Gordon S. Mitchell - US grants

The overall objective of this project is to obtain a better understanding of the basic mechanisms subserving the control of ventilation and regulation of airway smooth muscle tone. The proposed experiments constitute an effort to control each of three sensory receptor systems independently (pulmonary stretch receptors and - and chemoreceptors) so that their interactions in modulationg phrenic nerve activity and tracheal smooth muscle tone may be investigated. We will determine the relationships of phrenic nerve activity and tracheal caliber to lung stretch at various constant levels of and chemical drive in anesthetized dogs. The role of the carotid bodies and the influence of various anesthetic agents on these relationships will be investigated. Finally, we will determine the effects of reduced serotonin metabolism via tryptophan hydroxylase inhibition on lung stretch reflexes and - and chemoreflexes.

The overall objective of this project is to obtain a better understanding of the basic mechanisms subserving the control of ventilation and regulation of airway smooth muscle tone. The first project constitutes and effort to control each of three sensory receptor systems independently (pulmonary stretch receptors and 02-and CO2-chemoreceptors) so that their interactions in modulating respiratory activity may be investigated. I will determine the relationships of phrenic and para-recurrent laryngeal nerve activities and tracheal caliber to lung stretch at various constant levels of 02- and CO2- chemical drive in anesthetized dogs. The role of carotid bodies and the influence of anesthesia on these relationships will be investigated. In a second project, interactions between exercise and other factors which affect the control of ventilation will be investigated. In awake goats, the ventilatory response to exercise will be assessed during various acute and chronic treatments which alter resting ventilation and blood gases. The role of chemoreceptors in these responses will be determined. Specific attention will be devoted to the mechanism of hypoxia-exercise interactions in ventilatory control using an experimental preparation which allows perfusion of the carotid body chemoreceptors to be isolated from the rest of the body in an awake exercising animal.

The long range objective of this project is to understand fundamental mechanisms of ventilatory control, particularly mechanisms underlying the ventilatory response to physical activity. In this project period, we will continue to investigate neural mechanisms causing short and long term modulation of the ventilatory response to mild or moderate exercise. Short term modulation causes immediate (within trial) changes in the exercise ventilatory response whereas long term modulation changes systems responses over a time span of many trials. We also propose to begin investigations concerning the significance of short and long term modulation in compensating for impaired lung function during disease. Experiments will be conducted using awake goats trained to run on a treadmill as an experimental model. The specific aims are: 1) to test the hypothesis that short term modulation (STM) with increased respiratory dead space requires changes in spinal respiratory neuron excitability via descending serotonergic mechanisms. Goats with chronic subarachnoid catheters in the thoracic spinal cord will be used to determine the roles of 5-HT1 and 5-HT2 serotonin receptor subtypes, and to determine if STM can be pharmacologically enhanced via spinal serotonin reuptake inhibition. 2) to test the hypothesis that repeated CO/2-chemoreceptor stimulation during exercise augments ventilatory responses during future exercise trials (i.e., long term modulation, LTM) via serotonergic mechanisms. We propose to determine if pretreatment with a serotonergic neurotoxin (5,7-DHT) blocks LTM and if serotonin reuptake inhibition augments LTM. 3) to test the hypothesis that spinal serotonergic mechanisms are critical in maintaining an adequate exercise ventilatory response during models of lung disease. This objective will be met using two models of reversible lung disease: 1) partial disruption of the pulmonary circulation with a balloon catheter, thus creating an endogenous dead space; and 2) bronchoconstriction elicited by inhalation of methacholine. Short and long term modulation of the exercise ventilatory response indicate that the system adapts to changing conditions (e.g., pregnancy, onset of pulmonary disease, etc.). An understanding of these mechanisms may provide insight into normal compensatory processes, and the rationale for therapeutic intervention during disease. The results of these studies also have important implications in the design and interpretation of many studies on ventilatory control, since the central integration of respiratory inputs is commonly assumed be to be "hard wired."

