Steven K. Fisher - US grants

This project has several specific goals aimed at increasing our understanding of the vertebrate retina and its interdependence on the retinal pigment epithelium. These studies will contribute to our knowledge of retinal organization and to our understanding of the responses of the retina to injury. The effect of increased light intensity on cyclic cone disc shedding will be studied in ground squirrels. Synaptic organization and circuitry of the human retina will be studied in serial sections by electron microscopy, and the morphology of human retinal neurons studied by Golgi impregnation techniques. We will use light and electron microscope autoradiography to determine the patterns of uptake of various molecules (uridine, proline, galactose, dopamine, Gamma-aminobutyric acid, muscimol, taurine) by the normal cat retina and retina injured by separation of neural retina and pigment epithelium. Changes in the uptake of these molecules with time after injury will be monitored to determine if we can find metabolic changes that parallel the morphological changes described by us and others. The distribution of cytoskeletal proteins (tubulin, microtubule-associated proteins, actin, neurofilaments, vimentin, glial fibrillary acidic protein) and surface saccharides will be studied in normal retina and isolated retinal cells by cytochemical studies using antibodies and lectins. Changes in the distribution of cytoskeletal proteins will also be studied after separation of neural retina and pigment epithelium and correlated with previously described morphological changes. We will use indirect fluorescence to detect lectin binding, indirect immunofluorescence, peroxidase-anti-peroxidase, protein A-gold, or immunogold techniques will be used to study antibody binding. Protein A-gold or immunogold techniques will be used to study antibody binding by electron microscopy. Monoclonal antibodies will be raised against cat retina and pigment epithelium and the localization of their specific antigens detemined immunocytochemically by light and electron microscopy. One of the most important long-term goals of this project is to use information obtained from these experiments to rescue retinal cells after retinal injury.

This is a proposal for a Philips 420 Transmission Electron Microscope with accessories for energy dispersive X-ray spectroscopy. The instrument will be used primarily by five investigators with various research projects in biological sciences, including: retinal cell biology, genetics and development of Tetrahymena, cytoskeleton, and the study of synaptic vesicles. All investigators have current NIH peer-reviewed research grants. It is estimated that their use can potentially account for 100% of a normal 40-hour week. However, since actual use will certainly vary, the microscope will be made available to other users, with priority given to those with current NIH support. The instrument will be maintained by the Institute of Environmental Stress. Technical support of the microscope and help with specimen preparation will be provided by career staff.

Retinal detachment is a serious cause of visual impairment. Gaining an understanding of its cellular effects can help improve management of the disease and contribute to our basic knowledge of the retina and its relationship to the pigment epithelium. We have recently identified specific cellular and biochemical changes in long-term detachments using an animal model. Gaining a further understanding of these changes using a combination of structural, biochemical, and molecular techniques is a major goal of this project. Specifically we will determine: 1) when the upregulation of intermediate filament proteins and the downregulation of enzymatic and vitamin A binding proteins occurs in Muller cells (by using immunocytochemistry and immunoblot analyses); 2) if the mRNA levels,for one protein in each of these classes, changes in concert with the changes in protein expression (by Northern and slot blot analyses; appropriate cellular expression of the mRNAs will be studied by in situ hybridization); 3) the onset and extent of opsin redistribution in photoreceptors (by immunocytochemistry); 4) if opsin synthesis and outer segment renewal continues (by immunoprecipitation and Lucifer yellow band displacement analyses); 5) if opsin mRNA levels change and if opsin mRNA distribution changes in cells that have redistributed opsin or whose morphology is severely disrupted (by Northern, slot blot and in situ hybridization studies); 6) the extent of the proliferative response and identification of the cell types involved (by continuous delivery of 3H-thymidine, LM and EM autoradiography); 7) if growth factors are involved (by immunocytochemistry) or if they can mimic the effects of detachment (by injection of growth factors into the eye or their addition to cultures of Muller cells); 8) if the changes in protein expression or redistribution or the proliferative response are arrested or modified by retinal reattachment. A greater understanding of the synaptic organization of the human retina will be gained by studying synaptic circuitry by serial-section electron microscopy of adult and developing human retinas and the analysis of Golgi- impregnated whole-mounts. In the latter, we will: 1) study the stained neurons in the 75 existing whole-mounts by standard Golgi descriptive techniques, and 2) analyze the synaptic input into the Golgi stained cells by electron microscopy (by obtaining new postmortem tissue from donor eyes and using a variety of fixation protocols to develop better structural preservation of the retina).

