Edmund R. Hollis, Ph.D. - US grants

Project Summary/Abstract The corticospinal pathway is a pivotal mediator of voluntary motor control in humans and restoring corticospinal motor function after spinal cord injury remains one of the most substantial challenges facing translational and clinical neuroscience today. Regardless of the approach taken to alleviate the interruption of corticospinal axons, functional recovery will rely on the plasticity of the cortical motor network to incorporate the remodeled or replaced circuit. The creation of novel therapeutic interventions for the recovery of function after injury will have to be coupled to an understanding of the cellular and subcellular mechanisms that support cortical motor network remodeling and incorporation of injured neurons. Only recently have the tools necessary to answer these critical questions been developed. The long-term goal is to develop novel therapeutic interventions for the recovery of function after spinal cord injury through an understanding of the cellular and subcellular mechanisms that drive neural circuit remodeling. The overall objective for this proposal is to identify neuromodulatory mechanisms of motor map plasticity that correlate with recovery of skilled motor function and to determine the relationship between cortical network changes and corticospinal circuit remodeling after cervical spinal cord injury. The central hypothesis is that cholinergic input directly to corticospinal motor neurons is required for functional integration of corticospinal circuit changes into de novo motor networks after spinal cord injury. The rationale for the proposed research is that once the role of cholinergic activity in rehabilitation after spinal cord injury is defined, it is likely to provide new opportunities for pharmacological modulation and pairing with treatments that enhance corticospinal axon regeneration. The first approach will be to use cytotoxic lesions and optogenetic/chemogenetic control of relevant subcortical circuitry to modulate known mediators of motor learning while using two-photon microscopy to assess the functional incorporation of injured corticospinal neurons by measuring activity and structural changes during rehabilitation in awake, behaving animals. The second approach will be to define the molecular mechanisms and the cellular location of signaling events that underlie motor learning, and likely motor rehabilitation. The proposed studies are innovative in that they shift the focus of spinal cord rehabilitation onto the circuit mechanisms of cortical network plasticity and will have far- reaching importance in translating treatments for both acute and chronic injuries to motor networks. The proposed studies are significant because they will elucidate the mechanisms by which circuit remodeling influences recovery, which will have far-reaching importance in translating treatments for both acute and chronic injuries to cortical motor networks. The expectation is that completion of the proposed research will generate new opportunities for pharmacological modulation and combinatorial treatments that enhance corticospinal axon regeneration while mediating cortical motor network reorganization.

Project Summary/Abstract Plasticity of cortical motor networks is a critical mediator of skilled motor learning and a potential mechanism for supporting the rehabilitation of motor function after neurological injury. Fundamental to the understanding of how the remodeling of these cortical networks supports skilled motor control is the ability to record from identified cell types during the stages of skill acquisition and rehabilitation. Recent advances in two-photon imaging and the development of genetically encoded calcium indicators has allowed for an understanding of the dynamics of neural networks during motor learning. However, execution of the simple motor tasks currently in use in head- fixed mice are not sensitive to disruption of cortical motor networks, while more complex tasks currently require high speed video analysis and post-experimental processing. There is a critical need to develop unbiased testing devices for skilled, corticospinal-dependent behaviors to use in concert with modern in vivo imaging techniques. The long-term goal is to develop novel therapeutic interventions for the recovery of function after spinal cord injury through an understanding of the cellular and subcellular mechanisms that drive neural circuit remodeling. The overall objective for this proposal is to develop a system for assessing a complex, corticospinal-dependent behavior for use in future in vivo imaging studies of skilled motor learning and rehabilitation. In order to achieve this objective, the automated, objective supination task will be adapted for use in head-fixed mice during two- photon imaging. The rationale for developing this tool for use during in vivo imaging in head-fixed mice is that it will allow for the study of cortical motor networks during both the learning and rehabilitation of skilled, corticospinal-dependent, stereotyped forelimb movements in an unbiased manner in real time. The objective for this proposal will be met by addressing the following two specific aims: 1) Adapt a supination task for mice and determine the effects of corticospinal tract injury; and 2) Adapt the supination task for use in head-fixed mice and validate its use for the study of corticospinal neuron activity in vivo. Under the first aim, a reduced scale version of the Vulintus, Inc. MotoTrak rat supination task will be adapted for mice in concert with Vulintus, Inc., after which the effects of corticospinal tract injury will be tested on the supination task. Under the second aim, the supination task will be adapted for head-fixed mice and calcium transients will be used to study the response of corticospinal neurons to injury. The proposed work is innovative in that it will result in novel tools for the study of skilled, corticospinal-dependent motor learning and the activity of cortical motor networks in vivo in a quantitative, reproducible, and unbiased manner. The proposed work is significant as it will allow for vertical advancements in the study of skilled motor learning and rehabilitation after neurological injury. Ultimately, these tools have the potential to be applied broadly to study both the mechanisms of motor learning and the effects of neurological dysfunction, whether developmental or induced by injury and disease.

