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Sample records for adult mouse retina

  1. 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

  2. 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.

  3. Nestin Expression in the Adult Mouse Retina with Pharmaceutically Induced Retinal Degeneration

    PubMed Central

    2017-01-01

    The present study investigated the temporal pattern and cellular localization of nestin in the adult mouse retina with pharmaceutically induced retinal degeneration using N-methyl-N-nitrosourea (MNU). After a single intraperitoneal injection of MNU in 8-week-old C57BL/6 mice, the animals were sacrificed at 1, 3, 5, 7, and 21 days (n = 6, in each stage). The eyes were examined by means of immunohistochemical tests using nestin, ionized calcium-binding adaptor molecule (Iba-1), CD11b, F4/80, and glial fibrillary acidic protein (GFAP). Western blot analysis and manual cell counting were performed for quantification. Nestin expression was increased after MNU administration. Nestin+/Iba-1+ cells were migrated into outer nuclear layer (ONL) and peaked at day 3 post injection (PI). Nestin+/CD11b+ cells were also mainly identified in ONL at day 3 PI and peaked at day 5. Nestin+/F4/80+ cells were shown in the subretinal space and peaked at day 3 PI. Nestin+/GFAP+ cells were distinctly increased at day 1 PI and peaked at day 5 PI. The up-regulation of nestin expression after MNU administration in adult mouse retinal microglia, and monocyte/macrophage suggests that when retinal degeneration progresses, these cells may revert to a more developmentally immature state. Müller cells also showed reactive gliosis and differentiational changes. PMID:28049248

  4. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    PubMed Central

    Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

  5. Microglial cells in organotypic cultures of developing and adult mouse retina and their relationship with cell death.

    PubMed

    Ferrer-Martín, Rosa M; Martín-Oliva, David; Sierra, Ana; Carrasco, Maria-Carmen; Martín-Estebané, María; Calvente, Ruth; Marín-Teva, José L; Navascués, Julio; Cuadros, Miguel A

    2014-04-01

    Organotypic cultures of retinal explants allow the detailed analysis of microglial cells in a cellular microenvironment similar to that in the in situ retina, with the advantage of easy experimental manipulation. However, the in vitro culture causes changes in the retinal cytoarchitecture and induces a microglial response that may influence the results of these manipulations. The purpose of this study was to analyze the influence of the retinal age on changes in retinal cytoarchitecture, cell viability and death, and microglial phenotype and distribution throughout the in vitro culture of developing and adult retina explants. Explants from developing (3 and 10 postnatal days, P3 and P10) and adult (P60) mouse retinas were cultured for up to 10 days in vitro (div). Dead or dying cells were recognized by TUNEL staining, cell viability was determined by flow cytometry, and the numbers and distribution patterns of microglial cells were studied by flow cytometry and immunocytochemistry, respectively. The retinal cytoarchitecture was better preserved at prolonged culture times (10 div) in P10 retina explants than in P3 or adult explants. Particular patterns of cell viability and death were observed at each age: in general, explants from developing retinas showed higher cell viability and lower density of TUNEL-positive profiles versus adult retinas. The proportion of microglial cells relative to the whole population of retinal cells was higher in explants fixed immediately after their dissection (i.e., non-cultured) from adult retinas than in those from developing retinas. This proportion was always higher in non-cultured explants than in explants at 10 div, suggesting the death of some microglial cells during the culture. Activation of microglial cells, as revealed by their phenotypical appearance, was observed in both developing and adult retina explants from the beginning of the culture. Immunofluorescence with the anti-CD68 antibody showed that some activated

  6. Expression Atlas of the Deubiquitinating Enzymes in the Adult Mouse Retina, Their Evolutionary Diversification and Phenotypic Roles

    PubMed Central

    Esquerdo, Mariona; Grau-Bové, Xavier; Garanto, Alejandro; Toulis, Vasileios; Garcia-Monclús, Sílvia; Millo, Erica; López-Iniesta, Ma José; Abad-Morales, Víctor; Ruiz-Trillo, Iñaki; Marfany, Gemma

    2016-01-01

    Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders. PMID:26934049

  7. Thyroid Hormone Signaling in the Mouse Retina

    PubMed Central

    Arbogast, Patrick; Flamant, Frédéric; Godement, Pierre; Glösmann, Martin

    2016-01-01

    Thyroid hormone is a crucial regulator of gene expression in the developing and adult retina. Here we sought to map sites of thyroid hormone signaling at the cellular level using the transgenic FINDT3 reporter mouse model in which neurons express β-galactosidase (β-gal) under the control of a hybrid Gal4-TRα receptor when triiodothyronine (T3) and cofactors of thyroid receptor signaling are present. In the adult retina, nearly all neurons of the ganglion cell layer (GCL, ganglion cells and displaced amacrine cells) showed strong β-gal labeling. In the inner nuclear layer (INL), a minority of glycineric and GABAergic amacrine cells showed β-gal labeling, whereas the majority of amacrine cells were unlabeled. At the level of amacrine types, β-gal labeling was found in a large proportion of the glycinergic AII amacrines, but only in a small proportion of the cholinergic/GABAergic ‘starburst’ amacrines. At postnatal day 10, there also was a high density of strongly β-gal-labeled neurons in the GCL, but only few amacrine cells were labeled in the INL. There was no labeling of bipolar cells, horizontal cells and Müller glia cells at both stages. Most surprisingly, the photoreceptor somata in the outer nuclear layer also showed no β-gal label, although thyroid hormone is known to control cone opsin expression. This is the first record of thyroid hormone signaling in the inner retina of an adult mammal. We hypothesize that T3 levels in photoreceptors are below the detection threshold of the reporter system. The topographical distribution of β-gal-positive cells in the GCL follows the overall neuron distribution in that layer, with more T3-signaling cells in the ventral than the dorsal half-retina. PMID:27942035

  8. Radioadaptive Cytoprotective Pathways in the Mouse Retina

    NASA Technical Reports Server (NTRS)

    Zanello, Susana B.; Wotring, V.; Theriot, C.; Ploutz-Snyder, R.; Zhang, Y.; Wu, H.

    2010-01-01

    Exposure to cosmic radiation implies a risk of tissue degeneration. Radiation retinopathy is a complication of radiotherapy and exhibits common features with other retinopathies and neuropathies. Exposure to a low radiation dose elicits protective cellular events (radioadaptive response), reducing the stress of a subsequent higher dose. To assess the risk of radiation-induced retinal changes and the extent to which a small priming dose reduces this risk, we used a mouse model exposed to a source of Cs-137-gamma radiation. Gene expression profiling of retinas from non-irradiated control C57BL/6J mice (C) were compared to retinas from mice treated with a low 50 mGy dose (LD), a high 6 Gy dose (HD), and a combined treatment of 50 mGy (priming) and 6 Gy (challenge) doses (LHD). Whole retina RNA was isolated and expression analysis for selected genes performed by RTqPCR. Relevant target genes associated with cell death/survival, oxidative stress, cellular stress response and inflammation pathways, were analyzed. Cellular stress response genes were upregulated at 4 hr after the challenge dose in LHD retinas (Sirt1: 1.5 fold, Hsf1: 1.7 fold, Hspa1a: 2.5 fold; Hif1a: 1.8 fold, Bag1: 1.7). A similar trend was observed in LD animals. Most antioxidant enzymes (Hmox1, Sod2, Prdx1, Cygb, Cat1) and inflammatory mediators (NF B, Ptgs2 and Tgfb1) were upregulated in LHD and LD retinas. Expression of the pro-survival gene Bcl2 was upregulated in LD (6-fold) and LHD (4-fold) retinas. In conclusion, cytoprotective gene networks activation in the retina suggests a radioadaptive response to a priming irradiation dose, with mitigation of the deleterious effects of a subsequent high dose exposure. The enhancement of these cytoprotective mechanisms has potential value as a countermeasure to ocular alterations caused by radiation alone or in combination with other factors in spaceflight environments.

  9. TRPM3 expression in mouse retina.

    PubMed

    Brown, R Lane; Xiong, Wei-Hong; Peters, James H; Tekmen-Clark, Merve; Strycharska-Orczyk, Iwona; Reed, Brian T; Morgans, Catherine W; Duvoisin, Robert M

    2015-01-01

    Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(‑/‑) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

  10. The role of histamine in the retina: studies on the Hdc knockout mouse.

    PubMed

    Greferath, Ursula; Vessey, Kirstan A; Jobling, Andrew I; Mills, Samuel A; Bui, Bang V; He, Zheng; Nag, Nupur; Ohtsu, Hiroshi; Fletcher, Erica L

    2014-01-01

    The role of histamine in the retina is not well understood, despite it regulating a number of functions within the brain, including sleep, feeding, energy balance, and anxiety. In this study we characterized the structure and function of the retina in mice that lacked expression of the rate limiting enzyme in the formation of histamine, histidine decarboxylase (Hdc-/- mouse). Using laser capture microdissection, Hdc mRNA expression was assessed in the inner and outer nuclear layers of adult C57Bl6J wildtype (WT) and Hdc(-/-)-retinae. In adult WT and Hdc(-/-)-mice, retinal fundi were imaged, retinal structure was assessed using immunocytochemistry and function was probed by electroretinography. Blood flow velocity was assessed by quantifying temporal changes in the dynamic fluorescein angiography in arterioles and venules. In WT retinae, Hdc gene expression was detected in the outer nuclear layer, but not the inner nuclear layer, while the lack of Hdc expression was confirmed in the Hdc-/- retina. Preliminary examination of the fundus and retinal structure of the widely used Hdc-/- mouse strain revealed discrete lesions across the retina that corresponded to areas of photoreceptor abnormality reminiscent of the rd8 (Crb1) mutation. This was confirmed after genotyping and the strain designated Hdcrd8/rd8. In order to determine the effect of the lack of Hdc-alone on the retina, Hdc-/- mice free of the Crb1 mutation were bred. Retinal fundi appeared normal in these animals and there was no difference in retinal structure, macrogliosis, nor any change in microglial characteristics in Hdc-/- compared to wildtype retinae. In addition, retinal function and retinal blood flow dynamics showed no alterations in the Hdc-/- retina. Overall, these results suggest that histamine plays little role in modulating retinal structure and function.

  11. Architecture of cannabinoid signaling in mouse retina

    PubMed Central

    Hu, Sherry Shu-Jung; Arnold, Andy; Hutchens, Jacqueline M.; Radicke, Josh; Cravatt, Benjamin F.; Wager-Miller, Jim; Mackie, Ken; Straiker, Alex

    2010-01-01

    Cannabinoid receptors and their ligands constitute an endogenous signaling system that is found throughout the body, including the eye. This system can be activated by Δ9-tetrahydrocannabinol, a major drug of abuse. Cannabinoids offer considerable therapeutic potential in modulating ocular immune and inflammatory responses and in regulating intraocular pressure. The location of cannabinoid receptors 1 (CB1) in the retina is known, but recently a constellation of proteins has been identified that produce and break down endocannabinoids (eCBs) and modulate CB1 function. Localization of these proteins is critical to defining specific cannabinoid signaling circuitry in the retina. Here we show the localization of diacylglycerol lipase α and β (DGLα/β), implicated in the production of the eCB 2-arachidonoyl glycerol (2-AG); monoacylglycerol lipase (MGL) and α/β-hydrolase domain 6 (ABHD6), both implicated in the breakdown of 2-AG; cannabinoid receptor interacting protein 1a (CRIP1a), a protein that may modulate CB1 function; Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) which have been shown to break down the eCB anandamide and related acyl amides. In our most prominent finding, DGLα is present in post-synaptic Type 1 OFF cone bipolar cells juxtaposed to CB1-containing cone photoreceptor terminals. Interestingly, CRIP1a is reliably presynaptic to DGLα, consistent with a possible role in cannabinoid signaling, NAAA is restricted to retinal pigment epithelium (RPE), while DGLβ is limited to retinal blood vessels. These results taken together with previous anatomic and functional studies define specific cannabinoid circuitry likely to modulate eCB signaling at the first synapse of the retina as well as in the inner plexiform layer (IPL). PMID:20653038

  12. Distinct patterns of expression of the RB gene family in mouse and human retina.

    PubMed

    Spencer, Clarellen; Pajovic, Sanja; Devlin, Hollie; Dinh, Quynh-Dao; Corson, Timothy W; Gallie, Brenda L

    2005-06-01

    Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.

  13. In vivo intrinsic optical signal imaging of mouse retinas

    NASA Astrophysics Data System (ADS)

    Wang, Benquan; Yao, Xincheng

    2016-03-01

    Intrinsic optical signal (IOS) imaging is a promising noninvasive method for advanced study and diagnosis of eye diseases. Before pursuing clinical applications, more IOS studies employing animal models are necessary to establish the relationship between IOS distortions and eye diseases. Ample mouse models are available for investigating the relationship between IOS distortions and eye diseases. However, in vivo IOS imaging of mouse retinas is challenging due to the small ocular lens (compared to frog eyes) and inevitable eye movements. We report here in vivo IOS imaging of mouse retinas using a custom-designed functional OCT. The OCT system provided high resolution (3 μm) and high speed (up to 500 frames/s) imaging of mouse retinas. An animal holder equipped with a custom designed ear bar and bite bar was used to minimize eye movement due to breathing and heartbeats. Residual eye movement in OCT images was further compensated by accurate image registration. Dynamic OCT imaging revealed rapid IOSs from photoreceptor outer segments immediately (<10 ms) after the stimulation delivery, and unambiguous IOS changes were also observed from inner retinal layers with delayed time courses compared to that of photoreceptor IOSs.

  14. [In vitro organotypic cultivation of adult newt and rat retinas].

    PubMed

    Novikova, Iu P; Aleĭnikova, K S; Krasnov, M S; Poplinskaia, V A; Grigorian, E N

    2010-01-01

    Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiG FAP antibody dye demonstrated an increase in the parent M@uller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the developing adult rat retinal spheroid in vitro; BrdU-positive cells and multiple mitoses were revealed in this zone. However, the source of proliferation was not in the peripheral retina, and stable macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt retinas.

  15. Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina*

    PubMed Central

    Du, Jianhai; Rountree, Austin; Cleghorn, Whitney M.; Contreras, Laura; Lindsay, Ken J.; Sadilek, Martin; Gu, Haiwei; Djukovic, Danijel; Raftery, Dan; Satrústegui, Jorgina; Kanow, Mark; Chan, Lawrence; Tsang, Stephen H.; Sweet, Ian R.; Hurley, James B.

    2016-01-01

    Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5′-GMP, ribose-5-phosphate, ketone bodies, and purines. PMID:26677218

  16. Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

    PubMed Central

    Jelcick, Austin S.; Yuan, Yang; Leehy, Barrett D.; Cox, Lakeisha C.; Silveira, Alexandra C.; Qiu, Fang; Schenk, Sarah; Sachs, Andrew J.; Morrison, Margaux A.; Nystuen, Arne M.; DeAngelis, Margaret M.; Haider, Neena B.

    2011-01-01

    Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases. PMID:21779340

  17. Folate antagonist, methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina.

    PubMed

    Hara, Akira; Taguchi, Ayako; Aoki, Hitomi; Hatano, Yuichiro; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro

    2010-06-25

    Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-D-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.

  18. Retina

    MedlinePlus

    ... biology of the retina. In: Tasman W, Jaeger EA, eds. Duane's Foundations of Clinical Ophthalmology . 2013 ed. ... and retina with ultrasound. In: Tasman W, Jaeger EA, eds. Duane's Ophthalmology . 2013 ed. Philadelphia, PA: Lippincott ...

  19. Effects and Responses to Spaceflight in the Mouse Retina

    NASA Technical Reports Server (NTRS)

    Zanello, Susana B.; Theriot, Corey; Westby, Christian; Boyle, Richard

    2011-01-01

    Several stress environmental factors are combined in a unique fashion during spaceflight, affecting living beings widely across their physiological systems. Recently, attention has been placed on vision changes in astronauts returning from long duration missions. Alterations include hyperoptic shift, globe flattening, choroidal folds and optic disc edema, which are probably associated with increased intracranial pressure. These observations justify a better characterization of the ocular health risks associated with spaceflight. This study investigates the impact of spaceflight on the biology of the mouse retina. Within a successful tissue sharing effort, eyes from albino Balb/cJ mice aboard STS-133 were collected for histological analysis and gene expression profiling of the retina at 1 and 7 days after landing. Both vivarium and AEM (Animal Enclosure Module) mice were used as ground controls. Oxidative stress-induced DNA damage was higher in the flight samples compared to controls on R+1, and decreased on R+7. A trend toward higher oxidative and cellular stress response gene expression was also observed on R+1 compared to AEM controls, and these levels decreased on R+7. Several genes coding for key antioxidant enzymes, namely, heme-oxygenase-1, peroxiredoxin, and catalase, were among those upregulated after flight. Likewise, NF B and TGFbeta1, were upregulated in one flight specimen that overall showed the most elevated oxidative stress markers on R+1. In addition, retinas from vivarium control mice evidenced higher oxidative stress markers, NF B and TGFbeta1, likely due to the more intense illumination in vivarium cages versus the AEM. These preliminary data suggest that spaceflight represents a source of environmental stress that translates into oxidative and cellular stress in the retina, which is partially reversible upon return to Earth. Further work is needed to dissect the contribution of the various spaceflight factors (microgravity, radiation) and to

  20. A comparison of some organizational characteristics of the mouse central retina and the human macula.

    PubMed

    Volland, Stefanie; Esteve-Rudd, Julian; Hoo, Juyea; Yee, Claudine; Williams, David S

    2015-01-01

    Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch's membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch's membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident.

  1. A Comparison of Some Organizational Characteristics of the Mouse Central Retina and the Human Macula

    PubMed Central

    Hoo, Juyea; Yee, Claudine; Williams, David S.

    2015-01-01

    Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch’s membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch’s membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident. PMID:25923208

  2. Automatic Counting of Microglial Cells in Healthy and Glaucomatous Mouse Retinas

    PubMed Central

    Rojas, Blanca; Ramírez, Ana I.; de Hoz, Rosa; Salazar, Juan J.; Triviño, Alberto; Ramírez, José M.

    2015-01-01

    Proliferation of microglial cells has been considered a sign of glial activation and a hallmark of ongoing neurodegenerative diseases. Microglia activation is analyzed in animal models of different eye diseases. Numerous retinal samples are required for each of these studies to obtain relevant data of statistical significance. Because manual quantification of microglial cells is time consuming, the aim of this study was develop an algorithm for automatic identification of retinal microglia. Two groups of adult male Swiss mice were used: age-matched controls (naïve, n = 6) and mice subjected to unilateral laser-induced ocular hypertension (lasered; n = 9). In the latter group, both hypertensive eyes and contralateral untreated retinas were analyzed. Retinal whole mounts were immunostained with anti Iba-1 for detecting microglial cell populations. A new algorithm was developed in MATLAB for microglial quantification; it enabled the quantification of microglial cells in the inner and outer plexiform layers and evaluates the area of the retina occupied by Iba-1+ microglia in the nerve fiber-ganglion cell layer. The automatic method was applied to a set of 6,000 images. To validate the algorithm, mouse retinas were evaluated both manually and computationally; the program correctly assessed the number of cells (Pearson correlation R = 0.94 and R = 0.98 for the inner and outer plexiform layers respectively). Statistically significant differences in glial cell number were found between naïve, lasered eyes and contralateral eyes (P<0.05, naïve versus contralateral eyes; P<0.001, naïve versus lasered eyes and contralateral versus lasered eyes). The algorithm developed is a reliable and fast tool that can evaluate the number of microglial cells in naïve mouse retinas and in retinas exhibiting proliferation. The implementation of this new automatic method can enable faster quantification of microglial cells in retinal pathologies. PMID:26580208

  3. CX3CL1 (Fractalkine) Protein Expression in Normal and Degenerating Mouse Retina: In Vivo Studies

    PubMed Central

    Zieger, Marina; Ahnelt, Peter K.; Uhrin, Pavel

    2014-01-01

    We aimed to investigate fractalkine (CX3CL1) protein expression in wild type (wt) retina and its alterations during retinal degeneration in mouse model (rd10) of retinitis pigmentosa. Forms of retinal protein CX3CL1, total protein and mRNA levels of CX3CL1 were analyzed at postnatal days (P) 5, 10, 14, 22, 30, 45, and 60 by Western blotting and real-time PCR. Cellular sources of CX3CL1 were investigated by in situ hybridization histochemistry (ISH) and using transgenic (CX3CL1cherry) mice. The immunoblots revealed that in both, wt and rd10 retinas, a membrane integrated ∼100 kDa CX3CL1 form and a cleaved ∼85 kDa CX3CL1 form were present at P5. At P10, accumulation of another presumably intra-neuronal ∼95 kDa form and a decrease in the ∼85-kDa form were observed. From P14, a ∼95 kDa form became principal in wt retina, while in rd10 retinas a soluble ∼85 kDa form increased at P45 and P60. In comparison, retinas of rd10 mice had significantly lower levels of total CX3CL1 protein (from P10 onwards) and lower CX3CL1 mRNA levels (from P14), even before the onset of primary rod degeneration. ISH and mCherry reporter fluorescence showed neurons in the inner retina layers as principal sites of CX3CL1 synthesis both in wt and rd10 retinas. In conclusion, our results demonstrate that CX3CL1 has a distinctive course of expression and functional regulation in rd10 retina starting at P10. The biological activity of CX3CL1 is regulated by conversion of a membrane integrated to a soluble form during neurogenesis and in response to pathologic changes in the adult retinal milieu. Viable mature neurons in the inner retina likely exhibit a dynamic intracellular storage depot of CX3CL1. PMID:25191897

  4. Transplanted neurons integrate into adult retinas and respond to light.

    PubMed

    Venugopalan, Praseeda; Wang, Yan; Nguyen, Tu; Huang, Abigail; Muller, Kenneth J; Goldberg, Jeffrey L

    2016-02-04

    Retinal ganglion cells (RGCs) degenerate in diseases like glaucoma and are not replaced in adult mammals. Here we investigate whether transplanted RGCs can integrate into the mature retina. We have transplanted GFP-labelled RGCs into uninjured rat retinas in vivo by intravitreal injection. Transplanted RGCs acquire the general morphology of endogenous RGCs, with axons orienting towards the optic nerve head of the host retina and dendrites growing into the inner plexiform layer. Preliminary data show in some cases GFP(+) axons extending within the host optic nerves and optic tract, reaching usual synaptic targets in the brain, including the lateral geniculate nucleus and superior colliculus. Electrophysiological recordings from transplanted RGCs demonstrate the cells' electrical excitability and light responses similar to host ON, ON-OFF and OFF RGCs, although less rapid and with greater adaptation. These data present a promising approach to develop cell replacement strategies in diseased retinas with degenerating RGCs.

  5. Onecut1 and Onecut2 Play Critical Roles in the Development of the Mouse Retina

    PubMed Central

    Chowdhury, Rebecca; Trimarchi, Jeffrey M.

    2014-01-01

    The entire repertoire of intrinsic factors that control the cell fate determination process of specific retinal neurons has yet to be fully identified. Single cell transcriptome profiling experiments of retinal progenitor cells revealed considerable gene expression heterogeneity between individual cells, especially among different classes of transcription factors. In this study, we show that two of those factors, Onecut1 and Onecut2, are expressed during mouse retinal development. Using mice that are deficient for each of these transcription factors, we further demonstrate a significant loss (∼70–80%) of horizontal cells in the absence of either of these proteins, while the other retinal cells appear at normal numbers. Microarray profiling experiments performed on knockout retinas revealed defects in horizontal cell genes as early as E14.5. Additional profiling assays showed an upregulation of several stress response genes in the adult Onecut2 knockout, suggesting that the integrity of the retina is compromised in the absence of normal numbers of horizontal cells. Interestingly, melanopsin, the gene coding for the photopigment found in photosensitive ganglion cells, was observed to be upregulated in Onecut1 deficient retinas, pointing to a possible regulatory role for Onecut1. Taken together, our data show that similar to Onecut1, Onecut2 is also necessary for the formation of normal numbers of horizontal cells in the developing retina. PMID:25313862

  6. Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

    PubMed Central

    GUO, CHENYING; STELLA, SALVATORE L.; HIRANO, ARLENE A.; BRECHA, NICHOLAS C.

    2009-01-01

    Plasmalemmal and vesicular γ-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. PMID:18975268

  7. [Damage and functional recovery of the mouse retina after exposure to ionizing radiation and methylnitrosourea].

    PubMed

    Vinogradova, Iu V; Tronov, V A; Liakhova, K N; Poplinskaia, V A; Ostrovskiĭ, M A

    2014-01-01

    The eye retina consists of terminally differentiated cells that have lost their ability to proliferate. The death of these cells leads tothe loss of sight. The mice retina is characterized by relatively high resistance to radiation, which is provided by its ability to repair damage caused by environmental factors. The aim of our work was to assess the damaging effect of ionizing radiation and methylnitrosourea (MNU) on the DNA structure in the mouse retina, the functional activity of the retina, and its ability to recover in vivo. The results confirm the ability of the mature retina to structural and functional recovery. Adapting influence of low dose chemical agent increases retina resistance to cytotoxic dose of genotoxicants and prevents degeneration of photoreceptor layer of the retina. The results show the possibility of neurohormesis effect in the mice retina after exposure to ionizing radiation and chemicals.

  8. TRP channel gene expression in the mouse retina.

    PubMed

    Gilliam, Jared C; Wensel, Theodore G

    2011-12-08

    In order to identify candidate cation channels important for retinal physiology, 28 TRP channel genes were surveyed for expression in the mouse retina. Transcripts for all TRP channels were detected by RT-PCR and sequencing. Northern blotting revealed that mRNAs for 12 TRP channel genes are enriched in the retina. The strongest signals were observed for TRPC1, TRPC3, TRPM1, TRPM3, and TRPML1, and clear signals were obtained for TRPC4, TRPM7, TRPP2, TRPV2, and TRPV4. In situ hybridization and immunofluorescence revealed widespread expression throughout multiple retinal layers for TRPC1, TRPC3, TRPC4, TRPML1, PKD1, and TRPP2. Striking localization of enhanced mRNA expression was observed for TRPC1 in the photoreceptor inner segment layer, for TRPM1 in the inner nuclear layer (INL), for TRPM3 in the INL, and for TRPML1 in the outer plexiform and nuclear layers. Strong immunofluorescence signal in cone outer segments was observed for TRPM7 and TRPP2. TRPC5 immunostaining was largely confined to INL cells immediately adjacent to the inner plexiform layer. TRPV2 antibodies stained photoreceptor axons in the outer plexiform layer. Expression of TRPM1 splice variants was strong in the ciliary body, whereas TRPM3 was strongly expressed in the retinal pigmented epithelium.

  9. Tgfbi/Bigh3 silencing activates ERK in mouse retina.

    PubMed

    Allaman-Pillet, Nathalie; Oberson, Anne; Bustamante, Mauro; Tasinato, Andrea; Hummler, Edith; Schorderet, Daniel F

    2015-11-01

    BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

  10. A Possible Role of Neuroglobin in the Retina After Optic Nerve Injury: A Comparative Study of Zebrafish and Mouse Retina.

    PubMed

    Sugitani, Kayo; Koriyama, Yoshiki; Ogai, Kazuhiro; Wakasugi, Keisuke; Kato, Satoru

    2016-01-01

    Neuroglobin (Ngb) is a new member of the family of heme proteins and is specifically expressed in neurons of the central and peripheral nervous systems in all vertebrates. In particular, the retina has a 100-fold higher concentration of Ngb than do other nervous tissues. The role of Ngb in the retina is yet to be clarified. Therefore, to understand the functional role of Ngb in the retina after optic nerve injury (ONI), we used two types of retina, from zebrafish and mice, which have permissible and non-permissible capacity for nerve regeneration after ONI, respectively. After ONI, the Ngb protein in zebrafish was upregulated in the amacrine cells within 3 days, whereas in the mouse retina, Ngb was downregulated in the retinal ganglion cells (RGCs) within 3 days. Zebrafish Ngb (z-Ngb) significantly enhanced neurite outgrowth in retinal explant culture. According to these results, we designed an overexpression experiment with the mouse Ngb (m-Ngb) gene in RGC-5 cells (retinal precursor cells). The excess of m-Ngb actually rescued RGC-5 cells under hypoxic conditions and significantly enhanced neurite outgrowth in cell culture. These data suggest that mammalian Ngb has positive neuroprotective and neuritogenic effects that induce nerve regeneration after ONI.

  11. Mouse Embryonic Retina Delivers Information Controlling Cortical Neurogenesis

    PubMed Central

    Bonetti, Ciro; Surace, Enrico Maria

    2010-01-01

    The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development [1]. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded [2], the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs) during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal). Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system. PMID:21170332

  12. Specific sphingolipid content decrease in Cerkl knockdown mouse retinas

    PubMed Central

    Garanto, Alejandro; Mandal, Nawajes A.; Egido-Gabás, Meritxell; Marfany, Gemma; Fabriàs, Gemma; Anderson, Robert E.; Casas, Josefina; Gonzàlez-Duarte, Roser

    2014-01-01

    Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell–cell interactions, membrane conductance, synaptic transmission, and apoptosis. Mutations in Ceramide Kinase-Like (CERKL) cause autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal etiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl −/− model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 60%) in the Cerkl −/− compared to WT retinas, which was particularly evident in the glucosyl/galactosyl ceramide species (Glc/GalCer) whereas the phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios concerning its function. PMID:23501591

  13. Function and Circuitry of VIP+ Interneurons in the Mouse Retina

    PubMed Central

    Park, Silvia J.H.; Borghuis, Bart G.; Rahmani, Pouyan; Zeng, Qiang

    2015-01-01

    Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to ∼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the ∼30–40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP+) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP+ cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP+ cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP+ cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing. SIGNIFICANCE STATEMENT The retina is a model system for understanding nervous system function. At the first stage, rod and cone photoreceptors encode light and

  14. Morphology and connectivity of the small bistratified A8 amacrine cell in the mouse retina

    PubMed Central

    Lee, Sammy C.S.; Meyer, Arndt; Schubert, Timm; Hüser, Laura; Dedek, Karin; Haverkamp, Silke

    2015-01-01

    Amacrine cells comprise ~30 morphological types in the mammalian retina. The synaptic connectivity and function of a few GABAergic wide-field amacrine cells have recently been studied, however, with the exception of the rod pathway-specific AII amacrine cell the connectivity of glycinergic small-field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small-field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A-type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. Taken together, the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway-specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A-type ganglion cells. PMID:25630271

  15. Network Analysis and Visualization of Mouse Retina Connectivity Data

    PubMed Central

    2016-01-01

    The largest available cellular level connectivity map, of a 0.1 mm sample of the mouse retina Inner Plexiform Layer, was analysed using network models and visualized using spectral graph layouts and observed cell coordinates. This allows key nodes in the network to be identified with retinal neurons. Their strongest synaptic links can trace pathways in the network, elucidating possible circuits. Modular decomposition of the network, by sampling signal flows over nodes and links using the InfoMap method, shows discrete modules of cone bipolar cells that form a tiled mosaic in the retinal plane. The highest flow nodes, calculated by InfoMap, proved to be the most useful landmarks for elucidating possible circuits. Their dominant links to high flow amacrine cells reveal possible circuits linking bipolar through to ganglion cells and show an Off-On discrimination between the Left-Right sections of the sample. Circuits suggested by this analysis confirm known roles for some cells and point to roles for others. PMID:27414405

  16. Resistivity profiles of wild-type, rd1, and rd10 mouse retina.

    PubMed

    Boshuo Wang; Weiland, James D

    2015-08-01

    Electrical impedance of the retina is a critical factor in retinal prostheses, determining the intraretinal current flow and potential distribution of electrical stimulation. Previous resistivity measurements in retina were limited to healthy retina, and didn't include mouse models, a common and important animal model in retinal research. This experimental study measured the resistivity profiles of wild-type, rd1, and rd10 mice, providing basis for computational simulations and predictive modeling studies. The peak resistance frequency method has been utilized to measure the resistivity profiles of the retina cross section, and the results show agreement with previous studies in retina of normal rats and embryonic chicks. Retinal degeneration affects the width of the profile, which is in agreement with histological measurements. Degeneration also results in lower peak resistivity. The results indicate that, on the mesoscopic scale, resistivity is dominated by spatial factors, while influence of remodeling on the cellular level is not apparent under such scale.

  17. Heterogeneous transgene expression in the retinas of the TH-RFP, TH-Cre, TH-BAC-Cre and DAT-Cre mouse lines

    PubMed Central

    Vuong, Helen E.; de Sevilla Müller, Luis Pérez; Hardi, Claudia N.; McMahon, Douglas G.; Brecha, Nicholas C.

    2015-01-01

    Transgenic mouse lines are essential tools for understanding the connectivity, physiology and function of neuronal circuits, including those in the retina. This report compares transgene expression in the retina of a tyrosine hydroxylase (TH)-red fluorescent protein (RFP) line with three catecholamine-related Cre recombinase lines [TH-bacterial artificial chromosome (BAC)-, TH-, and dopamine transporter (DAT)-Cre] that were crossed with a ROSA26-tdTomato reporter line. Retinas were evaluated and immunostained with commonly used antibodies including those directed to TH, GABA and glycine to characterize the RFP or tdTomato fluorescent-labeled amacrine cells, and an antibody directed to RNA-binding protein with multiple splicing to identify ganglion cells. In TH-RFP retinas, types 1 and 2 dopamine (DA) amacrine cells were identified by their characteristic cellular morphology and type 1 DA cells by their expression of TH immunoreactivity. In the TH-BAC-, TH-, and DAT-tdTomato retinas, less than 1%, ~6%, and 0%, respectively, of the fluorescent cells were the expected type 1 DA amacrine cells. Instead, in the TH-BAC-tdTomato retinas, fluorescently labeled AII amacrine cells were predominant, with some medium somal diameter ganglion cells. In TH-tdTomato retinas, fluorescence was in multiple neurochemical amacrine cell types, including four types of polyaxonal amacrine cells. In DAT-tdTomato retinas, fluorescence was in GABA immunoreactive amacrine cells, including two types of bistratified and two types of monostratified amacrine cells. Although each of the Cre lines were generated with the intent to specifically label DA cells, our findings show a cellular diversity in Cre expression in the adult retina and indicate the importance of careful characterization of transgene labeling patterns. These mouse lines with their distinctive cellular labeling patterns will be useful tools for future studies of retinal function and visual processing. PMID:26335381

  18. DSCAM promotes refinement in the mouse retina through cell death and restriction of exploring dendrites.

    PubMed

    Li, Shuai; Sukeena, Joshua M; Simmons, Aaron B; Hansen, Ethan J; Nuhn, Renee E; Samuels, Ivy S; Fuerst, Peter G

    2015-04-08

    In this study we develop and use a gain-of-function mouse allele of the Down syndrome cell adhesion molecule (Dscam) to complement loss-of-function models. We assay the role of Dscam in promoting cell death, spacing, and laminar targeting of neurons in the developing mouse retina. We find that ectopic or overexpression of Dscam is sufficient to drive cell death. Gain-of-function studies indicate that Dscam is not sufficient to increase spatial organization, prevent cell-to-cell pairing, or promote active avoidance in the mouse retina, despite the similarity of the Dscam loss-of-function phenotype in the mouse retina to phenotypes observed in Drosophila Dscam1 mutants. Both gain- and loss-of-function studies support a role for Dscam in targeting neurites; DSCAM is necessary for precise dendrite lamination, and is sufficient to retarget neurites of outer retinal cells after ectopic expression. We further demonstrate that DSCAM guides dendrite targeting in type 2 dopaminergic amacrine cells, by restricting the stratum in which exploring retinal dendrites stabilize, in a Dscam dosage-dependent manner. Based on these results we propose a single model to account for the numerous Dscam gain- and loss-of-function phenotypes reported in the mouse retina whereby DSCAM eliminates inappropriately placed cells and connections.

  19. Otx2 ChIP-seq Reveals Unique and Redundant Functions in the Mature Mouse Retina

    PubMed Central

    Fant, Bruno; Lamonerie, Thomas

    2014-01-01

    During mouse retinal development and into adulthood, the transcription factor Otx2 is expressed in pigment epithelium, photoreceptors and bipolar cells. In the mature retina, Otx2 ablation causes photoreceptor degeneration through a non-cell-autonomous mechanism involving Otx2 function in the supporting RPE. Surprisingly, photoreceptor survival does not require Otx2 expression in the neural retina, where the related Crx homeobox gene, a major regulator of photoreceptor development, is also expressed. To get a deeper view of mouse Otx2 activities in the neural retina, we performed chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on Otx2. Using two independent ChIP-seq assays, we identified consistent sets of Otx2-bound cis-regulatory elements. Comparison with our previous RPE-specific Otx2 ChIP-seq data shows that Otx2 occupies different functional domains of the genome in RPE cells and in neural retina cells and regulates mostly different sets of genes. To assess the potential redundancy of Otx2 and Crx, we compared our data with Crx ChIP-seq data. While Crx genome occupancy markedly differs from Otx2 genome occupancy in the RPE, it largely overlaps that of Otx2 in the neural retina. Thus, in accordance with its essential role in the RPE and its non-essential role in the neural retina, Otx2 regulates different gene sets in the RPE and the neural retina, and shares an important part of its repertoire with Crx in the neural retina. Overall, this study provides a better understanding of gene-regulatory networks controlling photoreceptor homeostasis and disease. PMID:24558479

  20. Transcriptome networks in the mouse retina: An exon level BXD RI database

    PubMed Central

    King, Rebecca; Lu, Lu; Williams, Robert W.

    2015-01-01

    Purpose Differences in gene expression provide diverse retina phenotypes and may also contribute to susceptibility to injury and disease. The present study defines the transcriptome of the retina in the BXD RI strain set, using the Affymetrix Mouse Gene 2.0 ST array to investigate all exons of traditional protein coding genes, non-coding RNAs, and microRNAs. These data are presented in a highly interactive database on the GeneNetwork website. Methods In the Normal Retina Database, the mRNA levels of the transcriptome from retinas was quantified using the Affymetrix Mouse Gene 2.0 ST array. This database consists of data from male and female mice. The data set includes a total of 52 BXD RI strains, the parental strains (C57BL/6J and DBA/2J), and a reciprocal cross. Results In combination with GeneNetwork, the Department of Defense (DoD) Congressionally Directed Medical Research Programs (CDMRP) Normal Retina Database provides a large resource for mapping, graphing, analyzing, and testing complex genetic networks. Protein-coding and non-coding RNAs can be used to map quantitative trait loci (QTLs) that contribute to expression differences among the BXD strains and to establish links between classical ocular phenotypes associated with differences in the genomic sequence. Using this resource, we extracted transcriptome signatures for retinal cells and defined genetic networks associated with the maintenance of the normal retina. Furthermore, we examined differentially expressed exons within a single gene. Conclusions The high level of variation in mRNA levels found among the BXD RI strains makes it possible to identify expression networks that underline differences in retina structure and function. Ultimately, we will use this database to define changes that occur following blast injury to the retina. PMID:26604663

  1. Wiring patterns in the mouse retina: collecting evidence across the connectome, physiology and light microscopy

    PubMed Central

    Dunn, Felice A; Wong, Rachel O L

    2014-01-01

    The visual system has often been thought of as a parallel processor because distinct regions of the brain process different features of visual information. However, increasing evidence for convergence and divergence of circuit connections, even at the level of the retina where visual information is first processed, chips away at a model of dedicated and distinct pathways for parallel information flow. Instead, our current understanding is that parallel channels may emerge, not from exclusive microcircuits for each channel, but from unique combinations of microcircuits. This review depicts diagrammatically the current knowledge and remaining puzzles about the retinal circuit with a focus on the mouse retina. Advances in techniques for labelling cells and genetic manipulations have popularized the use of transgenic mice. We summarize evidence gained from serial electron microscopy, electrophysiology and light microscopy to illustrate the wiring patterns in mouse retina. We emphasize the need to explore proposed retinal connectivity using multiple methods to verify circuits both structurally and functionally. PMID:25172948

  2. Isoaspartyl Protein Damage and Repair in Mouse Retina

    PubMed Central

    Qin, Zhenxia; Yang, Jing; Klassen, Henry J.; Aswad, Dana W.

    2014-01-01

    Purpose. To determine the propensity of retinal proteins for spontaneous damage via formation of isoaspartyl sites, a common type of protein damage that could contribute to retinal disease. Methods. Tissue extracts were obtained from retinas and brains of control mice and from mice in which the gene for protein L-isoaspartate O-methyltransferase (PIMT; an enzyme that repairs isoaspartyl protein damage) was knocked out. PIMT expression in these extracts was measured by Western blot, and its specific activity was assayed by monitoring the rate of [3H]methyl transfer from S-adenosyl-[methyl-3H]L-methionine to γ-globulin. Isoaspartate levels in extracts were measured by their capacity to accept [3H]methyl groups via the PIMT-catalyzed methylation reaction. To compare molecular weight distributions of isoaspartyl-rich proteins in retina versus brain, proteins from PIMT knockout (KO) and control mice were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF). Isoaspartyl proteins were 3H-labeled on-blot using a PIMT overlay and imaged by autoradiography. Results. When normalized to the β-actin content of each tissue, retina was found to be nearly identical to brain with regard to expression and activity of PIMT and its propensity to accumulate isoaspartyl sites when PIMT is absent. The two tissues show distinct differences in the molecular weight distribution of isoaspartyl proteins. Conclusions. The retina is rich in PIMT activity and contains a wide range of proteins that are highly susceptible to this type of protein damage. Recoverin may be one such protein. Isoaspartate formation, along with oxidation, should be considered as a potential source of protein dysfunction and autoimmunity in retinal disease. PMID:24550364

  3. Transplantation of cultured adult porcine full-thickness retina.

    PubMed

    Engelsberg, Karl; Ghosh, Fredrik

    2007-01-01

    In this study we wanted to examine how an adult neuroretina from an animal with an eye similar to the human one survives in vitro. We also wanted to investigate how the culture process affects the adult retina when used in a transplantation paradigm. Full-thickness neuroretinal sheets from adult porcine eyes were dissected into pieces measuring 3 mm in diameter. These were kept in culture for 1-3 days. After this time, the explants were fixed or transplanted subretinally to adult pigs, which were killed after 72-74 days. Transplanted eyes, as well as tissue kept in culture only, were processed for hematoxylin and eosin staining and immunohistochemistry. Explants kept 1 day in vitro (DIV) displayed the normal morphology. In these specimens, single pyknotic cells were evident in the outer nuclear layer (ONL) and ganglion cell layer, but were more frequent in the inner nuclear layer (INL). After longer times in vitro, severe degenerative changes appeared. Transplanted explants kept 1 DIV prior to transplantation exhibited normal retinal lamination in two out of four specimens. Transducin and recoverin labeling revealed photoreceptors with inner segments in these grafts. Rod bipolar cells displayed a normal morphology. Vertically arranged Müller cells were also seen in the laminated grafts. Two of the three transplants kept 2 DIV displayed minimal lamination. Eyes with transplants kept 3 DIV prior to transplantation displayed degenerated grafts in all eyes. This study shows that adult porcine neuroretinal explants kept in culture for 1 day display a normal morphology in their major part. Additionally, 1-day explants can survive transplantation with retained morphology even after several months. This indicates the possibility of storing adult donor tissue between harvest and transplantation. The culture system may also be used in the future as a tool for manipulating retinal donor tissue prior to transplantation.

  4. Cholesterol in mouse retina originates primarily from in situ de novo biosynthesis.

    PubMed

    Lin, Joseph B; Mast, Natalia; Bederman, Ilya R; Li, Yong; Brunengraber, Henri; Björkhem, Ingemar; Pikuleva, Irina A

    2016-02-01

    The retina, a thin tissue in the back of the eye, has two apparent sources of cholesterol: in situ biosynthesis and cholesterol available from the systemic circulation. The quantitative contributions of these two cholesterol sources to the retinal cholesterol pool are unknown and have been determined in the present work. A new methodology was used. Mice were given separately deuterium-labeled drinking water and chow containing 0.3% deuterium-labeled cholesterol. In the retina, the rate of total cholesterol input was 21 μg of cholesterol/g retina • day, of which 15 μg of cholesterol/g retina • day was provided by local biosynthesis and 6 μg of cholesterol/g retina • day was uptaken from the systemic circulation. Thus, local cholesterol biosynthesis accounts for the majority (72%) of retinal cholesterol input. We also quantified cholesterol input to mouse brain, the organ sharing important similarities with the retina. The rate of total cerebral cholesterol input was 121 μg of cholesterol/g brain • day with local biosynthesis providing 97% of total cholesterol input. Our work addresses a long-standing question in eye research and adds new knowledge to the potential use of statins (drugs that inhibit cholesterol biosynthesis) as therapeutics for age-related macular degeneration, a common blinding disease.

  5. Differential Expression of Melanopsin Isoforms Opn4L and Opn4S during Postnatal Development of the Mouse Retina

    PubMed Central

    Hughes, Steven; Welsh, Laura; Katti, Christiana; González-Menéndez, Irene; Turton, Michael; Halford, Stephanie; Sekaran, Sumathi; Peirson, Stuart N.; Hankins, Mark W.; Foster, Russell G.

    2012-01-01

    Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development. PMID:22496826

  6. Differential expression of melanopsin isoforms Opn4L and Opn4S during postnatal development of the mouse retina.

    PubMed

    Hughes, Steven; Welsh, Laura; Katti, Christiana; González-Menéndez, Irene; Turton, Michael; Halford, Stephanie; Sekaran, Sumathi; Peirson, Stuart N; Hankins, Mark W; Foster, Russell G

    2012-01-01

    Photosensitive retinal ganglion cells (pRGCs) respond to light from birth and represent the earliest known light detection system to develop in the mouse retina. A number of morphologically and functionally distinct subtypes of pRGCs have been described in the adult retina, and have been linked to different physiological roles. We have previously identified two distinct isoforms of mouse melanopsin, Opn4L and Opn4S, which are generated by alternate splicing of the Opn4 locus. These isoforms are differentially expressed in pRGC subtypes of the adult mouse retina, with both Opn4L and Opn4S detected in M1 type pRGCs, and only Opn4L detected in M2 type pRGCs. Here we investigate the developmental expression of Opn4L and Opn4S and show a differential profile of expression during postnatal development. Opn4S mRNA is detected at relatively constant levels throughout postnatal development, with levels of Opn4S protein showing a marked increase between P0 and P3, and then increasing progressively over time until adult levels are reached by P10. By contrast, levels of Opn4L mRNA and protein are low at birth and show a marked increase at P14 and P30 compared to earlier time points. We suggest that these differing profiles of expression are associated with the functional maturation of M1 and M2 subtypes of pRGCs. Based upon our data, Opn4S expressing M1 type pRGCs mature first and are the dominant pRGC subtype in the neonate retina, whereas increased expression of Opn4L and the maturation of M2 type pRGCs occurs later, between P10 and P14, at a similar time to the maturation of rod and cone photoreceptors. We suggest that the distinct functions associated with these cell types will develop at different times during postnatal development.

  7. [Radiation preconditioning of mouse retina results in tolerance to MNU-induced degeneration and stimulates retinal recovery].

    PubMed

    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 studies have revealed a non-linear dose-response relationship for cytotoxic effect 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 the present study we used the mouse model for MNU-induced retinal degeneration to evaluate the adaptive response of the retina to proton irradiation and implication of glial Muller cells in this response. In this paper, we have shown that the recovery of the retina after exposure to genotoxic agents is associated with an increased efficiency of DNA damage repair and lowered death of retinal photoreceptors.

  8. The mouse retina in 3D: quantification of vascular growth and remodeling.

    PubMed

    Milde, Florian; Lauw, Stephanie; Koumoutsakos, Petros; Iruela-Arispe, M Luisa

    2013-12-01

    The mouse retina has become a prominent model for studying angiogenesis. The easy access and well-known developmental progression have significantly propelled our ability to examine and manipulate blood vessels in vivo. Nonetheless, most studies have restricted their evaluations to the superficial plexus (an upper vascular layer in contact with the vitreous). Here we present experimental data and quantification for the developmental progression of the full retina including the intermediate and deeper plexus that sprouts from the superficial layer. We analyze the origin and advancement of vertical sprouting and present the progression of vascular perfusion within the tissue. Furthermore, we introduce the use of Minkowsky functionals to quantify remodeling in the superficial and deeper plexus. The work expands information on the retina towards a 3D structure. This is of particular interest, as recent data have demonstrated differential effects of gene deletion on the upper and deeper plexus, highlighting the concept of distinct operational pathways during sprouting angiogenesis.

  9. The sarcoglycan-sarcospan complex localization in mouse retina is independent from dystrophins

    PubMed Central

    Fort, Patrice; Estrada, Francisco-Javier; Bordais, Agnès; Mornet, Dominique; Sahel, José-Alain; Picaud, Serge; Vargas, Haydeé Rosas; Coral-Vázquez, Ramón M.; Rendon, Alvaro

    2005-01-01

    The sarcoglycan–sarcospan (SG–SSPN) complex is part of the dystrophin-glycoprotein complex that has been extensively characterized in muscle. To establish the framework for functional studies of sarcoglycans in retina here, we quantified sarcoglycans mRNA levels with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and performed immunohistochemistry to determine their cellular and subcellular distribution. We showed that the β-, δ-, γ-, ε-sarcoglycans and sarcospan are expressed in mouse retina. They are localized predominantly in the outer and the inner limiting membranes, probably in the Müller cells and also in the ganglion cells axons where the expression of dystrophins have never been reported. We also investigated the status of the sarcoglycans in the retina of mdx3cv mutant mice for all Duchene Muscular Dystrophy (DMD) gene products. The absence of dystrophin did not produce any change in the sarcoglycan–sarcospan components expression and distribution. PMID:15993965

  10. Genetic and immunohistochemical analysis of HSPA5 in mouse and human retinas

    PubMed Central

    Chintalapudi, Sumana R.; Wang, XiaoFei; Li, Huiling; Lau, Yin H. Chan; Williams, Robert W.; Jablonski, Monica M.

    2016-01-01

    Purpose Photoreceptor degenerative diseases are among the leading causes of vision loss. Although the causative genetic mutations are often known, mechanisms leading to photoreceptor degeneration remain poorly defined. We have previously demonstrated that the photoreceptor membrane-associated protein XAP-1 antigen is a product of the HSPA5 gene. In this study, we used systems genetic methods, statistical modeling, and immunostaining to identify and analyze candidate genes that modulate Hspa5 expression in the retina. Methods Quantitative trait locus (QTL) mapping was used to map the genomic region that regulates Hspa5 in the cross between C57BL/6J X DBA/2J mice (BXD) genetic reference panel. The stepwise refinement of candidate genes was based on expression QTL mapping, gene expression correlation analyses (direct and partial), and analysis of regional sequence variants. The subcellular localization of candidate proteins and HSPA5 in mouse and human retinas was evaluated by immunohistochemistry. Differences in the localization of extracellular HSPA5 were assessed between healthy human donor and atrophic age-related macular degeneration (AMD) donor eyes. Results In the eyes of healthy mice, extracellular HSPA5 was confined to the area around the cone photoreceptor outer segments. Mapping variation in Hspa5 mRNA expression levels in the retina revealed a statistically significant trans-acting expression QTL (eQTL) on Chromosome 2 (Chr 2) and a suggestive locus on Chr 15. Sulf2 on Chr 2 was the strongest candidate gene based on partial correlation analysis, Pearson correlation with Hspa5, expression levels in the retina, a missense variant in exon 14, and its reported function in the extracellular matrix and interphotoreceptor matrix. SULF2 is localized to the rod and cone photoreceptors in both human and mouse retinas. In human retinas with no pathology, extracellular HSPA5 was localized around many cones within the macular area. In contrast, fewer HSPA5

  11. Imaging pulse wave velocity in mouse retina using swept-source OCT (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Song, Shaozhen; Wei, Wei; Wang, Ruikang K.

    2016-03-01

    Blood vessel dynamics has been a significant subject in cardiology and internal medicine, and pulse wave velocity (PWV) on artery vessels is a classic evaluation of arterial distensibility, and has never been ascertained as a cardiovascular risk marker. The aim of this study is to develop a high speed imaging technique to capture the pulsatile motion on mouse retina arteries with the ability to quantify PWV on any arterial vessels. We demonstrate a new non-invasive method to assess the vessel dynamics on mouse retina. A Swept-source optical coherence tomography (SS-OCT) system is used for imaging micro-scale blood vessel motion. The phase-stabilized SS-OCT provides a typical displacement sensitivity of 20 nm. The frame rate of imaging is ~16 kHz, at A-line rate of ~1.62 MHz, which allows the detection of transient pulse waves with adequate temporal resolution. Imaging volumes with repeated B-scans are obtained on mouse retina capillary bed, and the mouse oxymeter signal is recorded simultaneously. The pulse wave on artery and vein are resolved, and with the synchronized heart beat signal, the temporal delay on different vessel locations is determined. The vessel specific measurement of PWV is achieved for the first time with SS-OCT, for pulse waves propagating more than 100 cm/s. Using the novel methodology of retinal PWV assessment, it is hoped that the clinical OCT scans can provide extended diagnostic information of cardiology functionalities.

  12. Cardinal Orientation Selectivity Is Represented by Two Distinct Ganglion Cell Types in Mouse Retina

    PubMed Central

    Nath, Amurta

    2016-01-01

    Orientation selectivity (OS) is a prominent and well studied feature of early visual processing in mammals, but recent work has highlighted the possibility that parallel OS circuits might exist in multiple brain locations. Although both classic and modern work has identified an OS mechanism in selective wiring from lateral geniculate nucleus (LGN) to primary visual cortex, OS responses have now been found upstream of cortex in mouse LGN and superior colliculus, suggesting a possible origin in the retina. Indeed, retinal OS responses have been reported for decades in rabbit and more recently in mouse. However, we still know very little about the properties and mechanisms of retinal OS in the mouse, including whether there is a distinct OS ganglion cell type, which orientations are represented, and what are the synaptic mechanisms of retinal OS. We have identified two novel types of OS ganglion cells in the mouse retina that are highly selective for horizontal and vertical cardinal orientations. Reconstructions of the dendritic trees of these OS ganglion cells and measurements of their synaptic conductances offer insights into the mechanism of the OS computation at the earliest stage of the visual system. SIGNIFICANCE STATEMENT Orientation selectivity (OS) is one of the most well studied computations in the brain and has become a prominent model system in various areas of sensory neuroscience. Although the cortical mechanism of OS suggested by Hubel and Wiesel (1962) has been investigated intensely, other OS cells exist upstream of cortex as early as the retina and the mechanisms of OS in subcortical regions are much less well understood. We identified two ON retinal ganglion cells (RGCs) in mouse that compute OS along the horizontal (nasal–temporal) and vertical (dorsoventral) axes of visual space. We show the relationship between dendritic morphology and OS for each RGC type and reveal new synaptic mechanisms of OS computation in the retina. PMID:26985031

  13. Developmental and mutational changes of glycoproteins in the mouse neuronal retina: studies with bovine galactosyltransferase.

    PubMed

    Wallenfels, B

    1979-07-01

    Bovine galactosyltransferase (lactose synthase; EC 2.4.1.22) which catalyzes the transfer of galactose from UDPgalactose to glycoproteins with N-acetylglucosamine as the terminal residue of their oligosaccharide side chains was used to label glycoproteins of mouse retina with [14C]galactose. The glycoproteins were separated by isoelectric focusing in the first dimension and by sodium dodecyl sulfate gel electrophoresis in the second dimension. Their position on the gel was determined by autofluorography. With this method, quantitative as well as qualitative changes in the glycoprotein composition of the neuronal mouse retina during postnatal development were observed. Furthermore, it was found that the photoreceptor loss in mice with retinal degeneration was paralleled by the disappearance of certain glycoprotein bands.

  14. Developmental and mutational changes of glycoproteins in the mouse neuronal retina: studies with bovine galactosyltransferase.

    PubMed Central

    Wallenfels, B

    1979-01-01

    Bovine galactosyltransferase (lactose synthase; EC 2.4.1.22) which catalyzes the transfer of galactose from UDPgalactose to glycoproteins with N-acetylglucosamine as the terminal residue of their oligosaccharide side chains was used to label glycoproteins of mouse retina with [14C]galactose. The glycoproteins were separated by isoelectric focusing in the first dimension and by sodium dodecyl sulfate gel electrophoresis in the second dimension. Their position on the gel was determined by autofluorography. With this method, quantitative as well as qualitative changes in the glycoprotein composition of the neuronal mouse retina during postnatal development were observed. Furthermore, it was found that the photoreceptor loss in mice with retinal degeneration was paralleled by the disappearance of certain glycoprotein bands. Images PMID:290997

  15. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina

    NASA Astrophysics Data System (ADS)

    Ooto, Sotaro; Akagi, Tadamichi; Kageyama, Ryoichiro; Akita, Joe; Mandai, Michiko; Honda, Yoshihito; Takahashi, Masayo

    2004-09-01

    It has long been believed that the retina of mature mammals is incapable of regeneration. In this study, using the N-methyl-D-aspartate neurotoxicity model of adult rat retina, we observed that some Müller glial cells were stimulated to proliferate in response to a toxic injury and produce bipolar cells and rod photoreceptors. Although these newly produced neurons were limited in number, retinoic acid treatment promoted the number of regenerated bipolar cells. Moreover, misexpression of basic helix-loop-helix and homeobox genes promoted the induction of amacrine, horizontal, and rod photoreceptor specific phenotypes. These findings demonstrated that retinal neurons regenerated even in adult mammalian retina after toxic injury. Furthermore, we could partially control the fate of the regenerated neurons with extrinsic factors or intrinsic genes. The Müller glial cells constitute a potential source for the regeneration of adult mammalian retina and can be a target for drug delivery and gene therapy in retinal degenerative diseases.