DESCRIPTION: The fundamental goal of this project is to understand mechanisms whereby early life experiences profoundly influence mechanisms of ventilatory control in adult mammals (i.e. developmental plasticity). In the first three years of this project, it was demonstrated that the ventilatory control system is subject to developmental plasticity in the hypoxic ventilatory response. Specifically, one month of perinatal hyperoxia (60 percent) causes a persistent attenuation of the hypoxic ventilatory response in adult rats, two to four months after the hyperoxic exposure had ended. Since similar effects are not observed in rats exposed to the same duration and level of hyperoxia as adults, this functional impairment is unique to development. The aims of this proposal for competitive renewal are to extend these observations by testing the following hypotheses: 1) that perinatal hyperoxia causes persistent functional impairment of the hypoxic ventilatory response only within a limited developmental "window," approximately two weeks in length; 2) that slow, partial functional recovery occurs spontaneously with advancing age; and 3) that functional recovery of the hypoxic ventilatory response can be induced by sustained exposure to hypoxia, either within the developmental window or after the developmental window has expired. Experiments will combine neurophysiological recordings of hypoxic phrenic and carotid chemoafferent responses in anesthetized rats with measurements of the hypoxic ventilatory response in awake rats. These studies may have important clinical implications for infants subjected to oxygen therapy during critical care; they may suffer impaired chemoreflexes throughout their lives if excessive arterial oxygenation occurs during the developmental window. Further understanding of mechanism(s) that underlie developmental plasticity with its associated functional impairments may provide the rationale for therapeutic intervention, thereby enhancing functional recovery.

DESCRIPTION (Adapted from the applicant's abstract):This proposal hypothesizes that intermittent hypoxia elicits unique, serotonin-dependent mechanisms of plasticity in the central neural control of breathing. These forms of plasticity are unique since they are elicited by intermittent hypoxia, but not by an equivalent duration of sustained hypoxia. The PI proposes to investigate cellular and molecular mechanisms of two specific forms of plasticity elicited by intermittent hypoxia: 1) long term facilitation (LTF) of phrenic motor output following three brief hypoxic episodes; and 2) enhanced LTF in rats previously exposed to chronic intermittent hypoxia. A working model has been developed suggesting that, although these forms of plasticity differ in time course and in their requirement for gene transcription, they are initiated by the same events. The PI postulates that the common initiating event is repeated activation of serotonergic 5-HT2A receptors on phrenic motoneurons that increases intracellular kinase activity. As a result, glutamatergic receptors associated with descending respiratory drive and the gene transcription factor cyclic AMP response element binding protein (CREB) are phosphorylated, leading to LTF and enhanced LTF, respectively. Five specific aims are proposed to test the hypotheses that: 1) phrenic LTF requires spinal 5-HT2A receptor activation; 2) chronic intermittent (but not sustained) hypoxia enhances LTF; 3) enhanced LTF is associated with gene transcription regulated by CREB; 4) serotonergic 5-HT7 receptor induction is necessary for enhanced LTF; and 5) brain derived neurotrophic factor (BDNF) induction is necessary for enhanced LTF. A multidisciplinary approach will be used to test key elements of the model.

DESCRIPTION (provided by applicant): The fundamental hypothesis guiding this proposal is that chronic treatments, known to enhance serotonergic modulation of respiratory motor output, strengthen respiratory synaptic pathways to spinal (phrenic) motoneurons, thereby improving respiratory function during recovery from spinal cord injury. In specific, we will investigate the effects of Chronic Intermittent Hypoxia (CIH) and spinal deafferentation via Cervical Dorsal Rhizotomy (CDR) on synaptic pathways to phrenic motoneurons prior to acute spinal hemisection or following chronic spinal hemisection. Our laboratory has previously shown that both CIH and CDR enhance serotonergic modulation of phrenic motor output, but appear to do so by different mechanisms. We have also shown that spinal serotonin receptor activation enhances both functional and ineffective (crossed-spinal) synaptic pathways in rats. Thus, we will apply these unique models of serotonin-dependent respiratory plasticity to test the hypothesis that they will restore respiratory drive to phrenic motoneurons on the injured (hemisected) side. In Aims 1 and 2, we will test the hypotheses that pretreatment with either CIH or CDR enhances evoked and spontaneous phrenic activity in intact and crossed-spinal pathways in anesthetized rats. In the next three aims, we will apply CIH following chronic spinal hemisection to test the hypotheses that CIH enhances evoked and spontaneous phrenic activity in anesthetized rats (Aims 3), restores ventilatory responses to chemoreceptor stimulation in unanesthetized rats (Aims 4), and increases ventral spinal concentrations of brain derived neurotrophic factor below the hemisection (Aim 5). This study provides an unprecedented opportunity to determine whether two experimental treatments restore respiratory motor function below a well-defined cervical spinal injury, provides the basis for highly novel therapeutic approaches in the treatment of respiratory insufficiency following spinal cord injury.

