Jay Neitz - US grants

Recently developed experimental methods in which functional foreign genes are introduced into the germ line of mammals provide a powerful new tool for dissecting complex biological processes. These techniques have great potential for providing a new approach for the study of basic visual mechanisms. The research proposed here will introduce human cone pigment genes into mongolian gerbils (Meriones unguiculatus) with the aim of producing gerbils that express human pigments in their cone outer segments. The gerbil is uniquely suited for this project. It has a robust photopic system but only a single type of cone photopigment. The spectral peak of the gerbil cone pigment (494 nm) is well separated from that of any of the human cone photopigments. Thus, the presence of introduced human cone pigments will be easily detected in the gerbil even at low levels of expression. The proposed research will employ methods involving the production of transgenic gerbils and the subsequent assessment of the consequences of the introduced genes on visual function as follows: Cone pigment genes isolated from a human genomic library will be microinjected into gerbil embryos. The expression of the introduced genes in the resulting gerbils will be assayed by a retinal gross potential, the electroretinogram. Gerbils that express the human photopigment transgene will be further examined in behavioral experiments and direct photopigment measurements will be made using microspectrophotometry of cone outer segments. If human cone pigment genes can be expressed in gerbils, this work will provide the first step toward future experiments designed to elucidate the mechanisms involved in the developmental regulation of cone pigment gene expression with the particular aim of understanding errors in photopigment gene expression that underlie visual defects. This work will also provide a starting point for experiments designed to further illuminate the neural mechanisms of color vision.

DESCRIPTION (provided by applicant): The long-term goal of this research program is to understand the role of amino acid sequence polymorphisms in the long- and middle-wavelength cone opsins in vision disorders. All known amino acid substitutions observed in human rhodopsin or in the human S cone opsin are associated with photoreceptor abnormalities and disease. The question we will address in this proposal is - what is the role of amino acid substitutions in the L and M cone opsins in vision disorders? We propose the following specific aims: Specific Aim 1: Examine the relationship between cone opsin variants and age related macular degeneration (AMD). Using DNA samples from several hundred subjects with AMD and several hundred matched control subjects we will quantify the association between sequence variations in the L and M opsin genes and risk of AMD. Specific Aim 2: A high frequency opsin variant, designated LVAVA, has never been observed in a male with normal vision. Its occurrence is always associated with vision disorder. Individuals with this variant exhibit pathological myopia, cone ERG abnormalities and optic nerve hypoplasia indicating a reduced number of ganglion cells. We will examine the effects of the LVAVA variant on the structure and physiology of cone photoreceptors and other retinal cells in a mouse model and determine the mechanism by which the variant opsin produces its wide spectrum of abnormalities in the eye. Specific Aim 3: A second cone opsin variant which is never observed in males with normal vision, designated LIAVA, is generated at a high rate. Function is disrupted in cones expressing this variant and adaptive optics imaging demonstrates that cones expressing it are damaged or lost. We will examine the effects of the LIAVA mutation on the structure and physiology of cone photoreceptors in a mouse model and determine the mechanism by which the variant opsin disrupts photoreceptor function.

image processing; vision; eye; biomedical facility; digital imaging;

Image acquisition, processing, analysis and display play an increasingly important role in biomedical research. Our Image Analysis Module has continuously evolved in response to everchanging technology and to the expanding needs of our group. To aid our Core investigators with their imaging needs, the Module performs three interrelated functions: (1) it serves as the conduit for Core faculty to access expensive imaging equipment located in institutional core facilities and in our Core Modules, (2) it maintains the image acquisition instruments and the computer systems in the Core Modules, and (3) it helps our eye and vision researchers use imaging technologies in their individual and collaborative research. The third function has two components that take advantage of the exceptional capabilities of the engineer who staffs the Module. He is proficient in the use of the imaging equipment and its software and therefore can assist users in all aspects of image capture, analysis, manipulation and display. Further, he is competent to assist with both hardware and software development for investigators who do not use or who modify off-the-shelf equipment. This latter function was added to the Module during this current cycle in response to a growing need identified by our investigator group, which precipitated the hiring of an experienced Module engineer.

