Benjamin E Reese, Ph.D. - US grants

Color vision is a sensory capability that is extremely valuable for discriminating objects. The retina of the eye detects colors using the cone cells, containing special pigments that are the expression of certain genes. New discoveries in genetics have led to some proposed generalizations about the distribution and evolution of color vision in primates, which are considered to be the only mammals with exceptional color vision. But very few primate species have been rigorously studied for this attribute, to either confirm or refute the generalized picture. Earlier work revealed a large variability both among individual squirrel monkeys in making color matching tests, and in the pigments found in the cone cells of the retina of individual animals. The variation was discovered to be related to expression of color pigments based on a single gene on the X-chromosome, so there is a fundamentally different variability between male and female color perception. Preliminary studies have extended this finding to a few other "new-world monkey" species, emphasizing the difference from humans and "old-world monkeys" so far studied, which have a second cone pigment gene on the X-chromosome. This project will use behavioral measurements of color vision, and external recording of retinal potentials that convey information about the cone pigments, from a variety of monkey and prosimian species. The data will be used to test how valid the generalization may be about color vision in new-world and old-world monkeys, and how early in the evolutionary branching of primates the genetic split may have occurred. This novel work, using psychology, physiology, genetics, and zoology, will have impact on visual neuroscience and behavioral ecology, and may even provide some insight into human origins.

Ganglion cells in the retina may project to either the ipsilateral or contralateral side of the brain, depending upon their class and on their retinal position. The long-long-term objective is to understand the determinants of the "decussation pattern" of the distinct retinal ganglion cell classes in mammals. The specific aim of this proposal is to define the relative roles of two hypothesized mechanisms: 1) decussation patterns are produced as a consequence of regressive events that selectively eliminate cells projecting to the inappropriate side of the brain; or 2) decussation patterns are produced as a consequence of selective axonal navigation at the chiasmatic region, where some timeand/or position-dependent event determines the side of choice. Studies will use the visual systems of adult and developing cats and ferrets, in which there are well-characterized differences in decussation patterns between the retinal ganglion cell classes and between the species. These species and cell class differences are the basis for the proposed experiments. The experimental approach employed is to assess either the nature of the crossed and uncrossed ganglion cell distributions on the retina (the "decussation pattern"), or to determine the organization of optic fibers in the chiasmatic region, either a) during development, b) in the normal adult, or c) as a consequence of some early manipulation to the visual pathway. The experiments will make use of the techniques of Horseradish peroxidase histochemistry or carbocyanine dye diffusion, with which cells in the temporal retina can be identified according to their side of projection; tritiated thymidine autoradiography, with which the order of genesis of the retinal cells projecting to the different hemispheres can be determined; myelin staining of axonal classes, with which the fiber organization of the adult chiasm can be determined; and electron microscopy, with which the position of growth cones at different regions in the developing chiasmatic region can be defined. Finally, a novel tissue culture technique is described for studying chiasmatic pathway decisions during development.

Ganglion cells in the retina may project to either the ipsilateral or contralateral side of the brain, depending upon their class and on their retinal position. The long-long-term objective is to understand the determinants of the "decussation pattern" of the distinct retinal ganglion cell classes in mammals. The specific aim of this proposal is to define the relative roles of two hypothesized mechanisms: 1) decussation patterns are produced as a consequence of regressive events that selectively eliminate cells projecting to the inappropriate side of the brain; or 2) decussation patterns are produced as a consequence of selective axonal navigation at the chiasmatic region, where some timeand/or position-dependent event determines the side of choice. Studies will use the visual systems of adult and developing cats and ferrets, in which there are well-characterized differences in decussation patterns between the retinal ganglion cell classes and between the species. These species and cell class differences are the basis for the proposed experiments. The experimental approach employed is to assess either the nature of the crossed and uncrossed ganglion cell distributions on the retina (the "decussation pattern"), or to determine the organization of optic fibers in the chiasmatic region, either a) during development, b) in the normal adult, or c) as a consequence of some early manipulation to the visual pathway. The experiments will make use of the techniques of Horseradish peroxidase histochemistry or carbocyanine dye diffusion, with which cells in the temporal retina can be identified according to their side of projection; tritiated thymidine autoradiography, with which the order of genesis of the retinal cells projecting to the different hemispheres can be determined; myelin staining of axonal classes, with which the fiber organization of the adult chiasm can be determined; and electron microscopy, with which the position of growth cones at different regions in the developing chiasmatic region can be defined. Finally, a novel tissue culture technique is described for studying chiasmatic pathway decisions during development.

