Timothy C. Cope - US grants

Diversity in motor skills is achieved in large part by a process that selects for the activation, or recruitment, of motor units that are specialized with respect to their tension producing characteristics. A major aim of this proposal is to continue investigation of the patterns observed in this selection process, i.e. to study recruitment order. "Size" and type characteristics will be measured from motor units recruited in sequence in segmental reflexes. These characteristics are central to the two most prominant hypotheses proposed to explain recruitment order, and their simultaneous measurement will help to resolve the current controversy over which scheme actually produces orderly recruitment. Evidence to support the hypothesis favored by this study will be obtained in a parallel investigation using intracellular recording and stimulation techniques to measure biophysical properties of motoneurons, their monosynaptic connections and their adjunct muscle fibers. As a result of these studies, the process that accounts for the appropriate matching of motor capabilities with motor tasks will be much better understood. With the functional organization of normal motor units clearly described, the project will proceed to meet another major aim, namely to describe some chronic effects of motor nerve transection. Very little information is available concerning the recoverability of recruitment order. A single unconfirmed study demonstrates that volitional recruitment of motor units belonging to a reinnervated muscle in the human hand is disordered. Other studies indicate changes in motor-unit functional properties that may account for the abnormal recruitment order. Both methods used to study normal motor units as described above will also be applied in an investigation of the chronic changes in recruitment order and in biophysical properties of motor units in a muscle self-reinnervated after nerve section. These data will provide a more complete prognosis of the potential for recovery of normal motor function in victims of nerve injury. Furthermore, they will provide a much more complete picture of the extent to which motor unit properties are restored following nerve transection.

Persistent modification of synaptic strength is a central in most conceptual models of learning, memory and recovery form injury, yet it has received surprisingly little attention in the CNS of the adult mammal. Our long term objectives are to identify cellular processes involved in persistent alteration in the efficacy of synapses on spinal motoneurons and to relate the findings to changes in motor-unit recruitment behavior. A major aim of this proposal is to determine whether transmission at the synapses made by Ia fibers and alpha motoneurons can be modified by chronically suppressing their activity. Synaptic transmission form Ia fibers will be chronically inhibited by preventing impulses from reaching their synaptic terminals. After 2 weeks of verified impulse blockade we will apply intracellular recording techniques to measure the size of the monosynaptic excitatory postsynaptic potentials (EPSPs) produced in motoneurons by the inactivated Ia fibers. If these studies indicate that inhibition of synaptic usage does alter synaptic efficacy as our preliminary data suggest, then we can begin to assess the underlying mechanism and realize it as a potential means of altering monosynaptic reflex behavior. Alternatively, if no change can be identified, then this synapse would provide a unusual example of one whose efficacy is independent of usage, and through comparison with others that are sensitive to use help to identify the necessary elements for plasticity. Another aim of this proposal is to continue studying the recruitment of motor units in a muscle reinnervated by its own served nerve. Recent studies suggest that changes in synaptic strength are not observed under these conditions and cannot, therefore, account for observations that motor units in reinnervated muscles are not recruited in order by their tension as they are normally. It is instead suggested that recruitment order can be recovered if the muscle is not reinnervated by foreign nerves. Our experiment is designed to test that hypothesis. Using intra-axonal recording and stimulation we will determine the relative recruitment threshold and various electromechanical properties of single motor units in self-reinnervated muscle of anesthetized cats. These data will and in characterizing the performance of reinnervated muscle with respect to fundamental properties such as tension and endurance, and show the extent to which interrelations among motoneurons, their synaptic input and muscle units are collectively restored.