DESCRIPTION (provided by applicant): Myopia is a major problem worldwide with the number of affected individuals estimated to be as high as 90% for some Asian countries. The prevalence of myopia in the US is on the rise, up from 25% in 1971-1972 to 41.6% in1999-2004, with some underserved ethnic groups such as Native Americans and Alaskan Eskimos being particularly susceptible. The annual cost of treatment approximates 2-3 billion dollars for the estimated 40-50 million affected individuals in the US. In addition, myopia can lead to secondary complications that cause severely reduced vision. Myopia is caused both by genetic and environmental factors. Humans are normally born hyperopic, with eyes too short for the optics. During development, visual experience regulates eye growth so that the eye stops growing when the length is optimal for the optics (emmetropia). Myopia occurs when the eye grows past the point of emmetropia, becoming too long. The long- (L) and middle- (M) wavelength-sensitive cones mediate visually guided eye growth. Preliminary data is presented suggesting that 1) rare variants of the L and M cone opsin genes underlie a severe inherited form of myopia 2) there is an association between common myopia and variants of L and M opsin genes and 3) the L to M cone ratio influences visually guided eye growth. The L and M cone opsin genes are highly variable, encoding a tremendous amount of amino acid sequence variation in the opsins, making them excellent candidates for causing common forms of myopia. The ratio of L to M cones is also highly variable across individuals, which produces variability in the response of retinal circuits to environmental stimuli, which in turn influences eye growth. The involvement of the L/M cone pigments and cone ratio in the mechanism regulating eye growth suggests that axial elongation can be controlled by modifying visual experience. In a pilot study, children wore special eyeglasses containing one experimental and one control lens for three months. Both lenses had the individual's optimal correction. The experimental lens had a color-blocking filter to remove red light, and a holographic diffuser to blur the image slightly. The control lens passed red and green light equally but ensured that both eyes were exposed to the same light intensity throughout the study. Axial length measurements were taken at two week intervals. Eyes wearing the experimental treatment lens grew significantly slower than eyes wearing the control lens (p=0.001), making this a very promising method for preventing myopia. This application addresses the stated objective in NEI's Health Disparities Strategic Plan to "determine the etiology of human myopia and identify the risk factors associated with this and other refractive errors so as to prevent their occurrence or progression." Specific aim 1 will evaluate the role of cone ratio, axial length, and L and M cone opsin gene variants in the etiology of myopia. Aim 2 will investigate the role of L: M cone ratio in the etiology of myopia by comparing ratios across ethnic groups particularly at risk for myopia. Aim 3 will evaluate the potential of lenses that block specific wavelengths of light and introduce image blur in slowing axial elongation in myopic children. PUBLIC HEALTH RELEVANCE: The prevalence of myopia in the US is on the rise, up from 25% in 1971-1972 to 41.6% in1999-2004, with some underserved ethnic groups such as Native Americans and Alaskan Eskimos being particularly susceptible. The annual cost of treatment approximates 2-3 billion dollars for the estimated 40-50 million affected individuals in the US, in addition, myopia can lead to secondary complications that severely impair vision. The major objectives of this grant are to investigate newly identified genes and environmental cues in the etiology of nearsightedness and to establish a newly identified treatment that, in a small pilot study, showed great promise as an effective, inexpensive, non-invasive, non-pharmacological means of slowing or stopping abnormal eye growth.

DESCRIPTION (provided by applicant): Myopia is a major problem worldwide with the number of affected individuals estimated to be as high as 90% for some Asian countries. The prevalence of myopia in the US is on the rise, up from 25% in 1971-1972 to 41.6% in1999-2004, with some underserved ethnic groups such as Native Americans and Alaskan Eskimos being particularly susceptible. The annual cost of treatment approximates 2-3 billion dollars for the estimated 40-50 million affected individuals in the US. In addition, myopia can lead to secondary complications that cause severely reduced vision. Myopia is caused both by genetic and environmental factors. Humans are normally born hyperopic, with eyes too short for the optics. During development, visual experience regulates eye growth so that the eye stops growing when the length is optimal for the optics (emmetropia). Myopia occurs when the eye grows past the point of emmetropia, becoming too long. The long- (L) and middle- (M) wavelength-sensitive cones mediate visually guided eye growth. Preliminary data is presented suggesting that 1) rare variants of the L and M cone opsin genes underlie a severe inherited form of myopia 2) there is an association between common myopia and variants of L and M opsin genes and 3) the L to M cone ratio influences visually guided eye growth. The L and M cone opsin genes are highly variable, encoding a tremendous amount of amino acid sequence variation in the opsins, making them excellent candidates for causing common forms of myopia. The ratio of L to M cones is also highly variable across individuals, which produces variability in the response of retinal circuits to environmental stimuli, which in turn influences eye growth. The involvement of the L/M cone pigments and cone ratio in the mechanism regulating eye growth suggests that axial elongation can be controlled by modifying visual experience. In a pilot study, children wore special eyeglasses containing one experimental and one control lens for three months. Both lenses had the individual's optimal correction. The experimental lens had a color-blocking filter to remove red light, and a holographic diffuser to blur the image slightly. The control lens passed red and green light equally but ensured that both eyes were exposed to the same light intensity throughout the study. Axial length measurements were taken at two week intervals. Eyes wearing the experimental treatment lens grew significantly slower than eyes wearing the control lens (p=0.001), making this a very promising method for preventing myopia. This application addresses the stated objective in NEI's Health Disparities Strategic Plan to determine the etiology of human myopia and identify the risk factors associated with this and other refractive errors so as to prevent their occurrence or progression. Specific aim 1 will evaluate the role of cone ratio, axial length, and L and M cone opsin gene variants in the etiology of myopia. Aim 2 will investigate the role of L: M cone ratio in the etiology of myopia by comparing ratios across ethnic groups particularly at risk for myopia. Aim 3 will evaluate the potential of lenses that block specific wavelengths of light and introduce image blur in slowing axial elongation in myopic children.

