluorescence maximum at 550 nm and to appearance of a distinct, new maximum at 445-460 nm. The rate of photogeneration of reactive oxygen forms by both model lipofuscins was almost the same and approximately two times less than that of RPE lipofuscin granules. CONCLUSION These data suggest that fluorescent characteristics and phototoxicity of lipofuscin granules depend only to an insignificant degree on the oxidative modification of POS proteins and lipids, and generally are defined by the bisretinoid fluorophores contained in them. PMID:27909686

  16. Erythropoietin Slows Photoreceptor Cell Death in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa

    PubMed Central

    Kasmala, Lorraine; Bond, Wesley S.; de Lucas Cerrillo, Ana M.; Wynn, Kristi; Lewin, Alfred S.

    2016-01-01

    Purpose To test the efficacy of systemic gene delivery of a mutant form of erythropoietin (EPO-R76E) that has attenuated erythropoietic activity, in a mouse model of autosomal dominant retinitis pigmentosa. Methods Ten-day old mice carrying one copy of human rhodopsin with the P23H mutation and both copies of wild-type mouse rhodopsin (hP23H RHO+/-,mRHO+/+) were injected into the quadriceps with recombinant adeno-associated virus (rAAV) carrying either enhanced green fluorescent protein (eGFP) or EpoR76E. Visual function (electroretinogram) and retina structure (optical coherence tomography, histology, and immunohistochemistry) were assessed at 7 and 12 months of age. Results The outer nuclear layer thickness decreased over time at a slower rate in rAAV.EpoR76E treated as compared to the rAAV.eGFP injected mice. There was a statistically significant preservation of the electroretinogram at 7, but not 12 months of age. Conclusions Systemic EPO-R76E slows death of the photoreceptors and vision loss in hP23H RHO+/-,mRHO+/+ mice. Treatment with EPO-R76E may widen the therapeutic window for retinal degeneration patients by increasing the number of viable cells. Future studies might investigate if co-treatment with EPO-R76E and gene replacement therapy is more effective than gene replacement therapy alone. PMID:27299810

  17. Hessian-LoG filtering for enhancement and detection of photoreceptor cells in adaptive optics retinal images.

    PubMed

    Lazareva, Anfisa; Liatsis, Panos; Rauscher, Franziska G

    2016-01-01

    Automated analysis of retinal images plays a vital role in the examination, diagnosis, and prognosis of healthy and pathological retinas. Retinal disorders and the associated visual loss can be interpreted via quantitative correlations, based on measurements of photoreceptor loss. Therefore, it is important to develop reliable tools for identification of photoreceptor cells. In this paper, an automated algorithm is proposed, based on the use of the Hessian-Laplacian of Gaussian filter, which allows enhancement and detection of photoreceptor cells. The performance of the proposed technique is evaluated on both synthetic and high-resolution retinal images, in terms of packing density. The results on the synthetic data were compared against ground truth as well as cone counts obtained by the Li and Roorda algorithm. For the synthetic datasets, our method showed an average detection accuracy of 98.8%, compared to 93.9% for the Li and Roorda approach. The packing density estimates calculated on the retinal datasets were validated against manual counts and the results obtained by a proprietary software from Imagine Eyes and the Li and Roorda algorithm. Among the tested methods, the proposed approach showed the closest agreement with manual counting.

  18. Rod and cone photoreceptor cells produce ROS in response to stress in a live retinal explant system

    PubMed Central

    Bhatt, Lavinia; Groeger, Gillian; McDermott, Kieran

    2010-01-01

    Purpose The production of reactive oxygen species (ROS) can lead to oxidative stress, which is a strong contributory factor to many ocular diseases. In this study, the removal of trophic factors is used as a model system to investigate the effects of stress in the retina. The aims were to determine if both rod and cone photoreceptor cells produce ROS when they are deprived of trophic factor support and to demonstrate if the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes are responsible for this ROS production. Methods Retinas were explanted from mice aged between postnatal days 8–10 and cultured overnight. The following morning, confocal microscopy combined with various fluorescent probes was used to detect the production of ROS. Each time peanut agglutinin (PNA), a cone photoreceptor marker, was used to facilitate orientation of the retina. Dihydroethidium and dihydrorhodamine 123 (DHR123) were used to determine which cells produce ROS. Subsequently, western blots of retinal serial sections were used to detect the presence of Noxs in the different retinal layers. The Nox inhibitor apocynin was then tested to determine if it altered the production of ROS within these cells. Results Live retinal explants, viewed at high magnifications using confocal microscopy, displayed an increase in the fluorescent products of dihydroethidium and DHR123 upon serum removal when compared to controls. DHR123 fluorescence, once oxidized, localized to mitochondria and was found in the same focal plane as the PNA staining. This showed that cones and rods produced ROS when stressed. Retinal serial sectioning established that the photoreceptor layer expressed Nox4, dual oxidase (Duox) 1, and Duox2 at varying levels. Finally, the Nox inhibitor apocynin decreased the burst stimulated by the stress of serum removal. Conclusions Confocal microscopy and PNA staining allowed differentiation of cell types within the outermost layers of the retina, demonstrating that

  19. Photoreceptor transplantation in inherited and environmentally induced retinal degeneration: anatomy, immunohistochemistry and function.

    PubMed

    Silverman, M S; Hughes, S E

    1989-01-01

    In conclusion, we have shown that photoreceptors can be transplanted to retina in which the host's photoreceptors are lost by environmental (constant light) or inherited deficits. Furthermore transplanted photoreceptor cells maintain basic characteristics of normal photoreceptor cells by producing opsin and maintaining an intercellular organization and apposition to the host retina that is similar to that seen in the normal outer nuclear layer. To accomplish this we have devised a method to isolate the intact photoreceptor layer. This is significant because it will be necessary to maintain tight matrix organization if coherent vision is to be restored to the retina compromised by the loss of photoreceptors. We have further developed a surgical approach which minimizes trauma to the eye and allows controlled positioning of sheets of transplanted photoreceptors to their homotopic location within the eye. In addition these methods for transplantation and isolation of photoreceptors could be utilized to prepare and transplant other retinal layers so that selected populations of retinal cells can be used in other neurobiological investigations. Photoreceptors can be transplanted when developing or when mature. Not only can mature rat photoreceptors can be transplanted, but we have shown that mature photoreceptors from human donors can be transplanted as well. This is significantly different from neurons which must be immature in order to be transplanted. At present the reason for this difference is not known but has obvious importance for retinal and neural transplantation research in general. Finally, we have shown that transplanted photoreceptors activate the host's dystrophic retina in a light dependent manner that closely resembles the activation pattern seen in normal retina. This finding taken together with our results showing that human photoreceptors can be transplanted presents the possibility that some forms of human blindness might eventually be ameliorated

  20. The protective effects of bilberry and lingonberry extracts against UV light-induced retinal photoreceptor cell damage in vitro.

    PubMed

    Ogawa, Kenjirou; Tsuruma, Kazuhiro; Tanaka, Junji; Kakino, Mamoru; Kobayashi, Saori; Shimazawa, Masamitsu; Hara, Hideaki

    2013-10-30

    Bilberry extract (B-ext) and lingonberry extract (L-ext) are currently used as health supplements. We investigated the protective mechanisms of the B-ext and L-ext against ultraviolet A (UVA)-induced retinal photoreceptor cell damage. Cultured murine photoreceptor (661W) cells were exposed to UVA following treatment with B-ext and L-ext and their main constituents (cyanidin, delphinidin, malvidin, trans-resveratrol, and procyanidin). B-ext, L-ext, and constituents improved cell viability and suppressed ROS generation. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and protein kinase B (Akt) were analyzed by Western blotting. B-ext and cyanidin inhibited phosphorylation of p38 MAPK, and B-ext also inhibited phosphorylation of JNK by UVA. L-ext, trans-resveratrol, and procyanidin alleviated the reduction of phosphorylated Akt levels by UVA. Finally, a cotreatment with B-ext and L-ext showed an additive effect on cell viability. Our findings suggest that both B-ext and L-ext endow protective effects against UVA-induced retinal damage.

  1. Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

    PubMed Central

    2011-01-01

    Background Retinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined. Results Treatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA. Conclusions These data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor

  2. Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

    PubMed

    Xie, Bin-Bin; Zhang, Xiang-Mei; Hashimoto, Takao; Tien, Amy H; Chen, Andrew; Ge, Jian; Yang, Xian-Jie

    2014-01-01

    The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

  3. ERK-mediated activation of Fas apoptotic inhibitory molecule 2 (Faim2) prevents apoptosis of 661W cells in a model of detachment-induced photoreceptor cell death.

    PubMed

    Besirli, Cagri G; Zheng, Qiong-Duon; Reed, David M; Zacks, David N

    2012-01-01

    In this study, we examined the role of Fas apoptotic inhibitory molecule 2 (Faim2), an inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling during Fas-mediated apoptosis of 661W cells, an immortalized photoreceptor-like cell line Treatment of 661W cells with a Fas-activating antibody led to increased levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fas-treated 661W cells, but only the inhibition of the ERK pathway reduced the levels of Faim2. Blocking the ERK pathway using a pharmacological inhibitor increased the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis. When the levels of Faim2 were reduced in 661W cells by siRNA knockdown, Fas activating antibody treatment resulted in earlier and more robust caspase activation, and increased cell death. These results demonstrate that Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of 661W cells. The expression of Faim2 is triggered, at least in part, by Fas-receptor activation and subsequent ERK signaling. Our findings identify a novel protective pathway that auto-regulates Fas-induced photoreceptor apoptosis in vitro. Modulation of this pathway to increase Faim2 expression may be a potential therapeutic option to prevent photoreceptor death.

  4. Direct cell fate conversion of human somatic stem cells into cone and rod photoreceptor-like cells by inhibition of microRNA-203.

    PubMed

    Choi, Soon Won; Shin, Ji-Hee; Kim, Jae-Jun; Shin, Tae-Hoon; Seo, Yoojin; Kim, Hyung-Sik; Kang, Kyung-Sun

    2016-07-05

    Stem cell-based photoreceptor differentiation strategies have been the recent focus of therapies for retinal degenerative diseases. Previous studies utilized embryonic stem (ES) cells and neural retina differentiation cocktails, including DKK1 and Noggin. Here, we show a novel microRNA-mediated strategy of retina differentiation from somatic stem cells, which are potential allogeneic cell sources. Human amniotic epithelial stem cells (AESCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) treated with a retina differentiation cocktail induced gene expressions of retina development-relevant genes. Furthermore, microRNA-203 (miR-203) is abundantly expressed in human AESCs and human UCB-MSCs. This miR-203 is predicted to target multiple retina development-relevant genes, particularly DKK1, CRX, RORβ, NEUROD1, NRL and THRB. The inhibition of miR-203 induced a retina differentiation of AESCs and UCB-MSCs. Moreover, successive treatments of anti-miR-203 led to the expression of both mature photoreceptor (PR) markers, rhodopsin and opsin. In addition, we determined that CRX, NRL and DKK1 are direct targets of miR-203 using a luciferase assay. Thus, the work presented here suggests that somatic stem cells can potentially differentiate into neural retina cell types when treated with anti-miR-203. They may prove to be a source of both PR subtypes for future allogeneic stem cell-based therapies of non-regenerative retina diseases.

  5. Structural organization and function of mouse photoreceptor ribbon synapses involve the immunoglobulin protein synaptic cell adhesion molecule 1.

    PubMed

    Ribic, Adema; Liu, Xinran; Crair, Michael C; Biederer, Thomas

    2014-03-01

    Adhesive interactions in the retina instruct the developmental specification of inner retinal layers. However, potential roles of adhesion in the development and function of photoreceptor synapses remain incompletely understood. This contrasts with our understanding of synapse development in the CNS, which can be guided by select adhesion molecules such as the Synaptic Cell Adhesion Molecule 1 (SynCAM 1/CADM1/nectin-like 2 protein). This immunoglobulin superfamily protein modulates the development and plasticity of classical excitatory synapses. We show here by immunoelectron microscopy and immunoblotting that SynCAM 1 is expressed on mouse rod photoreceptors and their terminals in the outer nuclear and plexiform layers in a developmentally regulated manner. Expression of SynCAM 1 on rods is low in early postnatal stages (P3-P7) but increases after eye opening (P14). In support of functional roles in the photoreceptors, electroretinogram recordings demonstrate impaired responses to light stimulation in SynCAM 1 knockout (KO) mice. In addition, the structural integrity of synapses in the OPL requires SynCAM 1. Quantitative ultrastructural analysis of SynCAM 1 KO retina measured fewer fully assembled, triadic rod ribbon synapses. Furthermore, rod synapse ribbons are shortened in KO mice, and protein levels of Ribeye, a major structural component of ribbons, are reduced in SynCAM 1 KO retina. Together, our results implicate SynCAM 1 in the synaptic organization of the rod visual pathway and provide evidence for novel roles of synaptic adhesion in the structural and functional integrity of ribbon synapses.

  6. Phospholipid flippase ATP8A2 is required for normal visual and auditory function and photoreceptor and spiral ganglion cell survival.

    PubMed

    Coleman, Jonathan A; Zhu, Xianjun; Djajadi, Hidayat R; Molday, Laurie L; Smith, Richard S; Libby, Richard T; John, Simon W M; Molday, Robert S

    2014-03-01

    ATP8A2 is a P4-ATPase that is highly expressed in the retina, brain, spinal cord and testes. In the retina, ATP8A2 is localized in photoreceptors where it uses ATP to transport phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the exoplasmic to the cytoplasmic leaflet of membranes. Although mutations in ATP8A2 have been reported to cause mental retardation in humans and degeneration of spinal motor neurons in mice, the role of ATP8A2 in sensory systems has not been investigated. We have analyzed the retina and cochlea of ATP8A2-deficient mice to determine the role of ATP8A2 in visual and auditory systems. ATP8A2-deficient mice have shortened photoreceptor outer segments, a reduction in photoresponses and decreased photoreceptor viability. The ultrastructure and phagocytosis of the photoreceptor outer segment appeared normal, but the PS and PE compositions were altered and the rhodopsin content was decreased. The auditory brainstem response threshold was significantly higher and degeneration of spiral ganglion cells was apparent. Our studies indicate that ATP8A2 plays a crucial role in photoreceptor and spiral ganglion cell function and survival by maintaining phospholipid composition and contributing to vesicle trafficking.

  7. Neuroprotectin D1 is synthesized in the cone photoreceptor cell line 661W and elicits protection against light-induced stress.

    PubMed

    Kanan, Y; Gordon, W C; Mukherjee, P K; Bazan, N G; Al-Ubaidi, M R

    2015-03-01

    Docosahexaenoic acid (DHA), an omega-3 fatty acid family member, is obtained by diet or synthesized from dietary essential omega-3 linolenic acid and delivered systemically to the choriocapillaris, from where it is taken up by the retinal pigment epithelium (RPE). DHA is then transported to the inner segments of photoreceptors, where it is incorporated in phospholipids during the biogenesis of outer segment disk and plasma membranes. As apical photoreceptor disks are gradually shed and phagocytized by the RPE, DHA is retrieved and recycled back to photoreceptor inner segments for reassembly into new disks. Under uncompensated oxidative stress, the docosanoid neuroprotectin D1 (NPD1), a potent mediator derived from DHA, is formed by the RPE and displays its bioactivity in an autocrine and paracrine fashion. The purpose of this study was to determine whether photoreceptors have the ability to synthesize NPD1, and whether or not this lipid mediator exerts bioactivity on these cells. For this purpose, 661W cells (mouse-derived photoreceptor cells) were used. First we asked whether these cells have the ability to form NPD1 by incubating cells with deuterium (d4)-labeled DHA exposed to dark and bright light treatments, followed by LC-MS/MS-based lipidomic analysis to identify and quantify d4-NPD1. The second question pertains to the potential bioactivity of these lipids. Therefore, cells were incubated with 9-cis-retinal in the presence of bright light that triggers cell damage and death. Following 9-cis-retinal loading, DHA, NPD1, or vehicle were added to the media and the 661W cells maintained either in darkness or under bright light. DHA and NPD1 were then quantified in cells and media. Regardless of lighting conditions, 661W cells acquired DHA from the media and synthesized 4-9 times as much d4-NPD1 under bright light treatment in the absence and presence of 9-cis-retinal compared to cells in darkness. Viability assays of 9-cis-retinal-treated cells demonstrated that

  8. Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo.

    PubMed

    Fontaine, Valérie; Monteiro, Elodie; Brazhnikova, Elena; Lesage, Laëtitia; Balducci, Christine; Guibout, Louis; Feraille, Laurence; Elena, Pierre-Paul; Sahel, José-Alain; Veillet, Stanislas; Lafont, René

    2016-01-01

    The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9'-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9'-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial.

  9. Pharmacological inhibitions of glutamate transporters EAAT1 and EAAT2 compromise glutamate transport in photoreceptor to ON- bipolar cell synapses

    PubMed Central

    Tse, Dennis Y.; Chung, Inyoung; Wu, Samuel M.

    2015-01-01

    To maintain reliable signal transmission across a synapse, free synaptic neurotransmitters must be removed from the cleft in a timely manner. In the first visual synapse, this critical task is mainly undertaken by glutamate transporters (EAATs). Here we study the differential roles of the EAAT1, EAAT2 and EAAT5 subtypes in glutamate (GLU) uptake at the photoreceptor-to-depolarizing bipolar cell synapse in intact dark-adapted retina. Various doses of EAAT blockers and/or GLU were injected into the eye before the electroretinogram (ERG) was measured. Their effectiveness and potency in inhibiting the ERG b-wave were studied to determine their relative contributions to the GLU clearing activity at the synapse. The results showed that EAAT1 and EAAT2 plays different roles. Selectively blocking glial EAAT1 alone using UCPH101 inhibited the b-wave 2–24 hours following injection, suggesting a dominating role of EAAT1 in the overall GLU clearing capacity in the synaptic cleft. Selectively blocking EAAT2 on photoreceptor terminals had no significant effect on the b-wave, but increased the potency of exogenous GLU in inhibiting the b-wave. These suggest that EAAT2 play a secondary yet significant role in the GLU reuptake activity at the rod and the cone output synapses. Additionally, we have verified our electrophysiological findings with double-label immunohistochemistry, and extend the literature on the spatial distribution of EAAT2 splice variants in the mouse retina. PMID:25152321

  10. Norbixin Protects Retinal Pigmented Epithelium Cells and Photoreceptors against A2E-Mediated Phototoxicity In Vitro and In Vivo

    PubMed Central

    Monteiro, Elodie; Brazhnikova, Elena; Lesage, Laëtitia; Balducci, Christine; Guibout, Louis; Feraille, Laurence; Elena, Pierre-Paul; Sahel, José-Alain; Veillet, Stanislas; Lafont, René

    2016-01-01

    The accumulation of N-retinylidene-N-retinylethanolamine (A2E, a toxic by-product of the visual pigment cycle) in the retinal pigment epithelium (RPE) is a major cause of visual impairment in the elderly. Photooxidation of A2E results in retinal pigment epithelium degeneration followed by that of associated photoreceptors. Present treatments rely on nutrient supplementation with antioxidants. 9’-cis-Norbixin (a natural diapocarotenoid, 97% purity) was prepared from Bixa orellana seeds. It was first evaluated in primary cultures of porcine retinal pigment epithelium cells challenged with A2E and illuminated with blue light, and it provided an improved photo-protection as compared with lutein or zeaxanthin. In Abca4-/- Rdh8-/- mice (a model of dry AMD), intravitreally-injected norbixin maintained the electroretinogram and protected photoreceptors against light damage. In a standard rat blue-light model of photodamage, norbixin was at least equally as active as phenyl-N-tert-butylnitrone, a free radical spin-trap. Chronic experiments performed with Abca4-/- Rdh8-/- mice treated orally for 3 months with norbixin showed a reduced A2E accumulation in the retina. Norbixin appears promising for developing an oral treatment of macular degeneration. A drug candidate (BIO201) with 9’-cis-norbixin as the active principle ingredient is under development, and its potential will be assessed in a forthcoming clinical trial. PMID:27992460

  11. An essential role for RAX homeoprotein and NOTCH-HES signaling in Otx2 expression in embryonic retinal photoreceptor cell fate determination.

    PubMed

    Muranishi, Yuki; Terada, Koji; Inoue, Tatsuya; Katoh, Kimiko; Tsujii, Toshinori; Sanuki, Rikako; Kurokawa, Daisuke; Aizawa, Shinichi; Tamaki, Yasuhiro; Furukawa, Takahisa

    2011-11-16

    The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ∼500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.

  12. Analysis of the light-sensitivity of the photoreceptor cells of the ataxia and male sterility (AMS) mouse, an Nna1 mutant.

    PubMed

    Araki, Asuka; Maruyama, Riruke; Harada, Yuji; Ishikawa, Noriyoshi; Harada, Takayuki

    2012-11-01

    We confirmed retinal degeneration in the ataxia and male sterility (AMS) mouse, a mutant of the Nna1 gene, and examined the photosensitivity of the photoreceptors to determine how closely related the intrinsic and extrinsic factors were in triggering photoreceptor cell death. The AMS mice reared in a dark environment did not show atrophy of the outer nuclear layer (ONL) before 4 weeks of age, but in the older mice, retinal atrophy progressed in the same manner as in the AMS mice housed under normal light conditions. Examining the sensitivity to intentional light stimulation revealed the atrophy of the AMS retina to be exacerbated by a weak light. After administering strong light irradiation, equally severe ONL atrophy occurred in both the wild-type and AMS mice. These results indicate that in addition to functional loss of Nna1, another injurious stimulation is necessary to trigger death signals in photoreceptor cells during the postnatal period, but the cells die gradually and autonomously in older age, and that the mutation makes the cells vulnerable to a weak light, but does not increase the number of cells sensitive to strong light stimulation. Thus, these two factors are mutually independent death triggers in AMS photoreceptor cells.

  13. Differential autophagic effects of vital dyes in retinal pigment epithelial ARPE-19 and photoreceptor 661W cells

    PubMed Central

    Sheu, Shwu-Jiuan; Chen, Jiunn-Liang; Bee, Youn-Shen; Chen, Yi-An; Lin, Shi-Han; Shu, Chih-Wen

    2017-01-01

    Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage. PMID:28358857

  14. Large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells.

    PubMed

    Parinot, Célia; Rieu, Quentin; Chatagnon, Jonathan; Finnemann, Silvia C; Nandrot, Emeline F

    2014-12-12

    Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

  15. Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

    PubMed Central

    Parinot, Célia; Rieu, Quentin; Chatagnon, Jonathan; Finnemann, Silvia C.; Nandrot, Emeline F.

    2014-01-01

    Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells. PMID:25548986

  16. Niflumic acid reduces the hyperpolarization-activated current (I(h)) in rod photoreceptor cells.

    PubMed

    Satoh, T O; Yamada, M

    2001-08-01

    We examined the effects of niflumic acid (NFA), a chloride channel blocker, on the hyperpolarization-activated current (I(h)) in newt rod photoreceptors. At 100 microM, NFA delayed the activation of I(h) induced by hyperpolarizing voltage pulses to -83 mV from a holding potential of -43 mV, and reduced the steady-state current. However, reduction by NFA was weakened when I(h) was activated by hyperpolarizing steps to -123 mV, suggesting that these effects were voltage-dependent. The suppressive effects of NFA on I(h) were accompanied by a negative shift in activation voltage. NFA also delayed the relaxation of I(h) tail currents, showing that this drug also inhibited deactivation of the current. The reversal potential and the fully activated conductance were not affected. These observations suggest that NFA reduces I(h) by modifying the gating kinetics of the underlying channels. The suppressive actions of NFA remained when intracellular Ca2+ was strongly chelated, and the failure of suppression by NFA in inside-out patches suggests that the agent may act on the I(h) channel from the extracellular side. These results, obtained in rod photoreceptors, are consistent with similar effects of NFA on I(f) in cardiac myocytes, suggesting that both currents share similar pharmacological properties.

  17. Necrotic enlargement of cone photoreceptor cells and the release of high-mobility group box-1 in retinitis pigmentosa

    PubMed Central

    Murakami, Y; Ikeda, Y; Nakatake, S; Tachibana, T; Fujiwara, K; Yoshida, N; Notomi, S; Nakao, S; Hisatomi, T; Miller, J W; Vavvas, DG; Sonoda, KH; Ishibashi, T

    2015-01-01

    Retinitis pigmentosa (RP) refers to a group of inherited retinal degenerations resulting form rod and cone photoreceptor cell death. The rod cell death due to deleterious genetic mutations has been shown to occur mainly through apoptosis, whereas the mechanisms and features of the secondary cone cell death have not been fully elucidated. Our previous study showed that the cone cell death in rd10 mice, an animal model of RP, involves necrotic features and is partly mediated by the receptor interacting protein kinase. However, the relevancy of necrotic cone cell death in human RP patients remains unknown. In the present study, we showed that dying cone cells in rd10 mice exhibited cellular enlargement, along with necrotic changes such as cellular swelling and mitochondrial rupture. In human eyes, live imaging of cone cells by adaptive optics scanning laser ophthalmoscopy revealed significantly increased percentages of enlarged cone cells in the RP patients compared with the control subjects. The vitreous of the RP patients contained significantly higher levels of high-mobility group box-1, which is released extracellularly associated with necrotic cell death. These findings suggest that necrotic enlargement of cone cells is involved in the process of cone degeneration, and that necrosis may be a novel target to prevent or delay the loss of cone-mediated central vision in RP. PMID:27551484

  18. CNTF-mediated protection of photoreceptors requires initial activation of the cytokine receptor gp130 in Müller glial cells

    PubMed Central

    Rhee, Kun Do; Nusinowitz, Steven; Chao, Kevin; Yu, Fei; Bok, Dean; Yang, Xian-Jie

    2013-01-01

    Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective agent in multiple retinal degeneration animal models. Recently, CNTF has been evaluated in clinical trials for the inherited degenerative disease retinitis pigmentosa (RP) and for dry age-related macular degeneration (AMD). Despite its potential as a broad-spectrum therapeutic treatment for blinding diseases, the target cells of exogenous CNTF and its mechanism of action remain poorly understood. We have shown previously that constitutive expression of CNTF prevents photoreceptor death but alters the retinal transcriptome and suppresses visual function. Here, we use a lentivirus to deliver the same secreted human CNTF used in clinical trials to a mouse model of RP. We found that low levels of CNTF halt photoreceptor death, improve photoreceptor morphology, and correct opsin mislocalization. However, we did not detect corresponding improvement of retinal function as measured by the electroretinogram. Disruption of the cytokine receptor gp130 gene in Müller glia reduces CNTF-dependent photoreceptor survival and prevents phosphorylation of STAT3 and ERK in Müller glia and the rest of the retina. Targeted deletion of gp130 in rods also demolishes neuroprotection by CNTF and prevents further activation of Müller glia. Moreover, CNTF elevates the expression of LIF and endothelin 2, thus positively promoting Müller and photoreceptor interactions. We propose that exogenous CNTF initially targets Müller glia, and subsequently induces cytokines acting through gp130 in photoreceptors to promote neuronal survival. These results elucidate a cellular mechanism for exogenous CNTF-triggered neuroprotection and provide insight into the complex cellular responses induced by CNTF in diseased retinas. PMID:24191003

  19. Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation

    PubMed Central

    Chen, Holly Yu; Kaya, Koray Dogan; Dong, Lijin

    2016-01-01

    Purpose The generation of three-dimensional (3D) organoids with optic cup–like structures from pluripotent stem cells has created opportunities for investigating mammalian retinal development in vitro. However, retinal organoids in culture do not completely reflect the developmental state and in vivo architecture of the rod-dominant mouse retina. The goals of this study were to develop an efficient protocol for generating retinal organoids from stem cells and examine the morphogenesis of rods in vitro. Methods To assess rod photoreceptor differentiation in retinal organoids, we took advantage of Nrl-green fluorescent protein (GFP) mice that show rod-specific expression of GFP directed by the promoter of leucine zipper transcription factor NRL. Using embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively) derived from the Nrl-GFP mouse, we were successful in establishing long-term retinal organoid cultures using modified culture conditions (called High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids, or HIPRO). Results We demonstrated efficient differentiation of pluripotent stem cells to retinal structures. More than 70% of embryoid bodies formed optic vesicles at day (D) 7, >50% produced optic cups by D10, and most of them survived until at least D35. The HIPRO organoids included distinct inner retina neurons in a somewhat stratified architecture and mature Müller glia spanning the entire retina. Almost 70% of the cells in the retinal organoids were rod photoreceptors that exhibited elongated cilia. Transcriptome profiles of GFP+ rod photoreceptors, purified from organoids at D25–35, demonstrated a high correlation with the gene profiles of purified rods from the mouse retina at P2 to P6, indicating their early state of differentiation. Conclusions The 3D retinal organoids, generated by HIPRO method, closely mimic in vivo retinogenesis and provide an efficient in vitro model to investigate photoreceptor

  20. PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae.

    PubMed

    Zhang, Xin; Hong, Qin; Yang, Lei; Zhang, Min; Guo, Xirong; Chi, Xia; Tong, Meiling

    2015-08-01

    Polychlorinated biphenyls (PCBs), a group of highly toxic environmental pollutants, have been report to influence the visual system development in children. However, the underlying mechanism is unclear. The study was aim to investigate the effects of continuous PCBs exposure on optomotor response (OMR) and retinal photoreceptor cell development-related gene expression in zebrafish larvae. The fertilized zebrafish embryos were exposed to PCBs at concentrations of 0.125, 0.25, 0.5, and 1mg/L until 7 days post-fertilization. Control groups with blank and 0.01% methanol were also prepared. OMR test was used to detect the visual behavior. The mRNA expression of the CRX, RHO, SWS1, and SWS2 was assessed by the Quantitative Real-Time PCR. The OMR test showed that the visual behavior of the larvae was most sensitive when the grating spatial frequency was 0.20LP/mm and the moving speed was 25cm/s. Moreover, the proportion of positively swimming fish was significantly reduced in the 0.5 and 1mg/L PCB1254 treatment group (P<0.05) compared with the controls. In addition, the expression of SWS2 was significantly down-regulated in all PCB1254 treatment groups (P<0.05), whereas the decreased expression of the CRX, RHO and SWS1 was found in the 0.5 and 1mg/L PCB1254 groups (P<0.05). This is the first report to demonstrate that continue exposure of zebrafish larvae to PCBs causes photoreceptor cell development-related gene expression changes that lead to OMR behavioral alterations. Analysis of these visual behavioral paradigms may be useful in predicting the adverse effects of toxicants on visual function in fish.

  1. High irradiance responses involving photoreversible multiple photoreceptors as related to photoperiodic induction of cell division in Euglena.

    PubMed

    Bolige, Aoen; Goto, Ken

    2007-02-01

    Little is known about the photoreceptors involved in the photoperiodism of unicellular organisms, which we elucidated by deriving their action spectra. The flagellated alga Euglena gracilis exhibits photoperiodism, with a long-day response in cell reproduction. The underlying clock is a circadian rhythm with photoinductive capability, peaking at subjective dusk and occurring at the 26th hour in continuous darkness (DD) when transferred from continuous light (LL); it regulates photoinduction, a high-irradiance response (HIR), of a dark-capability of progressing through cell division. We derived the action spectra by irradiating E. gracilis with monochromatic light for 3h at around the 26th hour; the action maxima occurred at 380, 450-460, 480, 610, 640, 660, 680, and 740nm. Except for the maximum at 450-460nm, which was always a major maximum, the maxima greatly depended on the red (R)/far-red (FR) ratio of the prior LL. The high R/FR ratio resulted in a dominant major peak at 640nm and minor peaks at 480 and 680nm, whereas the low ratio resulted in dominant major peaks at 610 and 740nm and minor peaks at 380 and 660nm; the critical fluence was minimally about 60mmolm(-2). These HIRs resulted from the accumulation of corresponding low-fluence responses (LFRs) because we found that repetition of a 3-min light/dark cycle, with critical fluences of 1mmolm(-2), lasting for 3h resulted in the same photoinduction as the continuous 3-h irradiation. Moreover, these LFRs expressed photoreversibility. Thus, photoperiodic photoinduction involves Euglena-phytochrome (640 and 740nm) and blue photoreceptor (460nm). Although 380, 480, 610, 660, and 680nm may also represent Euglena-phytochrome, a definite conclusion awaits further study.

  2. p27KIP1 loss promotes proliferation and phagocytosis but prevents epithelial–mesenchymal transition in RPE cells after photoreceptor damage

    PubMed Central

    ul Quraish, Reeshan; Sudou, Norihiro; Nomura-Komoike, Kaori; Sato, Fumi

    2016-01-01

    Purpose p27KIP1 (p27), originally identified as a cell cycle inhibitor, is now known to have multifaceted roles beyond cell cycle regulation. p27 is required for the normal histogenesis of the RPE, but the role of p27 in the mature RPE remains elusive. To define the role of p27 in the maintenance and function of the RPE, we investigated the effects of p27 deletion on the responses of the RPE after photoreceptor damage. Methods Photoreceptor damage was induced in wild-type (WT) and p27 knockout (KO) mice with N-methyl-N-nitrosourea (MNU) treatment. Damage-induced responses of the RPE were investigated with bromodeoxyuridine (BrdU) incorporation assays, immunofluorescence, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays at different stages after MNU treatment. Subcellular localization of p27 in the WT RPE was also analyzed in vivo and in vitro. Results MNU treatment induced photoreceptor-specific degeneration in the WT and KO retinas. BrdU incorporation assays revealed virtually no proliferation of RPE cells in the WT retinas while, in the KO retinas, approximately 16% of the RPE cells incorporated BrdU at day 2 after MNU treatment. The RPE in the KO retinas developed aberrant protrusions into the outer nuclear layer in response to photoreceptor damage and engulfed outer segment debris, as well as TUNEL-positive photoreceptor cells. Increased phosphorylation of myosin light chains and their association with rhodopsin-positive phagosomes were observed in the mutant RPE, suggesting possible deregulation of cytoskeletal dynamics. In addition, WT RPE cells exhibited evidence of the epithelial–mesenchymal transition (EMT), including morphological changes, induction of α-smooth muscle actin expression, and attenuated expression of tight junction protein ZO-1 while these changes were absent in the KO retinas. In the normal WT retinas, p27 was localized to the nuclei of RPE cells while nuclear and cytoplasmic p27 was detected in RPE cells

  3. Circadian and Dopaminergic Regulation of Fatty Acid Oxidation Pathway Genes in Retina and Photoreceptor Cells

    PubMed Central

    Vancura, Patrick; Wolloscheck, Tanja; Baba, Kenkichi; Tosini, Gianluca; Iuvone, P. Michael; Spessert, Rainer

    2016-01-01

    The energy metabolism of the retina might comply with daily changes in energy demand and is impaired in diabetic retinopathy—one of the most common causes of blindness in Europe and the USA. The aim of this study was to investigate putative adaptation of energy metabolism in healthy and diabetic retina. Hence expression analysis of metabolic pathway genes was performed using quantitative polymerase chain reaction, semi-quantitative western blot and immunohistochemistry. Transcriptional profiling of key enzymes of energy metabolism identified transcripts of mitochondrial fatty acid β-oxidation enzymes, i.e. carnitine palmitoyltransferase-1α (Cpt-1α) and medium chain acyl-CoA dehydrogenase (Acadm) to display daily rhythms with peak values during daytime in preparations of the whole retina and microdissected photoreceptors. The cycling of both enzymes persisted in constant darkness, was dampened in mice deficient for dopamine D4 (D4) receptors and was altered in db/db mice—a model of diabetic retinopathy. The data of the present study are consistent with circadian clock-dependent and dopaminergic regulation of fatty acid oxidation in retina and its putative disturbance in diabetic retina. PMID:27727308

  4. Cell polarity: mechanochemical patterning.

    PubMed

    Goehring, Nathan W; Grill, Stephan W

    2013-02-01

    Nearly every cell type exhibits some form of polarity, yet the molecular mechanisms vary widely. Here we examine what we term 'chemical systems' where cell polarization arises through biochemical interactions in signaling pathways, 'mechanical systems' where cells polarize due to forces, stresses and transport, and 'mechanochemical systems' where polarization results from interplay between mechanics and chemical signaling. To reveal potentially unifying principles, we discuss mathematical conceptualizations of several prototypical examples. We suggest that the concept of local activation and global inhibition - originally developed to explain spatial patterning in reaction-diffusion systems - provides a framework for understanding many cases of cell polarity. Importantly, we find that the core ingredients in this framework - symmetry breaking, self-amplifying feedback, and long-range inhibition - involve processes that can be chemical, mechanical, or even mechanochemical in nature.

  5. Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development.

    PubMed

    Wong, Bernice H; Chan, Jia Pei; Cazenave-Gassiot, Amaury; Poh, Rebecca W; Foo, Juat Chin; Galam, Dwight L A; Ghosh, Sujoy; Nguyen, Long N; Barathi, Veluchamy A; Yeo, Sia W; Luu, Chi D; Wenk, Markus R; Silver, David L

    2016-05-13

    Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs.

  6. Molecular chaperones and photoreceptor function

    PubMed Central

    Kosmaoglou, Maria; Schwarz, Nele; Bett, John S.; Cheetham, Michael E.

    2008-01-01

    Molecular chaperones facilitate and regulate protein conformational change within cells. This encompasses many fundamental cellular processes: including the correct folding of nascent chains; protein transport and translocation; signal transduction and protein quality control. Chaperones are, therefore, important in several forms of human disease, including neurodegeneration. Within the retina, the highly specialized photoreceptor cell presents a fascinating paradigm to investigate the specialization of molecular chaperone function and reveals unique chaperone requirements essential to photoreceptor function. Mutations in several photoreceptor proteins lead to protein misfolding mediated neurodegeneration. The best characterized of these are mutations in the molecular light sensor, rhodopsin, which cause autosomal dominant retinitis pigmentosa. Rhodopsin biogenesis is likely to require chaperones, while rhodopsin misfolding involves molecular chaperones in quality control and the cellular response to protein aggregation. Furthermore, the specialization of components of the chaperone machinery to photoreceptor specific roles has been revealed by the identification of mutations in molecular chaperones that cause inherited retinal dysfunction and degeneration. These chaperones are involved in several important cellular pathways and further illuminate the essential and diverse roles of molecular chaperones. PMID:18490186

  7. Harmonin (Ush1c) is required in zebrafish Müller glial cells for photoreceptor synaptic development and function

    PubMed Central

    Phillips, Jennifer B.; Blanco-Sanchez, Bernardo; Lentz, Jennifer J.; Tallafuss, Alexandra; Khanobdee, Kornnika; Sampath, Srirangan; Jacobs, Zachary G.; Han, Philip F.; Mishra, Monalisa; Titus, Tom A.; Williams, David S.; Keats, Bronya J.; Washbourne, Philip; Westerfield, Monte

    2011-01-01

    SUMMARY Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C): one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients. PMID:21757509

  8. Harmonin (Ush1c) is required in zebrafish Müller glial cells for photoreceptor synaptic development and function.

    PubMed

    Phillips, Jennifer B; Blanco-Sanchez, Bernardo; Lentz, Jennifer J; Tallafuss, Alexandra; Khanobdee, Kornnika; Sampath, Srirangan; Jacobs, Zachary G; Han, Philip F; Mishra, Monalisa; Titus, Tom A; Williams, David S; Keats, Bronya J; Washbourne, Philip; Westerfield, Monte

    2011-11-01

    Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C): one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients.

  9. Effects of Ar-40 and Fe-56 ions on retinal photoreceptor cells of the rabbit: Implications for manned missions to Mars

    NASA Technical Reports Server (NTRS)

    Williams, G. R.; Lett, J. T.

    1994-01-01

    Losses of photoreceptor cells (rods) from the retinas of New Zealand white (NZW) rabbits were detectable within 2 years after localized acute irradiation of optic and proximal tissues with greater than or equal to 7 Gy of 530 MeV u(exp -1) Ar-40 ions or greater than or equal to 2 Gy of 465 MeV u(exp -1) Fe-56 ions in the Bragg plateau region of energy deposition. Those limits were determined only from an analysis of variance of dose groups because the shapes of the dose response curves at early post-irradiation times are not known, a concern being addressed by experiments in progress. Losses of photoreceptor cells for the period 0.5-2.5 years post-irradiation, determined by provisional linear regression analysis, were approxiamtely 1.7% Gy(exp -1) and 2.5% Gy(exp.-1) for Ar-40 and Fe-56 ions, respectively.

  10. Effects of 40Ar and 56Fe ions on retinal photoreceptor cells of the rabbit: implications for manned missions to Mars

    NASA Technical Reports Server (NTRS)

    Williams, G. R.; Lett, J. T.

    1994-01-01

    Losses of photoreceptor cells (rods) from the retinas of New Zealand white (NZW) rabbits were detectable within 2 years after localized acute irradiation of optic and proximal tissues with > or = 7 Gy of 530 MeV u-1 40Ar ions or > or = 2 Gy of 465 MeV u-1 56Fe ions in the Bragg plateau region of energy deposition. Those limits were determined only from an analysis of variance of dose groups because the shapes of the dose response curves at early post-irradiation times are not known, a concern being addressed by experiments in progress. Losses of photoreceptor cells for the period 0.5-2.5 years post-irradiation, determined by provisional linear regression analysis, were approximately 1.7% Gy-1 and 2.5% Gy-1 for 40Ar and 56Fe ions, respectively.

  11. The Influence of Photoreceptor Size and Distribution on Optical Sensitivity in the Eyes of Lanternfishes (Myctophidae)

    PubMed Central

    de Busserolles, Fanny; Fitzpatrick, John L.; Marshall, N. Justin; Collin, Shaun P.

    2014-01-01

    The mesopelagic zone of the deep-sea (200-1000 m) is characterised by exponentially diminishing levels of downwelling sunlight and by the predominance of bioluminescence emissions. The ability of mesopelagic organisms to detect and behaviourally react to downwelling sunlight and/or bioluminescence will depend on the visual task and ultimately on the eyes and their capacity for detecting low levels of illumination and intermittent point sources of bioluminescent light. In this study, we investigate the diversity of the visual system of the lanternfish (Myctophidae). We focus specifically on the photoreceptor cells by examining their size, arrangement, topographic distribution and contribution to optical sensitivity in 53 different species from 18 genera. We also examine the influence(s) of both phylogeny and ecology on these photoreceptor variables using phylogenetic comparative analyses in order to understand the constraints placed on the visual systems of this large group of mesopelagic fishes at the first stage of retinal processing. We report great diversity in the visual system of the Myctophidae at the level of the photoreceptors. Photoreceptor distribution reveals clear interspecific differences in visual specialisations (areas of high rod photoreceptor density), indicating potential interspecific differences in interactions with prey, predators and/or mates. A great diversity in photoreceptor design (length and diameter) and density is also present. Overall, the myctophid eye is very sensitive compared to other teleosts and each species seems to be specialised for the detection of a specific signal (downwelling light or bioluminescence), potentially reflecting different visual demands for survival. Phylogenetic comparative analyses highlight several relationships between photoreceptor characteristics and the ecological variables tested (depth distribution and luminous tissue patterns). Depth distribution at night was a significant factor in most of the

  12. A regulatory sequence from the retinoid X receptor γ gene directs expression to horizontal cells and photoreceptors in the embryonic chicken retina

    PubMed Central

    Blixt, Maria K. E.

    2016-01-01

    Purpose Combining techniques of episomal vector gene-specific Cre expression and genomic integration using the piggyBac transposon system enables studies of gene expression–specific cell lineage tracing in the chicken retina. In this work, we aimed to target the retinal horizontal cell progenitors. Methods A 208 bp gene regulatory sequence from the chicken retinoid X receptor γ gene (RXRγ208) was used to drive Cre expression. RXRγ is expressed in progenitors and photoreceptors during development. The vector was combined with a piggyBac “donor” vector containing a floxed STOP sequence followed by enhanced green fluorescent protein (EGFP), as well as a piggyBac helper vector for efficient integration into the host cell genome. The vectors were introduced into the embryonic chicken retina with in ovo electroporation. Tissue electroporation targets specific developmental time points and in specific structures. Results Cells that drove Cre expression from the regulatory RXRγ208 sequence excised the floxed STOP-sequence and expressed GFP. The approach generated a stable lineage with robust expression of GFP in retinal cells that have activated transcription from the RXRγ208 sequence. Furthermore, GFP was expressed in cells that express horizontal or photoreceptor markers when electroporation was performed between developmental stages 22 and 28. Electroporation of a stage 12 optic cup gave multiple cell types in accordance with RXRγ gene expression in the early retina. Conclusions In this study, we describe an easy, cost-effective, and time-efficient method for testing regulatory sequences in general. More specifically, our results open up the possibility for further studies of the RXRγ-gene regulatory network governing the formation of photoreceptor and horizontal cells. In addition, the method presents approaches to target the expression of effector genes, such as regulators of cell fate or cell cycle progression, to these cells and their progenitor. PMID

  13. Subcellular calcium localization and AT0-dependent Ca2+-uptake by smooth endoplasmic reticulum in an invertebrate photoreceptor cell. An ultrastrucutral, cytochemical and X-ray microanalytical study.

