Jonathan Horton - US grants

Approximately 1-2% of the American population suffer visual loss from amblyopia, despite improved standards of detection and treatment. Over the past quarter century, extensive scientific investigations performed in cats and monkeys have helped to elucidate the anatomical and physiological basis of amblyopia. However, little no direct information is available about the pathological changes that occur in the human visual cortex in amblyopia, in part because most of the experimental techniques developed for animal studies cannot be used in man. Cytochrome oxidase histochemistry was introduced by Wong-Riley in 1978 for mapping metabolic activity in the brain. This method has the special advantage that it can be applied to study patterns of metabolic activity in human post-mortem specimens. We have previously used the technique to reveal for the first time the arrangement of the ocular dominance columns in adult human visual cortex. We now propose to apply the cytochrome oxidase method to specimens of visual cortex obtained post-mortem from patients with a history of early visual loss. By examining a series of human cases involving actual loss of one eye (e.g. retinoblastoma, trauma) at various ages, it will be possible to determine the period of "plasticity" for the ocular dominance columns in humans. Cases from patients with a history of unilateral media opacity (e.g. cataract), strabismus, or anisometropia will provide new information about the anatomical and metabolic changes which occur in visual cortex from amblyopia. To interpret human autopsy studies reliably, it is important to conduct correlative experiments in animals, comparing patterns of cytochrome oxidase activity with other anatomical techniques. Despite the cost involved, the macaque monkey is the most appropriate species, because it has highly organized, well defined ocular dominance columns that appear extremely similar by cytochrome oxidase histochemistry to those in the human visual cortex. At one week of age, 2 monkeys will undergo monocular enucleation and 4 monkeys will undergo monocular eyelid suture. Six months later, the animals will receive anterograde tracer injections into single lamina of the lateral geniculate body to label the ocular dominance columns. Cytochrome oxidase activity will then be correlated with the labeled ocular dominance columns. These animal experiments will provide information essential for interpreting our parallel studies in human autopsy cases.

The Ischemic Optic Neuropathy Decompression Trial (IONDT) is a five year study designed to assess the safety and efficacy of optic nerve sheath decompression surgery (ONDS) for non-arteritic ischemic optic neuropathy, diagnosed within one month of onset of visual symptoms. A standardized, common protocol for treatment and data collection will be used by all centers. This multicenter, randomized clinical trial is designed to compare differences in patients randomized to one of two treatment groups as follows: Treatment Group Treatment Sequences No. of Patients 1. Test Treatment Optic Nerve Sheath 150 Decompression 2. Control Treatment Standard Medical Care 150 (no surgery) The primary hypotheses to be addressed by this trial is whether treatment with ONDS is more effective than standard care in (1) restoring or (2) maintaining central visual acuity in patients with NAION? The primary outcome for this trial (change in visual acuity) will be measured using the New York Lighthouse chart. A gain of three or more lines of visual acuity (doubling of the visual angle) will be considered improvement. Secondary outcomes include change in peripheral visual function (using standard Goldmann perimetry) and incidence of ophthalmic and systemic complications. Patients will be recruited over a two year period and followed for a minimum of one year. Study Centers will include a Chairman's Center, 26 Clinical Centers, and centralized facilities, including a Coordinating Center, a Reading Center, and a Project Office at the National Eye Institute.

Amblyopia causes visual loss in 2% of the American population, despite improved standards for detection and treatment. The broad objective of this proposal is to elucidate the structural basis of amblyopia in humans. Little direct information is available about the pathological changes induced by amblyopia in human visual cortex, in part because most of the invasive experimental techniques developed for animal studies cannot be used in humans. The cytochrome oxidase (CO) method of Wong-Riley has the special advantage that it is suitable for mapping patterns of metabolic activity in human autopsy tissues. In this proposal, CO histochemistry will be applied to specimens of visual cortex obtained post-mortem from patients with a history of early visual loss. The specific aim is to define the timing and duration of the critical period for the plasticity of ocular dominance columns, by examining cases involving loss of visual function (or actual loss of one eye, e.g., retinoblastoma, trauma) at various ages during childhood. In addition, the patterns of CO activity in striate cortex associated with different forms of amblyopia will be tested by examining specimens from patients with well-documented histories of early unilateral cataract, strabismus, or anisometropia. CO will also be used to determine if patches are present in human newborns. To interpret the CO data from human tissues, and to further explore the structural and physiological alterations in visual cortex responsible for amblyopia, a series of correlative experiments will be performed in macaque monkeys. The specific aims are: 1) To determine whether a gradient exists across the cortical representation of the visual field, from fovea to periphery, in the susceptibility of ocular dominance columns to shrinkage induced by visual deprivation. This will be achieved by reconstructing the entire mosaic of ocular dominance columns, labelled with CO histochemistry and proline autoradiography, in monkeys deprived at different ages by unilateral eyelid suture. 2) To determine whether ocular dominance columns in macaques are segregated at birth. This will be achieved by intraocular injection of [3H]-proline in monkeys delivered by Caesarean section 1 week before the end of normal gestation. 3) To characterize the receptive field properties and ocular dominance of cells within the foveal representation of amblyopic monkeys. This will be achieved by using quantitative electrophysiological recording techniques. 4) To determine whether visual deprivation reduces thalamic input to CO patches in V1 serving the amblyopic eye. This will be achieved by making large injections of [3H]-proline into the thalamus of visually deprived monkeys to label the patches in V1. 5) To determine whether visual deprivation causes a selective loss of projections from V1 -> V2 which serve the deprived eye. This will be achieved by injecting horseradish peroxidase into V2, and correlating the position of retrogradely labelled cells in V1 with the ocular dominance columns labelled by CO histochemistry.