  16. Wavefront sensorless approaches to adaptive optics for in vivo fluorescence imaging of mouse retina

    NASA Astrophysics Data System (ADS)

    Wahl, Daniel J.; Bonora, Stefano; Mata, Oscar S.; Haunerland, Bengt K.; Zawadzki, Robert J.; Sarunic, Marinko V.; Jian, Yifan

    2016-03-01

    Adaptive optics (AO) is necessary to correct aberrations when imaging the mouse eye with high numerical aperture. In order to obtain cellular resolution, we have implemented wavefront sensorless adaptive optics for in vivo fluorescence imaging of mouse retina. Our approach includes a lens-based system and MEMS deformable mirror for aberration correction. The AO system was constructed with a reflectance channel for structural images and fluorescence channel for functional images. The structural imaging was used in real-time for navigation on the retina using landmarks such as blood vessels. We have also implemented a tunable liquid lens to select the retinal layer of interest at which to perform the optimization. At the desired location on the mouse retina, the optimization algorithm used the fluorescence image data to drive a modal hill-climbing algorithm using an intensity or sharpness image quality metric. The optimization requires ~30 seconds to complete a search up to the 20th Zernike mode. In this report, we have demonstrated the AO performance for high-resolution images of the capillaries in a fluorescence angiography. We have also made progress on an approach to AO with pupil segmentation as a possible sensorless technique suitable for small animal retinal imaging. Pupil segmentation AO was implemented on the same ophthalmic system and imaging performance was demonstrated on fluorescent beads with induced aberrations.

  17. 670 nm light mitigates oxygen-induced degeneration in C57BL/6J mouse retina

    PubMed Central

    2013-01-01

    Background Irradiation with light wavelengths from the far red (FR) to the near infrared (NIR) spectrum (600 nm -1000 nm) has been shown to have beneficial effects in several disease models. In this study, we aim to examine whether 670 nm red light pretreatment can provide protection against hyperoxia-induced damage in the C57BL/6J mouse retina. Adult mice (90–110 days) were pretreated with 9 J/cm2 of 670 nm light once daily for 5 consecutive days prior to being placed in hyperoxic environment (75% oxygen). Control groups were exposed to hyperoxia, but received no 670 nm light pretreatment. Retinas were collected after 0, 3, 7, 10 or 14 days of hyperoxia exposure (n = 12/group) and prepared either for histological analysis, or RNA extraction and quantitative polymerase chain reaction (qPCR). Photoreceptor damage and loss were quantified by counting photoreceptors undergoing cell death and measuring photoreceptor layer thickness. Localization of acrolein, and cytochrome c oxidase subunit Va (Cox Va) were identified through immunohistochemistry. Expression of heme oxygenase-1 (Hmox-1), complement component 3 (C3) and fibroblast growth factor 2 (Fgf-2) genes were quantified using qPCR. Results The hyperoxia-induced photoreceptor loss was accompanied by reduction of metabolic marker, Cox Va, and increased expression of oxidative stress indicator, acrolein and Hmox-1. Pretreatment with 670 nm red light reduced expression of markers of oxidative stress and C3, and slowed, but did not prevent, photoreceptor loss over the time course of hyperoxia exposure. Conclusion The damaging effects of hyperoxia on photoreceptors were ameliorated following pretreatment with 670 nm light in hyperoxic mouse retinas. These results suggest that pretreatment with 670 nm light may provide stability to photoreceptors in conditions of oxidative stress. PMID:24134095

  18. Imaging translucent cell bodies in the living mouse retina without contrast agents

    PubMed Central

    Guevara-Torres, A.; Williams, D. R.; Schallek, J. B.

    2015-01-01

    The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina. PMID:26114032

  19. Imaging translucent cell bodies in the living mouse retina without contrast agents.

    PubMed

    Guevara-Torres, A; Williams, D R; Schallek, J B

    2015-06-01

    The transparency of most retinal cell classes typically precludes imaging them in the living eye; unless invasive methods are used that deploy extrinsic contrast agents. Using an adaptive optics scanning light ophthalmoscope (AOSLO) and capitalizing on the large numerical aperture of the mouse eye, we enhanced the contrast from otherwise transparent cells by subtracting the left from the right half of the light distribution in the detector plane. With this approach, it is possible to image the distal processes of photoreceptors, their more proximal cell bodies and the mosaic of horizontal cells in the living mouse retina.

  20. 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

  1. A model microfluidics-based system for the human and mouse retina.

    PubMed

    Mishra, Shawn; Thakur, Ankush; Redenti, Stephen; Vazquez, Maribel

    2015-12-01

    The application of microfluidics technologies to the study of retinal function and response holds great promise for development of new and improved treatments for patients with degenerative retinal diseases. Restoration of vision via retinal transplantation therapy has been severely limited by the low numbers of motile cells observed post transplantation. Using modern soft lithographic techniques, we have developed the μRetina, a novel and convenient biomimetic microfluidics device capable of examing the migratory behavior of retinal lineage cells within biomimetic geometries of the human and mouse retina. Coupled computer simulations and experimental validations were used to characterize and confirm the formation of chemical concentration gradients within the μRetina, while real-time images within the device captured radial and theta cell migration in response to concentration gradients of stromal derived factor (SDF-1), a known chemoattractant. Our data underscore how the μRetina can be used to examine the concentration-dependent migration of retinal progenitors in order to enhance current therapies, as well as develop novel migration-targeted treatments.

  2. Effect of light on global gene expression in the neuroglobin-deficient mouse retina

    PubMed Central

    ILMJÄRV, STEN; REIMETS, RIIN; HUNDAHL, CHRISTIAN ANSGAR; LUUK, HENDRIK

    2014-01-01

    Several previous studies have raised controversy over the functional role of neuroglobin (Ngb) in the retina. Certain studies indicate a significant impact of Ngb on retinal physiology, whereas others are conflicting. The present is an observational study that tested the effect of Ngb deficiency on gene expression in dark- and light-adapted mouse retinas. Large-scale gene expression profiling was performed using GeneChip® Mouse Exon 1.0 ST arrays and the results were compared to publicly available data sets. The lack of Ngb was found to have a minor effect on the light-induced retinal gene expression response. In addition, there was no increase in the expression of marker genes associated with hypoxia, endoplasmic reticulum-stress and oxidative stress in the Ngb-deficient retina. By contrast, several genes were identified that appeared to be differentially expressed between the genotypes when the effect of light was ignored. The present study indicates that Ngb deficiency does not lead to major alternations in light-dependent gene expression response, but leads to subtle systemic differences of a currently unknown functional significance. PMID:25279145

  3. Heterologous expression of the adenosine A1 receptor in transgenic mouse retina.

    PubMed

    Li, Ning; Salom, David; Zhang, Li; Harris, Tim; Ballesteros, Juan A; Golczak, Marcin; Jastrzebska, Beata; Palczewski, Krzysztof; Kurahara, Carole; Juan, Todd; Jordan, Steven; Salon, John A

    2007-07-17

    Traditional cell-based systems used to express integral membrane receptors have yet to produce protein samples of sufficient quality for structural study. Herein we report an in vivo method that harnesses the photoreceptor system of the retina to heterologously express G protein-coupled receptors in a biochemically homogeneous and pharmacologically functional conformation. As an example we show that the adenosine A1 receptor, when placed under the influence of the mouse opsin promoter and rhodopsin rod outer segment targeting sequence, localized to the photoreceptor cells of transgenic retina. The resulting receptor protein was uniformly glycosylated and pharmacologically well behaved. By comparison, we demonstrated in a control experiment that opsin, when expressed in the liver, had a complex pattern of glycosylation. Upon solubilization, the retinal adenosine A1 receptor retained binding characteristics similar to its starting material. This expression method may prove generally useful for generating high-quality G protein-coupled receptors for structural studies.

  4. Analysis of Alternative Pre-RNA Splicing in the Mouse Retina Using a Fluorescent Reporter.

    PubMed

    Murphy, Daniel; Kolandaivelu, Saravanan; Ramamurthy, Visvanathan; Stoilov, Peter

    2016-01-01

    In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.

  5. Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse

    PubMed Central

    Souza, Fred G. Oliveira; Bruce, Kady S.; Strang, Christianne E.; Morley, Barbara J.; Keyser, Kent T.

    2014-01-01

    Purpose The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer’s disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. Methods To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. Results In the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype

  6. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  7. Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo.

    PubMed

    Liu, Qian; Guan, Liping; Huang, Bing; Li, Weihua; Su, Qiao; Yu, Minbin; Xu, Xiaoping; Luo, Ting; Lin, Shaochun; Sun, Xuerong; Chen, Mengfei; Chen, Xigu

    2011-06-01

    Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate

  8. Diabetic retinopathy alters light-induced clock gene expression and dopamine levels in the mouse retina

    PubMed Central

    Lahouaoui, Hasna; Coutanson, Christine; Cooper, Howard M.; Bennis, Mohamed

    2016-01-01

    Purpose Diabetic retinopathy is one of the most common consequences of diabetes that affects millions of working-age adults worldwide and leads to progressive degeneration of the retina, visual loss, and blindness. Diabetes is associated with circadian disruption of the central and peripheral circadian clocks, but the mechanisms responsible for such alterations are unknown. Using a streptozotocin (STZ)-induced model of diabetes, we investigated whether diabetes alters 1) the circadian regulation of clock genes in the retina and in the central clocks, 2) the light response of clock genes in the retina, and/or 3) light-driven retinal dopamine (DA), a major output marker of the retinal clock. Methods To quantify circadian expression of clock and clock-controlled genes, retinas and suprachiasmatic nucleus (SCN) from the same animals were collected every 4 h in circadian conditions, 12 weeks post-diabetes. Induction of Per1, Per2, and c-fos mRNAs was quantified in the retina after the administration of a pulse of monochromatic light (480 nm, 1.17×1014 photons/cm2/s, 15 min) at circadian time 16. Gene expression was assessed with real-time reverse transcription PCR (RT–PCR). Pooled retinas from the control and STZ-diabetic mice were collected 2 h after light ON and light OFF (Zeitgeber time (ZT)2 and ZT14), and DA and its metabolite were analyzed with high-performance liquid chromatography (HPLC). Results We found variable effects of diabetes on the expression of clock genes in the retina and only slight differences in phase and/or amplitude in the SCN. c-fos and Per1 induction by a 480 nm light pulse was abolished in diabetic animals at 12 weeks post-induction of diabetes in comparison with the control mice, suggesting a deficit in light-induced neuronal activation of the retinal clock. Finally, we quantified a 56% reduction in the total number of tyrosine hydroxylase (TH) immunopositive cells, associated with a decrease in DA levels during the subjective day (ZT2

  9. Anatomical and Neurochemical Characterization of Dopaminergic Interplexiform Processes in Mouse and Rat Retinas

    PubMed Central

    WITKOVSKY, PAUL; GÁBRIEL, ROBERT; KRIŽAJ, DAVID

    2010-01-01

    Dopaminergic (DA) neurons of mouse and rat retinas are of the interplexiform subtype (DA-IPC), i.e., they send processes distally toward the outer retina, exhibiting numerous varicosities along their course. The primary question we addressed was whether distally located DA-IPC varicosities, identified by tyrosine hydroxylase (TH) immunoreactivity, had the characteristic presynaptic proteins associated with calcium-dependent vesicular release of neurotransmitter. We found that TH immunoreactive varicosities in the outer retina possessed vesicular monoamine transporter 2 and vesicular GABA transporter, but they lacked immunostaining for any of nine subtypes of voltage-dependent calcium channel. Immunoreactivity for other channels that may permit calcium influx such as certain ionotropic glutamate receptors and canonical transient receptor potential channels (TRPCs) was similarly absent, although DA-IPC varicosities did show ryanodine receptor immunoreactivity, indicating the presence of intracellular calcium stores. The synaptic vesicle proteins sv2a and sv2b and certain other proteins associated with the presynaptic membrane were absent from DA-IPC varicosities, but the vesicular SNARE protein, vamp2, was present in a fraction of those varicosities. We identified a presumed second class of IPC that is GABAergic but not dopaminergic. Outer retinal varicosities of this putative GABAergic IPC did colocalize synaptic vesicle protein 2a, suggesting they possessed a conventional vesicular release mechanism. PMID:18615559

  10. Structure of clonal and polyclonal cell arrays in chimeric mouse retina.

    PubMed Central

    Williams, R W; Goldowitz, D

    1992-01-01

    One of the most striking results of recent cell-lineage studies of vertebrate retina is the marked variability in the size and types of clones marked by retroviral transfection and dye injection of embryonic progenitor cells. Is this variability due to microenvironmental modulation of cell determination, to lineage restriction, or to experimental perturbation of the progenitor cells? We have taken advantage of species-specific DNA probes to mark groups of lineage-related cells in experimental mouse chimeras. This method of marking cells has two distinct advantages over previous methods: direct manipulation of progenitor cells is avoided, and clones are established at an earlier stage of retinal development. The most notable feature of retinal cohorts in chimeras is their structural uniformity--each is a solid radial array that contains the same ratio of major cell types as the retina itself. This is true even of the smallest monoclonal cohorts, which contain fewer than 200 cells. Our results provides compelling empirical support for the hypothesis that the murine retina is made up of hundreds of relatively homogeneous radial units, each derived from single retinal precursor cells. This finding is inconsistent with micro-environmental modulation of clone structure early in development. We raise the possibility that the heterogeneity among clones marked by dye injection and transfection is due to progressive lineage restriction or to experimental perturbation of the retinal progenitor cells. Images PMID:1741373

  11. A network comprising short and long noncoding RNAs and RNA helicase controls mouse retina architecture

    PubMed Central

    Krol, Jacek; Krol, Ilona; Alvarez, Claudia Patricia Patino; Fiscella, Michele; Hierlemann, Andreas; Roska, Botond; Filipowicz, Witold

    2015-01-01

    Brain regions, such as the cortex and retina, are composed of layers of uniform thickness. The molecular mechanism that controls this uniformity is not well understood. Here we show that during mouse postnatal development the timed expression of Rncr4, a retina-specific long noncoding RNA, regulates the similarly timed processing of pri-miR-183/96/182, which is repressed at an earlier developmental stage by RNA helicase Ddx3x. Shifting the timing of mature miR-183/96/182 accumulation or interfering with Ddx3x expression leads to the disorganization of retinal architecture, with the photoreceptor layer being most affected. We identify Crb1, a component of the adhesion belt between glial and photoreceptor cells, as a link between Rncr4-regulated miRNA metabolism and uniform retina layering. Our results suggest that the precise timing of glia–neuron interaction controlled by noncoding RNAs and Ddx3x is important for the even distribution of cells across layers. PMID:26041499

  12. Making the gradient: Thyroid hormone regulates cone opsin expression in the developing mouse retina

    PubMed Central

    Roberts, Melanie R.; Srinivas, Maya; Forrest, Douglas; Morreale de Escobar, Gabriella; Reh, Thomas A.

    2006-01-01

    Most mammals have two types of cone photoreceptors, which contain either medium wavelength (M) or short wavelength (S) opsin. The number and spatial organization of cone types varies dramatically among species, presumably to fine-tune the retina for different visual environments. In the mouse, S- and M-opsin are expressed in an opposing dorsal–ventral gradient. We previously reported that cone opsin patterning requires thyroid hormone β2, a nuclear hormone receptor that regulates transcription in conjunction with its ligand, thyroid hormone (TH). Here we show that exogenous TH inhibits S-opsin expression, but activates M-opsin expression. Binding of endogenous TH to TRβ2 is required to inhibit S-opsin and to activate M-opsin. TH is symmetrically distributed in the retina at birth as S-opsin expression begins, but becomes elevated in the dorsal retina at the time of M-opsin onset (postnatal day 10). Our results show that TH is a critical regulator of both S-opsin and M-opsin, and suggest that a TH gradient may play a role in establishing the gradient of M-opsin. These results also suggest that the ratio and patterning of cone types may be determined by TH availability during retinal development. PMID:16606843

  13. Glio-vascular modifications caused by Aquaporin-4 deletion in the mouse retina.

    PubMed

    Nicchia, Grazia Paola; Pisani, Francesco; Simone, Laura; Cibelli, Antonio; Mola, Maria Grazia; Dal Monte, Massimo; Frigeri, Antonio; Bagnoli, Paola; Svelto, Maria

    2016-05-01

    Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition. AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1β and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role

  14. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina

    PubMed Central

    Choi, Hannah; Zhang, Lei; Cembrowski, Mark S.; Sabottke, Carl F.; Markowitz, Alexander L.; Butts, Daniel A.; Kath, William L.; Singer, Joshua H.

    2014-01-01

    In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell. PMID:25008417

  15. Lgr5⁺ amacrine cells possess regenerative potential in the retina of adult mice.

    PubMed

    Chen, Mengfei; Tian, Shenghe; Glasgow, Nathan G; Gibson, Gregory; Yang, Xiaoling; Shiber, Christen E; Funderburgh, James; Watkins, Simon; Johnson, Jon W; Schuman, Joel S; Liu, Hongjun

    2015-08-01

    Current knowledge indicates that the adult mammalian retina lacks regenerative capacity. Here, we show that the adult stem cell marker, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), is expressed in the retina of adult mice. Lgr5(+) cells are generated at late stages of retinal development and exhibit properties of differentiated amacrine interneurons (amacrine cells). Nevertheless, Lgr5(+) amacrine cells contribute to regeneration of new retinal cells in the adult stage. The generation of new retinal cells, including retinal neurons and Müller glia from Lgr5(+) amacrine cells, begins in early adulthood and continues as the animal ages. Together, these findings suggest that the mammalian retina is not devoid of regeneration as previously thought. It is rather dynamic, and Lgr5(+) amacrine cells function as an endogenous regenerative source. The identification of such cells in the mammalian retina may provide new insights into neuronal regeneration and point to therapeutic opportunities for age-related retinal degenerative diseases.

  16. Amelioration of both functional and morphological abnormalities in the retina of a mouse model of ocular albinism following AAV-mediated gene transfer.

    PubMed

    Surace, Enrico Maria; Domenici, Luciano; Cortese, Katia; Cotugno, Gabriella; Di Vicino, Umberto; Venturi, Consuelo; Cellerino, Alessandro; Marigo, Valeria; Tacchetti, Carlo; Ballabio, Andrea; Auricchio, Alberto

    2005-10-01

    X-linked recessive ocular albinism type I (OA1) is due to mutations in the OA1 gene (approved gene symbol GPR143), which is expressed in the retinal pigment epithelium (RPE). The Oa1 (Gpr143) knockout mouse (Oa1(-/-)) model recapitulates many of the OA1 retinal morphological anomalies, including a lower number of melanosomes of increased size in the RPE. The Oa1(-/-) mouse also displays some of the retinal developmental abnormalities observed in albino patients such as misrouting of the optic tracts. Here, we show that these anomalies are associated with retinal electrophysiological abnormalities, including significant decrease in a- and b-wave amplitude and delayed recovery of b-wave amplitude from photoreceptor desensitization following bright light exposure. This suggests that lack of Oa1 in the RPE impacts on photoreceptor activity. More interestingly, adeno-associated viral vector-mediated Oa1 gene transfer to the retina of the Oa1(-/-) mouse model results in significant recovery of its retinal functional abnormalities. In addition, Oa1 retinal gene transfer increases the number of melanosomes in the Oa1(-/-) mouse RPE. Our data show that gene transfer to the adult retina unexpectedly rescues both functional and morphological abnormalities in a retinal developmental disorder, opening novel potential therapeutic perspectives for this and other forms of albinism.

  17. Activation of ganglion cells in wild-type and rd1 mouse retinas with monophasic and biphasic current pulses

    NASA Astrophysics Data System (ADS)

    Jensen, Ralph J.; Rizzo, Joseph F. III

    2009-06-01

    We and other research groups are designing an electronic retinal prosthesis to provide vision for patients who are blind due to photoreceptor degeneration. In this study, we examined the effect of stimulus waveform on the amount of current needed to activate retinal ganglion cells (RGCs) when the retinal neural network is stimulated. Isolated retinas of wild-type and rd1 mice were stimulated with cathodal and anodal monophasic current pulses of 1 ms duration and symmetric biphasic current pulses (1 ms per phase) delivered through an electrode that was located subretinally. For both wild-type and rd1 mouse retinas, cathodal current pulses were least effective in activating most RGCs. The median threshold current for a cathodal current pulse was 2.0-4.4 fold higher than the median threshold current for either an anodal or a biphasic current pulse. In wild-type mouse retinas, the median threshold current for activating RGCs with anodal current pulses was 23% lower than that with biphasic current pulses. In rd1 mouse retinas, the median threshold currents for anodal and biphasic current pulses were about the same. However, the variance in thresholds of rd1 RGCs for biphasic pulse stimulation was much smaller than for anodal pulse stimulation. Thus, a symmetric biphasic current pulse may be the best stimulus for activating the greatest number of RGCs in retinas devoid of photoreceptors.

  18. 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.

  19. Potential for neural regeneration after neurotoxic injury in the adult mammalian retina

    PubMed Central

    Ooto, Sotaro; Akagi, Tadamichi; Kageyama, Ryoichiro; Akita, Joe; Mandai, Michiko; Honda, Yoshihito; Takahashi, Masayo

    2004-01-01

    It has long been believed that the retina of mature mammals is incapable of regeneration. In this study, using the N-methyl-d-aspartate neurotoxicity model of adult rat retina, we observed that some Müller glial cells were stimulated to proliferate in response to a toxic injury and produce bipolar cells and rod photoreceptors. Although these newly produced neurons were limited in number, retinoic acid treatment promoted the number of regenerated bipolar cells. Moreover, misexpression of basic helix–loop–helix and homeobox genes promoted the induction of amacrine, horizontal, and rod photoreceptor specific phenotypes. These findings demonstrated that retinal neurons regenerated even in adult mammalian retina after toxic injury. Furthermore, we could partially control the fate of the regenerated neurons with extrinsic factors or intrinsic genes. The Müller glial cells constitute a potential source for the regeneration of adult mammalian retina and can be a target for drug delivery and gene therapy in retinal degenerative diseases. PMID:15353594

  20. Rod electrical coupling is controlled by a circadian clock and dopamine in mouse retina

    PubMed Central

    Jin, Nan Ge; Chuang, Alice Z; Masson, Philippe J; Ribelayga, Christophe P

    2015-01-01

    Key points Rod photoreceptors play a key role in vision in dim light; in the mammalian retina, although rods are anatomically connected or coupled by gap junctions, a type of electrical synapse, the functional importance and regulation of rod coupling has remained elusive. We have developed a new technique in the mouse: perforated patch-clamp recording of rod inner segments in isolated intact retinae maintained by superfusion. We find that rod electrical coupling is controlled by a circadian clock and dopamine, and is weak during the day and stronger at night. The results also indicate that the signal-to-noise ratio for a dim light response is increased at night because of coupling. Our observations will provide a framework for understanding the daily variations in human vision as well as the basis of specific retinal malfunctions. Abstract Rod single-photon responses are critical for vision in dim light. Electrical coupling via gap junction channels shapes the light response properties of vertebrate photoreceptors, but the regulation of rod coupling and its impact on the single-photon response have remained unclear. To directly address these questions, we developed a perforated patch-clamp recording technique and recorded from single rod inner segments in isolated intact neural mouse retinae, maintained by superfusion. Experiments were conducted at different times of the day or under constant environmental conditions, at different times across the circadian cycle. We show that rod electrical coupling is regulated by a circadian clock and dopamine, so that coupling is weak during the day and strong at night. Altogether, patch-clamp recordings of single-photon responses in mouse rods, tracer coupling, receptive field measurements and pharmacological manipulations of gap junction and dopamine receptor activity provide compelling evidence that rod coupling is modulated in a circadian manner. These data are consistent with computer modelling. At night, single

  1. Inner Retina Remodeling in a Mouse Model of Stargardt-like Macular Dystrophy (STGD3)

    PubMed Central

    Kuny, Sharee; Gaillard, Frédéric; Mema, Silvina C.; Freund, Paul R.; Zhang, Kang; MacDonald, Ian M.; Sparrow, Janet R.

    2010-01-01

    Purpose. To investigate the impact of progressive age-related photoreceptor degeneration on retinal integrity in Stargardt-like macular dystrophy (STGD3). Methods. The structural design of the inner retina of the ELOVL4 transgenic mouse model of STGD3 was compared with that of age-matched littermate wild-type (WT) mice from 1 to 24 months of age by using immunohistofluorescence and confocal microscopy and by relying on antibodies against cell-type–specific markers, synapse-associated proteins, and neurotransmitters. Results. Müller cell reactivity occurred at the earliest age studied, before photoreceptor loss. This finding is perhaps not surprising, considering the cell's ubiquitous roles in retina homeostasis. Second-order neurons displayed salient morphologic changes as a function of photoreceptoral input loss. Age-related sprouting of dendritic fibers from rod bipolar and horizontal cells into the ONL did not occur. In contrast, with the loss of photoreceptor sensory input, these second-order neurons progressively bore fewer synapses. After rod loss, the few remaining cones showed abnormal opsin expression, revealing tortuous branched axons. After complete ONL loss (beyond 18 months of age), localized areas of extreme retinal disruptions were observed in the central retina. RPE cell invasion, dense networks of strongly reactive Müller cell processes, and invagination of axons and blood vessels were distinctive features of these regions. In addition, otherwise unaffected cholinergic amacrine cells displayed severe perturbation of their cell bodies and synaptic plexi in these areas. Conclusions. Remodeling in ELOVL4 transgenic mice follows a pattern similar to that reported after other types of hereditary retinopathies in animals and humans, pointing to a potentially common pathophysiologic mechanism. PMID:19933199

  2. Heterogeneous Expression of the Core Circadian Clock Proteins among Neuronal Cell Types in Mouse Retina

    PubMed Central

    Liu, Xiaoqin; Zhang, Zhijing; Ribelayga, Christophe P.

    2012-01-01

    Circadian rhythms in metabolism, physiology, and behavior originate from cell-autonomous circadian clocks located in many organs and structures throughout the body and that share a common molecular mechanism based on the clock genes and their protein products. In the mammalian neural retina, despite evidence supporting the presence of several circadian clocks regulating many facets of retinal physiology and function, the exact cellular location and genetic signature of the retinal clock cells remain largely unknown. Here we examined the expression of the core circadian clock proteins CLOCK, BMAL1, NPAS2, PERIOD 1(PER1), PERIOD 2 (PER2), and CRYPTOCHROME2 (CRY2) in identified neurons of the mouse retina during daily and circadian cycles. We found concurrent clock protein expression in most retinal neurons, including cone photoreceptors, dopaminergic amacrine cells, and melanopsin-expressing intrinsically photosensitive ganglion cells. Remarkably, diurnal and circadian rhythms of expression of all clock proteins were observed in the cones whereas only CRY2 expression was found to be rhythmic in the dopaminergic amacrine cells. Only a low level of expression of the clock proteins was detected in the rods at any time of the daily or circadian cycle. Our observations provide evidence that cones and not rods are cell-autonomous circadian clocks and reveal an important disparity in the expression of the core clock components among neuronal cell types. We propose that the overall temporal architecture of the mammalian retina does not result from the synchronous activity of pervasive identical clocks but rather reflects the cellular and regional heterogeneity in clock function within retinal tissue. PMID:23189207

  3. Genetic Dissection of Rod and Cone Pathways in the Dark-Adapted Mouse Retina

    PubMed Central

    Abd-El-Barr, Muhammad M.; Pennesi, Mark E.; Saszik, Shannon M.; Barrow, Andrew J.; Lem, Janis; Bramblett, Debra E.; Paul, David L.; Frishman, Laura J.; Wu, Samuel M.

    2009-01-01

    A monumental task of the mammalian retina is to encode an enormous range (>109-fold) of light intensities experienced by the animal in natural environments. Retinal neurons carry out this task by dividing labor into many parallel rod and cone synaptic pathways. Here we study the operational plan of various rod- and cone-mediated pathways by analyzing electroretinograms (ERGs), primarily b-wave responses, in dark-adapted wildtype, connexin36 knockout, depolarizing rod–bipolar cell (DBCR) knockout, and rod transducin alpha-subunit knockout mice [WT, Cx36(−/−), Bhlhb4(−/−), and Trα(−/−)]. To provide additional insight into the cellular origins of various components of the ERG, we compared dark-adapted ERG responses with response dynamic ranges of individual retinal cells recorded with patch electrodes from dark-adapted mouse retinas published from other studies. Our results suggest that the connexin36-mediated rod–cone coupling is weak when light stimulation is weak and becomes stronger as light stimulation increases in strength and that rod signals may be transmitted to some DBCCs via direct chemical synapses. Moreover, our analysis indicates that DBCR responses contribute about 80% of the overall DBC response to scotopic light and that rod and cone signals contribute almost equally to the overall DBC responses when stimuli are strong enough to saturate the rod bipolar cell response. Furthermore, our study demonstrates that analysis of ERG b-wave of dark-adapted, pathway-specific mutants can be used as an in vivo tool for dissecting rod and cone synaptic pathways and for studying the functions of pathway-specific gene products in the retina. PMID:19587322

  4. The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina

    PubMed Central

    Xiong, Wei-Hong; Pang, Ji-Jie; Pennesi, Mark E.; Duvoisin, Robert M.; Wu, Samuel M.; Morgans, Catherine W.

    2015-01-01

    Purpose Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse. Methods Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice. Results Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration. Conclusions Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway. PMID:26230760

  5. Biological Characterization of Gene Response to Insulin-Induced Hypoglycemia in Mouse Retina

    PubMed Central

    Emery, Martine; Nanchen, Natacha; Preitner, Frédéric; Ibberson, Mark; Roduit, Raphaël

    2016-01-01

    Glucose is the most important metabolic substrate of the retina and maintenance of normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH) decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery) on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining “normal” GSH level, as well as a strict glycemic control, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy. PMID:26918849

  6. Increased neuronal death and disturbed axonal growth in the Polμ-deficient mouse embryonic retina

    PubMed Central

    Baleriola, Jimena; Álvarez-Lindo, Noemí; de la Villa, Pedro; Bernad, Antonio; Blanco, Luis; Suárez, Teresa; de la Rosa, Enrique J.

    2016-01-01

    Programmed cell death occurs naturally at different stages of neural development, including neurogenesis. The functional role of this early phase of neural cell death, which affects recently differentiated neurons among other cell types, remains undefined. Some mouse models defective in DNA double-strand break (DSB) repair present massive cell death during neural development, occasionally provoking embryonic lethality, while other organs and tissues remain unaffected. This suggests that DSBs occur frequently and selectively in the developing nervous system. We analyzed the embryonic retina of a mouse model deficient in the error-prone DNA polymerase μ (Polμ), a key component of the non-homologous end-joining (NHEJ) repair system. DNA DSBs were increased in the mutant mouse at embryonic day 13.5 (E13.5), as well as the incidence of cell death that affected young neurons, including retinal ganglion cells (RGCs). Polμ−/− mice also showed disturbed RGC axonal growth and navigation, and altered distribution of the axonal guidance molecules L1-CAM and Bravo (also known as Nr-CAM). These findings demonstrate that Polμ is necessary for proper retinal development, and support that the generation of DSBs and their repair via the NHEJ pathway are genuine processes involved in neural development. PMID:27172884

  7. 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

  8. CD1 Mouse Retina Is Shielded From Iron Overload Caused by a High Iron Diet

    PubMed Central

    Bhoiwala, Devang L.; Song, Ying; Cwanger, Alyssa; Clark, Esther; Zhao, Liang-liang; Wang, Chenguang; Li, Yafeng; Song, Delu; Dunaief, Joshua L.

    2015-01-01

    Purpose High RPE iron levels have been associated with age-related macular degeneration. Mutation of the ferroxidase ceruloplasmin leads to RPE iron accumulation and degeneration in patients with aceruloplasminemia; mice lacking ceruloplasmin and its homolog hephaestin have a similar RPE degeneration. To determine whether a high iron diet (HID) could cause RPE iron accumulation, possibly contributing to RPE oxidative stress in AMD, we tested the effect of dietary iron on mouse RPE iron. Methods Male CD1 strain mice were fed either a standard iron diet (SID) or the same diet with extra iron added (HID) for either 3 months or 10 months. Mice were analyzed with immunofluorescence and Perls' histochemical iron stain to assess iron levels. Levels of ferritin, transferrin receptor, and oxidative stress gene mRNAs were measured by quantitative PCR (qPCR) in neural retina (NR) and isolated RPE. Morphology was assessed in plastic sections. Results Ferritin immunoreactivity demonstrated a modest increase in the RPE in 10-month HID mice. Analysis by qPCR showed changes in mRNA levels of iron-responsive genes, indicating moderately increased iron in the RPE of 10-month HID mice. However, even by age 18 months, there was no Perls' signal in the retina or RPE and no retinal degeneration. Conclusions These findings indicate that iron absorbed from the diet can modestly increase the level of iron deposition in the wild-type mouse RPE without causing RPE or retinal degeneration. This suggests regulation of retinal iron uptake at the blood-retinal barriers. PMID:26275132

  9. Coupling ex vivo electroporation of mouse retinas and luciferase reporter assays to assess rod-specific promoter activity.

    PubMed

    Boulling, Arnaud; Escher, Pascal

    2016-07-01

    Ex vivo electroporation of mouse retinas is an established tool to modulate gene expression and to study cell type-specific gene expression. Here we coupled ex vivo electroporation to luciferase reporter assays to facilitate the study of rod-photoreceptor-specific gene promoters. The activity of the rod-specific proximal bovine rhodopsin promoter was significantly increased in C57BL/6J wild-type retinas at postnatal days 1 and 7 by 3.4-fold and 8.7-fold respectively. In C57BL/6J Nr2e3(rd7/rd7) retinas, where the rod photoreceptor-specific nuclear receptor Nr2e3 is not expressed, a significant increase by 2.5-fold was only observed at postnatal day 7. Cone-specific S-opsin promoter activity was not modulated in C57BL/6J wild-type and Nr2e3(rd7/rd7) retinas. Taken together, we describe an easily implementable protocol to assess rod-specific promoter activity in a physiological context resembling that of the developing postnatal mouse retina.

  10. Gsg1, Trnp1, and Tmem215 Mark Subpopulations of Bipolar Interneurons in the Mouse Retina

    PubMed Central

    Park, Ko Uoon; Randazzo, Grace; Jones, Kenneth L.; Brzezinski, Joseph A.

    2017-01-01

    Purpose How retinal bipolar cell interneurons are specified and assigned to specialized subtypes is only partially understood. In part, this is due to a lack of early pan- and subtype-specific bipolar cell markers. To discover these factors, we identified genes that were upregulated in Blimp1 (Prdm1) mutant retinas, which exhibit precocious bipolar cell development. Methods Postnatal day (P)2 retinas from Blimp1 conditional knock-out (CKO) mice and controls were processed for RNA sequencing. Genes that increased at least 45% and were statistically different between conditions were considered candidate bipolar-specific factors. Candidates were further evaluated by RT-PCR, in situ hybridization, and immunohistochemistry. Knock-in Tmem215-LacZ mice were used to better trace retinal expression. Results A comparison between Blimp1 CKO and control RNA-seq datasets revealed approximately 40 significantly upregulated genes. We characterized the expression of three genes that have no known function in the retina, Gsg1 (germ cell associated gene), Trnp1 (TMF-regulated nuclear protein), and Tmem215 (a predicted transmembrane protein). Germ cell associated gene appeared restricted to a small subset of cone bipolars while Trnp1 was seen in all ON type bipolar cells. Using Tmem215-LacZ heterozygous knock-in mice, we observed that β-galactosidase expression started early in bipolar cell development. In adults, Tmem215 was expressed by a subset of ON and OFF cone bipolar cells. Conclusions We have identified Gsg1, Tmem215, and Trnp1 as novel bipolar subtype-specific genes. The spatial and temporal pattern of their expression is consistent with a role in controlling bipolar subtype fate choice, differentiation, or physiology. PMID:28199486

  11. Identification of Radial Glia Progenitors in the Developing and Adult Retina of Sharks

    PubMed Central

    Sánchez-Farías, Nuria; Candal, Eva

    2016-01-01

    Neural stem cells give rise to transient progenitors termed neuroepithelial cells (NECs) and radial glial cells (RGCs). RGCs represent the major source of neurons, glia and adult stem cells in several regions of the central nervous system (CNS). RGCs are mostly transient in mammals, but they are widely maintained in the adult CNS of fishes, where they continue to be morphologically similar to RGCs in the mammalian brain and fulfill similar roles as progenitors and guide for migrating neurons. The retina of fishes offers an exceptional model to approach the study of adult neurogenesis because of the presence of constitutive proliferation from the ciliary marginal zone (CMZ), containing NECs, and from adult glial cells with radial morphology (the Müller glia). However, the cellular hierarchies and precise contribution of different types of progenitors to adult neurogenesis remain unsolved. We have analyzed the transition from NECs to RGCs and RGC differentiation in the retina of the cartilaginous fish Scyliorhinus canicula, which offers a particularly good spatial and temporal frame to investigate this process. We have characterized progenitor and adult RGCs by immunohistochemical detection of glial markers as glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). We have compared the emergence and localization of glial markers with that of proliferating cell nuclear antigen (PCNA, a proliferation maker) and Doublecortin (DCX, which increases at early stages of neuronal differentiation). During retinal development, GFAP-immunoreactive NECs located in the most peripheral CMZ (CMZp) codistribute with DCX-immunonegative cells. GFAP-immunoreactive RGCs and Müller cells are located in successive more central parts of the retina and codistribute with DCX- and DCX/GS-immunoreactive cells, respectively. The same types of progenitors are found in juveniles, suggesting that the contribution of the CMZ to adult neurogenesis implies a transition through the

  12. Patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

    PubMed Central

    Tu, Hung-Ya; Hsu, Chih-Chun; Chen, Yu-Jiun

    2016-01-01

    SHORT ABSTRACT This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation. LONG ABSTRACT The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs). PMID:27768050

  13. Genetically modified neural stem cells for a local and sustained delivery of neuroprotective factors to the dystrophic mouse retina.

    PubMed

    Jung, Gila; Sun, Jing; Petrowitz, Bettina; Riecken, Kristoffer; Kruszewski, Katharina; Jankowiak, Wanda; Kunst, Frank; Skevas, Christos; Richard, Gisbert; Fehse, Boris; Bartsch, Udo

    2013-12-01

    A continuous intraocular delivery of neurotrophic factors (NFs) is being explored as a strategy to rescue photoreceptor cells and visual functions in degenerative retinal disorders that are currently untreatable. To establish a cell-based intraocular delivery system for a sustained administration of NFs to the dystrophic mouse retina, we used a polycistronic lentiviral vector to genetically modify adherently cultivated murine neural stem (NS) cells. The vector concurrently encoded a gene of interest, a reporter gene, and a resistance gene and thus facilitated the selection, cloning, and in vivo tracking of the modified cells. To evaluate whether modified NS cells permit delivery of functionally relevant quantities of NFs to the dystrophic mouse retina, we expressed a secretable variant of ciliary neurotrophic factor (CNTF) in NS cells and grafted the cells into the vitreous space of Pde6b(rd1) and Pde6b(rd10) mice, two animal models of retinitis pigmentosa. In both mouse lines, grafted cells attached to the retina and lens, where they differentiated into astrocytes and some neurons. Adverse effects of the transplanted cells on the morphology of host retinas were not observed. Importantly, the CNTF-secreting NS cells significantly attenuated photoreceptor degeneration in both mutant mouse lines. The neuroprotective effect was significantly more pronounced when clonally derived NS cell lines selected for high expression levels of CNTF were grafted into Pde6b(rd1) mice. Intravitreal transplantations of modified NS cells may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based intraocular delivery of NFs in mouse models of photoreceptor degeneration.

  14. Neuroprotection of photoreceptors by direct delivery of erythropoietin to the retina of the retinal degeneration slow mouse.

    PubMed

    Rex, Tonia S; Wong, Ying; Kodali, Kishore; Merry, Shayla

    2009-11-01

    The primary objectives of this study were to determine if erythropoietin (EPO) is neuroprotective to the photoreceptors in the retinal degeneration slow (rds) mouse in the absence of an increase in hematocrit and to determine if deglycosylated EPO (DEPO) is less neuroprotective. We performed subretinal injections of 10U EPO, DEPO or hyperglycosylated EPO (HEPO) in postnatal day 7 rds mice. Whole eye EPO levels were quantified by ELISA at specified time points post-injection. TUNEL analysis, hematocrit, and immunohistochemistry were performed at postnatal day 20. Half of the amount of EPO measured immediately after injection was detected less than 1 h later. Twenty four hours later, EPO levels were 1000 times lower than the amount originally detected. Uninjected rds mice contained 36 +/- 2 TUNEL-positive cells/mm retina and PBS injected mice contained 17 +/- 3 TUNEL-positive cells/mm retina. EPO, DEPO, and HEPO treated rds retinas contained 5 +/- 2, 9 +/- 2, and 3 +/- 1 TUNEL-positive cells/mm retina, respectively. The hematocrit was 43% in control and 41% in treated rds mice Previous studies have shown neuroprotection of the retina by treatment with as little as 24-39 mU EPO/mg total protein in the eye. In this study, we detected 40 mU/mg EPO in the eye 11 h after injection of 10 U EPO. Treatment with all forms of EPO tested was neuroprotective to the photoreceptors without a concomitant increase in hematocrit.

  15. Doublecortin is widely expressed in the developing and adult retina of sharks.

    PubMed

    Sánchez-Farías, Nuria; Candal, Eva

    2015-05-01

    Doublecortin (DCX) is a microtubule-associated protein that has been considered a marker for neuronal precursors and young migrating neurons during the development of the central nervous system and in adult neurogenic niches. The retina of fishes represents an accessible, continuously growing and highly structured (layered) part of the central nervous system and, therefore, offers an exceptional model to extend our knowledge on the possible role of DCX in promoting neurogenesis and migration to appropriate layers. We have analyzed the distribution of DCX in the embryonic and postembryonic retina of a small shark, the lesser spotted dogfish Scyliorhinus canicula, by means of immunohistochemistry. We investigated the relationship between DCX expression and the neurogenic state of DCX-labeled cells by exploring its co-localization with the proliferation marker PCNA (proliferating cell nuclear antigen) and the marker of neuronal differentiation HuC/D. Since radially migrating neurons use radial glial fibers as substrate, we explored the possible correlation between DCX expression and cell migration along radial glia by comparing its expression with that of the glial marker GFAP (glial fibrillary acidic protein). Additionally, we characterized DCX-expressing cells by double immunocytochemistry using antibodies against Calbindin (a marker for mature bipolar and horizontal cells in this species) and Pax6, which has been proposed as a regulator of cell proliferation, cell differentiation, and neuron diversification in the neural retina of sharks. Strong DCX immunoreactivity was observed in immature cells and cell processes, at a time when retinal cells were not yet organized into different laminae. DCX was also found in subsets of mature ganglion, amacrine, bipolar and horizontal cells long after they had exited the cell cycle, a pattern that was maintained in juveniles and adults. Our results on DCX expression in the retina are compatible with a role for DCX in cell

  16. Pan-retinal characterisation of Light Responses from Ganglion Cells in the Developing Mouse Retina

    PubMed Central

    Hilgen, Gerrit; Pirmoradian, Sahar; Pamplona, Daniela; Kornprobst, Pierre; Cessac, Bruno; Hennig, Matthias H.; Sernagor, Evelyne

    2017-01-01

    We have investigated the ontogeny of light-driven responses in mouse retinal ganglion cells (RGCs). Using a large-scale, high-density multielectrode array, we recorded from hundreds to thousands of RGCs simultaneously at pan-retinal level, including dorsal and ventral locations. Responses to different contrasts not only revealed a complex developmental profile for ON, OFF and ON-OFF responses, but also unveiled differences between dorsal and ventral RGC responses. At eye-opening, dorsal RGCs of all types were more responsive to light, perhaps indicating an environmental priority to nest viewing for pre-weaning pups. The developmental profile of ON and OFF responses exhibited antagonistic behaviour, with the strongest ON responses shortly after eye-opening, followed by an increase in the strength of OFF responses later on. Further, we found that with maturation receptive field (RF) center sizes decrease, spike-triggered averaged responses to white noise become stronger, and centers become more circular while maintaining differences between RGC types. We conclude that the maturation of retinal functionality is not spatially homogeneous, likely reflecting ecological requirements that favour earlier maturation of the dorsal retina. PMID:28186129

  17. Degeneration stage-specific response pattern of retinal ganglion cell spikes in rd10 mouse retina.

    PubMed

    Park, D J; Senok, S S; Goo, Y S

    2015-01-01

    It is known that with retinal degeneration there is rewiring of retinal networks. Consequently, electrical stimulation of the degenerating retina produces responses that differ according to the stage of retinal degeneration. We sought to delineate a degeneration stage-specific parameter for the response pattern of retinal ganglion cell (RGC) spikes as a strategy for stage-specific electrical stimulation for perceptual efficiency of prosthetic vision devices. Electrically-evoked RGC spikes were recorded at different degeneration stages in the rd10 mouse model for human retinitis pigmentosa (RP). Retinal explants mounted on an 8×8 multi-electrode array were stimulated by applying 1 Hz cathodic-phase first biphasic current pulses. RGC firing rate during the first 100 ms post-stimulus was compared to that during the 100-1000 ms period and a response ratio of 100 ms (RR100 ms) was calculated through the different postnatal weeks. Our results show that during post-stimulus 100-1000 ms, the degree of correlation between pulse amplitude and evoked RGC spikes drastically decreases at PNW 4.5. This pattern was closely matched by the RR100 ms curve at this stage. We conclude that the RR100 ms might be a good indicator of the therapeutic potential of a retinal electrical prosthesis.

  18. Light-evoked synaptic activity of retinal ganglion and amacrine cells is regulated in developing mouse retina

    PubMed Central

    He, Quanhua; Wang, Ping; Tian, Ning

    2010-01-01

    Recent studies have shown a continued maturation of visual responsiveness and synaptic activity of retina after eye opening, including the size of receptive fields of retinal ganglion cells (RGCs), light-evoked synaptic output of RGCs, bipolar cell spontaneous synaptic inputs to RGCs, and the synaptic connections between RGCs and ON and OFF bipolar cells. Light deprivation retarded some of these age-dependent changes. However, many other functional and morphological features of RGCs are not sensitive to visual experience. To determine whether light-evoked synaptic responses of RGCs undergo developmental change, we directly examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to RGCs in developing retinas and found that both light-evoked excitatory and inhibitory synaptic currents decreased, but not increased, with age. We also examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to amacrine cells in developing retinas and found that the light-evoked synaptic input of amacrine cells is also down-regulated in developing mouse retina. Different from the developmental changes of RGC spontaneous synaptic activity, dark rearing has little effect on the developmental changes of light-evoked synaptic activity of both RGCs and amacrine cells. Therefore, we concluded that the synaptic mechanisms mediating spontaneous and light-evoked synaptic activity of RGCs and amacrine cells are likely to be different. PMID:21091802

  19. A critical analysis of Atoh7 (Math5) mRNA splicing in the developing mouse retina.

    PubMed

    Prasov, Lev; Brown, Nadean L; Glaser, Tom

    2010-08-24

    The Math5 (Atoh7) gene is transiently expressed during retinogenesis by progenitors exiting mitosis, and is essential for ganglion cell (RGC) development. Math5 contains a single exon, and its 1.7 kb mRNA encodes a 149-aa polypeptide. Mouse Math5 mutants have essentially no RGCs or optic nerves. Given the importance of this gene in retinal development, we thoroughly investigated the possibility of Math5 mRNA splicing by Northern blot, 3'RACE, RNase protection assays, and RT-PCR, using RNAs extracted from embryonic eyes and adult cerebellum, or transcribed in vitro from cDNA clones. Because Math5 mRNA contains an elevated G+C content, we used graded concentrations of betaine, an isostabilizing agent that disrupts secondary structure. Although approximately 10% of cerebellar Math5 RNAs are spliced, truncating the polypeptide, our results show few, if any, spliced Math5 transcripts exist in the developing retina (<1%). Rare deleted cDNAs do arise via RT-mediated RNA template switching in vitro, and are selectively amplified during PCR. These data differ starkly from a recent study (Kanadia and Cepko 2010), which concluded that the vast majority of Math5 and other bHLH transcripts are spliced to generate noncoding RNAs. Our findings clarify the architecture of the Math5 gene and its mechanism of action. These results have implications for all members of the bHLH gene family, for any gene that is alternatively spliced, and for the interpretation of all RT-PCR experiments.

  20. Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain

    PubMed Central

    Ye, Xin; Smallwood, Philip; Nathans, Jeremy

    2011-01-01

    The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (NdpAP). In the CNS, NdpAP expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of NdpAP expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, NdpAP expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. PMID:21055480

  1. Epigenetics of eu- and heterochromatin in inverted and conventional nuclei from mouse retina.

    PubMed

    Eberhart, Anja; Feodorova, Yana; Song, Congdi; Wanner, Gerhard; Kiseleva, Elena; Furukawa, Takahisa; Kimura, Hiroshi; Schotta, Gunnar; Leonhardt, Heinrich; Joffe, Boris; Solovei, Irina

    2013-08-01

    To improve light propagation through the retina, the rod nuclei of nocturnal mammals are uniquely changed compared to the nuclei of other cells. In particular, the main classes of chromatin are segregated in them and form regular concentric shells in order; inverted in comparison to conventional nuclei. A broad study of the epigenetic landscape of the inverted and conventional mouse retinal nuclei indicated several differences between them and several features of general interest for the organization of the mammalian nuclei. In difference to nuclei with conventional architecture, the packing density of pericentromeric satellites and LINE-rich chromatin is similar in inverted rod nuclei; euchromatin has a lower packing density in both cases. A high global chromatin condensation in rod nuclei minimizes the structural difference between active and inactive X chromosome homologues. DNA methylation is observed primarily in the chromocenter, Dnmt1 is primarily associated with the euchromatic shell. Heterochromatin proteins HP1-alpha and HP1-beta localize in heterochromatic shells, whereas HP1-gamma is associated with euchromatin. For most of the 25 studied histone modifications, we observed predominant colocalization with a certain main chromatin class. Both inversions in rod nuclei and maintenance of peripheral heterochromatin in conventional nuclei are not affected by a loss or depletion of the major silencing core histone modifications in respective knock-out mice, but for different reasons. Maintenance of peripheral heterochromatin appears to be ensured by redundancy both at the level of enzymes setting the epigenetic code (writers) and the code itself, whereas inversion in rods rely on the absence of the peripheral heterochromatin tethers (absence of code readers).

  2. Spatiotemporal features of early neuronogenesis differ in wild-type and albino mouse retina

    NASA Technical Reports Server (NTRS)

    Rachel, Rivka A.; Dolen, Gul; Hayes, Nancy L.; Lu, Alice; Erskine, Lynda; Nowakowski, Richard S.; Mason, Carol A.

    2002-01-01

    In albino mammals, lack of pigment in the retinal pigment epithelium is associated with retinal defects, including poor visual acuity from a photoreceptor deficit in the central retina and poor depth perception from a decrease in ipsilaterally projecting retinal fibers. Possible contributors to these abnormalities are reported delays in neuronogenesis (Ilia and Jeffery, 1996) and retinal maturation (Webster and Rowe, 1991). To further determine possible perturbations in neuronogenesis and/or differentiation, we used cell-specific markers and refined birth dating methods to examine these events during retinal ganglion cell (RGC) genesis in albino and pigmented mice from embryonic day 11 (E11) to E18. Our data indicate that relative to pigmented mice, more ganglion cells are born in the early stages of neuronogenesis in the albino retina, although the initiation of RGC genesis in the albino is unchanged. The cellular organization of the albino retina is perturbed as early as E12. In addition, cell cycle kinetics and output along the nasotemporal axis differ in retinas of albino and pigmented mice, both absolutely, with the temporal aspect of the retina expanded in albino, and relative to the position of the optic nerve head. Finally, blocking melanin synthesis in pigmented eyecups in culture leads to an increase in RGC differentiation, consistent with a role for melanin formation in regulating RGC neuronogenesis. These results point to spatiotemporal defects in neuronal production in the albino retina, which could perturb expression of genes that specify cell fate, number, and/or projection phenotype.

  3. Subretinal delivery and electroporation in pigmented and nonpigmented adult mouse eyes

    PubMed Central

    Nickerson, John M.; Goodman, Penny; Chrenek, Micah A.; Johnson, Christiana J.; Berglin, Lennart; Redmond, T. Michael.; Boatright, Jeffrey H.

    2013-01-01

    Subretinal injection offers one of the best ways to deliver many classes of drugs, reagents, cells and treatments to the photoreceptor, Müller, and retinal pigment epithelium (RPE) cells of the retina. Agents delivered to this space are placed within microns of the intended target cell, accumulating to high concentrations because there is no dilution due to transport processes or diffusion. Dilution in the interphotoreceptor space (IPS) is minimal because the IPS volume is only 10-20 microliters in the human eye and less than 1 microliter in the mouse eye. For gene delivery purposes, we wished to transfect the cells adjacent to the IPS in adult mouse eyes. Others transfect these cells in neonatal rats to study the development of the retina. In both neonates and adults, electroporation is found to be effective Here we describe the optimization of electroporation conditions for RPE cells in the adult mouse eye with naked plasmids. However, both techniques, subretinal injection and electroporation, present some technical challenges that require skill on the part of the surgeon to prevent untoward damage to the eye. Here we describe methods that we have used for the past ten years (1). PMID:22688698

  4. A simple method for in vivo labelling of infiltrating leukocytes in the mouse retina using indocyanine green dye

    PubMed Central

    Sim, Dawn A.; Chu, Colin J.; Selvam, Senthil; Powner, Michael B.; Liyanage, Sidath; Copland, David A.; Keane, Pearse A.; Tufail, Adnan; Egan, Catherine A.; Bainbridge, James W. B.; Lee, Richard W.; Dick, Andrew D.; Fruttiger, Marcus

    2015-01-01

    ABSTRACT We have developed a method to label and image myeloid cells infiltrating the mouse retina and choroid in vivo, using a single depot injection of indocyanine green dye (ICG). This was demonstrated using the following ocular models of inflammation and angiogenesis: endotoxin-induced uveitis, experimental autoimmune uveoretinitis and laser-induced choroidal neovascularization model. A near-infrared scanning ophthalmoscope was used for in vivo imaging of the eye, and flow cytometry was used on blood and spleen to assess the number and phenotype of labelled cells. ICG was administered 72 h before the induction of inflammation to ensure clearance from the systemic circulation. We found that in vivo intravenous administration failed to label any leukocytes, whereas depot injection, either intraperitoneal or subcutaneous, was successful in labelling leukocytes infiltrating into the retina. Progression of inflammation in the retina could be traced over a period of 14 days following a single depot injection of ICG. Additionally, bright-field microscopy, spectrophotometry and flow cytometric analysis suggest that the predominant population of cells stained by ICG are circulating myeloid cells. The translation of this approach into clinical practice would enable visualization of immune cells in situ. This will not only provide a greater understanding of pathogenesis, monitoring and assessment of therapy in many human ocular diseases but might also open the ability to image immunity live for neurodegenerative disorders, cardiovascular disease and systemic immune-mediated disorders. PMID:26398933

  5. The guanine nucleotide exchange factor Vav3 regulates differentiation of progenitor cells in the developing mouse retina.

    PubMed

    Luft, Veronika; Reinhard, Jacqueline; Shibuya, Masabumi; Fischer, Klaus D; Faissner, Andreas

    2015-02-01

    The seven main cell types in the mammalian retina arise from multipotent retinal progenitor cells, a process that is tightly regulated by intrinsic and extrinsic signals. However, the molecular mechanisms that control proliferation, differentiation and cell-fate decisions of retinal progenitor cells are not fully understood yet. Here, we report that the guanine nucleotide exchange factor Vav3, a regulator of Rho-GTPases, is involved in retinal development. We demonstrate that Vav3 is expressed in the mouse retina during the embryonic period. In order to study the role of Vav3 in the developing retina, we generate Vav3-deficient mice. The loss of Vav3 results in an accelerated differentiation of retinal ganglion cells and cone photoreceptors during early and late embryonic development. We provide evidence that more retinal progenitor cells express the late progenitor marker Sox9 in Vav3-deficient mice than in wild-types. This premature differentiation is compensated during the postnatal period and late-born cell types such as bipolar cells and Müller glia display normal numbers. Taken together, our data imply that Vav3 is a regulator of retinal progenitor cell differentiation, thus highlighting a novel role for guanine nucleotide exchange factors in retinogenesis.

  6. Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase

    SciTech Connect

    Hundahl, C.A.; Fahrenkrug, J.; Luuk, H.; Hay-Schmidt, A.; Hannibal, J.

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer Restricted Neuroglobin expression in the mouse retina. Black-Right-Pointing-Pointer Antibody validation using Neuroglobin-null mice. Black-Right-Pointing-Pointer Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. Black-Right-Pointing-Pointer No effect of Neuroglobin deficiency on neuronal survival. -- Abstract: Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

  7. Early Functional and Morphologic Abnormalities in the Diabetic Nyxnob Mouse Retina

    PubMed Central

    Tarchick, Matthew J.; Bassiri, Parastoo; Rohwer, Rebecca M.; Samuels, Ivy S.

    2016-01-01

    Purpose The electroretinogram c-wave is generated by the summation of the positive polarity hyperpolarization of the apical RPE membrane and a negative polarity slow PIII response of Müller glia cells. Therefore, the c-wave reduction noted in prior studies of mouse models of diabetes could reflect a reduction in the RPE component or an increase in slow PIII. The present study used a genetic approach to distinguish between these two alternatives. Methods Nyxnob mice lack the ERG b-wave, revealing the early phase of slow PIII. To visualize changes in slow PIII due to diabetes, Nyxnob mice were given streptozotocin (STZ) injections to induce diabetes or received vehicle as a control. After 1, 2, and 4 weeks of sustained hyperglycemia (>250 mg/dL), standard strobe flash ERG and dc-ERG testing were conducted. Histological analysis of the retina was performed. Results A reduced c-wave was noted at the 1 week time point, and persisted at later time points. In comparison, slow PIII amplitudes were unaffected after 1 week of hyperglycemia, but were significantly reduced in STZ mice at the 2-week time point. The decrease in amplitude occurred before any identifiable decrease to the a-wave. At the later time point, the a-wave became involved, although the slow PIII reductions were more pronounced. Morphological abnormalities in the RPE, including increased thickness and altered melanosome distribution, were identified in diabetic animals. Conclusions Because the c-wave and slow PIII were both reduced, these results demonstrated that diabetes-induced reductions to the c-wave cannot be attributed to an early increase in the Müller glia-derived potassium conductance. Furthermore, because the a-wave, slow PIII and c-wave reductions were not equivalent, and varied in their onset, the reductions cannot reflect the same mechanism, such as a change in membrane resistance. The presence of small changes to RPE architecture indicate that the c-wave reductions present in diabetic mice

  8. Regulation of Müller Glial Dependent Neuronal Regeneration in the Damaged Adult Zebrafish Retina

    PubMed Central

    Gorsuch, Ryne A.; Hyde, David R.