    PubMed

    Walz, B

    1979-10-01

    In Hirudo medicinalis an extensive and highly elaborate three dimensional network of smooth endoplasmic reticulum cisternae is found in very close structural relationship to the receptive (microvillar) membrane, as reported for many other invertebrates. A variant of the potassium pyroantimonate technique showed that these submicrovillar endoplasmic reticulum cisternae (SMC) and mitochondria are major intracellular calcium stores. Furthermore, using saponine-skinned photoreceptors for an in situ accumulation experiment, calcium oxalate precipitates in SMC demonstrate that this organelle is able to accumulate Ca2+ from a concentration of 2 x 10(-5) M, when ATP, Mg2+, and oxalate ions are present in the accumulation medium. This result provides direct evidence for the hypothesis that SMC may play a particularly important role in the regulation of intracellular ionized calcium in invertebrate photoreceptor cells. Morphological evidence supports this view.

  14. Photosensory transduction in unicellular eukaryotes: a comparison between related ciliates Blepharisma japonicum and Stentor coeruleus and photoreceptor cells of higher organisms.

    PubMed

    Sobierajska, Katarzyna; Fabczak, Hanna; Fabczak, Stanisław

    2006-06-01

    Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.

  15. Patterning of cells through patterning of biology.

    PubMed

    Kala, A; Jain, P K; Friedman, S H

    2014-07-01

    For the first time, cells have been patterned on surfaces through the spatial manipulation of native gene expression. By manipulating the inherent biology of the cell, as opposed to the chemical nature of the surfaces they are attached to, we have created a potentially more flexible way of creating patterns of cells that does not depend on the substrate. This was accomplished by bringing an siRNA that targets the expression of pten under the control of light, by modifying it with photocleavable groups. This pten-targeting siRNA has been previously demonstrated to induce dissociation of cells from surfaces. We modified this siRNA with dimethoxy nitro phenyl ethyl photocleavable groups (DMNPE) to allow the activity of the siRNA, and hence pten knockdown, to be toggled with light. Using this approach we demonstrated light dependent cell dissociation only with a DMNPE modified siRNA that targets pten and not with control siRNAs. In addition we demonstrated the ability to make simple patterns of cells through the application of masks during irradiation.

  16. Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga Volvox carteri and their potential role in cellular differentiation.

    PubMed

    Kianianmomeni, Arash

    2015-01-01

    The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in multicellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation.

  17. Cell-type specific photoreceptors and light signaling pathways in the multicellular green alga volvox carteri and their potential role in cellular differentiation

    PubMed Central

    Kianianmomeni, Arash

    2015-01-01

    The formation of multicellular organisms requires genetically predefined signaling pathways in various cell types. Besides differences in size, energy balance and life time, cell types should be enable to modulate appropriate developmental and adaptive responses in ever-changing surrounding environment. One of the most important environmental cues is light which regulates a variety of physiological and cellular processes. During evolution, diverse light-sensitive proteins, so-called photoreceptors, and corresponding signaling pathways have evolved, in almost all kingdoms of life, to monitor light continuously and adjust their growth and development accordingly. However, considering the fact that different cell types should be enable to trigger distinct light signaling pathways according to their needs, cell-type specific light signaling pathways are required to guarantee cell type-matched modulation of cellular and developmental processes in response to different light signals. The multicellular green alga Volvox carteri, which has only 2 cell types with clear division of labor, possesses cell-type specific photoreceptors and light signaling pathways which allow differential regulation of genes involved in various cellular and metabolic pathways in response to environmental light. The existence of cell-type specific light signaling pathways in muticellular organism like Volvox reflects an early development of cell-type specific signaling mechanisms during evolution to ensure maintenance of differentiation. PMID:25874475

  18. Role of Tau, a microtubule associated protein, in Drosophila photoreceptor morphogenesis.

    PubMed

    Nam, Sang-Chul

    2016-11-01

    Cell polarity genes have important functions in photoreceptor morphogenesis. Based on recent discovery of stabilized microtubule cytoskeleton in developing photoreceptors and its role in photoreceptor cell polarity, microtubule associated proteins might have important roles in controlling cell polarity proteins' localizations in developing photoreceptors. Here, Tau, a microtubule associated protein, was analyzed to find its potential role in photoreceptor cell polarity. Tau colocalizes with acetylated/stabilized microtubules in developing pupal photoreceptors. Although it is known that tau mutant photoreceptor has no defects in early eye differentiation and development, it shows dramatic disruptions of cell polarity proteins, adherens junctions, and the stable microtubules in developing pupal photoreceptors. This role of Tau in cell polarity proteins' localization in photoreceptor cells during the photoreceptor morphogenesis was further supported by Tau's overexpression studies. Tau overexpression caused dramatic expansions of apical membrane domains where the polarity proteins localize in the developing pupal photoreceptors. It is also found that Tau's role in photoreceptor cell polarity depends on Par-1 kinase. Furthermore, a strong genetic interaction between tau and crumbs was found. It is found that Tau has a crucial role in cell polarity protein localization during pupal photoreceptor morphogenesis stage, but not in early eye development including eye cell differentiation.

  19. Engineered photoreceptors as novel optogenetic tools.

    PubMed

    Möglich, Andreas; Moffat, Keith

    2010-10-28

    Cellular processes and indeed the survival of entire organisms crucially depend on precise spatiotemporal coordination of a multitude of molecular events. A new tool in cell biology is denoted "optogenetics" which describes the use of genetically encoded, light-gated proteins, i.e. photoreceptors, which perturb and control cellular and organismal behavior in a spatiotemporally exact manner. Photoreceptors resemble fluorescent reporter proteins such as GFP in being genetically encoded, non-invasive, and applicable to intact cells and organisms. They are explicitly intended to modulate activity; in contrast, fluorescent proteins generally do not disturb the processes under study. Fluorescent proteins have revolutionized cell biology because they allow the monitoring of such processes by imaging techniques that offer superb spatiotemporal resolution and sensitivity. Optogenetics extends these advantages to offer control. The scope of optogenetics has recently been expanded beyond the use of naturally occurring photoreceptors by the biologically-inspired design of engineered (or synthetic) photoreceptors. These photoreceptors are derived by fusion of one or more light-absorbing sensor domains with an output or effector domain displaying the activity to be controlled. Here, we focus on the design and application of such engineered photoreceptors. We treat basic signaling principles and discuss the two photosensor classes which are currently most widely used in fusion-based design: LOV domains and phytochromes. Based on these principles, we develop general strategies for the engineering of photoreceptors. Finally, we review recently successful examples of the design and application of engineered photoreceptors. Our perspective provides guidelines for researchers interested in developing and applying novel optogenetic tools.

  20. Sustained Delivery of Bioactive GDNF from Collagen and Alginate-Based Cell-Encapsulating Gel Promoted Photoreceptor Survival in an Inherited Retinal Degeneration Model

    PubMed Central

    Chan, Barbara P.; Lo, Amy C. Y.

    2016-01-01

    Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. However, CAC ECT properties and performance in the eye are not well-understood. Previously, we reported a cultured 3D CAC system that supported the growth of GDNF-secreting HEK293 cells with sustainable GDNF delivery. Here, the system was further developed into an intravitreally injectable gel with 1x104 or 2x105 cells encapsulated in 2mg/ml type I collagen and 1% alginate. Gels with lower alginate concentration yielded higher initial cell viability but faster spheroid formation while increasing initial cell density encouraged cell growth. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well-tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel-implanted in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those containing higher initial cell number yielded better photoreceptor survival. CAC ECT gels offers flexible system design and is a potential treatment option for posterior eye diseases. PMID:27441692

  1. RS9, a novel Nrf2 activator, attenuates light-induced death of cells of photoreceptor cells and Müller glia cells.

    PubMed

    Inoue, Yuki; Shimazawa, Masamitsu; Noda, Yasuhiro; Nagano, Ryota; Otsuka, Tomohiro; Kuse, Yoshiki; Nakano, Yukimichi; Tsuruma, Kazuhiro; Nakagami, Yasuhiro; Hara, Hideaki

    2017-03-27

    The retina is highly sensitive to oxidative stress because of its high consumption of oxygen associated with the phototransductional processes. Recent findings have suggested that oxidative stress is involved in the pathology of age-related macular degeneration (AMD), a progressive degeneration of the central retina. A well-known environmental risk factor is light exposure, as excessive and continuous light exposure can damage photoreceptors. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that controls antioxidative responses and phase 2 enzymes. Thus, we hypothesized that RS9, a specific activator of Nrf2, decreases light-induced retinal cell death in vivo and in vitro. Nrf2 was detected in the nucleus of the 661w cells exposed to RS9 and also after light exposure, and the Nrf2-antioxidant response element (ARE) binding was increased in 661w cells after exposure to RS9. Consequentially, the expression of the phase 2 enzyme's mRNAs of Ho-1, Nqo-1, and Gclm genes were increased in 661w cells after exposure to RS9. Further, RS9 decreased the light-induced death of 661W cells (2,500 lx, 24 h), and also reduced the functional damages and the histological degeneration of the nuclei in the outer nuclear layer (ONL) or the retina in the in vivo studies (8,000 lx, 3 h). HO-1 was increased after light exposure, and Nrf2 was translocated into the nucleus after light exposure in vivo. Silencing of Ho-1 reduced the protective effects of RS9 against light-induced death of 661w cells. These findings indicate that RS9 has therapeutic potential for retinal diseases that are aggravated by light exposure. This article is protected by copyright. All rights reserved.

  2. Hyperpolarization-activated current (I(h)) in ganglion-cell photoreceptors.

    PubMed

    Van Hook, Matthew J; Berson, David M

    2010-12-20

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and serve as the primary retinal drivers of non-image-forming visual functions such as circadian photoentrainment, the pupillary light reflex, and suppression of melatonin production in the pineal. Past electrophysiological studies of these cells have focused on their intrinsic photosensitivity and synaptic inputs. Much less is known about their voltage-gated channels and how these might shape their output to non-image-forming visual centers. Here, we show that rat ipRGCs retrolabeled from the suprachiasmatic nucleus (SCN) express a hyperpolarization-activated inwardly-rectifying current (I(h)). This current is blocked by the known I(h) blockers ZD7288 and extracellular cesium. As in other systems, including other retinal ganglion cells, I(h) in ipRGCs is characterized by slow kinetics and a slightly greater permeability for K(+) than for Na(+). Unlike in other systems, however, I(h) in ipRGCs apparently does not actively contribute to resting membrane potential. We also explore non-specific effects of the common I(h) blocker ZD7288 on rebound depolarization and evoked spiking and discuss possible functional roles of I(h) in non-image-forming vision. This study is the first to characterize I(h) in a well-defined population of retinal ganglion cells, namely SCN-projecting ipRGCs.

  3. Transmission from the dominant input shapes the stereotypic ratio of photoreceptor inputs onto horizontal cells

    PubMed Central

    Yoshimatsu, Takeshi; Williams, Philip R.; D’Orazi, Florence D.; Suzuki, Sachihiro C.; Fadool, James M.; Allison, W. Ted; Raymond, Pamela A.; Wong, Rachel O.

    2014-01-01

    Many neurons receive synapses in stereotypic proportions from converging but functionally distinct afferents. However, developmental mechanisms regulating synaptic convergence are not well understood. Here we describe a heterotypic mechanism by which one afferent controls synaptogenesis of another afferent, but not vice-versa. Like other CNS circuits, zebrafish retinal H3 horizontal cells undergo an initial period of remodeling, establishing synapses with UV and blue cones while eliminating red and green cone contacts. As development progresses, the horizontal cells selectively synapse with UV cones to generate a 5:1 UV-to-blue cone synapse ratio. Blue cone synaptogenesis increases in mutants lacking UV cones, and when transmitter release or visual stimulation of UV cones is perturbed. Connectivity is unaltered when blue cone transmission is suppressed. Moreover, there is no homotypic regulation of cone synaptogenesis by neurotransmission. Thus, biased connectivity in this circuit is established by an unusual activity-dependent, unidirectional control of synaptogenesis exerted by the dominant input. PMID:24832361

  4. Characterizing and modeling the intrinsic light response of rat ganglion-cell photoreceptors.

    PubMed

    Walch, Olivia J; Zhang, L Samantha; Reifler, Aaron N; Dolikian, Michael E; Forger, Daniel B; Wong, Kwoon Y

    2015-11-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate both image-forming vision and non-image-forming visual responses such as pupillary constriction and circadian photoentrainment. Five types of ipRGCs, named M1-M5, have been discovered in rodents. To further investigate their photoresponse properties, we made multielectrode array spike recordings from rat ipRGCs, classified them into M1, M2/M4, and M3/M5 clusters, and measured their intrinsic, melanopsin-based responses to single and flickering light pulses. Results showed that ipRGC spiking can track flickers up to ∼0.2 Hz in frequency and that flicker intervals between 5 and 14 s evoke the most spikes. We also learned that melanopsin's integration time is intensity and cluster dependent. Using these data, we constructed a mathematical model for each cluster's intrinsic photoresponse. We found that the data for the M1 cluster are best fit by a model that assumes a large photoresponse, causing the cell to enter depolarization block. Our models also led us to hypothesize that the M2/M4 and M3/M5 clusters experience comparable photoexcitation but that the M3/M5 cascade decays significantly faster than the M2/M4 cascade, resulting in different response waveforms between these clusters. These mathematical models will help predict how each ipRGC cluster might respond to stimuli of any waveform and could inform the invention of lighting technologies that promote health through melanopsin stimulation.

  5. Separation of function for classical and ganglion cell photoreceptors with respect to circadian rhythm entrainment and induction of photosomnolence.

    PubMed

    Morin, L P; Studholme, K M

    2011-12-29

    Four studies were performed to further clarify the contribution of rod/cone and intrinsically photoreceptive retinal ganglion cells to measures of entrainment, dark preference, light-induced locomotor suppression and photosomnolence. Wild type (WT), retinally degenerate (rd/rd), and melanopsin-less (OPN4⁻/⁻) mouse strains were compared. In Experiment 1, mice were exposed to a graded photoperiod in which approximately 0.26 μW/cm² irradiance diminished to dark over a 6-h interval. This method enabled "phase angle titration," with individual animals assuming activity onsets according to their sensitivity to light. WT and OPN4⁻/⁻ animals entrained with identical phase angles (effective irradiance=0.078 μW/cm²), but rd/rd mice required a more intense irradiance (0.161 μW/cm²) and entrainment occurred about 2.5 h earlier. In Experiment 2, all three strains preferred the dark side of a divided light-dark chamber until the irradiance dropped to 0.5 μW/cm² at which point, rd/rd mice no longer showed a preference. Experiments 3 and 4 determined that WT and rd/rd mice showed equivalent light-induced locomotor suppression, but the response was greatly impaired in OPN4⁻/⁻ mice. Closer examination of open field locomotion using infrared video-based methods and Any-maze(tm) software revealed two opposing effects of light. Locomotor suppression was equivalent in WT and rd/rd mice. Responses by OPN4⁻/⁻ mice varied from being absent (n=17) to normal (similar to WT and rd/rd mice; n=8). Light onset was associated with a significant, but brief, locomotion increase in WT and OPN4⁻/⁻ mice, but not in rd/rd mice. Any-maze(tm) analysis supports the view that light-induced locomotor quiescence is followed by behavioral sleep (photosomnolence), a fact that was visually validated from the raw video files. The data show that (a) classical photoreceptors, most likely rods, allow mice to prefer and entrain to very dim light such as found in natural twilight; (b) the

  6. Separation of Function for Classical and Ganglion Cell Photoreceptors with Respect to Circadian Rhythm Entrainment and Induction of Photosomnolence

    PubMed Central

    Morin, L.P.; Studholme, K.M.

    2011-01-01

    Four studies were performed to further clarify the contribution of rod/cone and intrinsically photoreceptive retinal ganglion cells to measures of entrainment, light-induced locomotor suppression and photosomnolence. Wildtype (WT), retinally degenerate (rd/rd) and melanopsin-less (OPN4−/−) mouse strains were compared. In Expt. 1, mice were exposed to a graded photoperiod in which approximately 0.26 μW/cm2 irradiance diminished to dark over a 6 hr interval. This method enabled “phase angle titration,” with individual animals assuming activity onsets according to their sensitivity to light. WT and OPN4−/− animals entrained with identical phase angles (effective irradiance=0.078 μW/cm2), but rd/rd mice required a more intense irradiance (0.161 μW/cm2) and entrainment occurred about 2.5 hr earlier. In Expt. 2, all three strains preferred the dark side of a divided light-dark chamber until the irradiance dropped to 0.5 μW/cm2 at which point, rd/rd mice no longer showed a preference. Expts. 3 and 4 determined that WT and rd/rd mice showed equivalent light-induced locomotor suppression, but the response was greatly impaired in OPN4−/− mice. Closer examination of open field locomotion using infrared video-based methods and Any-mazetm software revealed two opposing effects of light. Locomotor suppression was equivalent in WT and rd/rd mice. Responses by OPN4−/− mice varied from being absent (N=17) to normal (similar to WT and rd/rd mice; N=8). Light onset was associated with a significant, but brief, locomotion increase in WT and OPN4−/− mice, but not in rd/rd mice. Any-mazetm analysis supports the view that light-induced locomotor quiescence is followed by behavioral sleep (photosomnolence), a fact that was visually validated from the raw video files. The data show that a) classical photoreceptors, most likely rods, allow mice to prefer and entrain to very dim light such as found in natural twilight; b) the presence of melanopsin photopigment

  7. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex-vivo

    PubMed Central

    Singh, Mandeep S.; Lipinski, Daniel M.; Barnea-Cramer, Alona O.; Walker, Nathan J.; Barnard, Alun R.; Hankins, Mark W.; MacLaren, Robert E.

    2016-01-01

    Gene therapy using adeno-associated viral vectors (AAV) for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4-/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4-/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex-vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treating retinal degenerations in which high levels of transgene expression are required. PMID:27416076

  8. Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines

    PubMed Central

    Zhao, Zibo; Wang, Lu; Wen, Zhi; Ayaz-guner, Serife; Wang, Yidan; Ahlquist, Paul; Xu, Wei

    2013-01-01

    Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ERα expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound. PMID:24066170

  9. Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

    PubMed Central

    Mao, Mao; Zhou, Liang

    2017-01-01

    We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROα were the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rα were significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases. PMID:28194184

  10. Dense pattern multiple pass cells

    DOEpatents

    Silver, Joel A.; Bomse, David S.

    2010-09-21

    An optical cell and a method of operating an optical cell comprising employing a first mirror comprising a first hole therein at approximately a center of the first mirror and through which laser light enters the cell, employing a second mirror comprising a second hole therein at approximately a center of the second mirror and through which laser light exits the cell, and forming a Lissajous pattern of spots on the mirrors by repeated reflection of laser light entering the cell.

  11. The natural retinoprotectant chrysophanol attenuated photoreceptor cell apoptosis in an N-methyl-N-nitrosourea-induced mouse model of retinal degenaration

    PubMed Central

    Lin, Fan-Li; Lin, Cheng-Hui; Ho, Jau-Der; Yen, Jing-Lun; Chang, Hung-Ming; Chiou, George C. Y.; Cheng, Yu-Wen; Hsiao, George

    2017-01-01

    Retinitis pigmentosa (RP) is an inherited photoreceptor-degenerative disease, and neuronal degeneration in RP is exacerbated by glial activation. Cassia seed (Jue-ming-zi) is a traditional herbal medicine commonly used to treat ocular diseases in Asia. In this report, we investigated the retina-protective effect of chrysophanol, an active component of Cassia seed, in an N-methyl-N-nitrosourea (MNU)-induced mouse model of RP. We determined that chrysophanol inhibited the functional and morphological features of MNU-induced retinal degeneration using scotopic electroretinography (ERG), optical coherence tomography (OCT), and immunohistochemistry analysis of R/G opsin and rhodopsin. Furthermore, TUNEL assays revealed that chrysophanol attenuated MNU-induced photoreceptor cell apoptosis and inhibited the expression of the apoptosis-associated proteins PARP, Bax, and caspase-3. In addition, chrysophanol ameliorated reactive gliosis, as demonstrated by a decrease in GFAP immunolabeling, and suppressed the activation of matrix metalloproteinase (MMP)-9-mediated gelatinolysis. In vitro studies indicated that chrysophanol inhibited lipopolysaccharide (LPS)-induced iNOS and COX-2 expression in the BV2 mouse microglia cell line and inhibited MMP-9 activation in primary microglia. Our results demonstrate that chrysophanol provided neuroprotective effects and inhibited glial activation, suggesting that chrysophanol might have therapeutic value for the treatment of human RP and other retinopathies. PMID:28112220

  12. Selective Photoreceptor Gene Knock-out Reveals a Regulatory Role for the Growth Behavior of Pseudomonas syringae.

    PubMed

    Shah, Rashmi; Pathak, Gopal; Drepper, Thomas; Gärtner, Wolfgang

    2016-07-01

    The plant pathogen Pseudomonas syringae (Ps) is a well-established model organism for bacterial infection of plants. The genome sequences of two pathovars, pv. syringae and pv. tomato, revealed one gene encoding a blue and two genes encoding red/far red light-sensing photoreceptors. Continuing former molecular characterization of the photoreceptor proteins, we here report selective photoreceptor gene disruption for pv. tomato aiming at identification of potentially regulatory functions of these photoreceptors. Transformation of Ps cells with linear DNA constructs yielded interposon mutations of the corresponding genes. Cell growth studies of the generated photoreceptor knock-out mutants revealed their role in light-dependent regulation of cell growth and motility. Disruption of the blue-light (BL) receptor gene caused a growth deregulation, in line with an observed increased virulence of this mutant (Moriconi et al., Plant J., 2013, 76, 322). Bacterial phytochrome-1 (BphP1) deletion mutant caused unaltered cell growth, but a stronger swarming capacity. Inactivation of its ortholog, BphP2, however, caused reduced growth and remarkably altered dendritic swarming behavior. Combined knock-out of both bacteriophytochromes reproduced the swarming pattern observed for the BphP2 mutant alone. A triple knock-out mutant showed a growth rate between that of the BL (deregulation) and the phytochrome-2 mutant (growth reduction).

  13. [The role of the glial cells in the maintenance of the ionic environment of the photoreceptors of the retina of the drone (author's transl)].

    PubMed

    Tsacopoulos, M; Coles, J A

    1978-04-01

    A double-barrelled potassium sensitive microelectrode was used to record electrical potentials and K+ activities in the retina of the drone Apis Mellifera during stimulation with trains of flashes, 1 per sec, intense enough to produce receptor potentials of near maximal amplitude. During the stimulation photoreceptors lose about 25% of their intracellular potassium concentration. During stimulation the potassium activity in the extracellular space increased transitorily up to 20 mM and then fell to a plateau. By this time the potassium concentration increased by about 20% in the glial cells. These results suggest that the glial cells may participate in the regulation of K+ activity in the extracellular space. The increase of potassium activity in the glial cells may be a stimulus for activation of cellular metabolism.

  14. Machine learning approaches to supporting the identification of photoreceptor-enriched genes based on expression data

    PubMed Central

    Wang, Haiying; Zheng, Huiru; Simpson, David; Azuaje, Francisco

    2006-01-01

    Background Retinal photoreceptors are highly specialised cells, which detect light and are central to mammalian vision. Many retinal diseases occur as a result of inherited dysfunction of the rod and cone photoreceptor cells. Development and maintenance of photoreceptors requires appropriate regulation of the many genes specifically or highly expressed in these cells. Over the last decades, different experimental approaches have been developed to identify photoreceptor enriched genes. Recent progress in RNA analysis technology has generated large amounts of gene expression data relevant to retinal development. This paper assesses a machine learning methodology for supporting the identification of photoreceptor enriched genes based on expression data. Results Based on the analysis of publicly-available gene expression data from the developing mouse retina generated by serial analysis of gene expression (SAGE), this paper presents a predictive methodology comprising several in silico models for detecting key complex features and relationships encoded in the data, which may be useful to distinguish genes in terms of their functional roles. In order to understand temporal patterns of photoreceptor gene expression during retinal development, a two-way cluster analysis was firstly performed. By clustering SAGE libraries, a hierarchical tree reflecting relationships between developmental stages was obtained. By clustering SAGE tags, a more comprehensive expression profile for photoreceptor cells was revealed. To demonstrate the usefulness of machine learning-based models in predicting functional associations from the SAGE data, three supervised classification models were compared. The results indicated that a relatively simple instance-based model (KStar model) performed significantly better than relatively more complex algorithms, e.g. neural networks. To deal with the problem of functional class imbalance occurring in the dataset, two data re-sampling techniques were

  15. NAD+ maintenance attenuates light induced photoreceptor degeneration Δ

    PubMed Central

    Bai, Shi; Sheline, Christian T.

    2013-01-01

    Light-induced retinal damage (LD) occurs after surgery or sun exposure. We previously showed that zinc (Zn2+) accumulated in photoreceptors and RPE cells after LD but prior to cell death, and pyruvate or nicotinamide attenuated the resultant death perhaps by restoring nicotinamide adenine dinucleotide (NAD+) levels. We first examined the levels of NAD+ and the efficacy of pyruvate or nicotinamide in oxidative toxicities using primary retinal cultures. We next manipulated NAD+ levels in vivo and tested the affect on LD to photoreceptors and RPE. NAD+ levels cycle with a 24-h rhythm in mammals, which is affected by the feeding schedule. Therefore, we tested the affect of increasing NAD+ levels on LD by giving nicotinamide, inverting the feeding schedule, or using transgenic mice which overexpress cytoplasmic nicotinamide mononucleotide adenyl-transferase-1 (cytNMNAT1), an NAD+ synthetic enzyme. Zn2+ accumulation was also assessed in culture and in retinal sections. Retinas of light damaged animals were examined by OCT and plastic sectioning, and retinal NAD levels were measured. Day fed, or nicotinamide treated rats showed less NAD+ loss, and LD compared to night fed rats or untreated rats without changing the Zn2+ staining pattern. CytNMNAT1 showed less Zn2+ staining, NAD+ loss, and cell death after LD. In conclusion, intense light, Zn2+ and oxidative toxicities caused an increase in Zn2+, NAD+ loss, and cell death which were attenuated by NAD+ restoration. Therefore, NAD+ levels play a protective role in LD-induced death of photoreceptors and RPE cells. PMID:23274583

  16. Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor precursors

    PubMed Central

    Pearson, R. A.; Gonzalez-Cordero, A.; West, E. L.; Ribeiro, J. R.; Aghaizu, N.; Goh, D.; Sampson, R. D.; Georgiadis, A.; Waldron, P. V.; Duran, Y.; Naeem, A.; Kloc, M.; Cristante, E.; Kruczek, K.; Warre-Cornish, K.; Sowden, J. C.; Smith, A. J.; Ali, R. R.

    2016-01-01

    Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor–host nuclear or cell–cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders. PMID:27701378

  17. Clinical Light Exposure, Photoreceptor Degeneration, and AP-1 Activation: A Cell Death or Cell Survival Signal in the Rhodopsin Mutant Retina?

    PubMed Central

    Gu, Danian; Beltran, William A.; Li, Zexiao; Acland, Gregory M.; Aguirre, Gustavo D.

    2008-01-01

    Purpose The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. Methods Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), super-shift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. Results Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. Conclusions The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa. PMID:17962438

  18. The Role of Intraflagellar Transport in the Photoreceptor Sensory Cilium.

    PubMed

    Taub, Daniel G; Liu, Qin

    2016-01-01

    The photoreceptor is a complex specialized cell in which a major component responsible for visual transduction is the photoreceptor sensory cilium (PSC). Building and maintenance of the PSC requires the transport of large proteins along microtubules that extend from the inner segments to the outer segments. A key process, termed intraflagellar transport (IFT), has been recognized as an essential phenomenon for photoreceptor development and maintenance, and exciting new studies have highlighted its importance in retinal and cilia related diseases. This review focuses on the important roles of IFT players, including motor proteins, IFT proteins, and photoreceptor-specific cargos in photoreceptor sensory cilium. In addition, specific IFT components that are involved in inherited human diseases are discussed.

  19. Cellular and molecular events mediated by docosahexaenoic acid-derived neuroprotectin D1 signaling in photoreceptor cell survival and brain protection.

    PubMed

    Bazan, Nicolas G

    2009-01-01

    Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid) contributes in understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Abeta peptide 42 (Abeta42), calcium ionophore A23187, interleukin (IL)-1beta, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the antiapoptotic proteins of the Bcl-2 family and decreases the expression of proapoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1beta-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-beta (Abeta) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer's disease (AD) cornu ammonis region 1 (CA1) hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A(2) (cPLA(2)), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Abeta42-triggered activation of pro-inflammatory genes and upregulated the antiapoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these

  20. Cellular and molecular events mediated by docosahexaenoic acid-derived neuroprotectin D1 signaling in photoreceptor cell survival and brain protection

    PubMed Central

    Bazan, Nicolas G.

    2009-01-01

    Deficiency in docosahexaenoic acid (DHA) is associated with impaired visual and neurological postnatal development, cognitive decline, macular degeneration, and other neurodegenerative diseases. DHA is an omega-3 polyunsaturated fatty acyl chain concentrated in phospholipids of brain and retina, with photoreceptor cells displaying the highest content of DHA of all cell membranes. The identification and characterization of neuroprotectin D1 (NPD1, 10R, 17S-dihydroxy-docosa-4Z, 7Z, 11E, 13E, 15Z, 19Z-hexaenoic acid) contributes to understanding the biological significance of DHA. In oxidative stress-challenged human retinal pigment epithelial (RPE) cells, human brain cells, or rat brains undergoing ischemia-reperfusion, NPD1 synthesis is enhanced as a response for sustaining homeostasis. Thus, neurotrophins, Aβ peptide 42 (Aβ42), calcium ionophore A23187, interleukin (IL)-1 β, or DHA supply enhances NPD1 synthesis. NPD1, in turn, up-regulates the anti-apoptotic proteins of the Bcl-2 family and decreases the expression of pro-apoptotic Bcl-2 family members. Moreover, NPD1 inhibits IL-1 β-stimulated expression of cyclooxygenase-2 (COX-2). Because both RPE and photoreceptors are damaged and then die in retinal degenerations, elucidating how NPD1 signaling contributes to retinal cell survival may lead to a new understanding of disease mechanisms. In human neural cells, DHA attenuates amyloid-β (Aβ) secretion, resulting in concomitant formation of NPD1. NPD1 was found to be reduced in the Alzheimer’s disease (AD) CA1 hippocampal region, but not in other areas of the brain. The expression of key enzymes for NPD1 biosynthesis, cytosolic phospholipase A2 (cPLA2), and 15-lipoxygenase (15-LOX) was found altered in the AD hippocampal CA1 region. NPD1 repressed Aβ42-triggered activation of pro-inflammatory genes and upregulated the anti-apoptotic genes encoding Bcl-2, Bcl-xl, and Bfl-1(A1) in human brain cells in culture. Overall, these results support the concept that

  1. Multiple rod–cone and cone–rod photoreceptor transmutations in snakes: Evidence from visual opsin gene expression

    USGS Publications Warehouse

    Simoe, Bruno F; Sampaio, Filipa L.; Loew, Ellis R.; Sanders, Kate L.; Fisher, Robert N.; Hart, Nathan S.; Hunt, David M.; Partridge, Julian C.; Gower, David J.

    2016-01-01

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor ‘transmutation’. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels.

  2. Multiple rod-cone and cone-rod photoreceptor transmutations in snakes: evidence from visual opsin gene expression.

    PubMed

    Simões, Bruno F; Sampaio, Filipa L; Loew, Ellis R; Sanders, Kate L; Fisher, Robert N; Hart, Nathan S; Hunt, David M; Partridge, Julian C; Gower, David J

    2016-01-27

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor 'transmutation'. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels.

  3. Multiple rod–cone and cone–rod photoreceptor transmutations in snakes: evidence from visual opsin gene expression

    PubMed Central

    Sampaio, Filipa L.; Loew, Ellis R.; Sanders, Kate L.; Fisher, Robert N.; Hart, Nathan S.; Hunt, David M.; Partridge, Julian C.

    2016-01-01

    In 1934, Gordon Walls forwarded his radical theory of retinal photoreceptor ‘transmutation’. This proposed that rods and cones used for scotopic and photopic vision, respectively, were not fixed but could evolve into each other via a series of morphologically distinguishable intermediates. Walls' prime evidence came from series of diurnal and nocturnal geckos and snakes that appeared to have pure-cone or pure-rod retinas (in forms that Walls believed evolved from ancestors with the reverse complement) or which possessed intermediate photoreceptor cells. Walls was limited in testing his theory because the precise identity of visual pigments present in photoreceptors was then unknown. Subsequent molecular research has hitherto neglected this topic but presents new opportunities. We identify three visual opsin genes, rh1, sws1 and lws, in retinal mRNA of an ecologically and taxonomically diverse sample of snakes central to Walls' theory. We conclude that photoreceptors with superficially rod- or cone-like morphology are not limited to containing scotopic or photopic opsins, respectively. Walls' theory is essentially correct, and more research is needed to identify the patterns, processes and functional implications of transmutation. Future research will help to clarify the fundamental properties and physiology of photoreceptors adapted to function in different light levels. PMID:26817768

  4. Improved cell metabolism prolongs photoreceptor survival upon retinal-pigmented epithelium loss in the sodium iodate induced model of geographic atrophy.

    PubMed

    Zieger, Marina; Punzo, Claudio

    2016-03-01

    Age-related macular degeneration (AMD) is characterized by malfunction and loss of retinal-pigmented epithelium (RPE) cells. Because the RPE transfers nutrients from the choriocapillaris to photoreceptor (PR), PRs are affected as well. Geographic atrophy (GA) is an advanced form of AMD characterized by severe vision impairment due to RPE loss over large areas. Currently there is no treatment to delay the degeneration of nutrient deprived PRs once RPE cells die. Here we show that cell-autonomous activation of the key regulator of cell metabolism, the kinase mammalian target of rapamycin complex 1 (mTORC1), delays PR death in the sodium iodate induced model of RPE atrophy. Consistent with this finding loss of mTORC1 in cones accelerates cone death as cones fail to balance demand with supply. Interestingly, promoting rod survival does not promote cone survival in this model of RPE atrophy as both, rods and cones suffer from a sick and dying RPE. The findings suggest that activation of metabolic genes downstream of mTORC1 can serve as a strategy to prolong PR survival when RPE cells malfunction or die.

  5. Improved cell metabolism prolongs photoreceptor survival upon retinal-pigmented epithelium loss in the sodium iodate induced model of geographic atrophy

    PubMed Central

    Zieger, Marina; Punzo, Claudio

    2016-01-01

    Age-related macular degeneration (AMD) is characterized by malfunction and loss of retinal-pigmented epithelium (RPE) cells. Because the RPE transfers nutrients from the choriocapillaris to photoreceptor (PR), PRs are affected as well. Geographic atrophy (GA) is an advanced form of AMD characterized by severe vision impairment due to RPE loss over large areas. Currently there is no treatment to delay the degeneration of nutrient deprived PRs once RPE cells die. Here we show that cell-autonomous activation of the key regulator of cell metabolism, the kinase mammalian target of rapamycin complex 1 (mTORC1), delays PR death in the sodium iodate induced model of RPE atrophy. Consistent with this finding loss of mTORC1 in cones accelerates cone death as cones fail to balance demand with supply. Interestingly, promoting rod survival does not promote cone survival in this model of RPE atrophy as both, rods and cones suffer from a sick and dying RPE. The findings suggest that activation of metabolic genes downstream of mTORC1 can serve as a strategy to prolong PR survival when RPE cells malfunction or die. PMID:26883199

  6. Cell-differentiation rules that generate regular mosaic patterns: modelling motivated by cone mosaic formation in fish retina.

    PubMed

    Takesue, A; Mochizuki, A; Iwasa, Y

    1998-10-21

    We study characteristics of cell-differentiation rules that realize stable formation of regularly arranged checker-board patterns, exemplified by cone "mosaic" zebrafish retina, or the regular arrangement of cone photoreceptor cells. We consider the situation in which cells are arranged on a square lattice and are initially undifferentiated. Later each cell becomes one of the two differentiated states, affected by the state of the neighboring cells. The cells that undergo differentiation form a "morphogenetic cell row" which sweeps from one end to the other end of the lattice through time. This models an outward sweep of the margin of expanding mosaic region of the retina which occurs as undifferentiated photoreceptor cells become differentiated in concentric circles, joining the mosaic. We introduce an index to measure the ability of cell-differentiation rules to generate regular checker-board patterns from irregular initial patterns, and attempt to characterize the successful rules. We first show the importance of six "preservation conditions" which guarantee perfectly regular photoreceptor arrangement for all the rows after a regular row. Then we select an additional six "optimizing conditions" for responses to configuration that are consistently shown by the rules of high average scores. We also examine the effect of interaction between responses to different configurations. Finally we examine the concept of morphogenetic row precedence, i.e. that the successful rules generating a high score tend to treat the consistency with neighbors in the newly differentiated cells (those in the morphogenetic cell row) as more important that the consistency with previously differentiated neighbors.

  7. Cell patterning with mucin biopolymers

    PubMed Central

    Crouzier, T.; Jang, H.; Ahn, J.; Stocker, R.; Ribbeck, K.

    2014-01-01

    The precise spatial control of cell adhesion to surfaces is an endeavor that has enabled discoveries in cell biology and new possibilities in tissue engineering. The generation of cell-repellent surfaces currently requires advanced chemistry techniques and could be simplified. Here we show that mucins, glycoproteins of high structural and chemical complexity, spontaneously adsorb on hydrophobic substrates to form coatings that prevent the surface adhesion of mammalian epithelial cells, fibroblasts, and myoblasts. These mucin coatings can be patterned with micrometer precision using a microfluidic device, and are stable enough to support myoblast differentiation over seven days. Moreover, our data indicate that the cell-repellent effect is dependent on mucin-associated glycans because their removal results in a loss of effective cell-repulsion. Last, we show that a critical surface density of mucins, which is required to achieve cell-repulsion, is efficiently obtained on hydrophobic surfaces, but not on hydrophilic glass surfaces. However, this limitation can be overcome by coating glass with hydrophobic fluorosilane. We conclude that mucin biopolymers are attractive candidates to control cell adhesion on surfaces. PMID:23980712

  8. Transplanted photoreceptor precursors transfer proteins to host photoreceptors by a mechanism of cytoplasmic fusion

    PubMed Central

    Singh, Mandeep S.; Balmer, Jasmin; Barnard, Alun R.; Aslam, Sher A.; Moralli, Daniela; Green, Catherine M.; Barnea-Cramer, Alona; Duncan, Isabel; MacLaren, Robert E.

    2016-01-01

    Photoreceptor transplantation is a potential future treatment for blindness caused by retinal degeneration. Photoreceptor transplantation restores visual responses in end-stage retinal degeneration, but has also been assessed in non-degenerate retinas. In the latter scenario, subretinal transplantation places donor cells beneath an intact host outer nuclear layer (ONL) containing host photoreceptors. Here we show that host cells are labelled with the donor marker through cytoplasmic transfer—94±4.1% of apparently well-integrated donor cells containing both donor and host markers. We detect the occurrence of Cre-Lox recombination between donor and host photoreceptors, and we confirm the findings through FISH analysis of X and Y chromosomes in sex-discordant transplants. We do not find evidence of nuclear fusion of donor and host cells. The artefactual appearance of integrated donor cells in host retinas following transplantation is most commonly due to material transfer from donor cells. Understanding this novel mechanism may provide alternate therapeutic strategies at earlier stages of retinal degeneration. PMID:27901042

  9. Inhibition of the alternative complement pathway preserves photoreceptors after retinal injury

    PubMed Central

    Sweigard, J. Harry; Matsumoto, Hidetaka; Smith, Kaylee E.; Kim, Leo A.; Paschalis, Eleftherios I.; Okonuki, Yoko; Castillejos, Alexandra; Kataoka, Keiko; Hasegawa, Eiichi; Yanai, Ryoji; Husain, Deeba; Lambris, John D.; Vavvas, Demetrios; Miller, Joan W.; Connor, Kip M.

    2015-01-01

    Degeneration of photoreceptors is a primary cause of vision loss worldwide, making the underlying mechanisms surrounding photoreceptor cell death critical to developing new treatment strategies. Retinal detachment, characterized by the separation of photoreceptors from the underlying retinal pigment epithelium, is a sight-threatening event that can happen in a number of retinal diseases. The detached photoreceptors undergo apoptosis and programmed necrosis. Given that photoreceptors are nondividing cells, their loss leads to irreversible visual impairment even after successful retinal reattachment surgery. To better understand the underlying disease mechanisms, we analyzed innate immune system regulators in the vitreous of human patients with retinal detachment and correlated the results with findings in a mouse model of retinal detachment. We identified the alternative complement pathway as promoting early photoreceptor cell death during retinal detachment. Photoreceptors down-regulate membrane-bound inhibitors of complement, allowing for selective targeting by the alternative complement pathway. When photoreceptors in the detached retina were removed from the primary source of oxygen and nutrients (choroidal vascular bed), the retina became hypoxic, leading to an up-regulation of complement factor B, a key mediator of the alternative pathway. Inhibition of the alternative complement pathway in knockout mice or through pharmacological means ameliorated photoreceptor cell death during retinal detachment. Our current study begins to outline the mechanism by which the alternative complement pathway facilitates photoreceptor cell death in the damaged retina. PMID:26203084

  10. Evolution of clitellate phaosomes from rhabdomeric photoreceptor cells of polychaetes – a study in the leech Helobdella robusta (Annelida, Sedentaria, Clitellata)

    PubMed Central

    2013-01-01

    Introduction In Annelida two types of photoreceptor cells (PRCs) are regarded as generally present, rhabdomeric and ciliary PRCs. In certain taxa, however, an additional type of PRC may occur, the so called phaosomal PRC. Whereas the former two types of PRCs are always organized as an epithelium with their sensory processes projecting into an extracellular cavity formed by the PRCs and (pigmented) supportive cells, phaosomes are seemingly intracellular vacuoles housing the sensory processes. Phaosomal PRCs are the only type of PRC found in one major annelid group, Clitellata. Several hypotheses have been put forward explaining the evolutionary origin of the clitellate phaosomes. To elucidate the evolution of clitellate PRC and eyes the leech Helobdella robusta, for which a sequenced genome is available, was chosen. Results TEM observations showed that extraocular and ocular PRCs are structurally identical. Bioinformatic analyses revealed predictions for four opsin genes, three of which could be amplified. All belong to the rhabdomeric opsin family and phylogenetic analyses showed them in a derived position within annelid opsins. Gene expression studies showed two of them expressed in the eye and in the extraocular PRCs. Polychaete eye-typic key enzymes for ommochromme and pterin shading pigments synthesis are not expressed in leech eyes. Conclusions By comparative gene-expression studies we herein provide strong evidence that the phaosomal PRCs typical of Clitellata are derived from the rhabdomeric PRCs characteristic for polychaete adult eyes. Thus, they represent a highly derived type of PRC that evolved in the stem lineage of Clitellata rather than another, primitive type of PRC in Metazoa. Evolution of these PRCs in Clitellata is related to a loss of the primary eyes and most of their photoreceptive elements except for the rhabdomeric PRCs. Most likely this happened while changing to an endobenthic mode of life. This hypothesis of PRC evolution is in accordance

  11. CaV1.3 L-type channels, maxiK Ca(2+)-dependent K(+) channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells.

    PubMed

    Müller, Claudia; Más Gómez, Néstor; Ruth, Peter; Strauss, Olaf

    2014-05-01

    Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca(2+) regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca(2+) channels (Ca1.3(-/-)) and maxiK Ca(2+)-dependent K(+) channels (BK(-/-)). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca(2+) homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (-)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca(2+) channel in RPE cells, varied at different times of day. CaV1.3(-/-) RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK(-/-) RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK(-/-) mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca(2+) channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.

