Thomas A. Woolsey - US grants

The goal of this project is to study the relationship of neuronal form to function. All projects depend upon computer associated analysis of neuronal morphology (core unit) to aid in this. Specific studies are: (1) To examine the mammalian muscle spindle as a model of localized inputs to a branched neuronal structure (Project 1); (2) To examine amacrine and ganglion cells in the vertebrate retina to evaluate models foar neuronal activation ( Project 2); and (3) to examine neurons in the mouse cerebral cortex as a model for "mapping" intracortical connectivity (Project 3). In each case the relevant structures are revealed by selective optical and anatomical methods including Nomarski optics, intracellular injection of dyes (lucifer yellow and HRP), the Golgi method and 3H-2-DG auto-radiography. All preparations are analyzed with a computer based image processing system which allows for accurate measurement and display of three dimensional parocess lengths and the on-lin alignment of sequential images for either serial sections on adjacent frames of movie films (core unit). We hope to gain fundamental insites into the details of neuronal form as related to activity and form this to provide a basis of understanding which is a necessary prerequisite for understanding cellular changes which occur in diseases of the nevous system.

Local cerebral blood flow (LCBF) changes with stimulation of whiskers on the rat's face. Responses are related to individual functional columns of neurons of which the barrels in layer IV are a part. Vascular modules that correspond to cortical barrels have been demonstrated. The general hypothesis we will test is that vascular modules are anatomically coincident with and functionally linked to columns in the cerebral cortex. In particular, focal increases in neural activity stimulates capillaries from which signals propagate upstream through electrically coupled endothelium to relax arteriolar smooth muscle and to change LCBF depending on the activity of the neuropil. The experiments are designed to examine this hypothesis directly. We will: l) Characterize the local vascular responses to controlled stimulation of single whiskers or single whisker rows during in vivo videomicroscopy through closed cranial windows in rats by multiple quantitative dynamic parameters. This tests the hypothesis that stimulation of a single whisker increases flow in a single cerebral arteriole. 2) Define directly the vascular module by microperfusion of single terminal arterioles the vascular units of rat barrel cortex. This tests the hypothesis that a single cerebral arteriole supplies a single cortical barrel. And 3) Demonstrate neuron to vessel signalling mechanisms and propagation in cortical slices. This tests the hypotheses that active neural elements in layer IV barrels stimulate capillaries that then transmit signals through electrical coupling to arterioles for control of LCBF. We propose that conjoint neural and vascular modules: (a) provide an optimal mechanism for precise control of LCBF, (b) occur more generally in the brain, and (c) share common features of local microcirculation control with other capillary beds. Understanding this mechanism in the cerebral cortex will improve our appreciation of functional studies (PET, MRI) in normal individuals that depend on changes in LCBF and of microvascular occlusions in stroke.

This Program Project is a multidisciplinary approach to understanding the relationship between function and form in vertebrate neurons. There are eight research projects and a CORE. These are 1) studies retinal cells to establish a correlation between biophysical and connectional models and cellular anatomy and function; 2) studies the development of patterned projections in the trigeminal system, how they can be altered and how they can be modeled mathematically; 3) visualizes populations of cortical neurons activated with precisely controlled tactile stimulation; 4) looks at the formation of patterned somatosensory inputs to cortex transplanted from other cortical areas; 5) examines the distribution of individual fibers to the frontal cortex and thalamus from the basal forebrain; 6) studies the three dimensional characteristics of intracellularly stained brainstem neurons concerned with eye movement control; 7) studies the sensory and motor innervation of muscle; 8) investigates the role of growth factors in the development and maintenance of dendritic arbors of autonomic ganglion cells and spinal motor neurons. CORE provides resources for quantitation of neuronal structure and modelling functional and developmental aspects of structure particularly as relates to function.

CORE-C permits investigators to record three dimensional images of overlapping microscopic fields for analysis and archival storage. Software allows investigators indicate and "tease" out objects from masses of other stained elements such as axonal fibers. this is a new resource modeled on developments by Dr. Senft in present grant period. It consists of a high resolution confocal microscope with scanning stage, a Silicon Graphics Indigo work station with large working memory as a Volume rendering device; sufficient efficient storage for fleeting fluorescent images; VoxelView Software with support; and communications to other resources in this Program Project. this CORE will be used by investigators in projects 1-7 to digitize at varying resolutions fluorescent and other (HRP, Golgi, etc.) images through large tissue volumes in single and serial slices and to store these for future analysis. Investigators can make montages of adjacent overlapping fields for large high resolution images for visualization labeling patterns. A unique capability is the extraction, by segmentation, of three dimensional aspects of single elements in the tissue such as cell bodies and processes. Software will permit analysis of these properties for quantitative characteristics such as branch angles, branch frequencies, process diameters. We will develop protocols for moving these data to other CORE resources (COREs B and E) for analyses.