    2013-01-01

    This article examines our current knowledge underlying the mechanisms involved in neuronal regeneration in the adult zebrafish retina. Zebrafish, which has the capacity to regenerate a wide variety of tissues and organs (including the fins, kidney, heart, brain, and spinal cord), has become the premier model system to study retinal regeneration due to the robustness and speed of the response and the variety of genetic tools that can be applied to study this question. It is now well documented that retinal damage induces the resident Müller glia to dedifferentiate and reenter the cell cycle to produce neuronal progenitor cells that continue to proliferate, migrate to the damaged retinal layer and differentiate into the missing neuronal cell types. Increasing our understanding of how these cellular events are regulated and occur in response to neuronal damage may provide critical information that can be applied to stimulating a regeneration response in the mammalian retina. In this review, we will focus on the genes/proteins that regulate zebrafish retinal regeneration and will attempt to critically evaluate how these factors may interact to correctly orchestrate the definitive cellular events that occur during regeneration. PMID:23880528

  9. Regulation of Müller glial dependent neuronal regeneration in the damaged adult zebrafish retina.

    PubMed

    Gorsuch, Ryne A; Hyde, David R

    2014-06-01

    This article examines our current knowledge underlying the mechanisms involved in neuronal regeneration in the adult zebrafish retina. Zebrafish, which has the capacity to regenerate a wide variety of tissues and organs (including the fins, kidney, heart, brain, and spinal cord), has become the premier model system to study retinal regeneration due to the robustness and speed of the response and the variety of genetic tools that can be applied to study this question. It is now well documented that retinal damage induces the resident Müller glia to dedifferentiate and reenter the cell cycle to produce neuronal progenitor cells that continue to proliferate, migrate to the damaged retinal layer and differentiate into the missing neuronal cell types. Increasing our understanding of how these cellular events are regulated and occur in response to neuronal damage may provide critical information that can be applied to stimulating a regeneration response in the mammalian retina. In this review, we will focus on the genes/proteins that regulate zebrafish retinal regeneration and will attempt to critically evaluate how these factors may interact to correctly orchestrate the definitive cellular events that occur during regeneration.

  10. Expression of thromboxane synthase and the thromboxane-prostanoid receptor in the mouse and rat retina

    PubMed Central

    Wright, William S.; McElhatten, Robert M.; Harris, Norman R.

    2009-01-01

    Experimental models of the diabetic retina have suggested a pathological role for thromboxane. To date however, little information is available as to the cellular locations of retinal thromboxane synthase (TxS), or its receptor, even in non-diabetic controls. In this study, C57BL/6 mice and Wistar rats were injected with streptozotocin to induce diabetes, or with buffer for non-diabetic controls. Four weeks following the injection, eyes were enucleated and labeled for TxS and the thromboxane-prostanoid (TP) receptor. Immunofluorescent intensity was quantified in the ganglion cell plus inner plexiform layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, and photoreceptor inner segment. Even in control mice and rats, all layers of the retina showed immunoreactivity for TxS and the TP receptor: however, the pattern of expression demonstrated an inverse relationship, with the highest TxS staining in the inner retina, and the highest TP receptor staining in the outer retina (more specifically, in the photoreceptor inner segment). Four weeks of hyperglycemia did not increase the retinal levels of TxS or TP receptor; however, TP receptor intensities in the outer retina of diabetic rats were highly variable (mostly high but some low), with no values from the photoreceptor inner segment in the same range as obtained from controls. PMID:19523949

  11. Pathways of cholesterol homeostasis in mouse retina responsive to dietary and pharmacologic treatments.

    PubMed

    Zheng, Wenchao; Mast, Natalia; Saadane, Aicha; Pikuleva, Irina A

    2015-01-01

    Effects of serum cholesterol on cholesterol content in the retina are currently unknown. It is also unclear how cholesterol levels are controlled in the retina. High-cholesterol diet and oral administrations of simvastatin were used to modulate serum cholesterol in mice. These treatments only modestly affected cholesterol content in the retina and had no significant effect on retinal expression of the major cholesterol- and vision-related genes; the sterol-regulatory element binding protein pathway of transcriptional regulation does not seem to be operative in the retina under the experimental conditions used. Evidence is obtained that posttranslational mechanisms play a role in the control of retinal cholesterol. Retinal genes were only upregulated by oral administrations of TO901317 activating liver X receptors. Three of the upregulated genes could be of particular importance (apoD, Idol, and Rpe65) and have not yet been considered in the context of cholesterol homeostasis in the retina. Collectively, the data obtained identify specific features of retinal cholesterol maintenance and suggest additional therapies for age-related macular degeneration, a blinding disease characterized by cholesterol and lipid accumulations in chorioretinal tissues.

  12. Retinal ganglion cell responses to voltage and current stimulation in wild-type and rd1 mouse retinas

    NASA Astrophysics Data System (ADS)

    Goo, Yong Sook; Ye, Jang Hee; Lee, Seokyoung; Nam, Yoonkey; Ryu, Sang Baek; Kim, Kyung Hwan

    2011-06-01

    Retinal prostheses are being developed to restore vision for those with retinal diseases such as retinitis pigmentosa or age-related macular degeneration. Since neural prostheses depend upon electrical stimulation to control neural activity, optimal stimulation parameters for successful encoding of visual information are one of the most important requirements to enable visual perception. In this paper, we focused on retinal ganglion cell (RGC) responses to different stimulation parameters and compared threshold charge densities in wild-type and rd1 mice. For this purpose, we used in vitro retinal preparations of wild-type and rd1 mice. When the neural network was stimulated with voltage- and current-controlled pulses, RGCs from both wild-type and rd1 mice responded; however the temporal pattern of RGC response is very different. In wild-type RGCs, a single peak within 100 ms appears, while multiple peaks (approximately four peaks) with ~10 Hz rhythm within 400 ms appear in RGCs in the degenerated retina of rd1 mice. We find that an anodic phase-first biphasic voltage-controlled pulse is more efficient for stimulation than a biphasic current-controlled pulse based on lower threshold charge density. The threshold charge densities for activation of RGCs both with voltage- and current-controlled pulses are overall more elevated for the rd1 mouse than the wild-type mouse. Here, we propose the stimulus range for wild-type and rd1 retinas when the optimal modulation of a RGC response is possible.

  13. The Proteome of Native Adult Müller Glial Cells From Murine Retina*

    PubMed Central

    Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca

    2016-01-01

    To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial

  14. Identification of the mouse and rat orthologs of the gene mutated in Usher syndrome type IIA and the cellular source of USH2A mRNA in retina, a target tissue of the disease.

    PubMed

    Huang, Dali; Eudy, James D; Uzvolgyi, Eva; Davis, Jack R; Talmadge, Catherine B; Pretto, Dalyir; Weston, Michael D; Lehman, Janae E; Zhou, Ming; Seemayer, Thomas A; Ahmad, Iqbal; Kimberling, William J; Sumegi, Janos

    2002-08-01

    Usher syndrome type IIA (MIM: 27601) is an autosomal recessive disorder characterized by moderate to severe congenital deafness and progressive retinitis pigmentosa. We recently identified the human Usher syndrome type IIA gene (USH2A) on chromosome 1q41, which encodes a protein possessing 10 laminin epidermal growth factor and four fibronectin type 3 domains, both commonly observed in extracellular matrix proteins. To gain insight into the pathogenesis of Usher syndrome type IIA, we isolated and characterized the murine (Ush2a) and rat (rat Ush2a) orthologs of human USH2A. We mapped mouse Ush2a by fluorescence in situ hybridization to mouse chromosome 1 in the region syntenic to human chromosome 1q41. Rat Ush2a has been localized by radiation hybrid mapping to rat chromosome 13 between d13rat49 and d13rat76. The mouse and rat genes, similar to human USH2A, are expressed primarily in retina and cochlea. Mouse Ush2a encodes a 161-kDa protein that shows 68% identity and 9% similarity to the human USH2A protein. Rat Ush2a encodes a 167-kDa protein with 64% identity and 10% similarity to the human protein and 81% identity and 5% similarity to the mouse USH2A protein. The predicted amino acid sequence of the mouse and rat proteins, like their human counterpart, contains a leader sequence, an amino-terminal globular domain, 10 laminin epidermal growth factor domains, and four carboxy-terminal fibronectin type III motifs. With in situ hybridization, we compared the cellular expression of the USH2A gene in rat, mouse, and human retinas. USH2A mRNA in the adult rat, mouse, and human is expressed in the cells of the outer nuclear layer of the retina, one of the target tissues of the disease. In the developing rat retina, Ush2a mRNA expression appears in the neuroepithelium at embryonic day 17.

  15. TRIP8b Is Required for Maximal Expression of HCN1 in the Mouse Retina

    PubMed Central

    Pan, Yuan; Bhattarai, Sajag; Modestou, Modestos; Drack, Arlene V.; Chetkovich, Dane M.; Baker, Sheila A.

    2014-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1. PMID:24409334

  16. Transcription of the SCL/TAL1 Interrupting Locus (Stil) Is Required for Cell Proliferation in Adult Zebrafish Retinas*

    PubMed Central

    Sun, Lei; Li, Ping; Carr, Aprell L.; Gorsuch, Ryne; Yarka, Clare; Li, Jingling; Bartlett, Michael; Pfister, Delaney; Hyde, David R.; Li, Lei

    2014-01-01

    The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Previously, we identified a homolog of the Stil gene in zebrafish mutant (night blindness b, nbb), which showed neural defects in the retina (e.g. dopaminergic cell degeneration and/or lack of regeneration). In this research, we examined the roles of Stil in cell proliferation after degeneration in adult zebrafish retinas. We demonstrated that knockdown of Stil gene expression or inhibition of Sonic hedgehog (Shh) signaling transduction decreases the rate of cell proliferation. In contrast, activation of Shh signal transduction promotes cell proliferation. In nbb+/− retinas, inhibition of SUFU (a repressor in the Shh pathway) rescues the defects in cell proliferation due to down-regulation of Stil gene expression. The latter data suggest that Stil play a role in cell proliferation through the Shh signal transduction pathway. PMID:24469449

  17. Transcription of the SCL/TAL1 interrupting Locus (Stil) is required for cell proliferation in adult Zebrafish Retinas.

    PubMed

    Sun, Lei; Li, Ping; Carr, Aprell L; Gorsuch, Ryne; Yarka, Clare; Li, Jingling; Bartlett, Michael; Pfister, Delaney; Hyde, David R; Li, Lei

    2014-03-07

    The human oncogene SCL/TAL1 interrupting locus (Stil) is highly conserved in vertebrate species. Previously, we identified a homolog of the Stil gene in zebrafish mutant (night blindness b, nbb), which showed neural defects in the retina (e.g. dopaminergic cell degeneration and/or lack of regeneration). In this research, we examined the roles of Stil in cell proliferation after degeneration in adult zebrafish retinas. We demonstrated that knockdown of Stil gene expression or inhibition of Sonic hedgehog (Shh) signaling transduction decreases the rate of cell proliferation. In contrast, activation of Shh signal transduction promotes cell proliferation. In nbb(+/-) retinas, inhibition of SUFU (a repressor in the Shh pathway) rescues the defects in cell proliferation due to down-regulation of Stil gene expression. The latter data suggest that Stil play a role in cell proliferation through the Shh signal transduction pathway.

  18. Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina.

    PubMed

    Liu, Aihua; Terry, Ryan; Lin, Yanhua; Nelson, Kelly; Bernstein, Paul S

    2013-09-13

    Fatty acids (FAs), including long-chain and very long-chain polyunsaturated fatty acids (LC-PUFAs, C12-22; VLC-PUFAs, C24-38), play an important role in retinal function and health. Deficiencies in LC-PUFAs and VLC-PUFAs, as well as mutations in the enzyme responsible for elongation of very long-chain fatty acids (ELOVL4), have been associated with macular dystrophies and degenerations. Published analytical methods, including high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and gas chromatography-MS (GC-MS), can quantify VLC-PUFAs but require at least an entire human retina which limits the ability to understand physiologically relevant variations in lipids that can occur at a regional level within the retina. Until now, quantification of VLC-PUFAs in just the human macula, the cone-rich region of the central retina responsible for high acuity vision, has not been feasible due to its small size (4-5mm in diameter). In this study, we have developed a sensitive GC-MS method using newer generation enhanced GC-MS detector sensitivity which for the first time quantifies not only 14 VLC-PUFAs and 26 LC-FAs but also n-3/n-6 ratios of PUFAs in 4mm punches of human retina or a single pair of mouse retinas. Our results showed that saturated LC-FAs are higher in the human peripheral retina than in the macula, while unsaturated LC-FAs are higher in the macula than in the peripheral retina. On the other hand, the VLC-PUFAs are higher in the peripheral retina compared to macula. There is no difference in n-3/n-6 ratios of PUFAs observed between human macula and peripheral retina, while mouse retina has almost ten times more VLC-PUFAs than human macula and peripheral retina (2.27% versus 0.25% and 0.32%, respectively) and much higher n-3/n-6 ratios compared to human retina (9:1 versus ∼0.9:1). This high sensitivity analytical technique provides a valuable new tool for studies on the role of FAs in the pathological processes of macular

  19. Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina

    PubMed Central

    Vila, Alejandro; Huynh, Uyen-Chi N.; Brecha, Nicholas C.

    2008-01-01

    Purpose To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. Methods CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD67), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. Results CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 μm in diameter, and they had a density of 2636±347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD67, GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. Conclusions The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells. PMID:18728756

  20. Assessment of vascular regeneration in the CNS using the mouse retina.

    PubMed

    Miloudi, Khalil; Dejda, Agnieszka; Binet, François; Lapalme, Eric; Cerani, Agustin; Sapieha, Przemyslaw

    2014-06-23

    The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer's, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

  1. Spontaneous Oscillatory Rhythms in the Degenerating Mouse Retina Modulate Retinal Ganglion Cell Responses to Electrical Stimulation

    PubMed Central

    Goo, Yong Sook; Park, Dae Jin; Ahn, Jung Ryul; Senok, Solomon S.

    2016-01-01

    Characterization of the electrical activity of the retina in the animal models of retinal degeneration has been carried out in part to understand the progression of retinal degenerative diseases like age-related macular degeneration (AMD) and retinitis pigmentosa (RP), but also to determine optimum stimulus paradigms for use with retinal prosthetic devices. The models most studied in this regard have been the two lines of mice deficient in the β-subunit of phosphodiesterase (rd1 and rd10 mice), where the degenerating retinas exhibit characteristic spontaneous hyperactivity and oscillatory local field potentials (LFPs). Additionally, there is a robust ~10 Hz rhythmic burst of retinal ganglion cell (RGC) spikes on the trough of the oscillatory LFP. In rd1 mice, the rhythmic burst of RGC spikes is always phase-locked with the oscillatory LFP and this phase-locking property is preserved regardless of postnatal ages. However, in rd10 mice, the frequency of the oscillatory rhythm changes according to postnatal age, suggesting that this rhythm might be a marker of the stage of degeneration. Furthermore when a biphasic current stimulus is applied to rd10 mice degenerate retina, distinct RGC response patterns that correlate with the stage of degeneration emerge. This review also considers the significance of these response properties. PMID:26793063

  2. In vivo visualizing the dynamics of bone marrow stem cells in mouse retina and choroidal-retinal circulation

    NASA Astrophysics Data System (ADS)

    Wang, Heuy-Ching H.; Zwick, Harry; Edsall, Peter R.; Cheramie, Rachel D.; Lund, David J.; Stuck, Bruce

    2007-02-01

    It has recently been shown that bone marrow cells can differentiate into various lineage cells including neural cells in vitro and in vivo. Therefore it is an attractive therapeutic intervention to apply autologous bone marrow-derived stem cells that may offer neuroprotection to laser-induced retinal injuries. The purpose of this study is to develop a method with which to visualize bone marrow stem cells dynamics in mouse retinal circulation. We have used a physiological method, confocal scanning laser ophthalmoscope (SLO), to track the highly enriched stem/progenitor cells circulating in the retina. Stem cells were enriched by immunomagnetic depletion of cells committed to the T- and B lymphocytic, myeloid and erythorid lineages. CellTracker TM Green-labeled stem cells were injected into the tail veins of mice with laser-induced focal retinal injuries. Bone marrow stem cells labeled with CellTracker TM Green were visible in the retinal circulation for as long as 1 hour and 30 minutes. These studies suggest that stem cell-enriched bone marrow cells may have the ability to mobilize into laser-induced retinal injuries and possibly further proliferate, differentiate and functionally integrate into the retina.

  3. Differential effects of charybdotoxin on the activity of retinal ganglion cells in the dark- and light-adapted mouse retina

    PubMed Central

    Nemargut, Joseph P.; Zhu, Junling; Savoie, Brian T.; Wang, Guo-Yong

    2009-01-01

    Patch-clamp recordings were made from retinal ganglion cells in the mouse retina. Under dark adaptation, blockage of BKCa channels increases the spontaneous excitatory postsynaptic currents (EPSCs) and light-evoked On-EPSCs, while it decreases the light-evoked Off inhibitory postsynaptic currents (IPSCs). However, under light adaptation it decreases the light-evoked On-EPSCs, the spontaneous IPSCs and the light-evoked On- and Off-IPSCs. Blockage of BKCa channels significantly altered the outputs of RGCs by changing their light-evoked responses into a bursting pattern and increasing the light-evoked depolarization of the membrane potentials, while it did not significantly change the peak firing rates of light-evoked responses. PMID:19084033

  4. Characterization of dsRed2-positive cells in the doublecortin-dsRed2 transgenic adult rat retina.

    PubMed

    Trost, A; Schroedl, F; Marschallinger, J; Rivera, F J; Bogner, B; Runge, C; Couillard-Despres, S; Aigner, L; Reitsamer, H A

    2014-12-01

    Doublecortin (DCX) is predominantly expressed in neuronal precursor cells and young immature neurons of the developing and adult brain, where it is involved in neuronal differentiation, migration and plasticity. Moreover, its expression pattern reflects neurogenesis, and transgenic DCX promoter-driven reporter models have been previously used to investigate adult neurogenesis. In this study, we characterize dsRed2 reporter protein-expressing cells in the adult retina of the transgenic DCX promoter-dsRed2 rat model, with the aim to identify cells with putative neurogenic activity. Additionally, we confirmed the expression of the dsRed2 protein in DCX-expressing cells in the adult hippocampal dentate gyrus. Adult DCX-dsRed2 rat retinas were analyzed by immunohistochemistry for expression of DCX, NF200, Brn3a, Sox2, NeuN, calbindin, calretinin, PKC-a, Otx2, ChAT, PSA-NCAM and the glial markers GFAP and CRALBP, followed by confocal laser-scanning microscopy. In addition, brain sections of transgenic rats were analyzed for dsRed2 expression and co-localization with DCX, NeuN, GFAP and Sox2 in the cortex and dentate gyrus. Endogenous DCX expression in the adult retina was confined to horizontal cells, and these cells co-expressed the DCX promoter-driven dsRed2 reporter protein. In addition, we encountered dsRed2 expression in various other cell types in the retina: retinal ganglion cells (RGCs), a subpopulation of amacrine cells, a minority of bipolar cells and in perivascular cells. Since also RGCs expressed dsRed2, the DCX-dsRed2 rat model might offer a useful tool to study RGCs in vivo under various conditions. Müller glial cells, which have previously been identified as cells with stem cell features and with neurogenic potential, did express neither endogenous DCX nor the dsRed2 reporter. However, and surprisingly, we identified a perivascular glial cell type expressing the dsRed2 reporter, enmeshed with the glia/stem cell marker GFAP and colocalizing with the

  5. In vitro and in vivo differentiation of human embryonic stem cells into retina-like organs and comparison with that from mouse pluripotent epiblast stem cells.

    PubMed

    Aoki, Hitomi; Hara, Akira; Niwa, Masayuki; Yamada, Yasuhiro; Kunisada, Takahiro

    2009-09-01

    Correctly inducing the differentiation of pluripotent hESCs to a specific lineage with high purity is highly desirable for regenerative cell therapy. Our first effort to perform in vitro differentiation of hESCs resulted in a limited recapitulation of the ocular tissue structures. When undifferentiated hESCs were placed in vivo into the ocular tissue, in this case into the vitreous cavity, 3-dimensional retina-like structures reminiscent of the invagination of the optic vesicle were generated. Immunohistochemical analysis confirmed the presence of both a neural retina-like cell layer and a retinal pigmented epithelium-like cell layer, possibly equivalent to the developing E12.5 mouse retina. Furthermore, mouse epiblast-derived stem cells, which are reported to share some characteristics with hESCs, but not with mouse ESCs, also generated retinal anlage-like structures in vivo. hESC-derived retina-like structures present a novel therapeutic possibility for retinal diseases and also provide a novel experimental system to study early human eye development.

  6. Image registration and averaging of low laser power two-photon fluorescence images of mouse retina

    PubMed Central

    Alexander, Nathan S.; Palczewska, Grazyna; Stremplewski, Patrycjusz; Wojtkowski, Maciej; Kern, Timothy S.; Palczewski, Krzysztof

    2016-01-01

    Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [PalczewskaG., Nat Med. 20, 785 (2014)24952647 SharmaR., Biomed. Opt. Express 4, 1285 (2013)24009992]. PMID:27446697

  7. Dicer inactivation leads to progressive functional and structural degeneration of the mouse retina

    PubMed Central

    Damiani, Devid; Alexander, John J; O'Rourke, Jason R; McManus, Mike; Jadhav, Ashutosh P; Cepko, Constance L; Hauswirth, William W; Harfe, Brian D; Strettoi, Enrica

    2009-01-01

    MicroRNAs (miRNAs) are small, highly conserved molecules that have been shown to regulate the expression of genes by binding to specific target mRNAs. Dicer, an RNase III endonuclease, is essential for the production and function of mature miRNAs and removal of Dicer has been shown to disrupt many developmental processes. In this report, Dicer was removed specifically from the retina using a floxed Dicer conditional allele and the retinal Chx10Cre transgene. Retinal Dicer knockout mice displayed a reproducible inability to respond to light. In addition, morphological defects were observed with the formation of photoreceptor rosettes at P16 which progressed to more general cellular disorganization and widespread degeneration of retinal cell types as the animals aged. This was accompanied by concomitant decrease in both scotopic and photopic ERG responses. Interestingly, removing a single allele of Dicer resulted in ERG deficits throughout life but not to morphological abnormalities. Northern blot analysis of Dicer depleted retinas showed a decrease in several microRNAs. The observation that progressive retinal degeneration occurred upon removal of Dicer raises the possibility that miRNAs are involved in retinal neurodegenerative disorders. PMID:18463241

  8. 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.

  9. 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

  10. AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy.

    PubMed

    Dinculescu, Astra; Stupay, Rachel M; Deng, Wen-Tao; Dyka, Frank M; Min, Seok-Hong; Boye, Sanford L; Chiodo, Vince A; Abrahan, Carolina E; Zhu, Ping; Li, Qiuhong; Strettoi, Enrica; Novelli, Elena; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Smith, W Clay; Hauswirth, William W

    2016-01-01

    Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

  11. Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia

    PubMed Central

    Obry, Antoine; Santin, Mathieu D.; Ben-Yacoub, Sirine; Pâques, Michel; Amsellem-Levera, Sabine; Bribian, Ana; Simonutti, Manuel; Augustin, Sébastien; Debeir, Thomas; Sahel, José Alain; Christ, Annabel; de Castro, Fernando; Lehéricy, Stéphane; Cosette, Pascal; Kozyraki, Renata

    2015-01-01

    Myopia is a common ocular disorder generally due to increased axial length of the eye-globe. Its extreme form high myopia (HM) is a multifactorial disease leading to retinal and scleral damage, visual impairment or loss and is an important health issue. Mutations in the endocytic receptor LRP2 gene result in Donnai-Barrow (DBS) and Stickler syndromes, both characterized by HM. To clearly establish the link between Lrp2 and congenital HM we inactivated Lrp2 in the mouse forebrain including the neural retina and the retinal and ciliary pigment epithelia. High resolution in vivo MRI imaging and ophthalmological analyses showed that the adult Lrp2-deficient eyes were 40% longer than the control ones mainly due to an excessive elongation of the vitreal chamber. They had an apparently normal intraocular pressure and developed chorioretinal atrophy and posterior scleral staphyloma features reminiscent of human myopic retinopathy. Immunomorphological and ultrastructural analyses showed that increased eye lengthening was first observed by post-natal day 5 (P5) and that it was accompanied by a rapid decrease of the bipolar, photoreceptor and retinal ganglion cells, and eventually the optic nerve axons. It was followed by scleral thinning and collagen fiber disorganization, essentially in the posterior pole. We conclude that the function of LRP2 in the ocular tissues is necessary for normal eye growth and that the Lrp2-deficient eyes provide a unique tool to further study human HM. PMID:26107939

  12. Alternative Splicing of the LIM-Homeodomain Transcription Factor Isl1 in the Mouse Retina

    PubMed Central

    Whitney, Irene E.; Kautzman, Amanda G.; Reese, Benjamin E.

    2015-01-01

    Islet-1 (Isl1) is a LIM-homeodomain (LIM-HD) transcription factor that functions in a combinatorial manner with other LIM-HD proteins to direct the differentiation of distinct cell types within the central nervous system and many other tissues. A study of pancreatic cell lines showed that Isl1 is alternatively spliced generating a second isoform, Isl1β, which is missing 23 amino acids within the C-terminal region. This study examines the expression of the canonical and alternative Isl1 transcripts across other tissues, in particular, within the retina, where Isl1 is required for the differentiation of multiple neuronal cell types. The alternative splicing of Isl1 is shown to occur in multiple tissues, but the relative abundance of Isl1α and Isl1 β expression varies greatly across them. In most tissues, Isl1α is the more abundant transcript, but in others the transcripts are expressed equally, or the alternative splice variant is dominant. Within the retina, differential expression of the two Isl1 transcripts increases as a function of development, with dynamic changes in expression peaking at E16.5 and again at P10. At the cellular level, individual retinal ganglion cells vary in their expression, with a subset of small-to-medium sized cells expressing only the alternative isoform. The functional significance of the difference in protein sequence between the two Isl1 isoforms was also assessed using a luciferase assay, demonstrating that the alternative isoform forms a less effective transcriptional complex for activating gene expression. These results demonstrate the differential presence of the canonical and alternative isoforms of Isl1 amongst retinal ganglion cell classes. As Isl1 participates in the differentiation of multiple cell types within the CNS, the present results support a role for alternative splicing in the establishment of cellular diversity in the developing nervous system. PMID:25752730

  13. Morphology and Immunoreactivity of Retrogradely Double-Labeled Ganglion Cells in the Mouse Retina

    PubMed Central

    Wu, Samuel M.

    2011-01-01

    Purpose. To examine the specificity and reliability of a retrograde double-labeling technique that was recently established for identification of retinal ganglion cells (GCs) and to characterize the morphology of displaced (d)GCs (dGs). Methods. A mixture of the gap-junction–impermeable dye Lucifer yellow (LY) and the permeable dye neurobiotin (NB) was applied to the optic nerve stump for retrograde labeling of GCs and the cells coupled with them. A confocal microscope was adopted for morphologic observation. Results. GCs were identified by LY labeling, and they were all clearly labeled by NB. Cells coupled to GCs contained a weak NB signal but no LY. LY and NB revealed axon bundles, somas and dendrites of GCs. The retrogradely identified GCs numbered approximately 50,000 per retina, and they constituted 44% of the total neurons in the ganglion cell layer (GCL). Somas of retrogradely identified dGs were usually negative for glycine, ChAT (choline acetyltransferase), bNOS (brain-type nitric oxidase), GAD (glutamate decarboxylase), and glial markers, and occasionally, they were weakly GABA-positive. dGs averaged 760 per retina and composed 1.7% of total GCs. Sixteen morphologic subtypes of dGs were encountered, three of which were distinct from known GCs. dGs sent dendrites to either sublaminas of the IPL, mostly sublamina a. Conclusions. The retrograde labeling is reliable for identification of GCs. dGs participate in ON and OFF light pathways but favor the OFF pathway. ChAT, bNOS, glycine, and GAD remain reliable AC markers in the GCL. GCs may couple to GABAergic ACs, and the gap junctions likely pass NB and GABA. PMID:21482641

  14. A unique role for Kv3 voltage-gated potassium channels in starburst amacrine cell signaling in mouse retina.

    PubMed

    Ozaita, Ander; Petit-Jacques, Jerome; Völgyi, Béla; Ho, Chi Shun; Joho, Rolf H; Bloomfield, Stewart A; Rudy, Bernardo

    2004-08-18

    Direction-selective retinal ganglion cells show an increased activity evoked by light stimuli moving in the preferred direction. This selectivity is governed by direction-selective inhibition from starburst amacrine cells occurring during stimulus movement in the opposite or null direction. To understand the intrinsic membrane properties of starburst cells responsible for direction-selective GABA release, we performed whole-cell recordings from starburst cells in mouse retina. Voltage-clamp recordings revealed prominent voltage-dependent K(+) currents. The currents were mostly blocked by 1 mm TEA, activated rapidly at voltages more positive than -20 mV, and deactivated quickly, properties reminiscent of the currents carried by the Kv3 subfamily of K+ channels. Immunoblots confirmed the presence of Kv3.1 and Kv3.2 proteins in retina and immunohistochemistry revealed their expression in starburst cell somata and dendrites. The Kv3-like current in starburst cells was absent in Kv3.1-Kv3.2 knock-out mice. Current-clamp recordings showed that the fast activation of the Kv3 channels provides a voltage-dependent shunt that limits depolarization of the soma to potentials more positive than -20 mV. This provides a mechanism likely to contribute to the electrical isolation of individual starburst cell dendrites, a property thought essential for direction selectivity. This function of Kv3 channels differs from that in other neurons where they facilitate high-frequency repetitive firing. Moreover, we found a gradient in the intensity of Kv3.1b immunolabeling favoring proximal regions of starburst cells. We hypothesize that this Kv3 channel gradient contributes to the preference for centrifugal signal flow in dendrites underlying direction-selective GABA release from starburst amacrine cells

  15. Dark-adapted response threshold of OFF ganglion cells is not set by OFF bipolar cells in the mouse retina.

    PubMed

    Arman, A Cyrus; Sampath, Alapakkam P

    2012-05-01

    The nervous system frequently integrates parallel streams of information to encode a broad range of stimulus strengths. In mammalian retina it is generally believed that signals generated by rod and cone photoreceptors converge onto cone bipolar cells prior to reaching the retinal output, the ganglion cells. Near absolute visual threshold a specialized mammalian retinal circuit, the rod bipolar pathway, pools signals from many rods and converges on depolarizing (AII) amacrine cells. However, whether subsequent signal flow to OFF ganglion cells requires OFF cone bipolar cells near visual threshold remains unclear. Glycinergic synapses between AII amacrine cells and OFF cone bipolar cells are believed to relay subsequently rod-driven signals to OFF ganglion cells. However, AII amacrine cells also make glycinergic synapses directly with OFF ganglion cells. To determine the route for signal flow near visual threshold, we measured the effect of the glycine receptor antagonist strychnine on response threshold in fully dark-adapted retinal cells. As shown previously, we found that response threshold for OFF ganglion cells was elevated by strychnine. Surprisingly, strychnine did not elevate response threshold in any subclass of OFF cone bipolar cell. Instead, in every OFF cone bipolar subclass strychnine suppressed tonic glycinergic inhibition without altering response threshold. Consistent with this lack of influence of strychnine, we found that the dominant input to OFF cone bipolar cells in darkness was excitatory and the response threshold of the excitatory input varied by subclass. Thus, in the dark-adapted mouse retina, the high absolute sensitivity of OFF ganglion cells cannot be explained by signal transmission through OFF cone bipolar cells.

  16. Characterization of Light Lesion Paradigms and Optical Coherence Tomography as Tools to Study Adult Retina Regeneration in Zebrafish

    PubMed Central

    Weber, Anke; Hochmann, Sarah; Cimalla, Peter; Gärtner, Maria; Kuscha, Veronika; Hans, Stefan; Geffarth, Michaela; Kaslin, Jan; Koch, Edmund; Brand, Michael

    2013-01-01

    Light-induced lesions are a powerful tool to study the amazing ability of photoreceptors to regenerate in the adult zebrafish retina. However, the specificity of the lesion towards photoreceptors or regional differences within the retina are still incompletely understood. We therefore characterized the process of degeneration and regeneration in an established paradigm, using intense white light from a fluorescence lamp on swimming fish (diffuse light lesion). We also designed a new light lesion paradigm where light is focused through a microscope onto the retina of an immobilized fish (focused light lesion). Focused light lesion has the advantage of creating a locally restricted area of damage, with the additional benefit of an untreated control eye in the same animal. In both paradigms, cell death is observed as an immediate early response, and proliferation is initiated around 2 days post lesion (dpl), peaking at 3 dpl. We furthermore find that two photoreceptor subtypes (UV and blue sensitive cones) are more susceptible towards intense white light than red/green double cones and rods. We also observed specific differences within light lesioned areas with respect to the process of photoreceptor degeneration: UV cone debris is removed later than any other type of photoreceptor in light lesions. Unspecific damage to retinal neurons occurs at the center of a focused light lesion territory, but not in the diffuse light lesion areas. We simulated the fish eye optical properties using software simulation, and show that the optical properties may explain the light lesion patterns that we observe. Furthermore, as a new tool to study retinal degeneration and regeneration in individual fish in vivo, we use spectral domain optical coherence tomography. Collectively, the light lesion and imaging assays described here represent powerful tools for studying degeneration and regeneration processes in the adult zebrafish retina. PMID:24303018

  17. Mutated olfactomedin 1 in the interphotoreceptor matrix of the mouse retina causes functional deficits and vulnerability to light damage.

    PubMed

    Koch, Marcus A; Rosenhammer, Bernd; Paper, Walter; Volz, Cornelia; Braunger, Barbara M; Hausberger, Johanna; Jägle, Herbert; Tamm, Ernst R

    2017-04-01

    Olfactomedin 1 (OLFM1) is a secreted glycoprotein and member of the olfactomedin protein family, which is preferentially expressed in various areas throughout the central nervous system. To learn about the functional properties of OLFM1 in the eye, we investigated its localization in the mouse and pig eye. In addition, we analyzed the ocular phenotype of Olfm1 mutant mice in which 52 amino acids were deleted in the central part (M2 region) of OLFM1. OLFM1 was detected in cornea, sclera, retina, and optic nerve of both wild-type and Olfm1 mutant littermates. By immunohistochemistry and double labeling with the lectin peanut agglutinin, OLFM1 was found in the interphotoreceptor matrix (IPM) of mouse and pig retina where it was directly localized to the inner segments of photoreceptors. Western blotting confirmed the presence of the OLFM1 isoforms pancortin 1 (BMY) and pancortin 2 (BMZ) in the IPM. The retinal phenotype of Olfm1 mutant mice did not obviously differ from that of wild-type littermates. In addition, outer nuclear layer (ONL) and total retinal thickness were not different, and the same was true for the area of the optic nerve in cross sections. Functional changes were observed though by electroretinography, which showed significantly lower a- and b-wave amplitudes in Olfm1 mutant mice when compared to age-matched wild-type mice. When light damage experiments were performed as an experimental paradigm of photoreceptor apoptosis, significantly more TUNEL-positive cells were observed in Olfm1 mutant mice 30 h after light exposure. One week after light exposure, the ONL was significantly thinner in Olfm1 mutant mice than in wild-type littermates indicating increased photoreceptor loss. No differences were observed when rhodopsin turnover or ERK1/2 signaling was investigated. We conclude that OLFM1 is a newly identified IPM molecule that serves an important role for photoreceptor homeostasis, which is significantly compromised in the eyes of Olfm1 mutant mice.

  18. Standardized bioenergetic profiling of adult mouse cardiomyocytes.

    PubMed

    Readnower, Ryan D; Brainard, Robert E; Hill, Bradford G; Jones, Steven P

    2012-12-18

    Mitochondria are at the crux of life and death and as such have become ideal targets of intervention in cardiovascular disease. Generally, current methods to measure mitochondrial dysfunction rely on working with the isolated organelle and fail to incorporate mitochondrial function in a cellular context. Extracellular flux methodology has been particularly advantageous in this respect; however, certain primary cell types, such as adult cardiac myocytes, have been difficult to standardize with this technology. Here, we describe methods for using extracellular flux (XF) analysis to measure mitochondrial bioenergetics in isolated, intact, adult mouse cardiomyocytes (ACMs). Following isolation, ACMs were seeded overnight onto laminin-coated (20 μg/ml) microplates, which resulted in high attachment efficiency. After establishing seeding density, we found that a commonly used assay medium (containing a supraphysiological concentration of pyruvate at 1 mmol/l) produced a maximal bioenergetic response. After performing a pyruvate dose-response, we determined that pyruvate titrated to 0.1 mmol/l was optimal for examining alternative substrate oxidation. Methods for measuring fatty acid oxidation were established. These methods lay the framework using XF analysis to profile metabolism of ACMs and will likely augment our ability to understand mitochondrial dysfunction in heart failure and acute myocardial ischemia. This platform could easily be extended to models of diabetes or other metabolic defects.

  19. 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

  20. Scotopic Visual Signaling in the Mouse Retina Is Modulated by High-Affinity Plasma Membrane Calcium Extrusion

    PubMed Central

    Duncan, Jacque L.; Yang, Haidong; Doan, Thuy; Silverstein, Robert S.; Murphy, Gabe J.; Nune, George; Liu, Xiaorong; Copenhagen, David; Tempel, Bruce L.; Rieke, Fred; Križaj, David

    2007-01-01

    Transmission of visual signals at the first retinal synapse is associated with changes in calcium concentration in photoreceptors and bipolar cells. We investigated how loss of plasma membrane Ca2+ ATPase isoform 2 (PMCA2), the calcium transporter isoform with the highest affinity for Ca2+/calmodulin, affects transmission of rod- and cone-mediated responses. PMCA2 expression in the neuroblast layer was observed soon after birth; in the adult, PMCA2 was expressed in inner segments and synaptic terminals of rod photoreceptors, in rod bipolar cells, and in most inner retinal neurons but was absent from cones. To determine the role of PMCA2 in retinal signaling, we compared morphology and light responses of retinas from control mice and deafwaddler dfw2J mice, which lack functional PMCA2 protein. The cytoarchitecture of retinas from control and dfw2J mice was indistinguishable at the light microscope level. Suction electrode recordings revealed no difference in the sensitivity or amplitude of outer segment light responses of control and dfw2J rods. However, rod-mediated ERG b-wave responses in dfw2J mice were ∼45% smaller and significantly slower than those of control mice. Furthermore, recordings from individual rod bipolar cells showed that the sensitivity of transmission at the rod output synapse was reduced by ∼50%. No changes in the amplitude or timing of cone-mediated ERG responses were observed. These results suggest that PMCA2-mediated Ca2+ extrusion modulates the amplitude and timing of the high-sensitivity rod pathway to a much greater extent than that of the cone pathway. PMID:16822977

  1. Synaptic Input of ON-Bipolar Cells onto the Dopaminergic Neurons of the Mouse Retina

    PubMed Central

    Contini, Massimo; Lin, Bin; Kobayashi, Kazuto; Okano, Hideyuki; Masland, Richard H.; Raviola, Elio

    2010-01-01

    In the retina, dopamine fulfills a crucial role in neural adaptation to photopic illumination, but the pathway that carries cone signals to the dopaminergic amacrine (DA) cells was not known. We identified the site of ON-cone bipolar input onto DA cells in transgenic mice in which both types of catecholaminergic amacrine (CA) cells were labeled with green fluorescent protein or human placental alkaline phosphatase (PLAP). In confocal Z series of retinal whole mounts stained with antibodies to tyrosine hydroxylase (TH), DA cells gave rise to varicose processes that descended obliquely through the scleral half of the inner plexiform layer (IPL) and formed a loose, tangential plexus in the middle of this layer. Comparison with the distribution of the dendrites of type 2 CA cells and examination of neurobiotin-injected DA cells proved that their vitreal processes were situated in stratum S3 of the IPL. Electron microscope demonstration of PLAP activity showed that bipolar cell endings in S3 established ribbon synapses onto a postsynaptic dyad in which one or both processes were labeled by a precipitate of lead phosphate and therefore belonged to DA cells. In places, the postsynaptic DA cell processes returned a reciprocal synapse onto the bipolar endings. Confocal images of sections stained with antibodies to TH, kinesin Kif3a, which labels synaptic ribbons, and glutamate or GABAA receptors, confirmed that ribbon-containing endings made glutamatergic synapses onto DA cells processes in S3 and received from them GABAergic synapses. The presynaptic ON-bipolar cells most likely belonged to the CB3 (type 5) variety. PMID:20394057

  2. Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina

    PubMed Central

    Tachibana, Nobuhiko; Zinyk, Dawn; Ringuette, Randy; Wallace, Valerie; Schuurmans, Carol

    2017-01-01

    All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7–10 days in vitro. During this time frame, RPCs will differentiate, and the fate of the BrdU+ RPCs can be assessed using cell type-specific markers. Investigators who developed this pellet assay initially demonstrated that neonatal RPCs give rise to rods on an accelerated schedule compared to embryonic RPCs when the two cell types are mixed together (Watanabe and Raff, 1990; Watanabe et al., 1997). We have used this assay to demonstrate that sonic hedgehog (Shh), which we found acts as a negative regulator of retinal ganglion cell (RGC) differentiation, promotes RPC proliferation (Jensen and Wallace, 1997; Ringuette et al., 2014). More recently we modified the heterochronic pellet assay to assess the role of feedback signals for retinal amacrine cells, identifying transforming growth factor β2 (Tgfβ2) as a negative feedback signal, and Pten as a modulator of the Tgfβ2 response (Ma et al., 2007; Tachibana et al., 2016). This assay can be adapted to other lineages and tissues to assess cell-cell interactions between two different cell-types (heterotypic) in either an isochronic or heterochronic manner.

  3. 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

  4. Functional and morphological effects of laser-induced ocular hypertension in retinas of adult albino Swiss mice

    PubMed Central

    Salinas-Navarro, Manuel; Alarcón-Martínez, Luis; Valiente-Soriano, Francisco Javier; Ortín-Martínez, Arturo; Jiménez-López, Manuel; Avilés-Trigueros, Marcelino; Villegas-Pérez, María Paz; de la Villa, Pedro

    2009-01-01

    Purpose To investigate the effects of laser photocoagulation (LP)-induced ocular hypertension (OHT) on the survival and retrograde axonal transport of retinal ganglion cells (RGC), as well as on the function of retinal layers. Methods Adult albino Swiss mice (35–45 g) received laser photocoagulation of limbal and episcleral veins in the left eye. Mice were sacrificed at 8, 17, 35, and 63 days. Intraocular pressure (IOP) in both eyes was measured with a Tono-Lab before LP and at various days after LP. Flash electroretinogram (ERG) scotopic threshold response (STR) and a- and b-wave amplitudes were recorded before LP and at various times after LP. RGCs were labeled with 10% hydroxystilbamidine methanesulfonate (OHSt) applied to both superior colliculi before sacrifice and in some mice, with dextran tetramethylrhodamine (DTMR) applied to the ocular stump of the intraorbitally transected optic nerve. Retinas were immunostained for RT97 or Brn3a. Retinas were prepared as whole-mounts and photographed under a fluorescence microscope. Labeled RGCs were counted using image analysis software, and an isodensity contour plot was generated for each retina. Results IOP increased to twice its basal values by 24 h and was maintained until day 5, after which IOP gradually declined to reach basal values by 1 wk. Similar IOP increases were observed in all groups. The mean total number of OHSt+ RGCs was 13,428±6,295 (n=12), 10,456±14,301 (n=13), 12,622±14,174 (n=21), and 10,451±13,949 (n=13) for groups I, II, III, and IV, respectively; these values represented 28%, 23%, 26%, and 22% of the values found in their contralateral fellow retinas. The mean total population of Brn3a+ RGCs was 24,343±5,739 (n=12) and 10,219±8,887 (n=9), respectively, for groups I and III; these values represented 49% and 20%, respectively, of the values found in their fellow eyes. OHT retinas showed an absence of OHSt+ and DTMR+ RGCs in both focal wedge-shaped and diffuse regions of the retina. By 1

  5. In vivo optical coherence tomography of stimulus-evoked intrinsic optical signals in mouse retinas

    NASA Astrophysics Data System (ADS)

    Wang, Benquan; Lu, Yiming; Yao, Xincheng

    2016-09-01

    Intrinsic optical signal (IOS) imaging promises a noninvasive method for advanced study and diagnosis of eye diseases. Before pursuing clinical applications, it is essential to understand anatomic and physiological sources of retinal IOSs and to establish the relationship between IOS distortions and eye diseases. The purpose of this study was designed to demonstrate the feasibility of in vivo IOS imaging of mouse models. A high spatiotemporal resolution spectral domain optical coherence tomography (SD-OCT) was employed for depth-resolved retinal imaging. A custom-designed animal holder equipped with ear bar and bite bar was used to minimize eye movements. Dynamic OCT imaging revealed rapid IOS from the photoreceptor's outer segment immediately after the stimulation delivery, and slow IOS changes were observed from inner retinal layers. Comparative photoreceptor IOS and electroretinography recordings suggested that the fast photoreceptor IOS may be attributed to the early stage of phototransduction before the hyperpolarization of retinal photoreceptor.

  6. The Lgr5 transgene is expressed specifically in glycinergic amacrine cells in the mouse retina.

    PubMed

    Sukhdeo, Kumar; Koch, Catherine E; Miller, Tyler E; Zhou, Hannah; Rivera, Maricruz; Yan, Kenneth; Cepko, Constance L; Lathia, Justin D; Rich, Jeremy N

    2014-02-01

    Retinal amacrine cells are a diverse set of interneurons within the inner nuclear layer. The canonical Wnt pathway is highly active within mature amacrine cells, but its role remains unclear. Leucine-rich repeat containing G-protein receptor 5 (Lgr5) is a newly identified component of the Wnt receptor complex that potentiates beta-catenin signaling. In multiple epithelial organs Lgr5 marks adult tissue stem cells. We investigated the expression of this gene using Lgr5-eGFP-IRES-CreER transgenic reporter mice. In the eye, Lgr5 was exclusively expressed in glycinergic amacrine cells in adult mice. Amacrine cells are post-mitotic and represent the first neuronal and non-stem cell lineage to express Lgr5. We further interrogated the spatiotemporal labeling of individual amacrine cells with controlled fluorophore expression. This "fluorofilling" technique provides a tool to study amacrine morphology and dissect neural networks.

  7. Raman spectroscopy reveals spectroscopic changes in histologically normal retinas in a mouse model of alpha-synucleinopathy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The retina is an extension of the nervous system and is accessible for in vivo assessments. We have previously demonstrated changes in retinal function and pathology associated with scrapie, TME and BSE. The purpose of this work was to determine the utility of the retina to identify early CNS change...

  8. [Stem cells in adult retina--current state of research, future therapeutic prospects].

    PubMed

    Machalińska, Anna; Zuba-Surma, Ewa K

    2009-01-01

    The latest research reports revealed the presence of stem/progenitor cells located in different regions of matured eye. They are able to differentiate into retinal pigment epithelium cells as well as neural structure of retina. These cells were identified in neurosensory retina, pigment epithelium and within cilliary body and iris epithelium. Moreover, it has been proved that Muller glia possess the potential of differentiation into retinal cells. These findings indicate the presence of potential mechanisms enabling retinal cell repopulation and retinal tissue regeneration. In the present work, the recent reports documenting the presence of different stem cell populations in eye have been reviewed, particularly focusing on recently identified very small embryonic-like stem cells (VSEL-SCs). The potential clinical applications of the residing stem cells and limitations of such therapeutic strategies have been also discussed.

  9. Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

    PubMed Central

    Lahne, Manuela; Li, Jingling; Marton, Rebecca M.

    2015-01-01

    Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. SIGNIFICANCE STATEMENT The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms

  10. Dystrophin Dp71 Isoforms Are Differentially Expressed in the Mouse Brain and Retina: Report of New Alternative Splicing and a Novel Nomenclature for Dp71 Isoforms.

    PubMed

    Aragón, Jorge; González-Reyes, Mayram; Romo-Yáñez, José; Vacca, Ophélie; Aguilar-González, Guadalupe; Rendón, Alvaro; Vaillend, Cyrille; Montañez, Cecilia

    2017-01-27

    Multiple dystrophin Dp71 isoforms have been identified in rats, mice, and humans and in several cell line models. These Dp71 isoforms are produced by the alternative splicing of exons 71 to 74 and 78 and intron 77. Three main groups of Dp71 proteins are defined based on their C-terminal specificities: Dp71d, Dp71f, and Dp71e. Dp71 is highly expressed in the brain and retina; however, the specific isoforms present in these tissues have not been determined to date. In this work, we explored the expression of Dp71 isoforms in the mouse brain and retina using RT-PCR assays followed by the cloning of PCR products into the pGEM-T Easy vector, which was used to transform DH5α cells. Dp71-positive colonies were later analyzed by PCR multiplex and DNA sequencing to determine the alternative splicing. We thus demonstrated the expression of Dp71 transcripts corresponding to Dp71, Dp71a, Dp71c, Dp71b, Dp71ab, Dp71 Δ110, and novel Dp71 isoforms spliced in exon 74; 71 and 74; 71, 73 and 74; and 74 and 78, which we named Dp71d Δ74 , Dp71d Δ71,74 , Dp71d Δ71,73-74 , and Dp71f Δ74 , respectively. Additionally, we demonstrated that the Dp71d group of isoforms is highly expressed in the brain, while the Dp71f group predominates in the retina, at both the cDNA and protein levels. These findings suggest that distinct Dp71 isoforms may play different roles in the brain and retina.

  11. Epibatidine application in vitro blocks retinal waves without silencing all retinal ganglion cell action potentials in developing retina of the mouse and ferret.

    PubMed

    Sun, Chao; Speer, Colenso M; Wang, Guo-Yong; Chapman, Barbara; Chalupa, Leo M

    2008-12-01

    Epibatidine (EPI), a potent cholinergic agonist, disrupts acetylcholine-dependent spontaneous retinal activity. Early patch-clamp recordings in juvenile ferrets suggested that EPI blocks all retinal ganglion cell (RGC) action potentials when applied to the retina. In contrast, recent experiments on the developing mouse that relied on multielectrode array (MEA) recordings reported that EPI application decorrelates the activity of neighboring RGCs and eliminates retinal waves while preserving the spiking activity of many neurons. The different techniques used in previous studies raise the question of whether EPI has different effects on RGC activity in mouse compared with that in ferret. A resolution of this issue is essential for interpreting the results of developmental studies that relied on EPI to manipulate retinal activity. Our goal was to compare the effects of EPI on the spontaneous discharges of RGCs in mouse and ferret using 60-electrode MEA as well as patch-clamp recordings during the developmental stage when retinal waves are driven by acetylcholine in both species. We found that in both mouse and ferret EPI decorrelates RGC activity and eliminates retinal waves. However, EPI does not block all spontaneous activity in either species. Instead, our whole cell recordings reveal that EPI silences more than half of all RGCs while significantly increasing the activity of the remainder. These results have important implications for interpreting the results of previous studies that relied on this cholinergic agonist to perturb retinal activity.

  12. MIO-M1 cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics.

    PubMed

    Lawrence, Jean M; Singhal, Shweta; Bhatia, Bhairavi; Keegan, David J; Reh, Thomas A; Luthert, Philip J; Khaw, Peng T; Limb, Gloria Astrid

    2007-08-01

    Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor-like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor-2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including betaIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S-opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell-based therapies to restore retinal function. Disclosure of potential conflicts of interest is found at the end of this article.

  13. A Comprehensive Atlas of the Adult Mouse Penis.

    PubMed

    Phillips, Tiffany R; Wright, David K; Gradie, Paul E; Johnston, Leigh A; Pask, Andrew J

    2015-01-01

    Mice are routinely used to study the development of the external genitalia and, in particular, the process of male urethral closure. This is because misplacement of the male penile urethra, or hypospadias, is amongst the most common birth defects reported in humans. While mice present a tractable model to study penile development, several structures differ between mice and humans, and there is a lack of consensus in the literature on their annotation and developmental origins. Defining the ontology of the mouse prepuce is especially important for the relevance and interpretation of mouse models of hypospadias to human conditions. We have developed a detailed annotation of the adult mouse penis that addresses these differences and enables an accurate comparison of murine and human hypospadias phenotypes. Through MRI data, gross morphology and section histology, we define the origin of the mouse external and internal prepuces, their relationship to the single human foreskin as well as provide a comprehensive view of the various structures of the mouse penis and their associated muscle attachments within the body. These data are combined to annotate structures in a novel 3D adult penis atlas that can be downloaded, viewed at any angle, and manipulated to examine the relationship of various structures.

  14. Functional study of two biochemically unusual mutations in GUCY2D Leber congenital amaurosis expressed via adenoassociated virus vector in mouse retinas

    PubMed Central

    Boye, Sanford L.; Olshevskaya, Elena V.; Peshenko, Igor V.; McCullough, K. Tyler; Boye, Shannon E.

    2016-01-01

    Purpose To test, in living photoreceptors, two mutations, S248W and R1091x, in the GUCY2D gene linked to Leber congenital amaurosis 1 (LCA1) that fail to inactivate the catalytic activity of a heterologously expressed retinal membrane guanylyl cyclase 1 (RetGC1). Methods GUC2YD cDNA constructs coding for wild-type human (hWT), R1091x, and S248W GUCY2D under the control of the human rhodopsin kinase promoter were expressed in Gucy2e−/−Gucy2f−/− knockout (GCdKO) mouse retinas, which lack endogenous RetGC activity. The constructs were delivered via subretinally injected adenoassociated virus (AAV) vector in one eye, leaving the opposite eye as the non-injected negative control. After testing with electroretinography (ERG), the retinas extracted from the AAV-treated and control eyes were used in guanylyl cyclase activity assays, immunoblotting, and anti-RetGC1 immunofluorescence staining. Results Cyclase activity in retinas treated with either hWT or R1091x GUCY2D transgenes was similar but was undetectable in the S248W GUCY2D-treated retinas, which starkly contrasts their relative activities when heterologously expressed in human embryonic kidney (HEK293) cells. Rod and cone ERGs, absent in GCdKO, appeared in the hWT and R1091x GUCY2D-injected eyes, while the S248W mutant failed to restore scotopic ERG response and enabled only rudimentary photopic ERG response. The hWT and R1091x GUCY2D immunofluorescence was robust in the rod and cone outer segments, whereas the S248W was detectable only in the sparse cone outer segments and sporadic photoreceptor cell bodies. Robust RetGC1 expression was detected with immunoblotting in the hWT and R1091x-treated retinas but was marginal at best in the S248W GUCY2D retinas, despite the confirmed presence of the S248W GUCY2D transcripts. Conclusions The phenotype of S248W GUCY2D in living retinas did not correlate with the previously described normal biochemical activity of this mutant when heterologously expressed in non

  15. Distribution of Müller stem cells within the neural retina: evidence for the existence of a ciliary margin-like zone in the adult human eye.

    PubMed

    Bhatia, Bhairavi; Singhal, Shweta; Lawrence, Jean M; Khaw, Peng T; Limb, G Astrid

    2009-09-01

    Much interest has been generated by the identification of neural stem cells in the human neural retina and ciliary body. However, it is not clear whether stem cells identified in these ocular compartments are of the same origin or whether they ontogenically derive from different cell populations. This study examined the in situ anatomical distribution of these cells within the neural retina and ciliary body, as well as their ability to proliferate in response to EGF. Human retinae and ciliary body were examined for co-expression of Nestin, cellular retinaldehyde binding (CRALBP) or Vimentin, and the stem cell markers SOX2, CHX10, NOTCH1 and SHH. Retinal explants were cultured with epidermal growth factor (EGF) to assess retinal cell proliferation. Intense Nestin and CRALBP staining was observed in the neural retinal margin, where cells formed bundles of spindle cells (resembling glial cells) that lacked lamination and co-stained for SOX2, CHX10 and SHH. This staining differentiated the neural retina from the ciliary epithelium, which expressed SOX2, CHX10 and NOTCH1 but not Nestin or CRALBP. Nestin and CRALBP expression decreased towards the posterior retina, where it anatomically identified a population of Müller glia. All Vimentin positive Müller glia co-stained for SOX2, but only few Vimentin positive cells expressed Nestin and SOX2. Cells of the retinal margin and the inner nuclear layer (INL), where the soma of Müller glia predominate, re-entered the cell cycle upon retinal explant culture with EGF. Lack of lamination and abundance of Müller glia expressing stem cell markers in the marginal region of the adult human retina resemble the ciliary marginal zone (CMZ) of fish and amphibians. The findings that cells in this CM-like zone, as well in the inner nuclear layer proliferate in response to EGF suggest that the adult human retina has regenerative potential. Identification of factors that may promote retinal regeneration in the adult human eye would

  16. Octreotide Protects the Mouse Retina against Ischemic Reperfusion Injury through Regulation of Antioxidation and Activation of NF-κB

    PubMed Central

    Wang, Jun; Sun, Ziqiang; Shen, Junsheng; Wu, Dongdong; Liu, Fang; Yang, Ruisheng; Ji, Shaoping; Ji, Ailing; Li, Yanzhang

    2015-01-01

    Somatostatin (SST), an endogenous peptide, may exert anti-inflammatory and neuroprotective effects on retinal injury induced by ischemia. Retinal ischemic reperfusion (I/R) injury always produces many reactive oxygen species (ROS), which can aggravate the tissue damage. The effects of octreotide (OCT), a SST analogue, on retinal I/R injury and ROS formation, are not very clear. In this study, we observed the effects of OCT on morphological changes, oxidative stress, and cell death, induced by retinal I/R injury. The activation of nuclear factor κB (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) were further evaluated in I/R retina treated with or without OCT. The retinal layer thickness was increased at 1 day after I/R and decreased at 7 days after I/R (P < 0.05). This effect was associated with increase in MDA and ROS levels (P < 0.05). The Tunel-positive cells increased and the number of ganglion cell layer (GCL) neurons decreased significantly after I/R injury. The expression of p-p65 and ICAM-1 increased significantly in I/R retinas (P < 0.05). Each effect was markedly attenuated by application of OCT. These data indicate that OCT protects the retina against retinal I/R damage, which could be through inhibition of oxidative stress and downregulation of NF-κB and ICAM-1 expression. PMID:26175842

  17. Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina.