What strategies of motor unit recruitment are u by the CNS to control the musculoskeletal system? The answer to this question is currently incomplete, but significant progress toward this answer will be made by meeting the four specific aims of this proposal. Each aim is to test a principle that may be used in the nervous system's selection of motor units for particular tasks. Specific Aim 1 tests the hypothesis that units are selected for the contribution they make to torque production at a joint. Specific Aim 2 tests the hypothesis that units are selected for their membership in functionally discreet neuromuscular compartments. Specific Aims 3 and 4 test the hypothesis that units are selected for their speeds of contraction and/or relaxation. Experiments planned to meet Aim 4 expand ongoing recruitment investigations in this laboratory to include human subjects. We will explore the as yet unconfirmed claim that slow contracting motor units are selectively inhibited during volitionally controlled lengthening contractions. This strategy is postulated to provide control over the speed of joint rotation during active muscle lengthening. By monitoring single units from a muscle in the hand, we will test the generality of earlier reported findings for ante extensor muscles. The remaining aims will be met using decerebrate cats and applying techniques similar to ones used previously in this laboratory to examine recruitment sequences of physiologically-characterized motor units. Aim 3 is designed to test the effects of stimulating the rubrospinal tract because this tract is predicted to selectively reverse the usual recruitment order. The supraspinal influences are of interest also because the selective recruitment behaviors observed in humans, but not by us thus far in cats, may depend upon these influences. The remaining Aims 1 and 2 will test the extent to which recruitment is organized around geometric relationships between motor units.and the skeletal system. This sort of- organization would be revealed if recruitment is found to be related to motor unit torque or motor unit membership in muscle compartments that produce different torques. The long term objective is to define neural strategies applied in producing normal movements. We believe that this information would be extremely valuable to rehabilitation medicine; it would assist in diagnosing motor problems and in assessing the effectiveness of various treatments, and would also provide a foundation for developing treatments to restore normal movement after debilitating injuries such as spinal cord trauma.

DESCRIPTION (adapted from applicant's abstract) This proposal is to study influences on synaptic strength in the spinal monosynaptic reflex pathway in rat hindlimb circuits. Particularly, the synapses between the Ia afferent fibers and the motoneurons change their behavior after nerve cut. The proposal addresses the questions whether the changes are (specific aim 1) due to changes in activity of the afferent fibers, (specific aim 2) due to changes in activity of the muscles, and/or (specific aim 3) due to changes in neurotrophin levels. The system lends itself to addressing such questions for several reasons, including: the accessibility of the postsynaptic neurons for recording excitatory post-synaptic potentials (EPSPs), divergence of afferent fibers from a given muscle to motoneurons innervating both the same (homonymous) and other (heteronymous) muscles, and effective separation of the elements of the circuit allowing independent manipulation at various points along it. Two approaches will be used to address the first specific aim, regarding the influence of changes in afferent activity in cut fibers on synaptic strength. The first approach involves comparison of EPSPs generated by cut afferent fibers that are silent vs. those that fire spontaneously, and the second approach involves measurement of EPSPs generated by afferent fibers electrically stimulated over several days after nerve cut. The hypothesis is that afferent activity normally prevents increased synaptic strength, which is then released when the activity is decreased by nerve cut. In addition, levels of neurotrophins will be tested in the associated tissues to assess their possible involvement. Specific aim 2, concerning the effect of muscle activity on synaptic strength, will be tested by paralyzing a peripheral muscle using tetrodotoxin applied to its efferent nerve, then stimulating to activate the muscle. Such stimulation should prevent the increase in synaptic strength associated with the paralysis, if muscle activity is an important factor. Again, levels of neurotrophic factors will be assessed for possible involvement. Specific aim 3 will more directly assess the influence of neurotrophic factors in the target muscles on central synaptic strength by manipulating the levels of endogenous factors by sequestration using receptor complexes. The hypothesis will be tested that the reduction in availability of these factors from the muscle when the nerve is cut results in an increase in synaptic strength (that the neurotrophic factors normally prevent such an increase in synaptic strength).