Significance Amblyopia ([unreadable]lazy eye[unreadable]) causes visual loss in 2% of the American population. Our research is directed at elucidating structural changes in the primate visual cortex which occur from visual deprivation. Objectives Visual deprivation induced by monocular eyelid suture, a laboratory model for congenital cataract, results in shrinkage of ocular dominance columns serving the closed eye. We performed monocular suture in macaques at ages 1,3,5,7, and 12 weeks to define the critical period for plasticity of ocular dominance columns. Results After a minimum survival of 8 months, complete montages of [3H]proline-labelled columns were reconstructed from flat-mounts of striate cortex in both hemispheres. Visual deprivation induced the columns throughout V1 to retract the same distance from their original borders in layer IVc(. At the earliest age we tested (1 week), visual deprivation reduced the columns to fragments. These fragments always coincided with a cytochrome oxidase patch, or a short string of patches, in the upper layers. More severe column shrinkage occurred in layer IVc( (parvo) than layer IVca (magno). The geniculate input to the patches in layer III (konio) appeared normal after deprivation, despite loss of CO activity. Surprisingly, the blind spot representation of the open eye was shrunken by monocular deprivation, although binocular competition is absent in this region. We found that eyelid suture at age 1 week caused the most severe column shrinkage. With suture at later ages, the degree of column shrinkage showed a progressive decline. This implies that primate visual cortex is most vulnerable to deprivation during the first weeks of life, and should provide further impetus for treatment of children with congenital cataract at the earliest possible age. Future Directions Analysis of the changes in connections between cortical layers which occur in amblyopia and initiation of new studies regarding the cortical basis of strabismus (crossed eyes). KEYWORDS ocular dominance column, critical period, amblyopia, visual deprivation, cytochrome oxidase patch, flat-mount, striate cortex, strabismus

Description (provided by applicant): Strabismus is a disease that develops in 1-2% of children, affecting many for the rest of their lives. It is characterized by misalignment of the eyes. The crucial point is that the ocular misalignment usually occurs without any abnormality of the cranial nerves or intrinsic disorder of the eyes. The primary culprit is a failure of the neural mechanisms responsible for maintaining binocular fusion. The goal of this project is to elucidate the defects in brain function responsible for strabismus. Children with strabismus avoid seeing two images by using visual suppression, at the cost of stereovision. If the suppression remains constant in one eye, rather than alternating between the eyes, they may also develop amblyopia. Visual suppression is a critical factor in the development of strabismus, because it eliminates the drive to compensate by fusing separate images. It is unknown how visual suppression occurs, or even where signals from the deviated eye are blocked in the visual pathway. The first specific aim in this grant is to map the visual fields in a large population of human subjects with common forms of strabismus: infantile esotropia, accommodative esotropia, and exotropia. The testing will be done under dichoptic conditions, by presenting different colored images to each eye of subjects wearing colored filters. These experiments will reveal which portions of the visual field are perceived by each eye. The second specific aim is to map dichoptic visual fields in macaques raised with strabismus. The macaque is extremely similar to the human in the organization and function of its visual system. This aim will determine if macaques with strabismus exhibit suppression scotomas similar to those in humans. The advantage of pursuing experiments in nonhuman primates is that one can perform microelectrode studies in the visual cortex to probe the neural basis of visual suppression. The third specific aim is to undertake electrophysiological recordings in awake, strabismic macaques while they are looking with both eyes at visual stimuli, to learn how binocular interactions at the level of single cells give rise to suppression scotomas. Recordings will be made in different regions of the primary visual cortex (V1) in both hemispheres, to correlate single cell recordings with previously mapped suppression scotomas. The fourth specific aim is to examine the pattern of metabolic activity in V1 of macaques with strabismus. Inputs serving each eye are organized into alternating bands called ocular dominance columns. Strabismus induces abnormal staining patterns of the mitochondrial enzyme, cytochrome oxidase (CO), in ocular dominance columns. The pattern of CO activity throughout each V1 will be documented and compared with the pattern of suppression in the visual field mapped behaviorally. If there is a match, it will establish that suppression is mediated by a reduction of activity in columns of cells serving the non-perceiving eye within a suppression scotoma. PUBLIC HEALTH RELEVANCE This project will determine how children with strabismus avoid double vision by suppressing signals emanating from local regions of the retina in each eye. Elucidating the mechanism of visual suppression may lead to better methods to prevent and treat strabismus.

