Michael C. Crair - US grants

DESCRIPTION (provided by applicant): Among the fundamental goals of neuroscience is to understand the form, function, and development of the system of sensory and motor maps that are ubiquitous in higher nervous systems. The ordered arrangement of afferent projections from the thalamus to the cortex is the most thoroughly studied model system for exploring the function and development of sensory maps in mammals. This system is characterized by an array of thalamic afferents that form organized projections into cortical columns serving different stimulus variables, such as ocular dominance in visual cortex or whisker preference in somatosensory cortex. The pattern of thalamocortical projections undergoes an activity dependent elaboration and refinement that leads to the development of cortical columns with very selective stimulus response features. This process is influenced by changes in sensory experience, such as monocular visual deprivation or whisker cauterization, which leads to dramatically altered sensory maps, particularly when the deprivation is started in an early 'critical period'during development. The overall goal of the proposed experiments is to elucidate the mechanisms that mediate the development and plasticity of sensory maps in cortex. These experiments will be executed with a combination of anatomical, biochemical and electrophysiological approaches using normal and mutant or transgenic mice with specific emphasis on examining the mechanisms of whisker 'barrel'column development and plasticity in the somatosensory cortex of the rodent. A detailed explanation of these mechanisms will allow us to understand at a cellular level how molecules, neural activity and sensory experience together guide the development of brain circuitry. Ultimately, these same mechanisms are likely to be involved in the pathogenesis of behavioral disorders with a complex combination of genetic and experiential etiologies, such as Autism and Schizophrenia. This research aims to understand the cellular and molecular mechanisms responsible for how the brain wires itself up during development. Several brain disorders, including Epilepsy, Autism and Schizophrenia are thought to be caused by errors in this process, with abnormal brain function and behavior the result of miswiring that occurs in the developing brain.

Project Summary The overall objective of the Yale Vision Research Core Program is to enhance vision research at Yale University. With four key modules, this Program will aid the ongoing research projects of vision investigators by providing facilities and expertise that cannot be readily supported by individual research grants. The modules also serve to promote and facilitate collaborative interactions between vision investigators throughout the Yale University campus. The Core grant will also facilitate pilot projects to encourage established investigators at Yale to enter the field of vision research and develop new vision research grant proposals. Four key modules are proposed in this application. A Fabrication core module will assist in the design, development and construction of devices and instrumentation that are specific to vision research. This service module will provide an essential resource in helping new and junior investigators in vision research at Yale get their labs up and running as quickly as possible. Three resource modules are also proposed. A Programming core module will support the development of custom data acquisition and analysis software and the integration of specialized electronic equipment into experimental platforms through the services of a scientific programmer. An Imaging core module will provide access to physical facilities and intellectual resources for state-of-the art static and dynamic imaging of fixed and living visual system tissue. Finally, a Genotyping and Virus Production core module will serve an essential role in modern biological research, with a focus on genotyping and the production of novel viral vectors, by providing broad access to molecular biological equipment, techniques and skills that are either outside the area of expertise for individual vision researchers, or beyond their capabilities to utilize. These four modules make up a combined vision core program that serves the needs of all vision researchers at Yale University.

Description (provided by applicant): The brain organizes information about the sensory world into maps. Prominent examples are the maps of eye-preference and retinotopy in the lateral geniculate nucleus, superior colliculus and visual cortex. Experiments in this proposal will advance our understanding of mechanisms responsible for the development of precise neural circuitry in the mammalian brain. We will use a broad combination of techniques, including molecular biological, cell biological and electrophysiological techniques both in vitro and in vivo. We focus our experiments on maps of eye-preference and retinotopy in the superior colliculus of the mouse, which is a sensory motor structure that has emerged as an ideal model system for the examination of neural circuit development and function. It is widely hypothesized that molecular cues are responsible for the establishment of coarse map structure in the superior colliculus, and activity dependent processes subsequently refine these sensory motor circuits to functional precision. We propose to examine the nature of activity that is necessary for the development of visual maps, and determine whether this activity is permissive or instructive in shaping circuit development. We also propose to examine the normal development of single retinal ganglion cell axon arbors in the superior colliculus of the mouse, and determine how disrupting retinal activity disrupts arbor development. We finally propose to investigate the synaptic mechanisms that mediate activity-dependent refinement of visual maps in the superior colliculus. In all, the experiments in this proposal are designed to investigate the mechanisms responsible for the development of precise neural circuits in the mammalian brain, with a specific emphasis on the emergence of visual maps in the mouse superior colliculus. PUBLIC HEALTH RELEVANCE: We are interested in understanding how complex brain circuits develop. We focus on the visual system, as its function is relatively well understood and it is especially important to human behavior. Our experiments have the potential to help develop techniques to restore visual function following eye trauma or disease, such as glaucoma or age related macular degeneration.