DESCRIPTION (Applicant's abstract): Despite the capacity of peripheral nerves to regenerate after injury or disease, functional recovery of the sensory system is only partially realized. The broad objective of this proposal is to determine the cellular mechanisms that constrain recovery of the sensory restoration of normal movement, and its profound ineffectiveness, even long after a cut peripheral nerve has regenerated into its original muscle, results in persistent motor disability. The impotence of sensory feedback from muscle is likely to be explained by limitations in the recovery of sensory transduction in the muscle and possibly by a diminished strength of central synaptic transmission are accounted for, as commonly accepted, by the inability of the sensory nerves to reconnect with their cognate muscle receptors. The absence of direct evidence for and the presence of strong challenges to this premise lead us to hypothesize that simple failure of a muscle afferent to reconnect with the appropriate muscle receptor is neither necessary nor sufficient to explain sensory dysfunction. This central hypothesis will be tested through combined electrophysiological and immunohistological studies of sensory nerves supplying long-term reinnervated muscles in living adult rats and cats. Three specific aims are proposed to determine whether the reconnection with a muscle receptor fully explains: (1) the normal response properties of some muscle afferents, (2) the abnormal response properties of others, and (3) the central synaptic constraints on recovery of sensory function under conditions in which sensory neuropathies, e.g. Guillain-Baree syndrome, diabetic neuropathy, or chronic inflammatory demyelinating polyneuropathy.

Disorders of the peripheral nervous systems, including trauma and peripheral neuropathies, create a disconnection between the central nervous system and its peripheral targets (i.e.-sensory receptors and muscle). Recovery from these disorders requires both regeneration of the peripheral nerve and reinnervation of targets. In patients with peripheral nerve disorders, this recovery is rarely complete, resulting in residual weakness, sensory dysfunction, and balance problems. The long term objectives of this program are to 1) advance our understanding of the constraints to full sensory and motor recovery after peripheral nervous system injury and disease, and 2) to develop strategies for enhancing recovery in peripheral nervous system disorders though scientifically- based interventions in biological processes. To achieve these goals we have formed a team of scientists and clinician-scientists to focus experimental attention on specific cellular and systems-level mechanisms that determine the responses to, and recovery from the effects of peripheral nerve injury and disease in experimental animals. There are 5 projects within this Program. The proposal begins where the biological problem begins, with the cellular mechanisms that underlie axonal degeneration. Two projects focus on the interactions between motor neurons and muscle, and two projects address the mechanisms and behavioral consequences of the poor recovery of sensory feedback in reinnervated muscles. Project 1 examines axonal degeneration in two models of nerve injury. Project 1 examines axonal degeneration in two models of nerve injury, axotomy and exposure to neurotoxins. This project will focus on the roles of calcium and proteases in these models. Project 2 tests the dependency of sensory afferent functions, including sensory transduction and central synaptic transmission, on reconnection with sensory receptor organs in muscle. This will be done by directly examining the physiology and morphology of individual regenerated sensory afferents. Project 3 asks whether altering the conditions or reinnervation of muscle fiber changes the properties of the reinnervating motoneurons. Project 4 examines the role of muscle activity in regulating synaptic transmission. Denervation-induced changes in muscle gene expression will be manipulate to test the effects on recovery. In Project 5, the manner in which abnormalities of proprioceptive pathways result in loss of coordination will be studied during locomotion and targeted reaching.

Our broad goal is to advance understanding of the strategies used by the central nervous system to organize muscular activity in accomplishing motor tasks. It is evident that there must be some provision for the functional organization of motor units, and our proposal is designed to test one possible scheme. We propose that the nervous system organizes motor units into cohesive groups that we call motor-unit synergies. In our model, each synergy is assembled from motor units that belong to more than one muscle but need not include all motor units in any one muscle. Motor unit members of each synergy are recruited in order by a modified version of the size principle. A significant advantage of this scheme is that motor units in the synergy are self-organizing, i.e.they initiate and co-modulate their firing in an orderly fashion as a consequence of their intrinsic properties and the common synaptic input that assembles them. We propose to test for motor-unit synergies by obtaining simultaneous records from approximately 20 motor units from selected muscles in sentient cats during treadmill locomotion. Specific Aim 1 is to test the hypothesis that the motor-unit synergy we find in reflexes is expressed in voluntary movements. Specific Aim 2 is to test the hypothesis that synergies are formed by the motor units of muscles that-are co-active but distinctive in their independent mechanical actions. Specific Aim 3 is to test hypothesis that proprioceptive feedback contributes to the formation of motor-unit synergies. Meeting these aims will identify the rules for organization for motor units that are active throughout a limb. Motor units may be organized into synergies of their own or as part of a muscle-based system. Our findings will distinguish these two strategies or reveal conditions under which they switch or superimpose. In any case, our studies will provide a more global view of the operation of motor units than is currently available. When this information is obtained it will be valuable to determine whether the functional organization of motor units can be modified to acquire new skills or adapt to injury.