SUBPROJECT ABSTRACT NOT PROVIDED

This award provides funds for purchase of a portable photosynthesis system to be used by faculty at this and other, nearby undergraduate institutions for field-based educational and research activities dealing with the photosynthetic physiology of plants. The instrument will be used for physiological characterization of invasive exotic species as part of efforts to understand characteristics that allow them to be successful invaders. Species to be studied include those that have successfully invaded at least one of a variety of ecosystems, including the forest understory in the southern Appalachians, the rangelands of the semi-arid West, and the riparian zones of the Southwest. Researchers at Appalachian State University and Great Smoky Mountains National Park, will use the instrument to assess the effects of ozone exposure on stomatal control of leaf gas exchange. In addition to its research applications, the instrument will be used in teaching and training activities in six classes taught at the grantee institution and, in addition, will be used in undergraduate research training. Educational use is expected to benefit up to 100 students a year.

National Science Foundation funding will allow the purchase of four controlled environment plant growth chambers for the University of North Carolina at Asheville?s Biology Department. This equipment will enhance the quality and quantity of both teaching and research for professors and undergraduate students in the natural sciences. Use of these chambers will be fully incorporated into the Biology Department?s undergraduate science curriculum, with students first exposed to the chambers in our sophomore-level plant biology class. Cohorts of talented students, particularly those from underrepresented groups, will then be recruited to participate in undergraduate research with the three principal investigators.

By controlling variables such as light intensity, day length, temperature, and humidity, students and faculty will be able to investigate how genetic differences among plants explain differences in growth, development, and reproduction. Growth chambers will also allow researchers to change environmental conditions to elucidate plants? adaptations to a shifting global climate. Finally, the growth chambers will be used in exploring the biology of exotic-invasive plant species. These non-native plants pose particular threats to the health of native plant communities and high quality natural areas, and understanding the ways in which they interact with abiotic environmental factors will help control their spread.

Biological Sciences (61). This plant-based life sciences curriculum project entails the design, implementation, and assessment of an innovative model for research-based pedagogy that allows all students to do original biological research across multiple levels of the biological hierarchy, from molecules to communities. The model introduces laboratory modules developed from ongoing undergraduate thesis work into core first-year courses, then incorporates undergraduate research projects vertically into biology curricula at the sophomore, junior, and senior levels. The modules supplement traditional instruction in basic plant science concepts (community ecology, taxonomy, physiology, and molecular population genetics), and facilitate development of experimental design and analysis skills. In addition, the modules are being designed to expose classroom researchers to several different facets of and approaches to studying the same problem during multiple time periods (lower- and upper-division courses), and to engender in them an appreciation of the scientific process. Rather than using only traditional one-on-one methods for mentoring undergraduate research, this approach provides first-hand, course-based research experiences for all biology students. The evaluation of the effectiveness of the modules at addressing goals for student learning incorporates use of several established and reliable instruments. The modules are designed to be flexible, allowing for adoption of the research-based pedagogy model at a variety of institutional types (including secondary schools, urban or rural universities, and schools with small or large student bodies), and are being shared through a website. This research-based pedagogy model also incorporates use of upper-level research students to mentor cohorts of underclassmen during laboratory exercises. The research students benefit by receiving invaluable instructional training; serving as mentors is giving them opportunities to become more knowledgeable and adept at communicating about their chosen research topics.

This project is being co-funded by funds from the Directorate for Biological Sciences, Emerging Frontiers Division.

Phenology, the study of seasonal biological events, tracks the effects of both year to year climate variability and long-term environmental change on both local and global ecology. It has considerable potential for serving as a base for research studies by undergraduate classes and developing cross-campus studies. Several recent studies, including the President's Council of Advisors on Science and Technology (2014), and Vision and Change in Undergraduate Biology Education (a 2011 document representing the views of over 500 biology faculty and administrators) cite the importance of undergraduate research for retaining science majors and helping students better understand key concepts and develop crucial competencies. Four institutions, the University of North Carolina at Asheville, Appalachian State University, East Tennessee State University, and Warren Wilson College, are combining forces to create common gardens and to develop modules that can guide students' research studies to address potential impacts of global change at multiple levels of the biological hierarchy, from genes to ecosystems. The cooperating institutions are unique in character, diverse in their student bodies, and located in varying climate zones. The project will contribute to our knowledge about ecological issues and will expand the ways in which institutions can combine forces to increase the STEM knowledge and competencies of their students. Environmental change issues to be investigated include monitoring effects of invasive exotic removal techniques on herbs, shrubs, vines, and trees within the areas studied (studies for lower division courses), and exploring questions about the spread of potentially invasive exotic plants, using a combination of GIS (Global Information Systems) mapping, herbarium collections, and online databases (upper division courses).