DESCRIPTION (provided by applicant): The goal of this application is to provide highly focused training in Systems Neuroscience at Yale University School of Medicine, with special emphasis on the mammalian cortex. The philosophy of this program is to preserve and foster integrative approaches to neurobiology that will interface with molecular genetics and clinical medicine with respect to development, organization, and plasticity of the mammalian brain. Forty-three faculties from 17 basic and clinical departments are participants in this multidisciplinary program. The program offers both depth and breadth. The depth derives from its unique substantive focus on cortical circuits of the rodent, primate and human brain. The breadth of the program derives from the diversity of approaches, spanning developmental, systems and cognitive neuroscience. Faculty interests span axonal guidance mechanisms in embryos to memory decline and stroke in elderly humans. Methodologies include cloning;cell culture;immunocytochemistry;in situ hybridization;electron and two photon microscopy;voltage clamp and whole cell recording;calcium imaging;biochemistry and molecular analyses;psycho-pharmacology;rodent, monkey and human behavior;in vivo extracellular recording in behaving animals;and fMRI and PET imaging in human subjects. Two predoctoral and two postdoctoral positions are requested. Trainees are selected from a variety of backgrounds in biological sciences on the basis of their potential for excellence and leadership in research by an Admissions Committee (predocs) or Executive Committee of the NCS (postdocs). Mentors are Ph.D.s and M.D.s. with NINDS grants and/or NINDS related research foci. Training includes coursework, intensive research apprentice-ship, structured seminar programs, and laboratory and departmental presentations of research progress.

DESCRIPTION (provided by applicant): The goal of this application is to provide highly focused training in Systems Neuroscience at Yale University School of Medicine, with special emphasis on the Mammalian Cortex. The philosophy of this program is to preserve and foster integrative approaches to neurobiology that will interface with molecular genetics and clinical medicine with respect to development, organization, function and plasticity of the mammalian brain. Forty-two (42) faculty from 12 basic and clinical departments are participants in this multidisciplinary program. The program offers both depth and breadth. The depth derives from its unique substantive focus on cortical circuits of the rodent, primate and human brain. The breadth of the program derives from the diversity of approaches, spanning molecular, developmental, systems, theory and cognitive neuroscience. Faculty interests span cortical morphogenesis and axon guidance mechanisms in embryos to memory decline and stroke in elderly humans. Methodologies include cloning;cell culture;immunocytochemistry;in situ hybridization;electron and two photon microscopy;voltage clamp and whole cell recording;calcium and other forms of optical imaging;biochemistry and molecular analyses;psycho-pharmacology;rodent, monkey and human behavior;in vivo extracellular recording in behaving animals;and fMRI and PET imaging in human subjects. Two predoctoral and two postdoctoral positions are requested. Trainees are selected from a variety of backgrounds in biological sciences on the basis of their potential for excellence and leadership in research by an Admissions Committee (predocs) or Executive Committee (postdocs). Mentors are Ph.D.s and M.D.s. with NINDS grants and/or NINDS related research foci. Training includes coursework, intensive research apprentice-ship, structured seminar programs, and laboratory and departmental presentations of research progress. RELEVANCE: This training program fosters education on brain disorders such as dyslexia, autism, Tourette's Syndrome, Attention Deficit Hyperactivity Disorder, epilepsy, cerebral palsy, dementias (e.g., Alzheimer's and Huntington's diseases), and stroke. We actively encourage and successfully accomplish the translation of basic research findings to further our understanding of the cause and cure of human brain disorders.

