Gordon M. Shepherd - US grants

The long-term objective of this research is to elucidate the structure and function of local circuits within cortical regions of the vertebrate central nervous system. These circuits are extremely difficult to analyze in most cortical systems. The vertebrate olfactory bulb has been a valuable simple model for this purpose, and we will continue to use it in the proposed research. The methods will be based primarily on intracellular analysis of single neurons in an in vitro preparation of the turtle olfactory bulb. A major focus will be on dendro-dendritic synaptic interactions of mitral cells, elicited by antidromic and orthodromic activation, and intracellular current injection. Very slow potentials, lasting up to one minute or more, will be analyzed using different stimulus repetition rates. Self-inhibition, miniature synaptic potentials, and excitable properties of mitral cell dendrites will be analyzed, together with impulse firing frequences elicited by current injection. Neuromodulator actions of enkephalin, dopamine and acetylcholine will be tested. Fluxes of K+ during the synaptic potentials will be studied using ion-selective electrodes. Models of these neuronal properties will be constructed on an electrical network analysis program to aid in the analysis. Patch clamp recordings will be made from neurons dissociated from the in vitro bulb surface. Parallel studies will characterize the properties of olfactory receptor cells. Similar studies will be carried out on the smaller output neurons (tufted cells), and the intrinsic neurons (granule and periglomerular short-axon cells). New methods for microstimulation of intrinsic bulbar pathways will be developed. A unique study will correlate single-neuron membrane properties with single-neuron changes in energy metabolism, as revealed by high-resolution localization of 2-deoxyglucose using methods of quick-freeze and freeze-substitution. These results should give a much clearer understanding of neuronal structure-function relations within different cortical regions of the nervous system. The results should provide insight into normal functions of microcircuits within the central nervous system, and abnormal functions related to epilepsy, schizophrenia, and Alzheimer's dementia; they should also provide crucial information relating to current controversies about the neuronal basis of energy metabolism underlying brain imaging techniques.

computational neuroscience; olfactions; neural information processing; model design /development; limbic system; sensory mechanism; neuroanatomy; biological signal transduction; synapses;

ABSTRACT NOT PROVIDED

DESCRIPTION (Applicant's Abstract): In the neurosciences, the generation of experimental data relating to neuronal properties - receptors, channels and neurotransmitters - currently far exceeds the means for archiving, analyzing and integrating these data in ways that give insight into the neural basis of brain function. There is an increasingly urgent need to develop a new generation of sophisticated databases and informatics tools for this purpose. The olfactory system is an attractive model for attacking these problems, and we have therefore set up a pilot project entitled SenseLab for this purpose. Over the past six years, progress in this pilot project has led to several types of web accessible databases and tools to enhance our work on the olfactory system. These include: Olfactory Receptor Database (ORDB), for unpublished as well as published sequence data, to aid the effort in the olfactory field to clone and sequence the very large gene family of olfactory receptor genes; Odor Database (OdorDB), a database of odor molecules used to test for receptor affinities in expression systems; NeuronDB, a database of receptor, channel and neurotransmitter properties, to aid in the integration of these data in different compartments of different types of neurons, and to allow searches for specific properties across different neuron types; and ModelDB, a database of neuronal models. Further development of these databases and tools requires an enhanced effort in which single current project needs to be expanded into three parallel projects supported by this Program Project. Project 1 will test specific hypotheses regarding olfactory function at several levels of the olfactory pathway, using current databases and several new databases. Project 2 will be devoted to fundamental informatics research to refine current databases and tools and develop new ones. Project 3 will focus on applying modeling methods to analyzing specific types of neuronal integration, and developing model databases for wider testing of neuronal models of the web. Automated database entry linked to journal publication of neuronal properties will be explored. A Core will provide administrative and programming support. These databases and tools will enhance the understanding of neural mechanisms in sensation. They will be generalizable to other systems, to provide tools of wider use for integrating and searching neuroscientific data.