DESCRIPTION (provided by applicant): We seek continued support for a pre- and post-doctoral training program in respiratory neurobiology. Our goal is to prepare trainees for independent careers in basic and applied biomedical research and teaching. A well-trained group of respiratory neurobiologists has important implications for human health;well-trained investigators are critical to advance our understanding of widespread and devastating disorders associated with ventilatory control, such as sleep disordered breathing, respiratory insufficiency during neurodegenerative disease (e.g. ALS) or spinal injury, mental retardation (e.g. Rhett Syndrome), or catastrophic ventilatory failure during development (e.g. SIDS). Four main research themes characterize our training program: 1) Cellular responses to hypoxia;2) Neuroplasticity in respiratory motor control;3) Neurobiology of sleep and sleep disordered breathing;and 4) Cardio-respiratory responses of humans and animal models to hypoxia, exercise and sleep. Each theme includes trainers working at multiple levels of biological organization. Common scientific foundations lend cohesiveness to the training program, which has a long track record of collaborative, multidisciplinary research and training. Key elements of the training program include: independent research conducted in a close working relationship with a faculty supervisor;cooperative, multidisciplinary mentoring;group interactions through lab meetings, seminars and an annual scientific retreat;and training in scientific speaking and writing. Pre-doctoral trainees are admitted to a graduate degree-granting program such as the Neuroscience, Physiology, Comparative Biomedical Science and/or the Cell and Molecular Biology graduate programs. The specific program is chosen based on trainee interests, and determines specific requirements such as coursework. Postdoctoral trainees enter directly into a research laboratory based on their interests, and focus on development as an independent investigator including firm foundations in research ethics and survival skills. Recent trainees have met with considerable success, with more than 80% of former trainees finding suitable positions in academics-many in tenured or tenure track faculty positions. We have also had success in minority recruitment (currently 50% of our available slots), and in the training of medically qualified scientists. Thus, we propose to continue a training program that meets important goals of the NIH research-training mission.

DESCRIPTION (provided by applicant): Plasticity is an important feature of neural systems, including the neural system controlling breathing. Despite its potential biological and clinical significance, our understanding of mechanisms giving rise to any form of respiratory plasticity is incomplete. In this revised application for competitive renewal, investigations will be continued concerning cellular mechanisms giving rise to phrenic long-term facilitation, an important model of respiratory plasticity induced by acute exposure to intermittent hypoxia. In a rat model mechanisms of long- term facilitation will be investigated using multiple experimental approaches, including nerve recordings in anesthetized rats, measurements of ventilation in unanesthetized rats, application of RNA interference, a novel method of regulating potentially important genes that play a role in respiratory plasticity, protein analysis and fluorescent methods in fixed tissues to determine expression changes in important molecules, such as reactive oxygen species. Using these approaches, five hypotheses will be tested in this application for competitive renewal. First, in Aim 1, the hypothesis that the proteins NADPH oxidase and protein phosphatase 2A function as key regulators of long term facilitation will be explored. Next, the possibility that distinct cellular mechanisms give rise to long-lasting facilitation of phrenic nerve activity, of which phrenic long term facilitation is only one, will be tested. In Aim 2, two of these mechanisms will be investigated, designated the Q and S pathways to phrenic motor facilitation. In the final three aims, the hypotheses will be tested that the Q and S pathways normally suppress one another (Aim 3), but that animals have the capacity to shift between the Q and S pathways phrenic motor facilitation (Aim 4), or that they can engage both pathways at the same time (Aim 5). Such flexibility in achieving facilitation of respiratory nerve activity may impart flexibility as an individual responds to physiological challenges throughout life, such as the onset of disease. A detailed understanding of cellular mechanisms giving rise to phrenic motor facilitation will guide the development of novel therapeutic strategies for severe ventilatory control disorders, including obstructive sleep apnea and respiratory insufficiency in patients with spinal injury or motor neuron disease (ALS). Thus, an important underlying goal of our research is to identify molecules regulating PMF as potential therapeutic targets.

