Christopher A. Del Negro, Ph.D. - US grants

Breathing behavior in humans, and all mammals, emanates from rhythmic activity in brainstem respiratory neurons. The key population of rhythm-generating neurons is contained in the preBotzinger complex (preBotC) of the ventral medulla. Neurons in the preBotC form excitatory networks and synchronously discharge bursts during the inspiratory phase of network activity. The intrinsic properties of preBotC neurons are hypothesized to function synergistically with glutamatergic synapses during inspiratory burst generation. This project aims to discover these integrated functions at the cellular and synaptic level, by studying preBotC neurons actively engaged in rhythm generation in brainstem slices from neonatal mice that retain the ability to generate inspiratory motor output in vitro. Critical questions regard the burst-generating role of the Ca2+-activated nonspecific cationic current (ICAN), which is putatively expressed in preBotC neuron dendrites and is activated by glutamatergic synaptic inputs. This project will examine the role of ICAN, discover its molecular identity, and characterize how the glutamate receptors cause Ca2+ influx and activate ICAN. Experimental data will be used to create a realistic mathematical model of preBotC neurons, which will be assembled into a network model that simulates respiratory rhythm generation. The overall framework for analysis is that preBotC neurons have active dendritic properties that are maximally evoked in the context of synaptically coupled network activity, which can explain how very small numbers of preBotC neurons generate neural rhythms that are robust and resilient. The new knowledge obtained will be of general interest in neuroscience and biology because respiratory regulation is a critical brain function that must operate continuously to sustain life and homeostasis in all mammals.

This project is a model of interdisciplinary research that addresses an important biological problem at several levels of analysis from the cellular/molecular level to the system/behavior level, and employs powerful tools from mathematical modeling to advance understanding. The research will be integrated with computational biology and neuroscience instruction at The College of William and Mary to promote interdisciplinary education and research training at the graduate and undergraduate level.

DESCRIPTION (provided by applicant): This R21 Developmental Research Grant harnesses multi-photon laser technology to image a working neuronal network in vitro, and then test how constituent neurons contribute to network function via cell-specific laser ablation. Specifically, a computer-controlled system has been developed to detect neurons using multi-photon fluorescence microscopy, store the locations of these cells in memory, and then laser-ablate the target neurons one at a time, in sequence, while monitoring the function of the neural circuit electrophysiologically. Use of a long- wavelength pulsed laser provides unprecedented specificity and control of the lesion in three- dimensional tissue. This significant technological development provides a powerful new tool for interrogating the cellular bases for network behaviors, as well as pathophysiological breakdown in network function. This project focuses on the neural generation and control of breathing behavior. In particular, the investigations probe the circuit properties of the specialized inspiratory rhythm-generating site called the preBotzinger Complex (preBotC) located in the lower brainstem of humans and all mammals. SPECIFIC AIM 1 will evaluate the cellular composition of the preBotC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct subpopulations of neurons in the preBotC, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. The hypothesis that neurons derived from the homeodomain transcription factor Dbx1 comprise the essential rhythm- generating kernel of the preBotC will be specifically tested. SPECIFIC AIM 2 will evaluate whether and how respiratory function deteriorates when rhythmically active preBotC neurons are sequentially deleted. Here the target neurons will be selected on the basis of inspiratory rhythmic activity, rather than genetic origin. This set of experiments will serve as a general disease model to examine the cellular mechanisms underlying respiratory pathologies that have a central etiology. In summary, this R21 project will provide significant new information regarding the neural generation and control of breathing. This new knowledge is important for human health and wellness given that breathing is a relentless and indispensable human behavior that maintains homeostasis and life itself. In subsequent projects this new technique - for detecting and then ablating neurons in a cumulative sequence in vitro - will be applied to interrogate locomotor and masticatory rhythm-generating networks that can be also be studied in spinal cord and hindbrain preparations in vitro. Indeed, this lesioning system will be broadly applicable to studying networks in vitro from any brain region. PUBLIC HEALTH RELEVANCE: Breathing is a vital human behavior that is essential to maintain homeostasis and life itself. This project will advance our understanding of the cellular composition of brainstem neural circuits that generate and control breathing rhythms, and examine how respiratory function breaks down as respiratory rhythm-generating neurons progressively die. The new knowledge obtained will serve as a foundation for the treatment and prevention of respiratory disorders with a central neural etiology, and elucidate circuit-level properties that underlie rhythmic motor behaviors in general.