    PubMed

    Zawadzki, Robert J; Zhang, Pengfei; Zam, Azhar; Miller, Eric B; Goswami, Mayank; Wang, Xinlei; Jonnal, Ravi S; Lee, Sang-Hyuck; Kim, Dae Yu; Flannery, John G; Werner, John S; Burns, Marie E; Pugh, Edward N

    2015-06-01

    Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

  18. Adaptive-optics SLO imaging combined with widefield OCT and SLO enables precise 3D localization of fluorescent cells in the mouse retina

    PubMed Central

    Zawadzki, Robert J.; Zhang, Pengfei; Zam, Azhar; Miller, Eric B.; Goswami, Mayank; Wang, Xinlei; Jonnal, Ravi S.; Lee, Sang-Hyuck; Kim, Dae Yu; Flannery, John G.; Werner, John S.; Burns, Marie E.; Pugh, Edward N.

    2015-01-01

    Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed. PMID:26114038

  19. Methylation of DNA in mouse early embryos, teratocarcinoma cells and adult tissues of mouse and rabbit.

    PubMed Central

    Singer, J; Roberts-Ems, J; Luthardt, F W; Riggs, A D

    1979-01-01

    The distribution and amount of 5-methylcytosine (5-MeCyt) in DNA was measured for early embryos of mouse strain CF1 (2 to 4 cell stage to blastocyst) and mouse teratocarcinoma cells. In each case, the pattern of methylation was examined by use of the restriction enzymes Hha I and HPA II HPA II, which cut DNA at the sites 5'GCGC and 5'CCGG respectively, when the cytosines at these sites are not methylated. Mouse embryo DNA was found to have the same level of methylation as adult mouse tissues, and no changes in methylation were seen during differentiation of the teratocarcinoma cells. The ratio of 5-MeCyt/Cyt in DNA was measured by high performance liquid chromatography for the differentiating teratocarcinoma cells and for several adult mouse and rabbit tissues. The variation between tissues or between teratocarcinoma cells at different stages of differentiation was less than 10 percent. These results are discussed in view of proposals that 5-MeCyt plays a role in differentiation. Images PMID:523320

  20. Retinal lesions induce fast intrinsic cortical plasticity in adult mouse visual system.

    PubMed

    Smolders, Katrien; Vreysen, Samme; Laramée, Marie-Eve; Cuyvers, Annemie; Hu, Tjing-Tjing; Van Brussel, Leen; Eysel, Ulf T; Nys, Julie; Arckens, Lutgarde

    2016-09-01

    Neuronal activity plays an important role in the development and structural-functional maintenance of the brain as well as in its life-long plastic response to changes in sensory stimulation. We characterized the impact of unilateral 15° laser lesions in the temporal lower visual field of the retina, on visually driven neuronal activity in the afferent visual pathway of adult mice using in situ hybridization for the activity reporter gene zif268. In the first days post-lesion, we detected a discrete zone of reduced zif268 expression in the contralateral hemisphere, spanning the border between the monocular segment of the primary visual cortex (V1) with extrastriate visual area V2M. We could not detect a clear lesion projection zone (LPZ) in areas lateral to V1 whereas medial to V2M, agranular and granular retrosplenial cortex showed decreased zif268 levels over their full extent. All affected areas displayed a return to normal zif268 levels, and this was faster in higher order visual areas than in V1. The lesion did, however, induce a permanent LPZ in the retinorecipient layers of the superior colliculus. We identified a retinotopy-based intrinsic capacity of adult mouse visual cortex to recover from restricted vision loss, with recovery speed reflecting the areal cortical magnification factor. Our observations predict incomplete visual field representations for areas lateral to V1 vs. lack of retinotopic organization for areas medial to V2M. The validation of this mouse model paves the way for future interrogations of cortical region- and cell-type-specific contributions to functional recovery, up to microcircuit level.

  1. Systemic administration of the iron chelator deferiprone protects against light-induced photoreceptor degeneration in the mouse retina

    PubMed Central

    Song, Delu; Song, Ying; Hadziahmetovic, Majda; Zhong, Yong; Dunaief, Joshua L.

    2012-01-01

    Oxidative stress plays a key role in the light damage (LD) model of retinal degeneration as well as in age-related macular degeneration (AMD). Since iron can promote oxidative stress, the iron chelator Deferiprone (DFP) was tested for protection against light-induced retinal degeneration. To accomplish this, A/J mice were treated with or without DFP in drinking water, and then were placed in constant bright white fluorescent light (10,000 lux) for 20 hours. Retinas were evaluated at several time points after light exposure. Photoreceptor apoptosis was assessed using the TUNEL assay. Retinal degeneration was assessed by histology 10 days after exposure to damaging white light. Two genes upregulated by oxidative stress, heme oxygenase 1 (Hmox1) and ceruloplasmin (Cp), as well as complement component 3 (C3) were quantified by RT-qPCR. Cryosections were immunolabeled for oxidative stress marker (nitrotyrosine), a microglial marker (Iba1) as well as both heavy (H) and light (L) ferritin. Light exposure resulted in substantial photoreceptor-specific cell death. Dosing with DFP protected photoreceptors, decreasing the numbers of TUNEL-positive photoreceptors and increasing the number of surviving photoreceptors. The retinal mRNA levels of oxidative stress related genes and C3 were upregulated following light exposure and diminished by DFP treatment. Immunostaining for nitrotyrosine indicated that DFP reduced the nitrative stress caused by light exposure. Robust H/L-ferritin-containing microglial activation and migration to the outer retina occurred after light exposure and DFP treatment reduced microglial invasion. DFP is protective against light-induced retinal degeneration and has the potential to diminish oxidative stress in the retina. PMID:22579919

  2. ADAPTATION OF GROUP B COXSACKIE VIRUS TO ADULT MOUSE PANCREAS

    PubMed Central

    Dalldorf, Gilbert; Gifford, Rebecca

    1952-01-01

    An alteration of tissue tropism of a Coxsackie virus has been observed following different methods of propagation of the virus in animals. Tropism for the adult mouse pancreas, as described by Pappenheimer, appeared to be irrevocably lost following prolonged brain-to-brain transfer. It was present in the same strain on reisolation from human feces, was intensified following pancreas transfers, and suppressed by brain transfers. Pancreatotropism may be correlated with the titer of virus in the pancreas. PMID:13000059

  3. Immunosuppression via Loss of IL2rγ Enhances Long-Term Functional Integration of hESC-Derived Photoreceptors in the Mouse Retina.

    PubMed

    Zhu, Jie; Cifuentes, Helen; Reynolds, Joseph; Lamba, Deepak A

    2017-03-02

    Loss of photoreceptors is a common endpoint in degenerative retinal diseases. Human pluripotent stem cells provide a potential source for photoreceptor replacement, but, even in mouse models, the efficiency and efficacy of transplantation-based repair remains poor. In this study, we examined the degree to which immune rejection contributes to these disappointing outcomes using an immunodeficient IL2 receptor γ (IL2rγ)-null mouse model. Our results show that prevention of cell rejection in the normal and degenerating retinal environment significantly improves long-term survival and integration of hESC-derived donor retinal cells. Transplanted cells are able to differentiate into mature photoreceptors expressing various opsins and can functionally integrate into congenitally blind mice. Our work suggests that even though the retina is often considered immune-privileged, suppression of host immune-mediated cell rejection may well be a useful approach for improving long-term integration of transplanted cells with a view to successful clinical outcomes.

  4. Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.

    PubMed

    Walston, Steven T; Chow, Robert H; Weiland, James D

    2015-01-01

    Retinitis pigmentosa is a family of inherited retinal diseases identified by the degeneration of photoreceptors, which leads to blindness. In efforts to restore vision lost to retinitis pigmentosa, retinal prostheses have been developed to generate visual percepts by electrically stimulating the surviving retinal bipolar and ganglion cells. The response of retinal ganglion cells to electrical stimulation has been characterized through direct measurement. However, the response of bipolar cells has only been inferred by measuring retinal ganglion cell activity. This investigation reports on a novel tissue preparation technique facilitating bipolar cell patch clamp recordings in wholemount retina. We find that bipolar cells respond to extracellular electrical stimuli with time-locked voltage spike depolarizations, which are likely mediated by voltage-gated calcium channels.

  5. The effect of high energy (HZE) particle radiation (Ar-40) on aging parameters of mouse hippocampus and retina

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Kato, K.; Corbett, R.; Stevenson, J.; Black, S.; Sapp, W.; Miquel, J.; Lindseth, K. A.; Benton, E. V.

    1985-01-01

    Eight month old C57BL6 mice were exposed (head only) to 0.5 rad or 50 rads of Argon particles at the Lawrence Berkeley Radiation Facility, CA. Neuromotor performance was assessed monthly for six months beginning twelve weeks post-irradiation using a 'string test'. The decline in motor performance was dose-related and none of the animals was able to complete the task after four months of testing. Morphological changes were monitored six and twelve months post-irradiation by light and electron microscopy. The synaptic density in the CA-1 area of the hippocampus decreased six and twelve months after irradiation. The decrease after twelve months was less than after six months. The width of the outer nuclear layer (ONL) of the retina increased with increasing dose. The number of blood vessels between the ONL and the ganglion layer decreased twelve months after irradiation and this area did not show significant accumulation of age pigment.

  6. Resveratrol Inhibits Neuronal Apoptosis and Elevated Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Diabetic Mouse Retina

    PubMed Central

    Kim, Young-Hee; Kim, Yoon-Sook; Kang, Sang-Soo; Cho, Gyeong-Jae; Choi, Wan-Sung

    2010-01-01

    OBJECTIVE This study investigated the effects of resveratrol, a natural polyphenol with neuroprotective properties, on retinal neuronal cell death mediated by diabetes-induced activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). RESEARCH DESIGN AND METHODS Diabetes was induced in C57BL/6 mice by five consecutive intraperitoneal injections of 55 mg/kg streptozotocin (STZ). Control mice received buffer. All mice were killed 2 months after the injections, and the extent of neuronal cell death, CaMKII, and phospho-CaMKII protein expression levels and CaMKII kinase activity were examined in the retinas. To assess the role of CaMKII in the death of retinal neurons, a small-interfering RNA (siRNA) or specific inhibitor of CaMKII was injected into the right vitreous humor, and vehicle only was injected into the left vitreous humor, 2 days before death. Resveratrol (20 mg/kg) was administered by oral gavage daily for 4 weeks, beginning 1 month after the fifth injection of either STZ or buffer. RESULTS The death of retinal ganglion cells (RGCs), CaMKII, phospho-CaMKII protein levels, and CaMKII activity were all greatly increased in the retinas of diabetic mice compared with controls, 2 months after induction of diabetes. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive signals co-localized with CaMKII- and phospho-CaMKII immunoreactive RGCs. However, in addition to CaMKII knockdown and inhibition by siRNA or a specific inhibitor, respectively, resveratrol provided complete protection from diabetes-induced retinal cell death. CONCLUSIONS In the present study, resveratrol prevented diabetes-induced RGC death via CaMKII downregulation, implying that resveratrol may have potential therapeutic applications for prevention of diabetes-induced visual dysfunction. PMID:20424226

  7. Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles.

    PubMed

    Apaolaza, P S; Del Pozo-Rodríguez, A; Solinís, M A; Rodríguez, J M; Friedrich, U; Torrecilla, J; Weber, B H F; Rodríguez-Gascón, A

    2016-06-01

    X-linked juvenile retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in the RS1 gene encoding a protein termed retinoschisin. The disease is an excellent candidate for gene replacement therapy as the majority of mutations have been shown to lead to a complete deficiency of the secreted protein in the retinal structures. In this work, we have studied the ability of non-viral vectors based on solid lipid nanoparticles (SLN) to induce the expression of retinoschisin in photoreceptors (PR) after intravitreal administration to Rs1h-deficient mice. We designed two vectors prepared with SLN, protamine, and dextran (DX) or hyaluronic acid (HA), bearing a plasmid containing the human RS1 gene under the control of the murin opsin promoter (mOPS). In vitro, the nanocarriers were able to induce the expression of retinoschisin in a PR cell line. After injection into the murine vitreous, the formulation prepared with HA induced a higher transfection level in PR than the formulation prepared with DX. Moreover, the level of retinoschisin in the inner nuclear layer (INL), where bipolar cells are located, was also higher. Two weeks after vitreal administration into Rs1h-deficient mice, both formulations showed significant improvement of the retinal structure by inducing a decrease of cavities and PR loss, and an increase of retinal and outer nuclear layer (ONL) thickness. HA-SLN resulted in a significant higher increase in the thickness of both retina and ONL, which can be explained by the higher transfection level of PR. In conclusion, we have shown the structural improvement of the retina of Rs1h-deficient mice with PR specific expression of the RS1 gene driven by the specific promoter mOPS, after successful delivery via SLN-based non-viral vectors.

  8. Shh/Boc Signaling Is Required for Sustained Generation of Ipsilateral Projecting Ganglion Cells in the Mouse Retina

    PubMed Central

    Sánchez-Camacho, Cristina; Carreres, M. Isabel; Herrera, Eloisa; Okada, Ami; Bovolenta, Paola

    2013-01-01

    Sonic Hedgehog (Shh) signaling is an important determinant of vertebrate retinal ganglion cell (RGC) development. In mice, there are two major RGC populations: (1) the Islet2-expressing contralateral projecting (c)RGCs, which both produce and respond to Shh; and (2) the Zic2-expressing ipsilateral projecting RGCs (iRGCs), which lack Shh expression. In contrast to cRGCs, iRGCs, which are generated in the ventrotemporal crescent (VTC) of the retina, specifically express Boc, a cell adhesion molecule that acts as a high-affinity receptor for Shh. In Boc−/− mutant mice, the ipsilateral projection is significantly decreased. Here, we demonstrate that this phenotype results, at least in part, from the misspecification of a proportion of iRGCs. In Boc−/− VTC, the number of Zic2-positive RGCs is reduced, whereas more Islet2/Shh-positive RGCs are observed, a phenotype also detected in Zic2 and Foxd1 null embryos. Consistent with this observation, organization of retinal projections at the dorsallateral geniculate nucleus is altered in Boc−/− mice. Analyses of the molecular and cellular consequences of introducing Shh into the developing VTC and Zic2 and Boc into the central retina indicate that Boc expression alone is in sufficient to fully activate the ipsilateral program and that Zic2 regulates Shh expression. Taking these data together, we propose that expression of Boc in cells from the VTC is required to sustain Zic2 expression, likely by regulating the levels of Shh signaling from the nearby cRGCs. Zic2, in turn, directly or indirectly, counteracts Shh and Islet2 expression in the VTC and activates the ipsilateral program. PMID:23678105

  9. Litsea japonica extract inhibits neuronal apoptosis and the accumulation of advanced glycation end products in the diabetic mouse retina.

    PubMed

    Kim, Junghyun; Kim, Chan-Sik; Lee, Yun Mi; Sohn, Eunjin; Jo, Kyuhyung; Kim, Jin Sook

    2015-07-01

    The retinal accumulation of advanced glycation end products (AGEs) is a condition, which is found in diabetic retinopathy. The purpose of the present study was to investigate the protective effect of Litsea japonica extract (LJE) and to elucidate its underlying protective mechanism in model diabetic db/db mice. Male, 7 -week-old db/db mice were treated with LJE (100 or 250 mg/kg body weight) once a day orally for 12 weeks. The expression levels of AGEs and their receptor (RAGE) were subsequently assessed by immunohistochemistry. An electrophoretic mobility shift assay and southwestern histochemistry were used to detect activated nuclear factor κB (NF-κB). The immunohistochemical analysis demonstrated that LJE significantly reduced the expression levels of the AGEs and RAGE in the neural retinas of the db/db mice. LJE markedly inhibited the apoptosis of retinal ganglion cells. In addition, LJE suppressed the activation of NF-κB. These results suggested that LJE may be beneficial for the treatment of diabetes-induced retinal neurodegeneration, and the ability of LJE to attenuate retinal ganglion cell loss may be mediated by inhibition of the accumulation of AGEs.

  10. In Vitro Spermatogenesis in Explanted Adult Mouse Testis Tissues.

    PubMed

    Sato, Takuya; Katagiri, Kumiko; Kojima, Kazuaki; Komeya, Mitsuru; Yao, Masahiro; Ogawa, Takehiko

    2015-01-01

    Research on in vitro spermatogenesis is important for elucidating the spermatogenic mechanism. We previously developed an organ culture method which can support spermatogenesis from spermatogonial stem cells up to sperm formation using immature mouse testis tissues. In this study, we examined whether it is also applicable to mature testis tissues of adult mice. We used two lines of transgenic mice, Acrosin-GFP and Gsg2-GFP, which carry the marker GFP gene specific for meiotic and haploid cells, respectively. Testis tissue fragments of adult GFP mice, aged from 4 to 29 weeks old, which express GFP at full extension, were cultured in medium supplemented with 10% KSR or AlbuMAX. GFP expression decreased rapidly and became the lowest at 7 to 14 days of culture, but then slightly increased during the following culture period. This increase reflected de novo spermatogenesis, confirmed by BrdU labeling in spermatocytes and spermatids. We also used vitamin A-deficient mice, whose testes contain only spermatogonia. The testes of those mice at 13-21 weeks old, showing no GFP expression at explantation, gained GFP expression during culturing, and spermatogenesis was confirmed histologically. In addition, the adult testis tissues of Sl/Sld mutant mice, which lack spermatogenesis due to Kit ligand mutation, were cultured with recombinant Kit ligand to induce spermatogenesis up to haploid formation. Although the efficiency of spermatogenesis was lower than that of pup, present results showed that the organ culture method is effective for the culturing of mature adult mouse testis tissue, demonstrated by the induction of spermatogenesis from spermatogonia to haploid cells.

  11. Long term effects of low doses of Fe-56 ions on the brain and retina of the mouse - Ultrastructural and behavioral studies

    NASA Technical Reports Server (NTRS)

    Philpott, Delbert E.; Miquel, Jaime

    1986-01-01

    Eight-month-old male C57BL6 mice were exposed without anesthesia to whole-body irradiation in circular holders. The mice were tested for behavioral decrements after 0.5 and 50 rads of Fe particle irradiation at 6 and 12 months postirradiation to obtain long-term results. A standard maze was used, and the animals were timed for completion thereof. A string test also was administered to the mice, testing their ability to grasp and move along a string to safety. The results from animals exposed to 50 rads were significantly different from control results to p = less than 0.001 in both systems of testing. The hippocampus (believed to be the location of environmental interaction in the brain) and the retina were examined for ultrastructural changes. The ultrastructural changes were similar to those found in the Cosmos 782, 936, and Argon experiments. The mouse data indicate that iron particles were able to induce long-term changes in the central nervous system which led to behavioral impairment.

  12. Long term effects of low doses of 56Fe ions on the brain and retina of the mouse: ultrastructural and behavioral studies

    NASA Technical Reports Server (NTRS)

    Philpott, D. E.; Miquel, J.

    1986-01-01

    Eight month old male C57BL6 mice were exposed without anesthesia to whole-body irradiation in circular holders. The mice were tested for behavioral decrements after 0.5 and 50 rads of Fe particle irradiation at 6 and 12 months post irradiation to obtain long term results. A standard maze was used and the animals were timed for completion thereof. A string test also was administered to the mice, testing their ability to grasp and move along a string to safety. The results from animals exposed to 50 rads were significantly different from [correction of fron] control results to p = < .001 in both systems of testing. The hippocampus (believed to be the location of environmental interaction in the brain) and the retina were examined for ultrastructural changes. The ultrastructural changes were similar to those we found in our Cosmos 782, 936 and in our Argon experiments. The mouse data indicate that iron particles were able to induce long term changes in the central nervous system which lead to behavioral impairment.

  13. Electrophysiological Properties of Subventricular Zone Cells in Adult Mouse Brain

    PubMed Central

    Lai, Bin; Mao, Xiao Ou; Xie, Lin; Chang, Su-Youne; Xiong, Zhi-Gang; Jin, Kunlin; Greenberg, David A.

    2010-01-01

    The subventricular zone (SVZ) is a principal site of adult neurogenesis and appears to participate in the brain’s response to injury. Thus, measures that enhance SVZ neurogenesis may have a role in treatment of neurological disease. To better characterize SVZ cells and identify potential targets for therapeutic intervention, we studied electrophysiological properties of SVZ cells in adult mouse brain slices using patch-clamp techniques. Electrophysiology was correlated with immunohistochemical phenotype by injecting cells with lucifer yellow and by studying transgenic mice carrying green fluorescent protein under control of the doublecortin (DCX) or glial fibrillary acidic protein (GFAP) promoter. We identified five types of cells in the adult mouse SVZ: type 1 cells, with 4-aminopyridine (4-AP)/tetraethylammonium (TEA)-sensitive and CdCl2-sensitive inward currents; type 2 cells, with Ca2+-sensitive K+ and both 4-AP/TEA-sensitive and -insensitive currents; type 3 cells, with 4-AP/TEA-sensitive and -insensitive and small Na+ currents; type 4 cells, with slowly activating, large linear outward current and sustained outward current without fast-inactivating component; and type 5 cells, with a large outward rectifying current with a fast inactivating component. Type 2 and 3 cells expressed DCX, types 4 and 5 cells expressed GFAP, and type 1 cells expressed neither. We propose that SVZ neurogenesis involves a progression of electrophysiological cell phenotypes from types 4 and 5 cells (astrocytes) to type 1 cells (neuronal progenitors) to types 2 and 3 cells (nascent neurons), and that drugs acting on. ion channels expressed during neurogenesis might promote therapeutic neurogenesis in the injured brain. PMID:20434436

  14. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    SciTech Connect

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  15. Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

    SciTech Connect

    Wang, Jiying; Ohno-Matsui, Kyoko; Morita, Ikuo

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

  16. The alpha1 isoform of the Na+/K+ ATPase is up-regulated in dedifferentiated progenitor cells that mediate lens and retina regeneration in adult newts.

    PubMed

    Vergara, M Natalia; Smiley, Laura K; Del Rio-Tsonis, Katia; Tsonis, Panagiotis A

    2009-02-01

    Adult newts are able to regenerate their retina and lens after injury or complete removal through transdifferentiation of the pigmented epithelial tissues of the eye. This process needs to be tightly controlled, and several different mechanisms are likely to be recruited for this function. The Na(+)/K(+) ATPase is a transmembrane protein that establishes electrochemical gradients through the transport of Na(+) and K(+) and has been implicated in the modulation of key cellular processes such as cell division, migration and adhesion. Even though it is expressed in all cells, its isoform composition varies with cell type and is tightly controlled during development and regeneration. In the present study we characterize the expression pattern of Na(+)/K(+) ATPase alpha1 in the adult newt eye and during the process of lens and retina regeneration. We show that this isoform is up-regulated in undifferentiated cells during transdifferentiation. Such change in composition could be one of the mechanisms that newt cells utilize to modulate this process.

  17. Automatic segmentation of high pressure frozen and freeze-substituted mouse retina nuclei from FIB-SEM tomograms.

    PubMed

    Hoang, Thai V; Kizilyaprak, Caroline; Spehner, Danièle; Humbel, Bruno M; Schultz, Patrick

    2017-02-01

    Focused Ion Beam milling combined with Scanning Electron Microscopy is a powerful tool to determine the 3-D organization of whole cells and tissue at an isotropic resolution of 3-5nm. This opens the possibility to quantify several cellular parameters and to provide detailed phenotypic information in normal or disease states. Here we describe Biocomputing methods to extract in an automated way characteristic features of mouse rod photoreceptor nuclei such as the shape and the volume of the nucleus; the proportion of heterochromatin; the number, density and distribution of nuclear pore complexes (NPC). Values obtained on five nuclei show that the number of NPC (348±8) is the most conserved feature. Nuclei in higher eukaryotes show large variations in size and rod nuclei are amongst the smallest reported (32±3μm(3)). Despite large species- and cell-type-specific variations in size, the density of NPC (about 15/μm(2)) is highly conserved.

  18. Retina-specific activation of a sustained hypoxia-like response leads to severe retinal degeneration and loss of vision.

    PubMed

    Lange, Christina; Caprara, Christian; Tanimoto, Naoyuki; Beck, Susanne; Huber, Gesine; Samardzija, Marijana; Seeliger, Mathias; Grimm, Christian

    2011-01-01

    Loss of vision and blindness in human patients is often caused by the degeneration of neuronal cells in the retina. In mouse models, photoreceptors can be protected from death by hypoxic preconditioning. Preconditioning in low oxygen stabilizes and activates hypoxia inducible transcription factors (HIFs), which play a major role in the hypoxic response of tissues including the retina. We show that a tissue-specific knockdown of von Hippel-Lindau protein (VHL) activated HIF transcription factors in normoxic conditions in the retina. Sustained activation of HIF1 and HIF2 was accompanied by persisting embryonic vasculatures in the posterior eye and the iris. Embryonic vessels persisted into adulthood and led to a severely abnormal mature vessel system with vessels penetrating the photoreceptor layer in adult mice. The sustained hypoxia-like response also activated the leukemia inhibitory factor (LIF)-controlled endogenous molecular cell survival pathway. However, this was not sufficient to protect the retina against massive cell death in all retinal layers of adult mice. Caspases 1, 3 and 8 were upregulated during the degeneration as were several VHL target genes connected to the extracellular matrix. Misregulation of these genes may influence retinal structure and may therefore facilitate growth of vessels into the photoreceptor layer. Thus, an early and sustained activation of a hypoxia-like response in retinal cells leads to abnormal vasculature and severe retinal degeneration in the adult mouse retina.

  19. Doublecortin in Oligodendrocyte Precursor Cells in the Adult Mouse Brain

    PubMed Central

    Boulanger, Jenna J.; Messier, Claude

    2017-01-01

    Key Points Oligodendrocyte precursor cells express doublecortin, a microtubule-associated protein.Oligodendrocyte precursor cells express doublecortin, but at a lower level of expression than in neuronal precursor.Doublecortin is not associated with a potential immature neuronal phenotype in Oligodendrocyte precursor cells. Oligodendrocyte precursor cells (OPC) are glial cells that differentiate into myelinating oligodendrocytes during embryogenesis and early stages of post-natal life. OPCs continue to divide throughout adulthood and some eventually differentiate into oligodendrocytes in response to demyelinating lesions. There is growing evidence that OPCs are also involved in activity-driven de novo myelination of previously unmyelinated axons and myelin remodeling in adulthood. Considering these roles in the adult brain, OPCs are likely mobile cells that can migrate on some distances before they differentiate into myelinating oligodendrocytes. A number of studies have noted that OPCs express doublecortin (DCX), a microtubule-associated protein expressed in neural precursor cells and in migrating immature neurons. Here we describe the distribution of DCX in OPCs. We found that almost all OPCs express DCX, but the level of expression appears to be much lower than what is found in neural precursor. We found that DCX is downregulated when OPCs start expressing mature oligodendrocyte markers and is absent in myelinating oligodendrocytes. DCX does not appear to signal an immature neuronal phenotype in OPCs in the adult mouse brain. Rather, it could be involved either in cell migration, or as a marker of an immature oligodendroglial cell phenotype.

  20. Fate Tracing of neurogenin2-Expressing Cells in the Mouse Retina Using CreER™: LacZ

    PubMed Central

    Ma, Wenxin; Wang, Shu-Zhen

    2012-01-01

    Delineating the final fate of progenitor cells that transiently express a regulatory gene may shed light on how the gene participates in regulating retinal development. We describe the steps in tracing final fates of progenitor cells that once transiently express neurogenin2 (ngn2) during mouse retinal development with the binary, conditional Ngn2-CreER™—LacZ reporter system. Ngn2-CreER™ mice (Zirlinger et al. Proc Natl Acad Sci USA 99:8084–8089, 2002), in which ngn2 promoter drives the expression of Cre-estrogen receptor CreER™ (Littlewood et al. Nuc Acid Res 23:1686–1690, 1995; Hayashi and McMahon Dev Biol 244:305–318, 2002), are crossed with Rosa26-LoxP-LacZ reporter mice (Soriano Nat Genet 21:70–71, 1999), in which the expression of lacZ requires the removal of “stop” by Cre recombinase (Wagner et al. Transgenic Res 10:545–553, 2001). 4-hydroxytamoxifen (4-OHT), a synthetic ligand with high affinity for ER™, is administered to double transgenic embryos and/or neonatal mice. Binding of 4-OHT to Cre-ER™ activates Cre recombinase, which then catalyzes the removal of the “stop” sequence from the LoxP-LacZ transgene, leading to lacZ expression in cells that express ngn2. Retinal tissues are fixed at different time points after 4-OHT treatment and analyzed for LacZ activities by colorimetric reaction. Double-labeling with a cell type-specific marker can be used to define the identity of a LacZ+ cell. Combining persisted lacZ expression through the life of the cell and the short half-life (0.5–2 h) of 4-OHT (Danielian et al. Curr Biol 8:1323–1326, 1998), this system offers the opportunity to track the final fates of cells that have expressed ngn2 during the brief presence of 4-OHT administered during retinal development. PMID:22688703

  1. Temporal order of bipolar cell genesis in the neural retina

    PubMed Central

    Morrow, Eric M; Chen, C-M Amy; Cepko, Constance L

    2008-01-01

    Background Retinal bipolar cells comprise a diverse group of neurons. Cone bipolar cells and rod bipolar cells are so named for their connections with cone and rod photoreceptors, respectively. Morphological criteria have been established that distinguish nine types of cone bipolar cells and one type of rod bipolar cell in mouse and rat. While anatomical and physiological aspects of bipolar types have been actively studied, little is known about the sequence of events that leads to bipolar cell type specification and the potential relationship this process may have with synapse formation in the outer plexiform layer. In this study, we have examined the birth order of rod and cone bipolar cells in the developing mouse and rat in vivo. Results Using retroviral lineage analysis with the histochemical marker alkaline phosphatase, the percentage of cone and rod bipolar cells born on postnatal day 0 (P0), P4, and P6 were determined, based upon the well characterized morphology of these cells in the adult rat retina. In this in vivo experiment, we have demonstrated that cone bipolar genesis clearly precedes rod bipolar genesis. In addition, in the postnatal mouse retina, using a combination of tritiated-thymidine birthdating and immunohistochemistry to distinguish bipolar types, we have similarly found that cone bipolar genesis precedes rod bipolar genesis. The tritiated-thymidine birthdating studies also included quantification of the birth of all postnatally generated retinal cell types in the mouse. Conclusion Using two independent in vivo methodologies in rat and mouse retina, we have demonstrated that there are distinct waves of genesis of the two major bipolar cell types, with cone bipolar genesis preceding rod bipolar genesis. These waves of bipolar genesis correspond to the order of genesis of the presynaptic photoreceptor cell types. PMID:18215319

  2. Infrared retina

    DOEpatents

    Krishna, Sanjay [Albuquerque, NM; Hayat, Majeed M [Albuquerque, NM; Tyo, J Scott [Tucson, AZ; Jang, Woo-Yong [Albuquerque, NM

    2011-12-06

    Exemplary embodiments provide an infrared (IR) retinal system and method for making and using the IR retinal system. The IR retinal system can include adaptive sensor elements, whose properties including, e.g., spectral response, signal-to-noise ratio, polarization, or amplitude can be tailored at pixel level by changing the applied bias voltage across the detector. "Color" imagery can be obtained from the IR retinal system by using a single focal plane array. The IR sensor elements can be spectrally, spatially and temporally adaptive using quantum-confined transitions in nanoscale quantum dots. The IR sensor elements can be used as building blocks of an infrared retina, similar to cones of human retina, and can be designed to work in the long-wave infrared portion of the electromagnetic spectrum ranging from about 8 .mu.m to about 12 .mu.m as well as the mid-wave portion ranging from about 3 .mu.m to about 5 .mu.m.

  3. Notch signaling induces retinal stem-like properties in perinatal neural retina progenitors and promotes symmetric divisions in adult retinal stem cells.

    PubMed

    Balenci, Laurent; van der Kooy, Derek

    2014-02-01

    Understanding the mechanisms regulating retinal stem cell (RSC) activity is fundamental for future stem cell-based therapeutic purposes. By combining gain and loss of function approaches, we addressed whether Notch signaling may play a selective role in retinal stem versus retinal progenitor cells in both developing and adult eyes. Inhibition of either Notch or fibroblast growth factor signaling reduced proliferation of retinal stem and retinal progenitor cells, and inhibited RSC self-renewal. Conversely, exogenous Delta-like 3 and direct intrinsic Notch activation stimulated expansionary symmetric divisions in adult RSCs with the concomitant upregulation of Hes5. Knocking down Hes5 expression specifically decreased the numbers, but not the diameters, of adult RSC primary spheres, indicating that HES5 is the downstream effector of Notch receptor in controlling adult RSC proliferation. In addition, constitutive Notch activation induced retinal stem-like asymmetric self-renewal properties, with no expansion (no symmetrical division) in perinatal neural retina progenitor cells. These findings highlight central roles of Notch signaling activity in regulating the modes of division of retinal stem and retinal progenitor cells.

  4. Traumatic Brain Injury Severity Affects Neurogenesis in Adult Mouse Hippocampus.

    PubMed

    Wang, Xiaoting; Gao, Xiang; Michalski, Stephanie; Zhao, Shu; Chen, Jinhui

    2016-04-15

    Traumatic brain injury (TBI) has been proven to enhance neural stem cell (NSC) proliferation in the hippocampal dentate gyrus. However, various groups have reported contradictory results on whether TBI increases neurogenesis, partially due to a wide range in the severities of injuries seen with different TBI models. To address whether the severity of TBI affects neurogenesis in the injured brain, we assessed neurogenesis in mouse brains receiving different severities of controlled cortical impact (CCI) with the same injury device. The mice were subjected to mild, moderate, or severe TBI by a CCI device. The effects of TBI severity on neurogenesis were evaluated at three stages: NSC proliferation, immature neurons, and newly-generated mature neurons. The results showed that mild TBI did not affect neurogenesis at any of the three stages. Moderate TBI promoted NSC proliferation without increasing neurogenesis. Severe TBI increased neurogenesis at all three stages. Our data suggest that the severity of injury affects adult neurogenesis in the hippocampus, and thus it may partially explain the inconsistent results of different groups regarding neurogenesis following TBI. Further understanding the mechanism of TBI-induced neurogenesis may provide a potential approach for using endogenous NSCs to protect against neuronal loss after trauma.

  5. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer

    PubMed Central

    Ou, Jingxing; Vijayasarathy, Camasamudram; Ziccardi, Lucia; Chen, Shan; Zeng, Yong; Marangoni, Dario; Pope, Jodie G.; Bush, Ronald A.; Wu, Zhijian; Li, Wei; Sieving, Paul A.

    2015-01-01

    Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor–depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1–signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology. PMID:26098217

  6. Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina

    PubMed Central

    Meyer, Arndt; Tetenborg, Stephan; Greb, Helena; Segelken, Jasmin; Dorgau, Birthe; Weiler, Reto; Hormuzdi, Sheriar G.; Janssen-Bienhold, Ulrike; Dedek, Karin

    2016-01-01

    Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36—both expressed in AII amacrine cells—are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners. PMID:27303262

  7. Cykotine mRNA expression in mouse retina after laser injury by reverse transcriptase-polymerase chain reaction (RT-PCR)

    NASA Astrophysics Data System (ADS)

    Schuschereba, Steven T.; Bowman, Phillip D.; Ujimore, Veronica; Hoxie, Stephen W.; Pizarro, Jose M.; Cross, Michael E.; Lund, David J.

    1996-04-01

    The purpose of this study was to identify cytokines produced by the retina after laser injury. With the aid of a scanning laser ophthalmoscope (SLO), right eyes of mice received lesions from a continuous wave argon laser. Left eyes served as unirradiated controls. At 2, 4, 6, 12, 24, and 48 hr after laser irradiation groups of 3 mice were euthanized and retinas fixed for histology or isolated for RNA. Messenger RNA (mRNA) was reverse-transcribed into complementary DNA (cDNA) and subjected to polymerase chain reaction for the following cytokines: tumor necrosis factor-(alpha) (TNF-(alpha) ), interleukin-1(alpha) /(Beta) (IL- 1(alpha) /(Beta) ), interleukin-6 (IL-6), transforming growth factor-(Beta) 1 (TGF- (Beta) 1), macrophage colony stimulating factor (M-CSF), inducible nitric oxide synthase (iNOS), and glyceraldehyde 3-phosphate dehydrogenase (G3PDH). Histologically, lesions were confined to the photoreceptors, retinal pigment epithelium, and choroid. In laser-injured retinas, mRNA levels were elevated for IL-1(alpha) , TGF-(Beta) 1, iNOS, and G3PDH, but not TNF-(alpha) , IL-1(Beta) , or IL-6. It appears that the retina, in response to laser injury, upregulates a select number of cytokines in a time-course dependent fashion.

  8. Alzheimer’s Disease-Related Protein Expression in the Retina of Octodon degus

    PubMed Central

    Du, Lucia Y.; Chang, Lily Y-L.; Ardiles, Alvaro O.; Tapia-Rojas, Cheril; Araya, Joaquin; Inestrosa, Nibaldo C.

    2015-01-01

    New studies show that the retina also undergoes pathological changes during the development of Alzheimer’s disease (AD). While transgenic mouse models used in these previous studies have offered insight into this phenomenon, they do not model human sporadic AD, which is the most common form. Recently, the Octodon degus has been established as a sporadic model of AD. Degus display age-related cognitive impairment associated with Aβ aggregates and phosphorylated tau in the brain. Our aim for this study was to examine the expression of AD-related proteins in young, adult and old degus retina using enzyme-linked or fluorescence immunohistochemistry and to quantify the expression using slot blot and western blot assays. Aβ4G8 and Aβ6E10 detected Aβ peptides in some of the young animals but the expression was higher in the adults. Aβ peptides were observed in the inner and outer segment of the photoreceptors, the nerve fiber layer (NFL) and ganglion cell layer (GCL). Expression was higher in the central retinal region than in the retinal periphery. Using an anti-oligomer antibody we detected Aβ oligomer expression in the young, adult and old retina. Immunohistochemical labeling showed small discrete labeling of oligomers in the GCL that did not resemble plaques. Congo red staining did not result in green birefringence in any of the animals analyzed except for one old (84 months) animal. We also investigated expression of tau and phosphorylated tau. Expression was seen at all ages studied and in adults it was more consistently observed in the NFL-GCL. Hyperphosphorylated tau detected with AT8 antibody was significantly higher in the adult retina and it was localized to the GCL. We confirm for the first time that Aβ peptides and phosphorylated tau are expressed in the retina of degus. This is consistent with the proposal that AD biomarkers are present in the eye. PMID:26267479

  9. 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.

  10. Cancers Affecting the Retina

    MedlinePlus

    ... or ARMD) Epiretinal Membrane Detachment of the Retina Retinitis Pigmentosa Blockage of Central Retinal Veins and Branch Retinal ... or ARMD) Epiretinal Membrane Detachment of the Retina Retinitis Pigmentosa Blockage of Central Retinal Veins and Branch Retinal ...

  11. Sox2 and JAGGED1 expression in normal and drug-damaged adult mouse inner ear.

    PubMed

    Oesterle, Elizabeth C; Campbell, Sean; Taylor, Ruth R; Forge, Andrew; Hume, Clifford R

    2008-03-01

    Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.

  12. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    SciTech Connect

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  13. Photoreceptor organisation and phenotypic characterization in retinas of two diurnal rodent species: potential use as experimental animal models for human vision research.

    PubMed

    Bobu, Corina; Lahmam, Mohamed; Vuillez, Patrick; Ouarour, Ali; Hicks, David

    2008-02-01

    To characterize rod and cone distribution and composition in two diurnal mouse-like rodents, retinas from adult Arvicanthis ansorgei and Lemniscomys barbarus were processed for immunohistochemistry using multiple rod- and cone-specific antibodies. Antibodies tested included rhodopsin, cone opsins, pan-arrestin and cone arrestin, recoverin, and cGMP dependent ion channel. In both species, retinas were composed of approximately 33% cones, and most antibodies gave similar staining patterns. Data show these two diurnal rodents possess large numbers of cones, organised in a strict anatomical array. This suggests that diurnal rodents in general possess elevated cone numbers and could constitute valuable models for investigating cone pathophysiology.

  14. Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

    PubMed

    Weinl, Christine; Wasylyk, Christine; Garcia Garrido, Marina; Sothilingam, Vithiyanjali; Beck, Susanne C; Riehle, Heidemarie; Stritt, Christine; Roux, Michel J; Seeliger, Mathias W; Wasylyk, Bohdan; Nordheim, Alfred

    2014-01-01

    Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

  15. A comprehensive transcriptomic analysis of infant and adult mouse ovary.

    PubMed

    Pan, Linlin; Gong, Wei; Zhou, Yuanyuan; Li, Xiaonuan; Yu, Jun; Hu, Songnian

    2014-10-01

    Ovary development is a complex process involving numerous genes. A well-developed ovary is essential for females to keep fertility and reproduce offspring. In order to gain a better insight into the molecular mechanisms related to the process of mammalian ovary development, we performed a comparative transcriptomic analysis on ovaries isolated from infant and adult mice by using next-generation sequencing technology (SOLiD). We identified 15,454 and 16,646 transcriptionally active genes at the infant and adult stage, respectively. Among these genes, we also identified 7021 differentially expressed genes. Our analysis suggests that, in general, the adult ovary has a higher level of transcriptomic activity. However, it appears that genes related to primordial follicle development, such as those encoding Figla and Nobox, are more active in the infant ovary, whereas expression of genes vital for follicle development, such as Gdf9, Bmp4 and Bmp15, is upregulated in the adult. These data suggest a dynamic shift in gene expression during ovary development and it is apparent that these changes function to facilitate follicle maturation, when additional functional gene studies are considered. Furthermore, our investigation has also revealed several important functional pathways, such as apoptosis, MAPK and steroid biosynthesis, that appear to be much more active in the adult ovary compared to those of the infant. These findings will provide a solid foundation for future studies on ovary development in mice and other mammals and help to expand our understanding of the complex molecular and cellular events that occur during postnatal ovary development.

  16. Ascl3 marks adult progenitor cells of the mouse salivary gland

    PubMed Central

    Rugel-Stahl, Anastasia; Elliot, Marilyn; Ovitt, Catherine E.

    2012-01-01

    The Ascl3 transcription factor marks a subset of salivary gland duct cells present in the three major salivary glands of the mouse. In vivo, these cells generate both duct and secretory acinar cell descendants. Here, we have analyzed whether Ascl3-expressing cells retain this multipotent lineage potential in adult glands. Cells isolated from mouse salivary glands were cultured in vitro as non-adherent spheres. Lineage tracing of the Ascl3-expressing cells within the spheres demonstrates that Ascl3+ cells isolated from adult glands remain multipotent, generating both duct and acinar cell types in vitro. Furthermore, we demonstrate that the progenitor cells characterized by Keratin 5 expression are an independent population from Ascl3+ progenitor cells. We conclude that the Ascl3+ cells are intermediate lineage-restricted progenitor cells of the adult salivary glands. PMID:22370009

  17. Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues.

    PubMed Central

    Hooper, John D; Campagnolo, Luisa; Goodarzi, Goodarz; Truong, Tony N; Stuhlmann, Heidi; Quigley, James P

    2003-01-01

    We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial

  18. Fluoxetine increases plasticity and modulates the proteomic profile in the adult mouse visual cortex

    PubMed Central

    Ruiz-Perera, L.; Muniz, M.; Vierci, G.; Bornia, N.; Baroncelli, L.; Sale, A.; Rossi, F.M.

    2015-01-01

    The scarce functional recovery of the adult CNS following injuries or diseases is largely due to its reduced potential for plasticity, the ability to reorganize neural connections as a function of experience. Recently, some new strategies restoring high levels of plasticity in the adult brain have been identified, especially in the paradigmatic model of the visual system. A chronic treatment with the anti-depressant fluoxetine reinstates plasticity in the adult rat primary visual cortex, inducing recovery of vision in amblyopic animals. The molecular mechanisms underlying this effect remain largely unknown. Here, we explored fluoxetine effects on mouse visual cortical plasticity, and exploited a proteomic approach to identify possible candidates mediating the outcome of the antidepressant treatment on adult cortical plasticity. We showed that fluoxetine restores ocular dominance plasticity in the adult mouse visual cortex, and identified 31 differentially expressed protein spots in fluoxetine-treated animals vs. controls. MALDITOF/TOF mass spectrometry identification followed by bioinformatics analysis revealed that these proteins are involved in the control of cytoskeleton organization, endocytosis, molecular transport, intracellular signaling, redox cellular state, metabolism and protein degradation. Altogether, these results indicate a complex effect of fluoxetine on neuronal signaling mechanisms potentially involved in restoring plasticity in the adult brain. PMID:26205348

  19. Isolation and cultivation of stem cells from adult mouse testes.

    PubMed

    Guan, Kaomei; Wolf, Frieder; Becker, Alexander; Engel, Wolfgang; Nayernia, Karim; Hasenfuss, Gerd

    2009-01-01

    The successful isolation and cultivation of spermatogonial stem cells (SSCs) as well as induction of SSCs into pluripotent stem cells will allow us to study their biological characteristics and their applications in therapeutic approaches. Here we provide step-by-step procedures on the basis of previous work in our laboratory for: the isolation of testicular cells from adolescent mice by a modified enzymatic procedure; the enrichment of undifferentiated spermatogonia by laminin selection or genetic selection using Stra8-EGFP (enhanced green fluorescent protein) transgenic mice; the cultivation and conversion of undifferentiated spermatogonia into embryonic stem-like cells, so-called multipotent adult germline stem cells (maGSCs); and characterization of these cells. Normally, it will take about 16 weeks to obtain stable maGSC lines starting from the isolation of testicular cells.

  20. Adult Mouse Cortical Cell Taxonomy by Single Cell Transcriptomics

    PubMed Central

    Tasic, Bosiljka; Menon, Vilas; Nguyen, Thuc Nghi; Kim, Tae Kyung; Jarsky, Tim; Yao, Zizhen; Levi, Boaz; Gray, Lucas T.; Sorensen, Staci A.; Dolbeare, Tim; Bertagnolli, Darren; Goldy, Jeff; Shapovalova, Nadiya; Parry, Sheana; Lee, Changkyu; Smith, Kimberly; Bernard, Amy; Madisen, Linda; Sunkin, Susan M.; Hawrylycz, Michael; Koch, Christof; Zeng, Hongkui

    2016-01-01

    Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties. PMID:26727548

  1. Neurotransmitter properties of the newborn human retina

    SciTech Connect

    Hollyfield, J.G.; Frederick, J.M.; Rayborn, M.E.

    1983-07-01

    Human retinal tissue from a newborn was examined autoradiographically for the presence of high-affinity uptake and localization of the following putative neurotransmitters: dopamine, glycine, GABA, aspartate, and glutamate. In addition, the dopamine content of this newborn retina was measured by high pressure liquid chromatography. Our study reveals that specific uptake mechanisms for /sup 3/H-glycine, /sup 3/H-dopamine, and /sup 3/H-GABA are present at birth. However, the number and distribution of cells labeled with each of these /sup 3/H-transmitters are not identical to those observed in adult human retinas. Furthermore, the amount of endogenous dopamine in the newborn retina is approximately 1/20 the adult level. Photoreceptor-specific uptake of /sup 3/H-glutamate and /sup 3/H-aspartate are not observed. These findings indicate that, while some neurotransmitter-specific properties are present at birth, significant maturation of neurotransmitter systems occurs postnatally.

  2. Metabolic conversion of 12-O-tetradecanoylphorbol-13-acetate in adult and newborn mouse skin and mouse liver microsomes.

    PubMed

    Berry, D L; Bracken, W M; Fischer, S M; Viaje, A; Slaga, T J

    1978-08-01

    Tritiated 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to adult mouse skin; at specified time intervals the mice were killed, and the labeled phorbol was extracted and subjected to separation and quantitation by high-pressure liquid chromatography. After 24 hr, TPA comprised greater than 96% of the recovered label from the skin, and its apparent half-life was 17.8 hr. Pretreatment of adult skin with TPA for 4 weeks before treatment with labeled TPA resulted in an increase in the clearance rate of TPA from the skin. Skin from newborn mice was capable of converting TPA into monoesters and phorbol, but the clearance rate in the adult was about 12 times more rapid than it was in the newborn. Epidermal homogenates converted TPA into 12-O-tetradecanoylphorbol, phorbol-13-acetate, and phorbol. Hepatic homogenates were able to convert TPA to monoesters and phorbol at rates 14 to 15 times faster than were epidermal homogenates. Attempts to isolate any previously undescribed metabolites of TPA by use of liver homogenates were unsuccessful, and mixed-function oxidation did not contribute to the metabolism of TPA. From inhibitor studies it was judged that esterases were implicated in the conversion of TPA to monoesters and phorbol. The results support the hypothesis that the tumor-promoting activity of TPA is directly related to its concentration in a specific tissue and that conversion of TPA to an active metabolite probably does not occur.

  3. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.

    PubMed

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L; Polishchuk, Roman S; Auricchio, Alberto

    2015-12-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1.

  4. Expression and functions of ASIC1 in the zebrafish retina.

    PubMed

    Liu, Sha; Wang, Mei-Xia; Mao, Cheng-Jie; Cheng, Xiao-Yu; Wang, Chen-Tao; Huang, Jian; Zhong, Zhao-Min; Hu, Wei-Dong; Wang, Fen; Hu, Li-Fang; Wang, Han; Liu, Chun-Feng

    2014-12-12

    It has been demonstrated that acid sensing ionic channels (ASICs) are present in the central and peripheral nervous system of mammals, including the retina. However, it remains unclear whether the zebrafish retina also expresses ASICs. In the present study, the expression and distribution of zasic1 were examined in the retina of zebrafish. Both zasic1 mRNA and protein expressions were detected in the adult zebrafish retina. A wide distribution of ASIC1 in zebrafish retina was confirmed using whole mount in situ hybridization and immunohistochemistry study. Acidosis-induced currents in the isolated retinal ganglion cells (RGCs) were also recorded using whole cell patch clamping. Moreover, blockade of ASICs channel significantly reduced the locomotion of larval zebrafish in response to light exposure. In sum, our data demonstrate the presence of ASIC1 and its possible functional relevance in the retina of zebrafish.

  5. Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers.

    PubMed

    Guerrero-Bosagna, Carlos; Covert, Trevor R; Haque, Md M; Settles, Matthew; Nilsson, Eric E; Anway, Matthew D; Skinner, Michael K

    2012-12-01

    The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease.

  6. In Vivo Multimodal Imaging of NF-kappaB (pB) Spatial and Temporal Activation Following Light Injury in the Mouse Retina

    DTIC Science & Technology

    2014-01-15

    wet age-related macular degeneration (AMD), diabetic retinopathy, and other retinal disorders.1 There is a strong desire to find strate- gies to...the development of diabetic retinopathy.78 5 Conclusion Overall, we have introduced the cis-NF-κB-EGFP reporter mouse for in vivo studies of NF-κB...Predictive factors for visual acuity after intravitreal triamcinolone treatment for diabetic macular edema. Arch Ophthalmol 123, 1338–1343 (2005). 7

  7. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease

    PubMed Central

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V.; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L.; Polishchuk, Roman S.; Auricchio, Alberto

    2015-01-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4−/− mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5′-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4−/− mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. PMID:26420842

  8. Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

    PubMed

    Mollema, Nissa J; Yuan, Yang; Jelcick, Austin S; Sachs, Andrew J; von Alpen, Désirée; Schorderet, Daniel; Escher, Pascal; Haider, Neena B

    2011-03-08

    The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

  9. Receptor protein tyrosine phosphatase σ binds to neurons in the adult mouse brain

    PubMed Central

    Yi, Jae-Hyuk; Katagiri, Yasuhiro; Yu, Panpan; Lourie, Jacob; Bangayan, Nathanael J.; Symes, Aviva J.; Geller, Herbert M.

    2014-01-01

    The role of type IIA receptor protein tyrosine phosphatases (RPTPs), which includes LAR, RPTPσ and RPTPδ, in the nervous system is becoming increasingly recognized. Evidence supports a significant role for these RPTPs during the development of the nervous system as well as after injury, and mutations in RPTPs are associated with human disease. However, a major open question is the nature of the ligands that interact with type IIA RPTPs in the adult brain. Candidates include several different proteins as well as the glycosaminoglycan chains of proteoglycans. In order to investigate this problem, we used a receptor affinity probe assay with RPTPσ-AP fusion proteins on sections of adult mouse brain and to cultured neurons. Our results demonstrate that the major binding sites for RPTPσ in adult mouse brain are on neurons and are not proteoglycan GAG chains, as RPTPσ binding overlaps with the neuronal marker NeuN and was not significantly altered by treatments which eliminate chondroitin sulfate, heparan sulfate, or both. We also demonstrate no overlap of binding of RPTPσ with perineuronal nets, and a unique modulation of RPTPσ binding to brain by divalent cations. Our data therefore point to neuronal proteins, rather than CSPGs, as being the ligands for RPTPσ in the adult, uninjured brain. PMID:24530640

  10. Helping the Retina Regenerate

    MedlinePlus

    ... for RGC reprogramming is understanding the cues that direct their maturation and integration with other cells. The ... the retina. The report appears in Translational Vision Science and Technology. Learn more about the NEI AGI ...

  11. Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus.

    PubMed

    van Praag, H; Kempermann, G; Gage, F H

    1999-03-01

    Exposure to an enriched environment increases neurogenesis in the dentate gyrus of adult rodents. Environmental enrichment, however, typically consists of many components, such as expanded learning opportunities, increased social interaction, more physical activity and larger housing. We attempted to separate components by assigning adult mice to various conditions: water-maze learning (learner), swim-time-yoked control (swimmer), voluntary wheel running (runner), and enriched (enriched) and standard housing (control) groups. Neither maze training nor yoked swimming had any effect on bromodeoxyuridine (BrdU)-positive cell number. However, running doubled the number of surviving newborn cells, in amounts similar to enrichment conditions. Our findings demonstrate that voluntary exercise is sufficient for enhanced neurogenesis in the adult mouse dentate gyrus.

  12. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system

    PubMed Central

    Codeluppi, Simone; van Bruggen, David; Mendanha Falcão, Ana; Xiao, Lin; Li, Huiliang; Häring, Martin; Hochgerner, Hannah; Romanov, Roman A.; Gyllborg, Daniel; Muñoz Manchado, Ana; La Manno, Gioele; Lönnerberg, Peter; Floriddia, Elisa M.; Rezayee, Fatemah; Ernfors, Patrik; Arenas, Ernest; Hjerling-Leffler, Jens; Harkany, Tibor; Richardson, William D.; Linnarsson, Sten; Castelo-Branco, Gonçalo

    2016-01-01

    Oligodendrocytes have been considered as a functionally homogenous population in the central nervous system (CNS). We performed single-cell RNA-Seq on 5072 cells of the oligodendrocyte lineage from ten regions of the mouse juvenile/adult CNS. Twelve populations were identified, representing a continuum from Pdgfra+ oligodendrocyte precursors (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly-formed oligodendrocytes were found to be resident in the adult CNS and responsive to complex motor learning. A second Pdgfra+ population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS. PMID:27284195

  13. A case of adult cannibalism in the gray mouse lemur, Microcebus murinus.

    PubMed

    Hämäläinen, Anni

    2012-09-01

    Cannibalism, defined as the eating of conspecific flesh, has been observed in a number of primate species, although it is still a relatively rare phenomenon. In cases where primates were seen feeding on an individual of the same species, the victims have exclusively been infants or juveniles. Here, I report an event of a free-living, adult male gray mouse lemur, Microcebus murinus, cannibalizing an adult conspecific female that died of an unknown cause. This observation has implications for the basic ecology of the species and highlights the potential for great flexibility in diet and behavior by a primate. This is, to my knowledge, the first communication of cannibalistic behavior in this species, as well as the first reported case of a nonhuman primate cannibalizing an adult conspecific.

  14. Rapid and efficient gene delivery into the adult mouse brain via focal electroporation

    PubMed Central

    Nomura, Tadashi; Nishimura, Yusuke; Gotoh, Hitoshi; Ono, Katsuhiko

    2016-01-01

    In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner. PMID:27430903

  15. Histology and Ultrastructure of Transitional Changes in Skin Morphology in the Juvenile and Adult Four-Striped Mouse (Rhabdomys pumilio)

    PubMed Central

    Stewart, Eranée; Ajao, Moyosore Salihu

    2013-01-01

    The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin. PMID:24288469

  16. Histology and ultrastructure of transitional changes in skin morphology in the juvenile and adult four-striped mouse (Rhabdomys pumilio).

    PubMed

    Stewart, Eranée; Ajao, Moyosore Salihu; Ihunwo, Amadi Ogonda

    2013-01-01

    The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin.