DESCRIPTION (provided by applicant): The main aim of this grant is to develop databases and database tools in support of experimental neuroscience. We use as a model system the olfactory pathway, for which we have constructed a set of linked databases that is called SenseLab. Our project draws on expertise in three constituent projects. Project 1 will develop and apply the databases to experimental studies of successive stages in the olfactory pathway. These databases include Olfactory Receptor Database (ORDB) with an archive of over 5,000 chemical sensory receptors, and NeuronDB, which archives membrane properties that subserve dendritic integration. New initiatives include ORarrayDB to support genechip analysis of olfactory receptor gene expression, and OdorMapDB to support analysis of fMRI odor maps and correlation with maps generated by a variety of activity mapping methods. Project 2 is under the Yale Center for Medical Informatics, which provides the expertise needed for basic research in neuroinformatics and applications to the experiments and databases of Projects 1 and 3. It will expand a highly flexible database structure termed EAWCR not only to enhance our projects but also to launch new initiatives to provide for interoperability between Human Brain Project databases (HBPDB) and neuroscience databases more broadly (DBofNDB). Project 3 is responsible for development and maintenance of a database of neuronal models (ModelDB), primarily based on the software tool NEURON, a leading program for computational modeling of neuronal integration. Project 3 will collaborate with Project 1 on studies to model olfactory neurons and microcircuits, and will continue to develop ModelDB beyond the 100 models currently archived. The results of this program project should provide a model for how interlinked databases can be essential for experimental analysis of a model system, and should provide tools of general applicability to promote the use of databases throughout neuroscience.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): As a forebrain cortical region, the olfactory bulb is of special interest for the neural basis of smell perception. The new proposal builds on current work that has revealed new aspects of olfactory bulb circuits: their local organization in relatively narrow clusters of cells (columnar glomerular units) connected to individual glomeruli, the long distance interactions between these clusters to form glomerular unit ensembles for processing glomerular odor maps during odor stimulation, and their organization in the olfactory cortex. We will pursue analysis of these new aspects functional and anatomical approaches. First, we will use a new mouse model to monitor the stimulus of a single glomerulus, and record the spatiotemporal neural activity that arises from the local stimulus. Second, the Fos activation patterns after single glomerular stimulation will be examined. Third, we will use the pseudorabies virus as a retrograde tracer to reveal the route and reproducibility of distributed glomerular unit organization. Finally, the patterns of anatomical and functional connectivity will be examined using four complementary methods: calcium wave activation patterns, Fos staining after electrical stimulation, dextran dye tracing, and pseudorabies virus transsynaptic tracing. Together, these studies will provide a uniquely coordinated multidisciplinary test of the current hypothesis that odor processing involves activation of olfactory glomerular units in a widely distributed non-topographical manner, and the relationship of these patterns to olfactory cortex. The results will therefore suggest new principles for the functioning of cortical modules, and for the neural basis of normal odor processing.

DESCRIPTION (provided by applicant): The motor cortex (MC) functions as a major node in the cortical sensorimotor network. The specific circuits and synaptic mechanisms carrying long-range excitatory projections from sensory/association areas to key cell classes in MC such as corticospinal neurons have not yet been identified. Research on cortical sensory processing has previously established the concept of multiple pathways carrying spatial and non-spatial information from primary sensory to higher-order parietal areas, but how these excitatory projections from these areas converge on the cortical motor system remains poorly understood. Here we propose an experimental program designed to elucidate the cell-type specific connectivity underlying long-range corticocortical projections from higher-order sensory/association areas in parietal cortex to identified classes of MC neurons, focusing on corticospinal neurons. We will approach this overall goal using the mouse as our experimental model, retrograde and optogenetic labeling, and targeted opto-physiological recordings of functional synaptic connectivity in select pathways. Our aims are: (1) Determine the synaptic organization of retrosplenial cortex (RSC) projections to MC. (2) Define the input-output organization of RSC as a visuo-motor relay to MC. (3) Determine the synaptic organization of S2 inputs to MC. (4) Define the input-output organization of S2 corticospinal neurons. The proposed research program is highly innovative, we believe, because it brings together powerful new techniques to tackle an important, but experimentally previously inaccessible, issue in the field of sensorimotor research: the specific cellular mechanisms mediating corticocortical communication from higher-order sensory/association areas to motor cortical networks. The proposed research is significant because it will generate foundational knowledge about the macro- and microcircuit basis for feedforward corticocortical excitation of specific classes of MC neurons by higher-order sensory/association areas involved in sensorimotor integration.

(Cl.) Laser scanning microscopy (LSM), including confocal and 2-photon microscopy, continues to play a central role in many areas of biomedical research. In neuroscience alone, the number of applications continues to grow rapidly. However, in contrast to the ongoing advances made on the hardware aspects of LSM (eg. improved, cheaper light sources, detectors, and scanners), a major limitation to using LSM continues to be the cost and inflexibility of commercial software. Several years ago we introduced Scanlmage, an open source software package for LSM. Scanlmage has been highly successful and is now used by many labs, in particular for 2-photon microscopy. However, new types of cutting-edge applications require new functionality. In addition, it is usually important to combine LSM with other types of digital or analog stimulation or acquisition, such as electrophysiology, underscoring a related, more fundamental need, for an open-source general-purpose data acquisition program. Here, we propose (1) to continue to develop Scanlmage, with a focus on in vivo imaging and (2) to develop Ephus, a new and complementary program for general-purpose laser scanning and data acquisition, such as electrophysiological recording and video imaging. These programs are mostly written in Matlab, with an underlying C-backbone. Future releases of Scanimage will support new types of scanning hardware;improved region-of-interest and related scans;fast z-scanning;and, improved on-line multicolor display. Future releases of Ephus will include support for COD camera -based image acquisition and tools to facilitate easy creation of complex experimental paradigms, such as laser scanning photostimulation based mapping of neural circuits, We will maintain wiki-style documentation for Scanimage and Ephus. We believe that Scanlmage/Ephus will facilitate exciting biological discovery in numerous laboratories. v0, 0-0 0(Q --[unreadable] CAA N-0 .t? ,-,. .(D (n[unreadable] 0--u 6-0 aim ..c 11) (1) +-. (cu 07M O-> mom

