Research
Current Research Projects, Retinal Cell Biology Laboratory, Neuroscience Research Institute, UCSB
Photoreceptor Disk Membrane Morphogenesis
Funding Source: National Institutes of Health
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 different 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 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 wild-type mice, 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.
Central Serious Retinopathy: A Serious Cause of Visual Loss in Humans
Funding Source: Macula Vision Research Foundation
The goal of this study is to determine if a naturally occurring mutation in the mouse (called, nm3342) can be used to help us understand the human disease central serous retinopathy (also central serous chorioretinopathy or CSR). CSR can be relatively benign, and self-limiting, but can become recurrent or chronic with serious long-term visual consequences. The disease was first described by the Albrecht von Graefe in 1866, and more than 140 years later we know virtually nothing about its pathophysiology and there is no accepted treatment. There are no animal models for this disease.
Photoreceptors of the human retina must remain closely apposed to a single layer of cells known as the retinal pigmented epithelium (RPE) in order to remain healthy and functional. If these two become physically separated a sight-threatening condition known as a retinal detachment occurs. The most common form of detachment occurs when a hole or tear develops through the retina allowing fluid to move from the vitreous cavity and separate the two layers. Modern retinal surgery has about a 95% success rate for repairing this type of detachment. In CSR however, fluid accumulates between the two layers without a tear in the retina. The origin of the fluid in this case is assumed to occur due to a sudden “backward flow” across the RPE, usually at a highly focused location within the central, most visually critical part of the retina. Except in rare cases, CSR is left untreated because there is no effective therapeutic intervention. Presumably any treatment that would cause the retina to reattach more rapidly would improve visual recovery. Devising an effective therapy depends on understanding the physiological mechanisms underlying this mysterious disease. What causes this sudden accumulation of fluid behind the retina? Does it result from failure of some physiological mechanism in the RPE cells that normally keep fluid from accumulating in this space? Does it represent the death of a small patch of RPE cells, or a breakdown of complex junctions that link RPE cells together and normally form a barrier to fluid flow between the cells?
An understanding of CSR and the ability to develop rational therapies has suffered greatly from the lack of a satisfactory animal model for the disease. The recently discovered nm3342 mouse develops spontaneous serous detachment of the retina, and may provide the first animal in which to study mechanisms underlying this disease. In this project we are characterize the effect of this mutation on the RPE and retina and the expression of critical protein molecules b these cells. In a collaborative study with the laboratory of Dr. Sheldon Miller at the National Eye Institute we will compare the physiological properties of RPE cells from nm3342 mice to those derived from wild-type mice.
Wide-scale Imaging of Retinal and Optic Nerve Head Astrocytes After Retinal Detachment
Funding source: International Retinal Research Foundation
In this study we are testing the hypothesis that large-scale retinal injury, caused by retinal detachment, stimulates a large proportion of retinal and optic nerve head astrocytes to become “reactive”, thus providing a model system in which to study this process. We will characterize these cells and their relationship to nearby blood vessels and optic nerve axons and how this changes after injury of the retina by detachment. We will also test the hypothesis that a specific trophic factor (bFGF) is a key player in initiating astrocyte. The first part of this study will determine if retinal astrocytes become traditionally reactive in response to injury, an area virtually unstudied in the retina. In brain and spinal cord, astrocytic reactivity has many important implications for neuronal survival and regeneration. The growth factor bFGF, has been implicated in astrocytic reactivity elsewhere in the central nervous system, but whether it plays a similar mechanistic role in the retina is unknown. We will use confocal imaging of wholemounted mouse retinas to study astrocyte proliferation, migration, hypertrophy and changes in their relationship to other cell types at different times after retinal detachment. The morphology of individual astrocytes will be studied by confocal imaging single-cell dye filling techniques. Three-dimensional mosaiced images of the entire retinal surface should provide novel insight into the reactive capacity of retinal astrocytes and preliminary data for future studies of these cells and their role in retinal injury and repair.
The Use of Electron Image Tomography to Study the 3-Dimensional Structure of Photoreceptor Synapses at Sub-Cellular Resolution
Funding source: Santa Barbara Cottage Hospital
In a collaborative study with the National Center for Microscopy and Imaging Research at the University of California, San Diego we are using the ultra-high resolution of intermediate voltage electron microscope tomography to examine the change in morphology of photoreceptor terminals following retinal detachment. Retinal detachment is a serious injury that often has long-term consequences for vision. Our goal is to characterize the effects of detachment on photoreceptor synapses since they are the first step in the flow and processing of information through the retinal circuitry. We discovered several years ago that these synapses apparently “deconstruct” their complex architecture in reaction to detachment. We will use the innovative technology of intermediate voltage electron microscope (IVEM) tomography to generate high-resolution models of normal PR synapses and compare them to those degenerating after retinal detachment and regenerating after reattachment. The ability of PR cells to recover is remarkable, but the imperfect recovery of their immensely complex synapses may contribute to visual deficits that occur in many patients with otherwise successful retinal reattachment. The resulting data should expand our understanding of the organization of photoreceptor cell synapses, and reveal novel insight into the structural remodeling they can undergo. Electron tomography has many advantages over the use of traditional serial ultrathin sections and conventional electron microscopy starting with an order of magnitude increase in z-axis resolution. This will be the first time this technology has been used to study photoreceptor synapses and we believe that it will provide new levels of information about their structure and regenerative capacity.