PROJECT SUMMARY (See Instructions): Administration Core A. Mrs. Kimberly Hagler, Program Administrator, will work with Dr. Timothy Cope, Core A Principal Investigator, and Dr. Robert Fyffe, Core A Co-PI, to establish and manage Program Project business. This office will oversee the day-to-day operations of the Program, including: (1) disseminating all information; (2) organizing and managing group meetings and events; (3) ordering, receiving and distributing supplies; (4) facilitating access to secretarial and computer networking services; (5) assisting personnel actions; (6) maintaining all project accounts and preparing monthly budget reports with the assistance of a part-time accounting clerk; (7) preparing necessary reports for the Program. This core will also manage the subcontract for Project 2 with Emory University, and make all arrangements for the creation and utilization of an External Advisory Committee (EAC) that will meet at Wright State University annually. Decision Making Process is straightforward for this administrative core. Services listed above apply to all 3 Projects and to Core B. Should any special needs or adjustments be required, then all 4 Principal Investigators (projects 1, 2, 3, and Core B) will make the changes by consensus. Cost Effectiveness and Quality Control: The operating costs are heavily shared by the academic infrastructure established for Dept. Neuroscience, Cell Biology & Physiology at Wright State University. Departmental funds cover most of the salary for Mrs. Hagler, together with office space and supplies, computer support, FAX and photocopying. Quality control amounts to oversight by the Core PI, in consultation with the other PIs, of the effectiveness in delivery of services listed above.

PROJECT 1 Regeneration of a cut muscle nerve restores voluntary contraction but fails to re-establish normal sensorimotor function. We find that limb movements are uncoordinated and stretch reflexes are absent, not only in the reinnervated muscle but also in injury-spared synergists. Our recent findings point to multiple unexplored mechanisms within the spinal cord that might act, possibly in combination, to prevent stretch-reflex recovery that would otherwise be accomplished, at least partially, by regeneration. We propose to test two candidate mechanisms using electrophysiological measures applied to animals in which the nerves supplying a few muscles in the hindlimb are cut, rejoined surgically, and permitted to regenerate and reinnervate their original muscles. The first two aims focus on explaining the obstruction we observe in synaptic transmission between regenerated muscle stretch afferents and motoneurons (IA-MN synapses) within the spinal cord. Specific Aim 1 is designed to test the hypothesis that transmission at IA-MN synapses during physiologically-relevant firing rates is abnormally depressed because of cellular impairments in IA axons or synapses. Specific Aim 2 will test the hypothesis that transmission in regenerated IA-MN circuits is suppressed by active spinal networks. We will also study the temporal development of areflexia in muscles that are not subject to nerve injury. Specific Aim 3 will examine whether areflexia in injury-spared muscles develops independently of, or secondary to, reinnervation of its synergists. Meeting these aims will substantially increment our knowledge of post-regeneration limits on recovery of spinal synapses and circuits. This knowledge might also assist development of treatments for neurotrauma and neuropathies associated with sensory (proprioceptive) ataxia which share symptoms with the stretch areflexia under study in this project.