DESCRIPTION (provided by applicant): Corticofugal - top-down - pathways from primary auditory cortex (A1) to the auditory brainstem powerfully modulate auditory processing. One important top-down pathway is mediated by corticocollicular neurons: a subset of layer (L) 5 pyramidal neurons in A1 with long-range axonal projections to the inferior colliculus (IC). The corticocollicular projection shapes the response properties of IC neurons, and mediates sound localization learning. However, previous physiological studies of A1 corticofugal mechanisms have not specifically targeted corticocollicular neurons. Therefore, basic physiological properties of corticocollicular neurons, such as their synaptic organization and their cellular properties, remain largely unknown. Our major goal in this proposal is to identify the cell-specific cellular and synaptic mechanisms that determine how L5 corticocollicular and L5 corticocallosal neurons - L5 principal neurons projecting to contralateral cortex - differentially process synaptic and acoustic input. The proposed experimental program will provide fundamental new information about the synaptic, cellular, and microcircuit properties of corticocollicular and corticocallosal L5 neurons. We expect that our findings will establish a new framework for understanding the roles of A1 projection neurons in top-down corticocollicular modulation of auditory processing in normal and disease states, such as in tinnitus and in pathological sound and speech perception.

PROJECT SUMMARY An essential aspect of human behavior is the ability to perform skilled and purposive movements. This ability relies on an intact neocortex, and in particular on different classes of projection neurons that convey neural information from the neocortex to downstream circuits involved in behavior. Two classes of cortical projection neurons are of particular interest - corticospinal neurons, projecting to the spinal cord, and corticostriatal neurons, projecting to the striatum bilaterally. While much has been learned from brain slice experiments about the cellular and synaptic properties of corticospinal and corticostriatal neurons, knowledge about their activity patterns in vivo in relation to volitional movements has been impeded due to a lack of easy to use methods for selectively recording from and manipulating the activity of these projection neurons. Here, building on prior ex vivo experiments, we propose a research program to develop new approaches to address this gap. In one aim, we will develop multiple complementary approaches to record the activities of corticospinal and corticostriatal neurons in the awake, moving mouse. In one set of studies we will use in vivo whole-cell recordings to record from motor cortex neurons at high sensitivity and temporal resolution, with post-hoc identification of cell types. In parallel studies we will infect corticospinal and corticostriatal neurons with recombinant rabies viruses carrying genetically encoded calcium indicators (GECIs), and detect activity either at high resolution using two- photon calcium imaging or at a population level using optical fibers. In our second aim, we will develop methods to manipulate the activities of corticospinal and corticostriatal neurons in the awake, moving mice. By either up- or down-regulate projection neuron activity using optogenetic or pharmacogenetic tools, we will be able to begin to address the question of how corticospinal and corticostriatal neurons' activities are causally related to movement parameters. The proposed research program is innovative and significant, we believe, because it will generate multiple new experimental paradigms for probing the in vivo functions of cortical projection neurons, not only in motor areas but across all of neocortex, and because these tools will be readily applicable to mouse models of neurological disease.

