David Fitzpatrick - US grants

The goal is to gain an understanding of the morphological basis for inhibitory interactions within the visual cortex. Inhibitory mechanisms play a critical role in shaping the response properties of visual cortical neurons, and the identification of the neural elements involved in this process will provide an important contribution towards understanding the function of visual cortex. We have already successfully used an antiserum to glutamic acid decarboxylase (GAD, the synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid, GABA) to examine GABAergic neurons in the thalamus, and we now wish to use GAD immunocytochemistry along with other methods to address this issue in the striate cortex. Specifically, we propose to: 1) establish the number, type, and distribution of neurons and terminals which are immunoreactive for GAD and GABA within monkey striate cortex; 2) learn more about the morphology of distinct classes of GABAergic neurons by examining the types of synaptic contacts they receive on their somata, by correlating them with Golgi stained material, and by examining their relation to neuropeptide containing neurons; 3) examine the morphology and the source of prominent GAD and GABA immunoreactive terminals in the geniculate recipient laminae of striate cortex; and 4) establish the contribution made by GABA immunoreactive neurons to interlaminar and intralaminar connections within striate cortex and to projections outside of striate cortex. This research should provide valuable insights into the functional organization of visual cortex and may illustrate fundamental principles that apply to other regions of the cerebral cortex as well.

visual cortex;

The goal is to gain an understanding of the morphological basis for inhibitory interactions within the visual cortex. Inhibitory mechanisms play a critical role in shaping the response properties of visual cortical neurons, and the identification of the neural elements involved in this process will provide an important contribution towards understanding the function of visual cortex. We have already successfully used an antiserum to glutamic acid decarboxylase (GAD, the synthetic enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid, GABA) to examine GABAergic neurons in the thalamus, and we now wish to use GAD immunocytochemistry along with other methods to address this issue in the striate cortex. Specifically, we propose to: 1) establish the number, type, and distribution of neurons and terminals which are immunoreactive for GAD and GABA within monkey striate cortex; 2) learn more about the morphology of distinct classes of GABAergic neurons by examining the types of synaptic contacts they receive on their somata, by correlating them with Golgi stained material, and by examining their relation to neuropeptide containing neurons; 3) examine the morphology and the source of prominent GAD and GABA immunoreactive terminals in the geniculate recipient laminae of striate cortex; and 4) establish the contribution made by GABA immunoreactive neurons to interlaminar and intralaminar connections within striate cortex and to projections outside of striate cortex. This research should provide valuable insights into the functional organization of visual cortex and may illustrate fundamental principles that apply to other regions of the cerebral cortex as well.

DESCRIPTION: (Adapted from the applicant's abstract.) One of the major functions of the striate cortex is to bring together the information carried by separate populations of neurons in the lateral geniculate nucleus (LGN). Neurons in the LGN are monocular and respond selectivity to either ran increase in the level of illumination that falls on their receptive field centers (ON-center cells) or a decrease (OFF-center cells). In contrast, most neurons in the striate cortex are binocular and show both ON and OFF responses. While the mixing of these inputs is a well established fact, the intracortical circuitry that underlies these changes remains largely unknown. The goal of this research plan is to increase our understanding of the functional organization of striate cortex by examining some of the intracortical circuits that are responsible for ON/OFF and binocular convergence. The input-output organization of layer IV in the tree shrew striate cortex provides a unique opportunity to gain insights into this problem. With funds from our current NIH award we found evidence for two parallel streams that convey ON and OFF information to the superficial layers and we think these systems differ in the way they integrate ON and OFF information from the two eyes. The goal of this application is to test this hypothesis using combination of anatomical and physiological methods. Four specific aims are proposed: 1. To examine the response properties of neurons in different tiers of layer IV; 2. To determine the morphology and the response properties of individual layers IV neurons that project to two tiers within layer III (layers IIIa-b and IIIc); 3. To examine the response properties of neurons in layers IIIa-b and IIIc; 4. To determine whether layers IIIa-b and IIIc project to separate regions of extrastriate cortex. These experiments should provide valuable insights into the functional organization of striate cortex by offering an effective method for relating receptive field transformations to cortical circuitry.