PROJECT SUMMARY/ABSTRACT Factors that undermine the neural system controlling breathing diminish the capacity to compensate for pathology, threatening life itself. Plasticity is an essential feature of neural systems, including the neural system controlling breathing. The fundamental hypothesis guiding this proposal is that systemic inflammation impairs respiratory motor plasticity, undermining the ability to compensate for multiple pathologies, including chronic lung disease, traumatic, ischemic and degenerative neural disorders, and obstructive sleep apnea. We propose to investigate mechanisms whereby inflammation impairs a well-studied model of respiratory motor plasticity, phrenic long-term facilitiation (pLTF) following acute intermittent hypoxia. We will contrast inflammation induced by lipopolysaccharide (LPS) with that induced by one day of severe intermittent hypoxia (sIH); sIH simulates aspects of obstructive sleep apnea, a widespread clinical disorder with major implications for human health. Exciting preliminary data suggest that both LPS and sIH block pLTF via spinal inflammation. Since LPS and sIH elicit differential gene expression in different spinal cell types, yet have similar effects on pLTF, we propose a unifying hypothesis whereby multiple inflammatory molecules converge on a common downstream signaling cascade that constrains respiratory motor plasticity. An innovative, multidisciplinary approach will be used to test our hypotheses; experimental approaches include: phrenic nerve recordings in anesthetized rats, diaphragm EMG recordings in unanesthetized rats, immunohistochemical analysis of proteins in labeled phrenic motor neurons, analysis of inflammatory gene expression in freshly-isolated spinal astrocytes and microglia, and flow cytometry to assess proteins in identified cell types. Five specific hypotheses will be tested to advance our understanding: 1) Systemic LPS and sIH elicit spinal inflammation, thereby impairing phrenic and diaphragm LTF; 2) LPS and sIH differentially impair distinct pathways to phrenic motor facilitation (pMF). We will determine LPS and sIH effects on ERK- dependent (eg. pLTF), Akt-dependent and ERK/Akt-dependent pMF; 3) LPS and sIH elicit distinct inflammatory profiles. sIH affects only spinal microglia, whereas LPS also affects astrocytes; 4) Despite different inflammatory profiles, LPS and sIH impair pLTF by a common downstream mechanism involving p38 MAP kinase activation in phrenic motor neurons; and 5) Spinal p38 activity increases protein phosphatase 2A activity in phrenic motor neurons, thereby inhibiting ERK and constraining pLTF. Understanding mechanisms whereby inflammation undermines respiratory plasticity is of fundamental importance since inflammation may diminish the capacity for natural, compensatory plasticity during pathological states. Our long-range goal is to harness and promote respiratory plasticity as a therapeutic strategy to treat devastating breathing disorders, such as during cervical spinal injury or motor neuron disease.

ABSTRACT In this revised application, we propose a new pre- and post-doctoral training program at the University of Florida: Breathing REsearch And THErapeutics (BREATHE). Our proposal is to create a training program focused on the respiratory neuromuscular system, emphasizing the discovery of new knowledge and its translation to neuromuscular disorders that compromise breathing and airway defense. Diminished breathing capacity, unstable breathing and/or aspiration pneumonia from inadequate airway defense are hallmarks of many neuromuscular disorders, and respiratory failure is the most common cause of death. However, despite the fundamental importance of breathing and airway defense to the quality and duration of life in patients with degenerative neuromuscular diseases (eg. Muscular Dystrophy, Pompe Disease, ALS) or neural injury (eg. spinal cord injury), we know of no other training program with similar focus on breathing and airway defense in in these conditions. Our proposal directly addresses this critical gap in NHLBI funded research training. The ultimate goal of the BREATHE Training Program is to develop a unique cohort of researchers with strong foundations in basic research on respiratory neuromuscular biology, and a strong appreciation for the needs/realities of translational research in our attempts to develop treatments for impaired breathing and/or airway defense. Our perspective is that we can accelerate progress towards development of effective treatments for impaired breathing capacity/stability in diverse neuromuscular disorders by training investigators with: 1) a well-developed and comprehensive conceptual framework embracing similarities and differences between neuromuscular clinical disorders; and 2) a state-of-the-art ?tool kit,? providing the technological know- how to perform meaningful investigations in animal models and humans with spontaneous disease. We will train individuals with diverse academic backgrounds, including neuroscience, muscle biology, engineering and/or clinical training (physicians, physical or speech therapists and veterinarians; including multiple specialties in each group). A hallmark of our program will be ?cross-training,? where basic scientists are exposed to meaningful clinical experiences, and clinician scientists establish strong basic research foundations. We will capitalize on existing, and building strengths at the University of Florida to provide an integrated research-training program in respiratory neuromuscular biology and translational research. The UF Health Science Center has demonstrated its commitment to this area of research by creating a new Center for Respiratory Research and Rehabilitation. This Center, and other strengths at the UF Health Science Center, will provide an exceptional academic environment for the BREATHE Training Program.

