Ronald M. Harris-Warrick - US grants

The general goal of this proposal is to study the role of neuromodulators in the generation and modification of behavior. Simple behaviors are generated within the central nervous system by limited circuits of neurons called Central Pattern Generators (CPGs). CPGs can produce stereotyped motor patterns in isolation, but they depend on extensive modulatory input to produce the normal variety of related behaviors. The cellular mechanisms involved in this modulation have not been studied. I propose that modulatory inputs change the active components of the CPG by adding and subtracting cells from the circuit, changing firing characteristics of neurons, and changing synaptic efficacy within the circuit. To test this hypothesis, I will analyze the cellular and ionic mechanisms whereby serotonin, octopamine and dopamine modulate the well-characterized 14-neuron CPG for the pyloric rhythm in the stomatogastric ganglion of the lobster. Using electrophysiological and pharmacological techniques, I will answer several questions: 1) How many of the 14 identified neurons are directly excited or inhibited by an amine? 2) Are synaptic connections in the circuit modified by amines? 3) What are the ionic mechanisms for amine excitation and inhibition of each target neuron? This work will describe how generalized CPGs are sculpted by modulatory inputs to produce specific functional circuits generating a wide spectrum of behaviors.

Investigations into the control of nerve fiber growth and survival by local variations in extracellular potassium are proposed. A compartmentalized culture preparation will be employed in which axons from cell bodies of rat neurons located in one compartment grow across silicone grease barriers to enter separate compartments. In this way the fluid environment of the distal axons can be controlled independently of the fluid environment of the cell bodies and proximal axons. The distal axons can be removed at any time and promptly regenerate. This allows axonal regeneration of the same population of neurons to be observed sequentially under different experimental conditions and as the neurons develop and age in vitro. Micrometric measurements of neurite extension and photomicrographic comparisons of neurite density will be used to evaluate growth. Also, neurons in compartmentalized cultures can be chronically electrically stimulated, permitting the performance of experiments concerned with the role of electrical activity in growth and development. In addition to work with compartmentalized cultures, it is proposed to measure changes in [K+]e that occur in vivo by means of K+-selective microelectrodes. The questions addressed in this proposal arise because of observations made in this laboratory employing compartmentalized cultures. Neurites of sympathetic neurons, dorsal root ganglion neurons and spinal cord neurons can be excluded from entering a region of locally increased extracellular potassium ([K+]e). Also, a proximo-distal increase in [K+]e along the neurites has a variety of regressive effects on neurite growth, regeneration, and survival: elongation can be slowed, an extreme retraction of neurites can ensue, and complete degeneration can occur. These observations have implications for the mechanism of nerve fiber elongation, and they also raise the far-reaching possibility that natural variations in [K+]e in vivo during normal development may strongly influence the establishment and/or maintenance of connections between neurons. For example, K+ released by nerve fibers during activity may mediate competitive interactions for postsynaptic sites. It is also possible that regressive changes induced by variations in [K+]e may have implications for the problems of regeneration, aging, and seizure activity in the nervous system. Experiments are proposed to: (1) characterize the effects of locally elevated K+ on peripheral and central neurons with particular emphasis on changes that occur with development and aging in vivo and in vitro, (2) investigate the mechanisms of these effects, (3) begin to collect information about what magnitude of changes in [K+]e can occur during development in vivo, and (4) establish an in vitro system to study the long-term effects of activity on synapse formation and maintenance.

