William H. Merigan - US grants

DESCRIPTION: We propose to use lesion studies to determine whether there is similar processing of color and form information in human and macaque visual cortex. Studies with macaque monkeys will continue to explore the contribution of individual cortical visual areas, with the goal of determining capabilities that are uniquely reflective of each area's activity. We have found that localized lesions in near-extrastriate visual areas V2 and V4, cause precisely retintopic losses of complex, but not fundamental, visual functions. These studies will be extended to the effects of lesions of inferotemporal visual areas TEO and TE, which receive much of their input from the above cortical areas. We will use the cumulated information we have obtained about extrastriate macaque visual areas to explore parallel organization in the human visual system. Particular attention will be paid to lesions of near extrastriate visual areas, since the visual distribution of losses can help explore parallels between human and macaque visual cortex. Our studies will focus on human cerebral achromatopsia and visual form agnosias, which are among the most dramatic disorders suggesting that human visual cortex is organized into functional modules.

There is now substantial evidence of two anatomically and physiologically distinct pathways through primate striate and extra-striate visual cortex. These pathways have been labelled the "motion" and "color and form" pathways, suggesting a detailed knowledge of their individual functions and interactions. However, to date, there is little direct evidence either of the role these pathways play in visual perception, or of their strict independence. The studies proposed here are designed to determine the functional role of these separate pathways in primates by measuring visual loss resulting from damage to one or the other of them. The "motion" pathway will be damaged in one study by lesions of cortical area V2, which will interrupt one of the major projections to area MT, and in a later study by inactivation of MT and/or MST. The :color and form" pathway will be damaged at an early stage in its cortical path by lesions of area V2, and in a separate experiment, by lesions of area V4 itself. Lesions will be produced by localized injections of ibotenic acid into visual field specific regions determined by physiological mapping. Visual thresholds will then be tested during controlled fixation in the visual field locations that correspond to these injections. 1. The first group of studies will assess the effects of V2 lesions on visual capacities basic to both color and motion perception. Visual acuity, luminance and chromatic contrast sensitivity, color matching, and speed and direction difference thresholds will be measured. 2. A second group of studies will examine the role of visual area V4 in the processing of color information. It is expected that removal of this area should disrupt higher level but not simpler aspects of color perception. This hypothesis will be evaluated by testing both basic capacities (luminance and chromatic contrast sensitivity) and more complex abilities (color matching) within the visual field locus corresponding to the lesion. 3. A third set of studies will examine the effect of lesions of visual areas MT and MST on the perception of visual motion. Again both basic (opposite direction discrimination and velocity difference thresholds) and higher level (direction difference thresholds and manipulations of direction coherence and noise masking in dynamic dot patterns) capabilities will be tested. An additional group of studies will be conducted to compare transitory visual effects of MT and MST lesions with permanent effects.

Our previous work has using the block-copying task demonstrated unexpected trade-offs between observer eye and head movements and the use of visual memory in ongoing tasks. Understanding the operation of visual memory is crucial in making the transition from an understanding of low level perceptual machinery to understanding of how perception mediates ongoing sensori-motor bahavior. Recently we developed a new paradigm for exploring simple sensori-motor behaviors, in which observers construct wooden models of objects. Initial results confirm the crucial role of fixation that we observed with the simpler copying task, indicating that these memory/eye movement tradeoffs are a ubiquitous feature of normal sensori-motor behavior. During the past year we studied the way tradeoffs between eye fixations and memory change in the context of familiar visual objects, and how complex tasks become automatised". A crucial component of behavioral flexibility is our ability to learn sub-components of complex tasks (automati-sation) in order to perform the task more efficiently with minimal high level control. Initial observations suggest that automatization is revealed in the fixation patterns to the model. As learning progresses, each fixation gives more visual information. This corresponds to the process of "chunking," or consolidation of referents. Although this process has long been hypothesised and is a crucial component of cognitive models, it has proven elusive to study with standard psychological techniques. These experiments now allow us to look at the development of the chunking process. This project dovetails with a major research project at the University of Bielefeld, Germany, that uses the same wooden construction pieces in the development of machine recognition systems for language and vision.