  17. Cranial irradiation induces bone marrow-derived microglia in adult mouse brain tissue.

    PubMed

    Okonogi, Noriyuki; Nakamura, Kazuhiro; Suzuki, Yoshiyuki; Suto, Nana; Suzue, Kazutomo; Kaminuma, Takuya; Nakano, Takashi; Hirai, Hirokazu

    2014-07-01

    Postnatal hematopoietic progenitor cells do not contribute to microglial homeostasis in adult mice under normal conditions. However, previous studies using whole-body irradiation and bone marrow (BM) transplantation models have shown that adult BM cells migrate into the brain tissue and differentiate into microglia (BM-derived microglia; BMDM). Here, we investigated whether cranial irradiation alone was sufficient to induce the generation of BMDM in the adult mouse brain. Transgenic mice that express green fluorescent protein (GFP) under the control of a murine stem cell virus (MSCV) promoter (MSCV-GFP mice) were used. MSCV-GFP mice express GFP in BM cells but not in the resident microglia in the brain. Therefore, these mice allowed us to detect BM-derived cells in the brain without BM reconstitution. MSCV-GFP mice, aged 8-12 weeks, received 13.0 Gy irradiation only to the cranium, and BM-derived cells in the brain were quantified at 3 and 8 weeks after irradiation. No BM-derived cells were detected in control non-irradiated MSCV-GFP mouse brains, but numerous GFP-labeled BM-derived cells were present in the brain stem, basal ganglia and cerebral cortex of the irradiated MSCV-GFP mice. These BM-derived cells were positive for Iba1, a marker for microglia, indicating that GFP-positive BM-derived cells were microglial in nature. The population of BMDM was significantly greater at 8 weeks post-irradiation than at 3 weeks post-irradiation in all brain regions examined. Our results clearly show that cranial irradiation alone is sufficient to induce the generation of BMDM in the adult mouse.

  18. Transcriptomic analysis of the developing and adult mouse cochlear sensory epithelia.

    PubMed

    Smeti, Ibtihel; Assou, Said; Savary, Etienne; Masmoudi, Saber; Zine, Azel

    2012-01-01

    The adult mammalian cochlea lacks regenerative ability and the irreversible degeneration of cochlear sensory hair cells leads to permanent hearing loss. Previous data show that early postnatal cochlea harbors stem/progenitor-like cells and shows a limited regenerative/repair capacity. These properties are progressively lost later during the postnatal development. Little is known about the genes and pathways that are potentially involved in this difference of the regenerative/repair potentialities between early postnatal and adult mammalian cochlear sensory epithelia (CSE). The goal of our study is to investigate the transcriptomic profiles of these two stages. We used Mouse Genome 430 2.0 microarray to perform an extensive analysis of the genes expressed in mouse postnatal day-3 (P3) and adult CSE. Statistical analysis of microarray data was performed using SAM (Significance Analysis of Microarrays) software. We identified 5644 statistically significant differentially expressed transcripts with a fold change (FC) >2 and a False Discovery Rate (FDR) ≤0.05. The P3 CSE signature included 3,102 transcripts, among which were known genes in the cochlea, but also new transcripts such as, Hmga2 (high mobility group AT-hook 2) and Nrarp (Notch-regulated ankyrin repeat protein). The adult CSE overexpressed 2,542 transcripts including new transcripts, such as Prl (Prolactin) and Ar (Androgen receptor), that previously were not known to be expressed in the adult cochlea. Our comparative study revealed important genes and pathways differentially expressed between the developing and adult CSE. The identification of new candidate genes would be useful as potential markers of the maintenance or the loss of stem cells and regenerative/repair ability during mammalian cochlear development.

  19. Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

    PubMed Central

    Monteiro, Ana; Milne, Laura; Cruickshanks, Lyndsey; Jeffrey, Nathan; Guillou, Florian; Freeman, Tom C.; Mitchell, Rod T.; Smith, Lee B.

    2014-01-01

    The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health. PMID:25144714

  20. Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation

    PubMed Central

    Korogod, Natalya; Petersen, Carl CH; Knott, Graham W

    2015-01-01

    Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions. DOI: http://dx.doi.org/10.7554/eLife.05793.001 PMID:26259873

  1. Differential regulation of laminin b1 transgene expression in the neonatal and adult mouse brain.

    PubMed

    Sharif, K A; Baker, H; Gudas, L J

    2004-01-01

    Laminins are the major glycoproteins present in basement membrane, a type of extracellular matrix. We showed that the LAMB1 gene, which encodes the laminin beta1 subunit, is transcriptionally activated by retinoic acid in embryonic stem cells. However, little information is available concerning LAMB1 developmental regulation and spatial expression in the adult mouse brain. In this study we used transgenic mice expressing different lengths of LAMB1 promoter driving beta-galactosidase to investigate developmental and adult transcriptional regulation in the regions of the brain in which the laminin beta1 protein is expressed. CNS expression was not observed in transgenic mice carrying a 1.4LAMB1betagal construct. Mice carrying a 2.5LAMB1betagal construct expressed the LAMB1 transgene, as assayed by X-gal staining, only in the molecular layer of the neonatal cerebellum. In contrast, a 3.9LAMB1betagal transgene showed broad regional expression in the adult mouse brain, including the hippocampus, entorhinal cortex, colliculi, striatum, and substantia nigra. Similar expression patterns were observed for the endogenous laminin beta1 protein and for the 3.9LAMB1betagal transgene, analyzed with an antibody against the beta-galactosidase protein. The 3.9LAMB1betagal transgene expression in the hippocampal tri-synaptic circuit suggests a role for the LAMB1 gene in learning and memory.

  2. A novel mouse model that recapitulates adult-onset glycogenosis type 4

    PubMed Central

    Orhan Akman, H.; Emmanuele, Valentina; Kurt, Yasemin Gülcan; Kurt, Bülent; Sheiko, Tatiana; DiMauro, Salvatore; Craigen, William J.

    2015-01-01

    Glycogen storage disease type IV (GSD IV) is a rare autosomal recessive disorder caused by deficiency of the glycogen-branching enzyme (GBE). The diagnostic hallmark of the disease is the accumulation of a poorly branched form of glycogen known as polyglucosan (PG). The disease is clinically heterogeneous, with variable tissue involvement and age at onset. Complete loss of enzyme activity is lethal in utero or in infancy and affects primarily the muscle and the liver. However, residual enzyme activity as low as 5–20% leads to juvenile or adult onset of a disorder that primarily affects the central and peripheral nervous system and muscles and in the latter is termed adult polyglucosan body disease (APBD). Here, we describe a mouse model of GSD IV that reflects this spectrum of disease. Homologous recombination was used to knock in the most common GBE1 mutation p.Y329S c.986A > C found in APBD patients of Ashkenazi Jewish decent. Mice homozygous for this allele (Gbe1ys/ys) exhibit a phenotype similar to APBD, with widespread accumulation of PG. Adult mice exhibit progressive neuromuscular dysfunction and die prematurely. While the onset of symptoms is limited to adult mice, PG accumulates in tissues of newborn mice but is initially absent from the cerebral cortex and heart muscle. Thus, PG is well tolerated in most tissues, but the eventual accumulation in neurons and their axons causes neuropathy that leads to hind limb spasticity and premature death. This mouse model mimics the pathology and pathophysiologic features of human adult-onset branching enzyme deficiency. PMID:26385640

  3. Survival of glucose phosphate isomerase null somatic cells and germ cells in adult mouse chimaeras.

    PubMed

    Keighren, Margaret A; Flockhart, Jean H; West, John D

    2016-05-15

    The mouse Gpi1 gene encodes the glycolytic enzyme glucose phosphate isomerase. Homozygous Gpi1(-/-) null mouse embryos die but a previous study showed that some homozygous Gpi1(-/-) null cells survived when combined with wild-type cells in fetal chimaeras. One adult female Gpi1(-/-)↔Gpi1(c/c) chimaera with functional Gpi1(-/-) null oocytes was also identified in a preliminary study. The aims were to characterise the survival of Gpi1(-/-) null cells in adult Gpi1(-/-)↔Gpi1(c/c) chimaeras and determine if Gpi1(-/-) null germ cells are functional. Analysis of adult Gpi1(-/-)↔Gpi1(c/c) chimaeras with pigment and a reiterated transgenic lineage marker showed that low numbers of homozygous Gpi1(-/-) null cells could survive in many tissues of adult chimaeras, including oocytes. Breeding experiments confirmed that Gpi1(-/-) null oocytes in one female Gpi1(-/-)↔Gpi1(c/c) chimaera were functional and provided preliminary evidence that one male putative Gpi1(-/-)↔Gpi1(c/c) chimaera produced functional spermatozoa from homozygous Gpi1(-/-) null germ cells. Although the male chimaera was almost certainly Gpi1(-/-)↔Gpi1(c/c), this part of the study is considered preliminary because only blood was typed for GPI. Gpi1(-/-) null germ cells should survive in a chimaeric testis if they are supported by wild-type Sertoli cells. It is also feasible that spermatozoa could bypass a block at GPI, but not blocks at some later steps in glycolysis, by using fructose, rather than glucose, as the substrate for glycolysis. Although chimaera analysis proved inefficient for studying the fate of Gpi1(-/-) null germ cells, it successfully identified functional Gpi1(-/-) null oocytes and revealed that some Gpi1(-/-) null cells could survive in many adult tissues.

  4. Human tau expression reduces adult neurogenesis in a mouse model of tauopathy.

    PubMed

    Komuro, Yutaro; Xu, Guixiang; Bhaskar, Kiran; Lamb, Bruce T

    2015-06-01

    Accumulation of hyperphosphorylated and aggregated microtubule-associated protein tau (MAPT) is a central feature of a class of neurodegenerative diseases termed tauopathies. Notably, there is increasing evidence that tauopathies, including Alzheimer's disease, are also characterized by a reduction in neurogenesis, the birth of adult neurons. However, the exact relationship between hyperphosphorylation and aggregation of MAPT and neurogenic deficits remains unclear, including whether this is an early- or late-stage disease marker. In the present study, we used the genomic-based hTau mouse model of tauopathy to examine the temporal and spatial regulation of adult neurogenesis during the course of the disease. Surprisingly, hTau mice exhibited reductions in adult neurogenesis in 2 different brain regions by as early as 2 months of age, before the development of robust MAPT pathology in this model. This reduction was found to be due to reduced proliferation and not because of enhanced apoptosis in the hippocampus. At these same time points, hTau mice also exhibited altered MAPT phosphorylation with neurogenic precursors. To examine whether the effects of MAPT on neurogenesis were cell autonomous, neurospheres prepared from hTau animals were examined in vitro, revealing a growth deficit when compared with non-transgenic neurosphere cultures. Taken together, these studies provide evidence that altered adult neurogenesis is a robust and early marker of altered, cell-autonomous function of MAPT in the hTau mouse mode of tauopathy and that altered adult neurogenesis should be examined as a potential marker and therapeutic target for human tauopathies.

  5. A detailed characterization of the adult mouse model of glycogen storage disease Ia.

    PubMed

    Salganik, Susan V; Weinstein, David A; Shupe, Thomas D; Salganik, Max; Pintilie, Dana G; Petersen, Bryon E

    2009-09-01

    Glycogen storage disease type Ia (GSDIa) is caused by a genetic defect in the hepatic enzyme glucose-6-phosphatase (G6Pase-alpha), which manifests as life-threatening hypoglycemia with related metabolic complications. A G6Pase-alpha knockout (KO) mouse model was generated to study potential therapies for correcting this disorder. Since then, gene therapy studies have produced promising results, showing long-term improvement in liver histology and glycogen metabolism. Under existing protocols, however, untreated KO pups seldom survived weaning. Here, we present a thorough characterization of the G6Pase-alpha KO mouse, as well as the husbandry protocol for rearing this strain to adulthood. These mice were raised with only palliative care, and characterized from birth through 6 months of age. Once KO mice have survived the very frail weaning period, their size, agility, serum lipids and glycemic control improve dramatically, reaching levels approaching their wild-type littermates. In addition, our data reveal that adult mice lacking G6Pase-alpha are able to mate and produce viable offspring. However, liver histology and glycogen accumulation do not improve with age. Overall, the reliable production of mature KO mice could provide a critical tool for advancing the GSDIa field, as the availability of a robust enzyme-deficient adult offers a new spectrum of treatment avenues that would not be tolerated by the frail pups. Most importantly, our detailed characterization of the adult KO mouse provides a crucial baseline for accurately gauging the efficacy of experimental therapies in this important model.

  6. In vivo Editing of the Human Mutant Rhodopsin Gene by Electroporation of Plasmid-based CRISPR/Cas9 in the Mouse Retina

    PubMed Central

    Latella, Maria Carmela; Di Salvo, Maria Teresa; Cocchiarella, Fabienne; Benati, Daniela; Grisendi, Giulia; Comitato, Antonella; Marigo, Valeria; Recchia, Alessandra

    2016-01-01

    The bacterial CRISPR/Cas system has proven to be an efficient tool for genetic manipulation in various organisms. Here we show the application of CRISPR-Cas9 technology to edit the human Rhodopsin (RHO) gene in a mouse model for autosomal dominant Retinitis Pigmentosa. We designed single or double sgRNAs to knock-down mutant RHO expression by targeting exon 1 of the RHO gene carrying the P23H dominant mutation. By delivering Cas9 and sgRNAs in a single plasmid we induced an efficient gene editing in vitro, in HeLa cells engineered to constitutively express the P23H mutant RHO allele. Similarly, after subretinal electroporation of the CRISPR/Cas9 plasmid expressing two sgRNAs into P23H RHO transgenic mice, we scored specific gene editing as well as significant reduction of the mutant RHO protein. Successful in vivo application of the CRISPR/Cas9 system confirms its efficacy as a genetic engineering tool in photoreceptor cells. PMID:27874856

  7. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    SciTech Connect

    Sun, Li; Wu, Zhou; Baba, Masashi; Peters, Christoph; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2010-08-27

    Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy

  8. IGF-1R Regulates the Extracellular Level of Active MMP-2, Pathological Neovascularization, and Functionality in Retinas of OIR Mouse Model.

    PubMed

    Lorenc, Valeria E; Subirada Caldarone, Paula V; Paz, María C; Ferrer, Darío G; Luna, José D; Chiabrando, Gustavo A; Sánchez, María C

    2017-01-17

    In ischemic proliferative diseases such as retinopathies, persistent hypoxia leads to the release of numerous neovascular factors that participate in the formation of abnormal vessels and eventually cause blindness. The upregulation and activation of metalloproteinases (MMP-2 and MMP-9) represent a final common pathway in this process. Although many regulators of the neovascular process have been identified, the complete role of the insulin-like growth factor 1 (IGF-1) and its receptor (IGF-1R) appears to be significantly more complex. In this study, we used an oxygen-induced retinopathy (OIR) mouse model as well as an in vitro model of hypoxia to study the role of MMP-2 derived from Müller glial cells (MGCs) and its relation with the IGF-1/IGF-1R system. We demonstrated that MMP-2 protein expression increased in P17 OIR mice, which coincided with the active phase of the neovascular process. Also, glutamine synthetase (GS)-positive cells were also positive for MMP-2, whereas IGF-1R was expressed by GFAP-positive cells, indicating that both proteins were expressed in MGCs. In addition, in the OIR model a single intravitreal injection of the IGF-1R blocking antibody (αIR3) administered at P12 effectively prevented pathologic neovascularization, accelerated physiological revascularization, and improved retinal functionality at P17. Finally, in MGC supernatants, the blocking antibody abolished the IGF-1 effect on active MMP-2 under normoxic and hypoxic conditions without affecting the extracellular levels of pro-MMP-2. These results demonstrate, for the first time, that the IGF-1/IGF-1R system regulates active MMP-2 levels in MGCs, thus contributing to MEC remodeling during the retinal neovascular process.

  9. Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus

    PubMed Central

    Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Koyama, Hiroyuki; Hosoda, Kiminori; Kangawa, Kenji; Nakao, Kazuwa

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A—positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability. PMID:26849804

  10. Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus.

    PubMed

    Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Koyama, Hiroyuki; Hosoda, Kiminori; Kangawa, Kenji; Nakao, Kazuwa

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus-derived cell lines, termed adult mouse hypothalamus (AMH) cells, by developing transgenic mice in which SV40 Tag was overexpressed in chromogranin A-positive cells in a tamoxifen-dependent manner. In order to obtain leptin-responsive clones, we selected clones based on the phosphorylation levels of STAT3 induced by leptin. The selected clones were fairly responsive to leptin in terms of STAT3, ERK, and Akt phosphorylation and induction of c-Fos mRNA induction. Pretreatment with leptin, insulin, and palmitate attenuated the c-Fos mRNA response to leptin, suggesting that certain aspects of leptin resistance might be reconstituted in this cellular model. These cell lines are useful tools for understanding the molecular nature of the signal disturbance in the leptin-resistant state and for identifying potential target molecules for drugs that relieve leptin resistance, although they have drawbacks including de-differentiated nature and lack of long-time stability.

  11. Ultrastructural evidence of exosome secretion by progenitor cells in adult mouse myocardium and adult human cardiospheres.

    PubMed

    Barile, Lucio; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Moccetti, Tiziano; Vassalli, Giuseppe

    2012-01-01

    The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres) that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34⁺ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an "off-the-shelf" product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  12. Rescue of Adult Hippocampal Neurogenesis in a Mouse Model of HIV Neurologic Disease

    PubMed Central

    Lee, Myoung-Hwa; Wang, Tongguang; Jang, Mi-Hyeon; Steiner, Joseph; Haughey, Norman; Ming, Guo-li; Song, Hongjun; Nath, Avindra; Venkatesan, Arun

    2011-01-01

    The prevalence of central nervous system (CNS) neurologic dysfunction associated with human immunodeficiency virus (HIV) infection continues to increase, despite the use of antiretroviral therapy. Previous work has focused on the deleterious effects of HIV on mature neurons and on development of neuroprotective strategies, which have consistently failed to show a meaningful clinical benefit. It is now well established that new neurons are continuously generated in discrete regions in the adult mammalian brain, and accumulating evidence supports important roles for these neurons in specific cognitive functions. In a transgenic mouse model of HIV neurologic disease with glial expression of the HIV envelope protein gp120, we demonstrate a significant reduction in proliferation of hippocampal neural progenitors in the dentate gyrus of adult animals, resulting in a dramatic decrease in the number of newborn neurons in the adult brain. We identify amplifying neural progenitor cells (ANPs) as the first class of progenitors affected by gp120, and we also demonstrate that newly generated neurons exhibit aberrant dendritic development. Furthermore, voluntary exercise and treatment with a selective serotonin reuptake inhibitor increase the ANP population and rescue the observed deficits in gp120 transgenic mice. Thus, during HIV infection, the envelope protein gp120 may potently inhibit adult hippocampal neurogenesis, and neurorestorative approaches may be effective in ameliorating these effects. Our study has significant implications for the development of novel therapeutic approaches for HIV-infected individuals with neurologic dysfunction and may be applicable to other neurodegenerative diseases in which hippocampal neurogenesis is impaired. PMID:21146610

  13. Early functional neural networks in the developing retina

    NASA Astrophysics Data System (ADS)

    Wong, R. O. L.; Chernjavsky, A.; Smith, S. J.; Shatz, C. J.

    1995-04-01

    IN the adult mammalian retina, the principal direction of information flow is along a vertical pathway from photoreceptors to retinal interneurons to ganglion cells, the output neurons of the retina. We report here, however, that initially in development, at a time when the photoreceptors are not yet even present, there are already functionally defined networks within the retina. These networks are spontaneously active rather than visually driven, and they involve horizontal rather than vertical pathways. By means of optical recording using the calcium-sensitive dye Fura-2, we have found that sets of retinal ganglion cells and amacrine cells, a type of retinal interneuron, undergo synchronized oscillations in intracellular calcium concentration. These oscillations are highly correlated among subgroups of neighbouring cells, and spread in a wave-like fashion tangentially across the retina. Thus, in development of retinal circuitry, the initial patterning of neuronal function occurs in the horizontal domain before the adult pattern of vertical information transfer emerges.

  14. Voluntary physical exercise promotes ocular dominance plasticity in adult mouse primary visual cortex.

    PubMed

    Kalogeraki, Evgenia; Greifzu, Franziska; Haack, Franziska; Löwel, Siegrid

    2014-11-12

    Ocular dominance (OD) plasticity in the mouse primary visual cortex (V1) declines during aging and is absent beyond postnatal day (P) 110 when mice are raised in standard cages (SCs; Lehmann and Löwel, 2008). In contrast, raising mice in an enriched environment (EE) preserved a juvenile-like OD plasticity into late adulthood (Greifzu et al., 2014). EE raising provides the mice with more social interactions, voluntary physical exercise, and cognitive stimulation compared with SC, raising the question whether all components are needed or whether one of them is already sufficient to prolong plasticity. To test whether voluntary physical exercise alone already prolongs the sensitive phase for OD plasticity, we raised mice from 7 d before birth to adulthood in slightly larger than normal SCs with or without a running wheel (RW). When the mice were older than P135, we visualized V1 activity before and after monocular deprivation (MD) using intrinsic signal optical imaging. Adult RW-raised mice continued to show an OD shift toward the open eye after 7 d of MD, while age-matched SC mice without a RW did not show OD plasticity. Notably, running just during the 7 d MD period restored OD plasticity in adult SC-raised mice. In addition, the OD shift of the RW mice was mediated by a decrease of deprived-eye responses in V1, a signature of "juvenile-like" plasticity. We conclude that voluntary physical exercise alone is sufficient to promote plasticity in adult mouse V1.

  15. Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain.

    PubMed

    Ikeda, Yayoi; Matsunaga, Yuko; Takiguchi, Masahito; Ikeda, Masa-Aki

    2011-01-01

    Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.

  16. Light-evoked S-nitrosylation in the retina

    PubMed Central

    Tooker, Ryan E; Vigh, Jozsef

    2015-01-01

    Nitric oxide (NO) synthesis in the retina is triggered by light stimulation. NO has been shown to modulate visual signal processing at multiple sites in the vertebrate retina, via activation of the most sensitive target of NO signaling, soluble guanylate cyclase. NO can also alter protein structure and function and exert biological effects directly by binding to free thiol groups of cysteine residues in a chemical reaction called S-nitrosylation. However, in the central nervous system, including the retina, this reaction has not been considered to be significant under physiological conditions. Here we provide immunohistochemical evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner, with a strikingly similar pattern in both species. Pre-treatment with NEM, which occludes S-nitrosylation, or with TRIM, an inhibitor of neuronal NO synthase, eliminated the light-evoked increase in S-nitrosylated protein immunofluorescence (SNI) in the retinas of both species. Similarly, light did not increase SNI, above basal levels, in retinas of transgenic mice lacking neuronal NO synthase. Qualitative analysis of the light-adapted mouse retina with mass spectrometry revealed more than 300 proteins that were S-nitrosylated upon illumination, many of which are known to participate directly in retinal signal processing. Our data strongly suggest that in the retina, light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant post-translational modification affecting a wide range of proteins under physiological conditions. PMID:25823749

  17. Light-evoked S-nitrosylation in the retina.

    PubMed

    Tooker, Ryan E; Vigh, Jozsef

    2015-10-01

    Nitric oxide (NO) synthesis in the retina is triggered by light stimulation. NO has been shown to modulate visual signal processing at multiple sites in the vertebrate retina, via activation of the most sensitive target of NO signaling, soluble guanylate cyclase. NO can also alter protein structure and function and exert biological effects directly by binding to free thiol groups of cysteine residues in a chemical reaction called S-nitrosylation. However, in the central nervous system, including the retina, this reaction has not been considered to be significant under physiological conditions. Here we provide immunohistochemical evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner, with a strikingly similar pattern in both species. Pretreatment with N-ethylmaleimide (NEM), which occludes S-nitrosylation, or with 1-(2-trifluromethylphenyl)imidazole (TRIM), an inhibitor of neuronal NO synthase, eliminated the light-evoked increase in S-nitrosylated protein immunofluorescence (SNI) in the retinas of both species. Similarly, light did not increase SNI, above basal levels, in retinas of transgenic mice lacking neuronal NO synthase. Qualitative analysis of the light-adapted mouse retina with mass spectrometry revealed more than 300 proteins that were S-nitrosylated upon illumination, many of which are known to participate directly in retinal signal processing. Our data strongly suggest that in the retina light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant posttranslational modification affecting a wide range of proteins under physiological conditions.

  18. Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung

    PubMed Central

    Galiger, Celimene; Kostin, Sawa; Golec, Anita; Ahlbrecht, Katrin; Becker, Sven; Gherghiceanu, Mihaela; Popescu, Laurentiu M; Morty, Rory E; Seeger, Werner; Voswinckel, Robert

    2014-01-01

    Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAMneg/CD45neg/Oct4-GFPpos that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration. PMID:24889158

  19. Growth Arrest Specific 1 (GAS1) Is Abundantly Expressed in the Adult Mouse Central Nervous System

    PubMed Central

    Zarco, Natanael; Bautista, Elizabeth; Cuéllar, Manola; Vergara, Paula; Flores-Rodriguez, Paola; Aguilar-Roblero, Raúl

    2013-01-01

    Growth arrest specific 1 (GAS1) is a pleiotropic protein that induces apoptosis and cell arrest in different tumors, but it is also involved in the development of the nervous system and other tissues and organs. This dual ability is likely caused by its capacity to interact both by inhibiting the intracellular signaling cascade induced by glial cell-line derived neurotrophic factor and by facilitating the activity of the sonic hedgehog pathway. The presence of GAS1 mRNA has been described in adult mouse brain, and here we corroborated this observation. We then proceeded to determine the distribution of the protein in the adult central nervous system (CNS). We detected, by western blot analysis, expression of GAS1 in olfactory bulb, caudate-putamen, cerebral cortex, hippocampus, mesencephalon, medulla oblongata, cerebellum, and cervical spinal cord. To more carefully map the expression of GAS1, we performed double-label immunohistochemistry and noticed expression of GAS1 in neurons in all brain areas examined. We also observed expression of GAS1 in astroglial cells, albeit the pattern of expression was more restricted than that seen in neurons. Briefly, in the present article, we report the widespread distribution and cellular localization of the GAS1 native protein in adult mammalian CNS. PMID:23813868

  20. Abca7 deletion does not affect adult neurogenesis in the mouse.

    PubMed

    Li, Hongyun; Karl, Tim; Garner, Brett

    2016-01-20

    ATP-binding cassette transporter A7 (ABCA7) is highly expressed in the brain. Recent genome-wide association studies (GWAS) have identified ABCA7 single nucleotide polymorphisms (SNPs) that increase Alzheimer's disease (AD) risk, however, the mechanisms by which ABCA7 may control AD risk remain to be fully elucidated. Based on previous research suggesting that certain ABC transporters may play a role in the regulation of neurogenesis, we conducted a study of cell proliferation and neurogenic potential using cellular bromodeoxyuridine (BrdU) incorporation and doublecortin (DCX) immunostaining in adult Abca7 deficient mice and wild-type-like (WT) littermates. In the present study counting of BrdU-positive and DCX-positive cells in an established adult neurogenesis site in the dentate gyrus (DG) indicated there were no significant differences when WT and Abca7 deficient mice were compared. We also measured the area occupied by immunohistochemical staining for BrdU and DCX in the DG and the subventricular zone (SVZ) of the same mice and this confirmed that ABCA7 does not play a significant role in the regulation of cell proliferation or neurogenesis in the adult mouse.

  1. Localization and regulation of PML bodies in the adult mouse brain.

    PubMed

    Hall, Małgorzata H; Magalska, Adriana; Malinowska, Monika; Ruszczycki, Błażej; Czaban, Iwona; Patel, Satyam; Ambrożek-Latecka, Magdalena; Zołocińska, Ewa; Broszkiewicz, Hanna; Parobczak, Kamil; Nair, Rajeevkumar R; Rylski, Marcin; Pawlak, Robert; Bramham, Clive R; Wilczyński, Grzegorz M

    2016-06-01

    PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.

  2. New Role of Adult Lung c-kit+ Cells in a Mouse Model of Airway Hyperresponsiveness

    PubMed Central

    Cappetta, Donato; Urbanek, Konrad; Esposito, Grazia; Matteis, Maria; Sgambato, Manuela; Tartaglione, Gioia; Rossi, Francesco

    2016-01-01

    Structural changes contribute to airway hyperresponsiveness and airflow obstruction in asthma. Emerging evidence points to the involvement of c-kit+ cells in lung homeostasis, although their potential role in asthma is unknown. Our aim was to isolate c-kit+ cells from normal mouse lungs and to test whether these cells can interfere with hallmarks of asthma in an animal model. Adult mouse GFP-tagged c-kit+ cells, intratracheally delivered in the ovalbumin-induced airway hyperresponsiveness, positively affected airway remodeling and improved airway function. In bronchoalveolar lavage fluid of cell-treated animals, a reduction in the number of inflammatory cells and in IL-4, IL-5, and IL-13 release, along with an increase of IL-10, was observed. In MSC-treated mice, the macrophage polarization to M2-like subset may explain, at least in part, the increment in the level of anti-inflammatory cytokine IL-10. After in vitro stimulation of c-kit+ cells with proinflammatory cytokines, the indoleamine 2,3-dioxygenase and TGFβ were upregulated. These data, together with the increased apoptosis of inflammatory cells in vivo, indicate that c-kit+ cells downregulate immune response in asthma by influencing local environment, possibly by cell-to-cell contact combined to paracrine action. In conclusion, intratracheally administered c-kit+ cells reduce inflammation, positively modulate airway remodeling, and improve function. These data document previously unrecognized properties of c-kit+ cells, able to impede pathophysiological features of experimental airway hyperresponsiveness. PMID:28090152

  3. Comparison of melatonin with growth factors in promoting precursor cells proliferation in adult mouse subventricular zone

    PubMed Central

    Sotthibundhu, Areechun; Ekthuwapranee, Kasima; Govitrapong, Piyarat

    2016-01-01

    Melatonin, secreted mainly by the pineal gland, plays roles in various physiological functions including protecting cell death. We showed in previous study that the proliferation and differentiation of precursor cells from the adult mouse subventricular zone (SVZ) can be modulated by melatonin via the MT1 melatonin receptor. Since melatonin and epidermal growth factor receptor (EGFR) share some signaling pathway components, we investigated whether melatonin can promote the proliferation of precursor cells from the adult mouse SVZ via the extracellular signal-regulated protein kinase /mitogen-activated protein kinase (ERK/MAPK) pathways in comparison with epidermal growth factor (EGF). Melatonin-induced ERK/MAPK pathways compared with EGF were measured by using in vitro and vivo models. We used neurosphere proliferation assay, immunocytochemistry, and immuno-blotting to analyze significant differences between melatonin and growth factor treatment. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including luzindole and U0126. We found that significant increase in proliferation was observed when two growth factors (EGF+bFGF) and melatonin were used simultaneously compared with EGF + bFGF or compared with melatonin alone. In addition, the present result suggested the synergistic effect occurred of melatonin and growth factors on the activating the ERK/MAPK pathway. This study exhibited that melatonin could act as a trophic factor, increasing proliferation in precursor cells mediated through the melatonin receptor coupled to ERK/MAPK signaling pathways. Understanding the mechanism by which melatonin regulates precursor cells may conduct to the development of novel strategies for neurodegenerative disease therapy. PMID:28275319

  4. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion

    PubMed Central

    Miller, Michelle Erin; Rosten, Patty; Lemieux, Madeleine E.; Lai, Courteney; Humphries, R. Keith

    2016-01-01

    Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1’s importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1’s functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs) and implicate reactive oxygen species (ROS) as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA)26Sortm1(Cre/ERT)Nat/J or B6.Cg-Tg(Mx1-Cre)1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1’s role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation. PMID:26986211

  5. Functional adult acetylcholine receptor develops independently of motor innervation in Sol 8 mouse muscle cell line.

    PubMed Central

    Pinset, C; Mulle, C; Benoit, P; Changeux, J P; Chelly, J; Gros, F; Montarras, D

    1991-01-01

    We have defined culture conditions, using a feeder layer of cells from the embryonic mesenchymal cell line, 10T1/2 and a serum-free medium, which allow cells from the mouse myogenic cell line Sol 8 to form contracting myotubes for two weeks. Under these culture conditions, Sol 8 myotubes undergo a maturation process characterized by a sequential expression of two phenotypes. An early phenotype is typified by the expression of the nicotinic acetylcholine receptor (AChR) gamma-subunit transcripts and the presence of low conductance ACh-activated channels, typical of embryonic AChR. A late phenotype is characterized by the expression of AChR epsilon-subunit transcripts, the decreased accumulation of gamma-subunit transcripts and the appearance of high conductance ACh-activated channels, typical of adult AChR. These results indicate that the expression of functional adult type AChR does not require the presence of the motor nerve and therefore represents an intrinsic feature of the Sol 8 muscle cells. Chronic exposure of the cells to the voltage-sensitive Na+ channel blocking agent tetrodotoxin does not affect the appearance of the AChR epsilon-subunit transcripts but prevents the reduction of the steady-state level of the AChR gamma-subunit transcripts and yields a reduced proportion of the adult type channels. Thus, activity seems to facilitate the switch from the embryonic to the adult phenotype of the AChR protein. The Sol 8 cell system might be useful to analyse further the genetic and epigenetic regulation of muscle fibre maturation in mammals. Images PMID:1868829

  6. Oestradiol and Diet Modulate Energy Homeostasis and Hypothalamic Neurogenesis in the Adult Female Mouse

    PubMed Central

    Bless, E. P.; Reddy, T.; Acharya, K. D.; Beltz, B. S.; Tetel, M. J.

    2014-01-01

    Leptin and oestradiol have overlapping functions in energy homeostasis and fertility, and receptors for these hormones are localised in the same hypothalamic regions. Although, historically, it was assumed that mammalian adult neurogenesis was confined to the olfactory bulbs and the hippocampus, recent research has found new neurones in the male rodent hypothalamus. Furthermore, some of these new neurones are leptin-sensitive and affected by diet. In the present study, we tested the hypothesis that diet and hormonal status modulate hypothalamic neurogenesis in the adult female mouse. Adult mice were ovariectomised and implanted with capsules containing oestradiol (E2) or oil. Within each group, mice were fed a high-fat diet (HFD) or maintained on standard chow (STND). All animals were administered i.c.v. 5-bromo-2′-deoxyuridine (BrdU) for 9 days and sacrificed 34 days later after an injection of leptin to induce phosphorylation of signal transducer of activation and transcription 3 (pSTAT3). Brain tissue was immunohistochemically labelled for BrdU (newly born cells), Hu (neuronal marker) and pSTAT3 (leptin sensitive). Although mice on a HFD became obese, oestradiol protected against obesity. There was a strong interaction between diet and hormone on new cells (BrdU+) in the arcuate, ventromedial hypothalamus and dorsomedial hypothalamus. HFD increased the number of new cells, whereas E2 inhibited this effect. Conversely, E2 increased the number of new cells in mice on a STND diet in all hypothalamic regions studied. Although the total number of new leptin-sensitive neurones (BrdU-Hu-pSTAT3) found in the hypothalamus was low, HFD increased these new cells in the arcuate, whereas E2 attenuated this induction. These results suggest that adult neurogenesis in the hypothalamic neurogenic niche is modulated by diet and hormonal status and is related to energy homeostasis in female mice. PMID:25182179

  7. Circadian organization of the rodent retina involves strongly coupled, layer-specific oscillators.

    PubMed

    Jaeger, Catherine; Sandu, Cristina; Malan, André; Mellac, Katell; Hicks, David; Felder-Schmittbuhl, Marie-Paule

    2015-04-01

    Rhythmic physiology is central to retinal function and survival and adapts vision to daily light intensity changes. Mammalian retina rhythmically releases melatonin when cultured under constant conditions, and the occurrence of clock gene [e.g., Period (Per)] expression has been shown for most cellular layers. However, contribution of the distinct layers to genesis of circadian rhythms within the retina is still debated. To characterize their endogenous oscillatory capacity and their communication at the whole-tissue level, we used a vibratome-based method to isolate individual or paired retina cellular layers from the mPer2(Luc) mouse and Per1-luciferase (Per1-Luc) rat, and real-time recorded bioluminescence. We report that each layer of the mouse retina harbors a self-sustained oscillator whose period is significantly longer (∼ 26 hours) than in whole-retina explants (∼ 22.9 hours), indicating that the period is correlated with the degree of coupling. Accordingly, the maximal period (∼ 29 hours) is reached upon complete enzymatic dissociation of the retina. By using pharmacological approaches, we demonstrate that connection between retina oscillators involves gap junctions but only minor contribution from the main retina neurochemicals. Taken together with results from Per1-Luc rats, these data show that mammalian retina consists of a network of layer-specific oscillators whose period is determined by their connectivity.

  8. Drugs and the retina.

    PubMed

    Constable, Simon; Pirmohamed, Munir

    2004-05-01

    The retina is relatively protected from systemic drug administration because of the blood-retinal barrier, a highly selective mechanism adapted to providing a regulated homeostatic environment for this highly specialised tissue. However, a number of drugs have been associated with retinal toxicity. Vigabatrin, as an adjunctive therapy for the management of partial epilepsy, is associated with visual field defects in approximately 40% of patients. Hydroxychloroquine, used in the treatment of rheumatoid arthritis and systemic lupus erythematosus, is also associated with a retinopathy. In view of this, ophthalmological screening and monitoring is recommended during prescription of both of these drugs. In these cases, the retina is the site for an adverse drug reaction and the dose of therapy may be important in determining the likelihood of retinal toxicity. However, in the case of cytomegalovirus retinitis, the retina is the intended site for pharmacological action. The treatment of this condition with the antiviral agents ganciclovir, valganciclovir, foscarnet and cidofovir, can also be associated with significant systemic toxicity.

  9. Topography of ganglion cell production in the cat's retina

    SciTech Connect

    Walsh, C.; Polley, E.H.

    1985-03-01

    The ganglion cells of the cat's retina form several classes distinguishable in terms of soma size, axon diameter, dendritic morphology, physiological properties, and central connections. Labeling with (/sup 3/H)thymidine shows that the ganglion cells which survive in the adult are produced as several temporally shifted, overlapping waves: medium-sized cells are produced before large cells, whereas the smallest ganglion cells are produced throughout the period of ganglion cell generation. Large cells and medium-sized cells show the same distinctive pattern of production, forming rough spirals around the area centralis. The oldest cells tend to lie superior and nasal to the area centralis, whereas cells in the inferior nasal retina and inferior temporal retina are, in general, progressively younger. Within each retinal quadrant, cells nearer the area centralis tend to be older than cells in the periphery, but there is substantial overlap. The retinal raphe divides the superior temporal quadrant into two zones with different patterns of cell addition. Superior temporal retina near the vertical meridian adds cells only slightly later than superior nasal retina, whereas superior temporal retina near the horizontal meridian adds cells very late, contemporaneously with inferior temporal retina. The broader wave of production of smaller ganglion cells seems to follow this same spiral pattern at its beginning and end. The presence of the area centralis as a nodal point about which ganglion cell production in the retinal quadrants pivots suggests that the area centralis is already an important retinal landmark even at the earliest stages of retinal development.

  10. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  11. Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

    PubMed

    Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

    2016-07-29

    Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

  12. Research Resource: Comprehensive Expression Atlas of the Fibroblast Growth Factor System in Adult Mouse

    PubMed Central

    Fon Tacer, Klementina; Bookout, Angie L.; Ding, Xunshan; Kurosu, Hiroshi; John, George B.; Wang, Lei; Goetz, Regina; Mohammadi, Moosa; Kuro-o, Makoto; Mangelsdorf, David J.; Kliewer, Steven A.

    2010-01-01

    Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals. PMID:20667984

  13. Bmp4 from the optic vesicle specifies murine retina formation.

    PubMed

    Huang, Jie; Liu, Ying; Oltean, Alina; Beebe, David C

    2015-06-01

    Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

  14. Connecting the Retina to the Brain

    PubMed Central

    Herrera, Eloisa

    2014-01-01

    The visual system is beautifully crafted to transmit information of the external world to visual processing and cognitive centers in the brain. For visual information to be relayed to the brain, a series of axon pathfinding events must take place to ensure that the axons of retinal ganglion cells, the only neuronal cell type in the retina that sends axons out of the retina, find their way out of the eye to connect with targets in the brain. In the past few decades, the power of molecular and genetic tools, including the generation of genetically manipulated mouse lines, have multiplied our knowledge about the molecular mechanisms involved in the sculpting of the visual system. Here, we review major advances in our understanding of the mechanisms controlling the differentiation of RGCs, guidance of their axons from the retina to the primary visual centers, and the refinement processes essential for the establishment of topographic maps and eye-specific axon segregation. Human disorders, such as albinism and achiasmia, that impair RGC axon growth and guidance and, thus, the establishment of a fully functioning visual system will also be discussed. PMID:25504540

  15. Bipolar cells of the ground squirrel retina.

    PubMed

    Puller, Christian; Ondreka, Katharina; Haverkamp, Silke

    2011-03-01

    Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate.

  16. Age-Dependent Changes of Monocarboxylate Transporter 8 Availability in the Postnatal Murine Retina.

    PubMed

    Henning, Yoshiyuki; Szafranski, Karol

    2016-01-01

    The thyroid hormones (TH) triiodothyronine (T3) and its prohormone thyroxine (T4) are crucial for retinal development and function, and increasing evidence points at TH dysregulation as a cause for retinal degenerative diseases. Thus, precise regulation of retinal TH supply is required for proper retinal function, but knowledge on these mechanisms is still fragmentary. Several transmembrane transporters have been described as key regulators of TH availability in target tissues of which the monocarboxylate transporter 8 (MCT8), a high affinity transporter for T4 and T3, plays an essential role in the central nervous system. Moreover, in the embryonic chicken retina, MCT8 is highly expressed, but the postnatal availability of MCT8 in the mammalian retina was not reported to date. In the present study, spatiotemporal retinal MCT8 availability was examined in mice of different age. For this purpose, we quantified expression levels of Mct8 via Real-Time Reverse-Transcriptase PCR in mouse eyecups (C57BL/6) of juvenile and adult age groups. Additionally, age-dependent MCT8 protein levels were quantified via Western blotting and localized via immunofluorescence confocal microscopy. While no difference in Mct8 expression levels could be detected between age groups, MCT8 protein levels in juvenile animals were about two times higher than in adult animals based on Western blot analyses. Immunohistochemical analyses showed that MCT8 immunoreactivity in the eyecup was restricted to the retina and the retinal pigment epithelium. In juvenile mice, MCT8 was broadly observed along the apical membrane of the retinal pigment epithelium, tightly surrounding photoreceptor outer segments. Distinct immunopositive staining was also detected in the inner nuclear layer and the ganglion cell layer. However, in adult specimens, immunoreactivity visibly declined in all layers, which was in line with Western blot analyses. Since MCT8 was abundantly present in juvenile and about twofold lower in

  17. Age-Dependent Changes of Monocarboxylate Transporter 8 Availability in the Postnatal Murine Retina

    PubMed Central

    Henning, Yoshiyuki; Szafranski, Karol

    2016-01-01

    The thyroid hormones (TH) triiodothyronine (T3) and its prohormone thyroxine (T4) are crucial for retinal development and function, and increasing evidence points at TH dysregulation as a cause for retinal degenerative diseases. Thus, precise regulation of retinal TH supply is required for proper retinal function, but knowledge on these mechanisms is still fragmentary. Several transmembrane transporters have been described as key regulators of TH availability in target tissues of which the monocarboxylate transporter 8 (MCT8), a high affinity transporter for T4 and T3, plays an essential role in the central nervous system. Moreover, in the embryonic chicken retina, MCT8 is highly expressed, but the postnatal availability of MCT8 in the mammalian retina was not reported to date. In the present study, spatiotemporal retinal MCT8 availability was examined in mice of different age. For this purpose, we quantified expression levels of Mct8 via Real-Time Reverse-Transcriptase PCR in mouse eyecups (C57BL/6) of juvenile and adult age groups. Additionally, age-dependent MCT8 protein levels were quantified via Western blotting and localized via immunofluorescence confocal microscopy. While no difference in Mct8 expression levels could be detected between age groups, MCT8 protein levels in juvenile animals were about two times higher than in adult animals based on Western blot analyses. Immunohistochemical analyses showed that MCT8 immunoreactivity in the eyecup was restricted to the retina and the retinal pigment epithelium. In juvenile mice, MCT8 was broadly observed along the apical membrane of the retinal pigment epithelium, tightly surrounding photoreceptor outer segments. Distinct immunopositive staining was also detected in the inner nuclear layer and the ganglion cell layer. However, in adult specimens, immunoreactivity visibly declined in all layers, which was in line with Western blot analyses. Since MCT8 was abundantly present in juvenile and about twofold lower in

  18. Distribution of doublecortin expressing cells near the lateral ventricles in the adult mouse brain.

    PubMed

    Yang, Helen K C; Sundholm-Peters, Nikki L; Goings, Gwendolyn E; Walker, Avery S; Hyland, Kenneth; Szele, Francis G

    2004-05-01

    Doublecortin (Dcx) is a microtubule-associated protein expressed by migrating neuroblasts in the embryo and in the adult subventricular zone (SVZ). The adult SVZ contains neuroblasts that migrate in the rostral migratory stream (RMS) to the olfactory bulbs. We have examined the distribution and phenotype of Dcx-positive cells in the adult mouse SVZ and surrounding regions. Chains of Dcx-positive cells in the SVZ were distributed in a tight dorsal population contiguous with the RMS, with a separate ventral population comprised of discontinuous chains. Unexpectedly, Dcx-positive cells were also found outside of the SVZ: dorsally in the corpus callosum, and ventrally in the nucleus accumbens, ventromedial striatum, ventrolateral septum, and bed nucleus of the stria terminalis. Dcx-positive cells outside the SVZ had the morphology of migrating cells, occurred as individual cells or in chain-like clusters, and were more numerous anteriorly. Of the Dcx-positive cells found outside of the SVZ, 47% expressed the immature neuronal protein class III beta-tubulin, 8% expressed NeuN, a marker of mature neurons. Dcx-positive cells did not express molecules found in astrocytes, oligodendrocytes, or microglia. Structural and immunoelectron microscopy revealed that cells with the ultrastructural features of neuroblasts in the SVZ were Dcx+, and that clusters of neuroblasts emanated ventrally from the SVZ into the parenchyma. Our results suggest that the distribution of cells comprising the walls of the lateral ventricle are more heterogeneous than was thought previously, that SVZ cells may migrate dorsally and ventrally away from the SVZ, and that some emigrated cells express a neuronal phenotype.

  19. Expression of Npas4 mRNA in Telencephalic Areas of Adult and Postnatal Mouse Brain

    PubMed Central

    Damborsky, Joanne C.; Slaton, G. Simona; Winzer-Serhan, Ursula H.

    2015-01-01

    The transcription factor neuronal PAS domain-containing protein 4 (Npas4) is an inducible immediate early gene which regulates the formation of inhibitory synapses, and could have a significant regulatory role during cortical circuit formation. However, little is known about basal Npas4 mRNA expression during postnatal development. Here, postnatal and adult mouse brain sections were processed for isotopic in situ hybridization using an Npas4 specific cRNA antisense probe. In adults, Npas4 mRNA was found in the telencephalon with very restricted or no expression in diencephalon or mesencephalon. In most telencephalic areas, including the anterior olfactory nucleus (AON), piriform cortex, neocortex, hippocampus, dorsal caudate putamen (CPu), septum and basolateral amygdala nucleus (BLA), basal Npas4 expression was detected in scattered cells which exhibited strong hybridization signal. In embryonic and neonatal brain sections, Npas4 mRNA expression signals were very low. Starting at postnatal day 5 (P5), transcripts for Npas4 were detected in the AON, CPu and piriform cortex. At P8, additional Npas4 hybridization was found in CA1 and CA3 pyramidal layer, and in primary motor cortex. By P13, robust mRNA expression was located in layers IV and VI of all sensory cortices, frontal cortex and cingulate cortex. After onset of expression, postnatal spatial mRNA distribution was similar to that in adults, with the exception of the CPu, where Npas4 transcripts became gradually restricted to the most dorsal part. In conclusion, the spatial distribution of Npas4 mRNA is mostly restricted to telencephalic areas, and the temporal expression increases with developmental age during postnatal development, which seem to correlate with the onset of activity-driven excitatory transmission. PMID:26633966

  20. Stroke Increases Neural Stem Cells and Angiogenesis in the Neurogenic Niche of the Adult Mouse

    PubMed Central

    Zhang, Rui Lan; Chopp, Michael; Roberts, Cynthia; Liu, Xianshuang; Wei, Min; Nejad-Davarani, Siamak P.; Wang, Xinli; Zhang, Zheng Gang

    2014-01-01

    The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction. PMID:25437857

  1. Temporal profiles of synaptic plasticity-related signals in adult mouse hippocampus with methotrexate treatment.

    PubMed

    Yang, Miyoung; Kim, Juhwan; Kim, Sung-Ho; Kim, Joong-Sun; Shin, Taekyun; Moon, Changjong

    2012-07-25

    Methotrexate, which is used to treat many malignancies and autoimmune diseases, affects brain functions including hippocampal-dependent memory function. However, the precise mechanisms underlying methotrexate-induced hippocampal dysfunction are poorly understood. To evaluate temporal changes in synaptic plasticity-related signals, the expression and activity of N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, extracellular signal-regulated kinase 1/2, cAMP responsive element-binding protein, glutamate receptor 1, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor were examined in the hippocampi of adult C57BL/6 mice after methotrexate (40 mg/kg) intraperitoneal injection. Western blot analysis showed biphasic changes in synaptic plasticity-related signals in adult hippocampi following methotrexate treatment. N-methyl-D-aspartic acid receptor 1, calcium/calmodulin-dependent protein kinase II, and glutamate receptor 1 were acutely activated during the early phase (1 day post-injection), while extracellular signal-regulated kinase 1/2 and cAMP responsive element-binding protein activation showed biphasic increases during the early (1 day post-injection) and late phases (7-14 days post-injection). Brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression increased significantly during the late phase (7-14 days post-injection). Therefore, methotrexate treatment affects synaptic plasticity-related signals in the adult mouse hippocampus, suggesting that changes in synaptic plasticity-related signals may be associated with neuronal survival and plasticity-related cellular remodeling.

  2. Contributions of Mouse and Human Hematopoietic Cells to Remodeling of the Adult Auditory Nerve After Neuron Loss

    PubMed Central

    Lang, Hainan; Nishimoto, Eishi; Xing, Yazhi; Brown, LaShardai N; Noble, Kenyaria V; Barth, Jeremy L; LaRue, Amanda C; Ando, Kiyoshi; Schulte, Bradley A

    2016-01-01

    The peripheral auditory nerve (AN) carries sound information from sensory hair cells to the brain. The present study investigated the contribution of mouse and human hematopoietic stem cells (HSCs) to cellular diversity in the AN following the destruction of neuron cell bodies, also known as spiral ganglion neurons (SGNs). Exposure of the adult mouse cochlea to ouabain selectively killed type I SGNs and disrupted the blood-labyrinth barrier. This procedure also resulted in the upregulation of genes associated with hematopoietic cell homing and differentiation, and provided an environment conducive to the tissue engraftment of circulating stem/progenitor cells into the AN. Experiments were performed using both a mouse-mouse bone marrow transplantation model and a severely immune-incompetent mouse model transplanted with human CD34+ cord blood cells. Quantitative immunohistochemical analysis of recipient mice demonstrated that ouabain injury promoted an increase in the number of both HSC-derived macrophages and HSC-derived nonmacrophages in the AN. Although rare, a few HSC-derived cells in the injured AN exhibited glial-like qualities. These results suggest that human hematopoietic cells participate in remodeling of the AN after neuron cell body loss and that hematopoietic cells can be an important resource for promoting AN repair/regeneration in the adult inner ear. PMID:27600399

  3. High yield extraction of pure spinal motor neurons, astrocytes and microglia from single embryo and adult mouse spinal cord

    PubMed Central

    Beaudet, Marie-Josée; Yang, Qiurui; Cadau, Sébastien; Blais, Mathieu; Bellenfant, Sabrina; Gros-Louis, François; Berthod, François

    2015-01-01

    Extraction of mouse spinal motor neurons from transgenic mouse embryos recapitulating some aspects of neurodegenerative diseases like amyotrophic lateral sclerosis has met with limited success. Furthermore, extraction and long-term culture of adult mouse spinal motor neurons and glia remain also challenging. We present here a protocol designed to extract and purify high yields of motor neurons and glia from individual spinal cords collected on embryos and adult (5-month-old) normal or transgenic mice. This method is based on mild digestion of tissue followed by gradient density separation allowing to obtain two millions motor neurons over 92% pure from one E14.5 single embryo and more than 30,000 from an adult mouse. These cells can be cultured more than 14 days in vitro at a density of 100,000 cells/cm2 to maintain optimal viability. Functional astrocytes and microglia and small gamma motor neurons can be purified at the same time. This protocol will be a powerful and reliable method to obtain motor neurons and glia to better understand mechanisms underlying spinal cord diseases. PMID:26577180

  4. Doublecortin (DCX) is not Essential for Survival and Differentiation of Newborn Neurons in the Adult Mouse Dentate Gyrus

    PubMed Central

    Dhaliwal, Jagroop; Xi, Yanwei; Bruel-Jungerman, Elodie; Germain, Johanne; Francis, Fiona; Lagace, Diane C.

    2016-01-01

    In the adult brain, expression of the microtubule-associated protein Doublecortin (DCX) is associated with neural progenitor cells (NPCs) that give rise to new neurons in the dentate gyrus. Many studies quantify the number of DCX-expressing cells as a proxy for the level of adult neurogenesis, yet no study has determined the effect of removing DCX from adult hippocampal NPCs. Here, we use a retroviral and inducible mouse transgenic approach to either knockdown or knockout DCX from adult NPCs in the dentate gyrus and examine how this affects cell survival and neuronal maturation. Our results demonstrate that shRNA-mediated knockdown of DCX or Cre-mediated recombination in floxed DCX mice does not alter hippocampal neurogenesis and does not change the neuronal fate of the NPCs. Together these findings show that the survival and maturation of adult-generated hippocampal neurons does not require DCX. PMID:26793044

  5. Seeing double: visual physiology of double-retina eye ontogeny in stomatopod crustaceans.

    PubMed

    Feller, Kathryn D; Cohen, Jonathan H; Cronin, Thomas W

    2015-03-01

    Stomatopod eye development is unusual among crustaceans. Just prior to metamorphosis, an adult retina and associated neuro-processing structures emerge adjacent to the existing material in the larval compound eye. Depending on the species, the duration of this double-retina eye can range from a few hours to several days. Although this developmental process occurs in all stomatopod species observed to date, the retinal physiology and extent to which each retina contributes to the animal's visual sensitivity during this transition phase is unknown. We investigated the visual physiology of stomatopod double retinas using microspectrophotometry and electroretinogram recordings from different developmental stages of the Western Atlantic species Squilla empusa. Though microspectrophotometry data were inconclusive, we found robust ERG responses in both larval and adult retinas at all sampled time points indicating that the adult retina responds to light from the very onset of its emergence. We also found evidence of an increase in the response dynamics with ontogeny as well as an increase in sensitivity of retinal tissue during the double-retina phase relative to single retinas. These data provide an initial investigation into the ontogeny of vision during stomatopod double-retina eye development.

  6. Adult plasticity in the subcortical auditory pathway of the maternal mouse.

    PubMed

    Miranda, Jason A; Shepard, Kathryn N; McClintock, Shannon K; Liu, Robert C

    2014-01-01

    Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system - motherhood - is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered.

  7. Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine.

    PubMed

    Schreiber, Rainer; Faria, Diana; Skryabin, Boris V; Wanitchakool, Podchanart; Rock, Jason R; Kunzelmann, Karl

    2015-06-01

    Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca(2+) and opening of apical Cl(-) channels. In infants and young animals, but not in adults, Ca(2+)-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca(2+)-dependent Cl(-) channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca(2+)-dependent Cl(-) secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca(2+)-activated Cl(-) secretion. In contrast, Ano10 is essential for Ca(2+)-dependent Cl(-) secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl(-) secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca(2+) increase, while the essential and possibly only apical Cl(-) channel is CFTR. These results define a new role of Ano1 for intestinal Ca(2+)-dependent Cl(-) secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

  8. Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease

    PubMed Central

    Ambade, Aditya; Satishchandran, Abhishek; Gyongyosi, Benedek; Lowe, Patrick; Szabo, Gyongyi

    2016-01-01

    AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD). METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA). RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice. CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC. PMID:27122661

  9. Activity-dependent Notch signalling in the hypothalamic-neurohypophysial system of adult mouse brains.

    PubMed

    Mannari, T; Miyata, S

    2014-08-01

    Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta-like ligand 4 (DLL4) in the hypothalamic-neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double-labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)- and oxytocin (OXT)-containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT- but not AVP-containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP- and OXT-containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4-immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity-dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH.

  10. Distinct expression of Cbln family mRNAs in developing and adult mouse brains.

    PubMed

    Miura, Eriko; Iijima, Takatoshi; Yuzaki, Michisuke; Watanabe, Masahiko

    2006-08-01

    Cbln1 belongs to the C1q and tumour necrosis factor superfamily, and plays crucial roles as a cerebellar granule cell-derived transneuronal regulator for synapse integrity and plasticity in Purkinje cells. Although Cbln2-Cbln4 are also expressed in the brain and could form heteromeric complexes with Cbln1, their precise expressions remain unclear. Here, we investigated gene expression of the Cbln family in developing and adult C57BL mouse brains by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and high-resolution in situ hybridization (ISH) analyses. In the adult brain, spatial patterns of mRNA expression were highly differential depending on Cbln subtypes. Notably, particularly high levels of Cbln mRNAs were expressed in some nuclei and neurons, whereas their postsynaptic targets often lacked or were low for any Cbln mRNAs, as seen for cerebellar granule cells/Purkinje cells, entorhinal cortex/hippocampus, intralaminar group of thalamic nuclei/caudate-putamen, and dorsal nucleus of the lateral lemniscus/central nucleus of the inferior colliculus. In the developing brain, Cbln1, 2, and 4 mRNAs appeared as early as embryonic day 10-13, and exhibited transient up-regulation during the late embryonic and neonatal periods. For example, Cbln2 mRNA was expressed in the cortical plate of the developing neocortex, displaying a high rostromedial to low caudolateral gradient. In contrast, Cbln3 mRNA was selective to cerebellar granule cells throughout development, and its onset was as late as postnatal day 7-10. These results will provide a molecular-anatomical basis for future studies that characterize roles played by the Cbln family.