Retinal detachment is a serious cause of visual impairment. Gaining an understanding of its cellular effects can help improve management of the disease and contribute to our basic knowledge of the retina and its relationship to the pigment epithelium. We have recently identified specific cellular and biochemical changes in long-term detachments using an animal model. Gaining a further understanding of these changes using a combination of structural, biochemical, and molecular techniques is a major goal of this project. Specifically we will determine: 1) when the upregulation of intermediate filament proteins and the downregulation of enzymatic and vitamin A binding proteins occurs in Muller cells (by using immunocytochemistry and immunoblot analyses); 2) if the mRNA levels,for one protein in each of these classes, changes in concert with the changes in protein expression (by Northern and slot blot analyses; appropriate cellular expression of the mRNAs will be studied by in situ hybridization); 3) the onset and extent of opsin redistribution in photoreceptors (by immunocytochemistry); 4) if opsin synthesis and outer segment renewal continues (by immunoprecipitation and Lucifer yellow band displacement analyses); 5) if opsin mRNA levels change and if opsin mRNA distribution changes in cells that have redistributed opsin or whose morphology is severely disrupted (by Northern, slot blot and in situ hybridization studies); 6) the extent of the proliferative response and identification of the cell types involved (by continuous delivery of 3H-thymidine, LM and EM autoradiography); 7) if growth factors are involved (by immunocytochemistry) or if they can mimic the effects of detachment (by injection of growth factors into the eye or their addition to cultures of Muller cells); 8) if the changes in protein expression or redistribution or the proliferative response are arrested or modified by retinal reattachment. A greater understanding of the synaptic organization of the human retina will be gained by studying synaptic circuitry by serial-section electron microscopy of adult and developing human retinas and the analysis of Golgi- impregnated whole-mounts. In the latter, we will: 1) study the stained neurons in the 75 existing whole-mounts by standard Golgi descriptive techniques, and 2) analyze the synaptic input into the Golgi stained cells by electron microscopy (by obtaining new postmortem tissue from donor eyes and using a variety of fixation protocols to develop better structural preservation of the retina).

DESCRIPTION (Adapted from the applicant's abstract): Detachment of the neural retina from the retinal pigmented epithelium induces a cascade of events detectable within minutes through the activation of early response genes. This leads to the "retinopathy of detachment" (RD), a series of specific cellular events which have been detailed over the past funding period. This includes the death of some receptors by apoptosis, the "deconstruction" of surviving photoreceptor cells so that they assume a more primitive structure, changes in gene expression in photoreceptors and Muller cells (and other retinal cells as well), the proliferation of all non-neural cell types in the retina, and significant growth of Muller cells within and outside their normal retinal boundaries. Visual recovery even after successful reattachment is often less than optimal, especially if the macula is involved. Retinal detachments have serious visual consequences and are a complication of several retinal diseases as well as part of experimental therapies for blinding diseases: foveal translocation, retinal transplantation and subretinal injections of vectors for transfection of retinal cells. Thus, defining the biological mechanisms underlying the responses to detachment and reattachment and finding methods to optimize recovery of the retina would seem to be of clear medical significance. This renewal application uses the investigator's established feline model of detachment/reattachment to study the ability of reattachment to stop or reverse fundamental cellular changes induced by detachment; to further study the use of neurotrophins such as CNTF and hyperoxia as ways to mitigate the degenerative effects of detachment or promote recovery after attachment; and to study the potential role of IL-1 and its receptor antagonist (IL-1ra) in the response to detachment. It is known that cone photoreceptors react differently to detachment than rods, but the study of cones in cat is difficult, and prohibitive in primates. The applicant proposes to develop a detachment/reattachment model in the California ground squirrel, a species with a retina compromised of about 85 percent cones, that is readily available, and that has been used extensively in vision research. The applicant will determine the feasibility of using ERG measures as a means of correlating the physiological and structural recovery of cone photoreceptors. The applicant believes that the use of this model will aid in understanding the responses of cones and to better determine if treatments (e.g., neurotrophins, hyperoxia) that mitigate the effects of detachments in rod-dominated retinas will do so in a cone-dominant retina as well. The applicant believes this information will provide a greater understanding of the retina's responses to injury, and may lead to ways of improving visual recovery in humans.

