Dual oscillator model of the respiratory neuronal network generating quantal slowing of respiratory rhythm

We developed a dual oscillator model to facilitate the understanding of dynamic interactions between the parafacial respiratory group (pFRG) and the preBötzinger complex (preBötC) neurons in the respiratory rhythm generation. Both neuronal groups were modeled as groups of 81 interconnected pacemaker neurons; the bursting cell model described by Butera and others [model 1 in Butera et al. (J Neurophysiol 81:382–397, 1999a)] were used to model the pacemaker neurons. We assumed (1) both pFRG and preBötC networks are rhythm generators, (2) preBötC receives excitatory inputs from pFRG, and pFRG receives inhibitory inputs from preBötC, and (3) persistent Na+ current conductance and synaptic current conductances are randomly distributed within each population. Our model could reproduce 1:1 coupling of bursting rhythms between pFRG and preBötC with the characteristic biphasic firing pattern of pFRG neurons, i.e., firings during pre-inspiratory and post-inspiratory phases. Compatible with experimental results, the model predicted the changes in firing pattern of pFRG neurons from biphasic expiratory to monophasic inspiratory, synchronous with preBötC neurons. Quantal slowing, a phenomena of prolonged respiratory period that jumps non-deterministically to integer multiples of the control period, was observed when the excitability of preBötC network decreased while strengths of synaptic connections between the two groups remained unchanged, suggesting that, in contrast to the earlier suggestions (Mellen et al., Neuron 37:821–826, 2003; Wittmeier et al., Proc Natl Acad Sci USA 105(46):18000–18005, 2008), quantal slowing could occur without suppressed or stochastic excitatory synaptic transmission. With a reduced excitability of preBötC network, the breakdown of synchronous bursting of preBötC neurons was predicted by simulation. We suggest that quantal slowing could result from a breakdown of synchronized bursting within the preBötC

Multi-Scale Modeling of the Neural Control of Respiration

The generation of respiration in mammals begins in the lower brainstem where groups of neurons, that together comprise the respiratory central pattern generator (CPG), interact to produce a motor output that controls breathing. The pre-Bötzinger complex (pre-BötC) in the ventrolateral respiratory column (VRC) is believed to be a major contributor to rhythmic inspiratory activity that interacts with other neural compartments within the VRC as well as with other brainstem areas, including the pons. Though there has been a substantial push to understand the exact cellular and network mechanisms operating within the pre-BötC, as well as the way it is incorporated into the larger respiratory network, there is still much to be resolved. The overarching goal of the work presented in this dissertation is to contribute to our understanding of the neural control of respiration at several hierarchical levels. It is my hope that better insight into the complexities of these multiscale neural control mechanisms will provide a more complete framework for understanding various respiratory pathologies, and ultimately guide the development of novel therapies that will improve patient outcomes. I applied techniques from the fields of mathematics and computer science to develop computational models that reproduced results from electrophysiological recordings (done by our collaborators) and generated verifiable predictions. The scale of my modeling work encompasses the interaction of neurons in a single population, several interconnected populations of neurons that encompass the core of the mammalian respiratory network, and an integration of the respiratory network into a larger control system that includes afferent feedback loops. At each level I address specific, but related, topics that add to the general understanding of the neural control of respiration. The aims of my thesis address specific issues at each of the scales mentioned above. These issues may be summarized as follows: (i) the characteristic rhythmic bursting behavior observed in the pre-BötC, which was studied at the cellular levels with a particular interest in how this behavior impacts respiratory rhythmogenesis; (ii) a respiratory network connectome that defines interactions between several populations of neurons that together form the VRC, which produces an alternating pattern of inspiration, post-inspiration and expiration, and, how such a pattern may be affected by changes in chemical environment, e.g. elevated carbon dioxide or diminished oxygen concentrations; and (iii) the role of afferent feedback to the VRC, from the pons and lungs, which was studied in the context of respiratory phase switching mechanisms.Ph.D., Biomedical Engineering -- Drexel University, 201