  12. Retinal remodeling in inherited photoreceptor degenerations.

    PubMed

    Marc, Robert E; Jones, Bryan W

    2003-10-01

    Photoreceptor degenerations initiated in rods or the retinal pigmented epithelium usually evoke secondary cone death and sensory deafferentation of the surviving neural retina. In the mature central nervous system, deafferentation evokes atrophy and connective re-patterning. It has been assumed that the neural retina does not remodel, and that it is a passive survivor. Screening of advanced stages of human and rodent retinal degenerations with computational molecular phenotyping has exposed a prolonged period of aggressive negative remodeling in which neurons migrate along aberrant glial columns and seals, restructuring the adult neural retina (1). Many neurons die, but survivors rewire the remnant inner plexiform layer (IPL), forming thousands of novel ectopic microneuromas in the remnant inner nuclear layer (INL). Bipolar and amacrine cells engage in new circuits that are most likely corruptive. Remodeling in human and rodent retinas emerges regardless of the molecular defects that initially trigger retinal degenerations. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, the exposure of intrinsic retinal remodeling by the removal of sensory control in retinal degenerations suggests that neuronal organization in the normal retina may be more plastic than previously believed.

  13. Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

    PubMed Central

    Rocco, Maria Luisa; Balzamino, Bijorn Omar; Petrocchi Passeri, Pamela; Micera, Alessandra; Aloe, Luigi

    2015-01-01

    A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies. PMID:25897972

  14. argos Is required for projection of photoreceptor axons during optic lobe development in Drosophila.

    PubMed

    Sawamoto, K; Okabe, M; Tanimura, T; Hayashi, S; Mikoshiba, K; Okano, H

    1996-02-01

    The Drosophila argos gene encodes a secreted protein with an epidermal growth factor (EGF) motif, which acts as an inhibitor of cell recruitment in the developing eye and wing. Here, we have analyzed the role of argos during optic lobe development. argos expression was observed in the optic lobes throughout the developmental stages. In argos mutants, neuropiles failed to develop normally during embryonic and larval stages, and photoreceptor axons did not project properly into the lamina. Ubiquitous expression of argos, under control of the hsp70 promoter, rescued the defects in optic lobes. We have found that glial cells failed to differentiate in the larval optic lobes of argos mutants. Correspondingly, in loss-of-function repo mutants, whose glial cells also fail to differentiate, photoreceptor axons showed the impaired projection pattern similar to the argos phenotype. These results suggest that glial cells play a role for guidance of photoreceptor axons. The loss-of-function Star mutation (StarX155) dominantly suppressed the defects in the argos optic lobes, suggesting that these two genes act in an antagonistic fashion during optic lobe development.

  15. Structural analysis of retinal photoreceptor ellipsoid zone and postreceptor retinal layer associated with visual acuity in patients with retinitis pigmentosa by ganglion cell analysis combined with OCT imaging

    PubMed Central

    Liu, Guodong; Li, Hui; Liu, Xiaoqiang; Xu, Ding; Wang, Fang

    2016-01-01

    Abstract The aim of this study was to examine changes in photoreceptor ellipsoid zone (EZ) and postreceptor retinal layer in retinitis pigmentosa (RP) patients by ganglion cell analysis (GCA) combined with optical coherence tomography (OCT) imaging to evaluate the structure–function relationships between retinal layer changes and best corrected visual acuity (BCVA). Sixty-eight eyes of 35 patients with RP and 65 eyes of 35 normal controls were analyzed in the study. The average length of EZ was 911.1 ± 208.8 μm in RP patients, which was shortened with the progression of the disease on the OCT images. The average ganglion cell–inner plexiform layer thickness (GCIPLT) was 54.7 ± 18.9 μm in RP patients, while in normal controls it was 85.6 ± 6.8 μm. The GCIPLT in all quarters became significantly thinner along with outer retinal thinning. There was a significantly positive correlation between BCVA and EZ (r = −0.7622, P < 0.001) and GCIPLT (r = −0.452, P < 0.001). Therefore, we assess the retinal layer changes from a new perspective in RP patients, which suggests that EZ and GCIPLT obtained by GCA combined with OCT imaging are the direct and valid indicators to diagnosis and predict the pathological process of RP. PMID:28033301

  16. The morpho-anatomy and histology of the pineal complex in a major Indian carp, Catla catla: identification of the pineal photoreceptor cells and their responsiveness to constant light and constant darkness during different phases of the annual reproductive cycle.

    PubMed

    Dey, R; Bhattacharya, S; Maitra, S K; Banerji, T K

    2003-11-01

    In contrast to mammals in which the pineal gland is a discrete structure situated dorsally in the brain, the "pineal gland" in teleost fishes is composed of a number of separate but connected constituent parts, collectively described as the "pineal complex." In this paper, we have described the pineal complex in a common Indian carp, Catla catla, which exhibits an annual reproductive cycle. Attempts have been made to (a) provide an in-depth description of the structure of the pineal complex; and (b) identify the photoreceptor cells of the pineal, by exposing the animals to constant light (LL) and constant darkness (DD). Furthermore, we examined any possible influence of the reproductive status of the fish on the responsiveness of the pineal photoreceptor cells in C. catla following exposure to LL and DD. To this end, a total of four experiments were carried out during the four different phases of the annual reproductive cycle that is characteristic of this species. Each of these four experiments was carried out for a period of 30 days after which the fishes were sacrificed, different parts of the pineal complex were dissected out, and processed for histological and karyometric studies. Our results showed that the pineal complex in this species is composed of three separate but connected parts, (a) an end vesicle (EV); (b) a dorsal sac (DS); and (c) a long and thin pineal stalk (PS) that attaches the EV to the DS. Detailed karyometric and histo-morphologic studies following exposure of the animals to DD and LL showed that constant darkness led to a stimulatory effect on the pineal photoreceptor cells of the EV as evident from a significant increase in the nuclear diameter. In contrast, the nuclear diameter of the photoreceptor cells in animals subjected to constant light showed a significant reduction. Furthermore, the observed cellular changes in the EV of fish exposed either to LL or DD were independent of the stage of the gonadal cycle. The apparent lack of any

  17. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  18. In vivo requirement of protein prenylation for maintenance of retinal cytoarchitecture and photoreceptor structure

    PubMed Central

    1995-01-01

    Recent studies have demonstrated that inhibition of mevalonate synthesis in cultured cells leads to altered cell morphology due to inhibition of protein prenylation. To investigate the effects in vivo of mevalonate deprivation in nondividing, terminally differentiated neural cells, we have analyzed the effects on retinal tissue of intravitreal injection of lovastatin, a potent inhibitor of the mevalonate-producing enzyme, HMG-CoA reductase. A single injection of lovastatin (0.25 mumol) produced profound dysplastic-like changes in adult rat retinas primarily involving the photoreceptor layer. Within 2 d after injection, photoreceptor nuclei migrated in a circular pattern resulting in the formation of rosette-like structures by 4 d. Also during this period, photoreceptor inner and outer segment degeneration was evident. By 21 d, intact photoreceptor nuclei with remnants of inner and outer segments were dispersed throughout all retinal layers. To investigate the biochemical specificity of the lovastatin-induced alterations, and to distinguish the relative importance of the various branches of the mevalonate pathway, the incorporation of [3H]acetate into retinal lipids was examined in the presence and absence of metabolic inhibitors. HPLC analysis of lovastatin-treated retinas revealed a dramatic reduction in the incorporation of intravitreally injected [3H]acetate into nonsaponifiable lipids, compared with controls. In contrast, intravitreal injection of NB-598, a specific inhibitor of squalene epoxidase, eliminated the conversion of newly synthesized squalene to sterols without obvious pathology. Hence, involvement to the sterol branch of isoprenoid metabolism in the lovastatin-induced morphologic disruption was obviated. Intravitreal injection of 0.27 mumol of N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), an inhibitor of carboxyl methyltransferase activity and prenylated protein function, produced morphologic changes that were virtually indistinguishable from

  19. Photoreceptor Processing Speed and Input Resistance Changes during Light Adaptation Correlate with Spectral Class in the Bumblebee, Bombus impatiens

    PubMed Central

    Skorupski, Peter; Chittka, Lars

    2011-01-01

    Colour vision depends on comparison of signals from photoreceptors with different spectral sensitivities. However, response properties of photoreceptor cells may differ in ways other than spectral tuning. In insects, for example, broadband photoreceptors, with a major sensitivity peak in the green region of the spectrum (>500 nm), drive fast visual processes, which are largely blind to chromatic signals from more narrowly-tuned photoreceptors with peak sensitivities in the blue and UV regions of the spectrum. In addition, electrophysiological properties of the photoreceptor membrane may result in differences in response dynamics of photoreceptors of similar spectral class between species, and different spectral classes within a species. We used intracellular electrophysiological techniques to investigate response dynamics of the three spectral classes of photoreceptor underlying trichromatic colour vision in the bumblebee, Bombus impatiens, and we compare these with previously published data from a related species, Bombus terrestris. In both species, we found significantly faster responses in green, compared with blue- or UV-sensitive photoreceptors, although all 3 photoreceptor types are slower in B. impatiens than in B. terrestris. Integration times for light-adapted B. impatiens photoreceptors (estimated from impulse response half-width) were 11.3±1.6 ms for green photoreceptors compared with 18.6±4.4 ms and 15.6±4.4 for blue and UV, respectively. We also measured photoreceptor input resistance in dark- and light-adapted conditions. All photoreceptors showed a decrease in input resistance during light adaptation, but this decrease was considerably larger (declining to about 22% of the dark value) in green photoreceptors, compared to blue and UV (41% and 49%, respectively). Our results suggest that the conductances associated with light adaptation are largest in green photoreceptors, contributing to their greater temporal processing speed. We suggest that the

  20. 2-deoxy-d-glucose uptake in the inner retina: an in vivo study in the normal rat and following photoreceptor degeneration.

    PubMed Central

    Wilson, David J

    2002-01-01

    PURPOSE: To evaluate, in vivo, at the cellular level, glucose metabolism in the rat inner retina, and to determine how inner retinal glucose metabolism is affected by photoreceptor degeneration. METHODS: Glucose metabolism was evaluated using the 2-deoxyglucose technique. This is an autoradiographic technique that permits evaluation of glucose uptake at the cellular level. The three experimental groups consisted of normal rats (n = 13), dystrophic Royal College of Surgeons rats (n = 3), and rats previously treated with argon green photocoagulation (n = 5). RESULTS: Deoxyglucose uptake in the normal rat was not uniform across the inner retina. Uptake was greatest at the junction of the outer plexiform and inner nuclear layers, and in the inner plexiform layer. Following focal or diffuse photoreceptor loss, there was a marked decrease in the amount of deoxyglucose uptake at the junction of the outer plexiform and inner nuclear layers. CONCLUSION: The pattern of uptake of deoxyglucose in the inner retina is consistent with abundant uptake of deoxyglucose by Müller cells and at sites of synaptic transmission. The decline in deoxyglucose uptake following diffuse or focal photoreceptor loss indicates that there is diminished inner retinal glucose uptake following photoreceptor loss. This change in inner retinal glucose metabolism following photoreceptor loss may help to explain the inner retinal vascular changes observed following photocoagulation and in retinal dystrophies. PMID:12545701

  1. A new functional role uncovered for RASGRF2 in control of nuclear migration in cone photoreceptors during postnatal retinal development.

    PubMed

    Jimeno, David; Santos, Eugenio

    2017-01-02

    Despite their homologous structure and central nervous system(CNS) expression patterns, the GRF1 and GRF2 guanine nucleotide exchange factors(GEF) appear to play distinct, non-overlapping functions in cellular excitability, synaptic plasticity or neuromodulation. We recently uncovered a new functional role of GRF2 controlling nuclear migration in cone photoreceptors during postnatal neuroepithelial differentiation of the mouse retina. Analyzing GRF2-KO mice, we detected the specific accumulation of abnormally located, "ectopic" cone photoreceptor nuclei in the photoreceptor segment(PS) layer of their retinas. This alteration was accompanied by defective electroretinograms(ERG) indicative of impaired cone-mediated visual function, and accumulation around the "ectopic" nuclei of signaling molecules known to be functionally relevant for intracellular organelle migration, cytoskeletal reorganization or cell polarity establishment including PAR3, PAR6, and the phosphorylated proteins pPAK, pMLC2 and pVASP. We propose a mechanism whereby the absence of a productive functional interaction between GRF2 and its downstream target CDC42 leads to altered formation/structure of PAR-containing, polarity-related macromolecular complexes and abnormal activation of downstream signaling mediated by activated, phosphorylated forms of PAK, VASP and MLC2. As cone photoreceptors are responsible for color vision and visual acuity, these observations are potentially relevant for degenerative diseases of the human retina, harboring almost double number of cones than mice.

  2. Pattern recognition monitoring of PEM fuel cell

    DOEpatents

    Meltser, Mark Alexander

    1999-01-01

    The CO-concentration in the H.sub.2 feed stream to a PEM fuel cell stack is monitored by measuring current and voltage behavior patterns from an auxiliary cell attached to the end of the stack. The auxiliary cell is connected to the same oxygen and hydrogen feed manifolds that supply the stack, and discharges through a constant load. Pattern recognition software compares the current and voltage patterns from the auxiliary cell to current and voltage signature determined from a reference cell similar to the auxiliary cell and operated under controlled conditions over a wide range of CO-concentrations in the H.sub.2 fuel stream.

  3. Pattern recognition monitoring of PEM fuel cell

    DOEpatents

    Meltser, M.A.

    1999-08-31

    The CO-concentration in the H{sub 2} feed stream to a PEM fuel cell stack is monitored by measuring current and voltage behavior patterns from an auxiliary cell attached to the end of the stack. The auxiliary cell is connected to the same oxygen and hydrogen feed manifolds that supply the stack, and discharges through a constant load. Pattern recognition software compares the current and voltage patterns from the auxiliary cell to current and voltage signature determined from a reference cell similar to the auxiliary cell and operated under controlled conditions over a wide range of CO-concentrations in the H{sub 2} fuel stream. 4 figs.

  4. Mouse Ganglion-Cell Photoreceptors Are Driven by the Most Sensitive Rod Pathway and by Both Types of Cones

    PubMed Central

    Weng, Shijun; Estevez, Maureen E.; Berson, David M.

    2013-01-01

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) are depolarized by light by two mechanisms: directly, through activation of their photopigment melanopsin; and indirectly through synaptic circuits driven by rods and cones. To learn more about the rod and cone circuits driving ipRGCs, we made multielectrode array (MEA) and patch-clamp recordings in wildtype and genetically modified mice. Rod-driven ON inputs to ipRGCs proved to be as sensitive as any reaching the conventional ganglion cells. These signals presumably pass in part through the primary rod pathway, involving rod bipolar cells and AII amacrine cells coupled to ON cone bipolar cells through gap junctions. Consistent with this interpretation, the sensitive rod ON input to ipRGCs was eliminated by pharmacological or genetic disruption of gap junctions, as previously reported for conventional ganglion cells. A presumptive cone input was also detectable as a brisk, synaptically mediated ON response that persisted after disruption of rod ON pathways. This was roughly three log units less sensitive than the rod input. Spectral analysis revealed that both types of cones, the M- and S-cones, contribute to this response and that both cone types drive ON responses. This contrasts with the blue-OFF, yellow-ON chromatic opponency reported in primate ipRGCs. The cone-mediated response was surprisingly persistent during steady illumination, echoing the tonic nature of both the rod input to ipRGCs and their intrinsic, melanopsin-based phototransduction. These synaptic inputs greatly expand the dynamic range and spectral bandpass of the non-image-forming visual functions for which ipRGCs provide the principal retinal input. PMID:23762490

  5. Using Zinc Finger Nuclease Technology to Generate CRX‐Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation

    PubMed Central

    Collin, Joseph; Mellough, Carla B; Dorgau, Birthe; Przyborski, Stefan; Moreno‐Gimeno, Inmaculada

    2015-01-01

    Abstract The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone‐Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX‐GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX‐GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation. Stem Cells 2016;34:311–321 PMID:26608863

  6. Rebuilding the Missing Part—A Review on Photoreceptor Transplantation

    PubMed Central

    Santos-Ferreira, Tiago F.; Borsch, Oliver; Ader, Marius

    2017-01-01

    Vision represents one of the main senses for humans to interact with their environment. Our sight relies on the presence of fully functional light sensitive cells – rod and cone photoreceptors — allowing us to see under dim (rods) and bright (cones) light conditions. Photoreceptor degeneration is one of the major causes for vision impairment in industrialized countries and it is highly predominant in the population above the age of 50. Thus, with the continuous increase in life expectancy it will make retinal degeneration reach an epidemic proportion. To date, there is no cure established for photoreceptor loss, but several therapeutic approaches, spanning from neuroprotection, pharmacological drugs, gene therapy, retinal prosthesis, and cell (RPE or photoreceptor) transplantation, have been developed over the last decade with some already introduced in clinical trials. In this review, we focus on current developments in photoreceptor transplantation strategies, its major breakthroughs, current limitations and the next challenges to translate such cell-based approaches toward clinical application. PMID:28105007

  7. An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid, Doryteuthis pealeii.

    PubMed

    Kingston, Alexandra C N; Wardill, Trevor J; Hanlon, Roger T; Cronin, Thomas W

    2015-01-01

    Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized.

  8. An Unexpected Diversity of Photoreceptor Classes in the Longfin Squid, Doryteuthis pealeii

    PubMed Central

    Kingston, Alexandra C. N.; Wardill, Trevor J.; Hanlon, Roger T.; Cronin, Thomas W.

    2015-01-01

    Cephalopods are famous for their ability to change color and pattern rapidly for signaling and camouflage. They have keen eyes and remarkable vision, made possible by photoreceptors in their retinas. External to the eyes, photoreceptors also exist in parolfactory vesicles and some light organs, where they function using a rhodopsin protein that is identical to that expressed in the retina. Furthermore, dermal chromatophore organs contain rhodopsin and other components of phototransduction (including retinochrome, a photoisomerase first found in the retina), suggesting that they are photoreceptive. In this study, we used a modified whole-mount immunohistochemical technique to explore rhodopsin and retinochrome expression in a number of tissues and organs in the longfin squid, Doryteuthis pealeii. We found that fin central muscles, hair cells (epithelial primary sensory neurons), arm axial ganglia, and sucker peduncle nerves all express rhodopsin and retinochrome proteins. Our findings indicate that these animals possess an unexpected diversity of extraocular photoreceptors and suggest that extraocular photoreception using visual opsins and visual phototransduction machinery is far more widespread throughout cephalopod tissues than previously recognized. PMID:26351853

  9. The evolution of rod photoreceptors.

    PubMed

    Morshedian, Ala; Fain, Gordon L

    2017-04-05

    Photoreceptors in animals are generally of two kinds: the ciliary or c-type and the rhabdomeric or r-type. Although ciliary photoreceptors are found in many phyla, vertebrates seem to be unique in having two distinct kinds which together span the entire range of vision, from single photons to bright light. We ask why the principal photoreceptors of vertebrates are ciliary and not rhabdomeric, and how rods evolved from less sensitive cone-like photoreceptors to produce our duplex retina. We suggest that the principal advantage of vertebrate ciliary receptors is that they use less ATP than rhabdomeric photoreceptors. This difference may have provided sufficient selection pressure for the development of a completely ciliary eye. Although many of the details of rod evolution are still uncertain, present evidence indicates that (i) rods evolved very early before the split between the jawed and jawless vertebrates, (ii) outer-segment discs make no contribution to rod sensitivity but may have evolved to increase the efficiency of protein renewal, and (iii) evolution of the rod was incremental and multifaceted, produced by the formation of several novel protein isoforms and by changes in protein expression, with no one alteration having more than a few-fold effect on transduction activation or inactivation.This article is part of the themed issue 'Vision in dim light'.

  10. Dense Pattern Optical Multipass Cell

    NASA Technical Reports Server (NTRS)

    Silver, Joel A. (Inventor)

    2009-01-01

    A multiple pass optical cell and method comprising providing a pair of opposed cylindrical mirrors having curved axes with substantially equal focal lengths, positioning an entrance hole for introducing light into the cell and an exit hole for extracting light from the cell, wherein the entrance hole and exit hole are coextensive or non-coextensive, introducing light into the cell through the entrance hole, and extracting light from the cell through the exit hole.

  11. Dense pattern optical multipass cell

    DOEpatents

    Silver, Joel A [Santa Fe, NM

    2009-01-13

    A multiple pass optical cell and method comprising providing a pair of opposed cylindrical mirrors having curved axes with substantially equal focal lengths, positioning an entrance hole for introducing light into the cell and an exit hole for extracting light from the cell, wherein the entrance hole and exit hole are coextensive or non-coextensive, introducing light into the cell through the entrance hole, and extracting light from the cell through the exit hole.

  12. Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

    PubMed Central

    Ramirez, Juanma; Martinez, Aitor; Lectez, Benoit; Lee, So Young; Franco, Maribel; Barrio, Rosa; Dittmar, Gunnar; Mayor, Ugo

    2015-01-01

    Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system. PMID:26460970

  13. Specialized photoreceptor composition in the raptor fovea.

    PubMed

    Mitkus, Mindaugas; Olsson, Peter; Toomey, Matthew B; Corbo, Joseph C; Kelber, Almut

    2017-02-15

    The retinae of many bird species contain a depression with high photoreceptor density known as the fovea. Many species of raptors have two foveae, a deep central fovea and a shallower temporal fovea. Birds have six types of photoreceptors: rods, active in dim light, double cones that are thought to mediate achromatic discrimination, and four types of single cones mediating color vision. To maximize visual acuity, the fovea should only contain photoreceptors contributing to high-resolution vision. Interestingly, it has been suggested that raptors might lack double cones in the fovea. We used transmission electron microscopy and immunohistochemistry to evaluate this claim in five raptor species: the common buzzard (Buteo buteo), the honey buzzard (Pernis apivorus), the Eurasian sparrowhawk (Accipiter nisus), the red kite (Milvus milvus) and the peregrine falcon (Falco peregrinus). We found that all species, except the Eurasian sparrowhawk, lack double cones in the center of the central fovea. The size of the double cone-free zone differed between species. Only the common buzzard had a double cone-free zone in the temporal fovea. In three species, we examined opsin expression in the central fovea and found evidence that rod opsin positive cells were absent and violet-sensitive cone and green-sensitive cone opsin positive cells were present. We conclude that not only double cones, but also single cones may contribute to high-resolution vision in birds, and that raptors may in fact possess high-resolution tetrachromatic vision in the central fovea. This article is protected by copyright. All rights reserved.

  14. Membrane current responses of skate photoreceptors

    PubMed Central

    1989-01-01

    Light-evoked membrane currents were recorded with suction electrodes from the outer segments of individual photoreceptors enzymatically dissociated from the skate retina. The intensity-response relation of dark-adapted cells closely followed a Michaelis function for which a half-saturating response was elicited by a flash intensity that produced about 36 photoisomerizations. Dim-light responses, as well as the early rising phase of the responses to a wide range of flash intensities, could be described by a reaction scheme that involved a series of four first-order delay stages. The number of delay stages required to model the rising phase of the photocurrents did not change in light adaptation. However, background illumination that reduced sensitivity by 1.5 log units, or a bleaching exposure that resulted in a nearly equivalent desensitization, shortened significantly the time scale of the responses. In both instances there were two- to threefold increases in the rate constants of the transitional delays, and almost complete suppression of the tail current that characterized the response of the dark-adapted cell. These findings suggest that although light adaptation alters the gain and kinetics of the transduction mechanism, the nature of the intervening processes is the same in dark- and light-adapted photoreceptors. Moreover, the results show clearly that there is no need to postulate the existence of a second class of cone-like rods to account for the remarkable ability of skate photoreceptors to respond to incremental stimuli presented on "saturating" background fields or after exposure to an intense bleaching light. PMID:2614369

  15. Customized color patterning of photovoltaic cells

    SciTech Connect

    Cruz-Campa, Jose Luis; Nielson, Gregory N.; Okandan, Murat; Lentine, Anthony L.; Resnick, Paul J.; Gupta, Vipin P.

    2016-11-15

    Photovoltaic cells and photovoltaic modules, as well as methods of making and using such photovoltaic cells and photovoltaic modules, are disclosed. More particularly, embodiments of the photovoltaic cells selectively reflect visible light to provide the photovoltaic cells with a colorized appearance. Photovoltaic modules combining colorized photovoltaic cells may be used to harvest solar energy while providing a customized appearance, e.g., an image or pattern.

  16. Glycine input induces the synaptic facilitation in salamander rod photoreceptors.

    PubMed

    Shen, Wen; Jiang, Zheng; Li, Baoqin

    2008-11-01

    Glycinergic synapses in photoreceptors are made by centrifugal feedback neurons in the network, but the function of the synapses is largely unknown. Here we report that glycinergic input enhances photoreceptor synapses in amphibian retinas. Using specific antibodies against a glycine transporter (GlyT2) and glycine receptor beta subunit, we identified the morphology of glycinergic input in photoreceptor terminals. Electrophysiological recordings indicated that 10 muM glycine depolarized rods and activated voltage-gated Ca(2+) channels in the neurons. The effects facilitated glutamate vesicle release in photoreceptors, meanwhile increased the spontaneous excitatory postsynaptic currents in Off-bipolar cells. Endogenous glycine feedback also enhanced glutamate transmission in photoreceptors. Additionally, inhibition of a Cl(-) uptake transporter NKCC1 with bumetanid effectively eliminated glycine-evoked a weak depolarization in rods, suggesting that NKCC1 maintains a high Cl(-) level in rods, which causes to depolarize in responding to glycine input. This study reveals a new function of glycine in retinal synaptic transmission.

  17. Sortilin Participates in Light-dependent Photoreceptor Degeneration in Vivo

    PubMed Central

    Martín-Oliva, David; de la Villa, Pedro; Cuadros, Miguel A.; Frade, José M.

    2012-01-01

    Both proNGF and the neurotrophin receptor p75 (p75NTR) are known to regulate photoreceptor cell death caused by exposure of albino mice to intense illumination. ProNGF-induced apoptosis requires the participation of sortilin as a necessary p75NTR co-receptor, suggesting that sortilin may participate in the photoreceptor degeneration triggered by intense lighting. We report here that light-exposed albino mice showed sortilin, p75NTR, and proNGF expression in the outer nuclear layer, the retinal layer where photoreceptor cell bodies are located. In addition, cone progenitor-derived 661W cells subjected to intense illumination expressed sortilin and p75NTR and released proNGF into the culture medium. Pharmacological blockade of sortilin with either neurotensin or the “pro” domain of proNGF (pro-peptide) favored the survival of 661W cells subjected to intense light. In vivo, the pro-peptide attenuated retinal cell death in light-exposed albino mice. We propose that an auto/paracrine proapoptotic mechanism based on the interaction of proNGF with the receptor complex p75NTR/sortilin participates in intense light-dependent photoreceptor cell death. We therefore propose sortilin as a putative target for intervention in hereditary retinal dystrophies. PMID:22558402

  18. Deterministic patterns in cell motility

    NASA Astrophysics Data System (ADS)

    Lavi, Ido; Piel, Matthieu; Lennon-Duménil, Ana-Maria; Voituriez, Raphaël; Gov, Nir S.

    2016-12-01

    Cell migration paths are generally described as random walks, associated with both intrinsic and extrinsic noise. However, complex cell locomotion is not merely related to such fluctuations, but is often determined by the underlying machinery. Cell motility is driven mechanically by actin and myosin, two molecular components that generate contractile forces. Other cell functions make use of the same components and, therefore, will compete with the migratory apparatus. Here, we propose a physical model of such a competitive system, namely dendritic cells whose antigen capture function and migratory ability are coupled by myosin II. The model predicts that this coupling gives rise to a dynamic instability, whereby cells switch from persistent migration to unidirectional self-oscillation, through a Hopf bifurcation. Cells can then switch to periodic polarity reversals through a homoclinic bifurcation. These predicted dynamic regimes are characterized by robust features that we identify through in vitro trajectories of dendritic cells over long timescales and distances. We expect that competition for limited resources in other migrating cell types can lead to similar deterministic migration modes.

  19. Rippled-pattern basal cell carcinoma.

    PubMed

    Misago, Noriyuki; Tsuruta, Noriko; Narisawa, Yutaka

    2012-07-01

    Basal cell carcinoma (BCC) is the most common malignant cutaneous neoplasm, however, there have been few studies on BCC with a "rippled pattern" so far. We reviewed the 650 BCC specimens from the archives of our institution, and only one example of BCC with a rippled pattern was found. We herein report the histopathological characteristics of this case. Within the lesion, which showed the typical histopathological features of nodular BCC, there was a noticeable area composed of 10-15 basaloid aggregations, which showed the rippled pattern. The rippled pattern was characterized by alternating bands of epithelial cords of spindle-shaped basaloid cells and mucinous spaces. Characteristically, around the rippled-pattern area, neoplastic aggregations with a mucinous reticulated or cystic pattern (pseudo-tubular structures), and many cord-like structures were seen. A review of the published work and the present case suggested that the histopathological characteristics of rippled-pattern BCC are: (i) a nodular type of BCC; (ii) considerably rare; (iii) have frequent intervention by mucinous spaces between the epithelial cords; and (iv) no apparent divergent differentiation with folliculosebaceous-apocrine lineage. The last three characteristics contrasted with those of the rippled-pattern sebaceoma/trichoblastoma. However, neoplastic germinative cells in rippled-pattern BCC may naturally form cord-like structures in a manner similar to rippled-pattern sebaceoma/trichoblastoma.

  20. Autophagy supports survival and phototransduction protein levels in rod photoreceptors

    PubMed Central

    Zhou, Z; Doggett, T A; Sene, A; Apte, R S; Ferguson, T A

    2015-01-01

    Damage and loss of the postmitotic photoreceptors is a leading cause of blindness in many diseases of the eye. Although the mechanisms of photoreceptor death have been extensively studied, few studies have addressed mechanisms that help sustain these non-replicating neurons for the life of an organism. Autophagy is an intracellular pathway where cytoplasmic constituents are delivered to the lysosomal pathway for degradation. It is not only a major pathway activated in response to cellular stress, but is also important for cytoplasmic turnover and to supply the structural and energy needs of cells. We examined the importance of autophagy in photoreceptors by deleting the essential autophagy gene Atg5 specifically in rods. Loss of autophagy led to progressive degeneration of rod photoreceptors beginning at 8 weeks of age such that by 44 weeks few rods remained. Cone photoreceptor numbers were only slightly diminished following rod degeneration but their function was significantly decreased. Rod cell death was apoptotic but was not dependent on daily light exposure or accelerated by intense light. Although the light-regulated translocation of the phototransduction proteins arrestin and transducin were unaffected in rods lacking autophagy, Atg5-deficient rods accumulated transducin-α as they degenerated suggesting autophagy might regulate the level of this protein. This was confirmed when the light-induced decrease in transducin was abolished in Atg5-deficient rods and the inhibition of autophagy in retinal explants cultures prevented its degradation. These results demonstrate that basal autophagy is essential to the long-term health of rod photoreceptors and a critical process for maintaining optimal levels of the phototransduction protein transducin-α. As the lack of autophagy is associated with retinal degeneration and altered phototransduction protein degradation in the absence of harmful gene products, this process may be a viable therapeutic target where rod

  1. Individual variations in human cone photoreceptor packing density

    PubMed Central

    Chui, Toco Yuen Ping; Song, HongXin; Burns, Stephen A.

    2008-01-01

    PURPOSE To measure the variation in human cone photoreceptor packing density across the retina both within an individual and between individuals with different refractive errors. METHODS A high resolution adaptive optics scanning laser ophthalmoscope was used to image the cones of eleven human eyes. Five emmetropes and six myopes were tested (+0.50D to -7.50D). For each subject we obtained four approximately 10 degree by 1.5 degree strips of cone images. Each strip started at the fovea, and proceeded towards the periphery along the four primary meridians. The position of each cone within the sampling windows was digitized manually by the investigator. From these cone counts, the density of cones was calculated for a set of fixed distances from the fovea for locations throughout the image. RESULTS Cone photoreceptor packing density decreased from 27,712 cells/mm2 to 7,070 cells/mm2 from the retinal eccentricity of 0.30mm to 3.40mm along the superior meridian in five emmetropic eyes. Cone photoreceptor packing density in cells/mm2 was significantly lower in myopic eyes than in emmetropic eyes. At a given location, there was considerable individual variation in cone photoreceptor packing density, although more than 20% of the variance could be accounted for by differences in axial length. CONCLUSIONS Our results provide a baseline analysis of individual difference in cone photoreceptor packing density in healthy human eyes. As predicted by retinal stretching models, cone photoreceptor packing density is lower in highly myopic eyes than in emmetropic eyes. PMID:18552378

  2. Ionic currents underlying difference in light response between type A and type B photoreceptors.

    PubMed

    Blackwell, K T

    2006-05-01

    In Hermissenda crassicornis, the memory of light associated with turbulence is stored as changes in intrinsic and synaptic currents in both type A and type B photoreceptors. These photoreceptor types exhibit qualitatively different responses to light and current injection, and these differences shape the spatiotemporal firing patterns that control behavior. Thus the objective of the study was to identify the mechanisms underlying these differences. The approach was to develop a type B model that reproduced characteristics of type B photoreceptors recorded in vitro, and then to create a type A model by modifying a select number of ionic currents. Comparison of type A models with characteristics of type A photoreceptors recorded in vitro revealed that type A and type B photoreceptors have five main differences, three that have been characterized experimentally and two that constitute hypotheses to be tested with experiments in the future. The three differences between type A and type B photoreceptors previously characterized include the inward rectifier current, the fast sodium current, and conductance of calcium-dependent and transient potassium channels. Two additional changes were required to produce a type A photoreceptor model. The very fast firing frequency observed during the first second after light onset required a faster time constant of activation of the delayed rectifier. The fast spike adaptation required a fast, noninactivating calcium-dependent potassium current. Because these differences between type A and type B photoreceptors have not been confirmed in comparative experiments, they constitute hypotheses to be tested with future experiments.

  3. Patterning proteins and cells using soft lithography.

    PubMed

    Kane, R S; Takayama, S; Ostuni, E; Ingber, D E; Whitesides, G M

    1999-12-01

    This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.

  4. Tissue-specific regulation of flowering by photoreceptors.

    PubMed

    Endo, Motomu; Araki, Takashi; Nagatani, Akira

    2016-02-01

    Plants use various kinds of environmental signals to adjust the timing of the transition from the vegetative to reproductive phase (flowering). Since flowering at the appropriate time is crucial for plant reproductive strategy, several kinds of photoreceptors are deployed to sense environmental light conditions. In this review, we will update our current understanding of light signaling pathways in flowering regulation, especially, in which tissue do photoreceptors regulate flowering in response to light quality and photoperiod. Since light signaling is also integrated into other flowering pathways, we also introduce recent progress on how photoreceptors are involved in tissue-specific thermosensation and the gibberellin pathway. Finally, we discuss the importance of cell-type-specific analyses for future plant studies.

  5. Intravitreal injection or topical eye-drop application of a μ-calpain C2L domain peptide protects against photoreceptor cell death in Royal College of Surgeons' rats, a model of retinitis pigmentosa.

    PubMed

    Ozaki, Taku; Nakazawa, Mitsuru; Yamashita, Tetsuro; Sorimachi, Hiroyuki; Hata, Shoji; Tomita, Hiroshi; Isago, Hitomi; Baba, Ayaka; Ishiguro, Sei-Ichi

    2012-11-01

    Mitochondrial μ-calpain initiates apoptosis-inducing factor (AIF)-dependent apoptosis in retinal photoreceptor degeneration. Mitochondrial μ-calpain inhibitors may represent therapeutic targets for the disease. Therefore, we sought to identify inhibitors of mitochondrial calpains and determine their effects in Royal College of Surgeons' (RCS) rats, an animal model of retinitis pigmentosa (RP). We synthesized 20-mer peptides of the C2-like (C2L) domain of μ-calpain. Two μ-calpain peptides N2 and N9 inhibited mitochondrial μ-calpain activity (IC(50); 892 and 498nM, respectively), but not other proteases. Western blotting showed that 50μM of both μ-calpain peptides caused specific degradation of mitochondrial μ-calpain. Three-dimensional structure of calpains suggested that the peptides N2 and N9 corresponded to the regions forming salt bridges between the protease core domain 2 and the C2L domain. We determined the inhibitory regions of μ-calpain peptides N2 and N9 using 10-mers, and one peptide, N2-10-2, inhibited the activity of mitochondrial μ-calpain (IC(50); 112nM). We next conjugated the peptide N2-10-2 to the C-terminal of HIV-1 tat (HIV), a cell-penetrating peptide. Using isolated rat liver mitochondria, 50μM HIV-conjugated μ-calpain N2-10-2 peptide (HIV-Nμ, IC(50); 285nM) significantly inhibited AIF truncation. The intravitreal injection of 20mM HIV-Nμ also prevented retinal photoreceptor apoptosis determined by TUNEL staining, and preserved retinal function assessed by electroretinography in RCS rats. Topical application of 40mM HIV-Nμ also prevented apoptosis of retinal photoreceptors in RCS rats. Our results demonstrate that HIV-Nμ, a peptide inhibitor of mitochondrial μ-calpain, offers a new modality for treating RP.

  6. C-opsin expressing photoreceptors in echinoderms.

    PubMed

    Ullrich-Lüter, Esther M; D'Aniello, Salvatore; Arnone, Maria I

    2013-07-01

    Today's progress in molecular analysis and, in particular, the increased availability of genome sequences have enabled us to investigate photoreceptor cells (PRCs) in organisms that were formerly inaccessible to experimental manipulation. Our studies of marine non-chordate deuterostomes thus aim to bridge a gap of knowledge regarding the evolution of deuterostome PRCs prior to the emergence of vertebrates' eyes. In this contribution, we will show evidence for expression of a c-opsin photopigment, which, according to our phylogenetic analysis, is closely related to an assemblage of chordate visual c-opsins. An antibody raised against sea urchins' c-opsin protein (Sp-Opsin1) recognizes epitopes in a variety of tissues of different echinoderms. While in sea urchins this c-opsin is expressed in locomotory and buccal tube feet, spines, pedicellaria, and epidermis, in brittlestars and starfish we found the immuno-reaction to be located exclusively in cells within the animals' spines. Structural characteristics of these c-opsin+ PRC types include the close vicinity/connection to nerve strands and a, so far unexplored, conspicuous association with the animals' calcite skeleton, which previously has been hypothesized to play a role in echinoderm photobiology. These features are discussed within the context of the evolution of photoreceptors in echinoderms and in deuterostomes generally.

  7. Differential expression of photoreceptor-specific genes in the retina of a zebrafish cadherin2 mutant glass onion and zebrafish cadherin4 morphants.

    PubMed

    Liu, Q; Frey, R A; Babb-Clendenon, S G; Liu, B; Francl, J; Wilson, A L; Marrs, J A; Stenkamp, D L

    2007-01-01

    Cadherins are Ca2+ -dependent transmembrane molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin2 (also called N-cadherin) and cadherin4 (also called R-cadherin), members of the classic cadherin subfamily, have been shown to be involved in development of a variety of tissues and organs including the visual system. To gain insight into cadherin2 and cadherin4 function in differentiation of zebrafish photoreceptors, we have analyzed expression patterns of several photoreceptor-specific genes (crx, gnat1, gnat2, irbp, otx5, rod opsin, rx1, and uv opsin) and/or a cone photoreceptor marker (zpr-1) in the retina of a zebrafish cadherin2 mutant, glass onion (glo) and in zebrafish embryos injected with a cadherin4 specific antisense morpholino oligonucleotide (cdh4MO). We find that expression of all these genes, and of zpr-1, is greatly reduced in the retina of both the glo and cadherin4 morphants. Moreover, in these embryos, expression of some genes (e.g. gnat1, gnat2 and irbp) is more affected than others (e.g. rod opsin and uv opsin). In embryos with both cadherins functions blocked (glo embryos injected with the cdh4MO), the eye initially formed, but became severely and progressively disintegrated and expressed little or no crx and otx5 as development proceeded. Our results suggest that cadherin2 and cadherin4 play important roles in the differentiation of zebrafish retinal photoreceptors.

  8. Chloroplasts continuously monitor photoreceptor signals during accumulation movement.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2013-07-01

    Under low light conditions, chloroplasts gather at a cell surface to maximize light absorption for efficient photosynthesis, which is called the accumulation response. Phototropin1 (phot1) and phototropin2 (phot2) were identified as blue light photoreceptors in the accumulation response that occurs in Arabidopsis thaliana and Adiantum capillus-veneris with neochrome1 (neo1) as a red light photoreceptor in A. capillus-veneris. However, the signal molecule that is emitted from the photoreceptors and transmitted to the chloroplasts is not known. To investigate this topic, the accumulation response was induced by partial cell irradiation with a microbeam of red, blue and far-red light in A. capillus-veneris gametophyte cells. Chloroplasts moved towards the irradiated region and were able to sense the signal as long as its signal flowed. The signal from neo1 had a longer life than the signal that came from phototropins. When two microbeams with the same wavelength and the same fluence rate were placed 20 μm apart from each other and were applied to a dark-adapted cell, chloroplasts at an equidistant position always moved towards the center (midpoint) of the two microbeams, but not towards either one. This result indicates that chloroplasts are detecting the concentration of the signal but not the direction of signal flow. Chloroplasts repeatedly move and stop at roughly 10 s intervals during the accumulation response, suggesting that they monitor the intermittent signal waves from photoreceptors.

  9. Synthesis of docosahexaenoic acid from eicosapentaenoic acid in retina neurons protects photoreceptors from oxidative stress

    PubMed Central

    Simón, María Victoria; Agnolazza, Daniela L.; German, Olga Lorena; Garelli, Andrés; Politi, Luis E.; Agbaga, Martin-Paul; Anderson, Robert E.; Rotstein, Nora P.

    2015-01-01

    Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat (PQ) and hydrogen peroxide (H2O2). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. PMID:26662863

  10. Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors

    PubMed Central

    Xu, Ying; An, Futing; Borycz, Jolanta A.; Borycz, Janusz; Meinertzhagen, Ian A.; Wang, Tao

    2015-01-01

    Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals. PMID:26713872

  11. Histamine Recycling Is Mediated by CarT, a Carcinine Transporter in Drosophila Photoreceptors.

    PubMed

    Xu, Ying; An, Futing; Borycz, Jolanta A; Borycz, Janusz; Meinertzhagen, Ian A; Wang, Tao

    2015-12-01

    Histamine is an important chemical messenger that regulates multiple physiological processes in both vertebrate and invertebrate animals. Even so, how glial cells and neurons recycle histamine remains to be elucidated. Drosophila photoreceptor neurons use histamine as a neurotransmitter, and the released histamine is recycled through neighboring glia, where it is conjugated to β-alanine to form carcinine. However, how carcinine is then returned to the photoreceptor remains unclear. In an mRNA-seq screen for photoreceptor cell-enriched transporters, we identified CG9317, an SLC22 transporter family protein, and named it CarT (Carcinine Transporter). S2 cells that express CarT are able to take up carcinine in vitro. In the compound eye, CarT is exclusively localized to photoreceptor terminals. Null mutations of cart alter the content of histamine and its metabolites. Moreover, null cart mutants are defective in photoreceptor synaptic transmission and lack phototaxis. These findings reveal that CarT is required for histamine recycling at histaminergic photoreceptors and provide evidence for a CarT-dependent neurotransmitter trafficking pathway between glial cells and photoreceptor terminals.

  12. Diffraction pattern study for cell type identification.

    PubMed

    Mihailescu, M; Costescu, J

    2012-01-16

    This paper presents our study regarding diffracted intensity distribution in Fresnel and Fraunhofer approximation from different cell types. Starting from experimental information obtained through digital holographic microscopy, we modeled the cell shapes as oblate spheroids and built their phase-only transmission functions. In Fresnel approximation, the experimental and numerical diffraction patterns from mature and immature red blood cells have complementary central intensity values at different distances. The Fraunhofer diffraction patterns of deformed red blood cells were processed in the reciprocal space where, the isoamplitude curves were formed independently for each degree of cell deformation present within every sample; the values on each separate isoamplitude curve are proportional with the percentage of the respective cell type within the sample.