This collaborative project brings together a strong multi-institutional interdisciplinary team of investigators to study and advance the current understanding of cellular and sub-cellular events. Continuing technological advances in fluorescence and atomic-force microscopy allow scientists to observe molecular function, distribution, and interrelationships in living cells. However, a full understanding of tens of thousands of proteins and the complex molecular processes they engage in requires a voluminous amount of image data, which currently must be analyzed by visual inspection. To facilitate such an analysis, researchers from the four participating institutions are focusing on three main research thrusts. First, next-generation intelligent imaging involves information processing at the sensor level to enable high-speed and super-resolution imaging. The goal is to enable biologists to study cellular processes at resolutions in time and space that are not possible with current technologies. The second research thrust is pattern recognition and data mining as applied to bio-molecular image collections. Salient features that characterize the underlying patterns in cells and tissues need to be computed for the vast volumes of images acquired through automated microscopy. Third, a distributed database of bio-molecular images is being created. The merging of pattern-recognition and data-mining tools with new, powerful methods for indexing, data modeling, and collaboration, is aimed at creating a unique infrastructure that greatly facilitates image bioinformatics, thus complementing recent revolutionary advances in genomics.

The outcome of this research will lead to new and novel information-processing methods for bio-molecular image data. Efficient and effective representation of such data will enable researchers to search and browse through large collections of image and video data and look for similar patterns in such datasets, thus facilitating information discovery. During its five-year duration, this project will develop, test, and deploy a distributed database of bio-molecular image data accessible to researchers around the world. The impact of the distributed database will be through large-scale biology in which the results of a single experiment can be globally correlated with the results from other groups of scientists, thus accelerating discovery of dynamic relationships between structure and function in complex biological systems.

The project will develop new courses, and will facilitate student exchanges, semi-annual meetings, and workshops, benefiting students at all levels. This project will train a new generation of biologists, computer scientists and engineers well versed in the imaging and information-processing sciences at the forefront of next-generation biotechnology. Partnership will be established with institutions with large populations of students from groups underrepresented in science and engineering, such as the California State Universities at Fresno and San Bernardino and the Universidad Metropolitan in Puerto Rico, for undergraduate recruitment and outreach. An effective mode of outreach for students is to educate their teachers, and the project will offer summer fellowships for elementary, high-school, college, and university teachers.

DESCRIPTION (provided by applicant): Funds are requested for the purchase of an Olympus Fluoview FV500 confocal laser scanning microscope, which will be integrated into the UCSB Shared Microscopy Facility. This established facility has been providing core microscopic equipment and technical support to UCSB investigators for over 15 years. The proposed confocal microscope will perpetuate the commitment of the Shared Microscopy Facility to provide state-of-the art tools for the UCSB research community. The instrument requested will replace a seven-year-old BioRad 1024 microscope that is now seriously limited by poor reliability, decreasing repairability, and lack of potential for upgrade. The new microscope will have increased capabilities in terms of the number of available laser excitation wavelengths, the capacity for quantitative analyses, and the imaging of living cells. In addition, the new instrument affords advantages in terms of improved computational hardware and software for laser control and image capture, superior resolution, and larger field of view capabilities. The eleven principal investigators sponsoring this proposal represent five different academic departments or research units including the Neuroscience Research Institute, Department of Molecular, Cellular and Developmental Biology, Department of Psychology, Department of Chemistry and Biochemistry, and the Interdepartmental Program in Biomolecular Sciences and Engineering. Each has current NIH-supported research projects in which confocal microscopy is a critical investigational component. These investigations focus upon biomedical issues, including neuronal degenerative diseases such as Alzheimer's and Parkinson's, ocular pathologies such as age-related macular degeneration and retina] detachment, cancer biology and pharmacology, cell and molecular biology of infectious disease, as well as neuronal physiology and development. The confocal microscope in the Shared Microscopy Facility is the only one on the UCSB campus available to these investigators. The current microscope has been extensively utilized, generating data that has been presented in over 40 peer-reviewed publications in the last five years. It is anticipated that this level of productivity will continue to expand as a consequence of an increasing number of applications for confocal studies afforded by the proposed instrumentation, as well as its streamlined data handling capabilities and superior reliability characteristics. The enhanced multi-labeling capabilities and capacity to support living cell studies will allow investigators to explore novel research avenues that are unapproachable with our current instrumentation.