ABSTRACT Plasticity is a hallmark of the neural system controlling breathing. One extensively studied form of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic motor output after acute intermittent hypoxia (AIH). Multiple, distinct cellular mechanisms contribute to AIH-induced pLTF. Unfortunately, our understanding of how these mechanisms are regulated is limited. Two distinct mechanisms of AIH-induced phrenic motor facilitation (pMF) are known as the Q and S pathways. The Q pathway requires phrenic motor neuron 5-HT2 receptor activation, whereas the S pathway is initiated by phrenic motor neuron 5-HT7 receptors. Q and S pathway co-activation elicits powerful cross-talk inhibition; in fact, equal Q and S pathway activation cancels pMF expression. With moderate AIH (mAIH), the Q pathway dominates but is constrained by S pathway inhibition; S pathway inhibition releases this ?brake,? doubling mAIH-induced pLTF. Repetitive AIH (rAIH) preconditioning enhances mAIH-induced pLTF through unknown mechanisms. This property is essential in our translational efforts to harness rAIH as a treatment to improve breathing in people with cervical spinal injury or neuromuscular disease. The fundamental goal of this proposal is to understand how these cumulative rAIH benefits arise. Our central hypothesis is that rAIH minimizes Q-S pathway cross-talk interactions, enabling both to contribute to AIH-induced phrenic motor plasticity. AIH-induced phrenic motor plasticity exhibits profound age-dependent sexual dimorphism. However, we know essentially nothing concerning how age and sex alter differentially affect pMF mechanisms, or their response to rAIH preconditioning. Thus, we will compare Q and S pathway interactions in young (3 month) and middle-aged (12 month) female vs male rats (when sexual dimorphisms are greatest). We will also investigate differential rAIH preconditioning effects on diaphragm LTF in unanesthetized young and middle-aged female vs male rats. Increased understanding of age and sex effects in normal rats will establish the ?ground rules? for translation to clinical disorders that afflict men and women of different ages. We propose a working cellular model of rAIH-enhanced pLTF based on literature and exciting preliminary data. Based on this model, we propose four specific aims to test the hypotheses that rAIH preconditioning: 1) decreases Q and S pathway cross-talk inhibition, enabling contributions from both (Aim 1); and 2) strengthens the Q pathway to pMF by increasing the expression of key pathway molecules (Aim 2). Since AIH-induced pLTF exhibits profound age-dependent sexual dimorphisms, we will test the hypotheses that: 1) the Q and S pathways to pMF are differentially affected by age and the estrus cycle female rats (Aim 3); and 2) age and sex are key determinants of rAIH-enhanced diaphragm motor plasticity (Aim 4). These studies will greatly advance our understanding of rAIH-enhanced phrenic motor plasticity, and accelerate our ability to harness rAIH as a therapeutic modality to treat devastating clinical disorders that compromise breathing and threaten life itself.