Human Retinal Progenitor Cells as a Candidate Therapy for Retinitis Pigmentosa
Funding source: California Institute of Regenerative Medicine
Our laboratory is playing a specific role in this multidisciplinary collaborative study headed by Dr. Henry Klassen at the University of California, Irvine. This project was funded to determine the efficiency of survival and engraftment of human retinal progenitor cells surgically placed in the eyes of animals with genetic disorders equivalent to retinitis pigmentosa in humans. The ultimate goal is to use these cells to replace lost photoreceptors in people with retinitis pigmentosa. The current proposal is designed to optimize the probability of achieving an important stem cell milestone: the safe, therapeutic integration of a tissue-specific stem cell into the adult human CNS. The scientific feasibility of stem cell-mediated neuroprotection in retinal degeneration has been amply demonstrated in animals. The preeminent validity of this approach informed the unanimous conclusion of the Stem Cell Consortium (SCC) of the Foundation Fighting Blindness (FFB), at a meeting convened in December 2006 for the purpose of identifying the most feasible strategy available for treating retinal degenerations. The SCC also concluded that this approach represents the stem cell-based method closest to clinical application for RP, and that RP represents a near-term clinical target on both scientific and clinical considerations.
Computational Challenges in the Discovery and Understanding of Complex Biological Structures Through Multimodal Imaging
Funding source: National Science Foundation
This is a multidisciplinary study involving several investigators who are part of the Center for Bio-Image Informatics at UCSB and the University of Utah. It has as its major goal the first step toward creating a complete map of neuronal circuitry in a defined region of mammalian retina including all cell classes and connections. 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 bio-imaging, 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 development of a transformative image data-harvesting framework is the primary objective of this project. To focus our computational efforts, we have chosen a biological system that has a unique combination of high complexity and accessibility for imaging, the vertebrate retina. This choice is motivated by the experience of our interdisciplinary team consisting of computer scientists and neurobiologists who have been working together for over five years and collectively represent over a half-century of vision expertise. As a specific and ambitious goal, we propose creating a complete map of a mammalian retina, including all cell classes and connections. Such a mapping project compares in scale to the human genome project. The data sets involve complex cellular and subcellular building blocks, high-dimensional molecular signatures, intricate local and global 3D patterning rules, and rich heterocellular associations, with raw data collected from different animal samples at different times with different but overlapping probespectra and with different imaging modalities.
Uncertainty Management in Working with Scientific Images
Funding source: National Science Foundation
A collaborative project involving scientists from UCSB’s Center for Bio-Image Informatics. Images are ubiquitous in scientific applications, and are often the primary source of data for analysis, discovery and hypothesis validation. Typically, these images are processed to extract useful features, which are then used for detecting patterns of interest, building scientific models for data visualization, as well as for new pattern discovery. However, in each of these stages of data analysis there is inherent uncertainty. The premise of this research is that uncertainty is a universal reality in image informatics. Examples may range from remote-sensed imagery of wildfire evolution in ragged terrains, to microscope imagery of suspected breast cancer cells. Powerful models exist for analyzing imagery obtained in either application for determining the current condition (fire front location and propagation rate, cancer cell shape, size and density, etc.) and predicting future evolution. However, errors in parameter estimates are inevitable and inherent to the limitations of instruments and analysis methodologies. The uncertainties in these measures need to be explicitly taken into account in the modeling and recognition stages to optimize threat response strategies. Similar examples exist in virtually all disciplines that use images or video as main data sources. Thus, a highly critical requirement for a broad spectrum of applications is the ability to handle uncertainties in the context of new models and structures for data storage and to consider them in novel data query environments, for search, retrieval, and data mining over such data structures. This is coupled with the need to develop a new generation of visualization techniques to account for implicit uncertainties on multiple levels of analysis. This research project brings together researchers in image analysis, pattern recognition, databases, visualization, and neurosciences in addressing information processing challenges in the specific "testbed" context of bioimaging. The methods and solutions that will be developed cut across disciplinary boundaries and will benefit a wide range of applications. In addition to the basic research, there will be outreach activities that include high-school and undergraduate summer research and workshops that will be hosted at UCSB on this important topic and its implications.
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