[unreadable] DESCRIPTION (provided by applicant): Detachment of the neural retina from the retinal pigmented epithelium is a serious cause of visual loss. The retina has a remarkable ability to recover upon reattachment, although visual recovery, when the macula is involved, is often less than optimal. We have discovered over the course of this project that detachment initiates a complex series of biochemical and structural changes throughout the retina, including significant remodeling of both neurons and glia. We have also discovered that reattachment initiates its own set of changes beyond outer segment recovery, including rod neurite sprouting and the growth of Muller cells onto the vitreal surface of the retina. We have also discovered that the simple breathing of oxygen-enriched air can mitigate many of the degenerative changes associated with detachment. The better we understand these complex cellular responses and eventually the molecular mechanism underlying them, the better our position to developing new therapeutic approaches to this and other retinal degenerations becomes. The specific aims for this project are: 1) To study neuronal and glial remodeling in short and long-term detachments and reattachments and the effect of this remodeling on retinal circuitry. 2) To test the hypothesis that the intraretinal proliferative response induced by detachment produces a population of multipotent progenitor cells, and to determine the fate of the proliferating ceils. 3) To test the hypothesis that inhibiting this response will reduce the production of subretinal and/or epiretinal membranes (PVR) and alter the course of degeneration induced by detachment. 4) To test the hypothesis that hyperoxia will improve the cellular outcome of retinal reattachment. 5) To develop a mouse model of detachment and to study mice deficient in the two intermediate filament proteins, GFAP and vimentin to determine their roles in glial cell responsiveness to detachment. [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Recent advances in imaging have enabled multimodal/multiscale observations of complex natural systems. Annotating, harvesting, extracting, and correlating the information contained in these vast image volumes is critically dependent on new information-processing tools as well as robust workflow implementation of well-established tools. In cases such as bioimaging, image data comes from different physical samples from different specimens and needs to be statistically harmonized. Though piecewise computational workflows in data collection are often highly automated, little progress has been made in effective and efficient knowledge discovery. The lack of processing and discovery tools to navigate data of such complexity and magnitude is a critical bottleneck.

The project's computational efforts focus on a biological system that represents a unique combination of high complexity and accessibility for imaging: the vertebrate retina. The retina has a very complex yet highly structured architecture consisting of an unknown number of repeated neural circuits. Though it has been a focus of intense anatomical and physiological studies for over a century, no complete retinal map exists today. In fact, contrary to common misunderstanding, not even all of the retinal cell types have been discovered, much less mapped into functional circuitry. A retinal map is the critical anatomical ground truth that is needed in building realistic models of the earliest stage in visual processing. Building such a cell map requires advances in several areas, including imaging and molecular marker technologies, statistical pattern recognition, and databases. However, the critical barrier at this time is in analyzing the vast amount of images that will be generated from such a project, from samples coming from different retinal cross sections of different animals, and the need to integrate this information in a statistically robust manner to build a retinal map. It is expected that this project will advance not only the image-based information processing technologies but will also have a significant impact on neuroscience research.

The project is interdisciplinary and brings together researchers at the University of Utah and UCSB. Students on the project will get a broad training in retinal neurobiology, computer science and electrical engineering. This project will integrate research and education by introducing the results of the research into courses taught by the PIs on image processing, databases, bioinformatics, and pattern recognition.

? DESCRIPTION (provided by applicant): The photoreceptor outer segment is the site of phototransduction. The phototransductive disk membranes of each outer segment are continually renewed, resulting in a large amount of membrane trafficking from the photoreceptor inner segment to the photoreceptor outer segment, and, eventually, to the retinal pigmented epithelial cells. The formation of outer segment disk membranes is a major process of membrane remodeling, and the renewal is central to photoreceptor cell biology and disease. Competing hypotheses, resulting from differing EM observations, have been presented in the literature to account for how these disk membranes are formed. It has been argued that the different results are due to different methods of tissue fixation. However, in addition to differen methods of tissue preservation, the different studies have focused on different species, and, moreover, time of day has not been considered (yet, there is evidence of a daily cycle in disk membrane growth). The proposed research will advance our knowledge of the organization of nascent rod outer segment disks, and thus mechanisms of disk morphogenesis, by addressing the gaps in published work, and by applying novel technologies to this problem. We will study mouse and monkey retinas. With mouse, we will take the method of preservation of the basal disk membranes to a higher level by using high-pressure freezing/freeze substitution, and thus obviate potential artifacts introduced by chemical primary fixation. Further, we will perform EM serial tomography of the basal rod disks in mouse and monkey, in order to obtain nano-scale, 3D resolution of their organization. The superior resolution afforded by this method, together with the 3D imagery, will enable a complete analysis of membrane connections and continuities that has been unavailable by the methods used to date. This novel approach, will allow us to focus on defining the fundamentally important organization of newly formed basal disks in rod outer segments. Following the studies on WT mouse, we will perform comparable analyses, using three lines of mutant mice that possess defective disk morphogenesis. EM tomographic analyses of the aberrant disks in these mice will provide insight into the normal process, as well as increase our understanding of mechanisms underlying the forms of retinitis pigmentosa, cone-rod dystrophy, and macular degeneration that these mice model. Overall, this proposal will apply state-of-the-art EM technology and 3D image analyses that will provide a major advance in one of the most essential cell biological problems in photoreceptor biology and disease.