Francisco J. Alvarez, PhD - US grants

A fundamental property of animal cells is their ability to maintain a constant volume throughout life. In neurons, the possibility of changes in ionic composition and hence in cell volume is present at all times. This is a consequence of their small volume/surface ratio in conjunction with their synaptic and all-or-none electrical activity. Under physiologic conditions neurons are able to maintain and restore the ionic gradients which determine their resting membrane potential and osmotic balance. In spite of their importance, little is known about the mechanisms underlying cell volume regulation (CVR) and cell volume maintenance (CVM) in neurons. Furthermore, nerve cell swelling (cytotoxic edema), a dreaded complication of ischemia or trauma, has been postulated to result from loss of control of cell volume. Clearly further advances in pathophysiology of brain edema will require elucidation of the membrane mechanisms underlying CVR and CVM as well as the factors eliciting nerve cell swelling. This is a proposal to study the basic membrane mechanisms with which neurons are .equipped to regulate and maintain their volume in isosmotic and anisosmotic media. We aim to ascertain how nerve cells respond to hyposmotic and hyperosmotic challenges , whether they are endowed with mechanisms for regulatory volume adjustments, and what is the nature of these mechanisms. In isosmotic media we want to know: a) the role played by the Na+/K4+ pump in CVM, and the mechanism involved in volume regulation when the pump is inhibited; b) the role of anion transport in CVM; and c) whether cell volume can be altered by firing activity and how nerve cells restore their volume under these conditions. These studies will be conducted on identified land snail neurons and frog dorsal root ganglion cells. These cell types are chosen because: i) they are relatively large; ii) their cell bodies are nearly spherical; iii) many of their membrane carriers and channels have been characterized; iv) their intracellular ionic activities are known; v) they can be easily isolated and maintained in vitro for optical measurements; vi) they are examples of an invertebrate and a vertebrate neuron. Cell volume changes and membrane voltage will be measured with microelectrodes, using intracellular TMA+ as a volume marker. Intracellular ion -activities will be measured with ion-selective microelectrodes.

The aim of this project is to gain a better understanding of the mechanisms involved in the transmission of sensory information from primary afferent fibers to spinal cord neurons in the mammalian spinal cord. Primary afferent synaptic terminals are believed to use excitatory aminoacids (EAAs) as their principal neurotransmitters, and some of them contain in addition neuropeptides that function as more 'atypical' neuromodulators. Synaptic transmission from different primary afferents may have different consequences, e.g. a nociceptive terminal may induce long-lasting changes in the postsynaptic responses that differ from synaptic transmission from large mechanoreceptive fibers. Moreover, the same primary afferent may evoke different postsynaptic responses on different spinal cord neurons, suggesting the involvement of distinct synaptic mechanisms within a single axonal arborization. Our hypothesis is that these differences are partly related to the postsynaptic complement of EAA-receptors. The present proposal aims to use a multidisciplinary approach to identify the EAA receptors associated with primary afferent terminals from different classes of primary afferents and located in different laminae of the spinal cord. We will also test the presence or absence of neuropeptides within their synaptic terminals. Different classes of primary afferents will be electrophysiologically characterized by either axonal recordings in the spinal cord (for large diameter primary afferents, e.g. cutaneous mechanoreceptors, muscle spindle afferents) or by cell body recordings in the dorsal root ganglia (for small diameter afferents, including pain sensory afferents). Recorded elements will be intracellularly marked with electron-dense and/or fluorescent markers to we can then immunolocalize EAA receptors postsynaptic to the intracellularly labeled terminals using immunoelectron microscopic techniques, and/or assess neuropeptide presence using double fluorescent methods. In addition to providing powerful insights into basic mechanisms of sensory physiology ranging from mechanoreception to pain mechanisms, the data will provide valuable knowledge towards full understanding of sensory disorders like hyperalgesia, and other chronic pain pathologies, whose mechanisms are believed to reside partially in alterations of sensory transmission at the first few spinal cord synapses established between primary afferent fibers and spinal cord neurons. In more general terms, our results will also shed some light on the organization at individual synapses of the bewildering diversity of EAA receptors described in molecular biological studies.