Simple behaviors are generated within the central nervous system by limited neuronal networks call Central Pattern Generators (CPGS). In isolation, these networks produce stereotyped motor patterns, but in vivo, they interact with extensive modulatory and sensory inputs to produce the normal variety of related behaviors. A dysfunction of these modulatory inputs to produce the normal variety of related behaviors. A dysfunction of these modulatory inputs would seriously limit the spectrum of behavior generated by the CPG. The mechanisms involved in modulation of neuronal circuits are not well understood. Our hypothesis is that modulatory inputs alter the physiological organization of the network from moment to moment, producing a variety of different motor patterns from one network of cells. To test this hypothesis, we will study the actions of three amines, dopamine, octopamine and serotonin, as modulators of the CPG for the pyloric rhythm in the lobster stomatogastric ganglion. These amines can produce distinctive variants on the pyloric motor pattern when applied to the isolated stomatogastric ganglion. We will combine electrophysiological and pharmacological techniques to pursue the following goals: 1) Study the biophysical actions of amines on ion currents in different cells in the pyloric CPG, to determine the extent of diversity and convergence in modulator actions on different cells in a single circuit. 2) Study the second messenger mechanisms used by amines in different cells, to determine whether a modulator acts by single or multiple mechanisms within a neuronal circuit. 3) Analyze amine modulation of synaptic transmission in the circuit, to show that modulators can change the "wiring diagram" of the circuit from moment to moment. 4) Study the anatomy and physiological activity of cells that deliver the amines to the stomatogastric ganglion, to determine the behavioral context for amine modulation of the pyloric motor pattern. This work will further our understanding of how a single anatomically defined neuronal network can be modulated to produce a series of related functional circuits, each generating a unique variant on the basic motor pattern.

This project will explore the dynamics of a small neural network. There are three major aspects to the project: 1. modelling the neural network as a system of nonlinear differential equations with explicit representation of ion channels for each neuron within the network 2. experiments on the pyloric circuit of the stomatogastric ganglion of spiny lobsters that will be used to provide input data for parameter values of the models and to test predictions of the models 3. mathematical research to develop algorithms that will lead to tools for the explicit computation of geometric features within the models A mathematical software package called dstool, developed at Cornell for the exploration of dynamical systems, will be used, and will incorporate algorithmic advances into this environment. The initial emphasis will be the pacemaker neuron within the circuit. Guckenheimer will investigate how its response depends upon multiparameter variations of its environment that come from application of channel blockers and neuromodulators and from changes in extracellular ionic concentrations. Data from previous studies of synaptic efficacy and their dependencies upon amines will be used in modelling the interactions of neurons within the network.

We are studying the cellular basis for behavioral flexibility. Simple rhythmic behaviors are generated within the central nervous system by limited neural networks called Central Pattern Generators (CPGs). In isolation, a CPG can produce a stereotyped motor pattern, but in vivo, it interacts with an extensive network of modulatory and sensory inputs that can significantly change the properties of the neurons and synapses in the network. As a consequence, a single anatomically defined network can be "sculpted" to produce a variety of related behaviors. A loss of modulatory input would severely limit the behavioral flexibility of the animal by limiting the number of modes in which the motor network can operate. The cellular mechanisms that cause this sculpting of neural networks are not well understood. We are studying how three monoamines, dopamine, serotonin and octopamine, modulate the 14-neuron pyloric network in the crustacean stomatogastric ganglion. Each amine can induce a unique motor pattern when applied to the pyloric network. Our goal is to follow the complete process of behavioral modulation in this system, from the state of the animal that activates the monoaminergic inputs, through the cellular and ionic mechanisms that amines use to modify the pyloric network's functional circuit, to the altered motor pattern that evokes the changed behavior. We will combine current clamp, voltage clamp, and calcium imaging studies to determine the ionic mechanisms by which each amine alters the intrinsic electrophysiological properties of the neurons and their synaptic interactions. We will study the effects of these amines on the intact network in a variety of different states, and on its interactions with related networks, and try to interpret these effects based on the cellular and synaptic actions of the amines. Finally, we will study the identified neurons that provide the serotonin to the pyloric network, and analyze the behavioral context that activates them. This work will contribute to our understanding of how changes in the properties of cells and synapses in the nervous system can lead to changes in behavior. By showing how modulatory input sculpts a crustacean motor network to produce many behaviors, our work will serve as a model for studies of behavioral flexibility in other invertebrates and vertebrates alike.