DESCRIPTION (provided by applicant): This R01 project will advance our understanding of the brainstem neural circuits that generate and control breathing behavior in humans and all mammals. Breathing is an integral part of cardiopulmonary physiology and understanding its neural origins has significant implications for human health. Rhythmic breathing movements begin during embryonic development and emanate from coordinated activity in brainstem respiratory neurons. One key population of rhythm-generating neurons is contained in a site called the preB"tzinger complex (preB"tC). The discovery of the preB"tC made possible many powerful experiments that could be performed in vitro, and led to our contemporary understanding of the neurophysiology of respiration. Nevertheless, critical questions remain unanswered. Given the heterogeneity of respiratory-related and non-respiratory neurons in the preB"tC, can we discover which neurons are the key rhythm generators? If rhythmogenic neurons can be identified (and we argue that indeed they can), then can we ascertain the cellular, synaptic, and molecular-level mechanisms that underlie rhythm generation? Finally, the importance of peptidergic modulation of respiratory rhythm has been widely recognized in the past decade, but its underlying biophysical mechanisms remain incompletely understood. This project seeks answers to these specific questions by studying the preB"tC in thin brainstem slice preparations in vitro. SPECIFIC AIM 1 will evaluate the cellular composition of the preB"tC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct sub-populations, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. SPECIFIC AIM 2 will examine the synaptic-dendritic active membrane properties that generate inspiratory-related bursts. SPECIFIC AIM 3 will investigate whether presynaptic depression of excitatory transmission contributes to burst termination. SPECIFIC AIM 4 is designed to complement SPECIFIC AIM 3 by examining the postsynaptic membrane properties that also act to terminate inspiratory bursts. Finally, SPECIFIC AIM 5 will determine the ion channels that underlie respiratory modulation by key neuropeptides (and other neuromessengers). The new knowledge acquired during this project will aid in the treatment and prophylaxis of breathing disorders that result from failures in the brain and central nervous system. Moreover, studying a measurable behavior like breathing under controlled in vitro conditions helps reveal important principles that link neurons, synapses, and molecules to full-scale physiological behaviors, which will be of great interest in neuroscience as well as cardiopulmonary physiology. PUBLIC HEALTH RELEVANCE: Breathing is a vital human behavior that is essential to maintain homeostasis and life itself. This project will advance our understanding of the brainstem neural circuits that generate and control breathing rhythms by analyzing their function at multiple levels including: neuron type (i.e., genotype), cellular properties, ion channels, synaptic receptors, and intracellular signaling. The new knowledge obtained will serve as a foundation for the treatment and prevention of respiratory disorders with a central neural etiology, and elucidate circuit-level properties that underlie rhythmic motor behaviors in general.

DESCRIPTION (provided by applicant): This R01 project will advance our understanding of the brainstem neural circuits that generate and control breathing behavior in humans and all mammals. Breathing is an integral part of cardiopulmonary physiology and understanding its neural origins has significant implications for human health. Rhythmic breathing movements begin during embryonic development and emanate from coordinated activity in brainstem respiratory neurons. One key population of rhythm-generating neurons is contained in a site called the preBtzinger complex (preBtC). The discovery of the preBtC made possible many powerful experiments that could be performed in vitro, and led to our contemporary understanding of the neurophysiology of respiration. Nevertheless, critical questions remain unanswered. Given the heterogeneity of respiratory-related and non-respiratory neurons in the preBtC, can we discover which neurons are the key rhythm generators? If rhythmogenic neurons can be identified (and we argue that indeed they can), then can we ascertain the cellular, synaptic, and molecular-level mechanisms that underlie rhythm generation? Finally, the importance of peptidergic modulation of respiratory rhythm has been widely recognized in the past decade, but its underlying biophysical mechanisms remain incompletely understood. This project seeks answers to these specific questions by studying the preBtC in thin brainstem slice preparations in vitro. SPECIFIC AIM 1 will evaluate the cellular composition of the preBtC. Transgenic mouse models will be used to apply fluorescent tags to genetically distinct sub-populations, and then selectively and serially lesion them to test their respective roles in rhythmogenesis. SPECIFIC AIM 2 will examine the synaptic-dendritic active membrane properties that generate inspiratory-related bursts. SPECIFIC AIM 3 will investigate whether presynaptic depression of excitatory transmission contributes to burst termination. SPECIFIC AIM 4 is designed to complement SPECIFIC AIM 3 by examining the postsynaptic membrane properties that also act to terminate inspiratory bursts. Finally, SPECIFIC AIM 5 will determine the ion channels that underlie respiratory modulation by key neuropeptides (and other neuromessengers). The new knowledge acquired during this project will aid in the treatment and prophylaxis of breathing disorders that result from failures in the brain and central nervous system. Moreover, studying a measurable behavior like breathing under controlled in vitro conditions helps reveal important principles that link neurons, synapses, and molecules to full-scale physiological behaviors, which will be of great interest in neuroscience as well as cardiopulmonary physiology.