DESCRIPTION (provided by applicant): Spinal networks encode the motor outputs that generate all behaviors, therefore it is not surprising that spinal interneurons (INs) are highly complex and that our knowledge is rather incomplete about how many classes of spinal INs exist, their properties, connections and mechanisms of differentiation. This information is essential not only to understand motor function, but also how genetic alterations, disease or injury affects these circuits in adults and newborns. A few years ago a new conceptual framework to understand spinal INs was prompted by the discovery of a few canonical embryonic classes that diversify into the large variety of adult phenotypes. With previous funding we established that temporal control of neurogenesis and transcription factor (TF) expression correlates with specific IN phenotypes within a class known as V1. V1-INs include those that mediate recurrent inhibition of motoneurons (Renshaw cells) and many that mediate reciprocal inhibition of motoneurons with antagonist actions on a single joint (Ia inhibitory interneurons, IaINs). Thus, we divided V1s in an early generated group (that includes Renshaw cells and expression of MafB) and a late generated group (that includes IaINs and expression of Foxp2). However, V1 diversity is much larger and there is not yet a complete scheme of V1 IN variety and function, in part because lack of information about their output in terms of axon projections, connections and firing. Here we hypothesize that V1s of different birthdates express different combinations of TFs with specific roles in defining axonal projections and firing properties. In aim 1 we will analyze V1 axon projections and relate them to TF expression and time of birth. Aim 2 will analyze whether Foxp2 controls axon length and therefore the extent of rostro-caudal projections of different V1's. Aim 3 will analyze the firing properties of V1 groups, their relation to specific voltage-gated channels and whether these are determined by specific TFs. Validation of our hypotheses would suggest that birth-date and TF expression differentiate V1 groups with distinct connections and firing modulation. The results will also provide a more complete picture of V1s that will contribute to improve our understanding of spinal local motor circuits.

community; university

[unreadable] DESCRIPTION (provided by applicant): This proposal requests funds to purchase an Olympus Fluoview (FV) 1000 Spectral Unit Laser Scanning Confocal Microscope to be utilized by an interactive group of NIH-funded investigators, primarily located in the Department of Anatomy and Physiology at Wright State University. The unit will be also available for shared use with other investigators distributed throughout several other Departments of the Medical School and the College of Science and Mathematics. This new confocal system will serve two major purposes, 1) replacement of our six year-old Olympus FX and 2) upgrade our confocal instrumentation capabilities. Major advances provided by the new system include a) multiple fluorochrome labeling beyond our current two channel limitations; b) optimal controls of laser illumination; c) improved fluorochromes separation, even when emission curves are closely related and d) improved file handling, save and export for further quantitative analyses in 2D and 3D space. The instrument will be mostly used by a core group of five investigators that will form a collaborative unit with shared projects and common scientific interests on the modification of synapses and circuits during development, injury and recovery from injury. They have also accumulated a broad range of expertise with imaging equipment and quantitative methods, many of which require the improved capabilities of the Olympus FV 1000 system. The research topics from further users are varied and range from volume or pH control in neurons to cellular calcium transport mechanisms and chloride homeostasis in red blood cells and neurons to central nervous system control of respiratory function. The majority of investigators (major or minor users) are funded through NIH. To promote maintenance and equitable use, the instrument will be integrated within the microscopy and imaging facilities of the Center for Brain Research. This core facility was established in 2000 with the mission of providing imaging and histological needs (electron microscopy, image analysis, neuron tracing, confocal microscopy, microtomy and ultramicrotomy) to researchers located primarily (but not exclusively) in the School of Medicine and with an emphasis in Neuroscience research. As such, the acquisition of the Olympus FV 1000 systems will significantly enhance the research programs of the core investigators, but it will also have a beneficial impact on the broader research community at Wright State University. [unreadable] [unreadable] [unreadable]