Lung breathing in the bullfrog: generating respiratory rhythm and pattern

Thesis (M.S.) University of Alaska Fairbanks, 2008This research investigated location of the lung respiratory rhythm generator (RRG) in the bullfrog brainstem using neurokinin-1 (NK1R) and [mu]opioid ([mu]OR) receptor colocalization and characterized the role of these receptors in breathing pattern formation. colocalization was distinct near the facial nucleus in juvenile bullfrogs but not in tadpoles. NK1R intensity exhibited no developmental change, while [mu]OR intensity increased from late-stage tadpoles to juvenile frogs. Substance P (NK1R agonist; bath applied) increased lung burst frequency, lung burst cycle frequency (BCF), episode frequency, lung burst amplitude and area, but decreased number of lung bursts per episode and lung burst duration. Antagonist D decreased lung burst frequency and BCF, episode frequency, and the number of lung bursts per episode, and increased lung burst duration and area. DAMGO ([mu]OR agonist; bath applied) decreased lung burst frequency and BCF, episode frequency, and number of lung bursts per episode, but increased all lung burst parameters. Naloxone ([mu]OR antagonist) increased lung burst frequency and BCF, episode frequency, lung bursts per episode but decreased all lung burst parameters. Together these results indicate that NK1R and [mu]OR colocalization represents the lung RRG, and that episode formation is intrinsic to the respiratory control network but may or may not originate in the RRG

Developmental Origin of PreBotzinger Complex Respiratory Neurons

A subset of preBötzinger Complex (preBötC) neurokinin 1 receptor (NK1R) and somatostatin peptide (SST)-expressing neurons are necessary for breathing in adult rats, in vivo. Their developmental origins and relationship to other preBötC glutamatergic neurons are unknown. Here we show, in mice, that the “core” of preBötC SST+/NK1R+/SST 2a receptor+ (SST2aR) neurons, are derived from Dbx1-expressing progenitors. We also show that Dbx1-derived neurons heterogeneously coexpress NK1R and SST2aR within and beyond the borders of preBötC. More striking, we find that nearly all non-catecholaminergic glutamatergic neurons of the ventrolateral medulla (VLM) are also Dbx1 derived. PreBötC SST+ neurons are born between E9.5 and E11.5 in the same proportion as non-SST-expressing neurons. Additionally, preBötC Dbx1 neurons are respiratory modulated and show an early inspiratory phase of firing in rhythmically active slice preparations. Loss of Dbx1 eliminates all glutamatergic neurons from the respiratory VLM including preBötC NK1R+/SST+ neurons. Dbx1 mutant mice do not express any spontaneous respiratory behaviors in vivo. Moreover, they do not generate rhythmic inspiratory activity in isolated en bloc preparations even after acidic or serotonergic stimulation. These data indicate that preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1-derived neurons. These data indicate that Dbx1-derived neurons are essential for the expression and, we hypothesize, are responsible for the generation of respiratory behavior both in vitro and in vivo

Maternal Methadone Destabilizes Neonatal Breathing and Desensitizes Neonates to Opioid-Induced Respiratory Frequency Depression

16 pagesPregnant women and developing infants are understudied populations in the opioid crisis, despite the rise in opioid use during pregnancy. Maternal opioid use results in diverse negative outcomes for the fetus/newborn, including death; however, the effects of perinatal (maternal and neonatal) opioids on developing respiratory circuitry are not well understood. Given the profound depressive effects of opioids on central respiratory networks controlling breathing, we tested the hypothesis that perinatal opioid exposure impairs respiratory neural circuitry, creating breathing instability. Our data demonstrate maternal opioids increase apneas and destabilize neonatal breathing. Maternal opioids also blunted opioid-induced respiratory frequency depression acutely in neonates; a unique finding since adult respiratory circuity does not desensitize to opioids. This desensitization normalized rapidly between postnatal days 1 and 2 (P1 and P2), the same age quantal slowing emerged in respiratory rhythm. These data suggest significant reorganization of respiratory rhythm generating circuits at P1–2, the same time as the preBötzinger Complex (key site of respiratory rhythm generation) becomes the dominant respiratory rhythm generator. Thus, these studies provide critical insight relevant to the normal developmental trajectory of respiratory circuits and suggest changes to mutual coupling between respiratory oscillators, while also highlighting how maternal opioids alter these developing circuits. In conclusion, the results presented demonstrate neurorespiratory disruption by maternal opioids and blunted opioid-induced respiratory frequency depression with neonatal opioids, which will be important for understanding and treating the increasing population of neonates exposed to gestational opioids.Supported by the University of Oregon (AHu)

Atoh1-dependent rhombic lip neurons are required for temporal delay between independent respiratory oscillators in embryonic mice

