Robert B. Barlow - US grants

The project concerns the encoding of visual information at the retina and further processing of this information at higher levels in the visual pathway. The immediate objective is to understand as fully as possible the functional organization of the Limulus visual system in its natural state. The proposed experiments involve neuroanatomy, neurophysiology, biochemistry, and behavior. The results shall form the basis for extending and testing the present spatiotemporal model of neural integration in the retina and developing a quantitative description of neural interactions in the brain. Strong emphasis is placed on the mechanisms that underlie the efferent control of retinal sensitivity in Limulus. We recently discovered that efferent optic nerve fibers mediate circadian changes in photoreceptor structure and sensitivity. This discovery opens up a new area of research in the functional organization of the visual system. It provides a model system for studying the cellular mechanisms that control visual sensitivity. It also provides a system for investigating the neurosecretory mechanisms that mediate the effects of the endogenous circadian clock. The Limulus visual system will be investigated under as normal physiological conditions as possible. Experiments will be performed on the eye and brain in situ and in organ-culture media. Our ability to record intracellularly from single photoreceptors in situ for periods of up to 12 hours and extracellularly from single optic nerve fibers in situ for up to 6 days enables us to perform experiments that are not yet possible in any other visual system. Our long-range goal is to use the results of our research as a guide for understanding more complex visual systems.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

The long-range goal of our research is to understand the neural basis of visual behavior. We will work toward this goal by studying the Limulus visual system. Its retina contains the largest neural network for which a quantitative model exists, and we understand the animal's visual behavior well enough to determine precisely what it sees. our computational studies have yielded remarkable, new insights about how the retinal network encodes important visual information. We will combine theoretical and experimental techniques to investigate what information the eye must send to the brain for the animal to see and how the brain decodes this information.

Modulation of the sensitivity of the retina by circadian clocks and efferent input for the brains is the main focus of the project. Circadian clocks in the Limulus brain and Japanese quail's eye modulate the structure and sensitivity of retinas in both animals. Noncircadian efferent inputs from the quail brain further modulate retinal sensitivity. Our long range goal is to understand how such modulatory inputs adapt vision for essential tasks. We propose to investigate the circadian modulations of the Japanese quail retina by putative neurotransmitters, dopamine and melatonin (Aim 1), that circadian modulation of the Limulus retina by multiple neurotransmitters (Aim 2), and the efferent modulation of the spatial and temporal response properties of the Japanese quail (Aim 3) and Limulus (Aim 4) retinas. We plan parallel studies with Japanese quail and Limulus involving methods of anatomy, physiology, pharmacology and molecular biology. Our physiological techniques allow long-term recordings of retinal responses in situ from both Limulus (-5 days) and Japanese quail (-2 days) as well as precise control of efferent inputs to both retinas. We can recorded clock activity from isolated Limulus brains (-2 days) and analyze properties of isolated Limulus photoreceptors (-12 hours). Our pharmacological techniques allow manipulation of the putative circadian signals (dopamine and melatonin) in the quail eye and the manipulation of the efferent neurotransmitters (octopamine, 2kD neuropeptide, and light-adapting hormone) in the Limulus eye. These experimental techniques are well suited for investigating the efferent and circadian modulation of retinal sensitivity and establish Limulus and Japanese quail as appropriate models for this research. The primary tenet of the project is that comprehensive studies of these animal models will yield new insights on basic mechanisms of retinal function.

DESCRIPTION (from the author's abstract): Circadian and efferent modulation of visual sensitivity is the main focus of this project. Circadian clocks in the Limulus brain and Japanese quail eye modulate retinal structure and function in both animals, and clock(s) in the eye and/or brain modulate visual sensitivity in humans. Non circadian efferent input from the brain further modulates the quail retina, and changes in blood glucose further influence human visual sensitivity. Our long range goal is to understand how these modulatory factors adapt vision for essential tasks. This project has three interrelated lines of research with Specific Aims to investigate: 1) circadian modulation of photoreceptor noise by rhodopsin stabilization; 2) circadian modulation of retinal sensitivity by dopamine and melatonin; 3) circadian and metabolic modulation of human vision. The PI proposes to study the circadian modulation of photoreceptor noise by analyzing rhodopsin stabilization in Limulus with a range of techniques including voltage clamp, site-specific mutagenesis, gene expression in Xenopus oocytes and Drosophila eyes. The PI will study the circadian modulation of retinal sensitivity by the putative transmitters dopamine and melatonin in Japanese quail using in situ hybridization , nuclear run-on assays, Rnase protection assays and other cell and molecular biological techniques. We will study the circadian and metabolic modulation of vision in humans of various ages and diabetic conditions using psycho physical methods and FMRI of the visual cortex. The primary tenet of this project is that comprehensive studies of human vision and two suitable animal models will yield new insights on how circadian mechanisms modulate visual sensitivity. These studies may shed light on a possible relationship between photoreceptor noise, circadian rhythms and some forms of degenerative retinal disease.