The rodent trigeminal system has favorable characteristics for elucidating mechanisms of development of brain pathways and order in them. Each whisker on the face projects to a discrete cell group in the brain. In the cerebral cortex these are called barrels. The ensemble of barrels makes a map of the animal that can be recognized in tissue sections with or without stains. We propose to exploit the map that the barrels provide, the postnatal development of the intracortical connections within the map and the sensitivity of that development to changes in sensory input to clarify how specific intracortical circuits required for integration of inputs from the whiskers develop. We will examine the following hypotheses: . Hypothesis I: there is a sequence of development of intracortical (interbarrel) projections that is lamina specific, . Hypothesis II: connection to the ordered periphery is necessary for the proper patterns to develop, . Hypothesis III: the effect of selected whisker removal is asymmetrical at the boundaries between connected barrels and disconnected barrels which are at a competitive disadvantage, . Hypothesis IV: normal neuronal activity plays a role in the development of interbarrel connections, . Hypothesis V: intracortical action potentials play the major role in the development of the interbarrel connections. We will use lipophilic dyes, targeted under direct vision to individual barrels or to particular cortical layers over and under individual layer IV barrels and other transported markers, to demonstrate intracortical connectivity. The project will use CORE C to digitize and visualize results of fluorescent dye transport, CORE B to quantitate these results and the results from transported marker studies and CORE E to align findings from serial sections. These studies relate directly to work in Projects 4, 6, and 7. The goal is to understand the manner in which intracortical communication. The work can contribute to clarifying mechanisms that may be important for understanding mental retardation, epilepsy and learning disabilities.

The central theme of this program project is a multidisciplinary quantitative approach to function, structure and expression in developing and adult nervous systems. There are 7 research Projects. Project 1, investigates the hypothesis that different functional classes of 8th nerve afferents project to and regenerate back to distinctive brainstem nuclei. Project 2, tests the hypothesis that pattern formation in the rodent trigeminal system depends on selective expression of growth factors and their receptors. Project 3, explores the hypothesis that different functional classes or dorsal root ganglion cells express markers for and respond to different growth factors. Project 4, poses the hypothesis that extracellular matrix molecules effect the migration of young neurons to and within the fetal cerebral cortex. Project 5, examines the hypothesis that intra-cortical connections in the mouse whisker barrels follow a specific sequence of development that depends on experience. Project 6, tests the hypothesis that behavioral performance on binocular tasks known to rely on the motion processing pathways in development is tied to development of connections into, in and from V1. Project 7, explores the hypothesis that the different patterns of activity of two classes of inhibitory neurons in the hamster barrel cortex correlate directly with connectivity in a network. These projects are supported by 6 COREs. CORE B - Scanning Stage Image Processing Computer Microscope, supports unique capabilities for quantitation, in anatomical context, of objects (cells, silver grains, stain intensities) from microscope slides. CORE C - Confocal Microscope with Volume Rendering and Object Segmentation, permits visualization of large areas of tissue in exquisite detail and extraction of relevant features from the masses of neural processes. CORE D - Eutectics Microscope - 3D Reconstruction System, continues to provide accurate, reliable means to collect three dimensional information from single and serial sections for measurement and visualization. CORE E - Laboratory of NeuroImaging (LONI), supports communications between the cores and provides expertise and hardware for reconstruction from serial sections and other tasks. CORE F - Growth Factor Support, provides growth factors, and antibodies, and performs in situ hybridization.

This project concerns two aspects of mouse specific thalamic relay cells (TCRs) from the whisker "barreloids" in the ventroposterior medial nucleus (VPM) to their targets the whisker "barrels" in the somatosensory cortex; specifically the denfrites and axon terminasl (thalamocortical afferents+TCAs) of these neurons. General Hypothesis I. TCR dendrites define different roles in development and maturity: Studies of TCR dendrite organization in VPM. We suggest that an early TCR role is to transmit pattern information from the brainstem to the cortex; a later rrole is to incorporate information from different whiskers. TCRs in fixed slices through VPM will be stained by intracellular injections of dye and reconstructed and measured with a computer microscope to: 1) determine directly the relationship of TCR dendrites to segregated afferents from brainstem trigeminal nuclei; 2) follow the postnatal development of TCR dendrites including documentating functional correlates of anatomical changes; and, 3) demonstrate the TCR dendrites are plastic and can be changed by altering the class or pattern of inputs to VPM from the brainstem. General hypothesis II. TCA grouping in development is a population phenomenon; Studies of TCA organization in barrel cortex. We suggest that the rich and dense afferent patterns from the barreloids to the barrels arises in part from interactions with each other. TCAs will be stained in bulk and signly by dyes in living and fixed materials and analyzed with quantitative confocal and ocmputer microscopy to; 1) determine the extent to which single fibers and groups of fibers to particular barrels share the same characteristics; 2) follow the development of the clustering of these fibers in early postnatal life; and, 30 demonstrate differences and similarities between fibers supplying the cortex of animals deafferented since bith and those where the pattern of TCA inputs is perturbed by selective whisker removal. The project will use the St. Louiis CORE for intracellular staining, three dimensional measurements of processes and for quantitation of TCAs. We will rely on the expertise of many of the other investigators in this project for advice, guidance and assistance with targeting markers, recording and collecting and analyzing the data. This project is related to higher-order processing of trigeminal information and its disturbances with environmental modifications to the nervous system.