All motor behaviors require precise temporal coordination of different muscle groups. Breathing, for example, involves the sequential activation of numerous muscles hypothesized to be driven by a primary respiratory oscillator, the preBotzinger Complex, and at least one other as-yet unidentified rhythmogenic population. We tested the roles of Atoh1-, Phox2b-, and Dbx1-derived neurons (three groups that have known roles in respiration) in the generation and coordination of respiratory output in embryonic mice. We found that Dbx1-derived neurons are necessary for all respiratory behaviors, whereas independent but coupled respiratory rhythms persist from at least three different motor pools after eliminating or silencing Phox2b-or Atoh1-expressing hindbrain neurons. Without Atoh1 neurons, however, the motor pools become temporally disorganized and coupling between independent respiratory oscillators decreases. We propose Atoh1 neurons tune the sequential activation of independent oscillators essential for the fine control of different muscles during breathing

Atoh1-dependent rhombic lip neurons are required for temporal delay between independent respiratory oscillators in embryonic mice

All motor behaviors require precise temporal coordination of different muscle groups. Breathing, for example, involves the sequential activation of numerous muscles hypothesized to be driven by a primary respiratory oscillator, the preBötzinger Complex, and at least one other as-yet unidentified rhythmogenic population. We tested the roles of Atoh1-, Phox2b-, and Dbx1-derived neurons (three groups that have known roles in respiration) in the generation and coordination of respiratory output. We found that Dbx1-derived neurons are necessary for all respiratory behaviors, whereas independent but coupled respiratory rhythms persist from at least three different motor pools after eliminating or silencing Phox2b- or Atoh1-expressing hindbrain neurons. Without Atoh1 neurons, however, the motor pools become temporally disorganized and coupling between independent respiratory oscillators decreases. We propose Atoh1 neurons tune the sequential activation of independent oscillators essential for the fine control of different muscles during breathing. DOI: http://dx.doi.org/10.7554/eLife.02265.00

Developmental, Physiological, and Transcriptomic Analyses of Neurons involved in the Generation of Mammalian Breathing

Breathing is a rhythmic motor behavior with obvious physiological importance: breathing movements are essential for respiration, which sustains homeostasis and life itself in a wide array of animals including humans and all mammals. The breathing rhythm is produced by interneurons of the brainstem preBötzinger complex (preBötC) whose progenitors express the transcription factor Dbx1. However, the cellular and synaptic neural mechanisms underlying respiratory rhythmogenesis remain unclear. The first chapter of this dissertation examines a Dbx1 transgenic mouse line often exploited to study the neural control of breathing. It emphasizes the cellular fate of progenitors that express Dbx1 at different times during development. I couple tamoxifen-inducible Dbx1 Cre-driver mice with Cre-dependent reporters, then show that Dbx1-expressing progenitors give rise to preBötC neurons and glia. Further, I quantify the temporal assemblage of Dbx1 neurons and glia in the preBötC and provide practical guidance on breeding and tamoxifen administration strategies to bias reporter protein expression toward neurons (or glia), which can aid researchers in targeting studies to unravel their functions in respiratory neurobiology. The second chapter of this dissertation exploits the mouse model characterized in the first chapter and then focuses on mechanisms of respiratory rhythmogenesis. The breathing cycle consists of inspiratory and expiratory phases. Inspiratory burst-initiation and burst-sustaining mechanisms have been investigated by many groups. Here, I specifically investigate the role of short-term synaptic depression in burst termination and the inspiratory-expiratory phase transition using rhythmically active medullary slice preparations from Dbx1 Cre-driver mice coupled with channelrhodopsin reporters. I demonstrate the existence of a post- inspiratory refractory period that precludes light-evoked bursts in channelrhodopsin-expressing Dbx1-derived preBötC neurons. I show that postsynaptic factors cannot account for the refractory period, and that presynaptic vesicle depletion most likely underlies the refractory period. The third chapter of this dissertation focuses on transcriptomic analysis of Dbx1 preBötC neurons, and differences in gene expression between Dbx1-derived and non- Dbx1-derived preBötC neurons. I analyze and quantify the expression of over 20,000 genes, and make the raw data publicly available for further analysis. I argue that this full transcriptome approach will enable our research group (and others) to devise physiological studies that target specific subunits and isoforms of ion channels and integral membrane proteins to examine the role(s) of Dxb1- derived neurons and glia at the molecular level of breathing behavior. In addition to predictable gene candidates (such as ion channels, etc) this transcriptome analysis delivers unanticipated novel gene candidates that can be investigated in future respiratory physiology experiments. Knowing the site (preBötC) and cell class (Dbx1) at the point of origin of respiration, this dissertation provides tools and specific investigations that advance understanding of the neural mechanisms of breathing