Tissue Processing and Imaging Core B The objective of the Tissue Processing and Imaging Core is to provide resources that will meet the needs of all investigators for confocal imaging, electron microscopy and immunohistochemical processing. The goals of the projects make it vital that critical tissue processing and analysis is performed in a consistent and uniform way, preferably in a single location. The location of this core is in the middles of a corridor surrounded by the labs of all faculty participants and therefore fully accessible to all projects. It will take advantage of the accumulated expertise of the core's director (Dr.Alvarez) in structural techniques and in the development of quantitative approaches and the electron microscopy technical help and expertise(Mr. Zerda). The core will provide technical help for the processing of the more sophisticated and demanding experiments with electron microscopy immunohistochemistry. In summary, the core will not only provide the instrumentation and human resources but also the expertise necessary for the successful completion of the most complex immunocytochemical and structural analyses proposed in all three projects

PROJECT 2 After peripheral nerve injuries motor and sensory axons can regenerate and reestablish connectivity with muscle. However, despite recovery of muscle strength restored motor function is frequently abnormal. One possibility is that alterations in motor circuits at level of the spinal cord prevent full functional recovery. In project 2 we will test the hypothesis that postsynaptic inhibitory inputs are reorganized over motoneurons axotomized after a nerve injury and this creates imbalances in inhibition that could generate abnormal motor output. Our preliminary data show that inhibitory and excitatory synapses are differentially remodeled over axotomized motoneurons. Imbalances might occur if synaptic remodeling results in different proportions of excitatory and inhibitory inputs or because newly formed inhibitory synapses display altered properties or if inhibitory inputs from different sources are remodeled differently. It is unknown if possible imbalances are more or less permanent or how they differ between motoneurons that regenerate and reinnervate muscle with those that do not regenerate. We will investigate each of these possibilities in three specific aims. Aim1 will test the hypothesis that imbalances in inhibitory/excitatory ratios are created because different properties of inhibitory and excitatory synapse remodeling on axotomized motoneurons. Aim2 will test the hypothesis that inhibitory synapses with altered properties arise because failures in recapitulating mechanisms of inhibitory synapse maturation. Aim3 will test the hypothesis that alterations might differentially affect inhibitory synapses from different interneurons and circuits. We will use the same nerve injury model used in Projects 1 & 5, resection of the tibialis nerve supplying triceps surae muscles, with or without resuture to prevent or allow regeneration. We will use rats as animal model in aims 1 and 2. In aim 3 we will use transgenic mice that genetically encode EGFP inside the axons of different spinal inhibitory interneurons and permits isolation of specific circuits for analysis. At the completion of these aims we will have tested the possibility that inhibitory inputs become altered over axotomized motoneurons before, during and after peripheral nerve regeneration. This information will suggest whether or not remodeling of inhibitory inputs is significant and could contribute to permanent alterations in motor function. It is expected this work will provide valuable information to understand mechanisms that hamper functional recovery after nerve injuries.