  11. 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

  12. 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.

  13. Tryptophan hydroxylase and serotonin receptor 1A expression in the retina of the sea lamprey.

    PubMed

    Cornide-Petronio, María Eugenia; Anadón, Ramón; Barreiro-Iglesias, Antón; Rodicio, María Celina

    2015-06-01

    The dual development of the retina of lampreys is exceptional among vertebrates and offers an interesting EvoDevo (evolutionary developmental biology) model for understanding the origin and evolution of the vertebrate retina. Only a single type of photoreceptor, ganglion cell and bipolar cell are present in the early-differentiated central retina of lamprey prolarvae. A lateral retina appears later in medium-sized larvae (about 3 years after hatching in the sea lamprey), growing and remaining largely neuroblastic until metamorphosis. In this lateral retina, only ganglion cells and optic fibers differentiate in larvae, whereas differentiation of amacrine, horizontal, photoreceptor and bipolar cells mainly takes place during metamorphosis, which gives rise to the adult retina. Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter found in the retina of vertebrates whose synthesis is mediated by the rate-limiting enzyme tryptophan hydroxylase (TPH). TPH is also the first enzyme in the biosynthetic pathways of melatonin in photoreceptor cells. The serotonin 1A receptor (5-HT1A) is a major determinant of the activity of both serotonergic cells and their targets due to its pre- and post-synaptic location. Here, we report the developmental pattern of expression of tph and 5-ht1a transcripts in the sea lamprey retina by means of in situ hybridization. In larvae, strong tph mRNA signal was observed in photoreceptors and putative ganglion cells of the central retina, and in some neuroblasts of the lateral retina. In adults, strong tph expression was observed in bipolar, amacrine and ganglion cells and in photoreceptors. In the prolarval (central) retina, all the differentiated retinal cells expressed 5-ht1a transcripts, which were not observed in undifferentiated cells. In larvae, photoreceptors, bipolar cells and ganglion cells in the central retina, and neuroblasts in the lateral retina, showed 5-ht1a expression. In the adult retina, expression of 5-ht1a transcript

  14. Layer-specific chromatin accessibility landscapes reveal regulatory networks in adult mouse visual cortex

    PubMed Central

    Gray, Lucas T; Yao, Zizhen; Nguyen, Thuc Nghi; Kim, Tae Kyung; Zeng, Hongkui; Tasic, Bosiljka

    2017-01-01

    Mammalian cortex is a laminar structure, with each layer composed of a characteristic set of cell types with different morphological, electrophysiological, and connectional properties. Here, we define chromatin accessibility landscapes of major, layer-specific excitatory classes of neurons, and compare them to each other and to inhibitory cortical neurons using the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). We identify a large number of layer-specific accessible sites, and significant association with genes that are expressed in specific cortical layers. Integration of these data with layer-specific transcriptomic profiles and transcription factor binding motifs enabled us to construct a regulatory network revealing potential key layer-specific regulators, including Cux1/2, Foxp2, Nfia, Pou3f2, and Rorb. This dataset is a valuable resource for identifying candidate layer-specific cis-regulatory elements in adult mouse cortex. DOI: http://dx.doi.org/10.7554/eLife.21883.001 PMID:28112643

  15. Neurotoxic effects of ochratoxin A on the subventricular zone of adult mouse brain.

    PubMed

    Paradells, Sara; Rocamonde, Brenda; Llinares, Cristina; Herranz-Pérez, Vicente; Jimenez, Misericordia; Garcia-Verdugo, Jose Manuel; Zipancic, Ivan; Soria, Jose Miguel; Garcia-Esparza, Ma Angeles

    2015-07-01

    Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner.

  16. Properties of doublecortin expressing neurons in the adult mouse dentate gyrus.

    PubMed

    Spampanato, Jay; Sullivan, Robert K; Turpin, Fabrice R; Bartlett, Perry F; Sah, Pankaj

    2012-01-01

    The dentate gyrus is a neurogenic zone where neurons continue to be born throughout life, mature and integrate into the local circuitry. In adults, this generation of new neurons is thought to contribute to learning and memory formation. As newborn neurons mature, they undergo a developmental sequence in which different stages of development are marked by expression of different proteins. Doublecortin (DCX) is an early marker that is expressed in immature granule cells that are beginning migration and dendritic growth but is turned off before neurons reach maturity. In the present study, we use a mouse strain in which enhanced green fluorescent protein (EGFP) is expressed under the control of the DCX promoter. We show that these neurons have high input resistances and some cells can discharge trains of action potentials. In mature granule cells, action potentials are followed by a slow afterhyperpolarization that is absent in EGFP-positive neurons. EGFP-positive neurons had a lower spine density than mature neurons and stimulation of either the medial or lateral perforant pathway activated dual component glutamatergic synapses that had both AMPA and NMDA receptors. NMDA receptors present at these synapses had slow kinetics and were blocked by ifenprodil, indicative of high GluN2B subunit content. These results show that EGFP-positive neurons in the DCX-EGFP mice are functionally immature both in their firing properties and excitatory synapses.

  17. Differential Distribution of Major Brain Gangliosides in the Adult Mouse Central Nervous System

    PubMed Central

    Vajn, Katarina; Viljetić, Barbara; Degmečić, Ivan Večeslav; Schnaar, Ronald L.; Heffer, Marija

    2013-01-01

    Gangliosides - sialic acid-bearing glycolipids - are major cell surface determinants on neurons and axons. The same four closely related structures, GM1, GD1a, GD1b and GT1b, comprise the majority of total brain gangliosides in mammals and birds. Gangliosides regulate the activities of proteins in the membranes in which they reside, and also act as cell-cell recognition receptors. Understanding the functions of major brain gangliosides requires knowledge of their tissue distribution, which has been accomplished in the past using biochemical and immunohistochemical methods. Armed with new knowledge about the stability and accessibility of gangliosides in tissues and new IgG-class specific monoclonal antibodies, we investigated the detailed tissue distribution of gangliosides in the adult mouse brain. Gangliosides GD1b and GT1b are widely expressed in gray and white matter. In contrast, GM1 is predominately found in white matter and GD1a is specifically expressed in certain brain nuclei/tracts. These findings are considered in relationship to the hypothesis that gangliosides GD1a and GT1b act as receptors for an important axon-myelin recognition protein, myelin-associated glycoprotein (MAG). Mediating axon-myelin interactions is but one potential function of the major brain gangliosides, and more detailed knowledge of their distribution may help direct future functional studies. PMID:24098718

  18. Adult pallium transcriptomes surprise in not reflecting predicted homologies across diverse chicken and mouse pallial sectors.

    PubMed

    Belgard, T Grant; Montiel, Juan F; Wang, Wei Zhi; García-Moreno, Fernando; Margulies, Elliott H; Ponting, Chris P; Molnár, Zoltán

    2013-08-06

    The thorniest problem in comparative neurobiology is the identification of the particular brain region of birds and reptiles that corresponds to the mammalian neocortex [Butler AB, Reiner A, Karten HJ (2011) Ann N Y Acad Sci 1225:14-27; Wang Y, Brzozowska-Prechtl A, Karten HJ (2010) Proc Natl Acad Sci USA 107(28):12676-12681]. We explored which genes are actively transcribed in the regions of controversial ancestry in a representative bird (chicken) and mammal (mouse) at adult stages. We conducted four analyses comparing the expression patterns of their 5,130 most highly expressed one-to-one orthologous genes that considered global patterns of expression specificity, strong gene markers, and coexpression networks. Our study demonstrates transcriptomic divergence, plausible convergence, and, in two exceptional cases, conservation between specialized avian and mammalian telencephalic regions. This large-scale study potentially resolves the complex relationship between developmental homology and functional characteristics on the molecular level and settles long-standing evolutionary debates.

  19. MicroRNA Clusters in the Adult Mouse Heart: Age-Associated Changes.

    PubMed

    Zhang, Xiaomin; Azhar, Gohar; Williams, Emmanuel D; Rogers, Steven C; Wei, Jeanne Y

    2015-01-01

    The microRNAs and microRNA clusters have been implicated in normal cardiac development and also disease, including cardiac hypertrophy, cardiomyopathy, heart failure, and arrhythmias. Since a microRNA cluster has from two to dozens of microRNAs, the expression of a microRNA cluster could have a substantial impact on its target genes. In the present study, the configuration and distribution of microRNA clusters in the mouse genome were examined at various inter-microRNA distances. Three important microRNA clusters that are significantly impacted during adult cardiac aging, the miR-17-92, miR-106a-363, and miR-106b-25, were also examined in terms of their genomic location, RNA transcript character, sequence homology, and their relationship with the corresponding microRNA families. Multiple microRNAs derived from the three clusters potentially target various protein components of the cdc42-SRF signaling pathway, which regulates cytoskeleton dynamics associated with cardiac structure and function. The data indicate that aging impacted the expression of both guide and passenger strands of the microRNA clusters; nutrient stress also affected the expression of the three microRNA clusters. The miR-17-92, miR-106a-363, and miR-106b-25 clusters are likely to impact the Cdc42-SRF signaling pathway and thereby affect cardiac morphology and function during pathological conditions and the aging process.

  20. Brain derived neurotrophic factor in the retina of the teleost N. furzeri.

    PubMed

    Gatta, Claudia; Castaldo, Luciana; Cellerino, Alessandro; de Girolamo, Paolo; Lucini, Carla; D'Angelo, Livia

    2014-07-01

    BDNF plays an important role in the development and maintenance of visual circuitries in the retina and brain visual centers. In adulthood, BDNF signaling is involved in neural protection and regeneration of retina. In this survey, we investigated the expression of BDNF in the retina of adult Nothobranchius furzeri, a teleost fish employed for age research. After describing the retina of N. furzeri and confirming that the structure is organized in layers as in all vertebrates, we have studied the localization of BDNF mRNA and protein throughout the retinal layers. BDNF mRNA is detectable in all layers, whereas the protein is lacking in the photoreceptors. The occurrence of BDNF provides new insights on its role in the retina, particularly in view of age-related disease of retina.

  1. Adenovirus Vectors Targeting Distinct Cell Types in the Retina

    PubMed Central

    Sweigard, J. Harry; Cashman, Siobhan M.

    2010-01-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5ΔRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5ΔRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5ΔRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5ΔRGD vectors. PMID:19892875

  2. 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

  3. The proteome of human retina.

    PubMed

    Zhang, Pingbo; Dufresne, Craig; Turner, Randi; Ferri, Sara; Venkatraman, Vidya; Karani, Rabia; Lutty, Gerard A; Van Eyk, Jennifer E; Semba, Richard D

    2015-02-01

    The retina is a delicate tissue that detects light, converts photochemical energy into neural signals, and transmits the signals to the visual cortex of the brain. A detailed protein inventory of the proteome of the normal human eye may provide a foundation for new investigations into both the physiology of the retina and the pathophysiology of retinal diseases. To provide an inventory, proteins were extracted from five retinas of normal eyes and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed in duplicate using LC-MS/MS on an Orbitrap Elite mass spectrometer. A total of 3436 nonredundant proteins were identified in the human retina, including 20 unambiguous protein isoforms, of which eight have not previously been demonstrated to exist at the protein level. The proteins identified in the retina included most of the enzymes involved in the visual cycle and retinoid metabolism. One hundred and fifty-eight proteins that have been associated with age-related macular degeneration were identified in the retina. The MS proteome database of the human retina may serve as a valuable resource for future investigations of retinal biology and disease. All MS data have been deposited in the ProteomeXchange with identifier PXD001242 (http://proteomecentral.proteomexchange.org/dataset/PXD001242).

  4. Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown.

    PubMed

    Ogura, Shuntaro; Kurata, Kaori; Hattori, Yuki; Takase, Hiroshi; Ishiguro-Oonuma, Toshina; Hwang, Yoonha; Ahn, Soyeon; Park, Inwon; Ikeda, Wataru; Kusuhara, Sentaro; Fukushima, Yoko; Nara, Hiromi; Sakai, Hideto; Fujiwara, Takashi; Matsushita, Jun; Ema, Masatsugu; Hirashima, Masanori; Minami, Takashi; Shibuya, Masabumi; Takakura, Nobuyuki; Kim, Pilhan; Miyata, Takaki; Ogura, Yuichiro; Uemura, Akiyoshi

    2017-02-09

    In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

  5. Dopamine D2 receptors preferentially regulate the development of light responses of the inner retina

    PubMed Central

    Tian, Ning; Xu, Hong-ping; Wang, Ping

    2014-01-01

    Retinal light responsiveness measured via electroretinography undergoes developmental modulation and is thought to be critically regulated by both visual experience and dopamine. The primary goal of this study is to determine whether the dopamine D2 receptor regulates the visual experience-dependent functional development of the retina. Accordingly, we recorded electroretinograms from wild type mice and mice with a genetic deletion of the gene that encodes the dopamine D2 receptor raised under normal cyclic light conditions and constant darkness. Our results demonstrate that mutation of the dopamine D2 receptors preferentially increases the amplitude of the inner retinal light responses evoked by high intensity light measured as oscillatory potentials in adult mice. During postnatal development, all three major components of electroretinograms, the a-wave, b-wave and oscillatory potentials, increase with age. Comparatively, mutation of the dopamine D2 receptors preferentially reduces the age-dependent increase of b-waves evoked by low intensity light. Light deprivation from birth reduces the amplitude of b-waves and completely diminishes the increased amplitude of oscillatory potentials. Taken together, these results demonstrate that the dopamine D2 receptor plays an important role in the activity-dependent functional development of the mouse retina. PMID:25393815

  6. Sustained inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown

    PubMed Central

    Ogura, Shuntaro; Kurata, Kaori; Hattori, Yuki; Takase, Hiroshi; Ishiguro-Oonuma, Toshina; Hwang, Yoonha; Ahn, Soyeon; Ikeda, Wataru; Kusuhara, Sentaro; Fukushima, Yoko; Nara, Hiromi; Sakai, Hideto; Fujiwara, Takashi; Matsushita, Jun; Ema, Masatsugu; Hirashima, Masanori; Shibuya, Masabumi; Takakura, Nobuyuki; Kim, Pilhan; Miyata, Takaki; Ogura, Yuichiro

    2017-01-01

    In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders. PMID:28194443

  7. Expression patterns of Slit and Robo family members in adult mouse spinal cord and peripheral nervous system.

    PubMed

    Carr, Lauren; Parkinson, David B; Dun, Xin-Peng

    2017-01-01

    The secreted glycoproteins, Slit1-3, are classic axon guidance molecules that act as repulsive cues through their well characterised receptors Robo1-2 to allow precise axon pathfinding and neuronal migration. The expression patterns of Slit1-3 and Robo1-2 have been most characterized in the rodent developing nervous system and the adult brain, but little is known about their expression patterns in the adult rodent peripheral nervous system. Here, we report a detailed expression analysis of Slit1-3 and Robo1-2 in the adult mouse sciatic nerve as well as their expression in the nerve cell bodies within the ventral spinal cord (motor neurons) and dorsal root ganglion (sensory neurons). Our results show that, in the adult mouse peripheral nervous system, Slit1-3 and Robo1-2 are expressed in the cell bodies and axons of both motor and sensory neurons. While Slit1 and Robo2 are only expressed in peripheral axons and their cell bodies, Slit2, Slit3 and Robo1 are also expressed in satellite cells of the dorsal root ganglion, Schwann cells and fibroblasts of peripheral nerves. In addition to these expression patterns, we also demonstrate the expression of Robo1 in blood vessels of the peripheral nerves. Our work gives important new data on the expression patterns of Slit and Robo family members within the peripheral nervous system that may relate both to nerve homeostasis and the reaction of the peripheral nerves to injury.

  8. BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

    PubMed Central

    Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y.

    2016-01-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. PMID:26796036

  9. BAG3 regulates contractility and Ca(2+) homeostasis in adult mouse ventricular myocytes.

    PubMed

    Feldman, Arthur M; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D; Tilley, Douglas G; Gao, Erhe; Hoffman, Nicholas E; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y

    2016-03-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure.

  10. Genetic influences on exercise-induced adult hippocampal neurogenesis across 12 divergent mouse strains

    PubMed Central

    Clark, Peter J.; Kohman, Rachel A.; Miller, Daniel S.; Bhattacharya, Tushar K.; Brzezinska, Weronika J.; Rhodes, Justin S.

    2011-01-01

    New neurons are continuously born in the hippocampus of several mammalian species throughout adulthood. Adult neurogenesis represents a natural model for understanding how to grow and incorporate new nerve cells into pre-existing circuits in the brain. Finding molecules or biological pathways that increase neurogenesis has broad potential for regenerative medicine. One strategy is to identify mouse strains that display large versus small increases in neurogenesis in response to wheel running so the strains can be contrasted to find common genes or biological pathways associated with enhanced neuron formation. Therefore, mice from 12 different isogenic strains were housed with or without running wheels for 43 days to measure the genetic regulation of exercise-induced neurogenesis. The first 10 days mice received daily injections of BrdU to label dividing cells. Neurogenesis was measured as the total number of BrdU cells co-expressing NeuN mature neuronal marker in the hippocampal granule cell layer by immunohistochemistry. Exercise increased neurogenesis in all strains, but the magnitude significantly depended on genotype. Strain means for distance run on wheels, but not distance traveled in cages without wheels, were significantly correlated with strain mean level of neurogenesis. Further, certain strains displayed greater neurogenesis than others for a fixed level of running. Strain means for neurogenesis under sedentary conditions were not correlated with neurogenesis under runner conditions suggesting that different genes influence baseline versus exercise-induced neurogenesis. Genetic contributions to exercise-induced hippocampal neurogenesis suggest that it may be possible to identify genes and pathways associated with enhanced neuroplastic responses to exercise. PMID:21223504

  11. Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures

    PubMed Central

    Gelain, Fabrizio; Bottai, Daniele; Vescovi, Angleo; Zhang, Shuguang

    2006-01-01

    Biomedical researchers have become increasingly aware of the limitations of conventional 2-dimensional tissue cell culture systems, including coated Petri dishes, multi-well plates and slides, to fully address many critical issues in cell biology, cancer biology and neurobiology, such as the 3-D microenvironment, 3-D gradient diffusion, 3-D cell migration and 3-D cell-cell contact interactions. In order to fully understand how cells behave in the 3-D body, it is important to develop a well-controlled 3-D cell culture system where every single ingredient is known. Here we report the development of a 3-D cell culture system using a designer peptide nanofiber scaffold with mouse adult neural stem cells. We attached several functional motifs, including cell adhesion, differentiation and bone marrow homing motifs, to a self-assembling peptide RADA16 (Ac-RADARADARADARADA-COHN2). These functionalized peptides undergo self-assembly into a nanofiber structure similar to Matrigel. During cell culture, the cells were fully embedded in the 3-D environment of the scaffold. Two of the peptide scaffolds containing bone marrow homing motifs significantly enhanced the neural cell survival without extra soluble growth and neurotrophic factors to the routine cell culture media. In these designer scaffolds, the cell populations with β-Tubulin+, GFAP+ and Nestin+ markers are similar to those found in cell populations cultured on Matrigel. The gene expression profiling array experiments showed selective gene expression, possibly involved in neural stem cell adhesion and differentiation. Because the synthetic peptides are intrinsically pure and a number of desired function cellular motifs are easy to incorporate, these designer peptide nanofiber scaffolds provide a promising controlled 3-D culture system for diverse tissue cells, and are useful as well for general molecular and cell biology. PMID:17205123

  12. Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult

    PubMed Central

    Carreira, Vinicius S.; Fan, Yunxia; Kurita, Hisaka; Wang, Qin; Ko, Chia-I; Naticchioni, Mindi; Jiang, Min; Koch, Sheryl; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Xia, Ying; Rubinstein, Jack; Puga, Alvaro

    2015-01-01

    The Developmental Origins of Health and Disease (DOHaD) Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease. PMID:26555816

  13. Comprehensive Analysis of Neonatal versus Adult Unilateral Decortication in a Mouse Model Using Behavioral, Neuroanatomical, and DNA Microarray Approaches

    PubMed Central

    Yoshikawa, Akira; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Shioda, Seiji

    2014-01-01

    Previously, studying the development, especially of corticospinal neurons, it was concluded that the main compensatory mechanism after unilateral brain injury in rat at the neonatal stage was due in part to non-lesioned ipsilateral corticospinal neurons that escaped selection by axonal elimination or neuronal apoptosis. However, previous results suggesting compensatory mechanism in neonate brain were not correlated with high functional recovery. Therefore, what is the difference among neonate and adult in the context of functional recovery and potential mechanism(s) therein? Here, we utilized a brain unilateral decortication mouse model and compared motor functional recovery mechanism post-neonatal brain hemisuction (NBH) with adult brain hemisuction (ABH). Three analyses were performed: (1) Quantitative behavioral analysis of forelimb movements using ladder walking test; (2) neuroanatomical retrograde tracing analysis of unlesioned side corticospinal neurons; and (3) differential global gene expressions profiling in unlesioned-side neocortex (rostral from bregma) in NBH and ABH on a 8 × 60 K mouse whole genome Agilent DNA chip. Behavioral data confirmed higher recovery ability in NBH over ABH is related to non-lesional frontal neocortex including rostral caudal forelimb area. A first inventory of differentially expressed genes genome-wide in the NBH and ABH mouse model is provided as a resource for the scientific community. PMID:25490135

  14. Accumulated quiescent neural stem cells in adult hippocampus of the mouse model for the MECP2 duplication syndrome

    PubMed Central

    Chen, Zhifang; Li, Xiao; Zhou, Jingjing; Yuan, Bo; Yu, Bin; Tong, Dali; Cheng, Cheng; Shao, Yinqi; Xia, Shengnan; Zhang, Ran; Lyu, Jingwen; Yu, Xiuya; Dong, Chen; Zhou, Wen-Hao; Qiu, Zilong

    2017-01-01

    Duplications of Methyl CpG binding protein 2 (MECP2) -containing segments lead to the MECP2 duplication syndrome, in which severe autistic symptoms were identified. Whether adult neurogenesis may play a role in pathogenesis of autism and the role of MECP2 on state determination of adult neural stem cells (NSCs) remain largely unclear. Using a MECP2 transgenic (TG) mouse model for the MECP2 duplication syndrome, we found that adult hippocampal quiescent NSCs were significantly accumulated in TG mice comparing to wild type (WT) mice, the neural progenitor cells (NPCs) were reduced and the neuroblasts were increased in adult hippocampi of MECP2 TG mice. Interestingly, we found that parvalbumin (PV) positive interneurons were significantly decreased in MECP2 TG mice, which were critical for determining fates of adult hippocampal NSCs between the quiescence and activation. In summary, we found that MeCP2 plays a critical role in regulating fate determination of adult NSCs. These evidences further suggest that abnormal development of NSCs may play a role in the pathogenesis of the MECP2 duplication syndrome. PMID:28139724

  15. Neuroprotective effects of lutein in the retina.

    PubMed

    Ozawa, Yoko; Sasaki, Mariko; Takahashi, Noriko; Kamoshita, Mamoru; Miyake, Seiji; Tsubota, Kazuo

    2012-01-01

    Although a large variety of pharmaceutical therapies for treating disease have been developed in recent years, there has been little progress in disease prevention. In particular, the protection of neural tissue is essential, because it is hardly regenerated. The use of nutraceuticals for maintaining the health has been supported by several clinical studies, including cross-sectional and interventional studies for age-related macular disease. However, mechanistic evidence for their effects at the molecular level has been very limited. In this review, we focus on lutein, which is a xanthophyll type of carotenoid. Lutein is not synthesized in mammals, and must be obtained from the diet. It is delivered to the retina, and in humans, it is concentrated in the macula. Here, we describe the neuroprotective effects of lutein and their underlying molecular mechanisms in animal models of vision-threatening diseases, such as innate retinal inflammation, diabetic retinopathy, and light-induced retinal degeneration. In lutein-treated mouse ocular disease models, oxidative stress in the retina is reduced, and its downstream pathological signals are inhibited. Furthermore, degradation of the functional proteins, rhodopsin (a visual substance) and synaptophysin (a synaptic vesicle protein also influenced in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease), the depletion of brain-derived neurotrophic factor (BDNF), and DNA damage are prevented by lutein, which preserves visual function. We discuss the possibility of using lutein, an antioxidant, as a neuroprotective treatment for humans.

  16. Neuroprotective Effects of Lutein in the Retina

    PubMed Central

    Ozawa, Yoko; Sasaki, Mariko; Takahashi, Noriko; Kamoshita, Mamoru; Miyake, Seiji; Tsubota, Kazuo

    2012-01-01

    Although a large variety of pharmaceutical therapies for treating disease have been developed in recent years, there has been little progress in disease prevention. In particular, the protection of neural tissue is essential, because it is hardly regenerated. The use of nutraceuticals for maintaining the health has been supported by several clinical studies, including cross-sectional and interventional studies for age-related macular disease. However, mechanistic evidence for their effects at the molecular level has been very limited. In this review, we focus on lutein, which is a xanthophyll type of carotenoid. Lutein is not synthesized in mammals, and must be obtained from the diet. It is delivered to the retina, and in humans, it is concentrated in the macula. Here, we describe the neuroprotective effects of lutein and their underlying molecular mechanisms in animal models of vision-threatening diseases, such as innate retinal inflammation, diabetic retinopathy, and light-induced retinal degeneration. In lutein-treated mouse ocular disease models, oxidative stress in the retina is reduced, and its downstream pathological signals are inhibited. Furthermore, degradation of the functional proteins, rhodopsin (a visual substance) and synaptophysin (a synaptic vesicle protein also influenced in other neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease), the depletion of brain-derived neurotrophic factor (BDNF), and DNA damage are prevented by lutein, which preserves visual function. We discuss the possibility of using lutein, an antioxidant, as a neuroprotective treatment for humans. PMID:22211688

  17. TMEM16A is associated with voltage-gated calcium channels in mouse retina and its function is disrupted upon mutation of the auxiliary α2δ4 subunit

    PubMed Central

    Caputo, Antonella; Piano, Ilaria; Demontis, Gian Carlo; Bacchi, Niccolò; Casarosa, Simona; Santina, Luca Della; Gargini, Claudia

    2015-01-01

    Photoreceptors rely upon highly specialized synapses to efficiently transmit signals to multiple postsynaptic targets. Calcium influx in the presynaptic terminal is mediated by voltage-gated calcium channels (VGCC). This event triggers neurotransmitter release, but also gates calcium-activated chloride channels (TMEM), which in turn regulate VGCC activity. In order to investigate the relationship between VGCC and TMEM channels, we analyzed the retina of wild type (WT) and Cacna2d4 mutant mice, in which the VGCC auxiliary α2δ4 subunit carries a nonsense mutation, disrupting the normal channel function. Synaptic terminals of mutant photoreceptors are disarranged and synaptic proteins as well as TMEM16A channels lose their characteristic localization. In parallel, calcium-activated chloride currents are impaired in rods, despite unaltered TMEM16A protein levels. Co-immunoprecipitation revealed the interaction between VGCC and TMEM16A channels in the retina. Heterologous expression of these channels in tsA-201 cells showed that TMEM16A associates with the CaV1.4 subunit, and the association persists upon expression of the mutant α2δ4 subunit. Collectively, our experiments show association between TMEM16A and the α1 subunit of VGCC. Close proximity of these channels allows optimal function of the photoreceptor synaptic terminal under physiological conditions, but also makes TMEM16A channels susceptible to changes occurring to calcium channels. PMID:26557056

  18. Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

    PubMed

    Al Alam, Denise; Danopoulos, Soula; Schall, Kathy; Sala, Frederic G; Almohazey, Dana; Fernandez, G Esteban; Georgia, Senta; Frey, Mark R; Ford, Henri R; Grikscheit, Tracy; Bellusci, Saverio

    2015-04-15

    Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

  19. Isolation of multipotent neural stem/progenitor cells from both the dentate gyrus and subventricular zone of a single adult mouse

    PubMed Central

    Guo, Weixiang; Patzlaff, Natalie E.; Jobe, Emily M.; Zhao, Xinyu

    2013-01-01

    In adult mammals, the subventricular zone of the lateral ventricles (SVZ) and the subgranular zone of the dentate gyrus (DG) demonstrate ongoing neurogenesis, and multipotent neural stem/progenitor cells (NSCs) in these two regions exhibit different intrinsic properties. However, investigation of the mechanisms underlying such differences has been limited by a lack of efficient methods for isolating NSCs, particularly from the adult DG. Here we describe a protocol that enables us to isolate self-renewing and multipotent NSCs from the SVZ and the DG of the same adult mouse. The protocol involves the microdissection of the SVZ and DG from one adult mouse brain, isolation of NSCs from specific regions, and cultivation of NSCs in vitro. The entire procedure takes 2 to 3 hours. Since only one mouse is needed for each cell isolation procedure, this protocol will be particularly useful for studies with limited availability of mice, such as mice that contain multiple genetic modifications. PMID:23080272

  20. P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone.

    PubMed

    Messemer, Nanette; Kunert, Christin; Grohmann, Marcus; Sobottka, Helga; Nieber, Karen; Zimmermann, Herbert; Franke, Heike; Nörenberg, Wolfgang; Straub, Isabelle; Schaefer, Michael; Riedel, Thomas; Illes, Peter; Rubini, Patrizia

    2013-10-01

    Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca(2+) and zero Mg(2+) concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7(-/-) mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca(2+)-imaging by means of Fura-2 revealed that in a Mg(2+)-deficient bath medium Bz-ATP causes [Ca(2+)](i) transients fully depending on the presence of external Ca(2+). The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.

  1. Comparative ultrastructural features of excitatory synapses in the visual and frontal cortices of the adult mouse and monkey.

    PubMed

    Hsu, Alexander; Luebke, Jennifer I; Medalla, Maria

    2017-03-03

    The excitatory glutamatergic synapse is the principal site of communication between cortical pyramidal neurons and their targets, a key locus of action of many drugs, and highly vulnerable to dysfunction and loss in neurodegenerative disease. A detailed knowledge of the structure of these synapses in distinct cortical areas and across species is a prerequisite for understanding the anatomical underpinnings of cortical specialization and, potentially, selective vulnerability in neurological disorders. We used serial electron microscopy to assess the ultrastructural features of excitatory (asymmetric) synapses in the layers 2-3 (L2-3) neuropil of visual (V1) and frontal (FC) cortices of the adult mouse and compared findings to those in the rhesus monkey (V1 and lateral prefrontal cortex [LPFC]). Analyses of multiple ultrastructural variables revealed four organizational features. First, the density of asymmetric synapses does not differ between frontal and visual cortices in either species, but is significantly higher in mouse than in monkey. Second, the structural properties of asymmetric synapses in mouse V1 and FC are nearly identical, by stark contrast to the significant differences seen between monkey V1 and LPFC. Third, while the structural features of postsynaptic entities in mouse and monkey V1 do not differ, the size of presynaptic boutons are significantly larger in monkey V1. Fourth, both presynaptic and postsynaptic entities are significantly smaller in the mouse FC than in the monkey LPFC. The diversity of synaptic ultrastructural features demonstrated here have broad implications for the nature and efficacy of glutamatergic signaling in distinct cortical areas within and across species.

  2. Deficits in adult neurogenesis, contextual fear conditioning, and spatial learning in a Gfap mutant mouse model of Alexander disease.

    PubMed

    Hagemann, Tracy L; Paylor, Richard; Messing, Albee

    2013-11-20

    Glial fibrillary acidic protein (GFAP) is the major intermediate filament of mature astrocytes in the mammalian CNS. Dominant gain of function mutations in GFAP lead to the fatal neurodegenerative disorder, Alexander disease (AxD), which is characterized by cytoplasmic protein aggregates known as Rosenthal fibers along with variable degrees of leukodystrophy and intellectual disability. The mechanisms by which mutant GFAP leads to these pleiotropic effects are unknown. In addition to astrocytes, GFAP is also expressed in other cell types, particularly neural stem cells that form the reservoir supporting adult neurogenesis in the hippocampal dentate gyrus and subventricular zone of the lateral ventricles. Here, we show that mouse models of AxD exhibit significant pathology in GFAP-positive radial glia-like cells in the dentate gyrus, and suffer from deficits in adult neurogenesis. In addition, they display impairments in contextual learning and spatial memory. This is the first demonstration of cognitive phenotypes in a model of primary astrocyte disease.

  3. Tunable retina encoders for retina implants: why and how

    NASA Astrophysics Data System (ADS)

    Eckmiller, Rolf; Neumann, Dirk; Baruth, Oliver

    2005-03-01

    Current research towards retina implants for partial restoration of vision in blind humans with retinal degenerative dysfunctions focuses on implant and stimulation experiments and technologies. In contrast, our approach takes the availability of an epiretinal multi-electrode neural interface for granted and studies the conditions for successful joint information processing of both retinal prosthesis and brain. Our proposed learning retina encoder (RE) includes information processing modules to simulate the complex mapping operation of parts of the 5-layered neural retina and to provide an iterative, perception-based dialog between RE and human subject. Alternative information processing technologies in the learning RE are being described, which allow an individual optimization of the RE mapping operation by means of iterative tuning with learning algorithms in a dialog between implant wearing subject and RE. The primate visual system is modeled by a retina module (RM) composed of spatio-temporal (ST) filters and a central visual system module (VM). RM performs a mapping 1 of an optical pattern P1 in the physical domain onto a retinal output vector R1(t) in a neural domain, whereas VM performs a mapping 2 of R1(t) in a neural domain onto a visual percept P2 in the perceptual domain. Retinal ganglion cell properties represent non-invertible ST filters in RE, which generate ambiguous output signals. VM generates visual percepts only if the corresponding R1(t) is properly encoded, contains sufficient information, and can be disambiguated. Based on the learning RE and the proposed visual system model, a novel retina encoder (RE*) is proposed, which considers both ambiguity removal and miniature eye movements during fixation. Our simulation results suggest that VM requires miniature eye movements under control of the visual system to retrieve unambiguous patterns P2 corresponding to P1. For retina implant applications, RE* can be tuned to generate optimal ganglion cell

  4. Expression patterns of Slit and Robo family members in adult mouse spinal cord and peripheral nervous system

    PubMed Central

    Carr, Lauren; Parkinson, David B.; Dun, Xin-peng

    2017-01-01

    The secreted glycoproteins, Slit1-3, are classic axon guidance molecules that act as repulsive cues through their well characterised receptors Robo1-2 to allow precise axon pathfinding and neuronal migration. The expression patterns of Slit1-3 and Robo1-2 have been most characterized in the rodent developing nervous system and the adult brain, but little is known about their expression patterns in the adult rodent peripheral nervous system. Here, we report a detailed expression analysis of Slit1-3 and Robo1-2 in the adult mouse sciatic nerve as well as their expression in the nerve cell bodies within the ventral spinal cord (motor neurons) and dorsal root ganglion (sensory neurons). Our results show that, in the adult mouse peripheral nervous system, Slit1-3 and Robo1-2 are expressed in the cell bodies and axons of both motor and sensory neurons. While Slit1 and Robo2 are only expressed in peripheral axons and their cell bodies, Slit2, Slit3 and Robo1 are also expressed in satellite cells of the dorsal root ganglion, Schwann cells and fibroblasts of peripheral nerves. In addition to these expression patterns, we also demonstrate the expression of Robo1 in blood vessels of the peripheral nerves. Our work gives important new data on the expression patterns of Slit and Robo family members within the peripheral nervous system that may relate both to nerve homeostasis and the reaction of the peripheral nerves to injury. PMID:28234971

  5. Distribution of Cones in Human and Monkey Retina: Individual Variability and Radial Asymmetry

    NASA Astrophysics Data System (ADS)

    Curcio, Christine A.; Sloan, Kenneth R.; Packer, Orin; Hendrickson, Anita E.; Kalina, Robert E.

    1987-05-01

    The distribution of photoreceptors is known for only one complete human retina and for the cardinal meridians only in the macaque monkey retina. Cones can be mapped in computer-reconstructed whole mounts of human and monkey retina. A 2.9-fold range in maximum cone density in the foveas of young adult human eyes may contribute to individual differences in acuity. Cone distribution is radially asymmetrical about the fovea in both species, as previously described for the distribution of retinal ganglion cells and for lines of visual isosensitivity. Cone density was greater in the nasal than in the temporal peripheral retina, and this nasotemporal asymmetry was more pronounced in monkey than in human retina.

  6. Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina.

    PubMed

    Matsuoka, Ryota L; Nguyen-Ba-Charvet, Kim T; Parray, Aijaz; Badea, Tudor C; Chédotal, Alain; Kolodkin, Alex L

    2011-02-10

    In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

  7. Transmembrane semaphorin signaling controls laminar stratification in the mammalian retina

    PubMed Central

    Matsuoka, Ryota L.; Nguyen-Ba-Charvet, Kim T.; Parray, Aijaz; Badea, Tudor C.; Chédotal, Alain; Kolodkin, Alex L.

    2010-01-01

    In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells, and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL)1–3: a laminar region that is conventionally divided into five major parallel sublaminae1,2. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here, we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs), and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo with respect to the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes. PMID:21270798

  8. Modifications of hippocampal circuits and early disruption of adult neurogenesis in the tg2576 mouse model of Alzheimer's disease.

    PubMed

    Krezymon, Alice; Richetin, Kevin; Halley, Hélène; Roybon, Laurent; Lassalle, Jean-Michel; Francès, Bernard; Verret, Laure; Rampon, Claire

    2013-01-01

    At advanced stages of Alzheimer's disease, cognitive dysfunction is accompanied by severe alterations of hippocampal circuits that may largely underlie memory impairments. However, it is likely that anatomical remodeling in the hippocampus may start long before any cognitive alteration is detected. Using the well-described Tg2576 mouse model of Alzheimer's disease that develops progressive age-dependent amyloidosis and cognitive deficits, we examined whether specific stages of the disease were associated with the expression of anatomical markers of hippocampal dysfunction. We found that these mice develop a complex pattern of changes in their dentate gyrus with aging. Those include aberrant expression of neuropeptide Y and reduced levels of calbindin, reflecting a profound remodeling of inhibitory and excitatory circuits in the dentate gyrus. Preceding these changes, we identified severe alterations of adult hippocampal neurogenesis in Tg2576 mice. We gathered converging data in Tg2576 mice at young age, indicating impaired maturation of new neurons that may compromise their functional integration into hippocampal circuits. Thus, disruption of adult hippocampal neurogenesis occurred before network remodeling in this mouse model and therefore may account as an early event in the etiology of Alzheimer's pathology. Ultimately, both events may constitute key components of hippocampal dysfunction and associated cognitive deficits occurring in Alzheimer's disease.

  9. A rapidly activating sustained K+ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle.

    PubMed Central

    Fiset, C; Clark, R B; Larsen, T S; Giles, W R

    1997-01-01

    1. The K+ currents which control repolarization in adult mouse ventricle, and the effects of changes in action potential duration on excitation-contraction coupling in this tissue, have been studied with electrophysiological methods using single cell preparations and by recording mechanical parameters from an in vitro working heart preparation. 2. Under conditions where Ca(2+)-dependent currents were eliminated by buffering intracellular Ca2+ with EGTA, depolarizing voltage steps elicited two rapidly activating outward K+ currents: (i) a transient outward current, and (ii) a slowly inactivating or 'sustained' delayed rectifier. 3. These two currents were separated pharmacologically by the K+ channel blocker 4-amino-pyridine (4-AP). 4-AP at concentrations between 3 and 200 microM resulted in (i) a marked increase in action potential duration and a large decrease in the sustained K+ current at plateau potentials, as well as (ii) a significant increase in left ventricular systolic pressure in the working heart preparation. 4. The current-voltage (I-V) relation, kinetics, and block by low concentrations of 4-AP strongly suggest that the rapid delayed rectifier in adult mouse ventricles is the same K+ current (Kv1.5) that has been characterized in detail in human and canine atria. 5. These results show that the 4-AP-sensitive rapid delayed rectifier is a very important repolarizing current in mouse ventricle. The enhanced contractility produced by 4-AP (50 microM) in the working heart preparation demonstrates that modulation of the action potential duration, by blocking a K+ current, is a very significant inotropic variable. PMID:9401964

  10. A physiologically based pharmacokinetic model for atrazine and its main metabolites in the adult male C57BL/6 mouse

    SciTech Connect

    Lin Zhoumeng; Fisher, Jeffrey W.; Ross, Matthew K.; Filipov, Nikolay M.

    2011-02-15

    Atrazine (ATR) is a chlorotriazine herbicide that is widely used and relatively persistent in the environment. In laboratory rodents, excessive exposure to ATR is detrimental to the reproductive, immune, and nervous systems. To better understand the toxicokinetics of ATR and to fill the need for a mouse model, a physiologically based pharmacokinetic (PBPK) model for ATR and its main chlorotriazine metabolites (Cl-TRIs) desethyl atrazine (DE), desisopropyl atrazine (DIP), and didealkyl atrazine (DACT) was developed for the adult male C57BL/6 mouse. Taking advantage of all relevant and recently made available mouse-specific data, a flow-limited PBPK model was constructed. The ATR and DACT sub-models included blood, brain, liver, kidney, richly and slowly perfused tissue compartments, as well as plasma protein binding and red blood cell binding, whereas the DE and DIP sub-models were constructed as simple five-compartment models. The model adequately simulated plasma levels of ATR and Cl-TRIs and urinary dosimetry of Cl-TRIs at four single oral dose levels (250, 125, 25, and 5 mg/kg). Additionally, the model adequately described the dose dependency of brain and liver ATR and DACT concentrations. Cumulative urinary DACT amounts were accurately predicted across a wide dose range, suggesting the model's potential use for extrapolation to human exposures by performing reverse dosimetry. The model was validated using previously reported data for plasma ATR and DACT in mice and rats. Overall, besides being the first mouse PBPK model for ATR and its Cl-TRIs, this model, by analogy, provides insights into tissue dosimetry for rats. The model could be used in tissue dosimetry prediction and as an aid in the exposure assessment to this widely used herbicide.

  11. Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain

    PubMed Central

    Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

    2013-01-01

    An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain. PMID:23440889

  12. Pharmacological Analysis of Intrinsic Neuronal Oscillations in rd10 Retina

    PubMed Central

    Biswas, Sonia; Haselier, Christine; Mataruga, Anja; Thumann, Gabriele; Walter, Peter; Müller, Frank

    2014-01-01

    In the widely used mouse model of retinal degeneration, rd1, the loss of photoreceptors leads to rhythmic electrical activity of around 10–16 Hz in the remaining retinal network. Recent studies suggest that this oscillation is formed within the electrically coupled network of AII amacrine cells and ON-bipolar cells. A second mouse model, rd10, displays a delayed onset and slower progression of degeneration, making this mouse strain a better model for human retinitis pigmentosa. In rd10, oscillations occur at a frequency of 3–7 Hz, raising the question whether oscillations have the same origin in the two mouse models. As rd10 is increasingly being used as a model to develop experimental therapies, it is important to understand the mechanisms underlying the spontaneous rhythmic activity. To study the properties of oscillations in rd10 retina we combined multi electrode recordings with pharmacological manipulation of the retinal network. Oscillations were abolished by blockers for ionotropic glutamate receptors and gap junctions. Frequency and amplitude of oscillations were modulated strongly by blockers of inhibitory receptors and to a lesser extent by blockers of HCN channels. In summary, although we found certain differences in the pharmacological modulation of rhythmic activity in rd10 compared to rd1, the overall pattern looked similar. This suggests that the generation of rhythmic activity may underlie similar mechanisms in rd1 and rd10 retina. PMID:24918437

  13. An Adult Mouse Model of Vibrio cholerae-induced Diarrhea for Studying Pathogenesis and Potential Therapy of Cholera

    PubMed Central

    Sawasvirojwong, Sutthipong; Srimanote, Potjanee; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2013-01-01

    Cholera is a diarrheal disease causing significant morbidity and mortality worldwide. This study aimed to establish an adult mouse model of Vibrio cholerae-induced diarrhea and to characterize its pathophysiology. Ligated ileal loops of adult mice were inoculated for 6, 9, 12 and 18 h with a classical O1 hypertoxigenic 569B strain of V. cholerae (107 CFU/loop). Time-course studies demonstrated that the optimal period for inducing diarrhea was 12 h post-inoculation, when peak intestinal fluid accumulation (loop/weight ratio of ∼0.2 g/cm) occurred with the highest diarrhea success rate (90%). In addition, pathogenic numbers of V. cholerae (∼109 CFU/g tissue) were recovered from ileal loops at all time points between 6–18 h post-inoculation with the diarrheagenic amount of cholera toxin being detected in the secreted intestinal fluid at 12 h post-inoculation. Interestingly, repeated intraperitoneal administration of CFTRinh-172 (20 µg every 6 h), an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR), completely abolished the V. cholerae-induced intestinal fluid secretion without affecting V. cholerae growth in vivo. As analyzed by ex vivo measurement of intestinal electrical resistance and in vivo assay of fluorescein thiocyanate (FITC)-dextran trans-intestinal flux, V. cholerae infection had no effect on intestinal paracellular permeability. Measurements of albumin in the diarrheal fluid suggested that vascular leakage did not contribute to the pathogenesis of diarrhea in this model. Furthermore, histological examination of V. cholerae-infected intestinal tissues illustrated edematous submucosa, congestion of small vessels and enhanced mucus secretion from goblet cells. This study established a new adult mouse model of V. cholerae-induced diarrhea, which could be useful for studying the pathogenesis of cholera diarrhea and for evaluating future therapeutics/cholera vaccines. In addition, our study confirmed the major role of CFTR in V

  14. An Adult Mouse Model of Vibrio cholerae-induced Diarrhea for Studying Pathogenesis and Potential Therapy of Cholera.

    PubMed

    Sawasvirojwong, Sutthipong; Srimanote, Potjanee; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2013-06-01

    Cholera is a diarrheal disease causing significant morbidity and mortality worldwide. This study aimed to establish an adult mouse model of Vibrio cholerae-induced diarrhea and to characterize its pathophysiology. Ligated ileal loops of adult mice were inoculated for 6, 9, 12 and 18 h with a classical O1 hypertoxigenic 569B strain of V. cholerae (10(7) CFU/loop). Time-course studies demonstrated that the optimal period for inducing diarrhea was 12 h post-inoculation, when peak intestinal fluid accumulation (loop/weight ratio of ∼0.2 g/cm) occurred with the highest diarrhea success rate (90%). In addition, pathogenic numbers of V. cholerae (∼10(9) CFU/g tissue) were recovered from ileal loops at all time points between 6-18 h post-inoculation with the diarrheagenic amount of cholera toxin being detected in the secreted intestinal fluid at 12 h post-inoculation. Interestingly, repeated intraperitoneal administration of CFTRinh-172 (20 µg every 6 h), an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR), completely abolished the V. cholerae-induced intestinal fluid secretion without affecting V. cholerae growth in vivo. As analyzed by ex vivo measurement of intestinal electrical resistance and in vivo assay of fluorescein thiocyanate (FITC)-dextran trans-intestinal flux, V. cholerae infection had no effect on intestinal paracellular permeability. Measurements of albumin in the diarrheal fluid suggested that vascular leakage did not contribute to the pathogenesis of diarrhea in this model. Furthermore, histological examination of V. cholerae-infected intestinal tissues illustrated edematous submucosa, congestion of small vessels and enhanced mucus secretion from goblet cells. This study established a new adult mouse model of V. cholerae-induced diarrhea, which could be useful for studying the pathogenesis of cholera diarrhea and for evaluating future therapeutics/cholera vaccines. In addition, our study confirmed the major role of CFTR in V

  15. Repair of liver mediated by adult mouse liver neuro-glia antigen 2-positive progenitor cell transplantation in a mouse model of cirrhosis

    PubMed Central

    Zhang, Hongyu; Siegel, Christopher T.; Shuai, Ling; Lai, Jiejuan; Zeng, Linli; Zhang, Yujun; Lai, Xiangdong; Bie, Ping; Bai, Lianhua

    2016-01-01

    NG2-expressing cells are a population of periportal vascular stem/progenitors (MLpvNG2+ cells) that were isolated from healthy adult mouse liver by using a “Percoll-Plate-Wait” procedure. We demonstrated that isolated cells are able to restore liver function after transplantation into a cirrhotic liver, and co-localized with the pericyte marker (immunohistochemistry: PDGFR-β) and CK19. Cells were positive for: stem cell (Sca-1, CD133, Dlk) and liver stem cell markers (EpCAM, CD14, CD24, CD49f); and negative for: hematopoietic (CD34, CD45) and endothelial markers (CD31, vWf, von Willebrand factor). Cells were transplanted (1 × 106 cells) in mice with diethylnitrosamine-induced cirrhosis at week 6. Cells showed increased hepatic associated gene expression of alpha-fetoprotein (AFP), Albumin (Alb), Glucose-6-phosphatase (G6Pc), SRY (sex determining region Y)-box 9 (Sox9), hepatic nuclear factors (HNF1a, HNF1β, HNF3β, HNF4α, HNF6, Epithelial cell adhesion molecule (EpCAM), Leucine-rich repeated-containing G-protein coupled receptor 5-positive (Lgr5) and Tyrosine aminotransferase (TAT). Cells showed decreased fibrogenesis, hepatic stellate cell infiltration, Kupffer cells and inflammatory cytokines. Liver function markers improved. In a cirrhotic liver environment, cells could differentiate into hepatic lineages. In addition, grafted MLpvNG2+ cells could mobilize endogenous stem/progenitors to participate in liver repair. These results suggest that MLpvNG2+ cells may be novel adult liver progenitors that participate in liver regeneration. PMID:26905303

  16. A hierarchical artificial retina architecture

    NASA Astrophysics Data System (ADS)

    Parker, Alice C.; Azar, Adi N.

    2009-05-01

    Connectivity in the human retina is complex. Over one hundred million photoreceptors transduce light into electrical signals. These electrical signals are sent to the ganglion cells through amacrine and bipolar cells. Lateral connections involving horizontal and amacrine cells span throughout the outer plexiform layer and inner plexiform layer respectively. Horizontal cells are important for photoreceptor regulation by depolarizing them after an illumination occurs. Horizontal cells themselves form an electrical network that communicates by gap junctions, and these cells exhibit plasticity (change in behavior and structure) with respect to glycine receptors. The bipolar and amacrine cells transfer electrical signals from photoreceptors to the ganglion cells. Furthermore, amacrine cells are responsible for further processing the retinal image. Finally, the ganglion cells receive electrical signals from the bipolar and amacrine cells and will spike at a faster rate if there is a change in the overall intensity for a group of photoreceptors, sending a signal to the brain. Dramatic progress is being made with respect to retinal prostheses, raising hope for an entire synthetic retina in the future. We propose a bio-inspired 3D hierarchical pyramidal architecture for a synthetic retina that mimics the overall structure of the human retina. We chose to use a 3D architecture to facilitate connectivity among retinal cells, maintaining a hierarchical structure similar to that of the biological retina. The first layer of the architecture contains electronic circuits that model photoreceptors and horizontal cells. The second layer contains amacrine and bipolar electronic cells, and the third layer contains ganglion cells. Layer I has the highest number of cells, and layer III has the lowest number of cells, resulting in a pyramidal architecture. In our proposed architecture we intend to use photodetectors to transduce light into electrical signals. We propose to employ

  17. Stimulation of adult hippocampal neurogenesis by physical exercise and enriched environment is disturbed in a CADASIL mouse model

    PubMed Central

    Klein, C.; Schreyer, S.; Kohrs, F. E.; Elhamoury, P.; Pfeffer, A.; Munder, T.; Steiner, B.

    2017-01-01

    In the course of CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), a dysregulated adult hippocampal neurogenesis has been suggested as a potential mechanism for early cognitive decline. Previous work has shown that mice overexpressing wild type Notch3 and mice overexpressing Notch3 with a CADASIL mutation display impaired cell proliferation and survival of newly born hippocampal neurons prior to vascular abnormalities. Here, we aimed to elucidate how the long-term survival of these newly generated neurons is regulated by Notch3. Knowing that adult neurogenesis can be robustly stimulated by physical exercise and environmental enrichment, we also investigated the influence of such stimuli as potential therapeutic instruments for a dysregulated hippocampal neurogenesis in the CADASIL mouse model. Therefore, young-adult female mice were housed in standard (STD), environmentally enriched (ENR) or running wheel cages (RUN) for either 28 days or 6 months. Mice overexpressing mutated Notch3 and developing CADASIL (TgN3R169C), and mice overexpressing wild type Notch3 (TgN3WT) were used. We found that neurogenic stimulation by RUN and ENR is apparently impaired in both transgenic lines. The finding suggests that a disturbed neurogenic process due to Notch3-dependent micromilieu changes might be one vascular-independent mechanism contributing to cognitive decline observed in CADASIL. PMID:28345617

  18. Combined 3DISCO clearing method, retrograde tracer and ultramicroscopy to map corneal neurons in a whole adult mouse trigeminal ganglion.

    PubMed

    Launay, Pierre-Serge; Godefroy, David; Khabou, Hanen; Rostene, William; Sahel, Jose-Alain; Baudouin, Christophe; Melik Parsadaniantz, Stéphane; Reaux-Le Goazigo, Annabelle

    2015-10-01

    Tissue clearing and subsequent imaging of intact transparent tissues have provided an innovative way to analyze anatomical pathways in the nervous system. In this study, we combined a recent 3-dimensional imaging of solvent cleared organ (3DISCO) procedure, light-sheet microscopy, fluorescent retrograde tracer, and Imaris software to 3D map corneal sensory neurons within a whole adult mouse trigeminal ganglion (TG). We first established the optimized steps to easily and rapidly clear a fixed TG. We found that the 3DISCO procedure gave excellent results and took less than 3 h to clear the TG. In a second set of experiments, a retrograde tracer (cholera toxin B Alexa 594-conjugated) was applied to de-epithelialized cornea to retrograde-labeled corneal sensory neurons. Two days later, TGs were cleared by the 3DISCO method and serial imaging was performed using light-sheet ultramicroscopic technology. High-resolution images of labeled neurons can be easily and rapidly obtained from a 3D reconstructed whole mouse TG. We then provided a 3D reconstruction of corneal afferent neurons and analyzed their precise localization in the TG. Thus, we showed that neurons supplying corneal sensory innervation exhibit a highly specific limited dorsomedial localization within the TG. We report that our combined method offers the possibility to perform manual (on 20 μm sections) and automated (on 3D reconstructed TG) counting of labeled cells in a cleared mouse TG. To conclude, we illustrate that the combination of the 3DISCO clearing method with light-sheet microscopy, retrograde tracer, and automatic counting represents a rapid and reliable method to analyze a subpopulation of neurons within the peripheral and central nervous system.

  19. "The preadipocyte factor" DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential.

    PubMed

    Andersen, Ditte Caroline; Jensen, Line; Schrøder, Henrik Daa; Jensen, Charlotte Harken

    2009-09-03

    Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as "the" preadipocyte marker, yet the phenotype of DLK1(+) cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1(+) cells encompass around 1-2% of the adult mouse adipose stromal vascular fraction (SVF). Unexpectedly, the DLK1(+)SVF population was enriched for cells expressing genes generally ascribed to the vascular lineage and did not possess any adipogenic differentiation potential in vitro. Instead, DLK1(+) cells comprised an immediate ability for cobblestone formation, generation of tube-like structures on matrigel, and uptake of Acetylated Low Density-Lipoprotein, all characteristics of endothelial cells. We therefore suggest that DLK1(+)SVF cells are of a vascular origin and not them-selves committed preadipocytes as assumed hitherto.

  20. Different tumours induced by benzo(a)pyrene and its 7,8-dihydrodiol injected into adult mouse salivary gland.

    PubMed Central

    Wigley, C. B.; Amos, J.; Brookes, P.

    1978-01-01

    A comparison has been made between the carcinogenic activities of benzo(a)pyrene and the proposed proximate carcinogen, benzo(a)pyrene 7,8-dihydrodiol, in the adult C57BL mouse submandibular salivary gland. In preliminary studies using a range of doses, the dihydrodiol was slightly less active than the parent hydrocarbon in this system. There was a difference in the type of tumour induced by the 2 compounds. Benzo(a)pyrene induced tumours of the salivary glands at the site of injection, whereas the dihydrodiol induced malignant lymphosarcomas, particularly of the thymus, which were often metastatic to other orgnas. Possible reasons for the different sites of action of the 2 compounds are discussed. PMID:580763

  1. Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease.

    PubMed

    Kim, Hye Yun; Kim, Hyunjin V; Yoon, Jin H; Kang, Bo Ram; Cho, Soo Min; Lee, Sejin; Kim, Ji Yoon; Kim, Joo Won; Cho, Yakdol; Woo, Jiwan; Kim, YoungSoo

    2014-12-12

    Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aβ. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aβ-related damages.

  2. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia

    PubMed Central

    Zhang, Qifeng; Smethurst, Elizabeth; Segonds-Pichon, Anne; Schrewe, Heinrich; Wakelam, Michael J. O.

    2016-01-01

    Phospholipase D2 (PLD2) is an enzyme that produces phosphatidic acid (PA), a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO) mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA) regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction. PMID:27658289

  3. Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease

    PubMed Central

    Kim, Hye Yun; Kim, Hyunjin V.; Yoon, Jin H.; Kang, Bo Ram; Cho, Soo Min; Lee, Sejin; Kim, Ji Yoon; Kim, Joo Won; Cho, Yakdol; Woo, Jiwan; Kim, YoungSoo

    2014-01-01

    Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aβ. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aβ-related damages. PMID:25502280

  4. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

    PubMed Central

    Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

    2015-01-01

    Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

  5. Mouse Models of Human T Lymphotropic Virus Type-1–Associated Adult T-Cell Leukemia/Lymphoma

    PubMed Central

    Zimmerman, B.; Niewiesk, S.; Lairmore, M. D.

