Matthew R. Hodges, Ph.D. - US grants

Acute adjustments in ventilation represent a powerful homeostatic mechanism for the maintenance of arterial and/or brain P02, PC02 and pH. Sensory feedback to the ventilatory control centers occurs via peripheral and central chemoreceptors, but there are several unresolved questions regarding the mechanisms and relative importance of specific subpopulations of neurons thought to act as central C02/pH chemoreceptors. What confers upon select neurons the intrinsic ability to "sense" pH? What are the signal transduction pathways for pH? Which neurons perform this task? Under which circumstances are they important (sleep wakefulness)? The overall goals ofthis proposal are to address some of these questions regarding central chemoreception by taking advantage of unique inbred rat strains with inherent, and large differences in C02 sensitivity;the Brown Nonway (BN: low responder) and Dahl Salt-sensitive (SS: high responder) rats. First, we will determine if the deficit in C02 sensitivity in BN rats is due to decreased cellular C02/pH sensitivity of individual chemosensitive neurons in vitro, which will be aided by the development of transgenic rats expressing fluorescent proteins in select neuron pools for direct patch clamp recordings. We will also determine the relative effects of creating focal acidosis in brainstem regions harboring these neuronal subpopulations in BN and SS rats in vivo. Finally, our preliminary data suggests that deficits in multiple neuromodulators in the brainstem may contribute to the blunted C02 sensitivity in the BN rats, and thus we will determine whether if augmenting these neuromodulators individually or collectively can restore C02 sensitivity in the BN rat. The data obtained from these unique rat strains will provide significant insights into the mechanisms and relative importance of specific chemoreceptor subpopulafions in regulafing eupneic ventilation and ventilatory C02 sensitivity. They will also provide insights into respiratory-related diseases, and provide a framework for future genomic investigations aimed at idenfifying the genefic determinants and the unique cellular mechanisms by which central chemoreception occurs.

DESCRIPTION (provided by applicant): It is estimated that addiction and its associated costs: crime, domestic violence and child abuse, health care costs, and loss of employment and family structure, exceed half of a trillion dollars per year. Fighting addiction is not a matter of willpower, it is a battle to understand how drugs affect the brain and can permanently alter its function. One of the key limitations to understanding the cellular and genetic basis of a complex disease like addiction is the availability of good models to test hypotheses related to cells and genes and their functions. The laboratory rat is one model which has tremendous value because of its intensely studied physiological, biochemical, and behavioral characteristics and their genome sequence similarities to humans. More than 2000 genetic determinants of disease-related traits have been initially described in the rat, however, it has been difficult to precisely correlate specific genes with these traits because of limitations in technology for manipulating the rat genome. In the past 4 years, we have been developing new tools to close this technology gap. We have developed new and efficient ways of making transgenic rats using transposable elements which are highly reproducible and we were the first to apply zinc-finger nuclease (ZFN) technology to target and disrupt, or knock out, specific genes in the rat. These are two very important technologies that allow us to do many things, but more work is needed. Many times, knocking out a gene in the whole animal precludes studying its role in a particular cell or tissue because the gene is essential for early embryo development and so knocking it out causes the animal to die. In other cases, knocking out the gene in the whole animal doesn't allow one to distinguish what organ (or part of the brain, for example) the gene is functioning in to cause the disease. In this pilot/feasibility study, we propose to develop the next key technological step toward this goal - to be able to specifically disrupt genes in a particular cell or tissue at a particular time. We will develop transgenic rats which express an inducible CRE/loxP recombinase system in specific tissues. This system will allow us to create conditional knockout rats where we can control where and when a gene function is removed from a cell. We will focus brain neuron systems known to be important for studying addiction and behavior - the dopaminergic and serotonergic neurons. As a proof of principal that the system is working, we will knock out the gene that produces serotonin, a hormone that is important in the reward system of the brain and an integral player in addiction and other psychiatric disorders, specifically in neurons of the hindbrain. If successful, this approach will change the way we can approach genes, cells and diseases in the laboratory rat model to impact the socioeconomic burdens of addiction and drug abuse. PUBLIC HEALTH RELEVANCE: The key to developing effective therapies for the treatment of addiction and other psychiatric disorders is the identification of genes and the cells they work in to target new drugs and approaches. Lab rats are the preferred model for studying addiction by many resources, but the technology to study a particular gene by knocking out its function in a particular cell or tissue has not been demonstrated. This pilot and feasibility study aims to develop that technology in the lab rat so we can enable studies of specific genes and their roles in addictive behavior and other diseases.