PROJECT SUMMARY Corticospinal axonal projections are critical for mammalian motor control. Their length and complexity makes them vulnerable to an exceptionally wide range of neurological disease processes including cerebrovascular disorders, demyelinating diseases, ALS, spinal cord injury, and more. Corticospinal research has naturally focused on cortical and spinal mechanisms. However, corticospinal axons, like those of other types of pyramidal tract neurons, can send branches to the midbrain, pons, and medulla along the way to the spinal cord. The anatomical ?projectome? of corticospinal axonal branching to brainstem targets has not been systematically investigated. Even less is known about the synaptic ?connectome?; i.e., the cellular circuits formed by corticospinal input to postsynaptic neurons in various brainstem nuclei. Elucidating the brainstem circuits of corticospinal axons will be important for understanding the cellular basis of mammalian motor control. For example, there very likely are as-yet unrecognized subtypes of corticospinal neurons that, through differential innervation of brainstem targets, mediate specific sensorimotor, neuromodulatory, or other functions essential for motor coordination and control. We propose a two-pronged approach to investigate the brainstem branches and circuits of corticospinal axons in the mouse. In Aim 1, we will use a high-throughput molecular barcoding technique, MAPseq, to characterize the diversity and complexity of corticospinal projections to the brainstem, with single-axon resolution and large-scale sampling. In Aim 2, we will use optogenetic, electrophysiological, imaging, and viral labeling tools to characterize the cell-type-specific synaptic connectivity in these circuits. The projections and connections of corticospinal axons? brainstem branches will be both broadly surveyed across regions (midbrain, pons, medulla) and analyzed in a more focused manner at the level of specific types of projection neurons in key nuclei, including those associated with sensorimotor, neuromodulatory, and other systems (reticular, pontine, cuneate, locus coeruleus, and more). Results from this discovery-oriented research program will lay the groundwork for future hypothesis-oriented studies to investigate ? at the mechanistically important level of specific cell types and their synaptic connectivity ? how the brainstem circuits of corticospinal neurons contribute to mammalian motor function.

PROJECT SUMMARY Dexterous food-handling is a critical but still poorly understood type of basic motor behavior. We propose an exploratory research program to investigate how the mouse uses its digits and forepaws to dexterously handle food. Preliminary results suggest that mouse food-handling movements, despite appearing extremely complex, can be decomposed into several types of simpler elements, tentatively identified as distinct and very rapid submovements that exhibit partly stereotyped and partly variable kinematic features. They also suggest that the mouse?s thumb (first digit, or D1; pollex) plays a surprisingly large and previously unrecognized role in dexterous food-handling. To explore these hypotheses and observations, we will first develop an approach for quantitatively characterizing seed-handling behaviors of freely moving mice. For this we will use high-speed, close-up video capture, combined with deep neural network methods for automated markerless tracking (DeepLabCut). We will then develop a novel trial-based paradigm for characterizing seed-handling under the more experimentally controlled conditions of head-fixation. We will combine this approach with tools for optogenetic silencing of either selectively targeted cortical areas or selectively labeled corticospinal neurons in motor cortex, as well as multi- probe/multi-channel linear array recordings of spiking activity in key nodes along sensorimotor pathways. Collectively this work will advance our understanding of the elemental movements involved in the dexterous manipulation of food, providing new insights into this process in a genetically tractable model organism, and opening the way for future studies to dissect the cellular mechanisms in sensorimotor circuits mediating specific aspects of forelimb and digit motor control.

PROJECT SUMMARY Sensory-guided movements of hands involve the integration and coordination of ascending somatosensory signals and descending motor commands at multiple levels of the nervous system. A great deal of knowledge about the neurophysiology of active touch and related aspects of forelimb somatosensation and motor control has been gained from studies using humans and monkeys as subjects. More recently, studies using mice are enabling extensive molecular and cellular characterizations of somatosensory neurons mediating forelimb- related functions. However, a limitation to neurophysiological analysis of mouse forelimb functions is that the vibrotactile and other mechanical types of stimuli that are commonly used to probe the system have inherent practical limitations in their spatiotemporal precision, speed, flexibility, and complexity. These limitations reflect the small dimensions of the mouse's forelimb anatomy and the need for mechanical stimuli to be applied through direct physical contact. In contrast, in the visual system, the physics of light permits tightly controlled delivery of complex stimuli. Our overall goal in this proposal is to develop new paradigms for investigating forelimb somatosensory and motor functions in the mouse based on ?light touch?: optogenetic activation of mechanosensory afferents of the forelimbs/hands, using spatiotemporally controlled light stimuli. For this, we will first use anesthetized mice to implement paradigms for optical delivery of light stimuli through optical fibers or laser-scanning photostimulation (LSPS). The stimulation technology will be used to activate the forelimbs/hands of mice expressing the excitatory opsin channelrhodopsin-2 (ChR2) in forelimb mechanoreceptor afferents, and integrated with hardware and software for recording kinematics, neuronal spiking in cortex and elsewhere, and forelimb muscular activity. In a second series of experiments we will develop multiple variants of this approach for use with awake behaving mice, enabling diverse types of experiments such as cortical silencing during peripheral activation, high-speed mapping of tactile responses, dynamic stimulus patterns, and more. The overall outcome will be to augment and expand the available experimental paradigms for investigating somatosensory-based functions of the mouse's forelimb, complementing the growing availability in mice of genetic and viral methods for accessing and manipulating in a cell-type-specific manner the somatosensory cell types and their circuits involved in sensory-guided motor control of the forelimb.