This project provides funds for expanding and improving undergraduate biopsychology teaching laboratory curriculum. Two upgraded versions of current courses (Laboratories in Neuroanatomy and Animal Learning) and two new ones (Laboratories in Hormones and Behavior and Human Psychophysiology) are being offered to approximately 200 biopsychology majors. A laboratory complement is provided to lecture course material and emphasizes the dynamic, i.e., ever-changing, nature of scientific discovery. Other goals of the laboratory curriculum are as follows: (1) to provide undergraduates with hands on experience in state-of-the-art neuroscience research methodologies through small group instruction in the laboratory, preparing them for careers in the health sciences; (2) to teach undergraduates the scientific method, by having them design and conduct independent experiments to test specific, self-generated scientific hypotheses; (3) to improve scientific literacy (including critical thinking) and communication, by requiring students to read the original scientific literature and present their own data in written and/or oral form; (4) to enhance our students' computer usage skills. Achievement of these goals enables graduates: (1) to be cognizant of their own physiology; (2) to evaluate critically the enormous amount of information (scientific or otherwise) presented by the media; (3) to compete successfully in the markeplace for jobs or additional postgraduate education; (4) to succeed in the increasingly technology-driven workplace of current and future society.

This research proposal will define the ways in which neuroblasts of distinct laminar and functional fate disperse within the retina. Transgenic mice carrying the bacterial lacZ gene inserted on the X chromosome will be used, and the natural, random phenomenon of X inactivation in hemizygous females will restrict expression of the transgene to half of all retinal neurons. Since X inactivation occurs before any retinal neuroblasts have been born, and because progeny inherit the X-active status of their progenitors, this approach permits the marking of large numbers of retinal clones. Chimeric mice, formed by combining lacZ-expressing embryonic cells with wild-type embryos, will also be used to label small numbers of retinal clones in order to confirm the results from the X-inactivation transgenic mosaic mice. Retinae from both transgenic and chimeric adult mice show a conspicuous columnar arrangement of such clones of cells, but also reveal that distinct types of retinal cell do not respect this columnar segregation. The distributions of the clonally related cells suggest that the dispersion pattern of a neuroblast from the germinal zone of the developing retina can include a tangential as well as a radial component, but that the expression of the tangential component is confined to distinct subsets of retinal neuroblast. The present investigation will examine two hypotheses for this tangential dispersion. First, is tangential dispersion due to a passive displacement of these cells as the remainder of the retinal cells proliferate? And second, is the tangential dispersion due to an active displacement of the postmitotic neuroblasts, playing a role in the establishment of the orderly spacing between neurons of particular classes? Adult and developing retinae will be examined, in which cohorts of retinal cells sharing common birthdates will have been labelled during embryogenesis. Distinct types of retinal cells will b identified using immunocytochemistry to correlate phenotype and intercellular spacing with extent of tangential dispersion. These studies will clarify the mechanisms underlying the creation of the mature retinal architecture.