Guckenheimer 9418041 In this multidisciplinary project, the investigator and his colleagues at Cornell University together with Peck at Ithaca College focus upon studies of the stomatogastric ganglion. This small neural network with approximately 30 neurons is a central pattern generator that coordinates movement of the gastric mill and pylorus of crustacea. The investigators construct compartmental models of individual neurons based upon experimental measurements and amalgamate these into models for subnetworks. These systems provide a testbed for the development of algorithms to explore the bifurcations of dynamical systems. Particular attention is given to those aspects of the systems that involve multiple time scales. The goal of the project is to improve understanding of dynamical processes in biological systems, with an emphasis upon neural systems. The investigators work in a multidisciplinary setting, combining experiment and mathematical modeling, giving due attention to both mathematics and neuroscience, and ensuring that the two interact at all times in the work. The larger goals are pursued in the context of specific qystems, namely the study of a small neural circuit that controls rhythmic motions in the foregut of lobsters. This model system has been chosen for its modest size and accessibility for experimental manipulation. The project aims to understand how neural systems switch among motor patterns and how they regulate these patterns. ***

Schneiderman 9422143 During the development of the neuromuscular system, a series of complex interactions are involved in the specification of the pattern of innervation of any specific muscle. This research is designed to elucidate the sequence of events that result in the development of the appropriate connections between neurons and muscles. To address this problem a novel invertebrate system will be employed. The innervation of muscles will be studied during the process of metamorphosis, when degenerating muscles are being replaced with newly formed muscles. In particular, the role of hypothesized "muscle pioneer cells" in neural specification will be examined. The results of this research will provide basic information about the major steps involved in the development of connections between neurons and muscles, as well as insight into the plasticity inherent in the developing and regenerating neuromuscular system.

DESCRIPTION: The meeting, to be held under the auspices of the NY Academy of Sciences in NYC, March 1998, concerns the neural basis of locomotion. The meeting will last for three days and includes 31 speakers from both North America and abroad. The meeting will be open to everyone, and each speaker will provide a paper which will be published as a special issue of the Annals of the NYAS. There will be several poster sessions to accommodate the wide variety of topics in the meeting. A novel aspect of these poster sessions is that the session chairperson will make a tour through the posters, allowing each poster-presenter a 1-2 minute presentation followed by a 3 minute discussion of the poster. The sessions begin with a description and new results from each of the best-characterized motor generator systems: from mollusks through frogs. The next session will present new results from motor systems in higher vertebrates, using electrophysiological recordings and activity-dependent imaging, in chicks, turtles, and mammals. Later sessions will examine control of locomotion generators by both neurotransmitters and afferent input, modeling of generator systems, molecular determinants of the components of pattern generators, the development of motor systems, and finally clinical aspects of locomotion and rehabilitation.

DESCRIPTION (Adapted from applicant's abstract) : A large number of voltage-sensitive K+ channels are coded by the four genes in the Shaker family. In Drosophila, three of these genes undergo alternative splicing of their mRNA to generate multiple proteins with different biophysical properties. In addition, splice variants of a single gene can interact to form heteromultimeric channels. The physiological roles of this potassium channel diversity are not well understood. It is difficult to associate a particular gene with an ionic current in a cell, and to show how that gene product helps to shape the electrophysiological properties of the cell and the neural network within which the cell functions. This proposal is aimed at filling this gap using the small, well-defined pyloric network in the crustacean stomatogastric ganglion. This network's 14 neurons are easily identified, and their unique physiological properties are well known. We are cloning the cDNAs of the four genes in the Shaker family (shaker, shab, shaw and shal), and determining their functions in each of the 14 identified cells in this pyloric network. To understand the roles of Shaker family currents in the generation of the pyloric network's motor pattern, we propose the following goals: 1) Clone the alternative splice variants of shaker, shab, shaw and shal form lobster. 2) Express these variants in Xenopus oocytes and lobster neurons, and compare their biophysical properties to endogenous currents in the stomatogastric neurons. 3) Map the expression of these variants in the individual identified neurons using a single cell PCR method that we have recently developed. 4) Artificially alter the expression of Shaker family genes by injecting sense or antisense RNA into identified STG neurons, and determine the effect or raising or lowering specific currents on the cellular and network properties and cellular responses to monamine modulators. The ultimate goal of this work is to answer why there are so many genes and splice variants for voltage-dependent K+ channels. Our studies will allow us to assign physiological roles to specific K+ channel genes in determining both the intrinsic firing properties of identified neurons and the motor pattern generated by the pyloric network.