DESCRIPTION: Despite an increasingly detailed picture of the organization of neural circuits in visual cortex, we are still far from understanding the rules that relate these complex patterns of connections to the response properties of individual neurons. The proposed experiments focus on a fundamental property exhibited by many visual cortical neurons--selectivity for direction of motion. Direction selective neurons respond vigorously to movement of an appropriately oriented edge in one direction across their receptive field, and weakly, if at all, to movement in the opposite direction. The goal of this grant is to determine whether specificity in the modular and topographic arrangement of intracortical connections plays a role in shaping the direction selective responses of cortical neurons. These experiments are made possibly by the recent demonstration of a systematic map of direction preference in area 17 of the ferret. Optical imaging of intrinsic signals will be used to visualize the map of direction preference in area 17, and a combination of anatomical tracing techniques and in vitro tissue slice experiments will be used to assess the spatial arrangement of excitatory and inhibitory inputs to populations of neurons with known direction preference. These experiments should provide new insights into the functional organization of local circuits in visual cortex-- information that is crucial for understanding the neural basis of visual perception.

Optical studies of the surface of the optical cortex of primates (cat, monkey, ferret) have shown that the neurons are organized in correlated groups having a 'blobby' appearance. Groups have a correlated response to, for example, stimuli by bar-shaped objects of a given orientation. Other, overlapped groups have similar response to differently oriented bars. The size of a group is on the order of 500 microns. The optical techniques are sensitive to the oxyhemoglobin-hemoglobin shift that occurs due to increased metabolism when the neurons are stimulated. The optical techniques are limited to viewing depths in the brain of about 100 microns. The goal of this research is to study this structure at a variety of depths in the cerebral cortex beyond those reached by optical means. Gradient echo 3D MR microscopy offers a unique opportunity as the only method to perform this kind of study. What is proposed is a pilot study to obtain 3D images (@ 7T) in the optical cortex of a ferret with 100 micron isotropic resolution while the animal is visually stimulated with an already validated stimulation protocol to evaluate the potential of MR microscopy in this application. The purpose of the pilot project will be to develop an MRI protocol for adequate functional imaging. We will try 3D projection imaging as well as 3D spin warp approaches. In addition, it will be necessary to ensure that the ferret can be adequately supported in the 7T system and that the optical stimulation system will function in/near the magnet. Note that the investigators already routinely use gas (halothane) anesthesia on these animals over the course of a several hour experiment and have experience and publications in the area.

Cleft lip and palate is a major birth defect commonly used as a model for complex genetic diseases of human embryogenesis. Our current understanding of the coordination and function of the pathways driving craniofacial development is limited and biased by particular well-studied examples. The ability to integrate site- and stage- specific expression patterns of individual genes in a comprehensive representation of the developing face in model organisms is a critical first step towards dissection of these pathways. The functional analysis/expression core will combine this approach with the recent advances in functional 'knockdown" analysis to assist the projects in examining evidence of non-redundant gene-function during clinically important morphogenetic events. This core will use innovative technologies to provide resources to the other projects and cores of this P50 to enhance the process of gene discovery and improve our understanding of how genes interact in complex pathways that play a role in normal and abnormal facial development. The core will present the resulting data in an intuitive, publicly-accessible manner that can be used for both research and educational purposes. Specific aims will be to: 1. To develop whole-mount RNA in situ hybridization data generated by Projects 0003, 0004, 0005 and Pilot into a 4-dimensional atlas of craniofacial development in mouse embryos. 2. To construct developmental expression profiles for all genes within a human genomic region (9q) with strong evidence of linkage to CL/P. 3. To assess the use of self-organizing maps in predicting candidate genes from transcriptional profiles generated by microarray analysis 4. To determine the function of specific candidate genes in palate development using a high-throughput gene knockdown approach.