DESCRIPTION (Adapted from applicant's abstract): The long-range goal of the research program is to understand the neural basis of visual behavior. The principal investigator proposes to work towards this goal by studying the visual system of the horseshoe crab (Limulus polyphemus). Its retina contains the one of the largest neural networks for which a quantitative model exists. In addition, a circadian clock modulates the function of the retina, and it is relatively well known what the animal can see underwater day and night. The computational models have yielded new insights about visual coding during the day. Building on this work, the principal investigator plans to combine theoretical and experimental techniques to investigate what information the eyes send to the brain for the animal to see at night. Moreover, he plans to study how the brain decodes the information it receives from the eye. Specific aims are to investigate: 1. Retinal coding of visual information underlying behavior at night. How does the animal see so well at night? During the day, across its ensemble of optic nerve fibers, the eye functions as a global feature detector generating "neural images" of mate-like objects. Does the eye use such a distributed code at night? 2. Brain networks for target detection. How do brain networks decipher the neural codes they receive from the eye day and night? From multiple recordings of brain cells, the principal investigator will develop simplified models of brain network and then drive the models with neural images generated by his cell-based models of the retina. 3. Visual information in the animal's natural environment. The principal investigator will determine whether natural fluctuations in environmental lighting once thought to be noise increase retinal signal-to-noise performance and enhance the visibility of objects important to the animal. Preliminary experiments indicate they do. The principal investigator plans a systematic study involving computation, electrophysiology, neuroanatomy and behavior. He has developed a realistic cell-based model of the daytime state of the 1000-neuron retina. Furthermore, he verifies model predictions by recording responses from single optic nerve fibers of a behaving animal, while recording what the animals sees with a miniature underwater video camera, "CrabCam." And he has developed parallel techniques for studying brain networks. His new results and new techniques establish Limulus as a good model for analyzing the link between neural coding and visual behavior. The proposed studies promise to reveal basic information-processing mechanisms in sizable neural networks. These studies may be applicable to even more complex networks--a major goal of neuroscience.

How do we see? This simple question does not have a simple answer. Vision is a complex process that begins with the absorption of light by the eye and ends with behavior such as perception and locomotion. Because of the complex nature of the vertebrate visual system, especially that of primates, this project uses the relatively simple visual system of the horseshoe crab, Limulus polyphemus. Recent work has uncovered the nature of the neural code for mate detection by this animal during the day, as the eye is "neurally tuned" for this important task. This work will now be extended by a combined anatomical, behavioural, and physiological study of neural coding in the eye and brain with emphasis on understanding how the animal can see so well at night, when light levels are a millionfold lower than in the day.
The animal's visual performance in its natural habitat (ocean) will be precisely measured in day and night. The project will quantify the neural information the eye sends to the brain when the animal sees in its natural habitat and then analyze how the brain processes the information it receives from the eye. New behavioural and physiological techniques developed under NSF support together with a wealth of information about Limulus vision establish the animal as an excellent model for studying the link between neural coding and behaviour.
Past studies with Limulus have provided milestones in vision research. The proposed studies should prove equally productive. They should reveal concepts of neural coding and behaviour common to other animals and contribute to our understanding of how sensory signals relate to behaviour. In addition, there are outstanding opportunities for undergraduate participation in this multidisciplinary research.

Metabolic stress appears to be a factor in late-onset retinal degeneration in transgenic mice. We discovered retinal degeneration and loss of vision in mice rendered chronically hypoglycemic from a null mutation of the glucagon receptor gene, Gcgr, or of the prohormone convertase 2 gene, PC2. Gcgr-/- and PC2-/- mice show the first signs of degeneration and loss of acuity at about 10 months of age and become functionally blind by about 14 months. The underlying molecular and cellular mechanisms are not known, but the availability of glucose appears to be critical. Specific aims are to investigate: Aim 1: Late onset retinal degeneration in hypoglycemic mice. Aim 2: Cellular and molecular mechanisms underlying late-onset retinal degeneration. Aim 1proposes a rigorous, systematic study of the following properties of Gcgr and PC2 null mice: retinal/RPE anatomy, retinal and photoreceptor sensitivity, retinal cell distribution and cell death, and visual function. We will track retinal degeneration in livingmice using new, innovative methods: optical coherence tomography (OCT) to image retinal structures with 3.5^im resolution and behavioral methods to measure visual function. Aim 2 proposes to test for changes in cytosolic/organellar pH from the downregulation of V-ATPase in Gcgr-/- mice. Aim 2 will also test the sensitivityof retinas to the combined stress of hypoglycemia and hypothermia, and test the efficacy of supplemental diets and antioxidants in rescuing vision of Gcgr-/- and PC2-/- mice. Potential medical benefits: (1) identification of metabolic stress as a factor in late-onset retinal degeneration, (2) development of a transgenic mouse model for retinal degeneration resulting from the null mutation of a gene, Gcgr, not expressed in the retina, (3) use of innovativemethods for noninvasive measures of the retina and vision in mice, and (4) potential for yielding insights about causes of late-onset retinal degeneration in humans.