    2011-01-01

    Human T-lymphotropic virus type-1 (HTLV-1), the first human retrovirus discovered, is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1–associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1–associated diseases has been problematic. Mechanisms of early infection and cell-to-cell transmission can be studied in rabbits and nonhuman primates, but lesion development and reagents are limited in these species. The mouse provides a cost-effective, highly reproducible model in which to study factors related to lymphoma development and the preclinical efficacy of potential therapies against ATL. The ability to manipulate transgenic mice has provided important insight into viral genes responsible for lymphocyte transformation. Expansion of various strains of immunodeficient mice has accelerated the testing of drugs and targeted therapy against ATL. This review compares various mouse models to illustrate recent advances in the understanding of HTLV-1–associated ATL development and how improvements in these models are critical to the future development of targeted therapies against this aggressive T-cell lymphoma. PMID:20442421

  6. Small Fractions of Muscular Dystrophy Embryonic Stem Cells Yield Severe Cardiac and Skeletal Muscle Defects in Adult Mouse Chimeras.

    PubMed

    Gonzalez, J Patrick; Kyrychenko, Sergii; Kyrychenko, Viktoriia; Schneider, Joel S; Granier, Celine J; Himelman, Eric; Lahey, Kevin C; Zhao, Qingshi; Yehia, Ghassan; Tao, Yuan-Xiang; Bhaumik, Mantu; Shirokova, Natalia; Fraidenraich, Diego

    2017-03-01

    Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

  7. PPARβ/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone.

    PubMed

    Bernal, Carolina; Araya, Claudia; Palma, Verónica; Bronfman, Miguel

    2015-01-01

    The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARβ/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARβ/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARβ/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARβ/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARβ/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis.

  8. PPARβ/δ and PPARγ maintain undifferentiated phenotypes of mouse adult neural precursor cells from the subventricular zone

    PubMed Central

    Bernal, Carolina; Araya, Claudia; Palma, Verónica; Bronfman, Miguel

    2015-01-01

    The subventricular zone (SVZ) is one of the main niches of neural stem cells in the adult mammalian brain. Stem and precursor cells in this region are the source for neurogenesis and oligodendrogesis, mainly in the olfactory bulb and corpus callosum, respectively. The identification of the molecular components regulating the decision of these cells to differentiate or maintain an undifferentiated state is important in order to understand the modulation of neurogenic processes in physiological and pathological conditions. PPARs are a group of transcription factors, activated by lipid ligands, with important functions in cellular differentiation and proliferation in several tissues. In this work, we demonstrate that mouse adult neural precursor cells (NPCs), in situ and in vitro, express PPARβ/δ and PPARγ. Pharmacological activation of both PPARs isoforms induces proliferation and maintenance of the undifferentiated phenotype. Congruently, inhibition of PPARβ/δ and PPARγ results in a decrease of proliferation and loss of the undifferentiated phenotype. Interestingly, PPARγ regulates the level of EGFR in adult NPCs, concurrent with it is function described in embryonic NPCs. Furthermore, we describe for the first time that PPARβ/δ regulates SOX2 level in adult NPCs, probably through a direct transcriptional regulation, as we identified two putative PPAR response elements in the promoter region of Sox2. EGFR and SOX2 are key players in neural stem/precursor cells self-renewal. Finally, rosiglitazone, a PPARγ ligand, increases PPARβ/δ level, suggesting a possible cooperation between these two PPARs in the control of cell fate behavior. Our work contributes to the understanding of the molecular mechanisms associated to neural cell fate decision and places PPARβ/δ and PPARγ as interesting new targets of modulation of mammalian brain homeostasis. PMID:25852474

  9. Morphometric changes in C57BL/6 mice retina infected by Toxoplasma gondii ME 49 strain.

    PubMed

    Rocha, Ana Cristina Higino; Calabrese, Kátia da Silva; Tedesco, Roberto Carlos; Campos, Wesley Ribeiro; Neto, Miguel Houri; Vasconcelos, Anilton Cezar; Oréfice, Fernando

    2014-01-01

    This study evaluated the morphometric implications in C57BL/6 mouse retina infected by Toxoplasma gondii, ME 49 strain. Twenty C57BL/6 female mice were divided into group 1 (n=8, intraperitoneally infected with 30 cysts of T. gondii ME 49 strain) and group 2 (n=12 non-infected controls). The eyes were enucleated on the 60th day after infection, fixed and processed for light microscopy. Changes in retinal thickness and in the perimeter/area ratio (P/A) of the retinal layers were analyzed by digital morphometry. We considered that P/A was the measurement of retinal architecture distortion induced by toxoplasmosis. This study considered the ganglion cells and nerve fiber layers as a monolayer, thus six layers of retina were evaluated: photoreceptors (PRL), outer nuclear (ONL), outer plexiform (OPL), inner nuclear (INL), inner plexiform (IPL) and ganglion cells/nerve fiber monolayer (GNL). Histological analysis of infected mouse retina showed inflammatory infiltrate, necrosis, glial reaction and distortion of the retina architecture. It also presented increased thickness (167.8±24.9μm versus 121.1±15.4μm, in controls) and increased retinal thickness within the retinitis foci (187.7±16.6μm versus 147.9±12.2μm out of the retinitis foci). A statistically significant difference in P/A was observed between infected and uninfected mouse retinas. The same was observed in PRL, OPL, INL and GNL. Retinal morphometry may be used to demonstrate differences between infected and uninfected mouse retinas.

  10. Species-specific wiring for direction selectivity in the mammalian retina.

    PubMed

    Ding, Huayu; Smith, Robert G; Poleg-Polsky, Alon; Diamond, Jeffrey S; Briggman, Kevin L

    2016-07-07

    Directionally tuned signalling in starburst amacrine cell (SAC) dendrites lies at the heart of the circuit that detects the direction of moving stimuli in the mammalian retina. The relative contributions of intrinsic cellular properties and network connectivity to SAC direction selectivity remain unclear. Here we present a detailed connectomic reconstruction of SAC circuitry in mouse retina and describe two previously unknown features of synapse distributions along SAC dendrites: input and output synapses are segregated, with inputs restricted to proximal dendrites; and the distribution of inhibitory inputs is fundamentally different from that observed in rabbit retina. An anatomically constrained SAC network model suggests that SAC–SAC wiring differences between mouse and rabbit retina underlie distinct contributions of synaptic inhibition to velocity and contrast tuning and receptive field structure. In particular, the model indicates that mouse connectivity enables SACs to encode lower linear velocities that account for smaller eye diameter, thereby conserving angular velocity tuning. These predictions are confirmed with calcium imaging of mouse SAC dendrites responding to directional stimuli.

  11. Species-specific wiring for direction selectivity in the mammalian retina

    PubMed Central

    Ding, Huayu; Smith, Robert G.; Poleg-Polsky, Alon; Diamond, Jeffrey S.; Briggman, Kevin L.

    2016-01-01

    Summary Directionally tuned signaling in starburst amacrine cell (SAC) dendrites lies at the heart of the direction selective (DS) circuit in the mammalian retina. The relative contributions of intrinsic cellular properties and network connectivity to SAC DS remain unclear. We present a detailed connectomic reconstruction of SAC circuitry in mouse retina and describe previously unknown features of synapse distributions along SAC dendrites: 1) input and output synapses are segregated, with inputs restricted to proximal dendrites; 2) the distribution of inhibitory inputs is fundamentally different from that observed in rabbit retina. An anatomically constrained SAC network model suggests that SAC-SAC wiring differences between mouse and rabbit retina underlie distinct contributions of synaptic inhibition to velocity and contrast tuning and receptive field structure. In particular, the model indicates that mouse connectivity enables SACs to encode lower linear velocities that account for smaller eye diameter, thereby conserving angular velocity tuning. These predictions are confirmed with calcium imaging of mouse SAC dendrites in response to directional stimuli. PMID:27350241

  12. MEMS technologies for artificial retinas

    NASA Astrophysics Data System (ADS)

    Mokwa, Wilfried

    2010-02-01

    The mostly cause of blindness in the developed countries is a degeneration of the retina. For restoring this loss of vision one possible approach is the substitution of the lost functions by means of an electronic implant. This approach is based on MEMS technologies. It has been shown that electrical stimulation of retinal ganglion cells yield visual sensations1. Therefore, an artificial retina for blind humans based on this concept seems to be feasible. Besides electrical stimulation of retinal ganglion cells also the direct electrical stimulation of the optic nerve2 and the visual cortex3 have been under investigation. This paper wants to give an overview about the activities on the retinal ganglion cell stimulation.

  13. Generation of a conditional mouse model to target Acvr1b disruption in adult tissues.

    PubMed

    Ripoche, Doriane; Gout, Johann; Pommier, Roxane M; Jaafar, Rami; Zhang, Chang X; Bartholin, Laurent; Bertolino, Philippe

    2013-02-01

    Alk4 is a type I receptor that belongs to the transforming growth factor-beta (TGF-β) family. It takes part in the signaling of TGF-β ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult-tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed-allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre-mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen-inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue-specific contexts.

  14. GC-MS metabolomic analysis reveals significant alterations in cerebellar metabolic physiology in a mouse model of adult onset hypothyroidism.

    PubMed

    Constantinou, Caterina; Chrysanthopoulos, Panagiotis K; Margarity, Marigoula; Klapa, Maria I

    2011-02-04

    Although adult-onset hypothyroidism (AOH) has been connected to neural activity alterations, including movement, behavioral, and mental dysfunctions, the underlying changes in brain metabolic physiology have not been investigated in a systemic and systematic way. The current knowledge remains fragmented, referring to different experimental setups and recovered from various brain regions. In this study, we developed and applied a gas chromatography-mass spectrometry (GC-MS) metabolomics protocol to obtain a holistic view of the cerebellar metabolic physiology in a Balb/cJ mouse model of prolonged adult-onset hypothyroidism induced by a 64-day treatment with 1% potassium perchlorate in the drinking water of the animals. The high-throughput analysis enabled the correlation between multiple parallel-occurring metabolic phenomena; some have been previously related to AOH, while others implicated new pathways, designating new directions for further research. Specifically, an overall decline in the metabolic activity of the hypothyroid compared to the euthyroid cerebellum was observed, characteristically manifested in energy metabolism, glutamate/glutamine metabolism, osmolytic/antioxidant capacity, and protein/lipid synthesis. These alterations provide strong evidence that the mammalian cerebellum is metabolically responsive to AOH. In light of the cerebellum core functions and its increasingly recognized role in neurocognition, these findings further support the known phenotypic manifestations of AOH into movement and cognitive dysfunctions.

  15. Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

    SciTech Connect

    Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.; Wilson, Amber; Xia, Linghui; Yu, Hong; Webster, Keith A.

    2010-06-11

    Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

  16. Comparative study of Pax2 expression in glial cells in the retina and optic nerve of birds and mammals.

    PubMed

    Stanke, Jennifer; Moose, Holly E; El-Hodiri, Heithem M; Fischer, Andy J

    2010-06-15

    Little is known about the expression of Pax2 in mature retina or optic nerve. Here we probed for the expression of Pax2 in late stages of embryonic development and in mature chick retina. We find two distinct Pax2 isoforms expressed by cells within the retina and optic nerve. Surprisingly, Müller glia in central regions of the retina express Pax2, and levels of expression are decreased with increasing distance from the nerve head. In Müller glia, the expression levels of Pax2 are increased by acute retinal damage or treatment with growth factors. At the optic nerve, Pax2 is expressed by peripapillary glia, at the junction of the neural retina and optic nerve head and by glia within the optic nerve. In addition, we assayed for Pax2 expression in glial cells in mammalian retinas. In mammalian retinas, unlike the case in chick retina, the Müller glia do not express Pax2. Pax2-expressing cells are found in the optic nerve and astrocytes within the mouse retina. By comparison, Pax2-positive cells are not found within the guinea pig retina; Pax2-expressing glia are confined to the optic nerve. In dog and monkey (Macaca fascicularis), Pax2 is expressed by astrocytes that are scattered across inner retinal layers and by numerous glia within the optic nerve. Interestingly, Pax2-positive glial cells are found at the peripheral edge of the dog retina, but only in older animals. We conclude that the expression of Pax2 in the vertebrate eye is restricted to retinal astrocytes, peripapillary glia, and glia within the optic nerve.

  17. The expression and function of midkine in the vertebrate retina

    PubMed Central

    Gramage, E; Li, J; Hitchcock, P

    2014-01-01

    The functional role of midkine during development, following injury and in disease has been studied in a variety of tissues. In this review, we summarize what is known about midkine in the vertebrate retina, focusing largely on recent studies utilizing the zebrafish (Danio rerio) as an animal model. Zebrafish are a valuable animal model for studying the retina, due to its very rapid development and amazing ability for functional neuronal regeneration following neuronal cell death. The zebrafish genome harbours two midkine paralogues, midkine-a (mdka) and midkine-b (mdkb), which, during development, are expressed in nested patterns among different cell types. mdka is expressed in the retinal progenitors and mdkb is expressed in newly post-mitotic cells. Interestingly, studies of loss-and gain-of-function in zebrafish larvae indicate that midkine-a regulates cell cycle kinetics. Moreover, both mdka and mdkb are expressed in different cell types in the normal adult zebrafish retina, but after light-induced death of photoreceptors, both are up-regulated and expressed in proliferating Müller glia and photoreceptor progenitors, suggesting an important and (perhaps) coincident role for these cytokines during stem cell-based neuronal regeneration. Based on its known role in other tissues and the expression and function of the midkine paralogues in the zebrafish retina, we propose that midkine has an important functional role both during development and regeneration in the retina. Further studies are needed to understand this role and the mechanisms that underlie it. Linked Articles This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4 PMID:24460673

  18. Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling.

    PubMed

    Yu, Zhi-Bin; Wei, Hongguang; Jin, J-P

    2012-07-01

    Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.

  19. Topographic differences in adult neurogenesis in the mouse hippocampus: a stereology-based study using endogenous markers.

    PubMed

    Jinno, Shozo

    2011-05-01

    The hippocampus plays a critical role in various cognitive and affective functions. Increasing evidence shows that these functions are topographically distributed along the dorsoventral (septotemporal) and transverse axes of the hippocampus. For instance, dorsal hippocampus is involved in spatial memory and learning whereas ventral hippocampus is related to emotion. Here, we examined the topographic differences (dorsal vs. ventral; suprapyramidal vs. infrapyramidal) in adult neurogenesis in the mouse hippocampus using endogenous markers. The optical disector was applied to estimate the numerical densities (NDs) of labeled cells in the granule cell layer. The NDs of radial glia-like progenitors labeled by brain lipid binding protein were significantly lower in the infrapyramidal blade of the ventral DG than in other subdivisions. The NDs of doublecortin-expressing cells presumed neural progenitors and immature granule cells were significantly higher in the suprapyramidal blade of the dorsal DG than in the other subdivisions. The NDs of calretinin-expressing cells presumed young granule cells at the postmitotic stage were significantly higher in the suprapyramidal blade than in the infrapyramidal blade in the dorsal DG. No significant regional differences were detected in the NDs of dividing cells identified by proliferating cell nuclear antigen. Taken together, these findings suggest that a larger pool of immature granule cells in dorsal hippocampus might be responsible for spatial learning and memory, whereas a smaller pool of radial glia-like progenitors in ventral hippocampus might be associated with the susceptibility to affective disorders. Cell number estimation using a 300-μm-thick hypothetical slice indicates that regional differences in immature cells might contribute to the formation of topographic gradients in mature granule cells in the adult hippocampus. Our data also emphasizes the importance of considering such differences when evaluating changes in

  20. HOXA5 localization in postnatal and adult mouse brain is suggestive of regulatory roles in postmitotic neurons.

    PubMed

    Lizen, Benoit; Hutlet, Bertrand; Bissen, Diane; Sauvegarde, Deborah; Hermant, Maryse; Ahn, Marie-Thérèse; Gofflot, Françoise

    2017-04-01

    Hoxa5 is a member of the Hox gene family, which plays critical roles in successive steps of the central nervous system formation during embryonic and fetal development. Hoxa5 expression in the adult mouse brain has been reported, suggesting that this gene may be functionally required in the brain after birth. To provide further insight into the Hoxa5 expression pattern and potential functions in the brain, we have characterized its neuroanatomical profile from embryonic stages to adulthood. While most Hox mapping studies have been based solely on transcript analysis, we extended our analysis to HOXA5 protein localization in adulthood using specific antibodies. Our results show that Hoxa5 expression appears in the most caudal part of the hindbrain at fetal stages, where it is maintained until adulthood. In the medulla oblongata and pons, we detected Hoxa5 expression in many precerebellar neurons and in several nuclei implicated in the control of autonomic functions. In these territories, the HOXA5 protein is present solely in neurons, specifically in γ-aminobutyric acid (GABA)ergic, glutamatergic, and catecholaminergic neurons. Finally, we also detected Hoxa5 transcripts, but not the HOXA5 protein, in the thalamus and the cortex, from postnatal stages to adult stages, and in the cerebellum at adulthood. We provide evidence that some larger variants of Hoxa5 transcripts are present in these territories. Our mapping analysis allowed us to build hypotheses regarding HOXA5 functions in the nervous system after birth, such as a potential role in the establishment and refinement/plasticity of precerebellar circuits during postnatal and adult life. J. Comp. Neurol. 525:1155-1175, 2017. © 2016 Wiley Periodicals, Inc.

  1. The Effects of Metformin on Obesity-Induced Dysfunctional Retinas

    PubMed Central

    Kim, Andy Jeesu; Chang, Janet Ya-An; Shi, Liheng; Chang, Richard Cheng-An; Ko, Michael Lee; Ko, Gladys Yi-Ping

    2017-01-01

    Purpose The purpose of this study was to determine the effects of metformin on dysfunctional retinas in obesity-induced type 2 diabetic mice. Methods A high-fat diet (HFD)-induced diabetic mouse model (C57BL/6J) was used in this study. After 2 months of the HFD regimen, HFD mice were given daily metformin through oral gavage. Body weights, glucose tolerance, and retinal light responses were monitored regularly. Fluorescein angiography (FA) was used to assess changes in retinal vasculature. Ocular tissues (retina, vitreous, and lens) were harvested and analyzed for molecular changes as determined by immunofluorescent staining, Western blot analysis, and cytokine profiling. Results Starting 1 month after the diet regimen, mice fed the HFD had mildly compromised retinal light responses as measured by electroretinography (ERG), which worsened over time compared to that in the control. In HFD mice treated with metformin, systemic glucose levels reverted back to normal, and their weight gain slowed. Metformin reversed HFD-induced changes in phosphorylated protein kinase B (pAKT), extracellular signal-regulated kinase (pERK), and 5′AMP-activated protein kinase (pAMPK) in the retina. However, metformin treatments for 3 months did not restore the retinal light responses nor lessen the HFD-induced retinal neovascularization, even though it did reduce intraocular inflammation. Conclusions Although metformin was able to reverse systemic changes induced by HFD, it was not able to restore HFD-caused retinal light responses or deter neovascularization. PMID:28114566

  2. Genistein Exposure Inhibits Growth and Alters Steroidogenesis in Adult Mouse Antral Follicles

    PubMed Central

    Patel, Shreya; Peretz, Jackye; Pan, Yuan-Xiang; Helferich, William G.; Flaws, Jodi A.

    2016-01-01

    Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18 – 96 hours (h). Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. PMID:26792615

  3. Characterization and isolation of immature neurons of the adult mouse piriform cortex.

    PubMed

    Rubio, A; Belles, M; Belenguer, G; Vidueira, S; Fariñas, I; Nacher, J

    2016-07-01

    Physiological studies indicate that the piriform or primary olfactory cortex of adult mammals exhibits a high degree of synaptic plasticity. Interestingly, a subpopulation of cells in the layer II of the adult piriform cortex expresses neurodevelopmental markers, such as the polysialylated form of neural cell adhesion molecule (PSA-NCAM) or doublecortin (DCX). This study analyzes the nature, origin, and potential function of these poorly understood cells in mice. As previously described in rats, most of the PSA-NCAM expressing cells in layer II could be morphologically classified as tangled cells and only a small proportion of larger cells could be considered semilunar-pyramidal transitional neurons. Most were also immunoreactive for DCX, confirming their immature nature. In agreement with this, detection of PSA-NCAM combined with that of different cell lineage-specific antigens revealed that most PSA-NCAM positive cells did not co-express markers of glial cells or mature neurons. Their time of origin was evaluated by birthdating experiments with halogenated nucleosides performed at different developmental stages and in adulthood. We found that virtually all cells in this paleocortical region, including PSA-NCAM-positive cells, are born during fetal development. In addition, proliferation analyses in adult mice revealed that very few cells were cycling in layer II of the piriform cortex and that none of them was PSA-NCAM-positive. Moreover, we have established conditions to isolate and culture these immature neurons in the adult piriform cortex layer II. We find that although they can survive under certain conditions, they do not proliferate in vitro either. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 748-763, 2016.

  4. Early exposure to ethanol differentially affects ethanol preference at adult age in two inbred mouse strains.

    PubMed

    Molet, Jenny; Bouaziz, Elodie; Hamon, Michel; Lanfumey, Laurence

    2012-08-01

    Although the acute effects of ethanol exposure on brain development have been extensively studied, the long term consequences of juvenile ethanol intake on behavior at adult age, regarding especially ethanol consumption, are still poorly known. The aim of this study was to analyze the consequences of ethanol ingestion in juvenile C57BL/6J and DBA/2J mice on ethanol intake and neurobiological regulations at adulthood. Mice were given intragastric ethanol at 4 weeks of age under different protocols and their spontaneous ethanol consumption was assessed in a free choice paradigm at adulthood. Both serotonin 5-HT(1A) and cannabinoid CB1 receptors were investigated using [(35)S]GTP-γ-S binding assay for the juvenile ethanol regimens which modified adult ethanol consumption. In DBA/2J mice, juvenile ethanol ingestion dose-dependently promoted adult spontaneous ethanol consumption. This early ethanol exposure enhanced 5-HT(1A) autoreceptor-mediated [(35)S]GTP-γ-S binding in the dorsal raphe nucleus and reduced CB1 receptor-mediated G protein coupling in both the striatum and the globus pallidus at adult age. In contrast, early ethanol ingestion by C57BL/6J mice transiently lowered spontaneous ethanol consumption and increased G protein coupling of postsynaptic 5-HT(1A) receptors in the hippocampus but had no effect on CB1 receptors at adulthood. These results show that a brief and early exposure to ethanol can induce strain-dependent long-lasting changes in both behavior toward ethanol and key receptors of central 5-HT and CB systems in mice.

  5. MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary.

    PubMed

    Mishima, Takuya; Takizawa, Takami; Luo, Shan-Shun; Ishibashi, Osamu; Kawahigashi, Yutaka; Mizuguchi, Yoshiaki; Ishikawa, Tomoko; Mori, Miki; Kanda, Tomohiro; Goto, Tadashi; Takizawa, Toshihiro

    2008-12-01

    MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ- and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10,852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10,000 clones per organ), which included 6630 (159 genes) and 10,192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10,852 small RNA clones) and 87% (10,192/11,744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs.

  6. Electrical and chemical synapses among parvalbumin fast-spiking GABAergic interneurons in adult mouse neocortex

    PubMed Central

    Galarreta, Mario; Hestrin, Shaul

    2002-01-01

    Networks of γ-aminobutyric acid (GABA)ergic interneurons connected via electrical and chemical synapses are thought to play an important role in detecting and promoting synchronous activity in the cerebral cortex. Although the properties of electrical and chemical synaptic interactions among inhibitory interneurons are critical for their function as a network, they have only been studied systematically in juvenile animals. Here, we have used transgenic mice expressing the enhanced green fluorescent protein in cells containing parvalbumin (PV) to study the synaptic connectivity among fast-spiking (FS) cells in slices from adult animals (2–7 months old). We have recorded from pairs of PV-FS cells and found that the majority of them were electrically coupled (61%, 14 of 23 pairs). In addition, 78% of the pairs were connected via GABAergic chemical synapses, often reciprocally. The average coupling coefficient for step injections was 1.5% (n = 14), a smaller value than that reported in juvenile animals. GABA-mediated inhibitory postsynaptic currents and potentials decayed with exponential time constants of 2.6 and 5.9 ms, respectively, and exhibited paired-pulse depression (50-ms interval). The inhibitory synaptic responses in the adult were faster than those observed in young animals. Our results indicate that PV-FS cells are highly interconnected in the adult cerebral cortex by both electrical and chemical synapses, establishing networks that can have important implications for coordinating activity in cortical circuits. PMID:12213962

  7. Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle.

    PubMed

    Lei, Hulong; Yu, Bing; Huang, Zhiqing; Yang, Xuerong; Liu, Zehui; Mao, Xiangbing; Tian, Gang; He, Jun; Han, Guoquan; Chen, Hong; Mao, Qian; Chen, Daiwen

    2013-02-01

    Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P < 0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P < 0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P < 0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14 days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P < 0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P < 0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.

  8. Changes in the neural representation of odorants after olfactory deprivation in the adult mouse olfactory bulb.

    PubMed

    Kass, Marley D; Pottackal, Joseph; Turkel, Daniel J; McGann, John P

    2013-01-01

    Olfactory sensory deprivation during development has been shown to induce significant alterations in the neurophysiology of olfactory receptor neurons (ORNs), the primary sensory inputs to the brain's olfactory bulb. Deprivation has also been shown to alter the neurochemistry of the adult olfactory system, but the physiological consequences of these changes are poorly understood. Here we used in vivo synaptopHluorin (spH) imaging to visualize odorant-evoked neurotransmitter release from ORNs in adult transgenic mice that underwent 4 weeks of unilateral olfactory deprivation. Deprivation reduced odorant-evoked spH signals compared with sham-occluded mice. Unexpectedly, this reduction was equivalent between ORNs on the open and plugged sides. Changes in odorant selectivity of glomerular subpopulations of ORNs were also observed, but only in ORNs on the open side of deprived mice. These results suggest that naris occlusion in adult mice produces substantial changes in primary olfactory processing which may reflect not only the decrease in olfactory stimulation on the occluded side but also the alteration of response properties on the intact side. We also observed a modest effect of true sham occlusions that included noseplug insertion and removal, suggesting that conventional noseplug techniques may have physiological effects independent of deprivation per se and thus require more careful controls than has been previously appreciated.

  9. Immunolocalization of the P2X4 receptor on neurons and glia in the mammalian retina.

    PubMed

    Ho, T; Vessey, K A; Fletcher, E L

    2014-09-26

    Extracellular adenosine 5'-triphosphate (eATP) acts as a neurotransmitter within the retina and brain, activating a range of ionotropic P2X and metabotropic P2Y receptors. In this study, the specific localization of the P2X4 receptor (P2X4-R) subunit was evaluated in the retina using fluorescence immunohistochemistry and pre-embedding immuno-electron microscopy. Punctate P2X4-R labeling was largely localized to the inner and outer plexiform layers of mouse, rat and cat retinae. In the mouse outer retina, double-labeling of P2X4-R with the horizontal cell marker, calbindin, revealed P2X4-R immunoreactivity (P2X4-R-IR) on horizontal cell somata and processes. In the inner retina, P2X4-R expression was found closely associated with rod and cone bipolar cell terminals, and the punctate labeling was observed on calretinin-positive amacrine cells. Using immuno-electron microscopy, P2X4-Rs were observed on processes post-synaptic to photoreceptor and bipolar cell terminals, likely representing horizontal, amacrine and ganglion cells, respectively. Furthermore, P2X4-R expression was also observed on Müller cells, astrocytes and microglia. These data suggest a role for P2X4-Rs in the lateral inhibitory pathways of the retina, modulating neuronal function of photoreceptors and bipolar cells. The expression on macro- and microglial cells implicates a role for P2X4-Rs in glial signaling, tissue homeostasis and immunosurveillance within the mammalian retina.

  10. Characterization of muscle spindle afferents in the adult mouse using an in vitro muscle-nerve preparation.

    PubMed

    Wilkinson, Katherine A; Kloefkorn, Heidi E; Hochman, Shawn

    2012-01-01

    We utilized an in vitro adult mouse extensor digitorum longus (EDL) nerve-attached preparation to characterize the responses of muscle spindle afferents to ramp-and-hold stretch and sinusoidal vibratory stimuli. Responses were measured at both room (24°C) and muscle body temperature (34°C). Muscle spindle afferent static firing frequencies increased linearly in response to increasing stretch lengths to accurately encode the magnitude of muscle stretch (tested at 2.5%, 5% and 7.5% of resting length [Lo]). Peak firing frequency increased with ramp speeds (20% Lo/sec, 40% Lo/sec, and 60% Lo/sec). As a population, muscle spindle afferents could entrain 1:1 to sinusoidal vibrations throughout the frequency (10-100 Hz) and amplitude ranges tested (5-100 µm). Most units preferentially entrained to vibration frequencies close to their baseline steady-state firing frequencies. Cooling the muscle to 24°C decreased baseline firing frequency and units correspondingly entrained to slower frequency vibrations. The ramp component of stretch generated dynamic firing responses. These responses and related measures of dynamic sensitivity were not able to categorize units as primary (group Ia) or secondary (group II) even when tested with more extreme length changes (10% Lo). We conclude that the population of spindle afferents combines to encode stretch in a smoothly graded manner over the physiological range of lengths and speeds tested. Overall, spindle afferent response properties were comparable to those seen in other species, supporting subsequent use of the mouse genetic model system for studies on spindle function and dysfunction in an isolated muscle-nerve preparation.

  11. A Computational Framework for Realistic Retina Modeling.

    PubMed

    Martínez-Cañada, Pablo; Morillas, Christian; Pino, Begoña; Ros, Eduardo; Pelayo, Francisco

    2016-11-01

    Computational simulations of the retina have led to valuable insights about the biophysics of its neuronal activity and processing principles. A great number of retina models have been proposed to reproduce the behavioral diversity of the different visual processing pathways. While many of these models share common computational stages, previous efforts have been more focused on fitting specific retina functions rather than generalizing them beyond a particular model. Here, we define a set of computational retinal microcircuits that can be used as basic building blocks for the modeling of different retina mechanisms. To validate the hypothesis that similar processing structures may be repeatedly found in different retina functions, we implemented a series of retina models simply by combining these computational retinal microcircuits. Accuracy of the retina models for capturing neural behavior was assessed by fitting published electrophysiological recordings that characterize some of the best-known phenomena observed in the retina: adaptation to the mean light intensity and temporal contrast, and differential motion sensitivity. The retinal microcircuits are part of a new software platform for efficient computational retina modeling from single-cell to large-scale levels. It includes an interface with spiking neural networks that allows simulation of the spiking response of ganglion cells and integration with models of higher visual areas.

  12. Odour enrichment increases adult-born dopaminergic neurons in the mouse olfactory bulb.

    PubMed

    Bonzano, Sara; Bovetti, Serena; Fasolo, Aldo; Peretto, Paolo; De Marchis, Silvia

    2014-11-01

    The olfactory bulb (OB) is the first brain region involved in the processing of olfactory information. In adult mice, the OB is highly plastic, undergoing cellular/molecular dynamic changes that are modulated by sensory experience. Odour deprivation induces down-regulation of tyrosine hydroxylase (TH) expression in OB dopaminergic interneurons located in the glomerular layer (GL), resulting in decreased dopamine in the OB. Although the effect of sensory deprivation is well established, little is known about the influence of odour enrichment on dopaminergic cells. Here we report that prolonged odour enrichment on C57BL/6J strain mice selectively increases TH-immunopositive cells in the GL by nearly 20%. Following odour enrichment on TH-green fluorescent protein (GFP) transgenic mice, in which GFP identified both mature TH-positive cells and putative immature dopaminergic cells expressing TH mRNA but not TH protein, we found a similar 20% increase in GFP-expressing cells, with no changes in the ratio between TH-positive and TH-negative cells. These data suggest that enriched conditions induce an expansion in the whole dopaminergic lineage. Accordingly, by using 5-bromo-2-deoxyuridine injections to label adult-generated cells in the GL of TH-GFP mice, we found an increase in the percentage of 5-bromo-2-deoxyuridine-positive dopaminergic cells in enriched compared with control conditions, whereas no differences were found for calretinin- and calbindin-positive subtypes. Strikingly, the fraction of newborn cells among the dopaminergic population doubled in enriched conditions. On the whole, our results demonstrate that odour enrichment drives increased integration of adult-generated dopaminergic cells that could be critical to adapt the OB circuits to the environmental incoming information.

  13. Mouse model of CADASIL reveals novel insights into Notch3 function in adult hippocampal neurogenesis.

    PubMed

    Ehret, Fanny; Vogler, Steffen; Pojar, Sherin; Elliott, David A; Bradke, Frank; Steiner, Barbara; Kempermann, Gerd

    2015-03-01

    Could impaired adult hippocampal neurogenesis be a relevant mechanism underlying CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy)? Memory symptoms in CADASIL, the most common hereditary form of vascular dementia, are usually thought to be primarily due to vascular degeneration and white matter lacunes. Since adult hippocampal neurogenesis, a process essential for the integration of new spatial memory occurs in a highly vascularized niche, we considered dysregulation of adult neurogenesis as a potential mechanism for the manifestation of dementia in CADASIL. Analysis in aged mice overexpressing Notch3 with a CADASIL mutation, revealed vascular deficits in arteries of the hippocampal fissure but not in the niche of the dentate gyrus. At 12 months of age, cell proliferation and survival of newborn neurons were reduced not only in CADASIL mice but also in transgenic controls overexpressing wild type Notch3. At 6 months, hippocampal neurogenesis was altered in CADASIL mice independent of overt vascular abnormalities in the fissure. Further, we identified Notch3 expression in hippocampal precursor cells and maturing neurons in vivo as well as in cultured hippocampal precursor cells. Overexpression and knockdown experiments showed that Notch3 signaling negatively regulated precursor cell proliferation. Notch3 overexpression also led to deficits in KCl-induced precursor cell activation. This suggests a cell-autonomous effect of Notch3 signaling in the regulation of precursor proliferation and activation and a loss-of-function effect in CADASIL. Consequently, besides vascular damage, aberrant precursor cell proliferation and differentiation due to Notch3 dysfunction might be an additional independent mechanism for the development of hippocampal dysfunction in CADASIL.

  14. Characterizing Newly Repopulated Microglia in the Adult Mouse: Impacts on Animal Behavior, Cell Morphology, and Neuroinflammation

    PubMed Central

    Elmore, Monica R. P.; Lee, Rafael J.; West, Brian L.; Green, Kim N.

    2015-01-01

    Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R)/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS) resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg) or phosphate buffered saline (PBS) was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR), as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function similarly to the

  15. Reduced Glutamate Release in Adult BTBR Mouse Model of Autism Spectrum Disorder.

    PubMed

    Wei, Hongen; Ma, Yuehong; Ding, Caiyun; Jin, Guorong; Liu, Jianrong; Chang, Qiaoqiao; Hu, Fengyun; Yu, Li

    2016-11-01

    Autism spectrum disorder (ASD) is a developmental disorder characterized by impairments in social and communication abilities, as well as by restricted and repetitive behaviors. The BTBR T (+) Itpr3 (tf) (BTBR) mice have emerged as a well characterized and widely used mouse model of a range of ASD-like phenotype, showing deficiencies in social behaviors and unusual ultrasonic vocalizations as well as increased repetitive self-grooming. However, the inherited neurobiological changes that lead to ASD-like behaviors in these mice are incompletely known and still under active investigation. The aim of this study was to further evaluate the structure and neurotransmitter release of the glutamatergic synapse in BTBR mice. C57BL/6J (B6) mice were used as a control strain because of their high level of sociability. The important results showed that the evoked glutamate release in the cerebral cortex of BTBR mice was significantly lower than in B6 mice. And the level of vesicle docking-related protein Syntaxin-1A was reduced in BTBR mice. However, no significant changes were observed in the number of glutamatergic synapse, level of synaptic proteins, density of dendritic spine and postsynaptic density between BTBR mice and B6 mice. Overall, our results suggest that abnormal vesicular glutamate activity may underlie the ASD relevant pathology in the BTBR mice.

  16. Chronic Social Defeat Stress Modulates Dendritic Spines Structural Plasticity in Adult Mouse Frontal Association Cortex

    PubMed Central

    Shu, Yu

    2017-01-01

    Chronic stress is associated with occurrence of many mental disorders. Previous studies have shown that dendrites and spines of pyramidal neurons of the prefrontal cortex undergo drastic reorganization following chronic stress experience. So the prefrontal cortex is believed to play a key role in response of neural system to chronic stress. However, how stress induces dynamic structural changes in neural circuit of prefrontal cortex remains unknown. In the present study, we examined the effects of chronic social defeat stress on dendritic spine structural plasticity in the mouse frontal association (FrA) cortex in vivo using two-photon microscopy. We found that chronic stress altered spine dynamics in FrA and increased the connectivity in FrA neural circuits. We also found that the changes in spine dynamics in FrA are correlated with the deficit of sucrose preference in defeated mice. Our findings suggest that chronic stress experience leads to adaptive change in neural circuits that may be important for encoding stress experience related memory and anhedonia. PMID:28197343

  17. Competition and Homeostasis of Excitatory and Inhibitory Connectivity in the Adult Mouse Visual Cortex.

    PubMed

    Saiepour, M Hadi; Chakravarthy, Sridhara; Min, Rogier; Levelt, Christiaan N

    2015-10-01

    During cortical development, synaptic competition regulates the formation and adjustment of neuronal connectivity. It is unknown whether synaptic competition remains active in the adult brain and how inhibitory neurons participate in this process. Using morphological and electrophysiological measurements, we show that expressing a dominant-negative form of the TrkB receptor (TrkB.T1) in the majority of pyramidal neurons in the adult visual cortex does not affect excitatory synapse densities. This is in stark contrast to the previously reported loss of excitatory input which occurs if the exact same transgene is expressed in sparse neurons at the same age. This indicates that synaptic competition remains active in adulthood. Additionally, we show that interneurons not expressing the TrkB.T1 transgene may have a competitive advantage and obtain more excitatory synapses when most neighboring pyramidal neurons do express the transgene. Finally, we demonstrate that inhibitory synapses onto pyramidal neurons are reduced when TrkB signaling is interfered with in most pyramidal neurons but not when few pyramidal neurons have this deficit. This adjustment of inhibitory innervation is therefore not a cell-autonomous consequence of decreased TrkB signaling but more likely a homeostatic mechanism compensating for activity changes at the population level.

  18. Differential genomic imprinting regulates paracrine and autocrine roles of IGF2 in mouse adult neurogenesis

    PubMed Central

    Ferrón, S. R.; Radford, E. J.; Domingo-Muelas, A.; Kleine, I.; Ramme, A.; Gray, D.; Sandovici, I.; Constancia, M.; Ward, A.; Menheniott, T. R.; Ferguson-Smith, A. C.

    2015-01-01

    Genomic imprinting is implicated in the control of gene dosage in neurogenic niches. Here we address the importance of Igf2 imprinting for murine adult neurogenesis in the subventricular zone (SVZ) and in the subgranular zone (SGZ) of the hippocampus in vivo. In the SVZ, paracrine IGF2 is a cerebrospinal fluid and endothelial-derived neurogenic factor requiring biallelic expression, with mutants having reduced activation of the stem cell pool and impaired olfactory bulb neurogenesis. In contrast, Igf2 is imprinted in the hippocampus acting as an autocrine factor expressed in neural stem cells (NSCs) solely from the paternal allele. Conditional mutagenesis of Igf2 in blood vessels confirms that endothelial-derived IGF2 contributes to NSC maintenance in SVZ but not in the SGZ, and that this is regulated by the biallelic expression of IGF2 in the vascular compartment. Our findings indicate that a regulatory decision to imprint or not is a functionally important mechanism of transcriptional dosage control in adult neurogenesis. PMID:26369386

  19. Vasoproliferative tumours of the retina

    PubMed Central

    Heimann, H.; Bornfeld, N.; Vij, O.; Coupland, S.; Bechrakis, N.; Kellner, U.; Foerster, M.

    2000-01-01

    BACKGROUND—Vasoproliferative tumours of the retina (VPTR) are benign tumours of unknown origin, occurring mostly in otherwise healthy patients. VPTR may be associated with other chorioretinal diseases, such as uveitis. The tumours, which histologically represent reactive gliovascular proliferations, are characterised by a pink to yellow appearance on funduscopy and are accompanied by exudative and haemorrhagic changes of the retina.
METHODS—22 cases of VPTR in 21 patients were examined with a follow up period between 1 month and 6 years. Ophthalmological changes associated with VPTR were intraretinal and subretinal exudations (n=18), exudative detachments of the surrounding sensory retina (n=13), intraretinal and subretinal haemorrhages (n=10), exudative changes within the macula (n=10), hyperpigmentation of the retinal pigment epithelium at the border of the exudative retinal changes (n=9), and vitreous haemorrhages (n=4). Tumour biopsy was performed in two cases. Treatment consisted of plaque radiotherapy (n=14), plaque radiotherapy and cryotherapy (two), cryotherapy only (two), observation (three), and enucleation in one case of a blind and painful eye.
RESULTS—Regression of the tumour and the associated exudative changes could be observed in all treated cases. Visual acuity at last follow up improved two lines or more in two cases, remained within two lines of the initial visual acuity in 15 cases, and worsened in the remaining five. Histopathological examination of the biopsy specimens and the tumour of the enucleated eye showed massive capillary proliferation with perivascular spindle-shaped glial cells of retinal origin.
CONCLUSION—The correct diagnosis of VPTR is of importance as these lesions may lead to visual loss. Further, VPTR must be differentiated from angiomas associated with von Hippel-Lindau disease as well as from ocular and systemic malignancies. Regression of tumour thickness and associated retinal changes can be achieved with

  20. Innate Immune Network in the Retina Activated by Optic Nerve Crush

    PubMed Central

    Templeton, Justin P.; Freeman, Natalie E.; Nickerson, John M.; Jablonski, Monica M.; Rex, Tonia S.; Williams, Robert W.; Geisert, Eldon E.

    2013-01-01

    Purpose. Innate immunity plays a role in many diseases, including glaucoma and AMD. We have used transcriptome profiling in the mouse to identify a network of genes involved in innate immunity that is present in the normal retina and that is activated by optic nerve crush (ONC). Methods. Using a recombinant inbred (RI) mouse strain set (BXD, C57BL/6 crossed with DBA/2J mice), we generate expression datasets (Illumina WG 6.2 arrays) in the normal mouse retina and 2 days after ONC. The normal dataset is constructed from retinas from 80 mouse strains and the ONC dataset is constructed from 62 strains. These large datasets are hosted by GeneNetwork.org, along with a series of powerful bioinformatic tools. Results. In the retina datasets, one intriguing network involves transcripts associated with the innate immunity. Using C4b to interrogate the normal dataset, we can identify a group of genes that are coregulated across the BXD RI strains. Many of the genes in this network are associated with the innate immune system, including Serping1, Casp1, C3, Icam1, Tgfbr2, Cfi, Clu, C1qg, Aif1, and Cd74. Following ONC, the expression of these genes is upregulated, along with an increase in coordinated expression across the BXD strains. Many of the genes in this network are risk factors for AMD, including C3, EFEMP1, MCDR2, CFB, TLR4, HTA1, and C1QTNF5. Conclusions. We found a retina-intrinsic innate immunity network that is activated by injury including ONC. Many of the genes in this network are risk factors for retinal disease. PMID:23493296

  1. Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors.

    PubMed

    Lorber, Barbara; Berry, Martin; Logan, Ann

    2005-04-01

    In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.

  2. A new method for visualization of endothelial cells and extravascular leakage in adult mouse brain using fluorescein isothiocyanate.

    PubMed

    Miyata, Seiji; Morita, Shoko

    2011-10-30

    We described a new method for the visualization of vasculature and endothelial cells and the assessment of extravascular leakage in adult mouse brain by using fluorescein isothiocyanate (FITC), or a reactive fluorescent dye. FITC is the fluorescein derivative that reacts covalently with amine groups at alkaline pH. In this method, strong fluorescence of FITC was seen at vasculature throughout the brain and spinal cord, when mice received intracardiac perfusion with FITC-containing saline at pH 7.0 followed by paraformaldehyde (PFA) fixative at pH 8.0. The fluorescence of FITC was faint when animals were fixed with PFA fixative at pH 7.0 after the perfusion of FITC-containing saline at pH 7.0. The fluorescence of FITC was not detected when mice was fixed with PFA fixative before the perfusion of FITC-containing saline. Double labeling immunohistochemistry using an endothelial cell marker CD31 or a pericyte marker desmin revealed that FITC was accumulated at nuclei of endothelial cells but not at those of pericytes. Extravascular leakage of FITC was prominent in the area postrema or a brain region of the circumventricular organs that lacks the blood-brain barrier. Moreover, strong extravascular leakage of FITC was detected at damaged sites of the cerebral cortex with cryoinjury. Thus, FITC method is useful technique for examining the architecture of brain vasculature and endothelial cells and the assessment of extravascular leakage in adult rodents. Moreover, FITC binds covalently to cellular components, so that makes it possible to perform double labeling immunohistochemistry and long-term storage of the preparation.

  3. Cre recombinase-regulated Endothelin1 transgenic mouse lines: novel tools for analysis of embryonic and adult disorders

    PubMed Central

    Tavares, Andre L.P.; Clouthier, David E.

    2015-01-01

    Endothelin-1 (EDN1) influences both craniofacial and cardiovascular development and a number of adult physiological conditions by binding to one or both of the known endothelin receptors, thus initiating multiple signaling cascades. Animal models containing both conventional and conditional loss of the Edn1 gene have been used to dissect EDN1 function in both embryos and adults. However, while transgenic Edn1 over-expression or targeted genomic insertion of Edn1 has been performed to understand how elevated levels of Edn1 result in or exacerbate disease states, an animal model in which Edn1 over-expression can be achieved in a spatiotemporal-specific manner has not been reported. Here we describe the creation of Edn1 conditional over-expression transgenic mouse lines in which the chicken β-actin promoter and an Edn1 cDNA are separated by a strong stop sequence flanked by loxP sites. In the presence of Cre, the stop cassette is removed, leading to Edn1 expression. Using the Wnt1-Cre strain, in which Cre expression is targeted to the Wnt1-expressing domain of the central nervous system (CNS) from which neural crest cells (NCCs) arise, we show that stable CBA-Edn1 transgenic lines with varying EDN1 protein levels develop defects in NCC-derived tissues of the face, though the severity differs between lines. We also show that Edn1 expression can be achieved in other embryonic tissues utilizing other Cre strains, with this expression also resulting in developmental defects. CBA-Edn1 transgenic mice will be useful in investigating diverse aspects of EDN1-mediated-development and disease, including understanding how NCCs achieve and maintain a positional and functional identity and how aberrant EDN1 levels can lead to multiple physiological changes and diseases. PMID:25725491

  4. Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse

    PubMed Central

    Zampieri, Alexandre; Champagne, Julien; Auzemery, Baptiste; Fuentes, Ivanna; Maurel, Benjamin; Bienvenu, Frédéric

    2015-01-01

    We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed “Tandem-HTRF”, can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material. PMID:26503526

  5. Build a better mouse: directly-observed issues in computer use for adults with SMI.

    PubMed

    Black, Anne C; Serowik, Kristin L; Schensul, Jean J; Bowen, Anne M; Rosen, Marc I

    2013-03-01

    Integrating information technology into healthcare has the potential to bring treatment to hard-to-reach people. Individuals with serious mental illness (SMI), however, may derive limited benefit from these advances in care because of lack of computer ownership and experience. To date, conclusions about the computer skills and attitudes of adults with SMI have been based primarily on self-report. In the current study, 28 psychiatric outpatients with co-occurring cocaine use were interviewed about their computer use and opinions, and 25 were then directly observed using task analysis and think aloud methods as they navigated a multi-component health informational website. Participants reported low rates of computer ownership and use, and negative attitudes towards computers. Self-reported computer skills were higher than demonstrated in the task analysis. However, some participants spontaneously expressed more positive attitudes and greater computer self-efficacy after navigating the website. Implications for increasing access to computer-based health information are discussed.

  6. Multiple Retinal Axons Converge onto Relay Cells in the Adult Mouse Thalamus.

    PubMed

    Hammer, Sarah; Monavarfeshani, Aboozar; Lemon, Tyler; Su, Jianmin; Fox, Michael Andrew

    2015-09-08

    Activity-dependent refinement of neural circuits is a fundamental principle of neural development. This process has been well studied at retinogeniculate synapses-synapses that form between retinal ganglion cells (RGCs) and relay cells within the dorsal lateral geniculate nucleus. Physiological studies suggest that shortly after birth, inputs from ∼20 RGCs converge onto relay cells. Subsequently, all but just one to two of these inputs are eliminated. Despite widespread acceptance, this notion is at odds with ultrastructural studies showing numerous retinal terminals clustering onto relay cell dendrites in the adult. Here, we explored this discrepancy using brainbow AAVs and serial block face scanning electron microscopy (SBFSEM). Results with both approaches demonstrate that terminals from numerous RGCs cluster onto relay cell dendrites, challenging the notion that only one to two RGCs innervate each relay cell. These findings force us to re-evaluate our understanding of subcortical visual circuitry.

  7. Cyclohexane produces behavioral deficits associated with astrogliosis and microglial reactivity in the adult hippocampus mouse brain.

    PubMed

    Campos-Ordonez, Tania; Zarate-Lopez, David; Galvez-Contreras, Alma Y; Moy-Lopez, Norma; Guzman-Muniz, Jorge; Gonzalez-Perez, Oscar

    2015-05-01

    Cyclohexane is a volatile substance that has been utilized as a safe substitute of several organic solvents in diverse industrial processes, such as adhesives, paints, paint thinners, fingernail polish, lacquers, and rubber industry. A number of these commercial products are ordinarily used as inhaled drugs. However, it is not well known whether cyclohexane has noxious effects in the central nervous system. The aim of this study was to analyze the effects of cyclohexane inhalation on motor behavior, spatial memory, and reactive gliosis in the hippocampus of adult mice. We used a model that mimics recreational drug use in male Balb/C mice (P60), divided into two groups: controls and the cyclohexane group (exposed to 9,000 ppm of cyclohexane for 30 days). Both groups were then evaluated with a functional observational battery (FOB) and the Morris water maze (MWM). Furthermore, the relative expression of AP endonuclease 1 (APE1), and the number of astrocytes (GFAP+ cells) and microglia (Iba1+ cells) were quantified in the hippocampal CA1 and CA3 areas. Our findings indicated that cyclohexane produced severe functional deficits during a recreational exposure as assessed by the FOB. The MWM did not show statistically significant changes in the acquisition and retention of spatial memory. Remarkably, a significant increase in the number of astrocytes and microglia cells, as well as in the cytoplasmic processes of these cells were observed in the hippocampal CA1 and CA3 areas of cyclohexane-exposed mice. This cellular response was associated with an increase in the expression of APE1 in the same brain regions. In summary, cyclohexane exposure produces functional deficits that are associated with an important increase in the APE1 expression as well as the number of astrocytes and microglia cells and their cytoplasmic complexity in the CA1 and CA3 regions of the adult hippocampus.

  8. Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures

    PubMed Central

    Contartese, Daniela S.; Rolón, Federico; Sarotto, Anibal; Dorfman, Veronica B.; Loidl, Cesar F.; Martínez, Alfredo

    2016-01-01

    Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina. PMID:27556928

  9. Cold Shock Proteins Are Expressed in the Retina Following Exposure to Low Temperatures.

    PubMed

    Larrayoz, Ignacio M; Rey-Funes, Manuel; Contartese, Daniela S; Rolón, Federico; Sarotto, Anibal; Dorfman, Veronica B; Loidl, Cesar F; Martínez, Alfredo

    2016-01-01

    Hypothermia has been proposed as a therapeutic intervention for some retinal conditions, including ischemic insults. Cold exposure elevates expression of cold-shock proteins (CSP), including RNA-binding motif protein 3 (RBM3) and cold inducible RNA-binding protein (CIRP), but their presence in mammalian retina is so far unknown. Here we show the effects of hypothermia on the expression of these CSPs in retina-derived cell lines and in the retina of newborn and adult rats. Two cell lines of retinal origin, R28 and mRPE, were exposed to 32°C for different time periods and CSP expression was measured by qRT-PCR and Western blotting. Neonatal and adult Sprague-Dawley rats were exposed to a cold environment (8°C) and expression of CSPs in their retinas was studied by Western blotting, multiple inmunofluorescence, and confocal microscopy. RBM3 expression was upregulated by cold in both R28 and mRPE cells in a time-dependent fashion. On the other hand, CIRP was upregulated in R28 cells but not in mRPE. In vivo, expression of CSPs was negligible in the retina of newborn and adult rats kept at room temperature (24°C). Exposure to a cold environment elicited a strong expression of both proteins, especially in retinal pigment epithelium cells, photoreceptors, bipolar, amacrine and horizontal cells, Müller cells, and ganglion cells. In conclusion, CSP expression rapidly rises in the mammalian retina following exposure to hypothermia in a cell type-specific pattern. This observation may be at the basis of the molecular mechanism by which hypothermia exerts its therapeutic effects in the retina.

  10. Adenosine 5' triphosphate evoked mobilization of intracellular calcium in central nervous system white matter of adult mouse optic nerve.

    PubMed

    James, G; Butt, A M

    1999-06-11

    Although it has been established that immature glial cells express functional purinergic receptors, the responsiveness of mature glial cells in vivo had not been elucidated. This question was addressed using fura-2 ratiometric measurements of [Ca2+]i in the adult mouse optic nerve, a central nervous system (CNS) white matter tract, taking advantage of the facts that (i), the optic nerve contains glial cells but not neurons and (ii), that fura-2 loads primarily astrocytes in isolated intact optic nerves. We show that adenosine 5' triphosphate (ATP) evoked an increase in [Ca2+]i in a concentration-dependent manner with a half-maximal effect at 3 microm ATP, and with a rank order of agonist potency of ATP > ADP > alpha,beta-methyline-ATP > UDP > adenosine. The results indicate mainly P2Y and P2X components, consistent with the in vitro astroglial purinergic receptor profile. The in vivo response of mature glia to ATP may be important in their response to CNS damage.

  11. Biodegradation of the ZnO:Eu nanoparticles in the tissues of adult mouse after alimentary application.

    PubMed

    Kielbik, Paula; Kaszewski, Jaroslaw; Rosowska, Julita; Wolska, Ewelina; Witkowski, Bartłomiej S; Gralak, Mikolaj A; Gajewski, Zdzisław; Godlewski, Marek; Godlewski, Michal M

    2016-11-21

    Biodegradable zinc oxide nanoparticles (ZnO NPs) are considered promising materials for future biomedical applications. To fulfil this potential, biodistribution and elimination patterns of ZnO NPs in the living organism need to be resolved. In order to investigate gastrointestinal absorption of ZnO NPs and their intra-organism distribution, water suspension of ZnO or fluorescent ZnO:Eu (Europium-doped zinc oxide) NPs (10mg/ml; 0.3ml/mouse) was alimentary-administered (IG: intra-gastric) to adult mice. Internal organs collected at key time-points after IG were evaluated by AAS for Zn concentration and analysed by cytometric techniques. We found that Zn-based NPs were readily absorbed and distributed (3 h post IG) in the nanoparticle form throughout the organism. Results suggest, that liver and kidneys were key organs responsible for NPs elimination, while accumulation was observed in the spleen and adipose tissues. We also showed that ZnO/ZnO:Eu NPs were able to cross majority of biological barriers in the organism (including blood-brain-barrier).

  12. Interneuron precursor transplants in adult hippocampus reverse psychosis-relevant features in a mouse model of hippocampal disinhibition.

    PubMed

    Gilani, Ahmed I; Chohan, Muhammad O; Inan, Melis; Schobel, Scott A; Chaudhury, Nashid H; Paskewitz, Samuel; Chuhma, Nao; Glickstein, Sara; Merker, Robert J; Xu, Qing; Small, Scott A; Anderson, Stewart A; Ross, Margaret Elizabeth; Moore, Holly

    2014-05-20

    GABAergic interneuron hypofunction is hypothesized to underlie hippocampal dysfunction in schizophrenia. Here, we use the cyclin D2 knockout (Ccnd2(-/-)) mouse model to test potential links between hippocampal interneuron deficits and psychosis-relevant neurobehavioral phenotypes. Ccnd2(-/-) mice show cortical PV(+) interneuron reductions, prominently in hippocampus, associated with deficits in synaptic inhibition, increased in vivo spike activity of projection neurons, and increased in vivo basal metabolic activity (assessed with fMRI) in hippocampus. Ccnd2(-/-) mice show several neurophysiological and behavioral phenotypes that would be predicted to be produced by hippocampal disinhibition, including increased ventral tegmental area dopamine neuron population activity, behavioral hyperresponsiveness to amphetamine, and impairments in hippocampus-dependent cognition. Remarkably, transplantation of cells from the embryonic medial ganglionic eminence (the major origin of cerebral cortical interneurons) into the adult Ccnd2(-/-) caudoventral hippocampus reverses these psychosis-relevant phenotypes. Surviving neurons from these transplants are 97% GABAergic and widely distributed within the hippocampus. Up to 6 mo after the transplants, in vivo hippocampal metabolic activity is lowered, context-dependent learning and memory is improved, and dopamine neuron activity and the behavioral response to amphetamine are normalized. These findings establish functional links between hippocampal GABA interneuron deficits and psychosis-relevant dopaminergic and cognitive phenotypes, and support a rationale for targeting limbic cortical interneuron function in the prevention and treatment of schizophrenia.