DESCRIPTION (provided by applicant): The goal of this proposal is to purchase/acquire and implement a Two-photon microscope which will primarily be utilized to strengthen the neuroscience research at Wright State, and permit key investigators to perform studies they would otherwise be unable to accomplish. We have assembled a team of highly accomplished researchers who will constitute the major users of the requested instrument and all of which are PIs on NIH funded grants (R01s). Dr. Francisco Alvarez will use the requested instrument to study Generation and functional role of Renshaw cells in the developing spinal cord. This study has broad implications on motor development and reconfiguration and recovery of function of neurons in adults after spinal cord injuries that disrupt these intraspinal circuits. Dr. Francisco Alvarez-Leefman will utilize the microscope to understand the cellular and molecular mechanisms underlying cell volume control in neurons and glial cells. Dr. Robert Fyffe will utilize the microscope to investigate the dynamics of surface membrane ion channel localization following altered activity or axonal injury, a project that also has implications in the field of spinal cord/nerve injury and recovery. Dr. Robert Putnam will use the requested instrument to study the role of Intracellular Ca and pH in chemosensitive signaling in dendrites of brainstem neurons. A greater understanding of central chemosensitive signaling will open new targets for treatments aimed at altering respiratory drive in such disorders as central hypoventilation syndrome, SIDS, and sleep apnea. The instrument will be housed and fully supported in a core facility in the recently created Comprehensive Neuroscience Center, where, in addition to these four major users, there will be opportunity for the instrument to be used by additional staff/Minor users/ Junior Investigators for cellular and molecular studies. Based on our current expertise as well as our collaboration with Dr Michael O'Donovan at NIH (who has invited Dr. Alvarez for a month to use a two-photon microscope at NIH) we believe that we will master the use of the Two-photon microscope and will be able to make the most of its potential. A Two-photon microscope will enhance the Imaging Core Facility at Wright State which currently houses two confocal microscopes and a transmission electron microscope, all networked to off-line image analysis workstations. The technical expertise to be gained will be utilized to further advance other research endeavors at WSU. PUBLIC HEALTH RELEVANCE: A Two-Photon Microscope will enhance the Neuroscience core at Wright State University and promote state- of-the art scientific research. The research proposed here will make use of this instrument to its full capacity, leading to an understanding of both neurophysiologic and neuropathologic mechanisms of high relevance to nervous system function in health and disease or injury conditions.

PROJECT SUMMARY (See Instructions): After peripheral nerve injuries axons can regenerate and reestablish connectivity in the periphery; however restored motor function is not normal. Previously we have shown that some deficits, like lack of monosynaptic reflexes, can be explained by the permanent retraction of la proprioceptive synapses from motoneurons. We now propose that circuit reorganizations are relatively global and affect also spinal intermeuronal circuits that exert control over not only injured motoneurons, but also other motor pools controlling the same limb. As a result, a novel limb control pattern emerges that allows some function, but is also clearly pathological. In the proposed work we will seek confirmation for structural changes in spinal interneuronal circuits. The work will parallel functional studies proposed in project 1. We will analyze in detail the synaptic organization of recurrent and reciprocal inhibition, two key inhibitory circuits that modulate and pattern motoneuron firing and therefore muscle contractions. Recurrent inhibition exerts feedback control of motor output through an interposed interneuron named the Renshaw cell that receives direct excitation from intraspinal collaterals of motor axons. Reciprocal inhibition is mediated by la inhibitory interneurons which receive common inputs with certain motor pools, including those from la afferents, and inhibit motoneurons with antagonist action allowing for example smooth flexion-extension alternation during movement. We hypothesize that both interneurons become denervated from respectively, motor axons and la afferents after nerve injury. We propose that these alterations cause major changes in spinal circuitry. In aim 1 we will test the hypothesis that denervation of Renshaw cells coupled to injured motor axons causes synaptic reorganizations of recurrent inhibition in the whole spinal segment. In aim 2 we will test the hypothesis that differential la de-afferentation of inhibitory and excitatory interneurons in reciprocal pathways causes a shift in balance favoring excitation. These could explain the excessive co-contraction of antagonists observed after nerve injuries. Detail analyses of connectivity will be performed with a combination of techniques, including novel retrograde transynaptic viral tracing that allows revealing microcircuit connectivity.