ABSTRACT Cervical spinal cord injury (cSCI) disrupts neural pathways to spinal respiratory motor neurons, causing respiratory impairment and even death. New treatment strategies are desperately needed to improve breathing ability after cSCI. Since most cSCI are incomplete, meaningful functional recovery can be induced by harnessing the intrinsic capacity for neuroplasticity, strengthening spared neural pathways to respiratory motor neurons. Repetitive acute intermittent hypoxia (rAIH) is a simple, safe and effective means to induce respiratory motor plasticity and improve breathing ability in rodent models of acute cSCI. Unfortunately, moderate rAIH is less effective with chronic cSCI. Thus, unknown factors associated with chronic (not acute) cSCI undermine rAIH efficacy. Candidates include cross-talk inhibition from competing mechanisms of adenosine-dependent plasticity, persistent neuroinflammation and age-dependent sexual dimorphisms. In this project, our fundamental goals are: 1) to understand factors limiting AIH-induced phrenic motor plasticity; and 2) use that understanding to develop refined rAIH protocols that optimize therapeutic efficacy with chronic cSCI. AIH elicits multiple distinct mechanisms of phrenic motor facilitation (pMF), including: 1) serotonin-dependent Q pathway initiated by carotid chemoreceptor activation; and 2) adenosine-dependent S pathway initiated by local hypoxia in the phrenic motor nucleus. Although each pathway has therapeutic potential if activated alone, co-activation leads to pathway competition and even pMF cancellation. We hypothesize that chronic cSCI shifts the balance towards equal Q & S pathway activation, undermining rAIH therapeutic efficacy. Minimizing spinal hypoxia and adenosine accumulation by shortening AIH hypoxic episodes is predicted to improve functional outcomes by removing the adenosine constraint to plasticity. Since inflammation undermines serotonin (not adenosine)-dependent pMF, we will also test the hypothesis that anti-inflammatory drugs improve rAIH efficacy with chronic cSCI. Finally, since AIH-induced phrenic motor plasticity exhibits profound age-dependent sexual dimorphisms, we will compare rAIH efficacy in middle-aged male vs female rats. By using two established models of chronic (> 8 weeks) cSCI (C2 hemisection and C4 spinal contusion), we anticipate more robust conclusions since each model has unique advantages/limitations. Five aims are proposed to test the hypotheses that: 1) cSCI decreases spinal PO2, increasing the adenosine constraint to pMF; 2) AIH with shorter hypoxic episodes lessens tissue hypoxia and adenosine accumulation, optimizing pMF; 3) in male rats, optimized rAIH improves breathing capacity more than ?conventional? rAIH; 4) anti-inflammatory drugs enhance rAIH efficacy; and 5) optimized rAIH improves breathing capacity more in middle-aged female versus male rats. Each aim is supported by exciting preliminary data demonstrating feasibility and proof of concept. By optimizing repetitive AIH- induced plasticity to improve breathing ability, we gain new mechanistic insights and move closer to comprehensive clinical trials in humans suffering from impaired breathing due to chronic cSCI.

ABSTRACT Plasticity is a hallmark feature of the neural system controlling breathing. One well-studied form of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic activity triggered by acute intermittent hypoxia (AIH). Multiple distinct cellular mechanisms contribute to AIH-induced pLTF, depending on the severity of hypoxic episodes. Whereas the Q pathway requires 5-HT2 receptor activation on phrenic motor neurons, the S pathway requires adenosine 2A receptor activation. These distinct intra-cellular signaling pathways interact via powerful cross-talk inhibition; indeed, concurrent pathway activation actually cancels pLTF expression. Although we have learned a great deal about intra-cellular signaling mechanisms of AIH-induced pLTF, we know little concerning the role of inter-cellular signaling. Recent reports demonstrate that glia regulate neuroplasticity in multiple neural systems, including microglia, the innate immune cells of the CNS. Since virtually nothing is known concerning the role of microglia in regulating AIH-induced phrenic motor plasticity, our primary goal is to explore this knowledge gap in normal rats and in rats with systemic inflammation. The fundamental hypothesis guiding our proposal is that microglia differentially regulate competing pLTF mechanisms elicited by moderate versus severe AIH (Aim 1). We propose a unified model to explain such differential microglial regulation of AIH-induced pLTF. During severe hypoxia, we propose that phrenic motor neurons release Fractalkine (a chemokine unique to neurons), activating microglial Fractalkine receptors (unique to microglia) and triggering the microglial adenosine release necessary for severe AIH-induced pLTF (Aim 2). With moderate AIH, diminished inter-cellular Fractalkine and adenosine signaling permit the expression of serotonin-dependent pLTF (ie. Q pathway), but with a persistent adenosine constraint (Aim 3). We further propose that even mild systemic inflammation enhances microglial adenosine release during moderate AIH, increasing cross-talk inhibition and suppressing pLTF expression (Aim 4). Finally, since AIH-induced pLTF exhibits a profound age-dependent sexual dimorphism, we will test the hypothesis that phrenic motor neuron- microglia interactions are differentially affected by age in female versus male rats (Aim 5). This project will be the first attempt to identify a specific role of microglia in any form of respiratory motor plasticity, greatly increasing our mechanistic understanding concerning the importance of inter-cellular signaling in respiratory motor plasticity. Since repetitive AIH is emerging as a novel therapeutic intervention to restore breathing (and other movements) in people with debilitating disorders such as cervical spinal injury or ALS, greater understanding of factors regulating AIH-induced plasticity will help optimize AIH protocols and improve chances for successful translation of this promising therapeutic modality. Increased understanding of age and sex effects will establish ?ground rules? for translation to clinical disorders that afflict both men and women.