DESCRIPTION: (Adapted from the Investigator's Abstract) Simple behaviors are generated by networks of neurons, interacting in complex and nonlinear ways. The output from a behavioral network is determined by the pattern and strength of synaptic interactions within the network as well as the unique intrinsic firing properties of the different network neurons. These synaptic and firing properties are generated in turn by the differing ratios of ion channels and receptor genes that are expressed by each neuron in the network. Our goal is to bridge the span from gene expression to neural network function, to show how single genes contribute to shaping the behavioral output from a neural network. To accomplish this, we are studying the roles of a set of potassium and calcium channel genes within the 14-neuron pyloric network in the lobster stomatogastric ganglion. This network is one of the best-described neural networks: all of the synaptic connections are known and the firing properties of all the neurons are well understood. We propose to manipulate gene expression for ion channels in single identified neurons in this network to better understand the rules of network function. There are 3 specific aims to accomplish this goal. First, we will complete our ongoing project to clone the genes for and analyze the biophysical properties of the Shaker family of voltage dependent K+ channels as well as two calcium channels. Second, we will determine which neurons in the pyloric network express these genes, and where in the neuron the proteins are targeted. Third, we will increase the expression of these genes in single neurons, using injections of sense RNA, or decrease expression with injections of dominant negative, antisense or double-stranded RNA, and determine how this alters the firing and synaptic properties of the neuron and the pattern of activity in the pyloric network. This "molecular neuroethological" approach will allow us to study the effects of one gene and thus one channel at a time on the behavioral output of a network, which in the past has only been possible with theoretical mathematical models.

DESCRIPTION (provided by applicant): The training program in Cellular and Molecular Biology at Cornell University is based on our belief that cellular and molecular approaches provide the best insights when integrated with other levels of analysis of behavior. The six predoctoral students supported by this training grant are a subset of students for the Ph.D. degree in the Graduate Field of Neurobiology and Behavior. All students must meet the requirements of this Field, which provides students with a broad training in both neurobiology and behavior. In addition, students in the training program take a series of laboratory rotations as well as advanced lecture and laboratory courses to provide strength in cellular and molecular neurobiology. These are combined with a series of joule clubs and seminar series to remain current with the latest results. Our students benefit from interactions with other academic programs at Cornell, including biochemistry, molecular biology, genetics, pharmacology, chemistry, applied physics, biophysics and psychology, which offer courses and seminars of interest. Trainees are selected from prospective students who apply to the Graduate Field of Neurobiology and Behavior. They typically have completed a major in biological sciences, and have taken at least one course in neurobiology. These students are in the top 15 percentile on the graduate records examination, and nearly all have done research during their undergraduate education. The research areas represented by the core faculty include focus groups in the function of ion channels in neuronal signaling, molecular mechanisms of synaptic function and neuromodulation, neurodevelopment, neuropharmacology, sensory systems and animal communication, and neuroethology. Fully equipped facilities for all of the areas are available to the students both in faculty laboratories and in a number of shared facilities.

Understanding the spinal circuits that organize rhythmic limb movements is critical for future treatment of patients with spinal cord injuries. The overall goal of this project is to further our understanding of a set of intemeurons that participate in the central pattern generator (CPG) for locomotion in the neonatal rat spinal cord. The commissural intemeurons (CINs) send their axons to the contralateral spinal cord and are thought to play critical roles in left-right coordination and rhythm generation during locomotion. I propose to characterize two of the ionic currents that control post-inhibitory rebound in three anatomically defined classes of CINs with ascending, descending or intrasegmental axons. Using whole cell voltage clamp recordings, I will measure the parameters of the hyperpolarization-activated inward current, Ih and the low-threshold calcium current, IT, in each cell type, and determine the effects of serotonin on these currents. I will perform single cell RT- PCR studies to determine which genes related to Ih and IT are expressed in each CIN class. These experiments should provide further criteria for rigorous identification of neuronal classes in the CPG for locomotion in the neonatal rat spinal cord.