Project Summary / Abstract Every year nearly one million Americans undergo surgery for nerve reattachment after nerve injuries, but despite continued improvements in microsurgical techniques, a majority of these patients are left with permanent motor deficits. Usually these are believed to result from poor regeneration of the peripheral nerve. However, deficits are still present when experimental nerve injuries are designed in animal models for rapid, specific and efficient nerve regeneration and muscle re-innervation. In the past we reported that structural remodeling of spinal cord circuitry after nerve lesions is in part responsible. After injury, the central synaptic branches of sensory proprioceptive axons and motor axons injured in the periphery are removed from the ventral horn of the spinal cord resulting in dysfunction of several critical motor control circuits. The mechanisms of synapse and axon removal are therefore clearly important, but unknown. Our preliminary data implicate the neuroinflammatory response that occurs inside the intact spinal cord around cell bodies of peripherally-injured motoneurons and along the central projections of peripherally-injured sensory axons. Microglia is activated in these regions and although their capacity for synapse phagocytosis has been frequently proposed, their exact function after nerve injury remains controversial. In addition, monocytes infiltrate the spinal cord and transform into macrophages. These cells were missed in previous studies because they become indistinguishable from microglia. Thus, their interactions with resident microglia and possible roles in synaptic plasticity are unknown. More recently we found that after nerve injuries triggering large synaptic circuit remodeling there is additional infiltration by T-cells. The roles T-cells play in synaptic remodeling are completely unknown. To investigate the relation between microglia and different immune cell infiltrates in relation to synapse plasticity we will use mice in which microglial cells are labeled with GFP and infiltrating immune cells by RFP and perform a number of genetic manipulations to interfere with the function of one or other cell and test possible signaling mechanisms leading to microglia activation, immune cell infiltration and synapse removal. In Aim 1 we will investigate the role of peripherally derived monocytes, macrophages and T-cells in the synaptic removal of inputs from muscle sensory afferents. In Aim 2 we will investigate whether microglia activation is necessary for recruitment of blood-derived immune cells and investigate the signals promoting these invasion. In Aim 3 we will use two- photon time-lapse imaging to directly observe and analyze the process of removal of sensory synapses and the mechanisms that facilitate specific recognition of axons injured in the peripheral nerve. Finally, in Aim 4 we will block the central neuroinflammatory response to preserve proprioceptive synaptic inputs and test patterns of muscle activation during treadmill locomotion after nerve regeneration with electromyography. The results will inform about the role of neuroinflammation and the cellular mechanisms involved in removing specific inputs from the spinal cord and will provide first insights into motor outcomes after interfering with this process.

Project Summary / Abstract Motor axon output is controlled by Renshaw cells forming an inhibitory feedback circuit with spinal motoneurons that was first described by Birdsey Renshaw in 1941. Since then a large number of investigators have defined Renshaw cells connectivity, electrophysiological properties, actions on spinal motoneurons and interneurons and more recently their development and molecular genetics. However, up to date there is no consensus about how this recurrent inhibitory feedback circuit alters motor output, ongoing movement and motor actions. This gap in knowledge is important especially considering that the recurrent inhibitory circuit has been shown to fail in patients with high levels of spasticity due to stroke, spinal cord injury, amyotrophic lateral sclerosis (ALS) or hereditary spastic paresis. The P.I. also recently discovered that this circuit is disconnected in animal models of ALS and participated in a study that involucrate Renshaw cell dysfunction in Spinal Muscular Atrophy. It is clearly necessary to reexamine the exact role this circuit plays on the output of motor commands from the spinal cord. In this proposal we leverage new knowledge on the molecular biology and genetics of the Renshaw cell to develop animal models for specific silencing these cells in adult. We propose to use commercially available mouse models in a genetic intersectional approach based on co-expression of the parvalbumin (Pvalb) and calbindin (Calb1) genes and taking advantage of mice in which we can direct expression of reporter proteins, GiDREADDs or tetanus toxin under a dual conditional strategy based on co- expression of both cre and flp, each recombinase respectively dependent on Calb1 and Pvalb. We will also test new scAVV9 vectors for directing expression of dual conditional transgenes specifically in spinal cord neurons. The aims of this exploratory R21 proposal is to develop and validate these models in Aims 1 and 2 and then use them to test the role of Renshaw cells mediated recurrent inhibition in desynchronization of motor output to diminish physiological tremor during muscle contractions (Aim 3). Validation of this animal model will be of great use to test in the future classical and new theories on Renshaw cells function and also their involvement in different diseases of the spinal cord motor system.