Our visual abilities arise from the capacity of photoreceptor cells to transduce a photic stimulus into a neural response and to transmit this message to second order neurons. Effective transmission of that message is, in turn, dependent on processes acting during development that orchestrate the formation of the retinal circuitry. However, relatively little is known about the developmental mechanisms responsible for establishing the precise connectivity of photoreceptor cells. The present investigation will identify cellular and molecular mechanisms controlling the formation of photoreceptor connectivity in the developing ferret retina. Preliminary data indicate that photoreceptor terminals initially grown beyond their normal targets in the outer plexiform layer and that the errant terminals are subsequently retracted as proper connections are formed. This proposal will define the transitional morphology of photoreceptor terminals during the period of synapse elimination. Moreover, cellular and molecular mechanisms controlling terminal outgrowth, target recognition, and synaptic connectivity will be identified using in vivo experimental manipulations and in vitro approaches. These studies should clarify the mechanisms by which the changing circuitry associated with photoreceptors is established during development.

cell proliferation; gene expression; retinal bipolar neuron; retina; cell differentiation; phenotype; cytogenetics; cell age; cell type; radionuclide double label; sex chromosomes; autoradiography; laboratory mouse; genetically modified animals; immunocytochemistry; lac operon;

DESCRIPTION (provided by applicant): Recent advances in the field of developmental neuroscience have revealed many of the mechanisms controlling proliferation, patterning, neurogenesis, fate determination, migration, differentiation, pathway navigation, synaptogenesis and cell death. What has been lacking, however, is an understanding of the mechanisms that control neuronal positioning: what are the determinants of neuronal position within a brain structure, and how is a cell related to the positioning of other cells of the same or different type? The present exploratory proposal will create software tools for describing the geometrical relationship between neurons in a volume of tissue, and for modeling their positioning in three-dimensional space. Drawing upon such tools that have been successfully employed to describe the geometry and spatial relationships between retinal neurons distributed in two dimensions, we will extend such Matlab-based scripts to enable the same sorts of analyses in three dimensions. X-Y-Z positional information will be extracted from samples of brain tissue labeled to reveal individual types of neuron and/or glial cell, upon which a variety of Voronoi domain-based computations will be performed, including the measurement of Voronoi domain volumes, Delaunay segment lengths, nearest neighbor distances, and Voronoi facet areas. Auto-correlation analysis will be performed to identify whether there is any consistent higher-order patterning in the relationship between cells of a given type, or any evidence of exclusion zones maintaining a minimal distance between like-type cells. Cross-correlation analysis will determine the relationship between different types of cell. Evidence of exclusion zones in the autocorrelograms is suggestive of minimal-distance spacing rules operating between cells, and modeling studies will seek to define those rules by comparing simulations with real biological data. This project will therefore establish new tools for describing the spatial relationship between cells within a brain structure, specifically, the spacing rules that may govern their relative positioning. These tools will be made freely available to the scientific community for downloading from a website. They will provide the basis for future studies in which researchers can quantitate and model the spatial relationships between cells in the CNS in the process of exploring the biological mechanisms underlying intercellular spacing.