  13. Light-dependent repetitive Ca2+ spikes induced by extracellular application of neomycin in honeybee drone photoreceptors.

    PubMed

    Walz, B; Zimmermann, B; Ukhanov, K

    2000-05-01

    Photoreceptor cells of the honeybee drone fire, in the presence of the polycationic aminoglycoside neomycin, repetitive slow spike-like potentials superimposed on the receptor potential plateau phase. We have used conventional intracellular recordings and microfluorometric intracellular Ca2+ measurements to characterize these spike potentials. We have shown that the spike frequency increases in a light-intensity-dependent manner. The spikes are fired only when light stimuli depolarize the cell from a resting potential of -50 to -60 mV to at least -40 to -45 mV; they are tetrodotoxin insensitive and blocked by the Ca2+ channel blockers Ni2+, Cd2+, omega-agatoxin TK, verapamil and methoxyverapamil. Depolarization of the photoreceptors with high extracellular K+ in the presence of neomycin in darkness does not generate spikes. Small intracellular Ca2+ oscillations superimposed on the plateau phase of the light-induced increase in intracellular free Ca2+ concentration have a similar temporal pattern as the spike-like potentials. We conclude that the spike-like potentials require stimulation by light and are generated by voltage-dependent Ca2+ channels localized on the soma of the photoreceptors, distal to the basal lamina.

  14. Differences in calcium homeostasis between retinal rod and cone photoreceptors revealed by the effects of voltage on the cGMP-gated conductance in intact cells

    PubMed Central

    1994-01-01

    We measured currents under voltage clamp in intact retinal rod photoreceptors with tight seal electrodes in the perforated patch mode. In the dark, membrane depolarization to voltages > or = +20 mV activates a time- and voltage-dependent outward current in the outer segment. This dark voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course that is voltage dependent. DVAC reaches its maximum enhancement of approximately 30% in 4-6 s at +60 mV. DVAC is entirely suppressed by light and its current-voltage curve and reversal potential are the same as those of the photocurrent. Therefore, DVAC arises from the opening in darkness of the cGMP-gated channels of the outer segment. DVAC is blocked by BAPTA loaded into the cell's cytoplasm and is enhanced by lowering extracellular Ca2+ concentration. Because the cGMP-gated channels are not directly gated by voltage and because BAPTA blocks DVAC, we suggest this signal arises from a voltage-dependent decrease in cytoplasmic Ca2+ concentration that, in turn, activates guanylyl cyclase and causes cGMP synthesis. In rods loaded with high cytoplasmic Na+, membrane depolarization in darkness to voltages > or = +20 mV inactivates the outward current in the outer segment with an exponential time course. We call this DVIC (dark, voltage-inactivated current). DVIC reflects voltage-dependent closing of the cGMP-gated channel in the dark. DVIC, too, is blocked by cytoplasmic BAPTA, and it arises from a voltage-dependent rise in cytoplasmic Ca2+ in darkness, which occurs only if cytoplasmic Na is high. We develop a quantitative model to calculate the rate and extent of the voltage-dependent change in cytoplasmic Ca2+ concentration in a normal rod. We assume that this concentration is controlled by the balance between Ca2+ influx through the cGMP-gated channels and its efflux through a Na+/Ca2+, K+ exchanger. Lowered cytoplasmic Ca2+ is linked to guanylyl cyclase activation with characteristics determined from

  15. Pattern formation in cell membrane adhesion

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Hategan, A.; Sengupta, K.; Sackmann, E.

    2004-03-01

    Strong adhesion of highly active cells often nucleates focal adhesions or related structures that are, over time, reinforced by cytoskeleton (actin, etc.). Red cells lack such complex adhesion systems, but they are shown here to also exhibit complex spatial patterns within an adhesive contact zone. While strong adhesion and spreading of the red cell to a dense poly-L-lysine surface appears complete in < 1 s by reflective interference microscopy, over longer times of 10-15 min or more distinct patterns in fluorescently labeled membrane components emerge. The fluorescent lipid Fl-PE (fluorescein phosphoethanolamine), in particular, is seen to diffuse and reorganize (eg. worm-like domains of <500 nm) within the contact zone, independent of whether the cell is intact or ruptured. Lipid patterns are accompanied by visible perturbations in band 3 distribution and weaker perturbations in membrane skeleton actin. Although fluorescent poly-L-lysine is shown to be uniform under cells, pressing down on the membrane quenches the lipid patterns and reveals the topographical basis for pattern formation. Regions of strong contact are thus separated by regions where the membrane is more distant from the surface.

  16. Synthesis of docosahexaenoic acid from eicosapentaenoic acid in retina neurons protects photoreceptors from oxidative stress.

    PubMed

    Simón, María Victoria; Agnolazza, Daniela L; German, Olga Lorena; Garelli, Andrés; Politi, Luis E; Agbaga, Martin-Paul; Anderson, Robert E; Rotstein, Nora P

    2016-03-01

    Oxidative stress is involved in activating photoreceptor death in several retinal degenerations. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, protects cultured retina photoreceptors from apoptosis induced by oxidative stress and promotes photoreceptor differentiation. Here, we investigated whether eicosapentaenoic acid (EPA), a metabolic precursor to DHA, had similar effects and whether retinal neurons could metabolize EPA to DHA. Adding EPA to rat retina neuronal cultures increased opsin expression and protected photoreceptors from apoptosis induced by the oxidants paraquat and hydrogen peroxide (H2 O2 ). Palmitic, oleic, and arachidonic acids had no protective effect, showing the specificity for DHA. We found that EPA supplementation significantly increased DHA percentage in retinal neurons, but not EPA percentage. Photoreceptors and glial cells expressed Δ6 desaturase (FADS2), which introduces the last double bond in DHA biosynthetic pathway. Pre-treatment of neuronal cultures with CP-24879 hydrochloride, a Δ5/Δ6 desaturase inhibitor, prevented EPA-induced increase in DHA percentage and completely blocked EPA protection and its effect on photoreceptor differentiation. These results suggest that EPA promoted photoreceptor differentiation and rescued photoreceptors from oxidative stress-induced apoptosis through its elongation and desaturation to DHA. Our data show, for the first time, that isolated retinal neurons can synthesize DHA in culture. Docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in retina photoreceptors, and its precursor, eicosapentaenoic acid (EPA) have multiple beneficial effects. Here, we show that retina neurons in vitro express the desaturase FADS2 and can synthesize DHA from EPA. Moreover, addition of EPA to these cultures protects photoreceptors from oxidative stress and promotes their differentiation through its metabolization to DHA.

  17. Photovoltaic cell with nano-patterned substrate

    DOEpatents

    Cruz-Campa, Jose Luis; Zhou, Xiaowang; Zubia, David

    2016-10-18

    A photovoltaic solar cell comprises a nano-patterned substrate layer. A plurality of nano-windows are etched into an intermediate substrate layer to form the nano-patterned substrate layer. The nano-patterned substrate layer is positioned between an n-type semiconductor layer composed of an n-type semiconductor material and a p-type semiconductor layer composed of a p-type semiconductor material. Semiconductor material accumulates in the plurality of nano-windows, causing a plurality of heterojunctions to form between the n-type semiconductor layer and the p-type semiconductor layer.

  18. Orally administered epigallocatechin gallate attenuates light-induced photoreceptor damage.

    PubMed

    Costa, Belmira Lara da Silveira Andrade da; Fawcett, Rebecca; Li, Guang-Yu; Safa, Rukhsana; Osborne, Neville N

    2008-07-01

    EGCG, a major component of green tea, has a number of properties which includes it being a powerful antioxidant. The purpose of this investigation was to deduce whether inclusion of EGCG in the drinking water of albino rats attenuates the effect of a light insult (2200lx, for 24h) to the retina. TUNEL-positive cells were detected in the outer nuclear layer of the retina, indicating the efficacy of the light insult in inducing photoreceptor degeneration. Moreover, Ret-P1 and the mRNA for rhodopsin located at photoreceptors were also significantly reduced as well as the amplitude of both the a- and b-waves of the electroretinogram was also reduced showing that photoreceptors in particular are affected by light. An increase in protein/mRNA of GFAP located primarily to Müller cells caused by light shows that other retinal components are also influenced by the light insult. However, antigens associated with bipolar (alpha-PKC), ganglion (Thy-1) and amacrine (GABA) cells, in contrast, appeared unaffected. The light insult also caused a change in the content of various proteins (caspase-3, caspase-8, PARP, Bad, and Bcl-2) involved in apoptosis. A number of the changes to the retina caused by a light insult were significantly attenuated when EGCG was in the drinking water. The reduction of the a- and b-waves and photoreceptor specific mRNAs/protein caused by light were significantly less. In addition, EGCG attenuated the changes caused by light to certain apoptotic proteins (especially at after 2 days) but did not appear to significantly influence the light-induced up-regulation of GFAP protein/mRNA. It is concluded that orally administered EGCG blunts the detrimental effect of light to the retina of albino rats where the photoreceptors are primarily affected.

  19. The Giant Mottled Eel, Anguilla marmorata, Uses Blue-Shifted Rod Photoreceptors during Upstream Migration

    PubMed Central

    Wang, Feng-Yu; Fu, Wen-Chun; Wang, I-Li

    2014-01-01

    Catadromous fishes migrate between ocean and freshwater during particular phases of their life cycle. The dramatic environmental changes shape their physiological features, e.g. visual sensitivity, olfactory ability, and salinity tolerance. Anguilla marmorata, a catadromous eel, migrates upstream on dark nights, following the lunar cycle. Such behavior may be correlated with ontogenetic changes in sensory systems. Therefore, this study was designed to identify changes in spectral sensitivity and opsin gene expression of A. marmorata during upstream migration. Microspectrophotometry analysis revealed that the tropical eel possesses a duplex retina with rod and cone photoreceptors. The λmax of rod cells are 493, 489, and 489 nm in glass, yellow, and wild eels, while those of cone cells are 508, and 517 nm in yellow, and wild eels, respectively. Unlike European and American eels, Asian eels exhibited a blue-shifted pattern of rod photoreceptors during upstream migration. Quantitative gene expression analyses of four cloned opsin genes (Rh1f, Rh1d, Rh2, and SWS2) revealed that Rh1f expression is dominant at all three stages, while Rh1d is expressed only in older yellow eel. Furthermore, sequence comparison and protein modeling studies implied that a blue shift in Rh1d opsin may be induced by two known (N83, S292) and four putative (S124, V189, V286, I290) tuning sites adjacent to the retinal binding sites. Finally, expression of blue-shifted Rh1d opsin resulted in a spectral shift in rod photoreceptors. Our observations indicate that the giant mottled eel is color-blind, and its blue-shifted scotopic vision may influence its upstream migration behavior and habitat choice. PMID:25101636

  20. The giant mottled eel, Anguilla marmorata, uses blue-shifted rod photoreceptors during upstream migration.

    PubMed

    Wang, Feng-Yu; Fu, Wen-Chun; Wang, I-Li; Yan, Hong Young; Wang, Tzi-Yuan

    2014-01-01

    Catadromous fishes migrate between ocean and freshwater during particular phases of their life cycle. The dramatic environmental changes shape their physiological features, e.g. visual sensitivity, olfactory ability, and salinity tolerance. Anguilla marmorata, a catadromous eel, migrates upstream on dark nights, following the lunar cycle. Such behavior may be correlated with ontogenetic changes in sensory systems. Therefore, this study was designed to identify changes in spectral sensitivity and opsin gene expression of A. marmorata during upstream migration. Microspectrophotometry analysis revealed that the tropical eel possesses a duplex retina with rod and cone photoreceptors. The λmax of rod cells are 493, 489, and 489 nm in glass, yellow, and wild eels, while those of cone cells are 508, and 517 nm in yellow, and wild eels, respectively. Unlike European and American eels, Asian eels exhibited a blue-shifted pattern of rod photoreceptors during upstream migration. Quantitative gene expression analyses of four cloned opsin genes (Rh1f, Rh1d, Rh2, and SWS2) revealed that Rh1f expression is dominant at all three stages, while Rh1d is expressed only in older yellow eel. Furthermore, sequence comparison and protein modeling studies implied that a blue shift in Rh1d opsin may be induced by two known (N83, S292) and four putative (S124, V189, V286, I290) tuning sites adjacent to the retinal binding sites. Finally, expression of blue-shifted Rh1d opsin resulted in a spectral shift in rod photoreceptors. Our observations indicate that the giant mottled eel is color-blind, and its blue-shifted scotopic vision may influence its upstream migration behavior and habitat choice.

  1. Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

    PubMed Central

    1983-01-01

    We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin- binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze- fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested

  2. Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol.

    PubMed

    Andrews, L D; Cohen, A I

    1983-09-01

    We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested

  3. Photoreceptor damage following exposure to excess riboflavin.

    PubMed

    Eckhert, C D; Hsu, M H; Pang, N

    1993-12-15

    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.

  4. Colocalization of retinal dystrophin and actin in postsynaptic dendrites of rod and cone photoreceptor synapses.

    PubMed

    Schmitz, F; Holbach, M; Drenckhahn, D

    1993-12-01

    In this paper we demonstrate immunostaining specific for dystrophin in photoreceptor synapses of human, bovine and rat retinas. Cryosections of retinas incubated with dystrophin-specific monoclonal antibodies displayed a punctuate staining pattern in the outer plexiform layer. This pattern resulted from binding of the antibodies to synaptic complexes of both rods and cones, shown by double-labelling with antibodies to either synaptophysin or actin. Confocal laser fluorescence microscopy demonstrated that dystrophin staining colocalized predominantly with actin, which is concentrated in the postsynaptic portions of the synaptic complex. No significant dystrophin immunolabel was seen in the presynaptic terminals labelled with antibodies to synaptophysin, a marker of synaptic vesicles. Immunoblot analysis confirmed the presence of approximately 420 kDa and approximately 360 kDa dystrophin-like polypeptide bands associated with membranes of the bovine retina. We speculate that retinal dystrophin is involved in the linkage of actin filaments to the postsynaptic plasma membrane. Such a linkage may be important for the generation of synaptic microdomains and for certain phenomena of synaptic plasticity. The absence of dystrophin in patients suffering from Duchenne's muscular dystrophy is accompanied by visual problems and abnormalities of the electroretinogram. Therefore it is likely that retinal dystrophin plays a role in certain stages of synaptic transmission between photoreceptors and the postsynaptic dendritic complex formed by horizontal and bipolar cells.

  5. The Cis-regulatory Logic of the Mammalian Photoreceptor Transcriptional Network

    PubMed Central

    Hsiau, Timothy H.-C.; Diaconu, Claudiu; Myers, Connie A.; Lee, Jongwoo; Cepko, Constance L.; Corbo, Joseph C.

    2007-01-01

    The photoreceptor cells of the retina are subject to a greater number of genetic diseases than any other cell type in the human body. The majority of more than 120 cloned human blindness genes are highly expressed in photoreceptors. In order to establish an integrative framework in which to understand these diseases, we have undertaken an experimental and computational analysis of the network controlled by the mammalian photoreceptor transcription factors, Crx, Nrl, and Nr2e3. Using microarray and in situ hybridization datasets we have produced a model of this network which contains over 600 genes, including numerous retinal disease loci as well as previously uncharacterized photoreceptor transcription factors. To elucidate the connectivity of this network, we devised a computational algorithm to identify the photoreceptor-specific cis-regulatory elements (CREs) mediating the interactions between these transcription factors and their target genes. In vivo validation of our computational predictions resulted in the discovery of 19 novel photoreceptor-specific CREs near retinal disease genes. Examination of these CREs permitted the definition of a simple cis-regulatory grammar rule associated with high-level expression. To test the generality of this rule, we used an expanded form of it as a selection filter to evolve photoreceptor CREs from random DNA sequences in silico. When fused to fluorescent reporters, these evolved CREs drove strong, photoreceptor-specific expression in vivo. This study represents the first systematic identification and in vivo validation of CREs in a mammalian neuronal cell type and lays the groundwork for a systems biology of photoreceptor transcriptional regulation. PMID:17653270

  6. Optics of cone photoreceptors in the chicken (Gallus gallus domesticus)

    PubMed Central

    Wilby, David; Toomey, Matthew B.; Olsson, Peter; Frederiksen, Rikard; Cornwall, M. Carter; Oulton, Ruth; Kelber, Almut; Corbo, Joseph C.; Roberts, Nicholas W.

    2015-01-01

    Vision is the primary sensory modality of birds, and its importance is evident in the sophistication of their visual systems. Coloured oil droplets in the cone photoreceptors represent an adaptation in the avian retina, acting as long-pass colour filters. However, we currently lack understanding of how the optical properties and morphology of component structures (e.g. oil droplet, mitochondrial ellipsoid and outer segment) of the cone photoreceptor influence the transmission of light into the outer segment and the ultimate effect they have on receptor sensitivity. In this study, we use data from microspectrophotometry, digital holographic microscopy and electron microscopy to inform electromagnetic models of avian cone photoreceptors to quantitatively investigate the integrated optical function of the cell. We find that pigmented oil droplets primarily function as spectral filters, not light collection devices, although the mitochondrial ellipsoid improves optical coupling between the inner segment and oil droplet. In contrast, unpigmented droplets found in violet-sensitive cones double sensitivity at its peak relative to other cone types. Oil droplets and ellipsoids both narrow the angular sensitivity of single cone photoreceptors, but not as strongly as those in human cones. PMID:26423439

  7. Optics of cone photoreceptors in the chicken (Gallus gallus domesticus).

    PubMed

    Wilby, David; Toomey, Matthew B; Olsson, Peter; Frederiksen, Rikard; Cornwall, M Carter; Oulton, Ruth; Kelber, Almut; Corbo, Joseph C; Roberts, Nicholas W

    2015-10-06

    Vision is the primary sensory modality of birds, and its importance is evident in the sophistication of their visual systems. Coloured oil droplets in the cone photoreceptors represent an adaptation in the avian retina, acting as long-pass colour filters. However, we currently lack understanding of how the optical properties and morphology of component structures (e.g. oil droplet, mitochondrial ellipsoid and outer segment) of the cone photoreceptor influence the transmission of light into the outer segment and the ultimate effect they have on receptor sensitivity. In this study, we use data from microspectrophotometry, digital holographic microscopy and electron microscopy to inform electromagnetic models of avian cone photoreceptors to quantitatively investigate the integrated optical function of the cell. We find that pigmented oil droplets primarily function as spectral filters, not light collection devices, although the mitochondrial ellipsoid improves optical coupling between the inner segment and oil droplet. In contrast, unpigmented droplets found in violet-sensitive cones double sensitivity at its peak relative to other cone types. Oil droplets and ellipsoids both narrow the angular sensitivity of single cone photoreceptors, but not as strongly as those in human cones.

  8. Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution.

    PubMed

    Liegertová, Michaela; Pergner, Jiří; Kozmiková, Iryna; Fabian, Peter; Pombinho, Antonio R; Strnad, Hynek; Pačes, Jan; Vlček, Čestmír; Bartůněk, Petr; Kozmik, Zbyněk

    2015-07-08

    Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora, a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans.

  9. Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution

    PubMed Central

    Liegertová, Michaela; Pergner, Jiří; Kozmiková, Iryna; Fabian, Peter; Pombinho, Antonio R.; Strnad, Hynek; Pačes, Jan; Vlček, Čestmír; Bartůněk, Petr; Kozmik, Zbyněk

    2015-01-01

    Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora, a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans. PMID:26154478

  10. The bHLH Transcription Factor NeuroD Governs Photoreceptor Genesis and Regeneration Through Delta-Notch Signaling

    PubMed Central

    Taylor, Scott M.; Alvarez-Delfin, Karen; Saade, Carole J.; Thomas, Jennifer L.; Thummel, Ryan; Fadool, James M.; Hitchcock, Peter F.

    2015-01-01

    Purpose Photoreceptor genesis in the retina requires precise regulation of progenitor cell competence, cell cycle exit, and differentiation, although information around the mechanisms that govern these events currently is lacking. In zebrafish, the basic helix-loop-helix (bHLH) transcription factor NeuroD governs photoreceptor genesis, but the signaling pathways through which NeuroD functions are unknown. The purpose of this study was to identify these pathways, and during photoreceptor genesis, Notch signaling was investigated as the putative mediator of NeuroD function. Methods In embryos, genetic mosaic analysis was used to determine if NeuroD functions is cell- or non–cell-autonomous. Morpholino-induced NeuroD knockdown, CRISPR/Cas9 mutation, and pharmacologic and transgenic approaches were used, followed by in situ hybridization, immunocytochemistry, and quantitative RT-PCR (qRT-PCR), to identify mechanisms through which NeuroD functions. In adults, following photoreceptor ablation and NeuroD knockdown, similar methods as above were used to identify NeuroD function during photoreceptor regeneration. Results In embryos, NeuroD function is non–cell-autonomous, NeuroD knockdown increases Notch pathway gene expression, Notch inhibition rescues the NeuroD knockdown-induced deficiency in cell cycle exit but not photoreceptor maturation, and Notch activation and CRISPR/Cas9 mutation of neurod recapitulate NeuroD knockdown. In adults, NeuroD knockdown prevents cell cycle exit and photoreceptor regeneration and increases Notch pathway gene expression, and Notch inhibition rescues this phenotype. Conclusions These data demonstrate that during embryonic development, NeuroD governs photoreceptor genesis via non–cell-autonomous mechanisms and that, during photoreceptor development and regeneration, Notch signaling is a mechanistic link between NeuroD and cell cycle exit. In contrast, during embryonic development, NeuroD governs photoreceptor maturation via mechanisms

  11. Migration, integration and maturation of photoreceptor precursors following transplantation in the mouse retina.

    PubMed

    Warre-Cornish, Katherine; Barber, Amanda C; Sowden, Jane C; Ali, Robin R; Pearson, Rachael A

    2014-05-01

    Retinal degeneration leading to loss of photoreceptors is a major cause of untreatable blindness. Recent research has yielded definitive evidence for restoration of vision following the transplantation of rod photoreceptors in murine models of blindness, while advances in stem cell biology have enabled the generation of transplantable photoreceptors from embryonic stem cells. Importantly, the amount of visual function restored is dependent upon the number of photoreceptors that migrate correctly into the recipient retina. The developmental stage of the donor cells is important for their ability to migrate; they must be immature photoreceptor precursors. Little is known about how and when donor cell migration, integration, and maturation occurs. Here, we have performed a comprehensive histological analysis of the 6-week period following rod transplantation in mice. Donor cells migrate predominately as single entities during the first week undergoing a stereotyped sequence of morphological changes in their translocation from the site of transplantation, through the interphotoreceptor matrix and into the recipient retina. This includes initial polarization toward the outer nuclear layer (ONL), followed by formation of an apical attachment and rudimentary segment during migration into the ONL. Strikingly, acquisition of a nuclear architecture typical of mature rods was accelerated compared with normal development and a feature of migrating cells. Once within the ONL, precursors formed synaptic-like structures and outer segments in accordance with normal maturation. The restoration of visual function mediated by transplanted photoreceptors correlated with the later expression of rod α-transducin, achieving maximal function by 5 weeks.

  12. Cannabinoid receptor activation differentially modulates ion channels in photoreceptors of the tiger salamander.

    PubMed

    Straiker, Alex; Sullivan, Jane M

    2003-05-01

    Cannabinoid CB1 receptors have been detected in retinas of numerous species, with prominent labeling in photoreceptor terminals of the chick and monkey. CB1 labeling is well-conserved across species, suggesting that CB1 receptors might also be present in photoreceptors of the tiger salamander. Synaptic transmission in vertebrate photoreceptors is mediated by L-type calcium currents-currents that are modulated by CB1 receptors in bipolar cells of the tiger salamander. Presence of CB1 receptors in photoreceptor terminals would therefore be consistent with presynaptic modulation of synaptic transmission, a role seen for cannabinoids in other parts of the brain. Here we report immunohistochemical and electrophysiological evidence for the presence of functional CB1 receptors in rod and cone photoreceptors of the tiger salamander. The cannabinoid receptor agonist WIN 55212-2 enhances calcium currents of rod photoreceptors by 39% but decreases calcium currents of large single cones by 50%. In addition, WIN 55212-2 suppresses potassium currents of rods and large single cones by 44 and 48%, respectively. Thus functional CB1 receptors, present in the terminals of rod and cone photoreceptors, differentially modulate calcium and potassium currents in rods and large single cones. CB1 receptors are therefore well positioned to modulate neurotransmitter release at the first synapse of the visual system.

  13. Olaparib significantly delays photoreceptor loss in a model for hereditary retinal degeneration

    PubMed Central

    Sahaboglu, Ayse; Barth, Melanie; Secer, Enver; Amo, Eva M. del; Urtti, Arto; Arsenijevic, Yvan; Zrenner, Eberhart; Paquet-Durand, François

    2016-01-01

    The enzyme poly-ADP-ribose-polymerase (PARP) mediates DNA-repair and rearrangements of the nuclear chromatin. Generally, PARP activity is thought to promote cell survival and in recent years a number of PARP inhibitors have been clinically developed for cancer treatment. Paradoxically, PARP activity is also connected to many diseases including the untreatable blinding disease Retinitis Pigmentosa (RP), where PARP activity appears to drive the pathogenesis of photoreceptor loss. We tested the efficacy of three different PARP inhibitors to prevent photoreceptor loss in the rd1 mouse model for RP. In retinal explant cultures in vitro, olaparib had strong and long-lasting photoreceptor neuroprotective capacities. We demonstrated target engagement by showing that olaparib reduced photoreceptor accumulation of poly-ADP-ribosylated proteins. Remarkably, olaparib also reduced accumulation of cyclic-guanosine-monophosphate (cGMP), a characteristic marker for photoreceptor degeneration. Moreover, intravitreal injection of olaparib in rd1 animals diminished PARP activity and increased photoreceptor survival, confirming in vivo neuroprotection. This study affirms the role of PARP in inherited retinal degeneration and for the first time shows that a clinically approved PARP inhibitor can prevent photoreceptor degeneration in an RP model. The wealth of human clinical data available for olaparib highlights its strong potential for a rapid clinical translation into a novel RP treatment. PMID:28004814

  14. Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace.

    PubMed

    Jagadish, Smitha; Barnea, Gilad; Clandinin, Thomas R; Axel, Richard

    2014-08-06

    In Drosophila, the four inner photoreceptor neurons exhibit overlapping but distinct spectral sensitivities and mediate behaviors that reflect spectral preference. We developed a genetic strategy, Tango-Trace, that has permitted the identification of the connections of the four chromatic photoreceptors. Each of the four stochastically distributed chromatic photoreceptor subtypes make distinct connections in the medulla with four different TmY cells. Moreover, each class of TmY cells forms a retinotopic map in both the medulla and the lobula complex, generating four overlapping topographic maps that could carry different color information. Thus, the four inner photoreceptors transmit spectral information through distinct channels that may converge in both the medulla and lobula complex. These projections could provide an anatomic basis for color vision and may relay information about color to motion sensitive areas. Moreover, the Tango-Trace strategy we used may be applied more generally to identify neural circuits in the fly brain.

  15. Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.

    PubMed

    Sato, Shigeru; Omori, Yoshihiro; Katoh, Kimiko; Kondo, Mineo; Kanagawa, Motoi; Miyata, Kentaro; Funabiki, Kazuo; Koyasu, Toshiyuki; Kajimura, Naoko; Miyoshi, Tomomitsu; Sawai, Hajime; Kobayashi, Kazuhiro; Tani, Akiko; Toda, Tatsushi; Usukura, Jiro; Tano, Yasuo; Fujikado, Takashi; Furukawa, Takahisa

    2008-08-01

    Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix-like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

  16. Topographical characterization of cone photoreceptors and the area centralis of the canine retina

    PubMed Central

    Mowat, Freya M.; Petersen-Jones, Simon M.; Williamson, Helen; Williams, David L.; Luthert, Philip J.; Ali, Robin R.

    2008-01-01

    Purpose The canine is an important large animal model of human retinal genetic disorders. Studies of ganglion cell distribution in the canine retina have identified a visual streak of high density superior to the optic disc with a temporal area of peak density known as the area centralis. The topography of cone photoreceptors in the canine retina has not been characterized in detail, and in contrast to the macula in humans, the position of the area centralis in dogs is not apparent on clinical funduscopic examination. The purpose of this study was to define the location of the area centralis in the dog and to characterize in detail the topography of rod and cone photoreceptors within the area centralis. This will facilitate the investigation and treatment of retinal disease in the canine. Methods We used peanut agglutinin, which labels cone matrix sheaths and antibodies against long/medium wavelength (L/M)- and short wavelength (S)-cone opsins, to stain retinal cryosections and flatmounts from beagle dogs. Retinas were imaged using differential interference contrast imaging, fluorescence, and confocal microscopy. Within the area centralis, rod and cone size and density were quantified, and the proportion of cones expressing each cone opsin subtype was calculated. Using a grid pattern of sampling in 9 retinal flatmounts, we investigated the distribution of cones throughout the retina to predict the location of the area centralis. Results We identified the area centralis as the site of maximal density of rod and cone photoreceptor cells, which have a smaller inner segment cross-sectional area in this region. L/M opsin was expressed by the majority of cones in the retina, both within the area centralis and in the peripheral retina. Using the mean of cone density distribution from 9 retinas, we calculated that the area centralis is likely to be centered at a point 1.5 mm temporal and 0.6 mm superior to the optic disc. For clinical funduscopic examination, this

  17. Phototransduction and the Evolution of Photoreceptors

    PubMed Central

    Fain, Gordon L.; Hardie, Roger; Laughlin, Simon B.

    2010-01-01

    Photoreceptors in metazoans can be grouped into two classes, with their photoreceptive membrane derived either from cilia or microvilli. Both classes use some form of the visual pigment protein opsin, which together with 11-cis retinaldehyde absorbs light and activates a G-protein cascade, resulting in the opening or closing of ion channels. Considerable attention has recently been given to the molecular evolution of the opsins and other photoreceptor proteins; much is also known about transduction in the various photoreceptor types. Here we combine this knowledge in an attempt to understand why certain photoreceptors might have conferred particular selective advantages during evolution. We suggest that microvillar photoreceptors became predominant in most invertebrate species because of their single-photon sensitivity, high temporal resolution, and large dynamic range, and that rods and a duplex retina provided primitive chordates and vertebrates with similar sensitivity and dynamic range, but with a smaller expenditure of ATP. PMID:20144772

  18. Categorical and prolonged potentials are evoked when brief, intermediate-intensity flashes stimulate horseshoe crab lateral eye photoreceptors during octopamine neuromodulation.

    PubMed

    Lim, C C; Wasserman, G S

    2001-01-01

    Octopamine, a major efferent neurotransmitter in the lateral eye of the horseshoe crab (Limulus polyphemus), has previously been shown to modulate photoreceptor responses evoked by long flashes. Quantification of these data indicates that this modulation produced a genuine increase in sensitivity to light which cannot be entirely due to an increase in optical efficiency consequent on an anatomical alteration. Other previous studies demonstrated that extrinsic current can modulate Limulus lateral eye photoreceptor cells by inducing a bistable membrane potential with two distinct states. The present study was therefore undertaken to find out if octopamine could modulate visual responses by inducing prolonged and bistable polarization shifts similar to those demonstrated in several other neural systems. Intracellular microelectrodes were used to execute an electrophysiological study of the receptor potentials evoked in the lateral eye of Limulus when brief (20-ms) flashes were delivered while 50 microM octopamine perfused dark-adapted photoreceptors. The combined chemical and optical stimuli prolonged photoreceptor responses to light to the degree that they often exceeded the duration of the brief stimulus by hundreds of milliseconds. Moreover, these prolonged potentials were clearly bistable because they were categorical--either a prolongation was perceptually clear-cut and present or it was not, with no intermediate patterns being observed. During seawater control perfusions, such prolongations were absent. This appears to be the first demonstration of such categorical and prolonged potentials in a photoreceptor neuron. This finding particularly suggests that efferent-driven neuromodulation can enable the development of a persisting short-term representation of a brief stimulus, with this representation being retained at the most distal possible neural site.

  19. Liprin-α is required for photoreceptor target selection in Drosophila

    PubMed Central

    Choe, Kwang-Min; Prakash, Saurabh; Bright, Ali; Clandinin, Thomas R.

    2006-01-01

    Classical cadherin-mediated interactions between axons and dendrites are critical to target selection and synapse assembly. However, the molecular mechanisms by which these interactions are controlled are incompletely understood. In the Drosophila visual system, N-cadherin is required in both photoreceptor (R cell) axons and their targets to mediate stabilizing interactions required for R cell target selection. Here we identify the scaffolding protein Liprin-α as a critical component in this process. We isolated mutations in Liprin-α in a genetic screen for mutations affecting the pattern of synaptic connections made by R1–R6 photoreceptors. Using eye-specific mosaics, we demonstrate a previously undescribed, axonal function for Liprin-α in target selection: Liprin-α is required to be cell-autonomous in all subtypes of R1–R6 cells for their axons to reach their targets. Because Liprin-α, the receptor tyrosine phosphatase LAR, and N-cadherin share qualitatively similar mutant phenotypes in R1–R6 cells and are coexpressed in R cells and their synaptic targets, we infer that these three genes act at the same step in the targeting process. However, unlike N-cadherin, neither Liprin-α nor LAR is required postsynaptically for R cells to project to their correct targets. Thus, these two proteins, unlike N-cadherin, are functionally asymmetric between axons and dendrites. We propose that the adhesive mechanisms that link pre- and postsynaptic cells before synapse formation may be differentially regulated in these two compartments. PMID:16864799

  20. Patterning Bacterial Communities on Epithelial Cells

    PubMed Central

    Dwidar, Mohammed; Leung, Brendan M.; Yaguchi, Toshiyuki; Takayama, Shuichi; Mitchell, Robert J.

    2013-01-01

    Micropatterning of bacteria using aqueous two phase system (ATPS) enables the localized culture and formation of physically separated bacterial communities on human epithelial cell sheets. This method was used to compare the effects of Escherichia coli strain MG1655 and an isogenic invasive counterpart that expresses the invasin (inv) gene from Yersinia pseudotuberculosis on the underlying epithelial cell layer. Large portions of the cell layer beneath the invasive strain were killed or detached while the non-invasive E. coli had no apparent effect on the epithelial cell layer over a 24 h observation period. In addition, simultaneous testing of the localized effects of three different bacterial species; E. coli MG1655, Shigella boydii KACC 10792 and Pseudomonas sp DSM 50906 on an epithelial cell layer is also demonstrated. The paper further shows the ability to use a bacterial predator, Bdellovibriobacteriovorus HD 100, to selectively remove the E. coli, S. boydii and P. sp communities from this bacteria-patterned epithelial cell layer. Importantly, predation and removal of the P. Sp was critical for maintaining viability of the underlying epithelial cells. Although this paper focuses on a few specific cell types, the technique should be broadly applicable to understand a variety of bacteria-epithelial cell interactions. PMID:23785519

  1. Immunocytochemical analysis of photoreceptors in the tiger salamander retina.

    PubMed

    Zhang, Jian; Wu, Samuel M

    2009-01-01

    In the tiger salamander retina, visual signals are transmitted to the inner retina via six morphologically distinct types of photoreceptors: large/small rods, large/small single cones, and double cones composed of principal and accessory members. The objective of this study was to determine the morphology of these photoreceptors and their synaptic interconnection with bipolar cells and horizontal cells in the outer plexiform layer (OPL). Here we showed that glutamate antibodies labeled all photoreceptors and recovering antibodies strongly labeled all cones and weakly labeled all rods. Antibodies against calbindin selectively stained accessory members of double cones. Antibodies against S-cone opsin stained small rods, a subpopulation of small single cones, and the outer segments of accessory double cones and a subtype of unidentified single cones. On average, large rods and small S-cone opsin positive rods accounted for 98.6% and 1.4% of all rods, respectively. Large/small cones, principle/accessory double cones, S-cone opsin positive small single cones, and S-cone opsin positive unidentified single cones accounted for about 66.9%, 23%, 4.5%, and 5.6% of the total cones, respectively. Moreover, the differential connection between rods/cones and bipolar/horizontal cells and the wide distribution of AMPA receptor subunits GluR2/3 and GluR4 at the rod/cone synapses were observed. These results provide anatomical evidence for the physiological findings that bipolar/horizontal cells in the salamander retina are driven by rod/cone inputs of different weights, and that AMPA receptors play an important role in glutamatergic neurotransmission at the first visual synapses. The different photoreceptors selectively contacting bipolar and horizontal cells support the idea that visual signals may be conveyed to the inner retina by different functional pathways in the outer retina.

  2. Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila

    PubMed Central

    Gambis, Alexis; Dourlen, Pierre; Steller, Hermann; Mollereau, Bertrand

    2011-01-01

    We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The “Tomato/GFP-FLP/FRT” method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single cell resolution. PMID:21215264

  3. The cadherin Flamingo mediates level-dependent interactions that guide photoreceptor target choice in Drosophila.

    PubMed

    Chen, Pei-Ling; Clandinin, Thomas R

    2008-04-10

    Quantitative differences in cadherin activity have been proposed to play important roles in patterning connections between pre- and postsynaptic neurons. However, no examples of such a function have yet been described, and the mechanisms that would allow such differences to direct growth cones to specific synaptic targets are unknown. In the Drosophila visual system, photoreceptors are genetically programmed to make a complex, stereotypic set of synaptic connections. Here we show that the atypical cadherin Flamingo functions as a short-range, homophilic signal, passing between specific R cell growth cones to influence their choice of postsynaptic partners. We find that individual growth cones are sensitive to differences in Flamingo activity through opposing interactions between neighboring cells and require these interactions to be balanced in order to extend along the appropriate trajectory.

  4. Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila.

    PubMed

    Gambis, Alexis; Dourlen, Pierre; Steller, Hermann; Mollereau, Bertrand

    2011-03-01

    We report a new two-color fluorescent imaging system to visualize the mosaic adult photoreceptor neurons (PRs) in real-time. Using this method, we examined a collection of 434 mutants and identified genes required for PR survival, planar cell polarity (PCP), patterning and differentiation. We could track the progression of PR degeneration in living flies. By introducing the expression of p35, a caspase inhibitor, we found mutations that specifically activate caspase-dependent death. Moreover, we showed that grh is required in R3 for correct PCP establishment. The "Tomato/GFP-FLP/FRT" method allows high-throughput, rapid and precise identification of survival and developmental pathways in living adult PRs at single-cell resolution.

  5. Adenosine and dopamine receptors co-regulate photoreceptor coupling via gap junction phosphorylation in mouse retina

    PubMed Central

    Li, Hongyan; Zhang, Zhijing; Blackburn, Michael R.; Wang, Steven W.; Ribelayga, Christophe P.; O’Brien, John

    2013-01-01

    Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibits adenylyl cyclase (AC) and reduces PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to co-regulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately down-regulated expression of D4R and AC type I mRNA. We conclude that adenosine and dopamine co-regulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function. PMID:23407968

  6. gdf6a Is Required for Cone Photoreceptor Subtype Differentiation and for the Actions of tbx2b in Determining Rod Versus Cone Photoreceptor Fate

    PubMed Central

    DuVal, Michèle G.; Oel, A. Phillip; Allison, W. Ted

    2014-01-01

    Functional vision restoration is within reach via stem cell therapy, but one of the largest obstacles is the derivation of colour-sensitive cone photoreceptors that are required for high-acuity daytime vision. To enhance progress made using nocturnal murine models, we instead utilize cone-rich zebrafish and herein investigate relationships between gdf6a and tbx2b in cone photoreceptor development. Growth/differentiation factor 6a (gdf6a), a bone morphogenetic protein family ligand, is an emerging factor in photoreceptor degenerative diseases. The T-box transcription factor tbx2b is required to specify UV cone photoreceptor fate instead of rod photoreceptor fate. Interactions between these factors in cone development would be unanticipated, considering the discrete phenotypes in their respective mutants. However, gdf6a positively modulates the abundance of tbx2b transcript during early eye morphogenesis, and we extended this conclusion to later stages of retinal development comprising the times when photoreceptors differentiate. Despite this, gdf6a−/− larvae possess a normal relative number of UV cones and instead present with a low abundance of blue cone photoreceptors, approximately half that of siblings (p<0.001), supporting a differential role for gdf6a amongst the spectral subtypes of cone photoreceptors. Further, gdf6a−/− larvae from breeding of compound heterozygous gdf6a+/−;tbx2b+/− mutants exhibit the recessive lots-of-rods phenotype (which also shows a paucity of UV cones) at significantly elevated rates (44% or 48% for each of two tbx2b alleles, χ2 p≤0.007 for each compared to expected Mendelian 25%). Thus the gdf6a−/− background sensitizes fish such that the recessive lots-of-rods phenotype can appear in heterozygous tbx2b+/− fish. Overall, this work establishes a novel link between tbx2b and gdf6a in determining photoreceptor fates, defining the nexus of an intricate pathway influencing the abundance of cone spectral subtypes and

  7. Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes

    PubMed Central

    Battelle, Barbara-Anne; Kempler, Karen E.; Saraf, Spencer R.; Marten, Catherine E.; Dugger, Donald R.; Speiser, Daniel I.; Oakley, Todd H.

    2015-01-01

    The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates. PMID:25524988

  8. Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes.

    PubMed

    Battelle, Barbara-Anne; Kempler, Karen E; Saraf, Spencer R; Marten, Catherine E; Dugger, Donald R; Speiser, Daniel I; Oakley, Todd H

    2015-02-01

    The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.

  9. ROM-1 potentiates photoreceptor specific membrane fusion processes.

    PubMed

    Boesze-Battaglia, Kathleen; Stefano, Frank P; Fitzgerald, Catherine; Muller-Weeks, Susan

    2007-01-01

    Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin's non-glycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-1/P/rds complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes.

  10. Cortical microtubule rearrangements and cell wall patterning

    PubMed Central

    Oda, Yoshihisa

    2015-01-01

    Plant cortical microtubules, which form a highly ordered array beneath the plasma membrane, play essential roles in determining cell shape and function by directing the arrangement of cellulosic and non-cellulosic compounds on the cell surface. Interphase transverse arrays of cortical microtubules self-organize through their dynamic instability and inter-microtubule interactions, and by branch-form microtubule nucleation and severing. Recent studies revealed that distinct spatial signals including ROP GTPase, cellular geometry, and mechanical stress regulate the behavior of cortical microtubules at the subcellular and supercellular levels, giving rise to dramatic rearrangements in the cortical microtubule array in response to internal and external cues. Increasing evidence indicates that negative regulators of microtubules also contribute to the rearrangement of the cortical microtubule array. In this review, I summarize recent insights into how the rearrangement of the cortical microtubule array leads to proper, flexible cell wall patterning. PMID:25904930

  11. The Limit of Photoreceptor Sensitivity

    PubMed Central

    Holcman, David; Korenbrot, Juan I.

    2005-01-01

    Detection threshold in cone photoreceptors requires the simultaneous absorption of several photons because single photon photocurrent is small in amplitude and does not exceed intrinsic fluctuations in the outer segment dark current (dark noise). To understand the mechanisms that limit light sensitivity, we characterized the molecular origin of dark noise in intact, isolated bass single cones. Dark noise is caused by continuous fluctuations in the cytoplasmic concentrations of both cGMP and Ca2+ that arise from the activity in darkness of both guanylate cyclase (GC), the enzyme that synthesizes cGMP, and phosphodiesterase (PDE), the enzyme that hydrolyzes it. In cones loaded with high concentration Ca2+ buffering agents, we demonstrate that variation in cGMP levels arise from fluctuations in the mean PDE enzymatic activity. The rates of PDE activation and inactivation determine the quantitative characteristics of the dark noise power density spectrum. We developed a mathematical model based on the dynamics of PDE activity that accurately predicts this power spectrum. Analysis of the experimental data with the theoretical model allows us to determine the rates of PDE activation and deactivation in the intact photoreceptor. In fish cones, the mean lifetime of active PDE at room temperature is ∼55 ms. In nonmammalian rods, in contrast, active PDE lifetime is ∼555 ms. This remarkable difference helps explain why cones are noisier than rods and why cone photocurrents are smaller in peak amplitude and faster in time course than those in rods. Both these features make cones less light sensitive than rods. PMID:15928405

  12. Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses

    PubMed Central

    Schaefer, Kellie A.; Toral, Marcus A.; Velez, Gabriel; Cox, Allison J.; Baker, Sheila A.; Borcherding, Nicholas C.; Colgan, Diana F.; Bondada, Vimala; Mashburn, Charles B.; Yu, Chen-Guang; Geddes, James W.; Tsang, Stephen H.; Bassuk, Alexander G.; Mahajan, Vinit B.