Motor and sensory nerves regenerate in the periphery after being axotomized following nerve injuries. This capacity allows some functional recovery, however this is frequently suboptimal being the relative inefficiency of axon regeneration a major problem, particularly for injuries at some distance from muscle. Axotomy induces genetic changes in motoneurons that promote axon growth, yet this is rather slow taking months or years for axons to reach their targets after limb injuries. This compounds with the fact that the regenerative capacity of motoneurons is limited to a short temporal window and that chronic denervation results in muscle atrophy, as well as changes in central circuits, all impairing recovery. Therefore there is renewed interest on mechanisms to promote axon regeneration. One mechanism recently highlighted and intensely studied by one of the P.I.s (Dr. A.W. English) is the effect of activity and exercise in promoting axon regeneration. However, this approach is limited since patients are frequently either with the affected limbs immobilized or in bed rest preventing implementation of adequate exercise programs. We now seek proof for a mechanistic explanation that could be recruited also with passive rehabilitation and/or pharmacology. After axotomy motoneurons increase their excitability and shed excitatory synapses while maintaining inhibitory synapses. In addition, the potassium chloride co-transporter isoform 2 (KCC2) is downregulated changing the nature of inhibitory synapses from hyperpolarizing to depolarizing. The other P.I. in this proposal (Dr. F.J. Alvarez) is an expert in spinal inhibitory interneurons and synapses. Together, both P.I.s hypothesized that after axotomy inhibitory synapses are the main drivers of motoneuron activity and could stimulate axon regeneration. There is a strong scientific premise for this hypothesis: GABA actions promote axon elongation during early development and manipulations that enhanced preservation of inhibitory synapses on axotomized motoneurons correlated with faster functional recovery. To directly test whether inhibitory synaptic activity on axotomized motoneurons promotes axon regeneration we propose in Aim 1 to block inhibitory synapses on axotomized motoneurons using tetanus neurotoxin A and study the effects on motor axon regeneration and muscle reinnervation. In Aim 2 we will use mouse models to genetically define the interneurons targeting the cell body of regenerating motoneurons and that could provide a depolarizing synaptic drive. We will then use genetically-encoded activity modifiers to examine whether their activity influences motor axon regeneration. Resolution of these aims will reveal for the first time whether inhibitory synapse activity is a driving force for axon regeneration in the adult and the spinal neurons that might be responsible. This is of high significance from a translational point of view since it will point to new approaches to enhance ?inhibitory? drive by either pharmacological means or by recruiting key interneurons through various manipulations, like stretching or stimulating the antagonist muscles and nerves.