DESCRIPTION (provided by applicant): The display of patterned spontaneous activity is an emergent property of the immature nervous system that is thought to mediate synaptic competition and instruct self- organization in many developing neural circuits. In the visual system, isolated (in vitro) preparations of developing retina exhibit propagating electrical activiy amongst neighboring retinal ganglion cells (RGCs), termed 'retinal waves'. Since RGCs relay visual information to higher order structures in the central nervous system, retinal waves are thought to play a key role in activity-dependent refinement of topographic neural maps in the superior colliculus (SC), lateral geniculate nucleus (LGN), and visual cortex (VCtx). However, the role of retinal waves in neural circuit development remains remarkably controversial, in part because their existence has never been demonstrated in vivo. Previous work using extracellular microelectrode recording techniques in vivo demonstrated limited and local correlated spiking between pairs of embryonic rat RGCs, but no assessment of wave activity has been undertaken in vivo, likely because of the methodological challenges associated with recording from a large cohort of RGCs in neonatal animals. In this proposal, we use a highly novel imaging approach to examine and characterize spontaneous activity throughout the visual neuraxis, including RGCs, the SC and VCtx, in neonatal mice in vivo. We seek to establish whether traveling waves of spontaneous activity occur in awake, behaving neonatal mice, and examine the spatiotemporal properties of waves throughout the developing visual system during the first two weeks after birth. Our preliminary data indicates that spontaneous retinal waves are present for at least a week of development in vivo and exhibit a similar profile of spatiotemporal properties as those described previously in vitro. Moreover, retinal waves generate matched activity patterns in the midbrain and visual cortex. Given the remarkable fidelity of retinal waves during the period prior to eye opening in mice we report here in vivo, together with previous work demonstrating that spontaneous waves within macaque retina are present in vitro before birth, it seems likely that the visual system experiences patterned activation by retinal waves for a substantial gestational period during human fetal development that may be crucial for shaping the functional maturation of neural circuits before the onset of sensory experience. In all, these experiments are designed to investigate the properties and role of patterned spontaneous activity in vivo in the development of neural circuits in the mammalian visual system.

DESCRIPTION (provided by applicant): The brain organizes information about the sensory world into maps. Prominent examples are the maps of eye-preference and retinotopy in the lateral geniculate nucleus, superior colliculus and visual cortex. Experiments in this proposal will advance our understanding of mechanisms responsible for the development of precise neural circuitry in the mammalian brain. We will employ a broad range of techniques, including molecular biological, cell biological, neuroanatomical, electrophysiological and advanced optical imaging techniques in vitro and in vivo. We focus our experiments on maps of eye preference and retinotopy in the superior colliculus and lateral geniculate nucleus of the mouse. These structures are the dominant target of retinal projections to the brain. The lateral geniculate nucleus is the primary relay of visual information to the visual cortex, and the superior colliculu is a sensory motor structure that has emerged as an ideal model system for the examination of neural circuit development and function. It is widely hypothesized that molecular cues are responsible for the establishment of coarse map structure in the lateral geniculate and superior colliculus, and activity dependent processes subsequently refine these sensory motor circuits to functional precision. We first propose to definitively establish whether patterned spontaneous activity is necessary for the development of visual maps. We next propose to manipulate the temporal pattern of activity in the developing retina to examine the dependence of retinocollicular development of retinal ganglion cell activity. We finally propose to manipulate th spatial pattern of activity in the developing retina to examine the dependence of visual map development on the spatial character of spontaneous retinal activity. In all, the experiments in this proposal are designed to investigate the mechanisms responsible for the development of precise neural circuits in the mammalian brain, with a specific emphasis on the emergence of visual maps in the mouse superior colliculus.

? DESCRIPTION (provided by applicant): The purpose of this RFA is to promote the integration of experimental, analytic and theoretical capabilities for the examination of neural circuits and systems. This proposal is highly responsive to the RFA in that it links several different neuroscience labs to develop new technologies that provide for simultaneous multistate imaging and applies these technologies to the examination of how neuronal dynamics in mammalian cortex varies as a function of brain state and development. Paired imaging modalities will bridge the gap from imaging activity in individual neurons to whole brain circuit level analyses. The different scales will be linked with a comprehensive model such that each level of experimentation can inform the other. We will develop the technology to allow simultaneous single cell (two-photon) Ca2+ imaging of a local circuit and cortex-wide mesoscopic (single-photon) Ca2+ imaging across the entire neocortex. In separate paired studies in the same animals we will develop simultaneous mesoscopic Ca2+ imaging across the cortex with whole-brain functional MRI. Whole cortex mesoscopic Ca2+ imaging represents an innovative technology developed by one of the PIs (Crair) that uses mice expressing a genetically encoded Ca2+ indicator (GCaMP6) in all neurons or in select populations of neurons to allow both local neuronal and transcranial population level mesoscopic scale imaging across the cortex in the intact, unanesthetized developing mouse brain. This Ca2+ imaging technique will allow us to directly link single cell imaging to gross circuit level activity across the cortex and whole brain An integrative model is proposed to link these different modalities in order to understand the neural source of macroscopic circuit changes and the factors that influence this organization through development and as a function of behavioral brain state. This work is innovative in the novel Ca2+ imaging strategies to be further developed, and in the design of paired scale imaging to establish links between single neuron activity and circuit level organization. The work is significant in that it will provide a set of tools for detailed investigations of the impact of specific neuronal cell populations on brain circuit functional organization in healthy development and disease models. It is also significant in that new insights into the source and flow of neuronal activity will be obtained that will improve our understanding of the principles guiding self-organization in the developing brain and its dynamic modulation by brain state.