Project Summary/Abstract The corticospinal pathway is a pivotal mediator of voluntary motor control in humans and restoring corticospinal motor function after spinal cord injury remains one of the most substantial challenges facing translational and clinical neuroscience today. Regardless of the approach taken to alleviate the interruption of corticospinal axons, functional recovery will rely on the plasticity of the cortical motor network to incorporate the remodeled or replaced circuit. The creation of novel therapeutic interventions for the recovery of function after injury will have to be coupled to an understanding of the cellular and subcellular mechanisms that support cortical motor network remodeling and incorporation of injured neurons. Only recently have the tools necessary to answer these critical questions been developed. The long-term goal is to develop novel therapeutic interventions for the recovery of function after spinal cord injury through an understanding of the cellular and subcellular mechanisms that drive neural circuit remodeling. The overall objective for this proposal is to identify neuromodulatory mechanisms of motor map plasticity that correlate with recovery of skilled motor function and to determine the relationship between cortical network changes and corticospinal circuit remodeling after cervical spinal cord injury. The central hypothesis is that cholinergic input directly to corticospinal motor neurons is required for functional integration of corticospinal circuit changes into de novo motor networks after spinal cord injury. The rationale for the proposed research is that once the role of cholinergic activity in rehabilitation after spinal cord injury is defined, it is likely to provide new opportunities for pharmacological modulation and pairing with treatments that enhance corticospinal axon regeneration. The first approach will be to use cytotoxic lesions and optogenetic/chemogenetic control of relevant subcortical circuitry to modulate known mediators of motor learning while using two-photon microscopy to assess the functional incorporation of injured corticospinal neurons by measuring activity and structural changes during rehabilitation in awake, behaving animals. The second approach will be to define the molecular mechanisms and the cellular location of signaling events that underlie motor learning, and likely motor rehabilitation. The proposed studies are innovative in that they shift the focus of spinal cord rehabilitation onto the circuit mechanisms of cortical network plasticity and will have far- reaching importance in translating treatments for both acute and chronic injuries to motor networks. The proposed studies are significant because they will elucidate the mechanisms by which circuit remodeling influences recovery, which will have far-reaching importance in translating treatments for both acute and chronic injuries to cortical motor networks. The expectation is that completion of the proposed research will generate new opportunities for pharmacological modulation and combinatorial treatments that enhance corticospinal axon regeneration while mediating cortical motor network reorganization.

PROJECT SUMMARY: Cortical motor networks are a critical, if often overlooked, mediator of motor recovery after spinal cord injury (SCI). Cortical networks are required for instructing output through the corticofugal projections to the brainstem and spinal cord, and the plasticity of these networks will be indispensable for re- learning how to use the spinal circuits altered by SCI or therapeutic intervention. Rehabilitation is necessary for both the recovery of corticospinal-dependent forelimb function and the commensurate reorganization of disrupted cortical motor maps. It remains unknown what the underlying circuit mechanisms are that support cortical reorganization after SCI, or whether such broad reorganization is necessary to support functional recovery. The long-term goal is to develop therapeutic interventions that take advantage of cortical plasticity to promote recovery from SCI. The overall objective for this proposal is to identify the intracortical circuitry responsible for restoring skilled forelimb function. The central hypothesis is that latent intracortical connections projecting from de-efferented hindlimb to forelimb areas are required for rehabilitation-mediated recovery of skilled forelimb function after cervical SCI. The rationale for the proposed research is that the knowledge of how the motor cortex incorporates circuit changes after SCI will help us to target new therapies for motor recovery. The following three specific aims are proposed: 1) Record the endogenous activity from intracortical neurons during rehabilitation-mediated recovery from SCI; 2) Determine the structural changes in horizontal connections during rehabilitation from SCI; and 3) Identify the contribution of horizontal connections to motor recovery after SCI. For the first aim, the approach will be to record activity from interconnected hindlimb and forelimb motor regions during skilled forelimb behavior in order to determine their response to rehabilitation from SCI. In the second aim, the approach will be to image structural changes of intracortical axons and dendritic spines in vivo longitudinally during rehabilitation from SCI. In the third aim, the approach will be to 1) silence interconnected neurons in awake, behaving mice to determine their contribution to recovery, and 2) stimulate interconnected neurons and measure forelimb motor evoked potentials. The proposed studies are innovative in that they shift the focus of spinal cord injury research from axon regeneration to the intracortical networks required for interpreting the changes in spinal cord circuitry. The proposed studies are significant because they will provide a detailed understanding of the mechanisms of circuit remodeling that influence recovery. The expectation is that completion of the proposed research will determine the extent to which intracortical neuron plasticity underlies cortical motor map reorganization and supports functional recovery after SCI. These findings will establish a foundation upon which to build therapeutic advances and dictate which strategies are most appropriate to pursue for both acute and chronic SCI.

This application is a request for funds to purchase a Nikon A1R HD25 confocal microscope. This microscope will be housed in the Structural and Functional Imaging Core at the Winifred Masterson Burke Medical Research Institute (DBA Burke Neurological Institute; BNI). The Burke Neurological Institute is a leading nonprofit scientific research institute with the mission to find cures for chronic neurological disabilities. The mandate of the imaging core facility is to provide access to a range of light microscopy systems and provide technical assistance with image acquisition, processing, and analysis to all research groups within BNI. Confocal microscopy is essential for the majority of the NIH-funded laboratories at BNI and the previous shared confocal, a Zeiss LSM 510, has exceeded its serviceable lifetime. The Nikon A1R HD25 is both a significant upgrade and a much-needed resource for the NIH-funded laboratories of BNI. The A1R HD25 has many advanced features that would benefit our research progress; however, there are three key features that make it most suitable for the diverse studies that are performed in the Imaging Core: 1) the largest available working field of 25 mm, 2) the industry-leading environmental chamber with multi-dish holder, temperature controller and built-in digital gas mixer, and 3) Nikon's versatile Perfect Focus System (PFS), which corrects for focus drift caused by temperature changes or mechanical disturbances. The institute is highly supportive of this application and has pledged to invest in the maintenance and operation throughout its useful lifetime, as it has done for 17 years on our previous multi-user confocal microscope. The requested microscope will exert a sustained, powerful influence on the conduct of the current NIH-funded research programs at BNI, and will be critical to the success of new studies as they mature to NIH funding.