DESCRIPTION (provided by applicant): We designed this program project to coordinate synergistic research efforts around the central theme that nerve regeneration is not synonymous with functional recovery. We are uniquely focused on changes occurring in the sensorimotor motor circuits that are responsible for coordinating muscle activity and purposeful limb movement. In the aftermath of peripheral nerve transection and regeneration, spinal circuits do not regain normal feedback about movement from the centrally-projecting axon branches of either primary afferents or motoneurons. In the previous funding period, we discovered that these centrally- projecting axons and their spinal connections are permanently lost or altered, even when peripheral axon branches successfully reconnect with appropriate targets. Three projects, each led by an established investigator who brings unique experimental expertise and conceptual insight to the program, will advance our knowledge of the response of spinal circuits to peripheral nerve injury and regeneration. Project 1 will apply electrophysiological methods in vivo to test a proposed treatment to improve outcome following peripheral nerve injury. Project 2 will use a novel viral retrograde labeling technique to ask questions about circuit reorganization that have previously been impossible to address. Together, Projects 1 and 2 will test the hypothesis that circuit changes in the spinal cord triggered by peripheral nerve injury are more global than the monosynaptic reflex. The results will shed light on the nature of the changes that explain the modification in motor control and behavior after injury. Project 3 will define the pathway that underlies in vivo signaling that occurs via spontaneous vesicle release at the neuromuscular junction and will determine whether this pathway also signals synaptic stripping from motoneurons following peripheral nerve injury. All three projects will be assisted by the Cellular Imaging, Surgery and Tissue Processing Core Facility (Core B). This core provides the support and expertise necessary to ensure consistency and quality of procedures for all three PPG research projects. An external advisory committee reviewing our program project in 2010 concluded, this is an unusually interactive group with significant intellectual interactions evidet in many of the projects. It is our hope that continued close collaboration between projects, will bring significant added value as we move towards development of therapy to promote recovery following nerve injury. Public Health Relevance: Our overarching goal is to improve recovery following injury to the nervous system. Our specific focus is recovery of spinal cord motor function following peripheral nerve injury. Because similar cellular and synaptic changes might also operate as consequence of central nervous injuries, this work will also inform efforts to promote recovery from nervous system injuries after insults such as stroke and spinal cord injury. Disclaimer: The critiques and criterion scores from individual reviewers are provided below in an essentially unedited form. These were prepared prior to the review meeting and may not have been updated or revised subsequent to the discussion at the meeting. Therefore, they may not fully reflect the final opinions of the individual reviewers at the close of group discussion o the final majority opinion of the group. The Resume and Summary of Discussion above summarizes the final outcome of the group discussion. OVERALL PROGRAM EVALUATION

Abstract Chemotherapy is often accompanied by neuropathic sensory disorders that can limit or end treatment and cause long-term disability. Current research supports axon degeneration and hyperexcitability as underlying mechanisms. However, our recent research reveals an additional, absolutely novel mechanism having the potential to account for loss of patient function in chemotherapy-related neuropathy. We obtained in vivo electrophysiological measures which showed functional impairment of neuronal signaling from sensory and motor neurons in rats several weeks after receiving a clinically-relevant regimen of oxaliplatin (OX) chemotherapy. Hypo-excitability was consistently expressed as conspicuous failure to sustain firing in response to fixed levels of stimulation. The specificity of this defect, which leaves transient firing unaffected, suggests that OX treatment may impair sodium persistent inward currents (NaPIC) in sensory and motor neurons. Recent findings published in our lab promote this notion by showing that pharmacological block of NaPIC mimics the effect of OX on sustained firing. While our findings isolate chronic effects of chemotherapy on neuronal excitability, there is no chemotherapy without cancer. Cancer and OX therapy may act synergistically on common signaling pathways (e.g. oxidative and inflammatory) to produce neuronal hypo- excitability. The possibility of an interaction between cancer and OX therapy gains excitement from our preliminary reports that discovered sensory and motor neuron hypo-excitability is significantly amplified in rats with colorectal cancer. Here we propose incisive tests of our working hypothesis that OX treatment chronically impairs static neuronal signaling by reducing NaPIC in a rat model of cancer. We will measure the firing behavior of sensory and motor neurons via in vivo electrophysiological studies of cancer rats treated with OX, in order to achieve the following four specific aims: 1) test the hypothesis that interactions with cancer-related processes exacerbate chemotherapy-induced hypo-excitability in sensory and motor neurons; 2) test the hypothesis that chronic defects in repetitive firing by motor neurons result from an OX-induced decrease in persistent inward current; 3) develop therapy that normalizes firing of sensory and motor neurons in rats treated for cancer with OX; 4) identify factors related to the development of hypo-excitability induced by OX in a rat model of colorectal cancer. Successful accomplishment of these studies will: 1) determine for the first time in the CIPN field, of the extent to which chronic deficits in neuronal excitability arise from OX therapy, colorectal cancer, and their combination; 2) identify biophysical mechanisms underlying firing deficits of a CNS neuron after OX treatment; 3) develop pre-clinically a viable therapy for rescuing neurons from OX-induced firing deficits; 4) take the first step forward in understanding the pathogenesis of OX-induced hypo-excitability by relating its development and underlying biophysics to changes in gene expression of sensory and MNs.