    2016-01-01

    Purpose We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments. Methods CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions. Results Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains. Conclusions CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles. PMID:27152965

  13. Photobiomodulation reduces photoreceptor death and regulates cytoprotection in early states of P23H retinal dystrophy

    NASA Astrophysics Data System (ADS)

    Kirk, Diana K.; Gopalakrishnan, Sandeep; Schmitt, Heather; Abroe, Betsy; Stoehr, Michele; Dubis, Adam; Carroll, Joseph; Stone, Jonathan; Valter, Krisztina; Eells, Janis

    2013-03-01

    Irradiation by light in the far-red to near-infrared (NIR) region of the spectrum (photobiomodulation, PBM) has been demonstrated to attenuate the severity of neurodegenerative disease in experimental and clinical studies. The purpose of this study was to test the hypothesis that 670 nm PBM would protect against the loss of retinal function and improve photoreceptor survival in a rodent model of retinitis pigmentosa, the P23H transgenic rat. P23H rat pups were treated once per day with a 670 nm LED array (180 sec treatments at 50 mW/cm2; fluence 9 joules/cm2) (Quantum Devices Inc., Barneveld WI) from postnatal day (p) 16-20 or from p10-20. Sham-treated rats were restrained, but not exposed to NIR light. The status of the retina was determined at p22 by assessment of mitochondrial function, oxidative stress and cell death. In a second series of studies, retinal status was assessed at p30 by measuring photoreceptor function by ERG and retinal morphology by Spectral Domain Optical Coherence Tomography (SD-OCT). 670 nm PBM increased retinal mitochondrial cytochrome oxidase activity and upregulated the retina's production of the key mitochondrial antioxidant enzyme, MnSOD. PBM also attenuated photoreceptor cell loss and improved photoreceptor function. PBM protects photoreceptors in the developing P23H retina, by augmenting mitochondrial function and stimulating antioxidant protective pathways. Photobiomodulation may have therapeutic potential, where mitochondrial damage is a step in the death of photoreceptors.

  14. Regulation of photoreceptor gene expression by Crx-associated transcription factor network

    PubMed Central

    Hennig, Anne K.; Peng, Guang-Hua; Chen, Shiming

    2008-01-01

    Rod and cone photoreceptors in the mammalian retina are special types of neurons that are responsible for phototransduction, the first step of vision. Development and maintenance of photoreceptors require precisely regulated gene expression. This regulation is mediated by a network of photoreceptor transcription factors centered on Crx, an Otx-like homeodomain transcription factor. The cell type (subtype) specificity of this network is governed by factors that are preferentially expressed by rods or cones or both, including the rod-determining factors neural retina leucine zipper protein (Nrl) and the orphan nuclear receptor Nr2e3; and cone-determining factors, mostly nuclear receptor family members. The best-documented of these include thyroid hormone receptor β2 (Trβ2), retinoid related orphan receptor Rorβ, and retinoid X receptor Rxrγ. The appropriate function of this network also depends on general transcription factors and co-factors that are ubiquitously expressed, such as the Sp zinc finger transcription factors and STAGA coactivator complexes. These cell type-specific and general transcription regulators form complex interactomes; mutations that interfere with any of the interactions can cause photoreceptor development defects or degeneration. In this manuscript, we review recent progress on the roles of various photoreceptor transcription factors and interactions in photoreceptor subtype development. We also provide evidence of auto-, para-, and feedback regulation among these factors at the transcriptional level. These protein-protein and protein-promoter interactions provide precision and specificity in controlling photoreceptor subtype-specific gene expression, development and survival. Understanding these interactions may provide insights to more effective therapeutic interventions for photoreceptor diseases. PMID:17662965

  15. Light-Evoked Responses of the Retinal Pigment Epithelium: Changes Accompanying Photoreceptor Loss in the Mouse

    PubMed Central

    Sturgill, Gwen M.; Grossman, Gregory H.; Rayborn, Mary E.; Hollyfield, Joe G.; Peachey, Neal S.

    2010-01-01

    Mutations in genes expressed in the retinal pigment epithelium (RPE) underlie a number of human inherited retinal disorders that manifest with photoreceptor degeneration. Because light-evoked responses of the RPE are generated secondary to rod photoreceptor activity, RPE response reductions observed in human patients or animal models may simply reflect decreased photoreceptor input. The purpose of this study was to define how the electrophysiological characteristics of the RPE change when the complement of rod photoreceptors is decreased. To measure RPE function, we used an electroretinogram (dc-ERG)-based technique. We studied a slowly progressive mouse model of photoreceptor degeneration (PrphRd2/+), which was crossed onto a Nyxnob background to eliminate the b-wave and most other postreceptoral ERG components. On this background, PrphRd2/+ mice display characteristic reductions in a-wave amplitude, which parallel those in slow PIII amplitude and the loss of rod photoreceptors. At 2 and 4 mo of age, the amplitude of each dc-ERG component (c-wave, fast oscillation, light peak, and off response) was larger in PrphRd2/+ mice than predicted by rod photoreceptor activity (RmP3) or anatomical analysis. At 4 mo of age, the RPE in PrphRd2/+ mice showed several structural abnormalities including vacuoles and swollen, hypertrophic cells. These data demonstrate that insights into RPE function can be gained despite a loss of photoreceptors and structural changes in RPE cells and, moreover, that RPE function can be evaluated in a broader range of mouse models of human retinal disease. PMID:20484527

  16. Cell sheet technology and cell patterning for biofabrication.

    PubMed

    Hannachi, Imen Elloumi; Yamato, Masayuki; Okano, Teruo

    2009-06-01

    We have developed cell sheet technology as a modern method for the fabrication of functional tissue-like and organ-like structures. This technology allows for a sheet of interconnected cells and cells in full contact with their natural extracellular environment to be obtained. A cell sheet can be patterned and composed according to more than one cell type. The key technology of cell sheet engineering is that a fabricated cell sheet can be harvested and transplanted utilizing temperature-responsive surfaces. In this review, we summarize different aspects of cell sheet engineering and provide a survey of the application of cell sheets as a suitable material for biofabrication and clinics. Moreover, since cell micropatterning is a key tool for cell sheet engineering, in this review we focus on the introduction of our approaches to cell micropatterning and cell co-culture to the principles of automation and how they can be subjected to easy robotics programming. Finally, efforts towards making cell sheet technology suitable for biofabrication and robotic biofabrication are also summarized.

  17. Targeting of GFP to newborn rods by Nrl promoter and temporal expression profiling of flow-sorted photoreceptors

    PubMed Central

    Akimoto, Masayuki; Cheng, Hong; Zhu, Dongxiao; Brzezinski, Joseph A.; Khanna, Ritu; Filippova, Elena; Oh, Edwin C. T.; Jing, Yuezhou; Linares, Jose-Luis; Brooks, Matthew; Zareparsi, Sepideh; Mears, Alan J.; Hero, Alfred; Glaser, Tom; Swaroop, Anand

    2006-01-01

    The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl−/− retinas, the GFP+ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP+ photoreceptors from wild-type and Nrl−/− retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies. PMID:16505381

  18. The locations of mitochondria in mammalian photoreceptors: relation to retinal vasculature.

    PubMed

    Stone, Jonathan; van Driel, Diana; Valter, Krisztina; Rees, Sandra; Provis, Jan

    2008-01-16

    Adult mammalian photoreceptors are elongated cells, and their mitochondria are sequestered to the ends of the cell, to the inner segments and (in some species) to axon terminals in the outer plexiform layer (OPL). We hypothesised that mitochondria migrate to these locations towards sources of oxygen, from the choroid and (in some species) from the deep capillaries of the retinal circulation. Six mammalian species were surveyed, using electron and light microscopy, including immunohistochemistry for the mitochondrial enzyme cytochrome oxidase (CO). In all 6 species, mitochondria were absent from photoreceptor somas and were numerous in inner segments. Mitochondria were prominent in axon terminals in 3 species (mouse, rat, human) with a retinal circulation and were absent from those terminals in 3 species (wallaby, rat, guinea pig) with avascular retinas. Further, in a human developmental series, it was evident that mitochondria migrate within rods and cones, towards and eventually past the outer limiting membrane (OLM), into the inner segment. In Müller and RPE cells also, mitochondria concentrated at the external surface of the cells. Neurones located in the inner layers of avascular retinas have mitochondria, but their expression of CO is low. Mitochondrial locations in photoreceptors, Müller and RPE cells are economically explained as the result of migration within the cell towards sources of oxygen. In photoreceptors, this migration results in a separation of mitochondria from the nuclear genome; this separation may be a factor in the vulnerability of photoreceptors to mutations, toxins and environmental stresses, which other retinal neurones survive.

  19. Regulation of photoreceptor gene expression by the retinal homeobox (Rx) gene product

    PubMed Central

    Pan, Yi; Martinez-De Luna, Reyna I.; Lou, Chih-Hong; Nekkalapudi, Srivamsi; Kelly, Lisa E.; Sater, Amy K.; El-Hodiri, Heithem M.

    2010-01-01

    The retinal homeobox (Rx) gene product is essential for eye development. However little is known about its molecular function. It has been demonstrated that Rx binds to photoreceptor conserved element (PCE-1), a highly conserved element found in the promoter region of photoreceptor-specific genes such as rhodopsin and red cone opsin. We verify that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx binds to the rhodopsin and red cone opsin promoters in vivo. We also find that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Finally, we demonstrate that reduction of Rx expression in tadpoles results in decreases in expression of several PCE-1 containing photoreceptor genes, abnormal photoreceptor morphology, and impaired vision. Our data suggests that Rx, in combination with other transcription factors, is necessary for normal photoreceptor gene expression, maintenance, and function. This establishes a direct role for Rx in regulation of genes expressed in a differentiated cell type. PMID:20060393

  20. Retinal remodeling triggered by photoreceptor degenerations.

    PubMed

    Jones, Bryan W; Watt, Carl B; Frederick, Jeanne M; Baehr, Wolfgang; Chen, Ching-Kang; Levine, Edward M; Milam, Ann H; Lavail, Matthew M; Marc, Robert E

    2003-09-08

    Many photoreceptor degenerations initially affect rods, secondarily leading to cone death. It has long been assumed that the surviving neural retina is largely resistant to this sensory deafferentation. New evidence from fast retinal degenerations reveals that subtle plasticities in neuronal form and connectivity emerge early in disease. By screening mature natural, transgenic, and knockout retinal degeneration models with computational molecular phenotyping, we have found an extended late phase of negative remodeling that radically changes retinal structure. Three major transformations emerge: 1) Müller cell hypertrophy and elaboration of a distal glial seal between retina and the choroid/retinal pigmented epithelium; 2) apparent neuronal migration along glial surfaces to ectopic sites; and 3) rewiring through evolution of complex neurite fascicles, new synaptic foci in the remnant inner nuclear layer, and new connections throughout the retina. Although some neurons die, survivors express molecular signatures characteristic of normal bipolar, amacrine, and ganglion cells. Remodeling in human and rodent retinas is independent of the initial molecular targets of retinal degenerations, including defects in the retinal pigmented epithelium, rhodopsin, or downstream phototransduction elements. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, it suggests that the neural retina may be more plastic than previously believed.

  1. Photoreceptor disc shedding in the living human eye

    PubMed Central

    Kocaoglu, Omer P.; Liu, Zhuolin; Zhang, Furu; Kurokawa, Kazuhiro; Jonnal, Ravi S.; Miller, Donald T.

    2016-01-01

    Cone photoreceptors undergo a daily cycle of renewal and shedding of membranous discs in their outer segments (OS), the portion responsible for light capture. These physiological processes are fundamental to maintaining photoreceptor health, and their dysfunction is associated with numerous retinal diseases. While both processes have been extensively studied in animal models and postmortem eyes, little is known about them in the living eye, in particular human. In this study, we report discovery of the optical signature associated with disc shedding using a method based on adaptive optics optical coherence tomography (AO-OCT) in conjunction with post-processing methods to track and monitor individual cone cells in 4D. The optical signature of disc shedding is characterized by an abrupt transient loss in the cone outer segment tip (COST) reflection followed by its return that is axially displaced anteriorly. Using this signature, we measured the temporal and spatial properties of shedding events in three normal subjects. Average duration of the shedding event was 8.8 ± 13.4 minutes, and average length loss of the OS was 2.1 μm (7.0% of OS length). Prevalence of cone shedding was highest in the morning (14.3%) followed by the afternoon (5.7%) and evening (4.0%), with load distributed across the imaged patch. To the best of our knowledge these are the first images of photoreceptor disc shedding in the living retina. PMID:27895995

  2. Adaptive potentiation in rod photoreceptors after light exposure.

    PubMed

    McKeown, Alex S; Kraft, Timothy W

    2014-06-01

    Photoreceptors adapt to changes in illumination by altering transduction kinetics and sensitivity, thereby extending their working range. We describe a previously unknown form of rod photoreceptor adaptation in wild-type (WT) mice that manifests as a potentiation of the light response after periods of conditioning light exposure. We characterize the stimulus conditions that evoke this graded hypersensitivity and examine the molecular mechanisms of adaptation underlying the phenomenon. After exposure to periods of saturating illumination, rods show a 10-35% increase in circulating dark current, an adaptive potentiation (AP) to light exposure. This potentiation grows as exposure to light is extended up to 3 min and decreases with longer exposures. Cells return to their initial dark-adapted sensitivity with a time constant of recovery of ∼7 s. Halving the extracellular Mg concentration prolongs the adaptation, increasing the time constant of recovery to 13.3 s, but does not affect the magnitude of potentiation. In rods lacking guanylate cyclase activating proteins 1 and 2 (GCAP(-/-)), AP is more than doubled compared with WT rods, and halving the extracellular Mg concentration does not affect the recovery time constant. Rods from a mouse expressing cyclic nucleotide-gated channels incapable of binding calmodulin also showed a marked increase in the amplitude of AP. Application of an insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitor (Tyrphostin AG1024) blocked AP, whereas application of an insulin receptor kinase inhibitor (HNMPA(AM)3) failed to do so. A broad-acting tyrosine phosphatase inhibitor (orthovanadate) also blocked AP. Our findings identify a unique form of adaptation in photoreceptors, so that they show transient hypersensitivity to light, and are consistent with a model in which light history, acting via the IGF-1R, can increase the sensitivity of rod photoreceptors, whereas the photocurrent overshoot is regulated by Ca-calmodulin and Ca(2

  3. Arap1 Deficiency Causes Photoreceptor Degeneration in Mice

    PubMed Central

    Moshiri, Ala; Humpal, Devin; Leonard, Brian C.; Imai, Denise M.; Tham, Addy; Bower, Lynette; Clary, Dave; Glaser, Thomas M.; Lloyd, K. C. Kent; Murphy, Christopher J.

    2017-01-01

    Purpose Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the control of GTPase-activating proteins (GAPs). Arap1 is an Arf-directed GAP that inhibits the trafficking of epidermal growth factor receptor (EGFR) to the early endosome, but the diversity of its functions is incompletely understood. The aim of this study was to determine the role of Arap1 in the mammalian retina. Methods Genetically engineered Arap1 knockout mice were screened for ocular abnormalities in the National Institutes of Health Knockout Mouse Production and Phenotyping (KOMP2) Project. Arap1 knockout and wild-type eyes were imaged using optical coherence tomography and fundus photography, and analyzed by immunohistochemistry. Results Arap1−/− mice develop a normal appearing retina, but undergo photoreceptor degeneration starting at 4 weeks postnatal age. The fundus appearance of mutants is notable for pigmentary changes, optic nerve pallor, vascular attenuation, and outer retinal thinning, reminiscent of retinitis pigmentosa in humans. Immunohistochemical studies suggest the cell death is predominantly in the outer nuclear layer. Functional evaluation of the retina by electroretinography reveals amplitudes are reduced. Arap1 is detected most notably in Müller glia, and not in photoreceptors, implicating a role for Müller glia in photoreceptor survival. Conclusions Arap1 is necessary for normal photoreceptor survival in mice, and may be a novel gene relevant to human retinal degenerative processes, although its mechanism is unknown. Further studies in this mouse model of retinal degeneration will give insights into the cellular functions and signaling pathways in which Arap1 participates. PMID:28324111

  4. Patterned Poly(dopamine) Films for Enhanced Cell Adhesion.

    PubMed

    Chen, Xi; Cortez-Jugo, Christina; Choi, Gwan H; Björnmalm, Mattias; Dai, Yunlu; Yoo, Pil J; Caruso, Frank

    2017-01-18

    Engineered materials that promote cell adhesion and cell growth are important in tissue engineering and regenerative medicine. In this work, we produced poly(dopamine) (PDA) films with engineered patterns for improved cell adhesion. The patterned films were synthesized via the polymerization of dopamine at the air-water interface of a floating bed of spherical particles. Subsequent dissolution of the particles yielded free-standing PDA films with tunable geometrical patterns. Our results show that these patterned PDA films significantly enhance the adhesion of both cancer cells and stem cells, thus showing promise as substrates for cell attachment for various biomedical applications.

  5. Structure and function of the photoreceptor stentorins in Stentor coeruleus. I. Partial characterization of the photoreceptor organelle and stentorins.

    PubMed

    Kim, I H; Rhee, J S; Huh, J W; Florell, S; Faure, B; Lee, K W; Kahsai, T; Song, P S; Tamai, N; Yamazaki, T

    1990-08-01

    The unicellular ciliary protozoan, Stentor coeruleus, exhibits photophobic and phototactic responses to visible light stimuli. The pigment granule contains the photoreceptor chromoproteins (stentorins). Stentorin localized in the pigment granules of the cell serves as the primary photoreceptor for the photophobic and phototactic responses in this organism. An initial characterization of the pigment granules has been described in terms of size, absorbance spectra and ATPase activity. Two forms of the stentorin pigments have been isolated from the pigment granules. Stentorin I has an apparent molecular weight of 68,600 and 52,000 by SDS-PAGE (at 10 and 13% gel, respectively) or 102,000 by steric exclusion HPLC, whereas stentorin II is a larger molecular assembly probably composed of several proteins (mol. wt. greater than 500,000). Stentorin I is composed of at least two heterologous subunits corresponding to apparent mol. wts. of 46,000 (fluorescent, Coomassie blue negative) and 52,000 (fluorescent, Coomassie blue positive) on SDS-PAGE (13% gel). However, these values were found to be strongly dependent on the degree of crosslinking in the acrylamide gel. Stentorin II appears to be the primary photoreceptor whose absorption and fluorescence properties are consistent with the action spectra for the photoresponses of the ciliate to visible light.

  6. The temporal topography of the N-Methyl- N-nitrosourea induced photoreceptor degeneration in mouse retina

    PubMed Central

    Tao, Ye; Chen, Tao; Fang, Wei; Peng, Guanghua; Wang, liqiang; Qin, Limin; Liu, Bei; Fei Huang, Yi

    2015-01-01

    Retinitis pigmentosa (RP) is a group of inherited neurodegenerative diseases characterized by the progressive photoreceptors apoptosis. The N-Methyl- N-nitrosourea (MNU) is an alkylating toxicant which could induce photoreceptor apoptosis resembling that of the hereditary RP. However, the detailed process pattern of this degeneration remains poorly characterized. We systemically explored the topography of the photoreceptor degeneration in the MNU treated mouse, and related these spatial data with the time-dependent characteristics of retinal pathology. These temporal topographic data delineated sequential scenes of the progressive photoreceptor degeneration in the MNU treated retinas: focal photoreceptors showed different vulnerabilities to the MNU toxicity and displayed a distinctive spatial- and time-dependent progression. Moreover, the positional asymmetry between the retinal quadrants firstly provided instructive information about the unique toxicology properties of the MNU. Further mechanism study suggested that the up-regulation of Bax and Calpain-2, rather than the Caspase-3, should be responsible for the asymmetry in the MNU induced photoreceptor degeneration. Together with the comparative sensitivities to the neurotoxicity of MNU between two photoreceptor populations, these topographic data would facilitate the standardization of analytic parameters related to the MNU induced RP model, and enhance its application in the therapeutic explorations of human RP. PMID:26685797

  7. In the Limelight: Photoreceptors in Cyanobacteria

    PubMed Central

    2016-01-01

    ABSTRACT Certain cyanobacteria look green if grown in red light and vice versa. This dramatic color change, called complementary chromatic adaptation (CCA), is caused by alterations of the major colored light-harvesting proteins. A major controller of CCA is the cyanobacteriochrome (CBCR) RcaE, a red-green reversible photoreceptor that triggers a complex signal transduction pathway. Now, a new study demonstrates that CCA is also modulated by DpxA, a CBCR that senses yellow and teal (greenish blue) light. DpxA acts to expand the range of wavelengths that can impact CCA, by fine-tuning the process. This dual control of CCA might positively impact the fitness of cells growing in the shade of competing algae or in a water column where light levels and spectral quality change gradually with depth. This discovery adds to the growing number of light-responsive phenomena controlled by multiple CBCRs. Furthermore, the diverse CBCRs which are exclusively found in cyanobacteria have significant biotechnological potential. PMID:27353763

  8. No evidence for a genetic blueprint: The case of the "complex" mammalian photoreceptor.

    PubMed

    Kumaramanickavel, G; Denton, M J; Legge, M

    2015-04-01

    Despite the intensity of the search for genes causing inherited retinal degenerations over the past 3 decades, of the approximately 200 disease genes identified to date, all appear to be ordinary housekeeping genes specifying proteins playing basic structural and functional roles in the mature photoreceptor cells. No genes or genetic elements have been identified which can be construed as having a specific morphogenic role, directing the development of the cytoarchitecture of any particular retinal cell. The evidence suggests that the cytoarchitecture of the retinal photoreceptors, although enormously complex, arises from the self-organization of the cells constituents without any regulation or direction from an external genetic blueprint.

  9. Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

    PubMed

    Beltran, William A; Cideciyan, Artur V; Lewin, Alfred S; Iwabe, Simone; Khanna, Hemant; Sumaroka, Alexander; Chiodo, Vince A; Fajardo, Diego S; Román, Alejandro J; Deng, Wen-Tao; Swider, Malgorzata; Alemán, Tomas S; Boye, Sanford L; Genini, Sem; Swaroop, Anand; Hauswirth, William W; Jacobson, Samuel G; Aguirre, Gustavo D

    2012-02-07

    Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

  10. Knockdown of poc1b causes abnormal photoreceptor sensory cilium and vision impairment in zebrafish.

    PubMed

    Zhang, Conghui; Zhang, Qi; Wang, Fang; Liu, Qin

    2015-10-02

    Proteomic analysis of the mouse photoreceptor sensory cilium identified a set of cilia proteins, including Poc1 centriolar protein b (Poc1b). Previous functional studies in human cells and zebrafish embryos implicated that Poc1b plays important roles in centriole duplication and length control, as well as ciliogenesis. To study the function of Poc1b in photoreceptor sensory cilia and other primary cilia, we expressed a tagged recombinant Poc1b protein in cultured renal epithelial cells and rat retina. Poc1b was localized to the centrioles and spindle bundles during cell cycle progression, and to the basal body of photoreceptor sensory cilia. A morpholino knockdown and complementation assay of poc1b in zebrafish showed that loss of poc1b led to a range of morphological anomalies of cilia commonly associated with human ciliopathies. In the retina, the development of retinal laminae was significantly delayed and the length of photoreceptor outer segments was shortened. Visual behavior studies revealed impaired visual function in the poc1b morphants. In addition, ciliopathy-associated developmental defects, such as small eyes, curved body axis, heart defects, and shortened cilia in Kupffer's vesicle, were observed as well. These data suggest that poc1b is required for normal development and ciliogenesis of retinal photoreceptor sensory cilia and other cilia. Furthermore, this conclusion is supported by recent findings that mutations in POC1B gene have been identified in patients with inherited retinal dystrophy and syndromic retinal ciliopathy.

  11. Genomic and gene regulatory signatures of cryptozoic adaptation: Loss of blue sensitive photoreceptors through expansion of long wavelength-opsin expression in the red flour beetle Tribolium castaneum

    PubMed Central

    Jackowska, Magdalena; Bao, Riyue; Liu, Zhenyi; McDonald, Elizabeth C; Cook, Tiffany A; Friedrich, Markus

    2007-01-01

    Background Recent genome sequence analysis in the red flour beetle Tribolium castaneum indicated that this highly crepuscular animal encodes only two single opsin paralogs: a UV-opsin and a long wavelength (LW)-opsin; however, these animals do not encode a blue (B)-opsin as most other insects. Here, we studied the spatial regulation of the Tribolium single LW- and UV-opsin gene paralogs in comparison to that of the five opsin paralogs in the retina of Drosophila melanogaster. Results In situ hybridization analysis reveals that the Tribolium retina, in contrast with other insect retinas, constitutes a homogenous field of ommatidia that have seven LW-opsin expressing photoreceptors and one UV-/LW-opsin co-expressing photoreceptor per eye unit. This pattern is consistent with the loss of photoreceptors sensitive to blue wavelengths. It also identifies Tribolium as the first example of a species in insects that co-expresses two different opsins across the entire retina in violation of the widely observed "one receptor rule" of sensory cells. Conclusion Broader studies of opsin evolution in darkling beetles and other coleopteran groups have the potential to pinpoint the permissive and adaptive forces that played a role in the evolution of vision in Tribolium castaneum. PMID:18154648

  12. A Versatile Method of Patterning Proteins and Cells.

    PubMed

    Shrirao, Anil B; Kung, Frank H; Yip, Derek; Firestein, Bonnie L; Cho, Cheul H; Townes-Anderson, Ellen

    2017-02-26

    Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

  13. Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish

    PubMed Central

    Song, Ping; Dudinsky, Lynn; Fogerty, Joseph; Gaivin, Robert; Perkins, Brian D.

    2016-01-01

    Purpose Mutations in the gene ARL13B cause the classical form of Joubert syndrome, an autosomal recessive ciliopathy with variable degrees of retinal degeneration. As second-site modifier alleles can contribute to retinal pathology in ciliopathies, animal models provide a unique platform to test how genetic interactions modulate specific phenotypes. In this study, we analyzed the zebrafish arl13b mutant for retinal degeneration and for epistatic relationships with the planar cell polarity protein (PCP) component vangl2. Methods Photoreceptor and cilia structure was examined by light and electron microscopy. Immunohistochemistry was performed to examine ciliary markers. Genetic interactions were tested by pairwise crosses of heterozygous animals. Genetic mosaic animals were generated by blastula transplantation and analyzed by fluorescence microscopy. Results At 5 days after fertilization, photoreceptor outer segments were shorter in zebrafish arl13b−/− mutants compared to wild-type larvae, no overt signs of retinal degeneration were observed by light or electron microscopy. Starting at 14 days after fertilization (dpf) and continuing through 30 dpf, cells lacking Arl13b died following transplantation into wild-type host animals. Photoreceptors of arl13b−/−;vangl2−/− mutants were more compromised than the photoreceptors of single mutants. Finally, when grown within a wild-type retina, the vangl2−/− mutant cone photoreceptors displayed normal basal body positioning. Conclusions We show that arl13b−/− mutants have shortened cilia and photoreceptor outer segments and exhibit a slow, progressive photoreceptor degeneration that occurs over weeks. The data suggest that loss of Arl13b leads to slow photoreceptor degeneration, but can be exacerbated by the loss of vangl2. Importantly, the data show that Arl13b can genetically and physically interact with Vangl2 and this association is important for normal photoreceptor structure. The loss of vangl2

  14. Regulation of photoreceptor gene transcription via a highly conserved transcriptional regulatory element by vsx gene products

    PubMed Central

    Pan, Yi; Comiskey, Daniel F.; Kelly, Lisa E.; Chandler, Dawn S.

    2016-01-01

    Purpose The photoreceptor conserved element-1 (PCE-1) sequence is found in the transcriptional regulatory regions of many genes expressed in photoreceptors. The retinal homeobox (Rx or Rax) gene product functions by binding to PCE-1 sites. However, other transcriptional regulators have also been reported to bind to PCE-1. One of these, vsx2, is expressed in retinal progenitor and bipolar cells. The purpose of this study is to identify Xenopus laevis vsx gene products and characterize vsx gene product expression and function with respect to the PCE-1 site. Methods X. laevis vsx gene products were amplified with PCR. Expression patterns were determined with in situ hybridization using whole or sectioned X. laevis embryos and digoxigenin- or fluorescein-labeled antisense riboprobes. DNA binding characteristics of the vsx gene products were analyzed with electrophoretic mobility shift assays (EMSAs) using in vitro translated proteins and radiolabeled oligonucleotide probes. Gene transactivation assays were performed using luciferase-based reporters and in vitro transcribed effector gene products, injected into X. laevis embryos. Results We identified one vsx1 and two vsx2 gene products. The two vsx2 gene products are generated by alternate mRNA splicing. We verified that these gene products are expressed in the developing retina and that expression resolves into distinct cell types in the mature retina. Finally, we found that vsx gene products can bind the PCE-1 site in vitro and that the two vsx2 isoforms have different gene transactivation activities. Conclusions vsx gene products are expressed in the developing and mature neural retina. vsx gene products can bind the PCE-1 site in vitro and influence the expression of a rhodopsin promoter-luciferase reporter gene. The two isoforms of vsx have different gene transactivation activities in this reporter gene system. PMID:28003732

  15. Photoreceptor waveguides and effective retinal image quality

    NASA Astrophysics Data System (ADS)

    Vohnsen, Brian

    2007-03-01

    Individual photoreceptor waveguiding suggests that the entire retina can be considered as a composite fiber-optic element relating a retinal image to a corresponding waveguided image. In such a scheme, a visual sensation is produced only when the latter interacts with the pigments of the outer photoreceptor segments. Here the possible consequences of photoreceptor waveguiding on vision are studied with important implications for the pupil-apodization method commonly used to incorporate directional effects of the retina. In the absence of aberrations, it is found that the two approaches give identical predictions for an effective retinal image only when the pupil apodization is chosen twice as narrow as suggested by the traditional Stiles-Crawford effect. In addition, phase variations in the retinal field due to ocular aberrations can delicately alter a waveguided image, and this may provide plausible justification for an improved visual sensation as compared with what should be expected on the grounds of a retinal image only.

  16. Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function

    PubMed Central

    Jiang, Li; Tam, Beatrice M.; Ying, Guoxing; Wu, Sen; Hauswirth, William W.; Frederick, Jeanne M.; Moritz, Orson L.; Baehr, Wolfgang

    2015-01-01

    In Caenorhabditis elegans, homodimeric [kinesin family (KIF) 17, osmotic avoidance abnormal-3 (OSM-3)] and heterotrimeric (KIF3) kinesin-2 motors are required to establish sensory cilia by intraflagellar transport (IFT) where KIF3 and KIF17 cooperate to build the axoneme core and KIF17 builds the distal segments. However, the function of KIF17 in vertebrates is unresolved. We expressed full-length and motorless KIF17 constructs in mouse rod photoreceptors using adeno-associated virus in Xenopus laevis rod photoreceptors using a transgene and in ciliated IMCD3 cells. We found that tagged KIF17 localized along the rod outer segment axoneme when expressed in mouse and X. laevis photoreceptors, whereas KIF3A was restricted to the proximal axoneme. Motorless KIF3A and KIF17 mutants caused photoreceptor degeneration, likely through dominant negative effects on IFT. KIF17 mutant lacking the motor domain translocated to nuclei after exposure of a C-terminal nuclear localization signal. Germ-line deletion of Kif17 in mouse did not affect photoreceptor function. A rod-specific Kif3/Kif17 double knockout mouse demonstrated that KIF17 and KIF3 do not act synergistically and did not prevent rhodopsin trafficking to rod outer segments. In summary, the nematode model of KIF3/KIF17 cooperation apparently does not apply to mouse photoreceptors in which the photosensory cilium is built exclusively by KIF3.—Jiang, L., Tam, B. M., Ying, G., Wu, S., Hauswirth, W. W., Frederick, J. M., Moritz, O. L., Baehr, W. Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function. PMID:26229057

  17. Bestrophinopathy: An RPE-photoreceptor interface disease.

    PubMed

    Guziewicz, Karina E; Sinha, Divya; Gómez, Néstor M; Zorych, Kathryn; Dutrow, Emily V; Dhingra, Anuradha; Mullins, Robert F; Stone, Edwin M; Gamm, David M; Boesze-Battaglia, Kathleen; Aguirre, Gustavo D

    2017-01-19

    Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

  18. The Retina of Ansorge's Cusimanse (Crossarchus ansorgei): Number, Topography and Convergence of Photoreceptors and Ganglion Cells in Relation to Ecology and Behavior.

    PubMed

    Coimbra, João Paulo; Kaswera-Kyamakya, Consolate; Gilissen, Emmanuel; Manger, Paul R; Collin, Shaun P

    2015-01-01

    The family Herpestidae (cusimanses and mongooses) is a monophyletic radiation of carnivores with remarkable variation in microhabitat occupation and diel activity, but virtually nothing is known about how they use vision in the context of their behavioral ecology. In this paper, we measured the number and topographic distribution of neurons (rods, cones and retinal ganglion cells) and estimated the spatial resolving power of the eye of the diurnal, forest-dwelling Ansorge's cusimanse (Crossarchus ansorgei). Using retinal wholemounts and stereology, we found that rods are more numerous (42,500,000; 92%) than cones (3,900,000; 8%). Rod densities form a concentric and dorsotemporally asymmetric plateau that matches the location and shape of a bright yellow tapetum lucidum located within the dorsal aspect of the eye. Maximum rod density (340,300 cells/mm(2)) occurs within an elongated plateau below the optic disc that corresponds to a transitional region between the tapetum lucidum and the pigmented choroid. Cone densities form a temporal area with a peak density of 44,500 cells/mm(2) embedded in a weak horizontal streak that matches the topographic distribution of retinal ganglion cells. Convergence ratios of cones to retinal ganglion cells vary from 50:1 in the far periphery to 3:1 in the temporal area. With a ganglion cell peak density of 13,400 cells/mm(2) and an eye size of 11 mm in axial length, we estimated upper limits of spatial resolution of 7.5-8 cycles/degree, which is comparable to other carnivores such as hyenas. In conclusion, we suggest that the topographic retinal traits described for Ansorge's cusimanse conform to a presumed carnivore retinal blueprint but also show variations that reflect its specific ecological needs.

  19. Lens Injury Has a Protective Effect on Photoreceptors in the RCS Rat.

    PubMed

    Heiduschka, Peter; Renninger, Daniel; Fischer, Dietmar; Müller, Adrienne; Hofmeister, Sabine; Schraermeyer, Ulrich

    2013-01-01

    Lens injury induced activation of retinal glia, and subsequent release of ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF) potently protect axotomised retinal ganglion cells from apoptosis and promotes axon regeneration in the injured optic nerve. The goal of the current study was to investigate if similar effects may also be applicable to rescue photoreceptors from degeneration in a model of retinitis pigmentosa. Lens injury was performed in the Royal College of Surgeons (RCS) rats at the age of one month. The survival of photoreceptors was evaluated histologically, and retinal function was analysed by electroretinography (ERG). Expression of CNTF was also analysed. Lens injury significantly enhanced the survival of photoreceptors 1 month after surgery compared to untreated controls, which was associated with an enhanced ERG response. In addition, lens injury significantly protected photoreceptors from degeneration in the contralateral eye, although to a much lesser extent. We could show that lens injury is sufficient to transiently delay the degeneration of photoreceptors in the RCS rat. The observed neuroprotective effects may be at least partially mediated by an upregulation of CNTF expression seen after lens injury.

  20. Use of Hydrogen as a Novel Therapeutic Strategy Against Photoreceptor Degeneration in Retinitis Pigmentosa Patients.

    PubMed

    Tao, Ye; Geng, Lei; Wang, Liqiang; Xu, Weiwei; Qin, Limin; Peng, Guanghua; Huang, Yi Fei; Yang, Ji xue

    2016-03-08

    Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterized by progressive photoreceptor apoptosis. Reactive oxygen species (ROS) have been recognized as critical initiators of the photoreceptor apoptosis in RP. Photoreceptor survival in RP mutants will not only require the inhibition of effectors of apoptotic machinery, but also the elimination of the initiating upstream signals, such as ROS. These cytotoxic ROS should be neutralized by the antioxidant defense system, otherwise they would interact with the macromolecules essential for photoreceptor survival. Hydrogen is a promising gaseous agent that has come to the forefront of therapeutic research over the last few years. It has been verified that hydrogen is capable of neutralizing the cytotoxic ROS selectively, rectifying abnormities in the apoptotic cascades, and attenuating the related inflammatory response. Hydrogen is so mild that it does not disturb the metabolic oxidation-reduction reactions or disrupt the physiologic ROS involved in cell signaling. Based on these findings, we hypothesize that hydrogen might be an effective therapeutic agent to slow or prevent photoreceptor degeneration in RP retinas. It is a logical step to test hydrogen for therapeutic use in multiple RP animal models, and ultimately in human RP patients.

  1. Photoreceptors mapping from past history till date.

    PubMed

    Parihar, Parul; Singh, Rachana; Singh, Samiksha; Tripathi, Durgesh Kumar; Chauhan, Devendra Kumar; Singh, Vijay Pratap; Prasad, Sheo Mohan

    2016-09-01

    The critical source of information in plants is light, which is perceived by receptors present in plants and animals. Receptors present in plant and animal system regulate important processes, and knowing the chromophores and signalling domains for each receptor could pave a way to trace out links between these receptors. The signalling mechanism for each receptor will give insight knowledge. This review has focussed on the photoreceptors from past history till date, that have evolved in the plant as well as in the animal system (to lesser extent). We have also focussed our attention on finding the links between the receptors by showing the commonalities as well as the differences between them, and also tried to trace out the links with the help of chromophores and signalling domain. Several photoreceptors have been traced out, which share similarity in the chromophore as well as in the signalling domain, which indicate towards the evolution of photoreceptors from one another. For instance, cryptochrome has been found to evolve three times from CPD photolyase as well as evolution of different types of phytochrome is a result of duplication and divergence. In addition, similarity between the photoreceptors suggested towards evolution from one another. This review has also discussed possible mechanism for each receptor i.e. how they regulate developmental processes and involve what kinds of regulators and also gives an insight on signalling mechanisms by these receptors. This review could also be a new initiative in the study of UVR8 associated studies.

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

    PubMed

    Cao, Dingcai; Barrionuevo, Pablo A

    2015-03-01

    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). 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

  3. Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness

    PubMed Central

    Kmoch, S.; Majewski, J.; Ramamurthy, V.; Cao, S.; Fahiminiya, S.; Ren, H.; MacDonald, I.M.; Lopez, I.; Sun, V.; Keser, V.; Khan, A.; Stránecký, V.; Hartmannová, H.; Přistoupilová, A.; Hodaňová, K.; Piherová, L.; Kuchař, L.; Baxová, A.; Chen, R.; Barsottini, O.G.P.; Pyle, A.; Griffin, H.; Splitt, M.; Sallum, J.; Tolmie, J.L.; Sampson, J.R.; Chinnery, P.; Canada, Care4Rare; Banin, E.; Sharon, D.; Dutta, S.; Grebler, R.; Helfrich-Foerster, C.; Pedroso, J.L.; Kretzschmar, D.; Cayouette, M.; Koenekoop, R.K.

    2015-01-01

    Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photo-receptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness. PMID:25574898

  4. Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities

    PubMed Central

    Altimus, C.M.; Güler, A.D.; Alam, N.M.; Arman, A.C.; Prusky, G.T.; Sampath, A.P.; Hattar, S

    2010-01-01

    In mammals, synchronization of the circadian pacemaker in the hypothalamus is achieved through direct input from the eyes conveyed by intrinsically photosensitive retinal ganglion cells (ipRGCs). Circadian photoentrainment can be maintained by rod and cone photoreceptors, but their functional contributions and their retinal circuits that impinge on ipRGCs are not well understood. We demonstrate in genetic mouse models lacking functional rods, or where rods are the only functional photoreceptors, that rods are solely responsible for photoentrainment at scotopic light intensities. Surprisingly, rods were also capable of driving circadian photoentrainment at photopic intensities where they were incapable of supporting a visually–guided behavior. Using animals in which cone photoreceptors were ablated, we demonstrate that rods signal through cones at high light intensities, but not low light intensities. Thus two distinct retinal circuits drive ipRGC function to support circadian photoentrainment across a wide range of light intensities. PMID:20711184

  5. AAV Vectors for FRET-Based Analysis of Protein-Protein Interactions in Photoreceptor Outer Segments

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong N. P.; Riedmayr, Lisa M.; Hammelmann, Verena; Schön, Christian; Butz, Elisabeth S.; Wahl-Schott, Christian; Biel, Martin; Michalakis, Stylianos

    2016-01-01

    Fluorescence resonance energy transfer (FRET) is a powerful method for the detection and quantification of stationary and dynamic protein-protein interactions. Technical limitations have hampered systematic in vivo FRET experiments to study protein-protein interactions in their native environment. Here, we describe a rapid and robust protocol that combines adeno-associated virus (AAV) vector-mediated in vivo delivery of genetically encoded FRET partners with ex vivo FRET measurements. The method was established on acutely isolated outer segments of murine rod and cone photoreceptors and relies on the high co-transduction efficiency of retinal photoreceptors by co-delivered AAV vectors. The procedure can be used for the systematic analysis of protein-protein interactions of wild type or mutant outer segment proteins in their native environment. Conclusively, our protocol can help to characterize the physiological and pathophysiological relevance of photoreceptor specific proteins and, in principle, should also be transferable to other cell types. PMID:27516733

  6. Förster resonance energy transfer as a tool to study photoreceptor biology

    NASA Astrophysics Data System (ADS)

    Hovan, Stephanie C.; Howell, Scott; Park, Paul S.-H.

    2010-11-01

    Vision is initiated in photoreceptor cells of the retina by a set of biochemical events called phototransduction. These events occur via coordinated dynamic processes that include changes in secondary messenger concentrations, conformational changes and post-translational modifications of signaling proteins, and protein-protein interactions between signaling partners. A complete description of the orchestration of these dynamic processes is still unavailable. Described in this work is the first step in the development of tools combining fluorescent protein technology, Förster resonance energy transfer (FRET), and transgenic animals that have the potential to reveal important molecular insights about the dynamic processes occurring in photoreceptor cells. We characterize the fluorescent proteins SCFP3A and SYFP2 for use as a donor-acceptor pair in FRET assays, which will facilitate the visualization of dynamic processes in living cells. We also demonstrate the targeted expression of these fluorescent proteins to the rod photoreceptor cells of Xenopus laevis, and describe a general method for detecting FRET in these cells. The general approaches described here can address numerous types of questions related to phototransduction and photoreceptor biology by providing a platform to visualize dynamic processes in molecular detail within a native context.

  7. Directionality of Individual Cone Photoreceptors in the Parafoveal Region

    PubMed Central

    Morris, Hugh J.; Blanco, Leonardo; Codona, Johanan L.; Li, Simone; Choi, Stacey S.; Doble, Nathan

    2015-01-01

    The pointing direction of cone photoreceptors can be inferred from the Stiles-Crawford Effect of the First Kind (SCE-I) measurement. Healthy retinas have tightly packed cones with a SCE-I function peak either centered in the pupil or with a slight nasal bias. Various retinal pathologies can change the profile of the SCE-I function implying that the arrangement or the light capturing properties of the cone photoreceptors are affected. Measuring the SCE-I may reveal early signs of photoreceptor change before actual cell apoptosis occurs. In vivo retinal imaging with adaptive optics (AO) was used to measure the pointing direction of individual cones at eight retinal locations in four control human subjects. Retinal images were acquired by translating an aperture in the light delivery arm through 19 different locations across a subject’s entrance pupil. Angular tuning properties of individual cones were calculated by fitting a Gaussian to the reflected intensity profile of each cone projected onto the pupil. Results were compared to those from an accepted psychophysical SCE-I measurement technique. The maximal difference in cone directionality of an ensemble of cones, ρ̄, between the major and minor axes of the Gaussian fit was 0.05 versus 0.29 mm−2 in one subject. All four subjects were found to have a mean nasal bias of 0.81 mm with a standard deviation of ±0.30 mm in the peak position at all retinal locations with mean ρ̄ value decreasing by 23% with increasing retinal eccentricity. Results show that cones in the parafoveal region converge towards the center of the pupillary aperture, confirming the anterior pointing alignment hypothesis. PMID:26494187

  8. Examination of VLC-PUFA–Deficient Photoreceptor Terminals

    PubMed Central

    Bennett, Lea D.; Hopiavuori, Blake R.; Brush, Richard S.; Chan, Michael; Van Hook, Matthew J.; Thoreson, Wallace B.; Anderson, Robert E.