? DESCRIPTION (provided by applicant): This R01 renewal project aims to explain the neural origins of breathing behavior at the cellular and synaptic level. It advances understanding of the brainstem pre-Bötzinger complex (preBötC), which is acknowledged to be the principal site driving respiration in humans and all terrestrial mammals so far studied. Also, this project examines interneurons of the intermediate reticular formation, adjacent to the preBötC, which may give rise to respiratory premotor neurons. The intellectual driving force for this project is te discovery by the PI's team - and French colleagues - that the key rhythmogenic preBötC interneurons in perinatal mice are derived from embryonic precursors that express transcription factor Dbx1 (i.e., Dbx1 preBötC neurons). This project exploits this new knowledge and by coupling Dbx1 Cre-driver mice with six different flox-STOP reporter strains to perform a spectrum of experiments in vivo and in vitro such as patch-clamp recordings, cell-specific laser ablations with physiological monitoring, and optogenetic manipulations that interrogate network properties. Aim 1 uses juvenile and adult mice (in vivo and in vitro) to examine whether Dbx1 preBötC neurons are rhythmogenic beyond embryonic and neonatal stages of development. Aim 2 uses embryonic and neonatal mice in vitro in conjunction with cell-specific laser ablation methods to test whether preBötC neurons with bursting-pacemaker properties are obligatory for respiratory rhythm generation, offering a fresh approach to a 24-year-old unsolved problem regarding `pacemaker' driven preBötC rhythms. Aim 3 uses perinatal mice in vitro to characterize synaptic interconnections among Dbx1 neurons and quantify the input-output relationship. These experiments elucidate recurrent synaptic excitation in Dbx1 preBötC neurons, which is also putatively rhythmogenic. Aim 4 uses perinatal through adult mice (in vivo and in vitro) to examine whether Dbx1 neurons in the adjacent intermediate reticular formation serve as the first layer of premotor neurons for respiratory movements of the tongue (genioglossus) and pharynx. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project is significant because it characterizes the cellular and synaptic mechanisms that animate the key genetic class of neurons (i.e., Dbx1) at the core of the respiratory oscillator, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who - with French colleagues - first characterized the role of Dbx1 neurons in the preBötC and now is poised to further discover their detailed properties and downstream premotor counterparts. If this project succeeds, neuroscience would finally know the cellular and synaptic origins of a significant central pattern- generating circuit in a mammal and the point of origin for an important behavior, breathing.

? DESCRIPTION (provided by applicant): We aim to explain the neural origins of breathing behavior. Dysfunctions in respiratory control circuits cause significant health problems including obstructive and central apneas, as well as respiratory failure and death. These conditions afflict premature infants, children, adults, and patients with neurodegenerative disorders. This project focuses on the core rhythm-generating circuit for inspiratory breathing movements in the pre-Bötzinger complex (preBötC) of the brainstem. The preBötC has been studied for 25 years. Its defining characteristics and its constituent rhythm-generating neurons have been documented in some detail, however, the molecular identity of the ion channels in preBötC neurons that generate inspiratory bursts remains unidentified. The specific aims of this proposal address this key knowledge gap. If we succeed then breathing would be the first mammalian motor behavior that can be explained at the level of networks, cells, and ion channels. In particular, this projec will test one leading hypothesis that a class of non-selective cation channels generates inspiratory bursts, and by extension, that these channels underlie the motor drive for breathing in living animals. It is widely acknowledged that a molecularly unidentified Ca2+-activated non-selective cationic current (ICAN) contributes to inspiratory burst generation in preBötC neurons. Non-selective cation channels of the transient receptor potential (TRP) superfamily are the best candidates for the channel(s) that give rise to ICAN in the preBötC based on evidence from several sources. We have three main objectives. 1) Determine which TRP channels are expressed in preBötC neurons using quantitative PCR, physiology, and immunohistochemistry. 2) Down-regulate these TRP channels by interfering with mRNA transcripts and overexpressing dominant negative channel mutants in rhythmically active slice cultures. Then, we will measure the degree to which the burst-generating capability of preBötC neurons is compromised. 3) Down-regulate the same TRP channels in the preBötC of living mice and measure the extent to which breathing movements are impaired. This project innovates by using contemporary molecular biology techniques to address a problem that has heretofore been approached primarily by electrophysiology. This project is significant because it identifies the ion channels responsible for inspiratory burst generation, aggregate motor drive, and real breathing behavior, which represents a transformative advance in our understanding that would inform new prevention and treatment strategies to combat respiratory pathologies. The PI is the ideal scientist for this job because of his track record as a leader in respiratory neurobiology, who firt characterized ICAN as well as the genetic identity of the core rhythmogenic preBötC neurons. If this project succeeds, then we will know the molecular-level mechanism for inspiratory burst generation. Medical science would then be able to therapeutically target those channels to ameliorate respiratory pathologies in many circumstances. Neuroscience would finally know the fundamental molecular point of origin for all respiratory physiology.