Project Summary / Abstract Nerve injury patients face life-long sensorimotor deficits despite continued improvements in microsurgical techniques and nerve regeneration. These are usually believed to result from poor or unspecific regeneration of the peripheral nerve. However, deficits are still present when experimental nerve injuries are designed in animal models for rapid, specific and efficient nerve regeneration and muscle re-innervation. We have proposed that structural remodeling of spinal cord circuitry after nerve lesions is in part responsible. Thus, future advances in nerve regeneration will predictably be limited by deficits caused by this much less studied central synaptic plasticity. Remarkably, the central synaptic branches of Ia afferent proprioceptive axons injured in the periphery are removed from the spinal cord ventral horn after nerve injury resulting in dysfunction of critical motor control circuits. We recently found that this synaptic plasticity is graded to the type of nerve injury and correlated with the more or less target specificity obtained during muscle reinnervation. Our preliminary data suggest that neuroinflammation occurring inside the otherwise intact spinal cord ventral horn, is critical for grading circuit remodeling to the severity of the nerve injury. Ventral horn microglia are activated after nerve injuries and although their capacity for synapse phagocytosis has been frequently proposed, their function inside the spinal cord after a remote nerve injury continues to be debated. Moreover, we found that microglia activation is followed by infiltration of cells from the adaptive and innate peripheral immune system, but this is variable depending on injury type. When occurs, it correlates with maximal Ia synapse and axon removal from the ventral horn. These cells, particularly monocyte/macrophages were missed in previous studies because they share many markers with activated microglia, preventing their identification. Thus, their function inside the spinal cord ventral horn after nerve injury is unexplored. We will use genetic approaches to distinguish microglia from blood-derived immune cells and investigate their significance for Ia afferent removal. In Aim 1 we will genetically label and manipulate each cell type to test their roles in Ia axon and synapse deletions and probe cellular signaling mechanisms. In Aim 2 we will visualize with time-lapse two-photon microscopy genetically labeled sensory afferents and microglia or monocyte-derived cells to directly observe and analyze their interactions. Finally, in Aim 3 we will test the relevance of this mechanism for motor function, whether is maladaptive, causing long-lasting motor deficits or adaptive, to preserve the best function possible when peripheral connectivity becomes highly scrambled after regeneration. The new knowledge generated will allow us to consider new methods for optimization of central circuitry function through modulation of central neuroinflammation. This will be critical for developing strategies to improve sensorimotor function recovery in conjunction with methods to improve the speed, efficiency and specify of axon regeneration in the periphery.

PROJECT SUMMARY / ABSTRACT Defining the spinal cord circuitry that controls muscles is key to our understanding of movement and movement-related disorders. However, elucidation of this spinal circuitry has been problematic due to the lack of track tracing methods capable of labeling muscle-specific motoneurons and synaptically-connected interneurons in spinal microcircuits. Recently, modified rabies virus (RV) has become the vector of choice for tracing neural circuits. The attenuated RV was modified by deletion of the glycoprotein (G) gene necessary for virus propagation and neuronal uptake and by the addition of a fluorescent protein gene in its place. When combined with approaches that supply G to the initially infected neurons (starter neurons) through trans- complementation, the RV can then move one synaptic step and repeat the replication/labelling process in synaptically connected neurons. Since the RV always lacks the G-gene and cannot acquire it, virus spread stops at this point in the circuit revealing just monosynaptic connections. We inoculate specific hind limb muscles with RV, retrogradely label the muscle-specific motor neurons and then transynaptically label monosynaptically connected interneurons. This approach holds the promise of revealing premotor networks that modulate function of specific motor pools and even single motoneurons. This information is essential to understand how these connections change after nerve or spinal cord injury, neurodegenerative diseases, or aging. However, experiments in our lab and others soon demonstrated several limitations of the technique when applied to the tracing of muscle-specific premotor interneuronal networks. The virus was, in fact, lethal to motoneurons and spinal interneurons reducing the temporal window for labeling. Second, it is not taken up by adult motor axons. Third, even when we were able to infect large numbers of motoneurons in mature animals the transynaptic transfer inside the spinal cord did not occur. We hypothesized that the virus lethality, a G protein with low neurotropism and the robust microglia reaction around infected motoneurons all contribute to the lack of consistency in neonates and its complete failure in mature animals. In this proposal we aim to test significant modifications to increase the reliability and replicability of the method for revealing premotor spinal networks in neonates and adults. We will specifically test the feasibility of a new strain of virus with a cre- dependent self-inactivation cassette that limits the time of viral amplification in infected neurons and that we show extends motoneuron viability for at least a month. We will combine this with an optimized G-protein that increases neurotoropism and virus packaging and we will interfere with the microglia reaction to overcome the limited transynaptic spread in mature animals. We believe these improvements could be transformative in the field by making the technique robust enough to be used by many labs, augmenting the scope towards analyses of different diseases and allowing validation in many different labs and experimental conditions.