? DESCRIPTION (provided by applicant): Dysfunction of homeostatic ventilatory chemoreflexes likely contribute to the genesis of or maladaptation to multiple respiratory-related diseases in humans, but potential treatments are hampered by a poor understanding of the fundamental CNS mechanisms responsible for detecting and responding to hypercapnia. There are multiple CNS sites containing cells with presumed intrinsic CO2/pH sensitivity, including medullary raphe (MR) serotoninergic (5-HT) and phox2b-expressing retrotrapezoid nucleus (RTN) neurons. The identity of molecules that underlie cellular CO2/pH sensitivity to these cells remains unknown. In addition, excitatory neuromodulators such as 5-HT, substance P and thyrotropin-releasing hormone (TRH) are critical to neural respiratory control, but the importance of these neuromodulators in the CO2 chemoreflex is unclear. To address these knowledge gaps, we will test two major hypotheses by completing three Specific Aims. We hypothesize that: 1) sub-populations of phox2b+ RTN and MR 5-HT neurons are intrinsically chemosensitive due to the selective expression of one or more pH-sensitive ion channels, and 2) raphe-derived neuromodulation of the RTN is a major determinant of the mammalian CO2 chemoreflex. To identify molecules that may underlie cellular CO2/pH sensitivity, we have developed a unique scientific approach utilizing fluorescence-assisted cell sorting (FACS) followed by Next-gen RNA sequencing (RNASeq) to identify differentially-expressed genes among neurochemically-defined brainstem neuronal subpopulations. We have demonstrated the feasibility of our approach, and identified two genes (Kir4.1 and Kir5.1) that may underlie cellular CO2 chemosensitivity of 5-HT neurons. In Aim 1 we will use this approach to identify genes/molecules that may underlie cellular CO2 sensitivity by comparing CO2-sensitive and CO2-insensitive 5-HT and RTN neurons using hypercapnia-induced c-Fos expression to identify CO2 sensitive neurons. In Aim 2 we will functionally validate genes identified in Aim 1, and specifically the roles of Kir4.1/5.1 K+ channels in vitro using patch clamp recordings and in vivo in genomic Kir4.1, Kir5.1 and combined Kir4.1/5.1 knockout rats. To further address the role of neuromodulators in the ventilatory CO2 chemoreflex, we will study Brown Norway (BN) rats, which have a severely blunted CO2 chemoreflex but normal breathing during eupnea, hypoxia and exercise. These CO2-insensitive BN rats are deficient in brainstem 5-HT and TRH, and stimulation of 5-HT or TRH receptors augments the CO2 chemoreflex in BN rats. Accordingly, we hypothesize that these neuromodulatory effects occur through direct modulation of the RTN, which we will test in Aim 3 by microdialysis of agonists and antagonists of 5-HT, substance P and TRH receptors within the RTN of CO2-insensitive BN and highly CO2- sensitive Salt-sensitive (SS) and Sprague Dawley (SD) rats. Our innovative studies will generate important new data regarding fundamental mechanisms of cellular CO2 chemoreception and the CO2 chemoreflex, and provide a framework for a molecular genetics approach to study other components of ventilatory control.