DESCRIPTION (provided by applicant): An exciting possibility, raised by the recent development of light-gated channels such as channelrhodopsin, is the restoration of visual sensitivity in patients that are blind due to degeneration of rod or cone photoreceptors or the retinal pigment epithelium (RPE) cells that nourish the photoreceptors. Development of such a therapeutic approach to blinding eye disease supports the mission of the National Eye Institute, which "supports research that helps .. treat eye diseases.. and ..leads to sight-saving treatment". Photoreceptor and RPE degeneration causes blindness in many common eye diseases, such as macular degeneration, as well as in blinding exposure to lasers or other intense lights. It is likely that the approach to restoring vision studied here is feasible, as shown by recent research in which light-gated channels were inserted into retinal cells of blind rodents and produced vision. The studies proposed here will test the quality of vision that can be produced by this approach in macaque monkeys, whose vision is virtually identical to human vision. The research plan involves creating small patches of blind retina in macaque monkeys, caused by photoreceptor/RPE damage due to prolonged exposure to a 568 nm laser. Initial studies will use visual testing to verify that the light-exposed regions of retina are completely blind. The light-gated channel channelrhodopsin2 will then be inserted into undamaged retinal ganglion cells overlying the regions of photoreceptor/RPE damage by intravitreal injection of AAV2 viral vector. Psychophysical testing will then measure the restored vision mediated by the channelrhodopsin and verify that there is no residual vision for wavelengths of light beyond that absorbed by channelrhodopsin. The actual restoration of vision by insertion of light-gated switches in humans will require a gene therapy approach, in which a virus containing the light switches was injected into the eye. This is the method being used in the present study, although with macaque monkeys rather than human subjects. If this approach is successful, applications to human vision could be initiated in the near future. PUBLIC HEALTH RELEVANCE: Degeneration of photoreceptor/RPE is the cause of blindness in common eye diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), prompting a search for a visual prostheses that can provide vision mediated by the surviving inner retinal neurons. The research proposed here will examine the possibility that visual perception can be restored by this prosthesis in macaque monkeys blinded by photoreceptor/RPE degeneration.

DESCRIPTION (provided by applicant): The primate retina contains more than 17 classes of ganglion cells, but the contribution to vision of all but a few of these classes is unknown. This large gap in understanding is due to the fact that most ganglion cell types form such sparse mosaics that it is difficult with a single microelectrode or even an array of microelectrodes to record from enough cells of any given class to characterize its functional role. Another limitation of microelectrode technology is that the recording process is invasive, requiring penetration of the globe or, in the case of an eyecup preparation, enucleation of the eye. This precludes the ability to repeat experiments on the same cells and limits behavioral experiments on the same animals in which electrical responses have been obtained. However, rapid advances are being made in the development of reporter molecules that allow optical monitoring of the electrical responses of single neurons with multiphoton fluorescence. Moreover, the recent development of adaptive optics for correcting the eye's aberrations now makes it possible to image individual ganglion cells at ~ 2 micron resolution in the living primate eye. We will develop a new technology for retinal physiology, Functional Adaptive-optics Cellular Imaging in the Living Eye (FACILE) that combines adaptive optics in vivo imaging with optical recording to map the electrical activity of each of the several hundred ganglion cells simultaneously in a patch of monkey retina. We will use viral transduction to insert a genetically encoded calcium indicator (GCaMP3) into ganglion cells, exploring two delivery methods to further improve viral transduction of macaque ganglion cells: intravitreal injection of adeno- associated virus (AAV) in collaboration with John Flannery at UC, Berkeley and retrograde transport of pseudotyped equine anemia immunodeficiency virus (EAIV) injected into retino-recipient nuclei in collaboration with Ed Callaway at the Salk Institute. The development of FACILE will accelerate the complete characterization of the many pathways from the retina to the brain and will reveal the full contribution the retina makes to visual information processing. We will undertake early development of FACILE in a mouse model, and deploy the mature technology in monkey retina. In years 4-5, we will demonstrate the value of the approach by resolving the long-standing debate about whether the macaque retina contains direction-selective neurons, such as those that have been identified in the retinas of several other mammals.

The Instrumentation Module provides expertise in optical, electronic, and mechanical engineering to design, assemble, maintain, and repair novel devices for vision research. This module has contributed to at least 211 publications during the last funding period. It will be used to a moderate to extensive degree by 15 of the 18 participating investigators during the next funding period.