ABSTRACT Cervical spinal cord injury (cSCI) disrupts neural pathways to spinal respiratory motor neurons, causing respiratory impairment and even death. New treatment strategies are desperately needed to improve breathing ability after cSCI. Since most cSCI are incomplete, meaningful functional recovery can be induced by harnessing the intrinsic capacity for neuroplasticity, strengthening spared neural pathways to respiratory motor neurons. Repetitive acute intermittent hypoxia (rAIH) is a simple, safe and effective means to induce respiratory motor plasticity and improve breathing ability in rodent models of acute cSCI. Unfortunately, moderate rAIH is less effective with chronic cSCI. Thus, unknown factors associated with chronic (not acute) cSCI undermine rAIH efficacy. Candidates include cross-talk inhibition from competing mechanisms of adenosine-dependent plasticity, persistent neuroinflammation and age-dependent sexual dimorphisms. In this project, our fundamental goals are: 1) to understand factors limiting AIH-induced phrenic motor plasticity; and 2) use that understanding to develop refined rAIH protocols that optimize therapeutic efficacy with chronic cSCI. AIH elicits multiple distinct mechanisms of phrenic motor facilitation (pMF), including: 1) serotonin-dependent Q pathway initiated by carotid chemoreceptor activation; and 2) adenosine-dependent S pathway initiated by local hypoxia in the phrenic motor nucleus. Although each pathway has therapeutic potential if activated alone, co-activation leads to pathway competition and even pMF cancellation. We hypothesize that chronic cSCI shifts the balance towards equal Q & S pathway activation, undermining rAIH therapeutic efficacy. Minimizing spinal hypoxia and adenosine accumulation by shortening AIH hypoxic episodes is predicted to improve functional outcomes by removing the adenosine constraint to plasticity. Since inflammation undermines serotonin (not adenosine)-dependent pMF, we will also test the hypothesis that anti-inflammatory drugs improve rAIH efficacy with chronic cSCI. Finally, since AIH-induced phrenic motor plasticity exhibits profound age-dependent sexual dimorphisms, we will compare rAIH efficacy in middle-aged male vs female rats. By using two established models of chronic (> 8 weeks) cSCI (C2 hemisection and C4 spinal contusion), we anticipate more robust conclusions since each model has unique advantages/limitations. Five aims are proposed to test the hypotheses that: 1) cSCI decreases spinal PO2, increasing the adenosine constraint to pMF; 2) AIH with shorter hypoxic episodes lessens tissue hypoxia and adenosine accumulation, optimizing pMF; 3) in male rats, optimized rAIH improves breathing capacity more than ?conventional? rAIH; 4) anti-inflammatory drugs enhance rAIH efficacy; and 5) optimized rAIH improves breathing capacity more in middle-aged female versus male rats. Each aim is supported by exciting preliminary data demonstrating feasibility and proof of concept. By optimizing repetitive AIH- induced plasticity to improve breathing ability, we gain new mechanistic insights and move closer to comprehensive clinical trials in humans suffering from impaired breathing due to chronic cSCI.