Systems Biology Services and Shared Instrumentation Component: Project Summary The objective of the Vision Research Core is to enhance the productivity and efficiency of the research programs of the UW vision scientists, with special priority given to investigators holding NEI R01 grants. The core achieves this objective by: (1) giving investigators and their laboratory personnel access to resources that are outside the resources of individual R01 grants, (2) giving investigators and their laboratory personnel access to technical expertise that is outside the scope of individual labs, (3) providing training of laboratory personnel to enhance the capacity of individual labs, and (4) providing a culture that promotes collaboration. The Systems Biology Services and Shared Instrumentation Component achieves this goal ? by providing access to state-of-the-art instrumentation for in vivo and ex vivo functional analysis of the visual system ? by providing access to state-of-the-art instrumentation for in vivo imaging of retina, including detection of fluorescent proteins ? providing state-of-the-art light sources and calibration instruments for developing visual stimuli for functional experiments ? by providing technical assistance and training in use of the component?s shared instruments

If therapeutic approaches are to provide meaningful vision, it is essential that we investigate and discover the links between human perception and the activity of neurons and circuits in the visual system. Here we propose to study circuits involving S-cone pathways in primates as a model for how the visual system is organized to serve diverse functions. Much of the effort in trying to understand the biology of the primate visual system has emphasized its role in perception, in particular with producing an internal visual representation of the outside world and the objects and events within it. However, comparative studies of more primitive visual systems indicate that vision did not originally evolve as a system for perceiving the world. Rather, the visual systems of lower vertebrates emphasize neural circuitry for directly triggering movements, moment-to-moment, in real time and pathways serving non-image forming functions such as circadian photoentrainment. Our visual system has maintained evolutionarily ancient functions that are not directly involved in conscious perception. Attempts to understand the biology of vision in primates (including humans) that have failed to recognize that much of the visual system is not concerned with perception have limited our understanding of how the neural pathways comprising the visual system are organized. Theories of blue-yellow color vison in primates have focused on the classic ?blue-ON? neuron, the small bistratified retinal ganglion cell. However, evidence has accumulated that these cells may not be directly involved in the conscious perception of blue and yellow hues; rather they are well suited for triggering spatially directed movements. As an alternative to the idea that small bistratified ganglion cells are the basis for blue-yellow sensations, we have made discoveries that point to previously unknown circuitry that may have evolved only in primates specifically for conscious color perception. The goal of the experiments proposed here is to understand how S-cone pathways in primates are organized to serve the diversity of functions of the visual system including non-image forming vision functions and the largely separate functions of constructing a perceptual representation of the world vs. controlling goal-directed actions. We propose two specific aims: Specific Aim 1: To work out the diversity of parallel pathways for processing of signals from S cones in the primate retina using Serial Block Face Scanning Electron Microscopy. Specific Aim 2: To use a combination of whole-cell and loose patch recording in combination with pharmacological manipulation of specific synaptic elements to directly measure the spatio-chromatic organization of the receptive fields of ganglion cells in the primate retina that process signals from S cones, and to correlate the results to those obtained from anatomical characterization of S-cone pathways.

Functional Analysis of the Visual System and In Vivo Ocular Imaging ? Abstract The main objectives of the Vision Research Core are to enhance the productivity and efficiency of the research programs of the UW vision scientists and to facilitate collaboration among investigators. The Functional Analysis of the Visual System and In Vivo Ocular Imaging Core Module enhances productivity and efficiency by providing access to shared equipment and facilities that are used by multiple labs. Shared equipment is cost effective when multiple labs have need for the same expensive technology, but individual labs lack the resources to purchase the technology, to support personnel with the necessary technical expertise to use or maintain equipment, or are unable to make full time use of such technology. Structural analyses of the eye using in vivo ocular imaging and functional analyses of the visual system with in vivo and ex vivo electrophysiological and psychophysical methods are fundamentally important for modern basic and translational vision research. This core module provides specialized support for these analytical approaches through access to shared instruments and to technical expertise in the acquisition and analysis of data. The shared instruments and technical expertise offered by this module expands the capacity of UW vision researchers in the structural and functional analysis of the visual system, which in turn facilitates sharing of applications through collaborative exchange.