The collaborating institutions will create and beta-test modules, identify and mitigate barriers to successful implementation of curricular changes, and disseminate curricular changes. The modules will have a direct impact on the learning of over 2000 undergraduates per year, including non-STEM majors. They will be developed and partially administered by undergraduate and graduate research students, and will be instrumental in developing pedagogical expertise for faculty members. The ability of modules to impart botanical knowledge while encouraging higher-order cognitive processes, advancing quantitative literacy, teaching analytical techniques, honing scientific communication skills, cultivating positive student attitudes towards plants and STEM, and improving persistence and graduation in STEM majors will be assessed using a mixed method approach (quantitative and qualitative data collection). Results will begin to document the effects of global change on an understudied but important bioregion while developing a future workforce with solid STEM training.

This project is funded jointly by the Directorate for Biological Sciences, Division of Biological Infrastructure and the Directorate for Education and Human Resources, Division of Undergraduate Education, in support of efforts to address the challenges posed in Vision and Change in Undergraduate Education: A Call to Action http://visionandchange.org/finalreport/.

Project Summary ?Look me straight in the eye? a parent exhorts, but for some children it is impossible because one eye is deviated. In this condition, known as strabismus, stereovision may be lost and the deviated eye may develop amblyopia. Long term consequences include reduced eye-hand coordination, diminished quality of life, employment discrimination, social prejudice, and psychological distress. The overarching goal of this project is to discover why normal binocular vision fails in some children. When children lose fusion, they avoid diplopia by suppressing portions of the visual field seen with each eye. The development of these regions, called suppression scotomas, blocks the error signal that would normally induce an adjustment in extraocular eye muscle tone to bring the eyes back together. Ophthalmologists perform surgery to align eyes, but success rates are far from satisfactory, largely because the persistence of suppression robs children of the drive to recover fusion. To find better treatments, it is imperative to understand the neural basis of suppression. This project uses a translational approach: patients with strabismus are studied to characterize their deficits, and then these deficits are probed in nonhuman primates raised with an experimental form of strabismus that closely resembles the real disease. In Aim #1, studies are focused on children with exotropia, an outwards deviation of one eye. It is usually intermittent, but ophthalmologists tend to recommend surgery, for fear that an intermittent exotropia may progress to become constant, resulting in permanent loss of binocular function. In a longitudinal observational cohort study, patients will be outfitted with a wearable eyetracker to document the frequency of episodes of exotropia during the course of daily activities. The feasibility of using this device to track disease severity in individual patients will be investigated. Data will be collected over 5 years to elucidate the natural history of this condition. In Aim #2, a new chemogenetic technique will be used to silence retinal ganglion cells that project to the superior colliculus. The role these cells play in generating eye movements is unknown, because until now, no method has existed to selectively and reversibly block them. Exotropic monkeys will be examined to determine the impact on receptive field properties and the ability to make alternating saccades. In Aim #3, suppression scotomas will be mapped dichoptically in monkeys with exotropia. Once their layout is established, recordings will be made in the primary visual cortex, to compare responses of single cells to monocular vs. binocular stimulation. For binocular testing, stimuli will be delivered to the receptive field in one eye and to a location in the other eye that is displaced by the magnitude of the ocular deviation. The hypothesis is that cells with their receptive field located in regions where perception is suppressed under binocular viewing conditions will respond more weakly during binocular stimulation than during monocular stimulation. These cortical recordings, the first conducted in alert, behaving monkeys with strabismus, may reveal the neural basis of suppression by correlating single cell firing with visual perception.

PROJECT SUMMARY Computer technology is now an integral part of research. They are vital for all routine tasks, such as communication, lab management, and database searches. Every lab uses computers to acquire, store and analyze data. Additionally, many UCSF vision labs use computers experimentally to present visual stimuli and to record neuronal responses from the retina or brain. Even more so needed in the laboratory where there is specialized equipment with dedicated software, organizational IT infrastructure, experimental rigs, and analysis workstations with specific requirements to work with. The computer/IT core provides outstanding computer and IT support, programming, and teaching by an experienced technician; as well as a centralized database for collaborative use of validated programs both within UCSF and throughout the vision community.