Administrative Core Module Abstract The Administrative Core Module provides support for overall administration of Yale's Vision Research Core Program. The organizational structure includes a Program Director, an Executive Committee composed of all core module Directors and Co-Directors, quarterly meetings of all Yale Vision Research Core Investigators and an annual meeting of the External Advisory Committee. This administrative structure will ensure equal and adequate access to core facilities for all Vision Research Investigators through quarterly surveys of core function and performance, monitoring of a web-based scheduling system of core use and external evaluation via the External Advisory Committee. The ultimate goal of the administrative core is to provide infrastructure to organize and coordinate the efficient running of the entire Yale Vision Research Core Program and facilitate the research projects of all vision core investigators at Yale.

Imaging Core Module Abstract Over the past decade there has been a revolution in imaging technology and its application to basic biomedical research. The Imaging Core Module will be a critical resource in bringing this technology to the vision community at Yale. Advanced imaging equipment is expensive and typically cannot be purchased and maintained with the resources of a single investigator. Moreover, with the nearly complete conversion of imaging to advanced optics and digital methods, sophisticated data acquisition and analysis techniques are required to quantitatively examine visual system structure, development and function. The Yale Vision Core Program, through this Imaging Module, will provide access to state-of-the-art imaging technologies and the necessary training in advanced imaging approaches for vision investigators at Yale. The Imaging Core Module?s primary goal is to facilitate the wide use of advanced optical and digital imaging techniques within the Yale vision research community. This is achieved by three means. First, Yale School of Medicine and the Department of Neurobiology has made and will continue to make a significant investment in advanced imaging microscopes, which vision core investigators will gain free access to through the Yale Vision Core. Second, a Support Scientist, Dr. Stacy Wilson, who is an expert in optics and digital imaging techniques, will provide technical support in the proper use of the microscopes and digital imaging. Third, the core module will train vision core scientists (students, postdocs) in appropriate microscopy techniques and advanced digital image analysis. We anticipate that the Imaging Core Module will play an important role in the research success of vision investigators at Yale University.

Project Summary This RFA is aimed at bringing together interdisciplinary teams to focus on novel, transformative and integrative efforts that will revolutionize our understanding of the biological and bioinformatics content of the data collected from non-invasive human functional brain imaging techniques. Our proposal does exactly this. We are a multidisciplinary team of scientists with combined expertise in optogenetics, two photon Ca2+ imaging, biomedical engineering, molecular biology, animal and human fMRI, network theory, data analysis and modeling. In this work, we will use a novel imaging device that combines mesoscopic imaging of genetically encoded Ca2+ indicators with very high (50?m) spatial and high temporal (25ms) resolution across the entire cortex and simultaneous fMRI in transgenic mouse models. These animal experiments are designed to complement similar experiments in healthy human subjects. The results from the animal experiments will answer several long-standing questions about the source of the fMRI signal. Specifically, using imaging, we will quantify the contributions of different cell populations (excitatory neurons, inhibitory neurons, and glial cells) to the fMRI signal observed. We will be able to test and validate, for the first time, the application of graph theory approaches to the analysis of human fMRI data, and we will develop and test a new approach based on control theory for extracting more information from the fMRI signal. A powerful set of carefully controlled imaging experiments in mice will inform several aspects of analysis of human data. The human data will contain a test/retest component to ensure replication of the results and to allow predictive models to be built in one data set and tested in another. This work truly bridges scale and modalities and the simultaneous nature of the animal experiments will allow unprecedented clarity on the underlying source of the signal changes observed in fMRI. These animal studies are essential for providing new insights into the basis of human fMRI signals and data of this nature has not previously been available. The work in this proposal is novel in that it will directly inform measures of both evoked and spontaneous activity in terms of the underlying cell signal sources revealing the relative contributions of excitatory, inhibitory and glial cells to the fMRI signal. The implications of the work are multifaceted. This work will provide a platform for evaluating neurological models of disease. For example, mouse models of disease can be used to link to human data in diseases such as PTSD, depression, and autism, to name a few. It will also provide a firmer biological basis for understanding the node and network measures used in assessing the functional organization of the brain and will have important implications for the design of therapeutic interventions across a range of diseases.