[unreadable] DESCRIPTION (provided by applicant): The architectural complexity of the retina is brought about by a series of developmental events controlling proliferation, fate determination, migration, process outgrowth, target recognition, synaptogenesis and cell death. These processes establish a precisely layered structure in which retinal neurons become positioned at different depths, connected via two intervening synaptic layers. Superimposed upon this layered organization, certain cell types are distributed as orderly arrays across a given layer so that they, and their processes, ensure a uniform sampling of the retinal surface as they establish connectivity with their afferent and target neurons. This research program is seeking to understand the cellular, molecular and genetic determinants of this patterning and connectivity in such arrays, focusing upon the population of horizontal cells. A quantitative trait analysis of horizontal cell number in recombinant inbred strains will first identify genes that control the size of this neuronal population. Chimeric mice will be produced from parental strains that differ in horizontal cell number, to determine whether dendritic field size is controlled by cell-intrinsic vs. environmental instructions. A comparable quantitative trait analysis of the cone photoreceptors will also be conducted, and the role played by the afferents in specifying dendritic patterning of horizontal cells will be examined in various knockout and recombinant inbred strains in which the convergence ratio between cones and horizontal cells is modulated, or in which neurotransmission between these afferents and the dendrites is altered or abolished. The roles played by both homotypic neighbors and by afferents in the establishment of this connectivity will be determined, examining the hypothesis that the onset of visual activity drives a competitive interaction between neighboring horizontal cells as they seek to colonize individual pedicles in the developing outer plexiform layer. Microarray analysis of embryonic retina from two parental strains showing a two-fold difference in both horizontal and cone cell number will be used to identify downstream genes critical for the establishment of these differences. These experiments will reveal the genes that regulate horizontal and cone cell number, as well as the biological mechanisms by which horizontal cells establish their morphological patterning, dendritic coverage and connectivity with cone afferents during development. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Two-photon microscopy is revolutionizing many aspects of modern microscopy and having a dramatic impact upon biomedical research in general. The major advantages provided by two-photon instruments are the ability to image deep within tissue samples and the greatly enhanced abilities for live-cell work. Taken together, these technical capabilities open up an enormous array of novel strategies for investigators.Funds are requested for the purchase of a 2- photon microscope, composed of a Zeiss (LSM 510) laser scanning confocal microscope and a Chameleon XR Ti:Sapphire laser acquired from Coherent, providing femtosecond pulses compatible with live-tissue imaging. The instrument will be integrated into the UCSB Shared Microscopy Facility. This communal core facility, operated jointly by our Neuroscience Research Institute (an interdisciplinary Organized Research Unit of the University of California) and our Department of Molecular, Cellular and Developmental Biology has been providing shared microscopy instrumentation and technical support to UCSB investigators for 17 years. The full-time Director of the Facility, Dr. Brian Matsumoto, is an acknowledged expert in multiple aspects of cutting-edge microscopy. Presently, the major instrumentation in the Facility includes an Olympus Fluoview Confocal Laser Scanning Microscope, a JEOL transmission electron microscope, and four state-of-the-art Olympus fluorescence microscopes with digital cameras. The confocal and electron microscopes were both acquired via the Shared Instrumentation Program, and both have been used extensively and maintained meticulously. Various NIH-funded investigators at UCSB will benefit from the expanded repertoire of applications provided by this instrument, including the ability to image deep within tissue samples and the in-vitro analysis of living cells or tissues across time in the absence of the phototoxicity typically associated with single-photon approaches. Acquisition of this 2-photon microscope will therefore enhance the diverse efforts of a number of investigators at UCSB in pursuit of their NIH-funded research objectives. [unreadable] [unreadable] [unreadable]

ABSTRACT The cellular architecture and connectivity of the vertebrate retina is remarkably conserved across species. What distinguishes these retinas most is the relative numbers of each of the different cell types. Even within a species, there is significant variation in the size of neuronal populations. Polymorphic genes controlling the processes regulating cellular production, fate assignment and survival contribute most of this variation between individuals, and the present investigation will seek to identify genes responsible for this variation, and to understand their role in those processes modulating proliferation, fate determination and apoptosis. Twenty-six recombinant inbred strains of mice derived from the A/J and C57BL/6J strains of mice will be used to determine the natural variation in four different types of retinal bipolar cell, and the degree of co-variation between cell types will be examined. The variation in bipolar cell number will also be compared with data for cone photoreceptor number and with new data collected for rod photoreceptors. Variation in cell number will be mapped to genomic loci where candidate polymorphic genes will be identified and tested using gene knock-out strategies. The role of cell death in establishing bipolar cell numbers, and its temporal occurrence, will be assessed directly in Bax knock-out mice, while its afferent-dependency will be defined in coneless and conefull mutant mice. Other cell types have been shown to have their morphological differentiation controlled by the density of neighboring like-type cells as well as by their afferents, and so each of these variables will be modulated to determine their effects upon the differentiation of bipolar cell dendrites. Finally, a developmental transcriptome analysis of the retina will be conducted in these each of these recombinant inbred strains, and made available to the scientific community on-line at NerveNetwork. This will enable the direct mapping of variations in gene expression to genomic loci, thereby aiding in the identification of candidate genes underlying the above variations in bipolar and photoreceptor cell number, and the detection of correlations in gene expression to identify regulatory networks that participate in the production of individual types of bipolar or photoreceptor cells. These experiments will reveal the determinants of nerve cell number and morphology, clarifying our understanding of retinal development, as well as identifying gene polymorphisms that may contribute to retinal disease.