DESCRIPTION (provided by applicant): Project summary: The objectives of the graduate training program in Cellular and Molecular Approaches to Behavior at Cornell University are to train students to combine cellular and molecular neurobiological tools with higher levels of analysis to understand the mechanistic bases underlying behavior. There is a growing appreciation of the necessary interactions between neurobiology and the behavioral sciences, and our students will be trained to be leaders in this new interface. The research areas represented by the core faculty include focus groups in the function of ion channels in neuronal signaling, molecular mechanisms of synaptic function and neuromodulation, neurodevelopment, neuropharmacology, sensory systems and animal communication, motor systems, and neuroethology. Fully equipped facilities for all of these areas are available to the students both in faculty laboratories and in a number of shared facilities. Six predoctoral students will each be supported for a maximum of two years;they are usually appointed at the beginning of their training in the Graduate Field of Neurobiology and Behavior. Most have completed a major in biological sciences, and have taken at least one course in neurobiology. These students are in the top 15th percentile on the graduate records examination, and nearly all have done research during their undergraduate education. All students must meet the general training requirements of the Field, which provides them with a broad training in both neurobiology and behavior. In addition, trainees in our program take a series of laboratory rotations, advanced lecture and laboratory courses to provide strength in cellular and molecular neurobiology. These are combined with journal clubs and weekly seminars which discuss recent advances in our field. Our students benefit from interactions with other academic programs at Cornell by completing the requirements for a Minor, usually in biochemistry, molecular biology, genetics, pharmacology, chemistry, applied physics, biophysics or psychology, which offer courses and seminars of interest. Relevance: Our students will be trained to use cellular and molecular approaches to study systems-level questions about how behavior is generated by neuronal interactions in discrete neural networks. With the rapid progress in genomics, we will soon know the molecular "parts list" that makes a brain;the research supported by this grant will study how these parts work together to generate perception, locomotion and other behaviors. It should also provide insights into the causes and possible treatments for psychiatric and other behavioral diseases in humans.