  13. Morphological and behavioural changes occur following the X-ray irradiation of the adult mouse olfactory neuroepithelium

    PubMed Central

    2012-01-01

    Background The olfactory neuroepithelium lines the upper nasal cavity and is in direct contact with the external environment and the olfactory bulbs. The ability to self-renew throughout life and the reproducible recovery after injury, make it a model tissue to study mechanisms underlying neurogenesis. In this study, X-rays were used to disrupt proliferating olfactory stem cell populations and to assess their role in the cellular and morphological changes involved in olfactory neurogenic processes. Results We have analysed the histological and functional effects of a sub-lethal dose of X-rays on the adult mouse olfactory neuroepithelium at 2 hours, 24 hours, 1 week, 2 weeks and 5 weeks. We have shown an immediate cessation of proliferating olfactory stem cells as shown by BrdU, Ki67 and pH3 expression. At 24 hours there was an increase in the neural transcription factors Mash1 and Pax6 expression, and a disruption of the basal lamina and increase in glandular cell marker expression at 1 week post-irradiation. Coincident with these changes was an impairment of the olfactory function in vivo. Conclusions We have shown significant changes in basal cell proliferation as well as morphological changes in the olfactory neuroepithelium following X-ray irradiation. There is involvement of the basal lamina as well as a clear role for glandular and sustentacular cells. PMID:23113950

  14. Short-Term Regulation of Excitation-Contraction Coupling by the β1a Subunit in Adult Mouse Skeletal Muscle

    PubMed Central

    García, María C.; Carrillo, Elba; Galindo, José M.; Hernández, Ascensión; Copello, Julio A.; Fill, Michael; Sánchez, Jorge A.

    2005-01-01

    The β1a subunit of the skeletal muscle voltage-gated Ca2+ channel plays a fundamental role in the targeting of the channel to the tubular system as well as in channel function. To determine whether this cytosolic auxiliary subunit is also a regulatory protein of Ca2+ release from the sarcoplasmic reticulum in vivo, we pressure-injected the β1a subunit into intact adult mouse muscle fibers and recorded, with Fluo-3 AM, the intracellular Ca2+ signal induced by the action potential. We found that the β1a subunit significantly increased, within minutes, the amplitude of Ca2+ release without major changes in its time course. β1a subunits with the carboxy-terminus region deleted did not show an effect on Ca2+ release. The possibility that potentiation of Ca2+ release is due to a direct interaction between the β1a subunit and the ryanodine receptor was ruled out by bilayer experiments of RyR1 single-channel currents and also by Ca2+ flux experiments. Our data suggest that the β1a subunit is capable of regulating E-C coupling in the short term and that the integrity of the carboxy-terminus region is essential for its modulatory effect. PMID:16183888

  15. Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

    PubMed

    Hemmer, Kathrin; Zhang, Mingyue; van Wüllen, Thea; Sakalem, Marna; Tapia, Natalia; Baumuratov, Aidos; Kaltschmidt, Christian; Kaltschmidt, Barbara; Schöler, Hans R; Zhang, Weiqi; Schwamborn, Jens C

    2014-09-09

    Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications.

  16. Astrocytic adaptation during cerebral angiogenesis follows the new vessel formation induced through chronic hypoxia in adult mouse cortex

    NASA Astrophysics Data System (ADS)

    Masamoto, Kazuto; Kanno, Iwao

    2014-03-01

    We examined longitudinal changes of the neuro-glia-vascular unit during cerebral angiogenesis induced through chronic hypoxia in the adult mouse cortex. Tie2-GFP mice in which the vascular endothelial cells expressed green fluorescent proteins (GFP) were exposed to chronic hypoxia, while the spatiotemporal developments of the cortical capillary sprouts and the neighboring astrocytic remodeling were characterized with repeated two-photon microscopy. The capillary sprouts appeared at early phases of the hypoxia adaptation (1-2 weeks), while the morphological changes of the astrocytic soma and processes were not detected in this phase. In the later phases of the hypoxia adaptation (> 2 weeks), the capillary sprouts created a new connection with existing capillaries, and its neighboring astrocytes extended their processes to the newly-formed vessels. The findings show that morphological adaptation of the astrocytes follow the capillary development during the hypoxia adaptation, which indicate that the newly-formed vessels provoke cellular interactions with the neighboring astrocytes to strengthen the functional blood-brain barrier.

  17. Enkephalin in the goldfish retina

    SciTech Connect

    Su, Y.Y.; Fry, K.R.; Lam, D.M.; Watt, C.B.

    1986-12-01

    Enkephalin-like immunoreactive amacrine cells were visualized using the highly sensitive avidin-biotin method. The somas of these cells were situated in the inner nuclear and ganglion cell layers. Enkephalin-stained processes were observed in layers 1, 3, and 5 of the inner plexiform layer. The biosynthesis of sulfur-containing compounds in the goldfish retina was studied by means of a pulse-chase incubation with /sup 35/S-methionine. A /sup 35/S-labeled compound, which comigrated with authentic Met5-enkephalin on high-performance liquid chromatography (HPLC), was synthesized and was bound competitively by antibodies to enkephalin and by opiate receptors. This compound was tentatively identified as Met5-enkephalin. The newly synthesized /sup 35/S-Met5-enkephalin was released upon depolarization of the retina with a high K+ concentration. This K+-stimulated release was greatly suppressed by 5 mM Co/sup 2 +/, suggesting that the release was Ca/sup 2 +/ dependent. Using a double-label technique, enkephalin immunoreactivity and gamma-aminobutyric acid (GABA) uptake were colocalized to some amacrine cells, whereas others labeled only for enkephalin or GABA. The possible significance of enkephalin-GABA interactions is also discussed.

  18. 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.

  19. The Functional Architecture of the Retina.

    ERIC Educational Resources Information Center

    Masland, Richard H.

    1986-01-01

    Examines research related to the retina's coding of visual input with emphasis on the organization of two kinds of ganglion cell receptive fields. Reviews current techniques for examining the shapes and arrangement in the retina of entire populations of nerve cells. (ML)

  20. Impaired adult hippocampal neurogenesis and cognitive ability in a mouse model of intrastriatal hemorrhage.

    PubMed

    Yang, Yuan; Zhang, Meikui; Kang, Xiaoni; Jiang, Chen; Zhang, Huan; Wang, Pei; Li, Jingjing

    2015-07-10

    Thrombin released by hematoma is an important mediator of the secondary injury of intracerebral hemorrhage (ICH), however, the effect of thrombin on adult neurogenesis and cognitive ability remains elusive. In this study, intrastriatal injection of 0.05 U thrombin didn't affect the neurogenesis at the subgranular zone (SGZ), which was distal to the injection site. 0.1 U thrombin increased the 5-bromo-2-deoxyuridine(+) (BrdU(+), S-phase proliferating cells)/doublecortin(+) (DCX(+), immature neurons) double labelled neurons, but decreased BrdU(+)/NeuN(+) double labelled mature neurons. Higher doses of thrombin (1 U, 2 U, and 5 U) significantly decreased the BrdU(+)/DCX(+) and BrdU(+)/NeuN(+) double labelled cells. After 1 U thrombin injection, cell apoptosis was found at the dentate gyrus of hippocampus at 3-24 h, but not 5 d post-injury. Thrombin infusion (1 U) induced spatial memory deficits in Morris water maze test; whereas, hirudin, the thrombin antagonist, significantly reversed both neurogenesis loss and spatial learning and memory impairment. In conclusion, at least at short term (5 days) after striatum ICH, the effect of high dose of thrombin on neurogenesis of SGZ, and the spatial learning and memory ability, is detrimental.

  1. Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis

    PubMed Central

    Ostrowski, Stephen M.; Wright, Margaret C.; Bolock, Alexa M.; Geng, Xuehui; Maricich, Stephen M.

    2015-01-01

    Merkel cells are mechanosensitive skin cells whose production requires the basic helix-loop-helix transcription factor Atoh1. We induced ectopic Atoh1 expression in the skin of transgenic mice to determine whether Atoh1 was sufficient to create additional Merkel cells. In embryos, ectopic Atoh1 expression drove ectopic expression of the Merkel cell marker keratin 8 (K8) throughout the epidermis. Epidermal Atoh1 induction in adolescent mice similarly drove widespread K8 expression in glabrous skin of the paws, but in the whisker pads and body skin ectopic K8+ cells were confined to hair follicles and absent from interfollicular regions. Ectopic K8+ cells acquired several characteristics of mature Merkel cells in a time frame similar to that seen during postnatal development of normal Merkel cells. Although ectopic K8+ cell numbers decreased over time, small numbers of these cells remained in deep regions of body skin hair follicles at 3 months post-induction. In adult mice, greater numbers of ectopic K8+ cells were created by Atoh1 induction during anagen versus telogen and following disruption of Notch signaling by conditional deletion of Rbpj in the epidermis. Our data demonstrate that Atoh1 expression is sufficient to produce new Merkel cells in the epidermis, that epidermal cell competency to respond to Atoh1 varies by skin location, developmental age and hair cycle stage, and that the Notch pathway plays a key role in limiting epidermal cell competency to respond to Atoh1 expression. PMID:26138479

  2. Plasticity of Astrocytic Coverage and Glutamate Transporter Expression in Adult Mouse Cortex

    PubMed Central

    Steiner, Pascal; Hirling, Harald; Welker, Egbert; Knott, Graham W

    2006-01-01

    Astrocytes play a major role in the removal of glutamate from the extracellular compartment. This clearance limits the glutamate receptor activation and affects the synaptic response. This function of the astrocyte is dependent on its positioning around the synapse, as well as on the level of expression of its high-affinity glutamate transporters, GLT1 and GLAST. Using Western blot analysis and serial section electron microscopy, we studied how a change in sensory activity affected these parameters in the adult cortex. Using mice, we found that 24 h of whisker stimulation elicited a 2-fold increase in the expression of GLT1 and GLAST in the corresponding cortical column of the barrel cortex. This returns to basal levels 4 d after the stimulation was stopped, whereas the expression of the neuronal glutamate transporter EAAC1 remained unaltered throughout. Ultrastructural analysis from the same region showed that sensory stimulation also causes a significant increase in the astrocytic envelopment of excitatory synapses on dendritic spines. We conclude that a period of modified neuronal activity and synaptic release of glutamate leads to an increased astrocytic coverage of the bouton–spine interface and an increase in glutamate transporter expression in astrocytic processes. PMID:17048987

  3. Ectopic Atoh1 expression drives Merkel cell production in embryonic, postnatal and adult mouse epidermis.

    PubMed

    Ostrowski, Stephen M; Wright, Margaret C; Bolock, Alexa M; Geng, Xuehui; Maricich, Stephen M

    2015-07-15

    Merkel cells are mechanosensitive skin cells whose production requires the basic helix-loop-helix transcription factor Atoh1. We induced ectopic Atoh1 expression in the skin of transgenic mice to determine whether Atoh1 was sufficient to create additional Merkel cells. In embryos, ectopic Atoh1 expression drove ectopic expression of the Merkel cell marker keratin 8 (K8) throughout the epidermis. Epidermal Atoh1 induction in adolescent mice similarly drove widespread K8 expression in glabrous skin of the paws, but in the whisker pads and body skin ectopic K8+ cells were confined to hair follicles and absent from interfollicular regions. Ectopic K8+ cells acquired several characteristics of mature Merkel cells in a time frame similar to that seen during postnatal development of normal Merkel cells. Although ectopic K8+ cell numbers decreased over time, small numbers of these cells remained in deep regions of body skin hair follicles at 3 months post-induction. In adult mice, greater numbers of ectopic K8+ cells were created by Atoh1 induction during anagen versus telogen and following disruption of Notch signaling by conditional deletion of Rbpj in the epidermis. Our data demonstrate that Atoh1 expression is sufficient to produce new Merkel cells in the epidermis, that epidermal cell competency to respond to Atoh1 varies by skin location, developmental age and hair cycle stage, and that the Notch pathway plays a key role in limiting epidermal cell competency to respond to Atoh1 expression.

  4. Acetylcholine receptors in the human retina

    SciTech Connect

    Hutchins, J.B.; Hollyfield, J.G.

    1985-11-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand TH-propylbenzilylcholine mustard (TH-PrBCM) to label muscarinic receptors. TH- or SVI-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that TH-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina.

  5. Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration

    PubMed Central

    Eastlake, Karen; Heywood, Wendy E.; Tracey-White, Dhani; Aquino, Erika; Bliss, Emily; Vasta, Gerardo R.; Mills, Kevin; Khaw, Peng T.; Moosajee, Mariya; Limb, G. Astrid

    2017-01-01

    Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease. PMID:28300160

  6. Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators.

    PubMed

    Weitz, Andrew C; Behrend, Matthew R; Lee, Nan Sook; Klein, Ronald L; Chiodo, Vince A; Hauswirth, William W; Humayun, Mark S; Weiland, James D; Chow, Robert H

    2013-04-01

    Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.

  7. AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

    PubMed

    Vacca, Ophélie; Charles-Messance, Hugo; El Mathari, Brahim; Sene, Abdoulaye; Barbe, Peggy; Fouquet, Stéphane; Aragón, Jorge; Darche, Marie; Giocanti-Aurégan, Audrey; Paques, Michel; Sahel, José-Alain; Tadayoni, Ramin; Montañez, Cecilia; Dalkara, Deniz; Rendon, Alvaro

    2016-07-15

    Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).

  8. CD74 indicates microglial activation in experimental diabetic retinopathy and exogenous methylglyoxal mimics the response in normoglycemic retina.

    PubMed

    Wang, Jing; Lin, Jihong; Schlotterer, Andreas; Wu, Liang; Fleming, Thomas; Busch, Stephanie; Dietrich, Nadine; Hammes, Hans-Peter

    2014-10-01

    Diabetes induces vasoregression, neurodegeneration and glial activation in the retina. Formation of advanced glycation endoproducts (AGEs) is increased in diabetes and contributes to the pathogenesis of diabetic retinopathy. CD74 is increased in activated microglia in a rat model developing both neurodegeneration and vasoregression. In this study, we aimed at investigating whether glucose and major AGE precursor methylglyoxal induce increased CD74 expression in the retina. Expression of CD74 in retinal microglia was analyzed in streptozotocin-diabetic rats by wholemount immunofluorescence. Nondiabetic mice were intravitreally injected with methylglyoxal. Expression of CD74 was studied by retinal wholemount immunofluorescence and quantitative real-time PCR, 48 h after the injection. CD74-positive cells were increased in diabetic 4-month retinas. These cells represented a subpopulation of CD11b-labeled activated microglia and were mainly located in the superficial vascular layer (13.7-fold increase compared to nondiabetic group). Methylglyoxal induced an 9.4-fold increase of CD74-positive cells in the superficial vascular layer and elevated gene expression of CD74 in the mouse retina 2.8-fold. In summary, we identified CD74 as a microglial activation marker in the diabetic retina. Exogenous methylglyoxal mimics the response in normoglycemic retina. This suggests that methylglyoxal is important in mediating microglial activation in the diabetic retina.

  9. Response of olfactory axons to loss of synaptic targets in the adult mouse

    PubMed Central

    Ardiles, Yona; de la Puente, Rafael; Toledo, Rafael; Isgor, Ceylan; Guthrie, Kathleen

    2007-01-01

    Glomerular convergence has been proposed to rely on interactions between like olfactory axons, however topographic targeting is influenced by guidance molecules encountered in the olfactory bulb. Disruption of these cues during development misdirects sensory axons, however little is known about the role of bulb-derived signals in later life, as new axons arise during turnover of the olfactory sensory neuron (OSN) population. To evaluate the contribution of bulb neurons in maintaining topographic projections in adults, we ablated them with N-methyl-D-aspartate (NMDA) in P2-IRES-tauLacZ mice and examined how sensory axons responded to loss of their postsynaptic partners. NMDA lesion eliminated bulb neurons without damage to sensory axons or olfactory ensheathing glia. P2 axons contained within glomeruli at the time of lesion maintained convergence at these locations; there was no evidence of compensatory growth into the remnant tissue. Delayed apoptosis of OSNs in the target-deprived epithelium led to declines in P2 neuron number as well as the gradual atrophy, and in some cases complete loss, of P2 glomeruli in lesioned bulbs by three weeks. Increased cell proliferation in the epithelium partially restored the OSN population, and by eight weeks, new P2 axons distributed within diverse locations in the bulb remnant and within the anterior olfactory nucleus. Prior studies have suggested that initial development of olfactory topography does not rely on synapse formation with target neurons, however the present data demonstrate that continued maintenance of the sensory map requires the presence of sufficient numbers and/or types of available bulbar synaptic targets. PMID:17674970

  10. GAP-43 overexpression in adult mouse Purkinje cells overrides myelin-derived inhibition of neurite growth.

    PubMed

    Gianola, Sara; Rossi, Ferdinando

    2004-02-01

    Up-regulation of growth-associated proteins in adult neurons promotes axon regeneration and neuritic elongation onto nonpermissive substrates. To investigate the interaction between these molecules and myelin-related inhibitory factors, we examined transgenic mice in which overexpression of the growth-associated protein GAP-43 is driven by the Purkinje cell-specific promoter L7. Contrary to their wild-type counterparts, which have extremely poor regenerative capabilities, axotomized transgenic Purkinje cells exhibit profuse sprouting along the intracortical neurite and at the severed stump [Buffo et al. (1997) J. Neurosci., 17, 8778-8791]. Here, we investigated the relationship between such sprouting axons and oligodendroglia to ask whether GAP-43 overexpression enables Purkinje neurites to overcome myelin-derived inhibition. Intact transgenic Purkinje axons display normal morphology and myelination. Following injury, however, many GAP-43-overexpressing neurite stumps are devoid of myelin cover and sprout into white matter regions containing densely packed myelin and Nogo-A- or MAG-immunopositive oligodendrocytes. The intracortical segments of these neurites show focal accumulations of GAP-43, which are associated with disrupted or retracted myelin sheaths. Numerous sprouts originate from such demyelinated segments and spread into the granular layer. Some myelin loss, though not axon sprouting, is also evident in wild-type mice, but this phenomenon is definitely more rapid and extensive in transgenic cerebella. Thus, GAP-43-overexpressing Purkinje axons are endowed with enhanced capabilities for growing into nonpermissive territories and show a pronounced tendency to lose myelin. Our observations suggest that accumulation of GAP-43 along precise axon segments disrupts the normal axon-glia interaction and enhances the retraction of oligodendrocytic processes to facilitate the outgrowth of neuritic sprouts.

  11. In vitro and ex vivo retina angiogenesis assays.

    PubMed

    Rezzola, Sara; Belleri, Mirella; Gariano, Giuseppina; Ribatti, Domenico; Costagliola, Ciro; Semeraro, Francesco; Presta, Marco

    2014-07-01

    Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Seminal studies in the early 1980 s about the angiogenic activity exerted by mammalian retinal tissue extracts on the chick embryo chorioallantoic membrane and the later discovery of vascular endothelial growth factor (VEGF) accumulation in eyes of patients with diabetic retinopathy paved the way for the development of anti-angiogenic VEGF blockers for the treatment of retinal neovascularization. Since then, numerous preclinical and clinical studies about diabetic retinopathy and other retinal disorders have opened new lines of angiogenesis inquiry, indicating that limitations to anti-VEGF therapies may exist. Moreover, the production of growth factors other than VEGF may affect the response to anti-VEGF approaches. Thus, experimental models of retinal angiogenesis remain crucial for investigating novel anti-angiogenic therapies and bringing them to patients. To this aim, in vitro and ex vivo angiogenesis assays may be suitable for a rapid screening of potential anti-angiogenic molecules before in vivo validation of the putative lead compounds. This review focuses on the different in vitro and ex vivo angiogenesis assays that have been developed over the years based on the isolation of endothelial cells from the retina of various animal species and ex vivo cultures of neonatal and adult retina explants. Also, recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryos, an in vivo animal platform experimentally analogous to in vitro/ex vivo models, may represent a novel target for the identification of angiogenesis inhibitors. When compared to in vivo assays, in vitro and ex vivo models of retina neovascularization, including zebrafish embryo, may represent cost-effective and rapid tools for the screening of novel anti

  12. Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia

    PubMed Central

    Ramirez, Grisela

    2016-01-01

    Abstract Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein (IKBKAP). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre (Tα1-Cre). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients. PMID:27699209

  13. Analysis of Adult Female Mouse (Mus musculus) Group Behavior on the International Space Station (ISS)

    NASA Technical Reports Server (NTRS)

    Solomides, P.; Moyer, E. L.; Talyansky, Y.; Choi, S.; Gong, C.; Globus, R. K.; Ronca, A. E.

    2016-01-01

    As interest in long duration effects of space habitation increases, understanding the behavior of model organisms living within the habitats engineered to fly them is vital for designing, validating, and interpreting future spaceflight studies. A handful of papers have previously reported behavior of mice and rats in the weightless environment of space. The Rodent Research Hardware and Operations Validation (Rodent Research-1; RR1) utilized the Rodent Habitat (RH) developed at NASA Ames Research Center to fly mice on the ISS (International Space Station). Ten adult (16-week-old) female C57BL/6 mice were launched on September 21st, 2014 in an unmanned Dragon Capsule, and spent 37 days in microgravity. Here we report group behavioral phenotypes of the RR1 Flight (FLT) and environment-matched Ground Control (GC) mice in the Rodent Habitat (RH) during this long-duration flight. Video was recorded for 33 days on the ISS, permitting daily assessments of overall health and well-being of the mice, and providing a valuable repository for detailed behavioral analysis. We previously reported that, as compared to GC mice, RR1 FLT mice exhibited the same range of behaviors, including eating, drinking, exploration, self- and allo-grooming, and social interactions at similar or greater levels of occurrence. Overall activity was greater in FLT as compared to GC mice, with spontaneous ambulatory behavior, including organized 'circling' or 'race-tracking' behavior that emerged within the first few days of flight following a common developmental sequence, and comprised the primary dark cycle activity persisting throughout the remainder of the experiment. Participation by individual mice increased dramatically over the course of the flight. Here we present a detailed analysis of 'race-tracking' behavior in which we quantified: (1) Complete lap rotations by individual mice; (2) Numbers of collisions between circling mice; (3) Lap directionality; and (4) Recruitment of mice into a group

  14. Analysis of MTHFR, CBS, Glutathione, Taurine, and Hydrogen Sulfide Levels in Retinas of Hyperhomocysteinemic Mice

    PubMed Central

    Cui, Xuezhi; Navneet, Soumya; Wang, Jing; Roon, Penny; Chen, Wei; Xian, Ming; Smith, Sylvia B.

    2017-01-01

    Purpose Hyperhomocysteinemia (Hhcy) is implicated in certain retinal neurovascular diseases, although whether it is causative remains uncertain. In isolated ganglion cells (GCs), mild Hhcy induces profound death, whereas retinal phenotypes in Hhcy mice caused by mutations in remethylation (methylene tetrahydrofolatereductase [Mthfr+/−]) or transsulfuration pathways (cystathionine β-synthase [Cbs+/−]) demonstrate mild GC loss and mild vasculopathy. The current work investigated compensation in vivo of one pathway for the other, and, because the transsulfuration pathway yields cysteine necessary for formation of glutathione (GSH), taurine, and hydrogen sulfide (H2S), they were analyzed also. Methods Retinas isolated from wild-type (WT), Mthfr+/−, and Cbs+/− mice (12 and 22 weeks) were analyzed for methylene tetrahydrofolate reductase (MTHFR), cystathionine-β-synthase (CBS), and cystathionase (CTH) RNA/protein levels. Retinas were evaluated for levels of reduced:oxidized GSH (GSH:GSSG), Slc7a11 (xCT), taurine, taurine transporter (TAUT), and H2S. Results Aside from decreased CBS RNA/protein levels in Cbs+/− retinas, there were minimal alterations in remethylation/transsulfuration pathways in the two mutant mice strains. Glutathione and taurine levels in Mthfr+/− and Cbs+/− retinas were similar to WT, which may be due to robust levels of xCT and TAUT in mutant retinas. Interestingly, levels of H2S were markedly increased in retinas of Mthfr+/− and Cbs+/− mice compared with WT. Conclusions Ganglion cell loss and vasculopathy observed in Mthfr+/− and Cbs+/− mouse retinas may be milder than expected, not because of compensatory increases of enzymes in remethylation/transsulfuration pathways, but because downstream transsulfuration pathway products GSH, taurine, and H2S are maintained at robust levels. Elevation of H2S is particularly intriguing owing to neuroprotective properties reported for this gasotransmitter. PMID:28384716

  15. Quantum biology of the retina.

    PubMed

    Sia, Paul Ikgan; Luiten, André N; Stace, Thomas M; Wood, John Pm; Casson, Robert J

    2014-08-01

    The emerging field of quantum biology has led to a greater understanding of biological processes at the microscopic level. There is recent evidence to suggest that non-trivial quantum features such as entanglement, tunnelling and coherence have evolved in living systems. These quantum features are particularly evident in supersensitive light-harvesting systems such as in photosynthesis and photoreceptors. A biomimetic strategy utilizing biological quantum phenomena might allow new advances in the field of quantum engineering, particularly in quantum information systems. In addition, a better understanding of quantum biological features may lead to novel medical diagnostic and therapeutic developments. In the present review, we discuss the role of quantum physics in biological systems with an emphasis on the retina.

  16. Mechanical spectroscopy of retina explants at the protein level employing nanostructured scaffolds.

    PubMed

    Mayazur Rahman, S; Reichenbach, Andreas; Zink, Mareike; Mayr, Stefan G

    2016-04-14

    Development of neuronal tissue, such as folding of the brain, and formation of the fovea centralis in the human retina are intimately connected with the mechanical properties of the underlying cells and the extracellular matrix. In particular for neuronal tissue as complex as the vertebrate retina, mechanical properties are still a matter of debate due to their relation to numerous diseases as well as surgery, where the tension of the retina can result in tissue detachment during cutting. However, measuring the elasticity of adult retina wholemounts is difficult and until now only the mechanical properties at the surface have been characterized with micrometer resolution. Many processes, however, such as pathological changes prone to cause tissue rupture and detachment, respectively, are reflected in variations of retina elasticity at smaller length scales at the protein level. In the present work we demonstrate that freely oscillating cantilevers composed of nanostructured TiO2 scaffolds can be employed to study the frequency-dependent mechanical response of adult mammalian retina explants at the nanoscale. Constituting highly versatile scaffolds with strong tissue attachment for long-term organotypic culture atop, these scaffolds perform damped vibrations as fingerprints of the mechanical tissue properties that are derived using finite element calculations. Since the tissue adheres to the nanostructures via constitutive proteins on the photoreceptor side of the retina, the latter are stretched and compressed during vibration of the underlying scaffold. Probing mechanical response of individual proteins within the tissue, the proposed mechanical spectroscopy approach opens the way for studying tissue mechanics, diseases and the effect of drugs at the protein level.

  17. Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina.

    PubMed

    Romano, Maria Rosaria; Di Menna, Luisa; Scarselli, Pamela; Mascio, Giada; Madonna, Michele; Notartomaso, Serena; Puliti, Aldamaria; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando

    2016-04-01

    mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5(-/-) mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

  18. Cytogenesis in the monkey retina

    SciTech Connect

    La Vail, M.M.; Rapaport, D.H.; Rakic, P. )

    1991-07-01

    Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Mueller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Mueller, and rod cells have a similar time of origin.

  19. Layer-Specific Functional and Anatomical MRI of the Retina with Passband Balanced SSFP

    PubMed Central

    Muir, Eric R; Duong, Timothy Q

    2011-01-01

    The retina consists of multiple cellular and synaptic layers and is nourished by two distinct (retinal and choroidal) circulations bounding the retina, separated by an avascular layer. High spatiotemporal resolution, layer-specific MRI of the retina remains challenging due to magnetic inhomogeneity-induced artifacts. This study reports passband balanced-steady-state free-precession (bSSFP) MRI at 45×45×500μm and 1.6s temporal resolution to image the mouse retina, overcoming geometric distortion and signal dropout while maintaining rapid acquisition and high signal-to-noise ratio. bSSFP images revealed multiple alternating dark-bright-dark-bright retinal layers. Hypoxic (10%O2) inhalation decreased bSSFP signals in the two layers bounding the retina, corresponding to the retinal and choroidal vasculatures. The layer in between showed no substantial response and was assigned the avascular photoreceptor layers. Choroidal responses (−25.9±6.4%, mean±SD, n=6) were significantly (P<0.05) larger than retinal vascular responses (−11.6±2.4%). bSSFP offers very high spatiotemporal resolution and could have important applications in imaging layer-specific changes in retinal diseases. PMID:21604296

  20. Stage-dependent requirement of neuroretinal Pax6 for lens and retina development.

    PubMed

    Klimova, Lucie; Kozmik, Zbynek

    2014-03-01

    The physical contact of optic vesicle with head surface ectoderm is an initial event triggering eye morphogenesis. This interaction leads to lens specification followed by coordinated invagination of the lens placode and optic vesicle, resulting in formation of the lens, retina and retinal pigmented epithelium. Although the role of Pax6 in early lens development has been well documented, its role in optic vesicle neuroepithelium and early retinal progenitors is poorly understood. Here we show that conditional inactivation of Pax6 at distinct time points of mouse neuroretina development has a different impact on early eye morphogenesis. When Pax6 is eliminated in the retina at E10.5 using an mRx-Cre transgene, after a sufficient contact between the optic vesicle and surface ectoderm has occurred, the lens develops normally but the pool of retinal progenitor cells gradually fails to expand. Furthermore, a normal differentiation program is not initiated, leading to almost complete disappearance of the retina after birth. By contrast, when Pax6 was inactivated at the onset of contact between the optic vesicle and surface ectoderm in Pax6(Sey/flox) embryos, expression of lens-specific genes was not initiated and neither the lens nor the retina formed. Our data show that Pax6 in the optic vesicle is important not only for proper retina development, but also for lens formation in a non-cell-autonomous manner.

  1. Deep-brain magnetic stimulation promotes adult hippocampal neurogenesis and alleviates stress-related behaviors in mouse models for neuropsychiatric disorders

    PubMed Central

    2014-01-01

    Background Repetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation (DMS) is an effective therapy for various neuropsychiatric disorders including major depression disorder. The molecular and cellular mechanisms underlying the impacts of rTMS/DMS on the brain are not yet fully understood. Results Here we studied the effects of deep-brain magnetic stimulation to brain on the molecular and cellular level. We examined the adult hippocampal neurogenesis and hippocampal synaptic plasticity of rodent under stress conditions with deep-brain magnetic stimulation treatment. We found that DMS promotes adult hippocampal neurogenesis significantly and facilitates the development of adult new-born neurons. Remarkably, DMS exerts anti-depression effects in the learned helplessness mouse model and rescues hippocampal long-term plasticity impaired by restraint stress in rats. Moreover, DMS alleviates the stress response in a mouse model for Rett syndrome and prolongs the life span of these animals dramatically. Conclusions Deep-brain magnetic stimulation greatly facilitates adult hippocampal neurogenesis and maturation, also alleviates depression and stress-related responses in animal models. PMID:24512669

  2. Glycogen metabolism in the rat retina.

    PubMed

    Coffe, Víctor; Carbajal, Raymundo C; Salceda, Rocío

    2004-02-01

    It has been reported that glycogen levels in retina vary with retinal vascularization. However, the electrical activity of isolated retina depends on glucose supply, suggesting that it does not contain energetic reserves. We determined glycogen levels and pyruvate and lactate production under various conditions in isolated retina. Ex vivo retinas from light- and dark-adapted rats showed values of 44 +/- 0.3 and 19.5 +/- 0.4 nmol glucosyl residues/mg protein, respectively. The glycogen content of retinas from light-adapted animals was reduced by 50% when they were transferred to darkness. Glycogen levels were low in retinas incubated in glucose-free media and increased in the presence of glucose. The highest glycogen values were found in media containing 20 mm of glucose. A rapid increase in lactate production was observed in the presence of glucose. Surprisingly, glycogen levels were the lowest and lactate production was also very low in the presence of 30 mm glucose. Our results suggest that glycogen can be used as an immediate accessible energy reserve in retina. We speculate on the possibility that gluconeogenesis may play a protective role by removal of lactic acid.

  3. Role of dopamine in distal retina.

    PubMed

    Popova, E

    2014-05-01

    Dopamine is the most abundant catecholamine in the vertebrate retina. Despite the description of retinal dopaminergic cells three decades ago, many aspects of their function in the retina remain unclear. There is no consensus among the authors about the stimulus conditions for dopamine release (darkness, steady or flickering light) as well as about its action upon the various types of retinal cells. Many contradictory results exist concerning the dopamine effect on the gross electrical activity of the retina [reflected in electroretinogram (ERG)] and the receptors involved in its action. This review summarized current knowledge about the types of the dopaminergic neurons and receptors in the retina as well as the effects of dopamine receptor agonists and antagonists on the light responses of photoreceptors, horizontal and bipolar cells in both nonmammalian and mammalian retina. Special focus of interest concerns their effects upon the diffuse ERG as a useful tool for assessment of the overall function of the distal retina. An attempt is made to reveal some differences between the dopamine actions upon the activity of the ON versus OFF channel in the distal retina. The author has included her own results demonstrating such differences.

  4. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse.

    PubMed

    Gocel, James; Larson, John

    2012-09-27

    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  5. Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.

    PubMed

    Golmohammadi, Mohammad G; Blackmore, Daniel G; Large, Beatrice; Azari, Hassan; Esfandiary, Ebrahim; Paxinos, George; Franklin, Keith B J; Reynolds, Brent A; Rietze, Rodney L

    2008-04-01

    The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 mum coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphere-forming cells detected in individual 400 mum sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 +/- 276) or colonies (4275 +/- 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 +/- 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 +/- 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki-67+) or competent to divide (BrdU+/Mcm-2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques.

  6. Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain.

    PubMed

    Morita, Shoko; Furube, Eriko; Mannari, Tetsuya; Okuda, Hiroaki; Tatsumi, Kouko; Wanaka, Akio; Miyata, Seiji

    2015-03-01

    The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1α were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.

  7. The transformation of synaptic to system plasticity in motor output from the sacral cord of the adult mouse

    PubMed Central

    Elbasiouny, Sherif M.; Collins, William F.; Heckman, C. J.

    2015-01-01

    Synaptic plasticity is fundamental in shaping the output of neural networks. The transformation of synaptic plasticity at the cellular level into plasticity at the system level involves multiple factors, including behavior of local networks of interneurons. Here we investigate the synaptic to system transformation for plasticity in motor output in an in vitro preparation of the adult mouse spinal cord. System plasticity was assessed from compound action potentials (APs) in spinal ventral roots, which were generated simultaneously by the axons of many motoneurons (MNs). Synaptic plasticity was assessed from intracellular recordings of MNs. A computer model of the MN pool was used to identify the middle steps in the transformation from synaptic to system behavior. Two input systems that converge on the same MN pool were studied: one sensory and one descending. The two synaptic input systems generated very different motor outputs, with sensory stimulation consistently evoking short-term depression (STD) whereas descending stimulation had bimodal plasticity: STD at low frequencies but short-term facilitation (STF) at high frequencies. Intracellular and pharmacological studies revealed contributions from monosynaptic excitation and stimulus time-locked inhibition but also considerable asynchronous excitation sustained from local network activity. The computer simulations showed that STD in the monosynaptic excitatory input was the primary driver of the system STD in the sensory input whereas network excitation underlies the bimodal plasticity in the descending system. These results provide insight on the roles of plasticity in the monosynaptic and polysynaptic inputs converging on the same MN pool to overall motor plasticity. PMID:26203107

  8. Early Social Enrichment Rescues Adult Behavioral and Brain Abnormalities in a Mouse Model of Fragile X Syndrome

    PubMed Central

    Oddi, Diego; Subashi, Enejda; Middei, Silvia; Bellocchio, Luigi; Lemaire-Mayo, Valerie; Guzmán, Manuel; Crusio, Wim E; D'Amato, Francesca R; Pietropaolo, Susanna

    2015-01-01

    Converging lines of evidence support the use of environmental stimulation to ameliorate the symptoms of a variety of neurodevelopmental disorders. Applying these interventions at very early ages is critical to achieve a marked reduction of the pathological phenotypes. Here we evaluated the impact of early social enrichment in Fmr1-KO mice, a genetic mouse model of fragile X syndrome (FXS), a major developmental disorder and the most frequent monogenic cause of autism. Enrichment was achieved by providing male KO pups and their WT littermates with enhanced social stimulation, housing them from birth until weaning with the mother and an additional nonlactating female. At adulthood they were tested for locomotor, social, and cognitive abilities; furthermore, dendritic alterations were assessed in the hippocampus and amygdala, two brain regions known to be involved in the control of the examined behaviors and affected by spine pathology in Fmr1-KOs. Enrichment rescued the behavioral FXS-like deficits displayed in adulthood by Fmr1-KO mice, that is, hyperactivity, reduced social interactions, and cognitive deficits. Early social enrichment also eliminated the abnormalities shown by adult KO mice in the morphology of hippocampal and amygdala dendritic spines, namely an enhanced density of immature vs mature types. Importantly, enrichment did not induce neurobehavioral changes in WT mice, thus supporting specific effects on FXS-like pathology. These findings show that early environmental stimulation has profound and long-term beneficial effects on the pathological FXS phenotype, thereby encouraging the use of nonpharmacological interventions for the treatment of this and perhaps other neurodevelopmental diseases. PMID:25348604

  9. Liver Progenitors Isolated from Adult Healthy Mouse Liver Efficiently Differentiate to Functional Hepatocytes In Vitro and Repopulate Liver Tissue.

    PubMed

    Tanimizu, Naoki; Ichinohe, Norihisa; Ishii, Masayuki; Kino, Junichi; Mizuguchi, Toru; Hirata, Koichi; Mitaka, Toshihiro

    2016-12-01

    It has been proposed that tissue stem cells supply multiple epithelial cells in mature tissues and organs. However, it is unclear whether tissue stem cells generally contribute to cellular turnover in normal healthy organs. Here, we show that liver progenitors distinct from bipotent liver stem/progenitor cells (LPCs) persistently exist in mouse livers and potentially contribute to tissue maintenance. We found that, in addition to LPCs isolated as EpCAM(+) cells, liver progenitors were enriched in CD45(-) TER119(-) CD31(-) EpCAM(-) ICAM-1(+) fraction isolated from late-fetal and postnatal livers. ICAM-1(+) liver progenitors were abundant by 4 weeks (4W) after birth. Although their number decreased with age, ICAM-1(+) liver progenitors existed in livers beyond that stage. We established liver progenitor clones derived from ICAM-1(+) cells between 1 and 20W and found that those clones efficiently differentiated into mature hepatocytes (MHs), which secreted albumin, eliminated ammonium ion, stored glycogen, and showed cytochrome P450 activity. Even after long-term culture, those clones kept potential to differentiate to MHs. When ICAM-1(+) clones were transplanted into nude mice after retrorsine treatment and 70% partial hepatectomy, donor cells were incorporated into liver plates and expressed hepatocyte nuclear factor 4α, CCAAT/enhancer binding protein α, and carbamoylphosphate synthetase I. Moreover, after short-term treatment with oncostatin M, ICAM-1(+) clones could efficiently repopulate the recipient liver tissues. Our results indicate that liver progenitors that can efficiently differentiate to MHs exist in normal adult livers. Those liver progenitors could be an important source of new MHs for tissue maintenance and repair in vivo, and for regenerative medicine ex vivo. Stem Cells 2016;34:2889-2901.

  10. Characterization of thrombopoietin (TPO)-responsive progenitor cells in adult mouse bone marrow with in vivo megakaryocyte and erythroid potential.

    PubMed

    Ng, Ashley P; Kauppi, Maria; Metcalf, Donald; Di Rago, Ladina; Hyland, Craig D; Alexander, Warren S

    2012-02-14

    Hematopoietic progenitor cells are the progeny of hematopoietic stem cells that coordinate the production of precise numbers of mature blood cells of diverse functional lineages. Identification of cell-surface antigen expression associated with hematopoietic lineage restriction has allowed prospective isolation of progenitor cells with defined hematopoietic potential. To clarify further the cellular origins of megakaryocyte commitment, we assessed the in vitro and in vivo megakaryocyte and platelet potential of defined progenitor populations in the adult mouse bone marrow. We show that megakaryocytes arise from CD150(+) bipotential progenitors that display both platelet- and erythrocyte-producing potential in vivo and that can develop from the Flt3(-) fraction of the pregranulocyte-macrophage population. We define a bipotential erythroid-megakaryocyte progenitor population, the CD150(+)CD9(lo)endoglin(lo) fraction of Lin(-)cKit(+)IL7 receptor alpha(-)FcγRII/III(lo)Sca1(-) cells, which contains the bulk of the megakaryocyte colony-forming capacity of the bone marrow, including bipotential megakaryocyte-erythroid colony-forming capacity, and can generate both erythrocytes and platelets efficiently in vivo. This fraction is distinct from the CD150(+)CD9(hi)endoglin(lo) fraction, which contains bipotential precursors with characteristics of increased megakaryocytic maturation, and the CD150(+)CD9(lo)endoglin(hi) fraction, which contains erythroid lineage-committed cells. Finally, we demonstrate that bipotential erythroid-megakaryocyte progenitor and CD150(+)CD9(hi)endoglin(lo) cells are TPO-responsive and that the latter population specifically expands in the recovery from thrombocytopenia induced by anti-platelet serum.

  11. [Molecular-physiological aspects of peptide regulation of function of the retina in retinitis pigmentosa].

    PubMed

    Khavinson, V Kh; Proniaeva, V E; Lin'kova, N S; Trofimova, S V; Umnpv, R S

    2014-01-01

    Peptide's bioregulators promotes restoration of the physiological activity of the retina in retinitis pigmentosa in older adults and in animal models. The molecular mechanism of physiological activity of peptides is connected with its ability to regulate synthesis of protein markers of differentiation of neurons and retinal pigment epithelium epigenetically.

  12. Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus.

    PubMed

    Bonci, D M O; Lima, S M A de; Grötzner, S R; Ribeiro, C A Oliveira; Hamassaki, D E; Ventura, D F

    2006-03-01

    To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 microg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 microg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 +/- 393 cells/mm2) compared to control (1886 +/- 892 cells/mm2; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 +/- 56 cells/mm2 (2 microg/g) and 845 +/- 82 cells/mm2 (6 microg/g), also lower than control (1312 +/- 31 cells/mm2; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 microg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

  13. A new genus and species of demodecid mites from the tongue of a house mouse Mus musculus: description of adult and immature stages with data on parasitism.

    PubMed

    Izdebska, J N; Rolbiecki, L

    2016-06-01

    The study of the parasitofauna of the house mouse Mus musculus (Rodentia: Muridae) Linnaeus is particularly important owing to its multiple relationships with humans - as a cosmopolitan, synanthropic rodent, bred for pets, food for other animals or laboratory animal. This article proposes and describes a new genus and species of the parasitic mite based on adult and immature stages from the house mouse. Glossicodex musculi gen. n., sp. n. is a medium-sized demodecid mite (adult stages on average 199 µm in length) found in mouse tissue of the tongue. It is characterized by two large, hooked claws on each tarsus of the legs; the legs are relatively massive, consisting of large, non-overlapping segments. The palps consist of three slender, clearly separated, relatively narrow segments, wherein their coxal segments are also quite narrow and spaced. Also, segments of the palps of larva and nymphs are clearly isolated, and on the terminal segment, trident claws that resemble legs' claws can be found. On the ventral side, in immature stages, triangular scuta, topped with sclerotized spur, can be also observed. Glossicodex musculi was noted in 10.8% of mice with a mean infection intensity of 2.2 parasites per host.

  14. Embryonic mouse STO cell-derived xenografts express hepatocytic functions in the livers of nonimmunosuppressed adult rats.

    PubMed

    Zhang, Mingjun; Joseph, Brigid; Gupta, Sanjeev; Guest, I; Xu, Meng; Sell, Stewart; Son, Kyung-Hwa; Koch, Katherine S; Leffert, Hyam L

    2005-02-01

    Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)-negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time. Detection of intrahepatic mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient rats indicated survival and differentiation of donor cells for at least 3 months. Mouse COX1 targets were also detected intrahepatically 4-9 weeks after STO cell injection into nonimmunosuppressed wild-type rats. In contrast to STO-transplanted rats, mouse DNA or RNA was not detectable in untreated or mock-transplanted rats or in rats injected with donor cell DNA. In cultured STO donor cells, DPPIV and glucose-6-phosphatase activities were observed in small clusters; in contrast, mouse major histocompatibility complex class I H-2Kq, H-2Dq, and H-2Lq and class II I-Aq markers were undetectable in vitro before or after interferon gamma treatment. Together with H-2K allele typing, which confirmed the Swiss mouse origin of the donor cells, these observations indicate that mouse-derived STO cell lines can differentiate along hepatocytic lineage and engraft into rat liver across major histocompatibility barriers.

  15. Distribution of caveolin isoforms in the lemur retina.

    PubMed

    Berta, Agnes I; Kiss, Anna L; Lukáts, Akos; Szabó, Arnold; Szél, Agoston

    2007-09-01

    The distribution of caveolin isoforms was previously evaluated in the retinas of different species, but has not yet been described in the primate retina. In this study, the distribution of caveolins was assessed via immunochemistry using isoform-specific antibodies in the retina of the black-and-white ruffed lemur. Here, we report the presence of a variety of caveolin isoforms in many layers of the lemur retina. As normal human retinas were not available for research and the retinas of primates are fairly similar to those of humans, the lemur retina can be utilized as a model for caveolin distribution in normal humans.

  16. Inflamed In Vitro Retina: Cytotoxic Neuroinflammation and Galectin-3 Expression

    PubMed Central

    Bauer, Patrik Maximilian; Zalis, Marina Castro; Abdshill, Hodan; Deierborg, Tomas; Johansson, Fredrik; Englund-Johansson, Ulrica

    2016-01-01

    Background Disease progression in retinal neurodegeneration is strongly correlated to immune cell activation, which may have either a neuroprotective or neurotoxic effect. Increased knowledge about the immune response profile and retinal neurodegeneration may lead to candidate targets for treatments. Therefore, we have used the explanted retina as a model to explore the immune response and expression of the immune modulator galectin-3 (Gal-3), induced by the cultivation per se and after additional immune stimulation with lipopolysaccharide (LPS), and how this correlates with retinal neurotoxicity. Methods Post-natal mouse retinas were cultured in a defined medium. One group was stimulated with LPS (100 ng/ml, 24 h). Retinal architecture, apoptotic cell death, and micro- and macroglial activity were studied at the time of cultivation (0 days in vitro (DIV)) and at 3, 4 and 7 DIV using morphological staining, biochemical- and immunohistochemical techniques. Results Our results show that sustained activation of macro- and microglia, characterized by no detectable cytokine release and limited expression of Gal-3, is not further inducing apoptosis additional to the axotomy-induced apoptosis in innermost nuclear layer. An elevated immune response was detected after LPS stimulation, as demonstrated primarily by release of immune mediators (i.e. interleukin 2 (IL-2), IL-6, KC/GRO (also known as CLCX1) and tumour necrosis factor-α (TNF-α)), increased numbers of microglia displaying morphologies of late activation stages as well as Gal-3 expression. This was accompanied with increased apoptosis in the two additional nuclear layers, and damage to retinal gross architecture. Conclusion We demonstrate that an immune response characterized by sustained and increased release of cytokines, along with an increase in Gal-3 expression, is accompanied by significant increased neurotoxicity in the explanted retina. Further investigations using the current setting may lead to

  17. Complex computation in the retina

    NASA Astrophysics Data System (ADS)

    Deshmukh, Nikhil Rajiv

    Elucidating the general principles of computation in neural circuits is a difficult problem requiring both a tractable model circuit as well as sophisticated measurement tools. This thesis advances our understanding of complex computation in the salamander retina and its underlying circuitry and furthers the development of advanced tools to enable detailed study of neural circuits. The retina provides an ideal model system for neural circuits in general because it is capable of producing complex representations of the visual scene, and both its inputs and outputs are accessible to the experimenter. Chapter 2 describes the biophysical mechanisms that give rise to the omitted stimulus response in retinal ganglion cells described in Schwartz et al., (2007) and Schwartz and Berry, (2008). The extra response to omitted flashes is generated at the input to bipolar cells, and is separable from the characteristic latency shift of the OSR apparent in ganglion cells, which must occur downstream in the circuit. Chapter 3 characterizes the nonlinearities at the first synapse of the ON pathway in response to high contrast flashes and develops a phenomenological model that captures the effect of synaptic activation and intracellular signaling dynamics on flash responses. This work is the first attempt to model the dynamics of the poorly characterized mGluR6 transduction cascade unique to ON bipolar cells, and explains the second lobe of the biphasic flash response. Complementary to the study of neural circuits, recent advances in wafer-scale photolithography have made possible new devices to measure the electrical and mechanical properties of neurons. Chapter 4 reports a novel piezoelectric sensor that facilitates the simultaneous measurement of electrical and mechanical signals in neural tissue. This technology could reveal the relationship between the electrical activity of neurons and their local mechanical environment, which is critical to the study of mechanoreceptors

  18. The RNA binding protein RBPMS is a selective marker of ganglion cells in the mammalian retina

    PubMed Central

    Rodriguez, Allen R.; de Sevilla Müller, Luis Pérez; Brecha, Nicholas C.

    2014-01-01

    There are few neurochemical markers that reliably identify retinal ganglion cells (RGCs), which are a heterogeneous population of cells that integrate and transmit the visual signal from the retina to the central visual nuclei. We have developed and characterized a new set of affinity purified guinea pig and rabbit antibodies against RNA-binding protein with multiple splicing (RBPMS). On Western blots these antibodies recognize a single band at ~24 kDa, corresponding to RBPMS, and they strongly label RGC and displaced RGC (dRGC) somata in mouse, rat, guinea pig, rabbit and monkey retina. RBPMS immunoreactive cells and RGCs identified by other techniques have a similar range of somal diameters and areas. The density of RBPMS cells in mouse and rat retina is comparable to earlier semi-quantitative estimates of RGCs. RBPMS is mainly expressed in medium and large DAPI-, DRAQ5-, NeuroTrace- and NeuN-stained cells in the ganglion cell layer (GCL), and RBPMS is not expressed in syntaxin (HPC-1) immunoreactive cells in the inner nuclear layer (INL) and GCL, consistent with their identity as RGCs, and not displaced amacrine cells. In mouse and rat retina, most RBPMS cells are lost following optic nerve crush or transection at three weeks, and all Brn3a, SMI-32 and melanopsin immunoreactive RGCs also express RBPMS immunoreactivity. RBPMS immunoreactivity is localized to CFP-fluorescent RGCs in the B6.Cg-Tg(Thy1-CFP)23Jrs/J mouse line. These findings show that antibodies against RBPMS are robust reagents that exclusively identify RGCs and dRGCs in multiple mammalian species, and they will be especially useful for quantification of RGCs. PMID:24318667

  19. Imaging Single Cells in the Living Retina

    PubMed Central

    Williams, David R.

    2011-01-01

    A quarter century ago, we were limited to a macroscopic view of the retina inside the living eye. Since then, new imaging technologies, including confocal scanning laser ophthalmoscopy, optical coherence tomography, and adaptive optics fundus imaging, transformed the eye into a microscope in which individual cells can now be resolved noninvasively. These technologies have enabled a wide range of studies of the retina that were previously impossible. PMID:21596053

  20. 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.

  1. Hepatic progenitor cell lines from allyl alcohol-treated adult rats are derived from gamma-irradiated mouse STO cells.

    PubMed

    Zhang, Mingjun; Sell, Stewart; Leffert, Hyam L

    2003-01-01

    In attempts to recharacterize several markers of putative rat liver progenitor cells, single-stage reverse transcription-polymerase chain reaction (RT-PCR) analyses failed to confirm the reported immunochemical detection of albumin, alpha(1)-fetoprotein, and cytochrome P450-1A2 in the clonal line, 3(8)#21, and the cloned derivative, 3(8)#21-EGFP (enhanced green fluorescent protein). Undetectable expression occurred whether or not both lines were cultured on or off feeder layers of gamma-irradiated mouse embryonic STO (SIM [Sandoz inbred Swiss mouse] thioguanine-resistant ouabain-resistant) cells. PCR amplification of liver progenitor cell chromosomal (rat and mouse Pigr, rat INS1, mouse INS2) and mitochondrial (rat and mouse COX1) genes revealed only mouse sequences. Further analyses of rat and mouse COX1 sequences in cells from untampered storage vials of all 11 reported liver progenitor cell lines and strains revealed only mouse sequences. In addition, uniquely similar metaphase spreads were observed in STO, 3(8)#21, and 3(8)#21-EGFP cells. The combined results suggest that the previously reported "rat" liver progenitor cell lines were most likely generated during early derivation in cell culture from gamma-radiation-resistant or ineffectively irradiated mouse STO cells used as the feeder layers. These findings reveal new types of artifacts encountered in cocultures of tissue progenitor cells and feeder layer cell lines, and they sound a cautionary note: phenotypic and genotypic properties of feeder layers should be well-characterized before and during coculture with newly derived stem cells and clonal derivatives.

  2. Assessing the use of immersive virtual reality, mouse and touchscreen in pointing and dragging-and-dropping tasks among young, middle-aged and older adults.

    PubMed

    Chen, Jiayin; Or, Calvin

    2017-04-07

    This study assessed the use of an immersive virtual reality (VR), a mouse and a touchscreen for one-directional pointing, multi-directional pointing, and dragging-and-dropping tasks involving targets of smaller and larger widths by young (n = 18; 18-30 years), middle-aged (n = 18; 40-55 years) and older adults (n = 18; 65-75 years). A three-way, mixed-factorial design was used for data collection. The dependent variables were the movement time required and the error rate. Our main findings were that the participants took more time and made more errors in using the VR input interface than in using the mouse or the touchscreen. This pattern applied in all three age groups in all tasks, except for multi-directional pointing with a larger target width among the older group. Overall, older adults took longer to complete the tasks and made more errors than young or middle-aged adults. Larger target widths yielded shorter movement times and lower error rates in pointing tasks, but larger targets yielded higher rates of error in dragging-and-dropping tasks. Our study indicated that any other virtual environments that are similar to those we tested may be more suitable for displaying scenes than for manipulating objects that are small and require fine control. Although interacting with VR is relatively difficult, especially for older adults, there is still potential for older adults to adapt to that interface. Furthermore, adjusting the width of objects according to the type of manipulation required might be an effective way to promote performance.

  3. Selective expression of hyaluronan and receptor for hyaluronan mediated motility (Rhamm) in the adult mouse subventricular zone and rostral migratory stream and in ischemic cortex.

    PubMed

    Lindwall, Charlotta; Olsson, Martina; Osman, Ahmed M; Kuhn, H Georg; Curtis, Maurice A

    2013-03-29

    Hyaluronan is a large glycosaminoglycan, which is abundant in the extracellular matrix of the developing rodent brain. In the adult brain however, levels of hyaluronan are significantly reduced. In this study, we used neurocan-GFP as a histochemical probe to analyze the distribution of hyaluronan in the adult mouse subventricular zone (SVZ), as well as in the rostral migratory stream (RMS). Interestingly, we observed that hyaluronan is generally downregulated in the adult brain, but notably remains at high levels in the SVZ and RMS; areas in which neural stem/progenitor cells (NSPCs) persist, proliferate and migrate throughout life. In addition, we found that the receptor for hyaluronan-mediated motility (Rhamm) was expressed in migrating neuroblasts in these areas, indicating that Rhamm could be involved in regulating hyaluronan-mediated cell migration. Hyaluronan levels are balanced by synthesis through hyaluronan synthases (Has) and degradation by hyaluronidases (Hyal). We found that Has1 and Has2, as well as Hyal1 and Hyal2 were expressed in GFAP positive cells in the adult rodent SVZ and RMS, indicating that astrocytes could be regulating hyaluronan-mediated functions in these areas. We also demonstrate that hyaluronan levels are substantially increased at six weeks following a photothrombotic stroke lesion to the adult mouse cortex. Furthermore, GFAP positive cells in the peri-infarct area express Rhamm. Thus, hyaluronan may be involved in regulating cell migration in the normal SVZ and RMS and could also be responsible for priming the peri-infarct area following an ischemic lesion for cell migration.

  4. Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina.

    PubMed

    Sierra, Ana; Navascués, Julio; Cuadros, Miguel A; Calvente, Ruth; Martín-Oliva, David; Ferrer-Martín, Rosa M; Martín-Estebané, María; Carrasco, María-Carmen; Marín-Teva, José L

    2014-01-01

    Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid

  5. Cadherin-mediated cell adhesion is critical for the closing of the mouse optic fissure.

    PubMed

    Chen, Shuyi; Lewis, Brandy; Moran, Andrea; Xie, Ting

    2012-01-01

    Coloboma is a congenital disease that contributes significantly to childhood blindness. It results from the failure in closing the optic fissure, a transient opening on the ventral side of the developing eye. Although human and mouse genetic studies have identified a number of genes associated with coloboma, the detailed cellular mechanisms underlying the optic fissure closure and coloboma formation remain largely undefined. N-cadherin-mediated cell adhesion has been shown to be important for the optic fissure closure in zebrafish, but it remains to be determined experimentally how cell-cell adhesions are involved in the mammalian optic fissure closing process. α-Catenin is required for cell adhesion mediated by all of the classic cadherin molecules, including N-cadherin. In this study, we used the Cre-mediated conditional knockout technique to specifically delete α-catenin from the developing mouse eye to show that it is required for the successful closing of the optic fissure. In α-catenin conditional mutant optic cups, the major cell fates, including the optic fissure margin, neural retina and retinal pigmented epithelium, are specified normally, and the retinal progenitor cells proliferate normally. However, adherens junctions components, including N-cadherin, β-catenin and filamentous actin, fail to accumulate on the apical side of α-catenin mutant retinal progenitor cells, where adherens junctions are normally abundant, and the organization of the neural retina and the optic fissure margin is disrupted. Finally, the α-catenin mutant retina gradually degenerates in the adult mouse eye. Therefore, our results show that α-catenin-mediated cell adhesion and cell organization are important for the fissure closure in mice, and further suggest that genes that regulate cell adhesion may underlie certain coloboma cases in humans.

  6. FLRF, a novel evolutionarily conserved RING finger gene, is differentially expressed in mouse fetal and adult hematopoietic stem cells and progenitors.

    PubMed

    Abdullah, J M; Li, X; Nachtman, R G; Jurecic, R

    2001-01-01

    Through differential screening of mouse hematopoietic stem cell (HSC) and progenitor subtracted cDNA libraries we have identified a HSC-specific transcript that represents a novel RING finger gene, named FLRF (fetal liver ring finger). FLRF represent a novel evolutionarily highly conserved RING finger gene, present in Drosophila, zebrafish, Xenopus, mouse, and humans. Full-length cDNA cl