    2014-01-01

    Purpose. Juvenile-onset autosomal dominant Stargardt-like macular dystrophy (STGD3) is caused by mutations in ELOVL4 (elongation of very long fatty acids-4), an elongase necessary for the biosynthesis of very long chain fatty acids (VLC-FAs ≥ C26). Photoreceptors are enriched with VLC polyunsaturated fatty acids (VLC-PUFAs), which are necessary for long-term survival of rod photoreceptors. The purpose of these studies was to determine the effect of deletion of VLC-PUFAs on rod synaptic function in retinas of mice conditionally depleted (KO) of Elovl4. Methods. Retina function was assessed in wild-type (WT) and KO by electroretinography. Outer plexiform structure was evaluated by immunofluorescence and transmission electron microscopy. Single-cell recordings measured rod ion channel operation and rod bipolar glutamate signaling. Sucrose gradient centrifugation was used to isolate synaptosomes from bovine retina. Proteins and lipids were analyzed by Western blotting and tandem mass spectroscopy, respectively. Results. Inner retinal responses (b-wave, oscillatory potentials, and scotopic threshold responses) of the ERG were decreased in the KO mice compared to controls. However the rod ion channel operation and bipolar glutamate responses were comparable between groups. Biochemical analysis revealed that conventional and ribbon synapses have VLC-PUFAs. Ultrastructural analysis showed that the outer plexiform layer was disorganized and the diameter of vesicles in rod terminals was smaller in the KO mice. Conclusions. Very long chain PUFAs affect rod function by contributing to synaptic vesicle size, which may alter the dynamics of synaptic transmission, ultimately resulting in a loss of neuronal connectivity and death of rod photoreceptors. PMID:24764063

  9. Photoreceptor types and distributions in the retinae of insectivores.

    PubMed

    Peichl, L; Künzle, H; Vogel, P

    2000-01-01

    The retinae of insectivores have been rarely studied, and their photoreceptor arrangements and expression patterns of visual pigments are largely unknown. We have determined the presence and distribution of cones in three species of shrews (common shrew Sorex araneus, greater white-toothed shrew Crocidura russula, dark forest shrew Crocidura poensis; Soricidae) and in the lesser hedgehog tenrec Echinops telfairi (Tenrecidae). Special cone types were identified and quantified in flattened whole retinae by antisera/antibodies recognizing the middle-to-long-wavelength-sensitive (M/L-)cone opsin and the short-wavelength-sensitive (S-)cone opsin, respectively. A combination of immunocytochemistry with conventional histology was used to assess rod densities and cone/rod ratios. In all four species the rods dominate at densities of about 230,000-260,000/mm2. M/L- and S-cones are present, comprising between 2% of the photoreceptors in the nocturnal Echinops telfairi and 13% in Sorex araneus that has equal diurnal and nocturnal activity phases. This suggests dichromatic color vision like in many other mammals. A striking feature in all four species are dramatically higher S-cone proportions in ventral than in dorsal retina (0.5% vs. 2.5-12% in Sorex, 5-15% vs. 30-45% in Crocidura poensis, 3-12% vs. 20-50% in Crocidura russula, 10-30% vs. 40-70% in Echinops). The functional and comparative aspects of these structural findings are discussed.

  10. Cluap1 is Essential for Ciliogenesis and Photoreceptor Maintenance in the Vertebrate Eye

    PubMed Central

    Lee, Chanjae; Wallingford, John B.; Gross, Jeffrey M.

    2014-01-01

    Purpose. To identify the mutation and cell biological underpinnings of photoreceptor defects in zebrafish au5 mutants. Methods. Whole genome sequencing and SNP mapping were used to determine the genomic interval that harbors the au5 mutation. A candidate mutation was cloned and sequenced, and mRNA rescue used to validate that the affected gene was correctly identified. In situ hybridization, immunohistochemistry, and confocal imaging were used to determine the effects on photoreceptor development and maintenance in mutant retinae, and to determine if ciliogenesis or cilia-dependent development was affected in mutant embryos. Expression of tagged proteins and high-speed in vivo confocal imaging was used to quantify intraflagellar transport (IFT) and IFT particle localization within multiciliated cells of the Xenopus epidermis. Results. The au5 mutants possess a nonsense mutation in cluap1, which encodes a component of the IFT machinery. Photoreceptor defects result from degeneration of photoreceptors, and defects in ciliogenesis precede degeneration. Cilia in the olfactory pit are absent, and left-right heart positioning is aberrant, consistent with a role for cluap1 during ciliogenesis and cilia-dependent development. High-speed in vivo imaging demonstrates that cluap1 undergoes IFT and that it moves along the cilium bidirectionally, with similar localization and kinetics as IFT20, an IFT-B complex component. Conclusions. We identified a novel mutation in cluap1 and determined that photoreceptor maintenance is dependent on cluap1. Imaging data support a model in which cluap1 is a component of the IFT-B complex, and cilia formation requires cluap1 function. These data may provide new insights into the mechanism of photoreceptor degeneration in retinal ciliopathies. PMID:24970261

  11. Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in Bardet–Biedl syndrome

    PubMed Central

    Datta, Poppy; Allamargot, Chantal; Hudson, Joseph S.; Andersen, Emily K.; Bhattarai, Sajag; Drack, Arlene V.; Sheffield, Val C.; Seo, Seongjin

    2015-01-01

    Compartmentalization and polarized protein trafficking are essential for many cellular functions. The photoreceptor outer segment (OS) is a sensory compartment specialized for phototransduction, and it shares many features with primary cilia. As expected, mutations disrupting protein trafficking to cilia often disrupt protein trafficking to the OS and cause photoreceptor degeneration. Bardet–Biedl syndrome (BBS) is one of the ciliopathies associated with defective ciliary trafficking and photoreceptor degeneration. However, precise roles of BBS proteins in photoreceptor cells and the underlying mechanisms of photoreceptor degeneration in BBS are not well understood. Here, we show that accumulation of non-OS proteins in the OS underlies photoreceptor degeneration in BBS. Using a newly developed BBS mouse model [Leucine zipper transcription factor-like 1 (Lztfl1)/Bbs17 mutant], isolated OSs, and quantitative proteomics, we determined 138 proteins that are enriched more than threefold in BBS mutant OS. In contrast, only eight proteins showed a more than threefold reduction. We found striking accumulation of Stx3 and Stxbp1/Munc18-1 and loss of polarized localization of Prom1 within the Lztfl1 and Bbs1 mutant OS. Ultrastructural analysis revealed that large vesicles are formed in the BBS OS, disrupting the lamellar structure of the OS. Our findings suggest that accumulation (and consequent sequestration) of non-OS proteins in the OS is likely the primary cause of photoreceptor degeneration in BBS. Our data also suggest that a major function of BBS proteins in photoreceptors is to transport proteins from the OS to the cell body or to prevent entry of non-OS proteins into the OS. PMID:26216965

  12. Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival

    PubMed Central

    Rajala, Ammaji; Wang, Yuhong; Rajala, Raju V.S.

    2016-01-01

    In humans, daylight vision is primarily mediated by cone photoreceptors. These cells die in age-related retinal degenerations. Prolonging the life of cones for even one decade would have an enormous beneficial effect on usable vision in an aging population. Photoreceptors are postmitotic, but shed 10% of their outer segments daily, and must synthesize the membrane and protein equivalent of a proliferating cell each day. Although activation of oncogenic tyrosine kinase and inhibition of tyrosine phosphatase signaling is known to be essential for tumor progression, the cellular regulation of this signaling in postmitotic photoreceptor cells has not been studied. In the present study, we report that a novel G-protein coupled receptor–mediated insulin receptor (IR) signaling pathway is regulated by non-receptor tyrosine kinase Src through the inhibition of protein tyrosine phosphatase IB (PTP1B). We demonstrated the functional significance of this pathway through conditional deletion of IR and PTP1B in cones, in addition to delaying the death of cones in a mouse model of cone degeneration by activating the Src. This is the first study demonstrating the molecular mechanism of a novel signaling pathway in photoreceptor cells, which provides a window of opportunity to save the dying cones in retinal degenerative diseases. PMID:27391439

  13. The Par-PrkC Polarity Complex Is Required for Cilia Growth in Zebrafish Photoreceptors

    PubMed Central

    Krock, Bryan L.; Perkins, Brian D.

    2014-01-01

    Specification and development of the apical membrane in epithelial cells requires the function of polarity proteins, including Pard3 and an atypical protein kinase C (PrkC). Many epithelial cells possess microtubule-based organelles, known as cilia, that project from their apical surface and the membrane surrounding the cilium is contiguous with the apical cell membrane. Although cilia formation in cultured cells required Pard3, the in vivo requirement for Pard3 in cilia development remains unknown. The vertebrate photoreceptor outer segment represents a highly specialized cilia structure in which to identify factors necessary for apical and ciliary membrane formation. Pard3 and PrkC localized to distinct domains within vertebrate photoreceptors. Using partial morpholino knockdown, photo-morpholinos, and pharmacological approaches, the function of Pard3 and PrkC were found to be required for the formation of both the apical and ciliary membrane of vertebrate photoreceptors. Inhibition of Pard3 or PrkC activity significantly reduced the size of photoreceptor outer segments and resulted in mislocalization of rhodopsin. Suppression of Pard3 or PrkC also led to a reduction in cilia size and cilia number in Kupffer’s Vesicle, which resulted in left-right asymmetry defects. Thus, the Par-PrkC complex functions in cilia formation in vivo and this likely reflects a general role in specifying non-ciliary and ciliary compartments of the apical domain. PMID:25144710

  14. Visual Responses of Photoreceptor-Degenerated Rats Expressing Two Different Types of Channelrhodopsin Genes

    PubMed Central

    Sato, Masatoshi; Sugano, Eriko; Tabata, Kitako; Sannohe, Kei; Watanabe, Yoshito; Ozaki, Taku; Tamai, Makoto; Tomita, Hiroshi

    2017-01-01

    Optogenetic technologies are expected to be applicable for clinical use in restoring vision. However, the degree of recovered visual function is highly dependent on the function of the chosen optogenetic gene. To investigate the effect on visual function of dual expression of genes with different wavelength sensitivities, we transduced a modified Volvox-derived channelrhodopsin gene (mVChR1) via an adeno-associated virus vector into transgenic rats harbouring the ChR2 gene in retinal ganglion cells. These transgenic rats were given an intraperitoneal injection of N-methyl-N-nitrosourea to induce the degeneration of native photoreceptor cells prior to transduction of mVChR1. Optical coherence tomography images indicated the degeneration of the native photoreceptor cells after the N-methyl-N-nitrosourea injection. Complete loss of function of the native photoreceptor cells was confirmed using electroretinograms. In the ChR2 transgenic rats, visually evoked potentials were clearly detectable in spite of native photoreceptor function abolishment; however the responses were limited to within blue wavelengths. In contrast, the limited wavelength sensitivities were improved by the additional transduction of mVChR1, which exhibited sensitivities to green and red. Thus, the transductions of dual genes encoding channelrhodopsins that exhibit different wavelength sensitivities represents a promising candidate method to expand and to enhance rescued wavelength sensitivities in blind subjects. PMID:28112267

  15. Cell patterning on poly(sodium 4-styrenesulfonate)-patterned fluoropolymer substrate

    NASA Astrophysics Data System (ADS)

    Kim, Wan-Joong; Jung, Chang-Hee; Hwang, In-Tae; Jung, Chan-Hee; Choi, Jae-Hak; Hong, Sung-Kwon

    2013-10-01

    The surface functionalization of bio-inert fluoropolymer films through ion beam-induced surface graft polymerization was investigated to control the cellular behavior. The surface of poly(tetrafluoroethylene-co-perfluoropropl vinyl ether) (PFA) films was selectively activated by 150 keV H+ ion implantation in the presence of a pattern mask and sodium 4-styrenesulfonate (SS) was then graft polymerized onto the implanted PFA films to form hydrophilic poly(sodium 4-styrenesulfonate) (PSS)-patterned PFA films. The surface of the resulting PSS-patterned PFA films was investigated in terms of the degree of graft polymerization, chemical structure, chemical composition, wettability, and morphology. The analytical results revealed that PSS was selectively grafted onto the implanted regions of the PFA films. Furthermore, in vitro cell culture on the PSS-patterned PFA films exhibited a preferential adhesion and growth of cells onto the PSS-grafted regions, resulting in well-organized 100 μm cell patterns.

  16. Unique photoreceptor arrangements in a fish with polarized light discrimination.

    PubMed

    Novales Flamarique, Iñigo

    2011-03-01

    In contrast to other vertebrates, some anchovies have cone photoreceptors with longitudinally oriented outer segment lamellae. These photoreceptors are axially dichroic (i.e., they are sensitive to the polarization of axially incident light) and form the basis of a polarization detection system in the northern anchovy, Engraulis mordax. Whether other cone types exist in the retina of this animal, and whether multiple cone opsins are expressed in the retinas of anchovies, is unknown. Likewise, a detailed examination of photoreceptor ultrastructure in nondichroic photoreceptors has not been carried out despite its importance to understand visual specializations within the retina and its use in the formulation of models to explain cellular structure. Here, I combined light and electron microscopy with immunohistochemical studies of opsin expression to infer mechanisms of lamellar formation and to evaluate the potential for color vision in the northern anchovy retina. Morphological observations revealed three cone formations: 1) continuous rows made up of alternating long and short (bilobed) cones with longitudinally oriented lamellae that are orthogonal between cone types; 2) continuous rows of alternating long and short cones in which only the short cones have longitudinally oriented lamellae; and 3) rows of triple cones with transversely oriented lamellae, each triple cone consisting of two lateral cones flanking a small central cone. Ultrastructure investigations supported two models of outer segment formation resulting in the longitudinally oriented lamellae of long and short cones. In the case of the long cone, lateral compression of the outer segment, potentially via the formation of guanine platelet stacks in neighboring pigment epithelium cells, results in a shape transformation from conical to cunate and a tilt from transverse to longitudinal lamellae. In the case of the short (bilobed) cone, membrane invaginations from the connecting ciliary structure grow

  17. The cell pattern correction through design-based metrology

    NASA Astrophysics Data System (ADS)

    Kim, Yonghyeon; Lee, Kweonjae; Chang, Jinman; Kim, Taeheon; Han, Daehan; Lee, Kyusun; Hong, Aeran; Kang, Jinyoung; Choi, Bumjin; Lee, Joosung; Yeom, Kyehee; Lee, Jooyoung; Hong, Hyeongsun; Lee, Kyupil; Jin, Gyoyoung

    2015-03-01

    Starting with the sub 2Xnm node, the process window becomes smaller and tighter than before. Pattern related error budget is required for accurate critical-dimension control of Cell layers. Therefore, lithography has been faced with its various difficulties, such as weird distribution, overlay error, patterning difficulty etc. The distribution of cell pattern and overlay management are the most important factors in DRAM field. We had been experiencing that the fatal risk is caused by the patterns located in the tail of the distribution. The overlay also induces the various defect sources and misalignment issues. Even though we knew that these elements are important, we could not classify the defect type of Cell patterns. Because there is no way to gather massive small pattern CD samples in cell unit block and to compare layout with cell patterns by the CD-SEM. The CD- SEM is used in order to gather these data through high resolution, but CD-SEM takes long time to inspect and extract data because it measures the small FOV. (Field Of View) However, the NGR(E-beam tool) provides high speed with large FOV and high resolution. Also, it's possible to measure an accurate overlay between the target layout and cell patterns because they provide DBM. (Design Based Metrology) By using massive measured data, we extract the result that it is persuasive by applying the various analysis techniques, as cell distribution and defects, the pattern overlay error correction etc. We introduce how to correct cell pattern, by using the DBM measurement, and new analysis methods.

  18. Patterning as a signature of human epidermal stem cell regulation

    PubMed Central

    Klein, Allon M.; Nikolaidou-Neokosmidou, Varvara; Doupé, David P.; Jones, Philip H.; Simons, Benjamin D.

    2011-01-01

    Understanding how stem cells are regulated in adult tissues is a major challenge in cell biology. In the basal layer of human epidermis, clusters of almost quiescent stem cells are interspersed with proliferating and differentiating cells. Previous studies have shown that the proliferating cells follow a pattern of balanced stochastic cell fate. This behaviour enables them to maintain homeostasis, while stem cells remain confined to their quiescent clusters. Intriguingly, these clusters reappear spontaneously in culture, suggesting that they may play a functional role in stem cell auto-regulation. We propose a model of pattern formation that explains how clustering could regulate stem cell activity in homeostatic tissue through contact inhibition and stem cell aggregation. PMID:21632613

  19. Developing photoreceptor-based models of visual attraction in riverine tsetse, for use in the engineering of more-attractive polyester fabrics for control devices

    PubMed Central

    2017-01-01

    Riverine tsetse transmit the parasites that cause the most prevalent form of human African trypanosomiasis, Gambian HAT. In response to the imperative for cheap and efficient tsetse control, insecticide-treated ‘tiny targets’ have been developed through refinement of tsetse attractants based on blue fabric panels. However, modern blue polyesters used for this purpose attract many less tsetse than traditional phthalogen blue cottons. Therefore, colour engineering polyesters for improved attractiveness has great potential for tiny target development. Because flies have markedly different photoreceptor spectral sensitivities from humans, and the responses of these photoreceptors provide the inputs to their visually guided behaviours, it is essential that polyester colour engineering be guided by fly photoreceptor-based explanations of tsetse attraction. To this end, tsetse attraction to differently coloured fabrics was recently modelled using the calculated excitations elicited in a generic set of fly photoreceptors as predictors. However, electrophysiological data from tsetse indicate the potential for modified spectral sensitivities versus the generic pattern, and processing of fly photoreceptor responses within segregated achromatic and chromatic channels has long been hypothesised. Thus, I constructed photoreceptor-based models explaining the attraction of G. f. fuscipes to differently coloured tiny targets recorded in a previously published investigation, under differing assumptions about tsetse spectral sensitivities and organisation of visual processing. Models separating photoreceptor responses into achromatic and chromatic channels explained attraction better than earlier models combining weighted photoreceptor responses in a single mechanism, regardless of the spectral sensitivities assumed. However, common principles for fabric colour engineering were evident across the complete set of models examined, and were consistent with earlier work. Tools for

  20. Cerebellar Zonal Patterning Relies on Purkinje Cell Neurotransmission

    PubMed Central

    White, Joshua J.; Arancillo, Marife; Stay, Trace L.; George-Jones, Nicholas A.; Levy, Sabrina L.; Heck, Detlef H.

    2014-01-01

    Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity. PMID:24920627

  1. Distinct roles of light-activated channels TRP and TRPL in photoreceptors of Periplaneta americana.

    PubMed

    Saari, Paulus; French, Andrew S; Torkkeli, Päivi H; Liu, Hongxia; Immonen, Esa-Ville; Frolov, Roman V

    2017-04-03

    Electrophysiological studies in Drosophila melanogaster and Periplaneta americana have found that the receptor current in their microvillar photoreceptors is generated by two light-activated cationic channels, TRP (transient receptor potential) and TRPL (TRP-like), each having distinct properties. However, the relative contribution of the two channel types to sensory information coding by photoreceptors remains unclear. We recently showed that, in contrast to the diurnal Drosophila in which TRP is the principal phototransduction channel, photoreceptors of the nocturnal P. americana strongly depend on TRPL. Here, we perform a functional analysis, using patch-clamp and intracellular recordings, of P. americana photoreceptors after RNA interference to knock down TRP (TRPkd) and TRPL (TRPLkd). Several functional properties were changed in both knockdown phenotypes: cell membrane capacitance was reduced 1.7-fold, light sensitivity was greatly reduced, and amplitudes of sustained light-induced currents and voltage responses decreased more than twofold over the entire range of light intensities. The information rate (IR) was tested using a Gaussian white-noise modulated light stimulus and was lower in TRPkd photoreceptors (28 ± 21 bits/s) than in controls (52 ± 13 bits/s) because of high levels of bump noise. In contrast, although signal amplitudes were smaller than in controls, the mean IR of TRPLkd photoreceptors was unchanged at 54 ± 29 bits/s(1) because of proportionally lower noise. We conclude that TRPL channels provide high-gain/high-noise transduction, suitable for vision in dim light, whereas transduction by TRP channels is relatively low-gain/low-noise and allows better information transfer in bright light.

  2. Direct patterning of mammalian cells in an ultrasonic heptagon stencil.

    PubMed

    Bernassau, A L; Gesellchen, F; Macpherson, P G A; Riehle, M; Cumming, D R S

    2012-06-01

    We describe the construction of a ultrasonic device suitable for micro patterning particles and cells for tissue engineering applications. The device is formed by seven transducers shaped into a heptagon cavity. By exciting two and three transducers simultaneously, lines or hexagonal shapes can be formed with beads and cells. Furthermore, phase control of the transducers allows shifting the standing waves and thus patterning at different positions on a surface in a controlled manner. The paper discusses direct patterning of mammalian cells by ultrasound "stencil".

  3. Cone Photoreceptors in Bass Retina Use Two Connexins to Mediate Electrical Coupling

    PubMed Central

    O’Brien, John; Bao Nguyen, H.; Mills, Stephen L.

    2008-01-01

    Electrical coupling via gap junctions is a common property of CNS neurons. In retinal photoreceptors, coupling plays important roles in noise filtering, intensity coding, and spatial processing. In many vertebrates, coupling is regulated during the course of light adaptation. To understand the mechanisms of this regulation, we studied photoreceptor gap junction proteins. We found that two connexins were expressed in bass cone photoreceptors. Connexin35 (Cx35) mRNA was present in many cell types including photoreceptors, amacrine, bipolar, and a few ganglion cells. Antibodies to Cx35 labeled abundant gap junctions in both the inner and outer plexiform layers. In the outer plexiform layer, numerous plaques co-localized with cone telodendria at crossing contacts and tip-to-tip contacts. Cx34.7 mRNA was found predominantly in the photoreceptor layer, primarily in cones. Cx34.7 immunolabeling was limited to small plaques immediately beneath cone pedicles, and did not co-localize with Cx35. Cx34.7 plaques were associated with a dense complex of cone membrane beneath the pedicles, including apparent contacts between telodendria and cone pedicles. Tracer coupling studies of the connexins expressed in HeLa cells showed that coupling through Cx35 gap junctions was reduced by protein kinase A (PKA) activation and enhanced by PKA inhibition through a more than five-fold activity range. Cx34.7 was too poorly expressed to study. PKA regulation suggests that coupling through Cx35 gap junctions can be controlled dynamically through dopamine receptor pathways during light adaptation. If Cx34.7 forms functional cell-cell channels between cones, it would provide a physically separate pathway for electrical coupling. PMID:15201336

  4. Report on the National Eye Institute Audacious Goals Initiative: Photoreceptor Regeneration and Integration Workshop.

    PubMed

    Gamm, David M; Wong, Rachel

    2015-11-01

    The National Eye Institute (NEI) hosted a workshop on May 2, 2015, as part of the Audacious Goals Initiative (AGI) to foster a concerted effort to develop novel therapies for outer retinal diseases. The central goal of this initiative is to "demonstrate by 2025 the restoration of usable vision in humans through the regeneration of neurons and neural connections in the eye and visual system." More specifically, the AGI identified two neural retinal cell classes-ganglion cells and photoreceptors-as challenging, high impact targets for these efforts. A prior workshop and subsequent white paper provided a foundation to begin addressing issues regarding optic nerve regeneration, whereas the major objective of the May 2015 workshop was to review progress toward photoreceptor replacement and identify research gaps and barriers that are limiting advancement of the field. The present report summarizes that discussion and input, which was gathered from a panel of distinguished basic science and clinical investigators with diverse technical expertise and experience with different model systems. Four broad discussion categories were put forth during the workshop, each addressing a critical area of need in the pursuit of functional photoreceptor regeneration: (1) cell sources for photoreceptor regeneration, (2) cell delivery and/or integration, (3) outcome assessment, and (4) preclinical models and target patient populations. For each category, multiple challenges and opportunities for research discovery and tool production were identified and vetted. The present report summarizes the dialogue that took place and seeks to encourage continued interactions within the vision science community on this topic. It also serves as a guide for funding to support the pursuit of cell and circuit repair in diseases leading to photoreceptor degeneration.

  5. Overlap of abnormal photoreceptor development and progressive degeneration in Leber congenital amaurosis caused by NPHP5 mutation.

    PubMed

    Downs, Louise M; Scott, Erin M; Cideciyan, Artur V; Iwabe, Simone; Dufour, Valerie; Gardiner, Kristin L; Genini, Sem; Marinho, Luis Felipe; Sumaroka, Alexander; Kosyk, Mychajlo S; Swider, Malgorzata; Aguirre, Geoffrey K; Jacobson, Samuel G; Beltran, William A; Aguirre, Gustavo D

    2016-10-01

    Ciliary defects can result in severe disorders called ciliopathies. Mutations in NPHP5 cause a ciliopathy characterized by severe childhood onset retinal blindness, Leber congenital amaurosis (LCA), and renal disease. Using the canine NPHP5-LCA model we compared human and canine retinal phenotypes, and examined the early stages of photoreceptor development and degeneration, the kinetics of photoreceptor loss, the progression of degeneration and the expression profiles of selected genes. NPHP5-mutant dogs recapitulate the human phenotype of very early loss of rods, and relative retention of the central retinal cone photoreceptors that lack function. In mutant dogs, rod and cone photoreceptors have a sensory cilium, but develop and function abnormally and then rapidly degenerate; L/M cones are more severely affected than S-cones. The lack of outer segments in mutant cones indicates a ciliary dysfunction. Genes expressed in mutant rod or both rod and cone photoreceptors show significant downregulation, while those expressed only in cones are unchanged. Many genes in cell-death and -survival pathways also are downregulated. The canine disease is a non-syndromic LCA-ciliopathy, with normal renal structures and no CNS abnormalities. Our results identify the critical time points in the pathogenesis of the photoreceptor disease, and bring us closer to defining a potential time window for testing novel therapies for translation to patients.

  6. Photoreceptor IFT complexes containing chaperones, guanylyl cyclase 1 and rhodopsin.

    PubMed

    Bhowmick, Reshma; Li, Mei; Sun, Jun; Baker, Sheila A; Insinna, Christine; Besharse, Joseph C

    2009-06-01

    Intraflagellar transport (IFT) provides a mechanism for the transport of cilium-specific proteins, but the mechanisms for linkage of cargo and IFT proteins have not been identified. Using the sensory outer segments (OS) of photoreceptors, which are derived from sensory cilia, we have identified IFT-cargo complexes containing IFT proteins, kinesin 2 family proteins, two photoreceptor-specific membrane proteins, guanylyl cyclase 1 (GC1, Gucy2e) and rhodopsin (RHO), and the chaperones, mammalian relative of DNAJ, DnajB6 (MRJ), and HSC70 (Hspa8). Analysis of these complexes leads to a model in which MRJ through its binding to IFT88 and GC1 plays a critical role in formation or stabilization of the IFT-cargo complexes. Consistent with the function of MRJ in the activation of HSC70 ATPase activity, Mg-ATP enhances the co-IP of GC1, RHO, and MRJ with IFT proteins. Furthermore, RNAi knockdown of MRJ in IMCD3 cells expressing GC1-green fluorescent protein (GFP) reduces cilium membrane targeting of GC1-GFP without apparent effect on cilium elongation.

  7. Dynamic Statistics of Crayfish Caudal Photoreceptors

    PubMed Central

    Hermann, Howard T.; Olsen, Richard E.

    1967-01-01

    Crayfish caudal photoreceptor units were monitored during transient and steady-state responses to light stimuli (step on, step off). A statistical analysis of interpulse interval distributions during quasi-stationary time periods was carried out. Firing statistics during transient conditions were superposable with statistics under whatever steady stimulation produced the same firing rate, indicating that mean firing rate is a sufficient statistic. Distributions encountered formed a continuum of possible shapes. Considerable variation in shape was found with temperature and also among species, with Orconectes clarkii tending to fire more regularly than Orconectes virilis. Some properties of O. virilis statistics are described, including a linear relation between mean and standard deviation, and a tendency for intervals to be nonindependent. The data are considered as constraints on closed form models of the photoreceptor nerve pulse generator. PMID:6035125

  8. Scaffold-independent Patterning of Cells using Magnetic Nanoparticles

    NASA Astrophysics Data System (ADS)

    Ghosh, Suvojit; Biswas, Moanaro; Elankumaran, Subbiah; Puri, Ishwar

    2013-03-01

    Spatial patterning of cells in vitro relies on direct contact of cells on to solid surfaces. Scaffold independent patterning of cells has never been achieved so far. Patterning of cells has wide applications including stem cell biology, tissue architecture and regenerative medicine besides fundamental biology. Magnetized cells in a suspension can be manipulated using an externally applied magnetic field enabling directed patterning. We magnetized mammalian cells by internalization of superparamagnetic nanoparticles coated with bovine serum albumin (BSA). A magnetic field is then used to arrange cells in a desired pattern on a substrate or in suspension. The control strategy is derived from the self-assembly of magnetic colloids in a liquid considering magnetostatic interactions. The range of achievable structural features promise novel experimental methods investigating the influence of tissue shape and size on cell population dynamics wherein Fickian diffusion of autocrine growth signals are known to play a significant role. By eliminating the need for a scaffold, intercellular adhesion mechanics and the effects of temporally regulated signals can be investigated. The findings can be applied to novel tissue engineering methods.

  9. Insulin-related signaling pathways elicited by light in photoreceptor nuclei from bovine retina.

    PubMed

    Natalini, Paola M; Mateos, Melina V; Ilincheta de Boschero, Mónica G; Giusto, Norma M

    2016-04-01

    Retina light stimulation triggers phototransduction events as well as different signaling mechanisms in outer segments (sensorial portion) of photoreceptor cells. We have recently reported a novel light-dependent activation of diacylglycerol kinase (DAGK) and protein kinase C (PKC) at the nuclear level of photoreceptor cells. The aim of the present study was to analyze whether ex-vivo light exposure of bovine retinas also modulates insulin-related signaling pathways in nuclei from photoreceptor cells. To this end, a nuclear fraction enriched in small nuclei from photoreceptor cells (PNF) was obtained using a modified nuclear isolation protocol. In PNF obtained from bovine retinas exposed to light or darkness, the presence of insulin receptor (IR) and phosphorylated insulin receptor (pIR), the activation of Akt, p38 and extracellular signal-regulated kinase (ERK1/2) and the local action of insulin on lipid kinases were studied. Immunofluorescence (IF) and Western blot (WB) studies revealed the presence of IR in photoreceptor nuclei. In PNF a light-dependent increase in IR total content was observed. The presence of activated IR (pIR) was also observed in PNF by WB, being its content higher in PNF from light than in to darkness. Light exposure also produced a significant increase in the content of p-Akt (3 fold) and p-p38 (60%) without changes in total Akt and p38. In addition, an increase in the content of total ERK1/2 (2 fold) was found without changes in p-ERK/total ERK ratio, indicating that light induces translocation of p-ERK to the nucleus. Polyphosphoinositide kinase and diacylglycerol kinase (DAGK) activities were measured in isolated nuclei from light-activated or darkness-adapted retinas through the formation of polyphosphoinositides (PPIs) and phosphatidic acid (PA) using nuclear lipid substrates and [γ-(32)P]ATP as radioactive substrate. A light-dependent increase in PPIs and PA formation was detected when isolated nuclei were exposed to 0.8

  10. A novel mechanism of cone photoreceptor adaptation.

    PubMed

    Howlett, Marcus H C; Smith, Robert G; Kamermans, Maarten

    2017-04-01

    An animal's ability to survive depends on its sensory systems being able to adapt to a wide range of environmental conditions, by maximizing the information extracted and reducing the noise transmitted. The visual system does this by adapting to luminance and contrast. While luminance adaptation can begin at the retinal photoreceptors, contrast adaptation has been shown to start at later stages in the retina. Photoreceptors adapt to changes in luminance over multiple time scales ranging from tens of milliseconds to minutes, with the adaptive changes arising from processes within the phototransduction cascade. Here we show a new form of adaptation in cones that is independent of the phototransduction process. Rather, it is mediated by voltage-gated ion channels in the cone membrane and acts by changing the frequency response of cones such that their responses speed up as the membrane potential modulation depth increases and slow down as the membrane potential modulation depth decreases. This mechanism is effectively activated by high-contrast stimuli dominated by low frequencies such as natural stimuli. However, the more generally used Gaussian white noise stimuli were not effective since they did not modulate the cone membrane potential to the same extent. This new adaptive process had a time constant of less than a second. A critical component of the underlying mechanism is the hyperpolarization-activated current, Ih, as pharmacologically blocking it prevented the long- and mid- wavelength sensitive cone photoreceptors (L- and M-cones) from adapting. Consistent with this, short- wavelength sensitive cone photoreceptors (S-cones) did not show the adaptive response, and we found they also lacked a prominent Ih. The adaptive filtering mechanism identified here improves the information flow by removing higher-frequency noise during lower signal-to-noise ratio conditions, as occurs when contrast levels are low. Although this new adaptive mechanism can be driven by

  11. Loss of Na(+)/K(+)-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration.

    PubMed

    Luan, Zhuo; Reddig, Keith; Li, Hong-Sheng

    2014-11-01

    The activity of Na(+)/K(+)-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na(+)/K(+)-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na(+)/K(+)-ATPase deficiency to in vivo neurodegeneration has been lacking. In this study, we use Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na(+)/K(+) ATPase. Loss of ATPα, an α subunit of Na(+)/K(+)-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K(+). Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na(+)/K(+)-ATPase β subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, the loss of Na(+)/K(+)-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na(+)/K(+) ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na(+)/K(+) ATPase deficiency-mediated neurodegeneration.

  12. Loss of Na+/K+-ATPase in Drosophila photoreceptors leads to blindness and age-dependent neurodegeneration

    PubMed Central

    Luan, Zhuo; Reddig, Keith; Li, Hong-Sheng

    2014-01-01

    The activity of Na+/K+-ATPase establishes transmembrane ion gradients and is essential to cell function and survival. Either dysregulation or deficiency of neuronal Na+/K+-ATPase has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and rapid-onset dystonia Parkinsonism. However, genetic evidence that directly links neuronal Na+/K+-ATPase deficiency to in vivo neurodegeneration has been lacking. In this study, we use Drosophila photoreceptors to investigate the cell-autonomous effects of neuronal Na+/K+ ATPase. Loss of ATPα, an α subunit of Na+/K+-ATPase, in photoreceptors through UAS/Gal4-mediated RNAi eliminated the light-triggered depolarization of the photoreceptors, rendering the fly virtually blind in behavioral assays. Intracellular recordings indicated that ATPα knockdown photoreceptors were already depolarized in the dark, which was due to a loss of intracellular K+. Importantly, ATPα knockdown resulted in the degeneration of photoreceptors in older flies. This degeneration was independent of light and showed characteristics of apoptotic/hybrid cell death as observed via electron microscopy analysis. Loss of Nrv3, a Na+/K+-ATPase β subunit, partially reproduced the signaling and degenerative defects observed in ATPα knockdown flies. Thus, loss of Na+/K+-ATPase not only eradicates visual function but also causes age-dependent degeneration in photoreceptors, confirming the link between neuronal Na+/K+ ATPase deficiency and in vivo neurodegeneration. This work also establishes Drosophila photoreceptors as a genetic model for studying the cell-autonomous mechanisms underlying neuronal Na+/K+ ATPase deficiency-mediated neurodegeneration. PMID:25205229

  13. Insect photoreceptor adaptations to night vision.

    PubMed

    Honkanen, Anna; Immonen, Esa-Ville; Salmela, Iikka; Heimonen, Kyösti; Weckström, Matti

    2017-04-05

    Night vision is ultimately about extracting information from a noisy visual input. Several species of nocturnal insects exhibit complex visually guided behaviour in conditions where most animals are practically blind. The compound eyes of nocturnal insects produce strong responses to single photons and process them into meaningful neural signals, which are amplified by specialized neuroanatomical structures. While a lot is known about the light responses and the anatomical structures that promote pooling of responses to increase sensitivity, there is still a dearth of knowledge on the physiology of night vision. Retinal photoreceptors form the first bottleneck for the transfer of visual information. In this review, we cover the basics of what is known about physiological adaptations of insect photoreceptors for low-light vision. We will also discuss major enigmas of some of the functional properties of nocturnal photoreceptors, and describe recent advances in methodologies that may help to solve them and broaden the field of insect vision research to new model animals.This article is part of the themed issue 'Vision in dim light'.

  14. Variability in mitochondria of zebrafish photoreceptor ellipsoids.

    PubMed

    Tarboush, R; Novales Flamarique, I; Chapman, G B; Connaughton, V P

    2014-01-01

    Ultrastructural examination of photoreceptor inner segment ellipsoids in larval (4, 8, and 15 days postfertilization; dpf) and adult zebrafish identified morphologically different types of mitochondria. All photoreceptors had mitochondria of different sizes (large and small). At 4 dpf, rods had small, moderately stained electron-dense mitochondria (E-DM), and two cone types could be distinguished: (1) those with electron-lucent mitochondria (E-LM) and (2) those with mitochondria of moderate electron density. These distinctions were also apparent at later ages (8 and 15 dpf). Rods from adult fish had fewer mitochondria than their corresponding cones. The ellipsoids of some fully differentiated single and double cones contained large E-DM with few cristae; these were surrounded by small E-LM with typical internal morphology. The mitochondria within the ellipsoids of other single cones showed similar electron density. Microspectrophotometry of cone ellipsoids from adult fish indicated that the large E-DM had a small absorbance peak (∼0.03 OD units) and did not contain cytochrome-c, but crocetin, a carotenoid found in old world monkeys. Crocetin functions to prevent oxidative damage to photoreceptors, suggesting that the ellipsoid mitochondria in adult zebrafish cones protect against apoptosis and function metabolically, rather than as a light filter.

  15. Self-Organizing Actomyosin Patterns on the Cell Cortex at Epithelial Cell-Cell Junctions

    PubMed Central

    Moore, Thomas; Wu, Selwin K.; Michael, Magdalene; Yap, Alpha S.; Gomez, Guillermo A.; Neufeld, Zoltan

    2014-01-01

    The behavior of actomyosin critically determines morphologically distinct patterns of contractility found at the interface between adherent cells. One such pattern is found at the apical region (zonula adherens) of cell-cell junctions in epithelia, where clusters of the adhesion molecule E-cadherin concentrate in a static pattern. Meanwhile, E-cadherin clusters throughout lateral cell-cell contacts display dynamic movements in the plane of the junctions. To gain insight into the principles that determine the nature and organization of these dynamic structures, we analyze this behavior by modeling the 2D actomyosin cell cortex as an active fluid medium. The numerical simulations show that the stability of the actin filaments influences the spatial structure and dynamics of the system. We find that in addition to static Turing-type patterns, persistent dynamic behavior occurs in a wide range of parameters. In the 2D model, mechanical stress-dependent actin breakdown is shown to produce a continuously changing network of actin bridges, whereas with a constant breakdown rate, more isolated clusters of actomyosin tend to form. The model qualitatively reproduces the dynamic and stable patterns experimentally observed at the junctions between epithelial cells. PMID:25468344

  16. Retinophilin is a light-regulated phosphoprotein required to suppress photoreceptor dark noise in Drosophila.

    PubMed

    Mecklenburg, Kirk L; Takemori, Nobuaki; Komori, Naoka; Chu, Brian; Hardie, Roger C; Matsumoto, Hiroyuki; O'Tousa, Joseph E

    2010-01-27

    Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions but retains the normal kinetics of the light response. Photoreceptors lacking neither inactivation nor afterpotential C (NINAC) myosin III, a motor protein/kinase, also display a similar dark-noise phenotype as the RTP deletion. We show that NINAC mutants are depleted for RTP. These results suggest the increase in dark noise in NINAC mutants is attributable to lack of RTP and, furthermore, defines a novel role for NINAC in the rhabdomere. We propose that RTP is a light-regulated phosphoprotein that organizes rhabdomeric components to suppress random activation of the phototransduction cascade and thus increases the signaling fidelity of dark-adapted photoreceptors.

  17. Human cone visual pigment deletions spare sufficient photoreceptors to warrant gene therapy.

    PubMed

    Cideciyan, Artur V; Hufnagel, Robert B; Carroll, Joseph; Sumaroka, Alexander; Luo, Xunda; Schwartz, Sharon B; Dubra, Alfredo; Land, Megan; Michaelides, Michel; Gardner, Jessica C; Hardcastle, Alison J; Moore, Anthony T; Sisk, Robert A; Ahmed, Zubair M; Kohl, Susanne; Wissinger, Bernd; Jacobson, Samuel G

    2013-12-01

    Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5-58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.

  18. Hypoxic preconditioning protects photoreceptors against light damage independently of hypoxia inducible transcription factors in rods.

    PubMed

    Kast, Brigitte; Schori, Christian; Grimm, Christian

    2016-05-01

    Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors.

  19. Potential of Small Molecule–Mediated Reprogramming of Rod Photoreceptors to Treat Retinitis Pigmentosa

    PubMed Central

    Nakamura, Paul A.; Tang, Shibing; Shimchuk, Andy A.; Ding, Sheng; Reh, Thomas A.

    2016-01-01

    Purpose Mutations in rod photoreceptor genes can cause retinitis pigmentosa (RP). Rod gene expression is regulated by the nuclear hormone receptor, Nr2e3. Genetic deletion of Nr2e3 reprograms rods into cells that resemble cone photoreceptors, and might therefore prevent their death from some forms of RP. There are no identified ligands for Nr2e3; however, reverse agonists might mimic the genetic rescue effect and may be therapeutically useful for the treatment of RP. Methods We screened for small molecule modulators of Nr2e3 using primary retinal cell cultures and characterized the most potent, which we have named photoregulin1 (PR1), in vitro and in vivo. We also tested the ability of PR1 to slow the progression of photoreceptor degeneration in two common mouse models of autosomal dominant RP, the RhoP23H and the Pde6brd1 mutations. Results In developing retina, PR1 causes a decrease in rod gene expression and an increase in S opsin+ cones. Photoregulin1 continues to inhibit rod gene expression in adult mice. When applied to two mouse models of RP, PR1 slows the degeneration of photoreceptors. Conclusions Chemical compounds identified as modulators of Nr2e3 activity may be useful for the treatment of RP through their effects on expression of disease-causing mutant genes. PMID:27893103

  20. Cell cycles and proliferation patterns in Haematococcus pluvialis

    NASA Astrophysics Data System (ADS)

    Zhang, Chunhui; Liu, Jianguo; Zhang, Litao

    2016-09-01

    Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation; far less attention has been paid to cell cycles and proliferation patterns. The purpose of this study was to clarify cell cycles and proliferation patterns in H. pluvialis microscopically using a camera and video recorder system. The complicated life history of H. pluvialis can be divided into two stages: the motile stage and the non-motile stage. All the cells can be classified into forms as follows: motile cell, non-motile cell, zoospore and aplanospore. The main cell proliferation, both in the motile phase and non-motile phase in H. pluvialis, is by asexual reproduction. Under normal growth conditions, a motile cell usually produces two, sometimes four, and exceptionally eight zoospores. Under unfavorable conditions, the motile cell loses its flagella and transforms into a non-motile cell, and the non-motile cell usually produces 2, 4 or 8 aplanospores, and occasionally 20-32 aplanospores, which further develop into non-motile cells. Under suitable conditions, the non-motile cell is also able to release zoospores. The larger non-motile cells produce more than 16 zoospores, and the smaller ones produce 4 or 8 zoospores. Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase. There is, as yet, no convincing direct evidence for sexual reproduction.