[unreadable] DESCRIPTION (provided by applicant): Both the perception of moving objects and the visual guidance of eye movements require that cortical circuits generate an accurate neural representation of moving objects in the visual scene. This process is critically dependent on cortical neurons that respond selectively to the direction of moving stimuli. The goal of the proposed research is to continue our inquiry into the role of visual experience in the development of direction selective responses in ferret primary visual cortex. Work during the previous period of support demonstrated that experience plays a unique and essential role in the early maturation of direction selective cortical responses. If visual experience is withheld for the 2 weeks following eye opening, direction selective responses fail to emerge, and are not recovered by subsequent visual experience. The proposed experiments extend this line of inquiry to address four questions. (1) Does experience with moving visual stimuli serve as an instructive cue for the construction of direction selective cortical responses? (2) What role do innate neuronal tuning properties play in the experience dependent emergence of direction selective responses? (3) What contribution do changes in cortical layer 4 circuits and cortical layer 2/3 circuits make to the experience dependent emergence of direction selective responses? (4) Is the emergence of direction selective responses accompanied by direction-specific alterations in the arrangement of anatomical connections? These experiments employ state of the art functional imaging techniques in a novel experimental paradigm that permits us to monitor the impact of visual experience on developing cortical circuits in vivo. These experiments will contribute to a better understanding of deficits in motion perception that are common in developmental disorders, and more broadly, they will provide new insights into the mechanisms by which experience shapes cortical circuit development. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The nascent interdisciplinary field of Neuroeconomics harnesses the computational rigor of economics and the technological advances of modern biology to advance discovery in the decision sciences. Since pathologies of decision making are endemic to addiction-they serve a causal role in its development, they exacerbate the biological and social consequences of the disorder, and they impair remediation and treatment ~ the National Institutes of Health has recognized Neuroeconomics as fundamental to solving this important societal problem. Here we propose a National Institute of Drug Abuse (NIDA) P30 Core Center uniting two areas of institutional strength at Duke, Neuroeconomics/Cognitive Neuroscience and Interdisciplinary Addiction Research, to support hiring two new faculty, each in partnership with the Duke Institute for Brain Sciences (DIBS) and an academic department: Duke is an internationally recognized leader in both of these fields, yet their collective strengths have not yet been brought to bear on core problems of decision making in addiction. There are several areas of existing institutional strength in these areas that include the Center for Neuroeconomic Studies (CNS), the Center for Cognitive Neuroscience (CCN), the NIDA-supported Translational Prevention Research Center (TPRC), the Duke Center for Nicotine and Smoking Cessation Research (CNSCR), The Fuqua School of Business, and the strong behavioral neuroscience groups within the Departments of Neurobiology, Psychiatry, and Psychology and Neuroscience. Despite these strengths, fundamental gaps remain. The proposed Core Center will fill important gaps in the Duke decision-making research community, will bring important and cutting-edge research competencies, and will strengthen Duke's position as one of the leading institutions in this important research area. It will bring new teaching capabilities to the undergraduate neuroscience major and to graduate training more broadly. Finally, it will provide an important new link for DIBS to socially relevant applications for neuroscience, consistent with Duke's strategic goal of "Knowledge in the Sen/ice of Society". A NIDA P30 Core Center will give Duke the leverage to immediately fill these gaps and catalyze integration of these disparate groups by hiring two new faculties that each brings new expertise bridging current Neuroeconomics and Addiction research at Duke.

DESCRIPTION (provided by applicant): New technologies that have made it possible to visualize specific classes of neurons, and to monitor and manipulate their activity in vivo, are transforming our understanding of neural circuit function and development. While the range of tools for molecular circuit visualization and manipulation continues to grow at an explosive rate in the mouse, the development of comparable tools for the study of circuits in non-murine mammals lags far behind. The inability to employ state of the art molecular tools for cell-type specific visualization and manipulation of neural circuits severely limits progress in understanding important aspects of cortical organization that are not well represented in the mouse brain. The goal of this application is to begin to bridge this technology gap by developing a recombinant viral vector for use in non-murine mammals that will permit in vivo expression of molecular reporters and effectors using cell-type specific promoters. As proof of principle, we will generate and characterize a helper-dependent adenovirus (HdAd) vector that will allow the selective expression of genetically encoded fluorescent proteins and light-sensitive channels in GABAergic neurons of the ferret visual cortex. The availability of a viral construct that yields expression selectively in GABAergic neurons of non-murine mammals will make it possible to address a host of questions that are critical for understanding cortical function in health and disease. Moreover, success in generating this construct will open the door to a battery of cell-type specific molecular tools that will be of tremendous value for the study of cortical and subcortical structures in a wide range of mammalian species including primates.

? DESCRIPTION (provided by applicant): Establishing how features of the visual world are represented in the activity of cortical circuits, and how these representations are constructed during development remain fundamental challenges for visual neuroscience and are central to understanding the neural basis of visual perception and disorders of cortical function. The experiments in this proposal continue our analysis of the functional organization and development of columnar representations in layer 2/3 of ferret visual cortex. Preliminary evidence suggests an important new dimension to the representation of visual stimuli by layer 2/3 cortical circuits: a columnar architecture composed of neurons that respond preferentially to diffuse luminance increments (ON) or luminance decrements (OFF). By using a combination of techniques for in vivo imaging of functional ON and OFF inputs at the columnar and cellular level of resolution, we will test the hypothesis that ON and OFF responses to diffuse illumination are arranged in a modular fashion in layer 2/3 of visual cortex, explore the cellular basis for thi columnar architecture and its relation to the representation of orientation preference, and probe the contribution of experience independent and dependent mechanisms to its development. Taken together these experiments will yield novel insights into the functional organization and development of the cortical circuits that mediate visual perception, providing the foundational knowledge for addressing a broad range of neurological and psychiatric disorders that impact cortical circuit function.