[unreadable] DESCRIPTION (provided by applicant): Funds are requested to help support the Eighth International Congress of Neuroethology, to be held July 23-27, 2007 in Vancouver, B.C., Canada. The International Congress of Neuroethology (ICN) serves as the scientific meeting of the International Society for Neuroethology (ISN), and has been held every three years since 1986. The focus of these meetings is to bring together diverse neuroscientists who are investigating the neural basis of behavior across a broad spectrum of taxa (both vertebrate and invertebrate). This meeting provides an outstanding venue for discussions among international scientists who have a broad range of perspectives, but who focus on common basic-science questions that have important implications for neural function and disease. A series of plenary lectures, symposia and poster sessions focus on topics including sensory and motor processing, central integrative processes, development, synaptic plasticity and learning, regeneration, and systems-level approaches to understanding plasticity and behavior. Comparative approaches and emerging technologies are emphasized. The program for the meeting represents both different levels of analysis (from biophysics to behavior), and also different techniques and approaches (neurophysiology, molecular biology, genomics, imaging). Support is requested to fund travel and registration for winners of the Young Investigator Awards, for student and minority neuroscientists, and for dissemination of the proceedings of the conference. The benefits to American scientists in general, and to young investigators in particular, will be the opportunity for exposure to cutting- edge research and techniques from around the world in a format and venue that encourage interactions between students and investigators at all levels of experience. This conference will address major themes in the neural mechanisms that underlie normal behavior, with an emphasis on understanding the pathways in the brain that perceive sensory inputs, make major decisions and encode the appropriate movements. Many neural diseases result from defects in these processing steps; to understand the functional bases of abnormal neural function and disease states, multiple perspectives drawn from a variety of organisms are often critical. The comparative viewpoints and emerging technologies that are emphasized at the ICN therefore have the potential to contribute to the diagnosis of nervous system diseases and the development of treatments. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In the neonatal rodent spinal cord, the combination of serotonin and NMDA is adequate to activate the central pattern generator (CPG) networks for locomotion, but the cellular and biophysical mechanisms by which these transmitters organize and activate the CPG are poorly understood. We propose that these neuromodulators reconfigure the intrinsic properties of the CPG neurons and the strengths of network synapses to organize the network into a functional "idling" state, so that descending glutamatergic or sensory input can rapidly initiate locomotion. The neonatal mouse spinal cord is an excellent preparation to test these hypotheses: several neuronal candidates for the CPG have been identified using transgenic and anatomical methods to label specific interneuron types. Two identified classes of interneurons are thought to play important roles in the organization of the mouse spinal locomotor CPG: commissural interneurons (CINs) which coordinate left-right movements, and the Hb9 interneurons which may participate in the rhythm-generating component of the CPG. Following a research approach we have pursued for many years in the crustacean stomatogastric ganglion, we propose to study how the intrinsic cellular properties of these neurons shape their activity patterns during fictive locomotion, and how serotonin and NMDA affect those intrinsic properties. Our first aim is at the cellular level: using a combination of whole cell recording and calcium imaging, we will study the intrinsic firing properties of synaptically isolated interneurons, their modulation by serotonin, and its interaction with NMDA. The goal of this aim is to better understand how modulators can alter the neurons'activity to activate the motor pattern. Second, at the biophysical level, we will use voltage clamp methods to identify the ionic currents affected by serotonin and NMDA in CINs and Hb9 interneurons, to understand the biophysical basis for modulatory changes in the neurons'intrinsic firing properties. Third, we will begin to explore the plasticity of the intrinsic properties of these spinal interneurons by studying how they change during postnatal development, during the time the animal learns to walk, and following spinal cord injury. These projects will elucidate some of the cellular and molecular mechanisms that neuromodulators use to shape the locomotor CPG. Spinal cord injury causes loss not only of the rapid activating signals for locomotion, but also of the slower modulatory inputs that enable the network to function at all. To learn how to restore movement after spinal cord injury, we must understand both the modulatory mechanisms that enable the network to function and the rapid activating mechanisms in the locomotor CPG. PUBLIC HEALTH RELEVANCE We hypothesize that serotonin and other modulators modify the firing properties of locomotor network neurons and their synapses to enable the spinal network to produce the commands for locomotion. When these inputs are lost following spinal cord lesions, the network becomes non-functional. An eventual goal of our work is to provide a rational basis for post-injury neuromodulator therapy, to help maintain the locomotor networks in a functional state until regrowth of axons can be accomplished across the lesion.

This award to Cornell University is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). Cornell University will establish am Undergraduate Research and Mentoring (URM) program to engage under-represented minority students in intensive mentoring and research experience and prepare them for graduate studies in the biological sciences. The Cornell Biology Research Fellowships program will serve 35-50 Cornell undergraduates (7-10 per cohort) during the five-year period of the grant. NSF funds will support seven students in each cohort, or a total of 35 students. Through participation in research seminars and by shadowing faculty, freshmen and sophomores will have the opportunity to explore different fields of biology before applying to the program. The Biology Research Fellowships will offer a carefully designed set of activities that includes two years of funded research internships in faculty laboratories, a seminar series focusing on reading the scientific literature, writing research proposals, preparing and delivering scientific presentations, GRE preparation, and choosing and applying to graduate programs. Students are also involved in community outreach and peer-mentoring. Faculty mentors, all of whom have strong records of successfully training undergraduates in their laboratories, include at least twenty scientists whose research fields span the range of biology. The students will participate in all aspects of life in an academic laboratory: learning the approaches and techniques of their field, analyzing experimental results and developing new questions, and preparing the results for publications. All students will write an honors thesis, and faculty mentors will encourage and facilitate co-authorship on publications arising from research in which the students have been involved. The Biology Research Fellowships will produce young scientists who can serve as role models or mentors to groups traditionally underrepresented in the sciences. During the program, students will begin to experience and contribute in those roles by working with underrepresented minority students (pre-freshmen, freshmen, sophomores) as well as through outreach to high school students and teachers. More information is available by contacting the URM Program Director, Dr Steven Kresovich (sk20@cornell.edu), or Dr. Myra Shulman (mjs59@cornell.edu), or by visiting the Cornell Office of Undergraduate Biology website at: http://www.biology.cornell.edu/brf.