PROJECT SUMMARY Our long-term goal is to identify neural mechanisms and the functional roles of sensorimotor signals in health and disease as needed to guide mechanistically targeted diagnoses, assessments, and treatments for neurological movement disorders. Here we address the scientific barriers to understanding and treating a broad class of movement disorder symptoms recently defined as joint hyper-resistance, which encompass spasticity in stroke, spinal cord injury, or cerebral palsy; parkinsonian rigidity, and hypertonia. The objective of this collaborative, interdisciplinary proposal is to identify neural mechanisms of hyper-resistance and dissociate their relative roles in abnormal movement. We will focus on the neural mechanisms underlying two clinically- defined neural contributions to hyper-resistance: non-velocity dependent involuntary background activation and velocity-dependent stretch hyper-reflexia. We hypothesize that increased spinal excitability in many neurological disorders causes involuntary background activation and velocity-dependent stretch hyper-reflexia via three dissociable neural mechanisms: 1) alpha-drive to extrafusal muscle fibers increasing background muscle tension, 2) gamma-drive to specialized intrafusal muscle fibers in muscle spindles sensory organs, increasing their sensitivity to muscle stretch, and 3) sensorimotor gain of the spinal transformation of monosynaptic sensory input into motor output. Our proposed tests of this hypothesis will advance understanding of the important, yet still unresolved relative contributions made by these neural mechanisms to hyper-resistance. Based on our neuromechanical and multiscale modeling advances in the prior funding period, in Aim 1 we will develop a multiscale in silico neuromuscular circuit model to predict how independent changes in alpha- drive, gamma-drive, and sensorimotor gain differentially affect clinically-relevant movements such as the tendon tap and pendulum test. In Aim 2, we will characterize the relative increases in alpha-drive, gamma-drive, and sensorimotor gain across clinically-relevant spinal excitability levels in a living biological neuromuscular circuit in vivo using a decerebrate rat preparation. In Aim 3 we will identify clinically-relevant movement abnormalities across spinal excitability levels in a novel biohybrid robotic system coupling the living neuromuscular circuit (in vivo) to a virtual biomechanical limb (in silico). A robotic controller will enforce the physics of dynamically changing inertial and gravitational forces, allowing movement to emerge from the causal interaction between the in vivo neuromuscular circuit and the virtual limb. Through the close coordination of these Aims, we will establish a computational and experimental framework to address clinical barriers (1) to determine how changes in neural mechanisms and the inertial properties of the limb could correct movement abnormalities, (2) to provide insight into how these mechanisms could be identified through different clinical assessment scenarios, and (3) to compare the relative effects of different treatment targets. The proposed work will likely impact both clinically-relevant human sensorimotor research and basic sensorimotor neuroscience.