ABSTRACT Cellular populations in the nervous system vary in their demographics: They differ in their size, positioning, intercellular spacing, dendritic overlap, and connectivity. This research program has been identifying the genetic sources of this variation and analyzing the interdependencies between these population dynamics, using the retina as a model system and a panel of twenty-six genetically distinct recombinant inbred mouse strains. Neuron number varies considerably across these strains of mice, for every different type of retinal neuron analyzed to date, and this variation maps to discrete and largely independent genomic loci for each cell type, showing minimal evidence for genetic co-regulation. The present proposal will continue to explore the genetic sources of such variation in cell number, focusing upon different populations of retinal interneurons, and how such variation in their cell number affects those other demographic traits, via four new specific aims. Specific Aim 1 will extend our use of quantitative trait locus (QTL) mapping strategies to identify epistatic interactions controlling the variation in retinal cell number. It will identify candidate genes at interacting genomic loci, and demonstrate their genetic interaction directly. Specific Aim 2 will examine the role of the Rbfox gene family in retinal development, and identify changes in alternative splice transcripts in the absence of RBFOX function. Specific Aim 3 will define the role of the transcription factor, Nfia, in the selective control of AII amacrine cell number. It will assess the alternative splicing of Nfia as a function of development, and examine the functional properties of developmentally regulated isoforms. Specific Aim 4 will define the degree of dependency of VGluT3 amacrine cell differentiation upon the density and intercellular spacing of these cells, seeking to understand the role played by homotypic interactions in regulating retinal coverage. The present research proposal will thereby identify the genetic determinants and intercellular interactions that underlie the demographic features of cellular populations in the retina. These studies will clarify our understanding of retinal development and identify novel genes and their variants that may contribute to developmental disorders of the nervous system, together informing the emerging field of regenerative medicine.

Project Summary Basic research has identified ?-amyloid plaque formation and tau aggregation as the primary biomarkers of Alzheimer?s Disease (AD), which are hypothesized to be causally involved in AD progression. Molecular interventions of primarily ?-amyloid aggregation have over the last decade, however, been met with overt failure. Thus, alternative targets are now essential. Several inhibitors of tau aggregation in vitro have been identified, but only one, methylene blue, is currently being assessed for efficacy in vivo. Most small molecule chemical inhibitors fail to enter the drug pipeline due to insolubility as well as inherent toxicity issues. Consequently, our laboratory has considered naturally occurring molecules present in edible plants as possible alternatives. Plant polyphenols have long been associated with various health beneficial effects owing classically to their anti-oxidant properties. However, distinct bio-activities of certain polyphenolic compounds are now believed to be responsible for a significant reduction in age-related diseases, cancer, cardiovascular diseases, and processes associated with neurodegeneration. We previously demonstrated that an extract of cinnamon containing a procyanidin A-linked trimer molecule (865 Da) was non-toxic and capable of inhibiting tau aggregation in vitro (Peterson et al., J. Alz. Dis., 2009, 17: 585-97). We now show that an aqueous extract of peanut skin (PSE) similarly inhibits tau aggregation. The bio-active component was isolated, and the purified compound contained intrinsic inhibitory activity. The compound was identified as a procyanidin A-linked dimer (576 Da). The corresponding, highly similar B-linked dimer did not inhibit, suggesting a specific interaction between tau and procyanidin oligomers of A-type linkage. Monomeric epicatechin, the molecule from which procyanidin oligomers are derived, do not inhibit, suggesting that the dimer is the smallest procyanidin molecule that retains tau aggregation inhibitory activity. Finally, peanut skin extract was orally administered to a mouse model of Alzheimer?s disease, and preliminary data suggests the potential of the extract to inhibit tau pathology in vivo. In this grant application, we propose to test if purified procyanidin A-linked dimer administered orally to Alzheimer?s disease mice displays efficacy to inhibit tau pathology and the behavioral symptoms associated with AD. We hypothesize that procyanidin A-linked dimer present in a common and inexpensive food source may offer potential as a novel and effective intervention of tau aggregation in humans.