PROJECT SUMMARY Over the last twenty years, training in Physiology departments throughout the country has undergone a transformation that creates a challenge for students seeking an understanding of the discipline at the whole animal to the cellular and molecular levels. One exception is the Physiology Department of the Medical College of Wisconsin (MCW), that offers exceptional research training emphasizing the integration of knowledge at all of these levels, and the relationship of this knowledge to human disease processes. In our current proposal, we will continue providing this outstanding training in cellular, molecular, and whole animal Physiology for 6 NIH- supported trainees each year. In addition, we request funds to provide stipends for 6 rising 2nd year medical students enrolled at MCW to spend 11 weeks during the summer engaged in research in the laboratory of one of our faculty to enhance recruitment of medical students into biomedical research. Unique aspects of the proposed PhD training are the multidisciplinary mentoring program and opportunities for translational research fostered by a highly collaborative basic science and clinical faculty. Graduate students will be recruited nationally and selected for admission on the basis of undergraduate academic credentials, previous research experience, and commitment to a career in research. Students must successfully complete the requirements of the first two years of graduate school before being considered for T32 support. Selection of T32 trainees will be based on performance in course work, the preliminary examination and in research. Trainees are full-time Ph.D. candidates in the MCW Graduate School, and will complete a research that includes use of the techniques in molecular, cellular, tissue, and whole-animal or clinical investigation. Research training is supervised by Physiology faculty along with co-mentors from other basic science and clinical departments. Trainees will undergo continuous evaluation by utilizing Individual Development Plans created upon matriculation, which are reviewed and updated yearly. The major objective is to provide trainees with a broad foundation in interdisciplinary basic science and translational research through developing critical thinking, integrative reasoning, and technical skills required to succeed in evolving research careers focused on the prevention of hypertension, stroke, and respiratory diseases. An innovative feature of the training is the emphasis on addressing the national need to train for the integrated- systems approaches that represent the future of biomedical research in the post-genomic era. The success of our program in research is indicated by the 47 first-authored publications from 24 T32 supported graduates over the past 10 years (Table 5A), and their contribution to 51 manuscripts coauthored with other students or faculty, indicative of our highly collaborative training program. Another measure of overall success of our program is that 94% of T32 trainees who completed our program over the past 15 years (Table 8A) have continued in a biomedical field, with 18 obtaining research, teaching, and/or patient care faculty positions and only two trainees withdrew from the program without obtaining the PhD (average duration of training was 4.96 years).

Project Summary More than 50 million people suffer from epilepsy globally. Current anti-epileptic drugs (AEDs) cannot prevent seizures in ~30% of these patients, leading to uncontrolled or refractory epilepsy. Unfortunately, these patients are at extreme risk of Sudden Unexpected Death in Epilepsy (SUDEP), which is the leading cause of death in this cohort. Based on landmark SUDEP studies, the current hypothesis is that recurrent seizures induce extreme cardiorespiratory suppression and/or failure through negative effects on the neural networks that regulate vital functions such as breathing, heart rate and blood pressure. However, it remains unclear how repeated seizures fundamentally affect cardiorespiratory control networks within the brainstem, and by what mechanisms these vital systems fail in SUDEP. Here we aim to characterize the pathophysiologic consequences of repeated seizures in a novel rat model with a known mutation in a potassium channel gene (kcnj16; SSkcnj16-/- rats), in which a specific sound of mild intensity readily and reproducibly causes generalized tonic-clonic seizures (GTCSs). Sound-induced GTCSs in SSkcnj16-/- rats led to a stereotypic pattern of events similar to that described in epilepsy patients, including post-ictal generalized EEG suppression and apnea, followed by respiratory rate (RR) and heart rate (HR) suppression. Repeated seizures (1/day for up to 10 days) led to: 1) augmented post- ictal suppression of RR and HR, reduced ventilatory responses to hypoxic and hypercapnic challenges, and unexpected mortality in ~33% of these rats. Brainstem tissue analyses of SSkcnj16-/- rats exposed to repeated seizures showed evidence of time-dependent increases in inflammation and dysregulation of adenosine (ADO) and serotonin (5-HT) ? two powerful modulators of cardiorespiratory neural networks. Finally, pharmacologically augmenting 5-HT with an SSRI (fluoxetine) prevented the progressive suppression of post-ictal RR with repeated seizures. Herein we will test our central hypothesis that repeated seizures cause a progressive brainstem pathology initiated by neuroinflammation and mediated by ADO and 5-HT dysfunction leading to cardiorespiratory suppression and/or failure in SUDEP. The proposed studies utilizing this novel mutant rat model will provide: 1) a comprehensive characterization of the progressive pathophysiological responses to repeated audiogenic seizures, and sequence of events leading to unexpected death (Aim 1), 2) identified mechanisms of dysfunction in inflammatory and/or neuromodulatory pathways within critical cardiorespiratory brainstem nuclei negatively affected by repeated seizures (Aim 2), and 3) interventions that functionally test our hypothesis by blocking neuroinflammation or modulating ADO or 5-HT system activity on the backdrop of repeated seizures (Aim 3). We will utilize a combination of established and cutting-edge technologies to provide unprecedented molecular, cellular and systems-level insights into the pathophysiological consequences repeated seizures on vital control systems in order to identify novel targets for therapeutic interventions aimed at preventing or mitigating the risk of SUDEP in human epilepsy patients.