DESCRIPTION (provided by applicant): This project combines electrophysiological and modeling approaches to study the organization and neuronal composition of the Central Pattern Generator (CPG) neural circuits in the mammalian spinal cord that coordinate rhythmic neural activity driving locomotion. This study will take a general approach that utilizes various spontaneous and evoked perturbations in the locomotor pattern (including deletions of motoneuron activity, spinal cord lesions and pharmacological manipulations) as probes to understand CPG organization and function. It is proposed that analysis of the influences of these perturbations on the rhythmic motor pattern and the activity of identified spinal interneurons will provide important insights on the spinal CPG organization and operation. These data will be used to develop a comprehensive computational model of the spinal locomotor CPG, and to refine and validate this model, so that it can reproduce both normal locomotor activity and the consequences of experimental perturbations. In turn, the model will serve as a computational framework to formulate predictions to guide subsequent experimental investigations. The project brings together two senior scientists with complementary and overlapping expertise in experimental (Dr. Harris-Warrick at Cornell University) and computational (Dr. Rybak at Drexel University) neuroscience. It has three interlocking objectives: 1) Explore alterations in behavior of, and synaptic drive to, motoneurons and genetically defined interneurons during spontaneous and evoked deletions in flexor and/or extensor rhythmic motor activity, to define the possible function of these interneurons in the locomotor CPG. 2) Explore the consequences of reducing CPG complexity by spinal cord hemisection and removal of spinal segments, and compare the behavior of identified interneurons in the reduced cord during deletions and after pharmacological blockade of synaptic inhibition. 3) Develop a comprehensive computational model of the neural circuits forming the locomotor CPG in the neonatal mouse spinal cord that includes genetically identified interneurons and suggests their roles in the generation of the locomotor pattern. Validate this model in simulations reproducing the specific transformations in motoneuron and interneuron activity and the entire locomotor pattern during experimental perturbations proposed in objectives 1 and 2.The model will be progressively developed by continuous interaction with the experimental studies, and will serve both as a testbed for working concepts on spinal cord organization and as a source of predictions for subsequent experimental validation. Intellectual Merit: This proposal represents an important step toward a mechanistic understanding of the organization of the neural circuits forming the spinal locomotor CPG in mammals. The proposed experimental and modeling studies will also provide novel insights into the general principles of neural control of rhythmic motor behaviors. Broader Impacts: (1) Integration of research and education: At Cornell and Drexel, this work will be included in several courses on basic neuroscience and neuroengineering for students at many levels, from undergraduate to graduate and medical students. Undergraduate and graduate students will participate in this project at both institutions. At Cornell, Dr. Harris-Warrick will invite an underrepresented minority student to work on this project each year, and the project will support two female scientists who will be given careful mentoring for a future career in science. (2) Enhance infrastructure for research and education: Two laboratories with mostly nonoverlapping technical expertise will collaborate to understand the neural circuitry for locomotion. This collaboration will help both laboratories to combine computational and electrophysiological approaches to the study of neural circuits. The simulation package NSM 3.0, developed at Drexel, and all models developed in this project will be shared between project participants and made available to other research groups via a specially developed website at Drexel. (3) Medical Impact: It is now clear that all vertebrates, including humans, have spinal CPGs that drive and coordinate locomotor movements. These CPGs survive upper spinal cord injuries, and are in principle capable of restoring locomotion after injury, as demonstrated in rodents and cats. Better understanding of the organization and function of such CPGs will provide essential insights into future clinical strategies for restoration of locomotor function after spinal cord injury.