DESCRIPTION (provided by applicant): The Spring Brain Conference (SBC) was first organized in late 1989 to foster interdisciplinary communication among scientists studying different aspects of brain function in a format that stimulates significant interaction and discussion. The objective of the conference is to bring together top neuroscientists with varied backgrounds, interests and approaches expressly to promote the development of new strategies to investigate and stimulate the development of new therapeutic approaches to disorders of the CNS. SBC is held in Sedona, Arizona in mid-March. The dates for the next conference are March 16-19, 2005. Support is requested for meetings in 2005, 2006, 2007 and 2008. Registration and participation in the meeting is open. The meeting site, however, limits participants to a maximum of 100 scientists. In 2005, the conference will consist of 10-12 plenary scientific sessions each organized around a theme or topic. Sessions are chosen on a competitive basis and are presented in 2-4 oral presentations by recognized experts followed by open discussion. We request support for 1 established scientist, 3 Young Investigators and 6 Trainee Fellows to participate. Several sessions will be co-organized with program personnel from the NIH. For 2005, as in 2004, we will seek participation from different institutes such as: NINDS, NIA, NICHD, NIDA and NIMH. Two plenary lecturers will be invited to present expert overviews on topics of broad interest. We request support for a Keynote Speaker who gives the opening talk of conference. There will be a poster session, focusing on posters presented by young investigators/trainees/graduate students particularly young scientists from diverse background minority trainees. Over 70% of the budget is to support travel of these participants. The Young Investigators and Trainee Fellows will organize and participate in hands-on presentations to 180 Native American High School Children (grades 9-12) who are residents of the Kinlani Bordertown Dormitory in Flagstaff, AZ. This targeted outreach will be repeated over all 4 years of the grant to build relationships with this underrepresented group, and to attract them as new talent to the exciting field of neuroscience. In 2004, there was a significant increase in the number of women scientists participating in the SBC. Further increases in their participation at all levels are goals for the coming year. A report of the 2004 conference highlights is in press and will be posted on the web site. Such documentation and publication will be a part of all future SBC's. A preliminary announcement for the 2005 meeting is also in press. Broader advertisement will be purchased once the program is set and funding for this is requested also. Finally, funds are requested to partially defray costs of organizing and conducting the conference, collecting follow-up assessments, and supporting the meeting's web site.

DESCRIPTION (provided by applicant): The Spring Brain Conference (SBC) was first organized in late 1989 to foster interdisciplinary communication among scientists studying different aspects of brain function in a format that stimulates significant interaction and discussion. The objective of the conference is to bring together top neuroscientists with varied backgrounds, interests and approaches expressly to promote the development of new strategies to investigate and stimulate the development of new therapeutic approaches to disorders of the CNS. SBC is held in Sedona, Arizona in mid-March. The dates for the next conference are March 15-18, 2006. Support is requested for meetings in 2006 and 2007. Registration and participation in the meeting is open. The meeting site, however, limits participants to a maximum of 100 scientists. In 2006, the conference will consist of 10- 12 plenary scientific sessions each organized around a theme or topic. Sessions are chosen on a competitive basis and are given in 2-4 oral presentations by recognized experts followed by open discussion. We request support for 1 established scientist, 3 Young Investigators and 5 Trainee Fellows to participate. Several sessions will be organized by or in collaboration with program personnel from the NIH. For 2006, as in 2005, we will again encourage participation by staff from different institutes such as: NINDS, NIA, NICHD, NIDA and NIMH. Two plenary lecturers will be invited to present expert overviews on topics of broad interest. We request support for a Keynote Speaker who gives the opening talk of conference. There will be a poster session, focusing on posters presented by young investigators/trainees/graduate students, particularly young scientists from diverse background minority trainees. Over 70% of the budget is to support participation by these participants. The Young Investigators and Trainee Fellows will organize and participate in hands-on presentations to 180 Native American High School Children (grades 9-12) who are residents of the Kinlani Bordertown Dormitory in Flagstaff, AZ. This targeted outreach will expand on the successful interactions in 2005. We will continue and deepen this unique effort over the next 2 years of the grant. We are in a position to build unique relationships with under-represented Native Americans. A goal is to attract new talent from this group to the exciting field of neuroscience, biology and medicine. In 2005 the increase in the number of women scientists participating in the SBC was sustained. Further increasing participation of women scientists in all aspects of this conference remains a major goal for coming years. A report of the 2005 conference highlights will be in press soon and will be posted on the web site. A preliminary announcement for the 2006 meeting is in press. Once the program is set, advertisements (and materials) will be purchased for which funding is requested. Finally, funds are requested to partially defray costs of organizing and conducting the conference, collecting follow-up assessments, and supporting the meeting's web site.