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Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction.
Asunto(s)
Distrofia del Cono/genética , Opsinas de los Conos/genética , Genes Ligados a X , Mutación Missense/genética , Animales , Femenino , Humanos , Masculino , Ratones , Retinitis Pigmentosa/genética , Rodopsina/genética , Opsinas de Bastones/genéticaRESUMEN
The Neuroscience Learning Community (LC) that Stonehill introduced to its curriculum grew out of the Great Recession of 2008 and the need for our students to gain hands-on, high-impact learning experiences, despite limited resources. This learning model was first reported in 2013, and since then it has undergone changes that were necessary due to the number of credits and amount of time required for that model. Curriculum changes are common, and Stonehill College changed its credit requirements for LCs to meet students' needs. As a result, the new Neuroscience LC model that we describe here reduced credit hours while leveraging new faculty expertise, collaborations, and new community partnerships. This paper reports student evaluations of an LC model adapted to demand fewer credits and less time, but to retain the community-based learning aspect and to increase faculty collaboration, while maintaining a high standard of learning fundamental neuroscience topics. Evaluations suggest that students valued the updated Neuroscience LC because it helped them understand neuroscience concepts and the impact of neuroscience in our world.
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There are advantages and limitations associated with a science, technology, engineering and math (STEM) education at small, liberal arts colleges relative to larger universities. While there may be increased opportunity for personal attention and access to faculty, students at liberal arts colleges may not always have the opportunity to gain experience with state-of-the-art equipment and technology. Herein, we describe a case study of an inter-institutional partnership between Stonehill College and two neuroscience research laboratories which are part of the Veterans Affairs Boston Healthcare System (VABHS). Both laboratories are affiliated with Harvard Medical School (HMS). We discuss the benefits as well as the challenges associated with the development and maintenance of this partnership. The experience with the use of sophisticated instrumentation and technology available in these laboratories may give students a competitive edge when applying to graduate school programs. However, we contend that the most important advantage of this research experience is the development of a sense of self-esteem and professional competence that will allow students to meet the many challenges that lie ahead in graduate school and beyond.
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Service learning is becoming a keystone of the undergraduate learning experience. At Stonehill College, we implemented a service learning course, called a Learning Community, in Neuroscience. This course was created to complement the basic research available to Stonehill Neuroscience majors with experience in a more applied and "clinical" setting. The Neuroscience Learning Community is designed to promote a deep understanding of Neuroscience by combining traditional classroom instruction with clinical perspectives and real-life experiences. This Neuroscience Learning Community helps students translate abstract concepts within the context of neurodevelopment by providing students with contextual experience in a real-life, unscripted setting. The experiential learning outside of the classroom enabled students to participate in informed discussions in the classroom, especially with regard to neurodevelopmental disorders. We believe that all students taking this course gain an understanding of the importance of basic and applied Neuroscience as it relates to the individual and the community. Students also have used this concrete, learning-by-doing experience to make informed decisions about career paths and choice of major.
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Serotonin initiates various rhythmic behaviors in vertebrates. Previously we have shown that serotonergic neurons innervate the central vocal pathway in the African clawed frog (Xenopus laevis). We also discovered that exogenous serotonin applied to isolated brains in vitro activates fictive vocalizations by activating 5-HT(2C)-like receptors. In this study, we examined the location of 5-HT(2C)-like receptors and determined whether endogenously released serotonin also initiates vocalizations by activating 5-HT(2C)-like receptors in male Xenopus brains. To this end, we first identified the specific location of 5-HT(2C)-like receptors using immunohistochemistry. We next examined which of the populations of neurons that express 5-HT(2C)-like receptors are functionally relevant for initiating fictive vocalizations by applying a 5-HT(2C) receptor agonist to brains transected at various levels. Of four populations of immunopositive neurons, we showed that 5-HT(2C)-like receptors located in two areas of the brain stem vocal circuit, the raphe nucleus and motor nucleus IX-X, initiate fictive vocalizations. We next showed that endogenous serotonin can also activate fictive vocalizations by increasing the extracellular concentration of endogenous serotonin using a selective serotonin reuptake inhibitor (SSRI). The SSRI-induced vocal initiation is also mediated by activation of 5-HT(2C)-like receptors because blockade of these receptors prevents fictive vocalization. The results suggest that in vivo release of serotonin initiates male vocalizations by activating 5-HT(2C)-like receptors in the brain stem vocal nuclei.