  1. The analysis method of the DRAM cell pattern hotspot

    NASA Astrophysics Data System (ADS)

    Lee, Kyusun; Lee, Kweonjae; Chang, Jinman; Kim, Taeheon; Han, Daehan; Hong, Aeran; Kim, Yonghyeon; Kang, Jinyoung; Choi, Bumjin; Lee, Joosung; Lee, Jooyoung; Hong, Hyeongsun; Lee, Kyupil; Jin, Gyoyoung

    2015-03-01

    It is increasingly difficult to determine degree of completion of the patterning and the distribution at the DRAM Cell Patterns. When we research DRAM Device Cell Pattern, there are three big problems currently, it is as follows. First, due to etch loading, it is difficult to predict the potential defect. Second, due to under layer topology, it is impossible to demonstrate the influence of the hotspot. Finally, it is extremely difficult to predict final ACI pattern by the photo simulation, because current patterning process is double patterning technology which means photo pattern is completely different from final etch pattern. Therefore, if the hotspot occurs in wafer, it is very difficult to find it. CD-SEM is the most common pattern measurement tool in semiconductor fabrication site. CD-SEM is used to accurately measure small region of wafer pattern primarily. Therefore, there is no possibility of finding places where unpredictable defect occurs. Even though, "Current Defect detector" can measure a wide area, every chip has same pattern issue, the detector cannot detect critical hotspots. Because defect detecting algorithm of bright field machine is based on image processing, if same problems occur on compared and comparing chip, the machine cannot identify it. Moreover this instrument is not distinguished the difference of distribution about 1nm~3nm. So, "Defect detector" is difficult to handle the data for potential weak point far lower than target CD. In order to solve those problems, another method is needed. In this paper, we introduce the analysis method of the DRAM Cell Pattern Hotspot.

  2. Rescue from photoreceptor degeneration in the rd mouse by human immunodeficiency virus vector-mediated gene transfer.

    PubMed

    Takahashi, M; Miyoshi, H; Verma, I M; Gage, F H

    1999-09-01

    Retinitis pigmentosa (RP) is the most common inherited retinal disease, in which photoreceptor cells degenerate, leading to blindness. Mutations in the rod photoreceptor cGMP phosphodiesterase beta subunit (PDEbeta) gene are found in patients with autosomal recessive RP as well as in the rd mouse. We have recently shown that lentivirus vectors based on human immunodeficiency virus (HIV) type 1 achieve stable and efficient gene transfer into retinal cells. In this study, we evaluated the potential of HIV vector-mediated gene therapy for RP in the rd mouse. HIV vectors containing a gene encoding a hemagglutinin (HA)-tagged PDEbeta were injected into the subretinal spaces of newborn rd mouse eyes. One to three rows of photoreceptor nuclei were observed in the eyes for at least 24 weeks postinjection, whereas no photoreceptor cells remained in the eyes of control animals at 6 weeks postinjection. Expression of HA-tagged PDEbeta in the rescued photoreceptor cells was confirmed by two-color confocal immunofluorescence analysis using anti-HA and anti-opsin antibodies. HIV vector-mediated gene therapy appears to be a promising means for the treatment of recessive forms of inherited retinal degeneration.

  3. Insulinoma-associated 1a (Insm1a) is required for photoreceptor differentiation in the zebrafish retina

    PubMed Central

    Forbes-Osborne, Marie A.; Wilson, Stephen G.; Morris, Ann C.

    2013-01-01

    The zinc-finger transcription factor Insulinoma-associated 1 (Insm1, previously IA-1) is expressed in the developing nervous and neuroendocrine systems, and is required for cell type specific differentiation. Expression of Insm1 is largely absent in the adult, although it is present in neurogenic regions of the adult brain and zebrafish retina. While expression of Insm1 has also been observed in the embryonic retina of numerous vertebrate species, its function during retinal development has remained unexplored. Here, we demonstrate that in the developing zebrafish retina, insm1a is required for photoreceptor differentiation. Insm1a-deficient embryos were microphthalmic and displayed defects in rod and cone photoreceptor differentiation. Rod photoreceptor cells were more sensitive to loss of insm1a expression than were cone photoreceptor cells. Additionally, we provide evidence that insm1a regulates cell cycle progression of retinoblasts, and functions upstream of the bHLH transcription factors ath5/atoh7 and neurod, and the photoreceptor specification genes crx and nr2e3. Finally, we show that insm1a is negatively regulated by Notch-Delta signaling. Taken together, our data demonstrate that Insm1 influences neuronal subtype differentiation during retinal development. PMID:23747542

  4. Cell patterning on biological gels via cell spraying through a mask.

    PubMed

    Nahmias, Yaakov; Arneja, Abhinav; Tower, Theodore T; Renn, Michael J; Odde, David J

    2005-01-01

    We present an easily applicable and inexpensive method for patterning cells on arbitrary surfaces including biological gels with little loss of viability or function. Single-cell suspensions of human umbilical vein endothelial cells and NIH 3T3 fibroblasts were sprayed with an off-the-shelf airbrush through a mask to create 100-microm scale patterns on collagen gels. Three-dimensional patterns were created by layering a collagen gel on top of the first pattern and patterning the top gel. Coculture of rat hepatocytes with NIH 3T3 patterns on collagen gels resulted in localized increased activity of cytochrome P-450 along the pattern. These results suggest that cell spraying is a useful tool for the study of heterotypic cellular interactions and tissue-engineering applications on biologically relevant matrices, and for the creation of three-dimensional cell patterns in vitro.

  5. NEURONATIN IS A STRESS-RESPONSIVE PROTEIN OF ROD PHOTORECEPTORS

    PubMed Central

    SHINDE, VISHAL; PITALE, PRIYAMVADA M.; HOWSE, WAYNE; GORBATYUK, OLEG; GORBATYUK, MARINA

    2016-01-01

    Neuronatin (NNAT) is a small transmembrane proteolipid that is highly expressed in the embryonic developing brain and several other peripheral tissues. This study is the first to provide evidence that NNAT is detected in the adult retina of various adult rod-dominant mammals, including wild-type (WT) rodents, transgenic rodents expressing mutant S334ter, P23H, or T17M rhodopsin, non-human primates, humans, and cone-dominant tree shrews. Immunohistochemical and quantitative real time polymerase chain reaction (qRT-PCR) analyses were applied to detect NNAT. Confocal microscopy analysis revealed that NNAT immunofluorescence is restricted to the outer segments (OSs) of photoreceptors without evidence of staining in other retinal cell types across all mammalian species. Moreover, in tree shrew retinas, we found NNAT to be co-localized with rhodopsin, indicating its predominant expression in rods. The rod-derived expression of NNAT was further confirmed by qRT-PCR in isolated rod photoreceptor cells. We also used these cells to mimic cellular stress in transgenic retinas by treating them with the endoplasmic reticulum stress inducer, tunicamycin. Thus, our data revealed accumulation of NNAT around the nucleus as compared to dispersed localization of NNAT within control cells. This distribution coincided with the partial intracellular mislocalization of NNAT to the outer nuclear layer observed in transgenic retinas. In addition, stressed retinas demonstrated an increase of NNAT mRNA and protein levels. Therefore, our study demonstrated that NNAT is a novel stress-responsive protein with a potential structural and/or functional role in adult mammalian retinas. PMID:27109921

  6. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats.

    PubMed

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-09-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision.

  7. Rod photoreceptors protect from cone degeneration-induced retinal remodeling and restore visual responses in zebrafish

    PubMed Central

    Saade, Carole J.; Alvarez-Delfin, Karen; Fadool, James M.

    2013-01-01

    Humans are largely dependent upon cone-mediated vision. However, death or dysfunction of rods, the predominant photoreceptor subtype, results in secondary loss of cones, remodeling of retinal circuitry and blindness. The changes in circuitry may contribute to the vision deficit and undermine attempts at restoring sight. We exploit zebrafish larvae as a genetic model to specifically characterize changes associated with photoreceptor degenerations in a cone-dominated retina. Photoreceptors form synapses with two types of second order neurons, bipolar cells and horizontal cells. Using cell-specific reporter gene expression and immunolabeling for postsynaptic glutamate receptors, significant remodeling is observed following cone degeneration in the pde6cw59 larval retina but not rod degeneration in the Xops:mCFPq13 line. In adults, rods and cones are present in approximately equal numbers, and in pde6cw59 mutants glutamate receptor expression and synaptic structures in the outer plexiform layer are preserved, and visual responses are gained in these once-blind fish. We propose that the abundance of rods in the adult protects the retina from cone degeneration-induced remodeling. We test this hypothesis by genetically manipulating the number of rods in larvae. We show that an increased number and uniform distribution of rods in lor/tbx2bp22bbtl or six7 morpholino-injected larvae protect from pde6cw59-induced secondary changes. The observations that remodeling is a common consequence of photoreceptor death across species, and that in zebrafish a small number of surviving photoreceptors afford protection from degeneration-induced changes provides a model for systematic analysis of factors that slow or even prevent the secondary deteriorations associated with neural degenerative disease. PMID:23365220

  8. Adenosine triphosphate-induced photoreceptor death and retinal remodeling in rats

    PubMed Central

    Vessey, Kirstan A; Greferath, Ursula; Aplin, Felix P; Jobling, Andrew I; Phipps, Joanna A; Ho, Tracy; De Iongh, Robbert U; Fletcher, Erica L

    2014-01-01

    Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five-week-old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age-related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2-year-old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP-induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision. J. Comp. Neurol. 522:2928–2950, 2014. © 2014 Wiley Periodicals, Inc. PMID:24639102

  9. Stable transgenesis in the marine annelid Platynereis dumerilii sheds new light on photoreceptor evolution.

    PubMed

    Backfisch, Benjamin; Veedin Rajan, Vinoth Babu; Fischer, Ruth M; Lohs, Claudia; Arboleda, Enrique; Tessmar-Raible, Kristin; Raible, Florian

    2013-01-02

    Research in eye evolution has mostly focused on eyes residing in the head. In contrast, noncephalic light sensors are far less understood and rather regarded as evolutionary innovations. We established stable transgenesis in the annelid Platynereis, a reference species for evolutionary and developmental comparisons. EGFP controlled by cis-regulatory elements of r-opsin, a characteristic marker for rhabdomeric photoreceptors, faithfully recapitulates known r-opsin expression in the adult eyes, and marks a pair of pigment-associated frontolateral eyelets in the brain. Unexpectedly, transgenic animals revealed an additional series of photoreceptors in the ventral nerve cord as well as photoreceptors that are located in each pair of the segmental dorsal appendages (notopodia) and project into the ventral nerve cord. Consistent with a photosensory function of these noncephalic cells, decapitated animals display a clear photoavoidance response. Molecular analysis of the receptors suggests that they differentiate independent of pax6, a gene involved in early eye development of many metazoans, and that the ventral cells may share origins with the Hesse organs in the amphioxus neural tube. Finally, expression analysis of opn4×-2 and opn4m-2, two zebrafish orthologs of Platynereis r-opsin, reveals that these genes share expression in the neuromasts, known mechanoreceptors of the lateral line peripheral nervous system. Together, this establishes that noncephalic photoreceptors are more widespread than assumed, and may even reflect more ancient aspects of sensory systems. Our study marks significant advance for the understanding of photoreceptor cell (PRC) evolution and development and for Platynereis as a functional lophotrochozoan model system.

  10. Heterogeneous differentiation patterns of individual CD8+ T cells.

    PubMed

    Gerlach, Carmen; Rohr, Jan C; Perié, Leïla; van Rooij, Nienke; van Heijst, Jeroen W J; Velds, Arno; Urbanus, Jos; Naik, Shalin H; Jacobs, Heinz; Beltman, Joost B; de Boer, Rob J; Schumacher, Ton N M

    2013-05-03

    Upon infection, antigen-specific CD8(+) T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8(+) T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8(+) T cell heterogeneity and demonstrates that reproducibility of CD8(+) T cell responses is achieved through population averaging.

  11. Autoimmune responses against photoreceptor antigens during retinal degeneration and their role in macrophage recruitment into retinas of RCS rats.

    PubMed

    Kyger, Madison; Worley, Aneta; Adamus, Grazyna

    2013-01-15

    Autoimmunity may contribute to retinal degeneration. The studies examined the evolution of autoimmune responses against retina in naïve dystrophic RCS rats over the course of retinal degeneration. We showed that anti-retinal autoantibodies and T cells are generated in response to the availability of antigenic material released from dying photoreceptor cells during retinal degeneration but with distinctive activation trends. Passive transfer of anti-retinal antibodies enhanced disease progression by disrupting the BRB, upregulating MCP-1, attracting blood macrophages into retina, and augmenting apoptotic photoreceptor cell death. Our findings directly link anti-retinal autoantibodies to activated macrophage entry and their possible role in neurodegeneration.

  12. Development of motorized plasma lithography for cell patterning.

    PubMed

    Deguchi, Shinji; Nagasawa, Yohei; Saito, Akira C; Matsui, Tsubasa S; Yokoyama, Sho; Sato, Masaaki

    2014-03-01

    The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.

  13. Myocardial Cell Pattern on Piezoelectric Nanofiber Mats for Energy Harvesting

    NASA Astrophysics Data System (ADS)

    Liu, X.; Wang, X.; Zhao, H.; Du, Y.

    2014-11-01

    The paper presents in vitro contractile myocardial cell pattern on piezoelectric nanofiber mats with applications in energy harvesting. The cell-based energy harvester consists of myocardial cell sheet and a PDMS substrate with a PVDF nanofiber mat on. Experimentally, cultured on specifically distributed nanofiber mats, neonatal rat ventricular cardiomyocytes are characterized with the related morphology and contraction. Previously, we have come up with the concept of energy harvesting from heart beating using piezoelectric material. A bio-hybrid energy harvester combined living cardiomyocytes, PDMS polymer substrate and piezoelectric PVDF film with the electrical output of peak current 87.5nA and peak voltage 92.3mV. However, the thickness of the cardiomyocyte cultured on a two-dimensional substrate is much less than that of the piezoelectric film. The Micro Contact Printing (μCP) method used in cell pattern on the PDMS thin film has tough requirement for the film surface. As such, in this paper we fabricated nanofiber-constructed PDMS thin film to realize cell pattern due to PVDF nanofibers with better piezoelectricity and microstructures of nanofiber mats guiding cell distribution. Living cardiomyocytes patterned on those distributed piezoelectric nanofibers with the result of the same distribution as the nanofiber pattern.

  14. Ire1 supports normal ER differentiation in developing Drosophila photoreceptors

    PubMed Central

    Xu, Zuyuan; Chikka, Madhusudana Rao; Xia, Hongai; Ready, Donald F.

    2016-01-01

    ABSTRACT The endoplasmic reticulum (ER) serves virtually all aspects of cell physiology and, by pathways that are incompletely understood, is dynamically remodeled to meet changing cell needs. Inositol-requiring enzyme 1 (Ire1), a conserved core protein of the unfolded protein response (UPR), participates in ER remodeling and is particularly required during the differentiation of cells devoted to intense secretory activity, so-called ‘professional’ secretory cells. Here, we characterize the role of Ire1 in ER differentiation in the developing Drosophila compound eye photoreceptors (R cells). As part of normal development, R cells take a turn as professional secretory cells with a massive secretory effort that builds the photosensitive membrane organelle, the rhabdomere. We find rough ER sheets proliferate as rhabdomere biogenesis culminates, and Ire1 is required for normal ER differentiation. Ire1 is active early in R cell development and is required in anticipation of peak biosynthesis. Without Ire1, the amount of rough ER sheets is strongly reduced and the extensive cortical ER network at the rhabdomere base, the subrhabdomere cisterna (SRC), fails. Instead, ER proliferates in persistent and ribosome-poor tubular tangles. A phase of Ire1 activity early in R cell development thus shapes dynamic ER. PMID:26787744

  15. Patterned hydrogel substrates for cell culture with electrohydrodynamic jet printing.

    PubMed

    Poellmann, Michael J; Barton, Kira L; Mishra, Sandipan; Johnson, Amy J Wagoner

    2011-09-09

    Cells respond to and are directed by physiochemical cues in their microenvironment, including geometry and substrate stiffness. The development of substrates for cell culture with precisely controlled physiochemical characteristics has the potential to advance the understanding of cell biology considerably. In this communication, E-jet printing is introduced as a method for creating high-resolution protein patterns on substrates with controlled elasticity. It is the first application of E-jet printing on a soft surface. Protein spots as small as 5 µm in diameter on polyacrylamide are demonstrated. The patterned hydrogels are shown to support cell attachment and spreading. Polyacrylamide substrates patterned by E-jet printing may be applied to further the study of cellular mechanobiology.

  16. The Classification of HEp-2 Cell Patterns Using Fractal Descriptor.

    PubMed

    Xu, Rudan; Sun, Yuanyuan; Yang, Zhihao; Song, Bo; Hu, Xiaopeng

    2015-07-01

    Indirect immunofluorescence (IIF) with HEp-2 cells is considered as a powerful, sensitive and comprehensive technique for analyzing antinuclear autoantibodies (ANAs). The automatic classification of the HEp-2 cell images from IIF has played an important role in diagnosis. Fractal dimension can be used on the analysis of image representing and also on the property quantification like texture complexity and spatial occupation. In this study, we apply the fractal theory in the application of HEp-2 cell staining pattern classification, utilizing fractal descriptor firstly in the HEp-2 cell pattern classification with the help of morphological descriptor and pixel difference descriptor. The method is applied to the data set of MIVIA and uses the support vector machine (SVM) classifier. Experimental results show that the fractal descriptor combining with morphological descriptor and pixel difference descriptor makes the precisions of six patterns more stable, all above 50%, achieving 67.17% overall accuracy at best with relatively simple feature vectors.

  17. An expanded set of photoreceptors in the Eastern Pale Clouded Yellow butterfly, Colias erate

    PubMed Central

    Arikawa, Kentaro; Stavenga, Doekele G.

    2010-01-01

    We studied the spectral and polarisation sensitivities of photoreceptors of the butterfly Colias erate by using intracellular electrophysiological recordings and stimulation with light pulses. We developed a method of response waveform comparison (RWC) for evaluating the effective intensity of the light pulses. We identified one UV, four violet-blue, two green and two red photoreceptor classes. We estimated the peak wavelengths of four rhodopsins to be at about 360, 420, 460 and 560 nm. The four violet-blue classes are presumably based on combinations of two rhodopsins and a violet-absorbing screening pigment. The green classes have reduced sensitivity in the ultraviolet range. The two red classes have primary peaks at about 650 and 665 nm, respectively, and secondary peaks at about 480 nm. The shift of the main peak, so far the largest amongst insects, is presumably achieved by tuning the effective thickness of the red perirhabdomal screening pigment. Polarisation sensitivity of green and red photoreceptors is higher at the secondary than at the main peak. We found a 20-fold variation of sensitivity within the cells of one green class, implying possible photoreceptor subfunctionalisation. We propose an allocation scheme of the receptor classes into the three ventral ommatidial types. PMID:20524001

  18. An expanded set of photoreceptors in the Eastern Pale Clouded Yellow butterfly, Colias erate.

    PubMed

    Pirih, Primoz; Arikawa, Kentaro; Stavenga, Doekele G

    2010-07-01

    We studied the spectral and polarisation sensitivities of photoreceptors of the butterfly Colias erate by using intracellular electrophysiological recordings and stimulation with light pulses. We developed a method of response waveform comparison (RWC) for evaluating the effective intensity of the light pulses. We identified one UV, four violet-blue, two green and two red photoreceptor classes. We estimated the peak wavelengths of four rhodopsins to be at about 360, 420, 460 and 560 nm. The four violet-blue classes are presumably based on combinations of two rhodopsins and a violet-absorbing screening pigment. The green classes have reduced sensitivity in the ultraviolet range. The two red classes have primary peaks at about 650 and 665 nm, respectively, and secondary peaks at about 480 nm. The shift of the main peak, so far the largest amongst insects, is presumably achieved by tuning the effective thickness of the red perirhabdomal screening pigment. Polarisation sensitivity of green and red photoreceptors is higher at the secondary than at the main peak. We found a 20-fold variation of sensitivity within the cells of one green class, implying possible photoreceptor subfunctionalisation. We propose an allocation scheme of the receptor classes into the three ventral ommatidial types.

  19. Rhodopsin coexpression in UV photoreceptors of Aedes aegypti and Anopheles gambiae mosquitoes.

    PubMed

    Hu, Xiaobang; Leming, Matthew T; Whaley, Michelle A; O'Tousa, Joseph E

    2014-03-15

    Differential rhodopsin gene expression within specialized R7 photoreceptor cells divides the retinas of Aedes aegypti and Anopheles gambiae mosquitoes into distinct domains. The two species express the rhodopsin orthologs Aaop8 and Agop8, respectively, in a large subset of these R7 photoreceptors that function as ultraviolet receptors. We show here that a divergent subfamily of mosquito rhodopsins, Aaop10 and Agop10, is coexpressed in these R7 photoreceptors. The properties of the A. aegypti Aaop8 and Aaop10 rhodopsins were analyzed by creating transgenic Drosophila expressing these rhodopsins. Electroretinogram recordings, and spectral analysis of head extracts, obtained from the Aaop8 strain confirmed that Aaop8 is an ultraviolet-sensitive rhodopsin. Aaop10 was poorly expressed and capable of eliciting only small and slow light responses in Drosophila photoreceptors, and electroretinogram analysis suggested that it is a long-wavelength rhodopsin with a maximal sensitivity near 500 nm. Thus, coexpression of Aaop10 rhodopsin with Aaop8 rhodopsin has the potential to modify the spectral properties of mosquito ultraviolet receptors. Retention of Op10 rhodopsin family members in the genomes of Drosophila species suggests that this rhodopsin family may play a conserved role in insect vision.

  20. BMP signaling orchestrates photoreceptor specification in the zebrafish pineal gland in collaboration with Notch.

    PubMed

    Quillien, Aurélie; Blanco-Sanchez, Bernardo; Halluin, Caroline; Moore, John C; Lawson, Nathan D; Blader, Patrick; Cau, Elise

    2011-06-01

    A variety of signaling pathways have been shown to regulate specification of neuronal subtype identity. However, the mechanisms by which future neurons simultaneously process information from multiple pathways to establish their identity remain poorly understood. The zebrafish pineal gland offers a simple system with which to address questions concerning the integration of signaling pathways during neural specification as it contains only two types of neurons - photoreceptors and projection neurons. We have previously shown that Notch signaling inhibits the projection neuron fate. Here, we show that BMP signaling is both necessary and sufficient to promote the photoreceptor fate. We also demonstrate that crosstalk between BMP and Notch signaling is required for the inhibition of a projection neuron fate in future photoreceptors. In this case, BMP signaling is required as a competence factor for the efficient activation of Notch targets. Our results indicate that both the induction of a photoreceptor fate and the interaction with Notch relies on a canonical BMP/Smad5 pathway. However, the activation of Notch-dependent transcription does not require a canonical Smad5-DNA interaction. Our results provide new insights into how multiple signaling influences are integrated during cell fate specification in the vertebrate CNS.

  1. Arf-like Protein 3 (ARL3) Regulates Protein Trafficking and Ciliogenesis in Mouse Photoreceptors.

    PubMed

    Hanke-Gogokhia, Christin; Wu, Zhijian; Gerstner, Cecilia D; Frederick, Jeanne M; Zhang, Houbin; Baehr, Wolfgang

    2016-03-25

    Arf-like protein 3 (ARL3) is a ubiquitous small GTPase expressed in ciliated cells of plants and animals. Germline deletion ofArl3in mice causes multiorgan ciliopathy reminiscent of Bardet-Biedl or Joubert syndromes. As photoreceptors are elegantly compartmentalized and have cilia, we probed the function of ARL3 (ADP-ribosylation factor (Arf)-like 3 protein) by generating rod photoreceptor-specific (prefix(rod)) and retina-specific (prefix(ret))Arl3deletions. In predegenerate(rod)Arl3(-/-)mice, lipidated phototransduction proteins showed trafficking deficiencies, consistent with the role of ARL3 as a cargo displacement factor for lipid-binding proteins. By contrast,(ret)Arl3(-/-)rods and cones expressing Cre recombinase during embryonic development formed neither connecting cilia nor outer segments and degenerated rapidly. Absence of cilia infers participation of ARL3 in ciliogenesis and axoneme formation. Ciliogenesis was rescued, and degeneration was reversed in part by subretinal injection of adeno-associated virus particles expressing ARL3-EGFP. The conditional knock-out phenotypes permitted identification of two ARL3 functions, both in the GTP-bound form as follows: one as a regulator of intraflagellar transport participating in photoreceptor ciliogenesis and the other as a cargo displacement factor transporting lipidated protein to the outer segment. Surprisingly, a farnesylated inositol polyphosphate phosphatase only trafficked from the endoplasmic reticulum to the Golgi, thereby excluding it from a role in photoreceptor cilia physiology.

  2. Longitudinal evaluation of expression of virally delivered transgenes in gerbil cone photoreceptors

    PubMed Central

    Mauck, Matthew C.; Mancuso, Katherine; Kuchenbecker, James A.; Connor, Thomas B.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

    2008-01-01

    Delivery of foreign opsin genes to cone photoreceptors using recombinant adeno-associated virus (rAAV) is a potential tool for studying the basic mechanisms underlying cone based vision and for treating vision disorders. We used an in vivo retinal imaging system to monitor, over time, expression of virally-delivered genes targeted to cone photoreceptors in the Mongolian gerbil (Meriones unguiculatus). Gerbils have a well-developed photopic visual system, with 11-14% of their photoreceptors being cones. We used replication deficient serotype 5 rAAV to deliver a gene for green fluorescent protein (GFP). In an effort to direct expression of the gene specifically to either S or M cones, the transgene was under the control of either the human X-chromosome opsin gene regulatory elements, i.e., an enhancer termed the Locus Control Region (LCR) and L promoter, or the human S-opsin promoter. Longitudinal fluorescence images reveal that gene expression is first detectable about 14 days post-injection, reaches a peak after about 3 months, and is observed more than a year post-injection if the initial viral concentration is sufficiently high. The regulatory elements are able to direct expression to a subpopulation of cones while excluding expression in rods and non-photoreceptor retinal cells. When the same viral constructs are used to deliver a human long-wavelength opsin gene to gerbil cones, stimulation of the introduced human photopigment with long-wavelength light produces robust cone responses. PMID:18598398

  3. Spatial Pattern of Cell Damage in Tissue from Heavy Ions

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem L.; Huff, Janice L.; Cucinotta, Francis A.

    2007-01-01

    A new Monte Carlo algorithm was developed that can model passage of heavy ions in a tissue, and their action on the cellular matrix for 2- or 3-dimensional cases. The build-up of secondaries such as projectile fragments, target fragments, other light fragments, and delta-rays was simulated. Cells were modeled as a cell culture monolayer in one example, where the data were taken directly from microscopy (2-d cell matrix). A simple model of tissue was given as abstract spheres with close approximation to real cell geometries (3-d cell matrix), as well as a realistic model of tissue was proposed based on microscopy images. Image segmentation was used to identify cells in an irradiated cell culture monolayer, or slices of tissue. The cells were then inserted into the model box pixel by pixel. In the case of cell monolayers (2-d), the image size may exceed the modeled box size. Such image was is moved with respect to the box in order to sample as many cells as possible. In the case of the simple tissue (3-d), the tissue box is modeled with periodic boundary conditions, which extrapolate the technique to macroscopic volumes of tissue. For real tissue, specific spatial patterns for cell apoptosis and necrosis are expected. The cell patterns were modeled based on action cross sections for apoptosis and necrosis estimated based on BNL data, and other experimental data.

  4. Modal content of living human cone photoreceptors

    PubMed Central

    Liu, Zhuolin; Kocaoglu, Omer P.; Turner, Timothy L.; Miller, Donald T.

    2015-01-01

    Decades of experimental and theoretical investigations have established that photoreceptors capture light based on the principles of optical waveguiding. Yet considerable uncertainty remains, even for the most basic prediction as to whether photoreceptors support more than a single waveguide mode. To test for modal behavior in human cone photoreceptors in the near infrared, we took advantage of adaptive-optics optical coherence tomography (AO-OCT, λc = 785 nm) to noninvasively image in three dimensions the reflectance profile of cones. Modal content of reflections generated at the cone inner segment and outer segment junction (IS/OS) and cone outer segment tip (COST) was examined over a range of cone diameters in 1,802 cones from 0.6° to 10° retinal eccentricity. Second moment analysis in conjunction with theoretical predictions indicate cone IS and OS have optical properties consistent of waveguides, which depend on segment diameter and refractive index. Cone IS was found to support a single mode near the fovea (≤3°) and multiple modes further away (>4°). In contrast, no evidence of multiple modes was found in the cone OSs. The IS/OS and COST reflections share a common optical aperture, are most circular near the fovea, show no orientation preference, and are temporally stable. We tested mode predictions of a conventional step-index fiber model and found that in order to fit our AO-OCT results required a lower estimate of the IS refractive index and introduction of an IS focusing/tapering effect. PMID:26417509

  5. Multimodal Imaging of Photoreceptor Structure in Choroideremia

    PubMed Central

    Johnson, Ryan D.; Williams, Vesper; Summerfelt, Phyllis; Dubra, Alfredo; Weinberg, David V.; Stepien, Kimberly E.; Fishman, Gerald A.; Carroll, Joseph

    2016-01-01

    Purpose Choroideremia is a progressive X-linked recessive dystrophy, characterized by degeneration of the retinal pigment epithelium (RPE), choroid, choriocapillaris, and photoreceptors. We examined photoreceptor structure in a series of subjects with choroideremia with particular attention to areas bordering atrophic lesions. Methods Twelve males with clinically-diagnosed choroideremia and confirmed hemizygous mutations in the CHM gene were examined. High-resolution images of the retina were obtained using spectral domain optical coherence tomography (SD-OCT) and both confocal and non-confocal split-detector adaptive optics scanning light ophthalmoscope (AOSLO) techniques. Results Eleven CHM gene mutations (3 novel) were identified; three subjects had the same mutation and one subject had two mutations. SD-OCT findings included interdigitation zone (IZ) attenuation or loss in 10/12 subjects, often in areas with intact ellipsoid zones; RPE thinning in all subjects; interlaminar bridges in the imaged areas of 10/12 subjects; and outer retinal tubulations (ORTs) in 10/12 subjects. Only split-detector AOSLO could reliably resolve cones near lesion borders, and such cones were abnormally heterogeneous in morphology, diameter and density. On split-detector imaging, the cone mosaic terminated sharply at lesion borders in 5/5 cases examined. Split-detector imaging detected remnant cone inner segments within ORTs, which were generally contiguous with a central patch of preserved retina. Conclusions Early IZ dropout and RPE thinning on SD-OCT are consistent with previously published results. Evidence of remnant cone inner segments within ORTs and the continuity of the ORTs with preserved retina suggests that these may represent an intermediate state of retinal degeneration prior to complete atrophy. Taken together, these results supports a model of choroideremia in which the RPE degenerates before photoreceptors. PMID:27936069

  6. Can subretinal microphotodiodes successfully replace degenerated photoreceptors?

    PubMed

    Zrenner, E; Stett, A; Weiss, S; Aramant, R B; Guenther, E; Kohler, K; Miliczek, K D; Seiler, M J; Haemmerle, H

    1999-07-01

    The idea of implanting microphotodiode arrays as visual prostheses has aroused controversy on its feasibility from the moment it appeared in print. We now present results which basically support the concept of replacing damaged photoreceptors with subretinally implanted stimulation devices. Network activity in degenerated rat retinae could be modulated through local electrical stimulation in vitro. We also investigated the long term stability and biocompatibility of the subretinal implants and their impact on retinal physiology in rats. Ganzfeld electroretinograms and histology showed no significant side effect of subretinal implants on retinal function or the architecture of the inner retina.

  7. R7 Photoreceptor Specification in the Developing Drosophila Eye: The Role of the Transcription Factor Deadpan.

    PubMed

    Mavromatakis, Yannis Emmanuel; Tomlinson, Andrew

    2016-07-01

    As cells proceed along their developmental pathways they make a series of sequential cell fate decisions. Each of those decisions needs to be made in a robust manner so there is no ambiguity in the state of the cell as it proceeds to the next stage. Here we examine the decision made by the Drosophila R7 precursor cell to become a photoreceptor and ask how the robustness of that decision is achieved. The transcription factor Tramtrack (Ttk) inhibits photoreceptor assignment, and previous studies found that the RTK-induced degradation of Ttk was critically required for R7 specification. Here we find that the transcription factor Deadpan (Dpn) is also required; it is needed to silence ttk transcription, and only when Ttk protein degradation and transcriptional silencing occur together is the photoreceptor fate robustly achieved. Dpn expression needs to be tightly restricted to R7 precursors, and we describe the role played by Ttk in repressing dpn transcription. Thus, Dpn and Ttk act as mutually repressive transcription factors, with Dpn acting to ensure that Ttk is effectively removed from R7, and Ttk acting to prevent Dpn expression in other cells. Furthermore, we find that N activity is required to promote dpn transcription, and only in R7 precursors does the removal of Ttk coincide with high N activity, and only in this cell does Dpn expression result.

  8. R7 Photoreceptor Specification in the Developing Drosophila Eye: The Role of the Transcription Factor Deadpan

    PubMed Central

    Mavromatakis, Yannis Emmanuel; Tomlinson, Andrew

    2016-01-01

    As cells proceed along their developmental pathways they make a series of sequential cell fate decisions. Each of those decisions needs to be made in a robust manner so there is no ambiguity in the state of the cell as it proceeds to the next stage. Here we examine the decision made by the Drosophila R7 precursor cell to become a photoreceptor and ask how the robustness of that decision is achieved. The transcription factor Tramtrack (Ttk) inhibits photoreceptor assignment, and previous studies found that the RTK-induced degradation of Ttk was critically required for R7 specification. Here we find that the transcription factor Deadpan (Dpn) is also required; it is needed to silence ttk transcription, and only when Ttk protein degradation and transcriptional silencing occur together is the photoreceptor fate robustly achieved. Dpn expression needs to be tightly restricted to R7 precursors, and we describe the role played by Ttk in repressing dpn transcription. Thus, Dpn and Ttk act as mutually repressive transcription factors, with Dpn acting to ensure that Ttk is effectively removed from R7, and Ttk acting to prevent Dpn expression in other cells. Furthermore, we find that N activity is required to promote dpn transcription, and only in R7 precursors does the removal of Ttk coincide with high N activity, and only in this cell does Dpn expression result. PMID:27427987

  9. Enhanced cell viability and cell adhesion using low conductivity medium for negative dielectrophoretic cell patterning.

    PubMed

    Puttaswamy, Srinivasu Valagerahally; Sivashankar, Shilpa; Chen, Rong-Jhe; Chin, Chung-Kuang; Chang, Hwan-You; Liu, Cheng Hsien

    2010-10-01

    Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.

  10. Plasma-Sprayed Titanium Patterns for Enhancing Early Cell Responses

    NASA Astrophysics Data System (ADS)

    Shi, Yunqi; Xie, Youtao; Pan, Houhua; Zheng, Xuebin; Huang, Liping; Ji, Fang; Li, Kai

    2016-06-01

    Titanium coating has been widely used as a biocompatible metal in biomedical applications. However, the early cell responses and long-term fixation of titanium implants are not satisfied. To obviate these defects, in this paper, micro-post arrays with various widths (150-1000 μm) and intervals (100-300 μm) were fabricated on the titanium substrate by template-assisted plasma spraying technology. In vitro cell culture experiments showed that MC3T3-E1 cells exhibited significantly higher osteogenic differentiation as well as slightly improved adhesion and proliferation on the micro-patterned coatings compared with the traditional one. The cell number on the pattern with 1000 µm width reached 130% after 6 days of incubation, and the expressions of osteopontin (OPN) as well as osteocalcin (OC) were doubled. No obvious difference was found in cell adhesion on various size patterns. The present micro-patterned coatings proposed a new modification method for the traditional plasma spraying technology to enhance the early cell responses and convenience for the bone in-growth.

  11. Cell patterning for liver tissue engineering via dielectrophoretic mechanisms.

    PubMed

    Yahya, Wan Nurlina Wan; Kadri, Nahrizul Adib; Ibrahim, Fatimah

    2014-07-02

    Liver transplantation is the most common treatment for patients with end-stage liver failure. However, liver transplantation is greatly limited by a shortage of donors. Liver tissue engineering may offer an alternative by providing an implantable engineered liver. Currently, diverse types of engineering approaches for in vitro liver cell culture are available, including scaffold-based methods, microfluidic platforms, and micropatterning techniques. Active cell patterning via dielectrophoretic (DEP) force showed some advantages over other methods, including high speed, ease of handling, high precision and being label-free. This article summarizes liver function and regenerative mechanisms for better understanding in developing engineered liver. We then review recent advances in liver tissue engineering techniques and focus on DEP-based cell patterning, including microelectrode design and patterning configuration.

  12. Cell Patterning for Liver Tissue Engineering via Dielectrophoretic Mechanisms

    PubMed Central

    Yahya, Wan Nurlina Wan; Kadri, Nahrizul Adib; Ibrahim, Fatimah

    2014-01-01

    Liver transplantation is the most common treatment for patients with end-stage liver failure. However, liver transplantation is greatly limited by a shortage of donors. Liver tissue engineering may offer an alternative by providing an implantable engineered liver. Currently, diverse types of engineering approaches for in vitro liver cell culture are available, including scaffold-based methods, microfluidic platforms, and micropatterning techniques. Active cell patterning via dielectrophoretic (DEP) force showed some advantages over other methods, including high speed, ease of handling, high precision and being label-free. This article summarizes liver function and regenerative mechanisms for better understanding in developing engineered liver. We then review recent advances in liver tissue engineering techniques and focus on DEP-based cell patterning, including microelectrode design and patterning configuration. PMID:24991941

  13. Intestinal stem cell replacement follows a pattern of neutral drift.

    PubMed

    Lopez-Garcia, Carlos; Klein, Allon M; Simons, Benjamin D; Winton, Douglas J

    2010-11-05

    With the capacity for rapid self-renewal and regeneration, the intestinal epithelium is stereotypical of stem cell-supported tissues. Yet the pattern of stem cell turnover remains in question. Applying analytical methods from population dynamics and statistical physics to an inducible genetic labeling system, we showed that clone size distributions conform to a distinctive scaling behavior at short times. This result demonstrates that intestinal stem cells form an equipotent population in which the loss of a stem cell is compensated by the multiplication of a neighbor, leading to neutral drift dynamics in which clones expand and contract at random until they either take over the crypt or they are lost. Combined with long-term clonal fate data, we show that the rate of stem cell replacement is comparable to the cell division rate, implying that neutral drift and symmetrical cell divisions are central to stem cell homeostasis.

  14. Patterned Thermoresponsive Microgel Coatings for Noninvasive Processing of Adherent Cells.

    PubMed

    Uhlig, Katja; Wegener, Thomas; He, Jian; Zeiser, Michael; Bookhold, Johannes; Dewald, Inna; Godino, Neus; Jaeger, Magnus; Hellweg, Thomas; Fery, Andreas; Duschl, Claus

    2016-03-14

    Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments.

  15. INTERACTION AND LOCALIZATION OF THE RETINITIS PIGMENTOSA PROTEIN RP2 AND NSF IN RETINAL PHOTORECEPTOR CELLS†

    PubMed Central

    Holopainen, Juha M.; Cheng, Christiana L.; Molday, Laurie L.; Johal, Gurp; Coleman, Jonathan; Dyka, Frank; Hii, Theresa; Ahn, Jinhi; Molday, Robert S.

    2010-01-01

    RP2 is a ubiquitously expressed protein encoded by a gene associated with X-linked retinitis pigmentosa (XLRP), a retinal degenerative disease that causes severe vision loss. Previous in vitro studies have shown that RP2 binds to ADP ribosylation factor-like 3 (Arl3) and activates its intrinsic GTPase activity, but the function of RP2 in the retina, and in particular photoreceptor cells, remains unclear. To begin to define the role of RP2 in the retina and XLRP, we have carried out biochemical studies to identify proteins in retinal cell extracts that interact with RP2. Here, we show that RP2 interacts with N-ethylmaleimide sensitive factor (NSF) in retinal cells as well as cultured embryonic kidney (HEK293) cells by mass spectrometry-based proteomics and biochemical analysis. This interaction is mediated by the N-terminal domain of NSF. The E138G and ΔI137 mutations of RP2 known to cause XLRP abolished the interaction of RP2 with the N-terminal domain of NSF. Immunofluorescence labeling studies further showed that RP2 co-localized with NSF in photoreceptors and other cells of the retina. Intense punctate staining of RP2 was observed close to the junction between the inner and outer segments beneath the connecting cilium, as well as within the synaptic region of rod and cone photoreceptors. Our studies indicate that RP2, in addition to serving as a regulator of Arl3, interacts with NSF and this complex may play an important role in membrane protein trafficking in photoreceptors and other cells of the retina. PMID:20669900

  16. NADPH Oxidase Contributes to Photoreceptor Degeneration in Constitutively Active RAC1 Mice

    PubMed Central

    Song, Hongman; Vijayasarathy, Camasamudram; Zeng, Yong; Marangoni, Dario; Bush, Ronald A.; Wu, Zhijian; Sieving, Paul A.

    2016-01-01

    Purpose The active form of small GTPase RAC1 is required for activation of NADPH oxidase (NOX), which in turn generates reactive oxygen species (ROS) in nonphagocytic cells. We explored whether NOX-induced oxidative stress contributes to rod degeneration in retinas expressing constitutively active (CA) RAC1. Methods Transgenic (Tg)–CA-RAC1 mice were given apocynin (10 mg/kg, intraperitoneal), a NOX inhibitor, or vehicle daily for up to 13 weeks. Superoxide production and oxidative damage were assessed by dihydroethidium staining and by protein carbonyls and malondialdehyde levels, respectively. Outer nuclear layer (ONL) cells were counted and electroretinogram (ERG) amplitudes measured in Tg-CA-RAC1 mice. Outer nuclear layer cells were counted in wild-type (WT) mice after transfer of CA-Rac1 gene by subretinal injection of AAV8-pOpsin-CA Rac1-GFP. Results Transgenic-CA-RAC1 retinas had significantly fewer photoreceptor cells and more apoptotic ONL cells than WT controls from postnatal week (Pw) 3 to Pw13. Superoxide accumulation and protein and lipid oxidation were increased in Tg-CA-RAC1 retinas and were reduced in mice treated with apocynin. Apocynin reduced the loss of photoreceptors and increased the rod ERG a- and b-wave amplitudes when compared with vehicle-injected transgenic controls. Photoreceptor loss was also observed in regions of adult WT retina transduced with AAV8-pOpsin-CA Rac1-GFP but not in neighboring regions that were not transduced or in AAV8-pOpsin-GFP–transduced retinas. Conclusions Constitutively active RAC1 promotes photoreceptor cell death by oxidative damage that occurs, at least partially, through NOX-induced ROS. Reactive oxygen species are likely involved in multiple forms of retinal degenerations, and our results support investigating RAC1 inhibition as a therapeutic approach that targets this disease pathway. PMID:27233035

  17. Modulation of cell adhesion complexes by surface protein patterns.

    PubMed

    Pesen, Devrim; Haviland, David B

    2009-03-01

    Cell adhesion is an important process in several biological phenomena. To investigate the formation and organization of focal adhesions, we developed a patterning approach based on electron beam lithography. Nanodots (radius <1230 nm) and nanorings (inner radius <320 nm) of fibronectin (FN) were patterned on a K-Casein background. Intracellular vinculin immunofluorescence mirrored the FN nanopatterns. Atomic force microscopy showed that FN nanodots and nanorings organize the immediate cytoskeleton into straight fibrils and diverging fibril bundles, respectively. Our results suggest that a minimum of approximately 40 FN molecules is required for a cell to form a focal adhesion.