This project aims to explain the neural mechanisms of breathing. Breathing in mammals consists of eupnea, periodic inspiratory pumping movements that draw air into the lungs for gas exchange, and sighs, larger less frequent breaths that periodically reinflate gas-exchange air sacs or express emotion, Eupnea and sigh rhythms are well coordinated and originate from the same set of brainstem neurons, but their underlying neural mechanisms remain incompletely understood. Using computational simulation and experimental tests of model predictions, this project will elucidate the mechanisms for eupnea and sigh rhythms in three SPECIFIC AIMS. In Aim 1, the project will ascertain the excitatory microcircuit dynamics for eupnea rhythm. An existing model of eupnea rhythm will be made mathematically tractable for geometric and bifurcation analyses, and its exclusive focus on synaptic dynamics will be augmented with biophysically realistic somatic membrane properties, In Aim 2, the project will explore the biochemical oscillatory mechanisms that give rise to sigh rhythm by developing and contrasting models of metabotropic signaling and intracellular Ca2+ oscillations that generate sigh-like network rhythm, In Aim 3, the project will examine the synaptic mechanisms that couple and coordinate the eupnea and sigh rhythms. Experiments will determine the synaptic transmission that coordinates eupnea and sigh, which will then constrain the models from Aims 1 and 2. This project will yield two deliverables of high intellectual merit: 1) an explanation of the cellular and synaptic mechanisms of eupnea- and sigh-related breathing rhythms, and ii) a biophysically realistic model for the core microcircuit that drives inspiratory breathing movements, both eupnea and sigh, suitable for inclusion within comprehensive models of the full behavior (e.g., with more motor phases and sensory feedback). Because rhythms are a ubiquitous aspect of brain function, the rhythmogenic mechanisms of breathing are of broad interest. This project will provide new knowledge regarding the cellular and synaptic neural origins of breathing that will inform the treatment and prevention of respiratory neuropathologies that afflict persons of all ages. The project will support STEM training of Ph.D. students and undergraduates, a thriving biomathematics consortium at William & Mary, and a summer internship program for public high schools, RELEVANCE (See instructions): Breathing consists of eupnea, regular breaths that pump oxygen into the lungs for gas exchange, and sighs, larger but less frequent breaths that reinflate gas-exchange air sacs or express emotion. This project applies mathematical models and experiments to explain how the mammalian brainstem generates breathing rhythms: eupnea and sigh. This knowledge will inform the treatment and prevention of respiratory neuropathologies that afflict persons of all ages from 'premies' to the elderly.