DESCRIPTION (provided by applicant): Restoration of vision has long been a goal of vision research. It has been studied sporadically since the 1700s, but first produced effective vision restoration when arrays of stimulating electrodes were implanted into the brain, initially by Brindley in 1968 and subsequently by William Dobelle in the 1980s. Since that time, there has been rapid development of a wide range of methods for vision restoration, most of which have been examined only in laboratory animals. These include such diverse approaches as implantation of stem cells into retina and the insertion of light-gated channels into inner retinal neurons. The restoration method that has had the most impact recently, however, is the implantation of electronic stimulation devices just below or above the retina, an approach which recently received CE Mark approval in Europe and FDA approval in the United States. The rapid progress in developing vision restoration technologies has resulted in conferences and publications on technical challenges facing each approach. However, there has been little direct comparison of which methods best meet the goals of vision restoration. Discussion between research groups involved with different approaches has been limited in part by the unique constraints on each methodology. For example, implantation of electronic retinal prostheses is routinely performed in blind humans, although much parallel research continues to examine electronic stimulation of retinal ganglion cells in laboratory animals. Human perception can be easily tested, thus this approach has been shown to produce perceptual benefits, unlike other methods such as viral vectors and pluripotent stem cells that involve great risks of pathology in humans and have therefore been conducted largely in laboratory animals. On the other hand, pluripotent stem cells and use of light sensitive prosthetics such as channelrhodopsin might provide important advantages over electronic prostheses, but analysis of this question requires clear identification of the goals of vision restoration. The 29th Symposium of the Center for Visual Science will bring vision restoration researchers together with low vision specialists who study the importance for daily living of such visual capabilities as: foveal versus peripheral vision, motion versus form perception, and high luminance cone versus low luminance rod mediated vision. It will examine vision restoration in light of the variety of disorders that damag vision and the type of information that partially or totally blind patients need to function normaly. It will also consider the role played by central visual pathways in restoring vision, limitations t recovery resulting from alteration of early visual development, and the importance of cortical plasticity in refinement of visual perception as patients adapt to their visual prosthesis. The conference will also provide an opportunity for students and post-docs to present their work in poster sessions, and will make competitive travel fellowships available to the best of the students and post- docs who wish to attend and present their work.

DESCRIPTION (provided by applicant): Photoreceptors and retinal pigment epithelium (RPE) cells in the outer retina are highly vulnerable to degenerative eye disease, but recent advances in optogenetics offer the possibility of restoring vision after loss of photoreceptor function. Ths approach has been demonstrated already in the mouse. This project will deploy a monkey model to explore the feasibility of a retinal prosthetic that bypasses degenerated photoreceptors by using optogenetic methods to make retinal ganglion cells light-sensitive. The monkey model is needed because the monkey has a fovea and human-like visual perception. To establish a monkey model of retinal degeneration, we will use phototoxicity to selectively destroy photoreceptors in localized retinal areas following exposure to bright light. We will then attempt to restore light sensitivity by transducing ganglion cells with an intravitreal injection of a vira vector designed to express channelrhodopsin. We will evaluate both the effectiveness of the light exposure in destroying photoreceptor function and the effectiveness of the optogenetic prosthesis in restoring function with two methods. First, we will measure the light responses of ganglion cells with a genetically encoded calcium indicator, imaged in the living eye with adaptive optics. The method will allow us to track ganglion cell function repeatedly over many weeks in the same monkey. Second, we will measure the visual effectiveness of the optogenetic prosthetic with visual psychophysical tasks. If these experiments are successful, they will clarify the photosensitivity, dynamic range, and information capacity of an optogenetic prosthetic applied to the primate fovea.

INSTRUMENTATION CORE SUMMARY The Instrumentation Module provides Center for Visual Science (CVS) faculty with the electrical, mechanical, and computer interface engineering expertise necessary to develop novel devices and instrumentation. The Instrumentation core is staffed by Martin Gira, an electronics engineer, and Dan Guarino, a mechanical engineer. Gira combines more than 29 years of experience in electrical and mechanical engineering in the Center for Visual Science with a strong work ethic and very positive interactions with the many investigators he supports. Guarino is a recently- trained mechanical engineer who complements Gira's expertise in electronics, working on the design and assembly of electronic devices for interfacing computers to both mechanical instruments and display/imaging hardware. The experience and specialized expertise of the module's staff are an important foundation of the CVS research effort, making the Instrumentation Module essential for CVS to continue breaking new ground in vision research.

Project summary Despite the current enthusiasm for optogenetic vision restoration, there is remarkably little direct evidence about whether it can reverse blindness in humans. The feasibility of optogenetics was first suggested by studies from many labs that showed restoration of neuronal visual responses and visually guided behaviors in previously blind mice or dogs. These studies have been very informative in exploring interventions to treat retinal diseases genetically identical to some human disorders. However, they have not addressed restoration in an animal model that closely matches human vision, nor have they examined the perceptual quality of the restored vision. In the studies proposed here, we will address these two issues, first by using macaque monkeys in all studies, the animal model that most resembles humans in visual structure and function, and second by examining the range of visual perceptual abilities made possible by optogenetics. We will use a recently developed in-vivo physiology method to study vision restoration at the level of individual retinal cells in macaque monkeys. This approach will be used in the first aim to examine at a cellular level the spatial, temporal and sensitivity responses of macaque retinal neurons produced by optogenetic restoration. In the second aim we will use controlled fixation psychophysical testing of macaques to examine the range of visual capabilities provided by channelrhodopsin restoration, something that has never been studied. Finally, we will examine the duration of optogenetic restoration, measuring any long-term decrease in function or practice-related improvement in visual function.