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Tronco Encefálico/fisiología , Red Nerviosa/fisiología , Receptor de Serotonina 5-HT2C/metabolismo , Serotonina/metabolismo , Vocalización Animal/fisiología , Xenopus laevis/fisiología , Animales , Masculino , Distribución TisularRESUMEN
Male and female African clawed frogs (Xenopus laevis) produce rhythmic, sexually distinct vocalizations as part of courtship and mating. We found that Xenopus vocal behavior is governed by a sexually dimorphic central pattern generator (CPG) and that fictive vocalizations can be elicited from an in vitro brain preparation by application of serotonin or by electrical stimulation of a premotor nucleus. Male brains produced fictive vocal patterns representing two calls commonly produced by males in vivo (advertisement and amplectant call), as well as one call pattern (release call) that is common for juvenile males and females in vivo but rare for adult males. Female brains also produced fictive release call. The production of male calls is androgen dependent in Xenopus; to test the effects of androgens on the CPG, we examined fictive calling in the brains of testosterone-treated females. Both fictive male advertisement call and release call were produced. This suggests that all Xenopus possess a sexually undifferentiated pattern generator for release call. Androgen exposure leads to a gain-of-function, allowing the production of male-specific call types without prohibiting the production of the undifferentiated call pattern. We also demonstrate that the CPG is located in the brainstem and seems to rely on the same nuclei in both males and females. Finally, we identified endogenous serotonergic inputs to both the premotor and motor nuclei in the brainstem that may regulate vocal activity in vivo.
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Caracteres Sexuales , Tegmento Mesencefálico/fisiología , Vocalización Animal/fisiología , Xenopus laevis/fisiología , Animales , Gonadotropina Coriónica/farmacología , Coito/fisiología , Cortejo , Estimulación Eléctrica , Femenino , Nervios Laríngeos/fisiología , Masculino , Proteínas del Tejido Nervioso/análisis , Ovariectomía , Periodicidad , Serotonina/farmacología , Tegmento Mesencefálico/efectos de los fármacos , Testosterona/farmacología , Triptófano Hidroxilasa/análisis , Vocalización Animal/efectos de los fármacos , Xenopus laevis/anatomía & histologíaRESUMEN
This paper applies the solutions to the fundamental issues addressed in Part I to specific design problems of embedding data in image and video. We apply multilevel embedding to allow the amount of embedded information that can be reliably extracted to be adaptive with respect to the actual noise conditions. When extending the multilevel embedding to video, we propose strategies for handling uneven embedding capacity from region to region within a frame as well as from frame to frame. We also embed control information to facilitate the accurate extraction of the user data payload and to combat such distortions as frame jitter. The proposed algorithm can be used for a variety of applications such as copy control, access control, robust annotation, and content-based authentication.
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BACKGROUND: Abnormal trophoblast differentiation and function is the basis of many placenta-based pregnancy disorders, including pre-eclampsia and fetal growth restriction. PPARgamma, a ligand-activated nuclear receptor, plays essential roles in placental development; null murine embryos die at midgestation due to abnormalities in all placental layers, in particular, small labyrinth and expanded giant cell layer. Previous studies have focused mostly on the role of PPARgamma in trophoblast invasion. Based on the previously reported role of PPARgamma in preadipocyte differentiation, we hypothesized that PPARgamma also plays a pivotal role in trophoblast differentiation. To test this hypothesis, we report derivation of wild-type and PPARgamma-null trophoblast stem (TS) cells. METHODOLOGY/PRINCIPAL FINDINGS: PPARgamma-null TS cells showed defects in both proliferation and differentiation, specifically into labyrinthine trophoblast. Detailed marker analysis and functional studies revealed reduced differentiation of all three labyrinthine lineages, and enhanced giant cell differentiation, particularly the invasive subtypes. In addition, rosiglitazone, a specific PPARgamma agonist, reduced giant cell differentiation, while inducing Gcm1, a key regulator in labyrinth. Finally, reintroducing PPARgamma into null TS cells, using an adenovirus, normalized invasion and partially reversed defective labyrinthine differentiation, as assessed both by morphology and marker analysis. CONCLUSIONS/SIGNIFICANCE: In addition to regulating trophoblast invasion, PPARgamma plays a predominant role in differentiation of labyrinthine trophoblast lineages, which, along with fetal endothelium, form the vascular exchange interface with maternal blood. Elucidating cellular and molecular mechanisms mediating PPARgamma action will help determine if modulating PPARgamma activity, for which clinical pharmacologic agonists already exist, might modify the course of pregnancy disorders associated with placental dysfunction.