PROJECT SUMMARY Our goal is to explain the neurophysiology of breathing. Breathing refers to periodic movements of the chest and airways that ventilate the lungs. To breathe, the brain must: ? Generate a rhythm, ? Produce a spatiotemporal pattern for breathing muscles, ? Integrate sensory feedback. Here we focus on jobs 1 and 2, discovering the ionic mechanisms underlying the rhythm and motor pattern. The brainstem preBötzinger complex (preBötC) generates the inspiratory breathing rhythm. Its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors (i.e., Dbx1 neurons). Because we know the site (preBötC) and the canonical cell-class (Dbx1) at the point of origin for breathing, we are well equipped to discover which ion channels influence normal inspiration (eupnea), ?sighs?, and gasps in hypoxia. We manipulate ion channels using genetic technologies. We assess breathing phenotypes in intact adult mice. We explain the biophysics of these phenotypes via patch-clamp recordings from adult Dbx1 preBötC neurons. Three types of ion channels are implicated in preBötC function: (1) Na+ channels that engender persistent Na+ current (INaP), (2) Transient receptor potential (Trp) channels that mediate Ca2+-activated nonspecific cationic current (ICAN), and (3) K+ channels that give rise to transient outward K+ current (i.e., A-current, IA). Specific aim 1 evaluates the role of INaP in Dbx1 preBötC neurons. A rhythmogenic role for INaP has been suspected for 27 years. We use intersectional mouse genetics and short-hairpin RNA (shRNA) to knockout or knock-down Na+ channel genes that give rise to INaP and evaluate its role in eupnea, sighing, and gasping. Specific aim 2 evaluates the role of ICAN in Dbx1 preBötC neurons. ICAN is a major charge carrier for inspiratory bursts. Next-generation RNA Seq technology provides us with a suite of Trp channel targets that we will acutely attenuate using shRNA to test the role of ICAN in eupnea, sighing, and gasping. Specific aim 3 evaluates the role of IA in Dbx1 preBötC neurons. IA-expressing preBötC neurons feature other key rhythmogenic properties so they may be specialized. We use intersectional mouse genetics and shRNA to knockout or knock-down K+ channel genes for IA to evaluate its role in eupnea, sighing, and gasping. Then we selectively kill the 56% of IA-expressing Dbx1 preBötC neurons to test whether they serve a specialized rhythmogenic role. The success of this project would be a watershed in terms of understanding the ion channel-level origins of real behavior. The new knowledge could be applicable to treatment and prophylaxis of respiratory pathologies with a central etiology. The new knowledge may provide general insights regarding the neural origins of motor rhythms.

PROJECT SUMMARY Locomotion is important behavior for humans and all animals. Rhythmic movements are executed by microcircuits of the spinal cord. Setting movement goals and selecting motor programs requires higher brain planning centers that funnel inputs through the basal ganglia. Intercalated between the basal ganglia and spinal cord is a critical midbrain region dubbed the mesencephalic locomotor region (MLR), which initiates movements and controls their form and intensity. At present, we know a great deal about spinal cord and basal ganglia microcircuits, and both are well represented in the neuroscience literature. However, the MLR is much less well understood, particularly at the level of ion channels and synapses that animate MLR function. This project steps in to fill that knowledge gap. Aim 1 examines the cellular ionic mechanisms used by excitatory MLR interneurons to initiate motor programs. We hypothesize that low voltage-activated Ca2+ currents produce low-threshold spike (LTS) bursts and plateau potentials to trigger motor programs. We will test this hypothesis in adult brain slices in vitro and via Cre- dependent short-hairpin RNA (shRNA) experiments to attenuate ion channel expression in excitatory MLR interneurons in unanaesthetized freely behaving adult mice. Aim 1 will also involve next-generation deep sequencing of the transcriptome of MLR neurons. We will harness those data for this project while also releasing them into the Commons for general use among neuroscientists. Aim 2 examines the synaptic mechanisms that activate MLR neurons, which we hypothesize is disinhibition via GABAB receptors. That mechanism aligns with expectations that disinhibition via the direct path of the basal ganglia evokes motor programs via the MLR. We will test our hypothesis in adult brain slices in vitro and via Cre-dependent shRNA experiments that attenuate GABAB-GIRK channel synaptic function in unanaesthetized freely behaving adult mice. If successful, this project will explain how key midbrain microcircuits initiate locomotor behaviors at the level of ion channels and synapses, and thus bridge a major knowledge gap in motor control. Given that basal ganglia, MLR, and spinal cord circuits are ubiquitous features in the brains of all vertebrates during 500 MY of evolution, the insights we develop in a genetically and electrophysiological advantageous mammalian animal model (mouse) will be generally applicable to understanding motor control across a wide array of vertebrate animals. There may be translational significance for the treatment of movement disorders as well. The PIs of the project share complementary expertise in electrophysiology and behavioral experimentation in adult mice. The PIs have an established collaboration with considerable pilot data, which led to the specific aims and hypotheses above. Regardless of experimental outcomes, this project will elucidate the cellular and synaptic bases of MLR function and advance understanding in motor control.