  18. Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization

    PubMed Central

    Mittelmeier, Telsa M.; Boyd, Joseph S.; Lamb, Mary Rose

    2011-01-01

    The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules. In wild-type cells, ChR1 was observed in an equatorial patch adjacent to D4 near the end of the acetylated microtubules and along the D4 rootlet. In cells with cytoskeletal protein mutations, supernumerary ChR1 patches remained adjacent to acetylated microtubules. In mlt1 (multieyed) mutant cells, supernumerary photoreceptor patches were not restricted to the D4 rootlet, and more anterior eyespots correlated with shorter acetylated microtubule rootlets. The data suggest a model in which photoreceptor localization is dependent on microtubule-based trafficking selective for the D4 rootlet, which is perturbed in mlt1 mutant cells. PMID:21555459

  19. Spectroscopic characterization of the Stentor photoreceptor.

    PubMed

    Walker, E B; Lee, T Y; Song, P S

    1979-09-20

    1. On the basis of chromatographic and spectroscopic (absorption, fluorescence and its polarization, fluorescence lifetime, circular dichroism) characterization of the Stentor photoreceptor (stentorin) for photophobic response, the photoreceptor chromophore released from mild acid hydrolysis has been identified as hypericin. 2. The native chromophore is apparently linked to a protein (65 K) containing Lys and several hydrophobic residues, which is soluble in acetone and n-pentane. The peptide-linked stentorin (I) chromophore exhibits circular dichroism in the visible region due to the induced optical activity provided by the peptide. 3. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of a 38% fraction of the sucrose density centrifugation has resolved stentorin II proteins having molecular weights of 13 000, 16 000, 65 000 and 130 000. These proteins, as well as the acetone-soluble peptide, have been spectroscopically characterized with particular emphasis on their primary photoreactivity as the photophobic receptor of Stentor coeruleus. 4. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium. A strong dependence upon pH in the fluorescence spectra of both synthetic and native chromophores is also evident, showing a significant drop in the pKa of one or more hydroxyl groups in the excited state. A mechanism for the photophobic response, based on this lowering of the pKa as the primary photoprocess, has been discussed.

  20. Modeling cell-death patterning during biofilm formation

    NASA Astrophysics Data System (ADS)

    Ghosh, Pushpita; Ben-Jacob, Eshel; Levine, Herbert

    2013-12-01

    Self-organization by bacterial cells often leads to the formation of a highly complex spatially-structured biofilm. In such a bacterial biofilm, cells adhere to each other and are embedded in a self-produced extracellular matrix (ECM). Bacillus substilis bacteria utilize localized cell-death patterns which focuses mechanical forces to form wrinkled sheet-like structures in three dimensions. A most intriguing feature underlying this biofilm formation is that vertical buckling and ridge location is biased to occur in region of high cell-death. Here we present a spatially extended model to investigate the role of the bacterial secreted ECM during the biofilm formation and the self-organization of cell-death. Using this reaction-diffusion model we show that the interaction between the cell's motion and the ECM concentration gives rise to a self-trapping instability, leading to variety of cell-death patterns. The resultant spot patterns generated by our model are shown to be in semi-quantitative agreement with recent experimental observation.

  1. Multiscale patterned transplantable stem cell patches for bone tissue regeneration.

    PubMed

    Kim, Jangho; Bae, Won-Gyu; Choung, Han-Wool; Lim, Ki Taek; Seonwoo, Hoon; Jeong, Hoon Eui; Suh, Khap-Yang; Jeon, Noo Li; Choung, Pill-Hoon; Chung, Jong Hoon

    2014-11-01

    Stem cell-based therapy has been proposed as an enabling alternative not only for the treatment of diseases but also for the regeneration of tissues beyond complex surgical treatments or tissue transplantation. In this study, we approached a conceptual platform that can integrate stem cells into a multiscale patterned substrate for bone regeneration. Inspired by human bone tissue, we developed hierarchically micro- and nanopatterned transplantable patches as synthetic extracellular matrices by employing capillary force lithography in combination with a surface micro-wrinkling method using a poly(lactic-co-glycolic acid) (PLGA) polymer. The multiscale patterned PLGA patches were highly flexible and showed higher tissue adhesion to the underlying tissue than did the single nanopatterned patches. In response to the anisotropically multiscale patterned topography, the adhesion and differentiation of human mesenchymal stem cells (hMSCs) were sensitively controlled. Furthermore, the stem cell patch composed of hMSCs and transplantable PLGA substrate promoted bone regeneration in vivo when both the micro- and nanotopography of the substrate surfaces were synergistically combined. Thus, our study concludes that multiscale patterned transplantable stem cell patches may have a great potential for bone regeneration as well as for various regenerative medicine approaches.

  2. Generation of Viable Cell and Biomaterial Patterns by Laser Transfer

    NASA Astrophysics Data System (ADS)

    Ringeisen, Bradley

    2001-03-01

    In order to fabricate and interface biological systems for next generation applications such as biosensors, protein recognition microarrays, and engineered tissues, it is imperative to have a method of accurately and rapidly depositing different active biomaterials in patterns or layered structures. Ideally, the biomaterial structures would also be compatible with many different substrates including technologically relevant platforms such as electronic circuits or various detection devices. We have developed a novel laser-based technique, termed matrix assisted pulsed laser evaporation direct write (MAPLE DW), that is able to direct write patterns and three-dimensional structures of numerous biologically active species ranging from proteins and antibodies to living cells. Specifically, we have shown that MAPLE DW is capable of forming mesoscopic patterns of living prokaryotic cells (E. coli bacteria), living mammalian cells (Chinese hamster ovaries), active proteins (biotinylated bovine serum albumin, horse radish peroxidase), and antibodies specific to a variety of classes of cancer related proteins including intracellular and extracellular matrix proteins, signaling proteins, cell cycle proteins, growth factors, and growth factor receptors. In addition, patterns of viable cells and active biomolecules were deposited on different substrates including metals, semiconductors, nutrient agar, and functionalized glass slides. We will present an explanation of the laser-based transfer mechanism as well as results from our recent efforts to fabricate protein recognition microarrays and tissue-based microfluidic networks.

  3. Postembryonic developmental changes in photoreceptors of the stick insect Carausius morosus enhance the shift to an adult nocturnal life-style.

    PubMed

    Frolov, Roman; Immonen, Esa-Ville; Vähäsöyrinki, Mikko; Weckström, Matti

    2012-11-21

    Optimization of sensory processing during development can be studied by using photoreceptors of hemimetabolous insects (with incomplete metamorphosis) as a research model. We have addressed this topic in the stick insect Carausius morosus, where retinal growth after hatching is accompanied by a diurnal-to-nocturnal shift in behavior, by recording from photoreceptors of first instar nymphs and adult animals using the patch-clamp method. In the nymphs, ommatidia were smaller and photoreceptors were on average 15-fold less sensitive to light than in adults. The magnitude of A-type K(+) current did not increase but the delayed rectifier doubled in adults compared with nymphs, the K(+) current densities being greater in the nymphs. By contrast, the density of light-induced current did not increase, although its magnitude increased 8.6-fold, probably due to the growth of microvilli. Nymph photoreceptors performed poorly, demonstrating a peak information rate (IR) of 2.9 ± 0.7 bits/s versus 34.1 ± 5.0 bits/s in adults in response to white-noise stimulation. Strong correlations were found between photoreceptor capacitance (a proxy for cell size) and IR, and between light sensitivity and IR, with larger and more sensitive photoreceptors performing better. In adults, IR peaked at light intensities matching irradiation from the evening sky. Our results indicate that biophysical properties of photoreceptors at each age stage and visual behavior are interdependent and that developmental improvement in photoreceptor performance may facilitate the switch from the diurnal to the safer nocturnal lifestyle. This also has implications for how photoreceptors achieve optimal performance.

  4. Patterned cell arrays and patterned co-cultures on polydopamine-modified poly(vinyl alcohol) hydrogels.

    PubMed

    Beckwith, Kai M; Sikorski, Pawel

    2013-12-01

    Live cell arrays are an emerging tool that expand traditional 2D in vitro cell culture, increasing experimental precision and throughput. A patterned cell system was developed by combining the cell-repellent properties of polyvinyl alcohol hydrogels with the cell adhesive properties of self-assembled films of dopamine (polydopamine). It was shown that polydopamine could be patterned onto spin-cast polyvinyl alcohol hydrogels by microcontact printing, which in turn effectively patterned the growth of several cell types (HeLa, human embryonic kidney, human umbilical vein endothelial cells (HUVEC) and prostate cancer). The cells could be patterned in geometries down to single-cell confinement, and it was demonstrated that cell patterns could be maintained for at least 3 weeks. Furthermore, polydopamine could be used to modify poly(vinyl alcohol) in situ using a cell-compatible deposition buffer (1 mg mL(-1) dopamine in 25 mM tris with a physiological salt balance). The treatment switched the PVA hydrogel from cell repellent to cell adhesive. Finally, by combining microcontact printing and in situ deposition of polydopamine, patterned co-cultures of the same cell type (HeLa/HeLa) and dissimilar cell types (HeLa/HUVEC) were realized through simple chemistry and could be studied over time. The combination of polyvinyl alcohol and polydopamine was shown to be an attractive route to versatile, patterned cell culture experiments with minimal infrastructure requirements and low complexity.

  5. Unique system of photoreceptors in sea urchin tube feet

    PubMed Central

    Ullrich-Lüter, Esther M; Dupont, Sam; Arboleda, Enrique; Hausen, Harald; Arnone, Maria Ina

    2011-01-01

    Different sea urchin species show a vast variety of responses to variations in light intensity; however, despite this behavioral evidence for photosensitivity, light sensing in these animals has remained an enigma. Genome information of the recently sequenced purple sea urchin (Strongylocentrotus purpuratus) allowed us to address this question from a previously unexplored molecular perspective by localizing expression of the rhabdomeric opsin Sp-opsin4 and Sp-pax6, two genes essential for photoreceptor function and development, respectively. Using a specifically designed antibody against Sp-Opsin4 and in situ hybridization for both genes, we detected expression in two distinct groups of photoreceptor cells (PRCs) located in the animal's numerous tube feet. Specific reactivity of the Sp-Opsin4 antibody with sea star optic cushions, which regulate phototaxis, suggests a similar visual function in sea urchins. Ultrastructural characterization of the sea urchin PRCs revealed them to be of a microvillar receptor type. Our data suggest that echinoderms, in contrast to chordates, deploy a microvillar, r-opsin–expressing PRC type for vision, a feature that has been so far documented only in protostome animals. Surprisingly, sea urchin PRCs lack any associated screening pigment. Indeed, one of the tube foot PRC clusters may account for directional vision by being shaded through the opaque calcite skeleton. The PRC axons connect to the animal internal nervous system, suggesting an integrative function beyond local short circuits. Because juveniles display no phototaxis until skeleton completion, we suggest a model in which the entire sea urchin, deploying its skeleton as PRC screening device, functions as a huge compound eye. PMID:21536888

  6. Mechanisms Underlying Stage-1 TRPL Channel Translocation in Drosophila Photoreceptors

    PubMed Central

    Lieu, Minh-Ha; Vallejos, Maximiliano J.; Michael, Emily; Tsunoda, Susan

    2012-01-01

    Background TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere), TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels. Methodology/Principal Findings We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content. Conclusions/Significance Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protein transport, and not degradation/re-synthesis. Re-localization from each stage to the rhabdomere likely involves different strategies. Since TRPL channels can translocate to stage-1 in the absence of ATP, with no major requirement of the cytoskeleton, we suggest that stage-1 translocation involves simple diffusion through the apical membrane, which may be regulated by release of a light-dependent anchor in

  7. Control of a four-color sensing photoreceptor by a two-color sensing photoreceptor reveals complex light regulation in cyanobacteria.

    PubMed

    Bussell, Adam N; Kehoe, David M

    2013-07-30

    Photoreceptors are biologically important for sensing changes in the color and intensity of ambient light and, for photosynthetic organisms, processing this light information to optimize food production through photosynthesis. Cyanobacteria are an evolutionarily and ecologically important prokaryotic group of oxygenic photosynthesizers that contain cyanobacteriochrome (CBCR) photoreceptors, whose family members sense nearly the entire visible spectrum of light colors. Some cyanobacteria contain 12 to 15 different CBCRs, and many family members contain multiple light-sensing domains. However, the complex interactions that must be occurring within and between these photoreceptors remain unexplored. Here we describe the regulation and photobiology of a unique CBCR called IflA (influenced by far-red light), demonstrating that a second CBCR called RcaE strongly regulates IflA abundance and that IflA uses two distinct photosensory domains to respond to four different light colors: blue, green, red, and far-red. The absorption of red or far-red light by one domain affects the conformation of the other domain, and the rate of relaxation of one of these domains is influenced by the conformation of the other. Deletion of iflA results in delayed growth at low cell density, suggesting that IflA accelerates growth under this condition, apparently by sensing the ratio of red to far-red light in the environment. The types of complex photobiological interactions described here, both between unrelated CBCR family members and within photosensory domains of a single CBCR, may be advantageous for species using these photoreceptors in aquatic environments, where light color ratios are influenced by many biotic and abiotic factors.

  8. Evolution of photosensory pineal organs in new light: the fate of neuroendocrine photoreceptors.

    PubMed Central

    Ekström, Peter; Meissl, Hilmar

    2003-01-01

    Pineal evolution is envisaged as a gradual transformation of pinealocytes (a gradual regression of pinealocyte sensory capacity within a particular cell line), the so-called sensory cell line of the pineal organ. In most non-mammals the pineal organ is a directly photosensory organ, while the pineal organ of mammals (epiphysis cerebri) is a non-sensory neuroendocrine organ under photoperiod control. The phylogenetic transformation of the pineal organ is reflected in the morphology and physiology of the main parenchymal cell type, the pinealocyte. In anamniotes, pinealocytes with retinal cone photoreceptor-like characteristics predominate, whereas in sauropsids so-called rudimentary photoreceptors predominate. These have well-developed secretory characteristics, and have been interpreted as intermediaries between the anamniote pineal photoreceptors and the mammalian non-sensory pinealocytes. We have re-examined the original studies on which the gradual transformation hypothesis of pineal evolution is based, and found that the evidence for this model of pineal evolution is ambiguous. In the light of recent advances in the understanding of neural development mechanisms, we propose a new hypothesis of pineal evolution, in which the old notion 'gradual regression within the sensory cell line' should be replaced with 'changes in fate restriction within the neural lineage of the pineal field'. PMID:14561326

  9. Two components of photoreceptor potential in phototaxis of the flagellated green alga Haematococcus pluvialis

    PubMed Central

    Sineshchekov, Oleg A.; Litvin, Felix F.; Keszthelyi, Lajos

    1990-01-01

    The kinetics of the photoreceptor potential of phototaxis in biflagellated green alga Haematococcus pluvialis in response to a 10-ns laser pulse of three wavelengths (465, 550, and 590 nm) were measured in single cells with 30 μs time resolution. The rise and the decay of photoinduced potential are both at least biphasic. The first component of the rise is very stable and has no measurable (<30 μs) time delay. The second component is triggered after a 120-400-μs lag period, depending on flash intensity. Its appearance is sensitive to the physiological state of the cell and the amplitude can be increased by phototactically ineffective red background illumination. The electrical generators for both components are localized in the same region of the cell membrane (on the stigma-bearing side) and these components have the same depolarizing sign. The results indicate that the photoreceptor potential in phototaxis comprises two components, which could be interpreted as light-induced charge movement within the photoreceptor molecules and changes in ion permeability of the cell membrane. PMID:19431753

  10. Biological surface engineering: a simple system for cell pattern formation.

    PubMed

    Zhang, S; Yan, L; Altman, M; Lässle, M; Nugent, H; Frankel, F; Lauffenburger, D A; Whitesides, G M; Rich, A

    1999-07-01

    Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.

  11. Ectopic Expression of a Microbial-Type Rhodopsin Restores Visual Responses in Mice with Photoreceptor Degeneration

    PubMed Central

    Bi, Anding; Cui, Jinjuan; Ma, Yu-Ping; Olshevskaya, Elena; Pu, Mingliang; Dizhoor, Alexander M.; Pan, Zhuo-Hua

    2006-01-01

    Summary The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration. PMID:16600853

  12. Functional photoreceptor loss revealed with adaptive optics: an alternate cause of color blindness.

    PubMed

    Carroll, Joseph; Neitz, Maureen; Hofer, Heidi; Neitz, Jay; Williams, David R

    2004-06-01

    There is enormous variation in the X-linked L/M (long/middle wavelength sensitive) gene array underlying "normal" color vision in humans. This variability has been shown to underlie individual variation in color matching behavior. Recently, red-green color blindness has also been shown to be associated with distinctly different genotypes. This has opened the possibility that there may be important phenotypic differences within classically defined groups of color blind individuals. Here, adaptive optics retinal imaging has revealed a mechanism for producing dichromatic color vision in which the expression of a mutant cone photopigment gene leads to the loss of the entire corresponding class of cone photoreceptor cells. Previously, the theory that common forms of inherited color blindness could be caused by the loss of photoreceptor cells had been discounted. We confirm that remarkably, this loss of one-third of the cones does not impair any aspect of vision other than color.

  13. Divergent T-Cell Cytokine Patterns in Inflammatory Arthritis

    NASA Astrophysics Data System (ADS)

    Simon, A. K.; Seipelt, E.; Sieper, J.

    1994-08-01

    A major immunoregulatory mechanism in inflammatory infections and allergic diseases is the control of the balance of cytokines secreted by Th1/Th2 subsets of T helper (Th) cells. This might also be true in autoimmune diseases; a Th2 pattern that prevents an effective immune response in infections with intracellular bacteria may favor immunosuppression in autoimmune diseases. The pattern of cytokine expression was compared in the synovial tissue from patients with a typical autoimmune disease, rheumatoid arthritis, and with a disorder with similar synovial pathology but driven by persisting exogenous antigen, reactive arthritis. We screened 12 rheumatoid and 9 reactive arthritis synovial tissues by PCR and in situ hybridization for their expression of T-cell cytokines. The cytokine pattern differs significantly between the two diseases; rheumatoid arthritis samples express a Th1-like pattern whereas in reactive arthritis interferon γ expression is accompanied by that of interleukin 4. Studying the expression of cytokines by in situ hybridization confirmed the results found by PCR; they also show an extremely low frequency of cytokine-transcribing cells. In a double-staining experiment, it was demonstrated that interleukin 4 is made by CD4 cells. These experiments favor the possibility of therapeutic intervention in inflammatory rheumatic diseases by means of inhibitory cytokines.

  14. Development of Spatial Distribution Patterns by Biofilm Cells

    PubMed Central

    Haagensen, Janus A. J.; Hansen, Susse K.; Christensen, Bjarke B.; Molin, Søren

    2015-01-01

    Confined spatial patterns of microbial distribution are prevalent in nature, such as in microbial mats, soil communities, and water stream biofilms. The symbiotic two-species consortium of Pseudomonas putida and Acinetobacter sp. strain C6, originally isolated from a creosote-polluted aquifer, has evolved a distinct spatial organization in the laboratory that is characterized by an increased fitness and productivity. In this consortium, P. putida is reliant on microcolonies formed by Acinetobacter sp. C6, to which it attaches. Here we describe the processes that lead to the microcolony pattern by Acinetobacter sp. C6. Ecological spatial pattern analyses revealed that the microcolonies were not entirely randomly distributed and instead were arranged in a uniform pattern. Detailed time-lapse confocal microscopy at the single-cell level demonstrated that the spatial pattern was the result of an intriguing self-organization: small multicellular clusters moved along the surface to fuse with one another to form microcolonies. This active distribution capability was dependent on environmental factors (carbon source and oxygen) and historical contingency (formation of phenotypic variants). The findings of this study are discussed in the context of species distribution patterns observed in macroecology, and we summarize observations about the processes involved in coadaptation between P. putida and Acinetobacter sp. C6. Our results contribute to an understanding of spatial species distribution patterns as they are observed in nature, as well as the ecology of engineered communities that have the potential for enhanced and sustainable bioprocessing capacity. PMID:26116674

  15. Regular Patterns in Cerebellar Purkinje Cell Simple Spike Trains

    PubMed Central

    Shin, Soon-Lim; Hoebeek, Freek E.; Schonewille, Martijn; De Zeeuw, Chris I.; Aertsen, Ad; De Schutter, Erik

    2007-01-01

    Background Cerebellar Purkinje cells (PC) in vivo are commonly reported to generate irregular spike trains, documented by high coefficients of variation of interspike-intervals (ISI). In strong contrast, they fire very regularly in the in vitro slice preparation. We studied the nature of this difference in firing properties by focusing on short-term variability and its dependence on behavioral state. Methodology/Principal Findings Using an analysis based on CV2 values, we could isolate precise regular spiking patterns, lasting up to hundreds of milliseconds, in PC simple spike trains recorded in both anesthetized and awake rodents. Regular spike patterns, defined by low variability of successive ISIs, comprised over half of the spikes, showed a wide range of mean ISIs, and were affected by behavioral state and tactile stimulation. Interestingly, regular patterns often coincided in nearby Purkinje cells without precise synchronization of individual spikes. Regular patterns exclusively appeared during the up state of the PC membrane potential, while single ISIs occurred both during up and down states. Possible functional consequences of regular spike patterns were investigated by modeling the synaptic conductance in neurons of the deep cerebellar nuclei (DCN). Simulations showed that these regular patterns caused epochs of relatively constant synaptic conductance in DCN neurons. Conclusions/Significance Our findings indicate that the apparent irregularity in cerebellar PC simple spike trains in vivo is most likely caused by mixing of different regular spike patterns, separated by single long intervals, over time. We propose that PCs may signal information, at least in part, in regular spike patterns to downstream DCN neurons. PMID:17534435

  16. Nerve Cell Patterning Using Polytetrafluoroethylene and Poly-D-Lysine

    NASA Astrophysics Data System (ADS)

    Kusunoki, Masanobu; Togo, Yoshitaka; Wada, Takuto; Hashimoto, Yoshiya; Nishikawa, Hiroaki; Hontsu, Shigeki

    We developed a patterning technique for nerve cells that uses polytetrafluoroethylene (PTFE) and poly-D-lysine (PDL). The PDL layer on the PTFE substrate acted as a buffer for nerve cell adhesion, while Ar ion-irradiated areas on PTFE were used for separation of nerve cells. This structure was then used as a scaffold. A matrix circuit of nerve cells with dendrites or axons was subsequently observed on the scaffold after a week of cell culture. The Ar ion-irradiated area exhibited graphite or amorphous carbon, and PDL was detached from carbon grains by mechanical stress during cell culture. A possible explanation for the adhesion between PDL and PTFE is the van der Waals force. The stress between PDL/PTFE was reduced by the flexibility of PDL gel, which prevented separation of PDL from PTFE.

  17. Hydrogel microfluidics for the patterning of pluripotent stem cells

    PubMed Central

    Cosson, S.; Lutolf, M. P.

    2014-01-01

    Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We demonstrate the spatiotemporally controlled neuronal commitment of mouse embryonic stem cells via delivery of retinoic acid gradients. This technique should be useful for testing the effect of dose and timing of biomolecules, singly or in combination, on stem cell fate. PMID:24662945

  18. Ordered Patterns of Cell Shape and Orientational Correlation during Spontaneous Cell Migration

    PubMed Central

    Iwaya, Suguru; Sano, Masaki

    2008-01-01

    Background In the absence of stimuli, most motile eukaryotic cells move by spontaneously coordinating cell deformation with cell movement in the absence of stimuli. Yet little is known about how cells change their own shape and how cells coordinate the deformation and movement. Here, we investigated the mechanism of spontaneous cell migration by using computational analyses. Methodology We observed spontaneously migrating Dictyostelium cells in both a vegetative state (round cell shape and slow motion) and starved one (elongated cell shape and fast motion). We then extracted regular patterns of morphological dynamics and the pattern-dependent systematic coordination with filamentous actin (F-actin) and cell movement by statistical dynamic analyses. Conclusions/Significance We found that Dictyostelium cells in both vegetative and starved states commonly organize their own shape into three ordered patterns, elongation, rotation, and oscillation, in the absence of external stimuli. Further, cells inactivated for PI3-kinase (PI3K) and/or PTEN did not show ordered patterns due to the lack of spatial control in pseudopodial formation in both the vegetative and starved states. We also found that spontaneous polarization was achieved in starved cells by asymmetric localization of PTEN and F-actin. This breaking of the symmetry of protein localization maintained the leading edge and considerably enhanced the persistence of directed migration, and overall random exploration was ensured by switching among the different ordered patterns. Our findings suggest that Dictyostelium cells spontaneously create the ordered patterns of cell shape mediated by PI3K/PTEN/F-actin and control the direction of cell movement by coordination with these patterns even in the absence of external stimuli. PMID:19011688

  19. Light-dependent channels from excised patches of Limulus ventral photoreceptors are opened by cGMP.

    PubMed Central

    Bacigalupo, J; Johnson, E C; Vergara, C; Lisman, J E

    1991-01-01

    The identity of the second messenger that directly activates the light-dependent conductance in invertebrate photoreceptors remains unclear; the available evidence provides some support for cGMP and Ca2+. To resolve this issue we have applied these second messengers to membrane patches excised from the light-sensitive lobe of Limulus ventral photoreceptors. Our results show that these patches contain channels that can be opened by cGMP, but not by Ca2+. These cGMP-activated channels closely resemble the channels activated by light in cell-attached patches. This evidence suggests that cGMP is the messenger that opens the light-dependent channel in invertebrate photoreceptors. PMID:1716765

  20. Pattern matching based active optical sorting of colloids/cells

    NASA Astrophysics Data System (ADS)

    Verma, R. S.; Dasgupta, R.; Ahlawat, S.; Kumar, N.; Uppal, A.; Gupta, P. K.

    2013-08-01

    We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.

  1. Regional differences in lectin binding patterns of vestibular hair cells

    NASA Technical Reports Server (NTRS)

    Baird, R. A.; Schuff, N. R.; Bancroft, J.

    1993-01-01

    Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

  2. Regional differences in lectin binding patterns of vestibular hair cells

    NASA Technical Reports Server (NTRS)

    Baird, Richard A.; Schuff, N. R.; Bancroft, J.

    1994-01-01

    Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not stain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type 1 hair cells while labeling, as in the bullfrog, Type 2 hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

  3. mTOR-mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice

    PubMed Central

    Zhao, Chen; Yasumura, Douglas; Li, Xiyan; Matthes, Michael; Lloyd, Marcia; Nielsen, Gregory; Ahern, Kelly; Snyder, Michael; Bok, Dean; Dunaief, Joshua L.; LaVail, Matthew M.; Vollrath, Douglas

    2010-01-01

    Retinal pigment epithelial (RPE) cell dysfunction plays a central role in various retinal degenerative diseases, but knowledge is limited regarding the pathways responsible for adult RPE stress responses in vivo. RPE mitochondrial dysfunction has been implicated in the pathogenesis of several forms of retinal degeneration. Here we have shown that postnatal ablation of RPE mitochondrial oxidative phosphorylation in mice triggers gradual epithelium dedifferentiation, typified by reduction of RPE-characteristic proteins and cellular hypertrophy. The electrical response of the retina to light decreased and photoreceptors eventually degenerated. Abnormal RPE cell behavior was associated with increased glycolysis and activation of, and dependence upon, the hepatocyte growth factor/met proto-oncogene pathway. RPE dedifferentiation and hypertrophy arose through stimulation of the AKT/mammalian target of rapamycin (AKT/mTOR) pathway. Administration of an oxidant to wild-type mice also caused RPE dedifferentiation and mTOR activation. Importantly, treatment with the mTOR inhibitor rapamycin blunted key aspects of dedifferentiation and preserved photoreceptor function for both insults. These results reveal an in vivo response of the mature RPE to diverse stressors that prolongs RPE cell survival at the expense of epithelial attributes and photoreceptor function. Our findings provide a rationale for mTOR pathway inhibition as a therapeutic strategy for retinal degenerative diseases involving RPE stress. PMID:21135502

  4. Patterning cells on optically transparent indium tin oxide electrodes.

    PubMed

    Shah, Sunny; Revzin, Alexander

    2007-01-01

    The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues. In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates. The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive. Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule. Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks. Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns. Micropatterning of several cell types was demonstrated on these substrates. In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

  5. Microspectrophotometry and the photoreceptor of Phycomyces I.

    PubMed

    Wolken, J J

    1969-11-01

    By applying microspectrophotometry to the sporangiophore of Phycomyces blakesleeanus wild-type and the albino car-10(-) type II, absorption spectra were obtained for 1- to 5-day cultures. Spectra in the growing-zone of the wild-type during Stage IVb, taken from 0.1 to 3 mm below the base of the sporangium, show two distinctly different spectra: one is more characteristic of a carotene, the other of a flavin. Combined, these absorption spectra reproduce closely the action spectrum. For the albino car-10(-), which is deficient in carotenes, only the spectrum characteristic of lumichrome or a reduced flavin was found. A c-type cytochrome was isolated from both strains which, if coupled with a flavin, could permit a photoreversible oxidation-reduction system. Birefringent crystals were observed to be aligned in the growing zone in which the photoreceptor is believed to lie. Micro-spectrophotometry of these crystals shows absorption peaks similar to those of riboflavin crystals.

  6. Conditional knockdown of DNA methyltransferase 1 reveals a key role of retinal pigment epithelium integrity in photoreceptor outer segment morphogenesis

    PubMed Central

    Nasonkin, Igor O.; Merbs, Shannath L.; Lazo, Kevin; Oliver, Verity F.; Brooks, Matthew; Patel, Krushangi; Enke, Raymond A.; Nellissery, Jacob; Jamrich, Milan; Le, Yun Z.; Bharti, Kapil; Fariss, Robert N.; Rachel, Rivka A.; Zack, Donald J.; Rodriguez-Boulan, Enrique J.; Swaroop, Anand

    2013-01-01

    Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease. PMID:23406904

  7. Visual transduction in human rod photoreceptors.

    PubMed

    Kraft, T W; Schneeweis, D M; Schnapf, J L

    1993-05-01

    1. Photocurrents were recorded with suction electrodes from rod photoreceptors of seven humans. 2. Brief flashes of light evoked transient outward currents of up to 20 pA. With increasing light intensity the peak response amplitude increased along an exponential saturation function. A half-saturating peak response was evoked by approximately sixty-five photoisomerizations. 3. Responses to brief dim flashes rose to a peak in about 200 ms. The waveform was roughly like the impulse response of a series of four to five low-pass filters. 4. The rising phases of the responses to flashes of increasing strength were found to fit with a biochemical model of phototransduction with an 'effective delay time' and 'characteristic time' of about 2 and 800 ms, respectively. 5. Spectral sensitivities were obtained over a wavelength range from 380 to 760 nm. The action spectrum, which peaked at 495 nm, followed the template described for photoreceptors in the macaque retina. Variation between rods in the position of the spectrum on the wavelength axis was small. 6. The scotopic luminosity function derived from human psychophysical experiments was found to agree well with the measured rod action spectrum after adjustments were made for lens absorption and photopigment self-screening in the intact eye. 7. Responses to steps of light rose monotonically to a maintained level, showing little or no relaxation. Nevertheless, the relationship between light intensity and steady-state response amplitude was shallower than that expected from simple response saturation. This is consistent with an adaptation mechanism acting on a rapid time scale. 8. Flash sensitivity fell with increasing intensities of background light according to Weber's law. Sensitivity was reduced twofold by lights evoking about 120 photoisomerizations per second. Background lights decreased the time to peak and the integration time of the flash response by up to 20%.

  8. Tuning cell adhesion by direct nanostructuring silicon into cell repulsive/adhesive patterns.

    PubMed

    Premnath, Priyatha; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

    2015-09-10

    Developing platforms that allow tuning cell functionality through incorporating physical, chemical, or mechanical cues onto the material surfaces is one of the key challenges in research in the field of biomaterials. In this respect, various approaches have been proposed and numerous structures have been developed on a variety of materials. Most of these approaches, however, demand a multistep process or post-chemical treatment. Therefore, a simple approach would be desirable to develop bio-functionalized platforms for effectively modulating cell adhesion and consequently programming cell functionality without requiring any chemical or biological surface treatment. This study introduces a versatile yet simple laser approach to structure silicon (Si) chips into cytophobic/cytophilic patterns in order to modulate cell adhesion and proliferation. These patterns are fabricated on platforms through direct laser processing of Si substrates, which renders a desired computer-generated configuration into patterns. We investigate the morphology, chemistry, and wettability of the platform surfaces. Subsequently, we study the functionality of the fabricated platforms on modulating cervical cancer cells (HeLa) behaviour. The results from in vitro studies suggest that the nanostructures efficiently repel HeLa cells and drive them to migrate onto untreated sites. The study of the morphology of the cells reveals that cells evade the cytophobic area by bending and changing direction. Additionally, cell patterning, cell directionality, cell channelling, and cell trapping are achieved by developing different platforms with specific patterns. The flexibility and controllability of this approach to effectively structure Si substrates to cell-repulsive and cell-adhesive patterns offer perceptible outlook for developing bio-functionalized platforms for a variety of biomedical devices. Moreover, this approach could pave the way for developing anti-cancer platforms that selectively repel

  9. Automatic cone photoreceptor segmentation using graph theory and dynamic programming.

    PubMed

    Chiu, Stephanie J; Lokhnygina, Yuliya; Dubis, Adam M; Dubra, Alfredo; Carroll, Joseph; Izatt, Joseph A; Farsiu, Sina

    2013-06-01

    Geometrical analysis of the photoreceptor mosaic can reveal subclinical ocular pathologies. In this paper, we describe a fully automatic algorithm to identify and segment photoreceptors in adaptive optics ophthalmoscope images of the photoreceptor mosaic. This method is an extension of our previously described closed contour segmentation framework based on graph theory and dynamic programming (GTDP). We validated the performance of the proposed algorithm by comparing it to the state-of-the-art technique on a large data set consisting of over 200,000 cones and posted the results online. We found that the GTDP method achieved a higher detection rate, decreasing the cone miss rate by over a factor of five.

  10. Laser-based techniques for living cell pattern formation

    NASA Astrophysics Data System (ADS)

    Hopp, Béla; Smausz, Tomi; Papdi, Bence; Bor, Zsolt; Szabó, András; Kolozsvári, Lajos; Fotakis, Costas; Nógrádi, Antal

    2008-10-01

    In the production of biosensors or artificial tissues a basic step is the immobilization of living cells along the required pattern. In this paper the ability of some promising laser-based methods to influence the interaction between cells and various surfaces is presented. In the first set of experiments laser-induced patterned photochemical modification of polymer foils was used to achieve guided adherence and growth of cells to the modified areas: (a) Polytetrafluoroethylene was irradiated with ArF excimer laser ( λ=193 nm, FWHM=20 ns, F=9 mJ/cm2) in presence of triethylene tetramine liquid photoreagent; (b) a thin carbon layer was produced by KrF excimer laser ( λ=248 nm, FWHM=30 ns, F=35 mJ/cm2) irradiation on polyimide surface to influence the cell adherence. It was found that the incorporation of amine groups in the PTFE polymer chain instead of the fluorine atoms can both promote and prevent the adherence of living cells (depending on the applied cell types) on the treated surfaces, while the laser generated carbon layer on polyimide surface did not effectively improve adherence. Our attempts to influence the cell adherence by morphological modifications created by ArF laser irradiation onto polyethylene terephtalate surface showed a surface roughness dependence. This method was effective only when the Ra roughness parameter of the developed structure did not exceed the 0.1 micrometer value. Pulsed laser deposition with femtosecond KrF excimer lasers ( F=2.2 J/cm2) was effectively used to deposit structured thin films from biomaterials (endothelial cell growth supplement and collagen embedded in starch matrix) to promote the adherence and growth of cells. These results present evidence that some surface can be successfully altered to induce guided cell growth.

  11. Spatially patterned matrix elasticity directs stem cell fate

    NASA Astrophysics Data System (ADS)

    Yang, Chun; DelRio, Frank W.; Ma, Hao; Killaars, Anouk R.; Basta, Lena P.; Kyburz, Kyle A.; Anseth, Kristi S.

    2016-08-01

    There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness.

  12. Spatially patterned matrix elasticity directs stem cell fate

    PubMed Central

    Yang, Chun; DelRio, Frank W.; Ma, Hao; Killaars, Anouk R.; Basta, Lena P.; Kyburz, Kyle A.; Anseth, Kristi S.

    2016-01-01

    There is a growing appreciation for the functional role of matrix mechanics in regulating stem cell self-renewal and differentiation processes. However, it is largely unknown how subcellular, spatial mechanical variations in the local extracellular environment mediate intracellular signal transduction and direct cell fate. Here, the effect of spatial distribution, magnitude, and organization of subcellular matrix mechanical properties on human mesenchymal stem cell (hMSCs) function was investigated. Exploiting a photodegradation reaction, a hydrogel cell culture substrate was fabricated with regions of spatially varied and distinct mechanical properties, which were subsequently mapped and quantified by atomic force microscopy (AFM). The variations in the underlying matrix mechanics were found to regulate cellular adhesion and transcriptional events. Highly spread, elongated morphologies and higher Yes-associated protein (YAP) activation were observed in hMSCs seeded on hydrogels with higher concentrations of stiff regions in a dose-dependent manner. However, when the spatial organization of the mechanically stiff regions was altered from a regular to randomized pattern, lower levels of YAP activation with smaller and more rounded cell morphologies were induced in hMSCs. We infer from these results that irregular, disorganized variations in matrix mechanics, compared with regular patterns, appear to disrupt actin organization, and lead to different cell fates; this was verified by observations of lower alkaline phosphatase (ALP) activity and higher expression of CD105, a stem cell marker, in hMSCs in random versus regular patterns of mechanical properties. Collectively, this material platform has allowed innovative experiments to elucidate a novel spatial mechanical dosing mechanism that correlates to both the magnitude and organization of spatial stiffness. PMID:27436901

  13. Effect of light intensity on flight control and temporal properties of photoreceptors in bumblebees.

    PubMed

    Reber, Therese; Vähäkainu, Antti; Baird, Emily; Weckström, Matti; Warrant, Eric; Dacke, Marie

    2015-05-01

    To control flight, insects rely on the pattern of visual motion generated on the retina as they move through the environment. When light levels fall, vision becomes less reliable and flight control thus becomes more challenging. Here, we investigated the effect of light intensity on flight control by filming the trajectories of free-flying bumblebees (Bombus terrestris, Linnaeus 1758) in an experimental tunnel at different light levels. As light levels fell, flight speed decreased and the flight trajectories became more tortuous but the bees were still remarkably good at centring their flight about the tunnel's midline. To investigate whether this robust flight performance can be explained by visual adaptations in the bumblebee retina, we also examined the response speed of the green-sensitive photoreceptors at the same light intensities. We found that the response speed of the photoreceptors significantly decreased as light levels fell. This indicates that bumblebees have both behavioural (reduction in flight speed) and retinal (reduction in response speed of the photoreceptors) adaptations to allow them to fly in dim light. However, the more tortuous flight paths recorded in dim light suggest that these adaptations do not support flight with the same precision during the twilight hours of the day.

  14. Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors

    SciTech Connect

    Baumann, O.; Walz, B. ); Somlyo, A.V.; Somlyo, A.P. )

    1991-02-01

    Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental compositon of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 {plus minus} 1.1 mmol/kg (dry weight). During a 3-sec nonsaturating light stimulus, {approximately}50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was {approximately}0.7. Our results show unambiguously that the ER is the source of Ca{sup 2+} release during cell stimulation and suggest the Mg{sup 2+} can nearly balance the charge movement of Ca{sup 2+}.

  15. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  16. Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces

    NASA Astrophysics Data System (ADS)

    Jeon, Hojeong; Koo, Sangmo; Reese, Willie Mae; Loskill, Peter; Grigoropoulos, Costas P.; Healy, Kevin E.

    2015-09-01

    Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells’ focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces.

  17. Volvoxrhodopsin, a light-regulated sensory photoreceptor of the spheroidal green alga Volvox carteri.

    PubMed Central

    Ebnet, E; Fischer, M; Deininger, W; Hegemann, P

    1999-01-01

    Somatic cells of the multicellular alga Volvox carteri contain a visual rhodopsin that controls the organism's phototactic behavior via two independent photoreceptor currents. Here, we report the identification of an opsinlike gene, designated as volvoxopsin (vop). The encoded protein exhibits homologies to the opsin of the unicellular alga Chlamydomonas reinhardtii (chlamyopsin) and to the entire animal opsin family, thus providing new perspectives on opsin evolution. Volvoxopsin accumulates within the eyes of somatic cells. However, the vop transcript is detectable only in the reproductive eyeless gonidia and embryos. vop mRNA levels increase 400-fold during embryogenesis, when embryos develop in darkness, whereas the vop transcript does not accumulate when embryos develop in the light. An antisense transformant, T3, was generated. This transformant produces 10 times less volvoxopsin than does the wild type. In T3, the vop transcript is virtually absent, whereas the antisense transcript is predominant and light regulated. It follows that vop expression is under light-dependent transcriptional control but that volvoxopsin itself is not the regulatory photoreceptor. Transformant T3 is phototactic, but its phototactic sensitivity is reduced 10-fold relative to the parental wild-type strain HK10. Thus, we offer definitive genetic evidence that a rhodopsin serves as the photoreceptor for phototaxis in a green alga. PMID:10449581

  18. Synthesis and patterning of tunable multiscale materials with engineered cells

    PubMed Central

    Chen, Allen Y.; Deng, Zhengtao; Billings, Amanda N.; Seker, Urartu O.S.; Lu, Michelle Y.; Citorik, Robert J.; Zakeri, Bijan; Lu, Timothy K.

    2014-01-01

    Many natural biological systems - such as biofilms, shells and skeletal tissues - are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells. PMID:24658114

  19. Tuning cell adhesion by direct nanostructuring silicon into cell repulsive/adhesive patterns

    SciTech Connect

    Premnath, Priyatha; Venkatakrishnan, Krishnan

    2015-09-10

    Developing platforms that allow tuning cell functionality through incorporating physical, chemical, or mechanical cues onto the material surfaces is one of the key challenges in research in the field of biomaterials. In this respect, various approaches have been proposed and numerous structures have been developed on a variety of materials. Most of these approaches, however, demand a multistep process or post-chemical treatment. Therefore, a simple approach would be desirable to develop bio-functionalized platforms for effectively modulating cell adhesion and consequently programming cell functionality without requiring any chemical or biological surface treatment. This study introduces a versatile yet simple laser approach to structure silicon (Si) chips into cytophobic/cytophilic patterns in order to modulate cell adhesion and proliferation. These patterns are fabricated on platforms through direct laser processing of Si substrates, which renders a desired computer-generated configuration into patterns. We investigate the morphology, chemistry, and wettability of the platform surfaces. Subsequently, we study the functionality of the fabricated platforms on modulating cervical cancer cells (HeLa) behaviour. The results from in vitro studies suggest that the nanostructures efficiently repel HeLa cells and drive them to migrate onto untreated sites. The study of the morphology of the cells reveals that cells evade the cytophobic area by bending and changing direction. Additionally, cell patterning, cell directionality, cell channelling, and cell trapping are achieved by developing different platforms with specific patterns. The flexibility and controllability of this approach to effectively structure Si substrates to cell-repulsive and cell-adhesive patterns offer perceptible outlook for developing bio-functionalized platforms for a variety of biomedical devices. Moreover, this approach could pave the way for developing anti-cancer platforms that selectively repel

  20. Control of cell nucleus shapes via micropillar patterns.

    PubMed

    Pan, Zhen; Yan, Ce; Peng, Rong; Zhao, Yingchun; He, Yao; Ding, Jiandong

    2012-02-01

    We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(D,L-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion.

  1. Estimating the Size of Onion Epidermal Cells from Diffraction Patterns

    NASA Astrophysics Data System (ADS)

    Groff, Jeffrey R.

    2012-10-01

    Bioscience and premedical profession students are a major demographic served by introductory physics courses at many colleges and universities. Exposing these students to biological applications of physical principles will help them to appreciate physics as a useful tool for their future professions. Here I describe an experiment suitable for introductory physics where principles of wave optics are applied to probe the size of onion epidermal cells. The epidermis tissue is composed of cells of relatively uniform size and shape (Fig. 1) so the tissue acts like a one-dimensional transmission diffraction grating. The diffraction patterns generated when a laser beam passes through the tissue (Fig. 2) are analyzed and an estimate of the average width of individual onion epidermal cells is calculated. The results are compared to direct measurements taken using a light microscope. The use of microscopes and plant-cell tissue slides creates opportunities for cross-discipline collaboration between physics and biology instructors.

  2. Innate Immune Pattern Recognition: A Cell Biological Perspective

    PubMed Central

    Brubaker, Sky W.; Bonham, Kevin S.; Zanoni, Ivan

    2016-01-01

    Receptors of the innate immune system detect conserved determinants of microbial and viral origin. Activation of these receptors initiates signaling events that culminate in an effective immune response. Recently, the view that innate immune signaling events rely on and operate within a complex cellular infrastructure has become an important framework for understanding the regulation of innate immunity. Compartmentalization within this infrastructure provides the cell with the ability to assign spatial information to microbial detection and regulate immune responses. Several cell biological processes play a role in the regulation of innate signaling responses; at the same time, innate si