See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/278690557 Respiration Chapter · November 2013 DOI: 10.1007/978-1-4614-1997-6_49 CITATIONS READS 0 4,736 2 authors: John J Greer Gregory D Funk University of Alberta University of Alberta 126 PUBLICATIONS 6,621 CITATIONS 141 PUBLICATIONS 4,513 CITATIONS SEE PROFILE All content following this page was uploaded by John J Greer on 05 February 2016. The user has requested enhancement of the downloaded file. SEE PROFILE Respiration 45 John J. Greer and Gregory D. Funk Abbreviations 5-HT AMPA ATP B€ otC cAMP CCHS CCK CNS cVRG Dbx1 DRG GABA KF LPBr LRt Mcp MeCP2 Mo5 MPBr NK1R NTS OSA P cells PaCO2 PAO2 Serotonin 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid Adenosine triphosphate B€ otzinger complex Cyclic adenosine monophosphate Congenital central hypoventilation syndrome Cholecystokinin Central nervous system Caudal division of ventral respiratory group Developing brain homeobox protein Dorsal respiratory group g-aminobutyric acid K€ olliker-Fuse nucleus Lateral parabrachial nucleus Lateral reticular nucleus Medial cerebellar peduncle Methyl-CpG-binding protein 2 Motor nucleus of the trigeminal nerve Medial parabrachial nucleus Neurokinin 1 receptor Nucleus of the solitary tractus Obstructive sleep apnea Pump cells Partial pressure of arterial carbon dioxide Partial pressure of alveolar oxygen J.J. Greer (*) • G.D. Funk Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada e-mail: john.greer@ualberta.ca, jgf@ualberta.ca D.W. Pfaff (ed.), Neuroscience in the 21st Century, DOI 10.1007/978-1-4614-1997-6_49, # Springer Science+Business Media, LLC 2013 1423 1424 PaO2 pFRG/RTN Pn preB€ otC PRG RAR REM Robo3 rVRG SAR scp SIDS SO Sol SolC SolVL sp5 SubP TASK TRH VE vlPons VRC J.J. Greer and G.D. Funk Partial pressure of arterial oxygen Parafacial respiratory group/retrotrapezoid nucleus complex Basilar pontine nuclei Pre-B€ otzinger complex Pontine respiratory group Rapidly adapting receptor Rapid eye movements Roundabout homolog 3 Rostral division of ventral respiratory group Slowly adapting receptor Superior cerebellar peduncle Sudden Infant Death Syndrome Superior olive Solitary tract Commissural subdivision of the nucleus of the solitary tract Ventrolateral subdivision of the nucleus of the solitary tract Spinal trigeminal tract Substance P Acid-sensitive two-pore domain K+ Thyrotropin-releasing hormone Minute ventilation Ventrolateral pontine region Ventral respiratory column Brief History Introduction and Historical Perspective Breathing is the “simple” rhythmic act of moving air through the airways and into and out of our lungs. A striking feature of the neuronal network controlling this behavior is that it is incredibly robust. In humans, it begins in utero with the onset of fetal breathing movements in the first trimester and continues largely uninterrupted after birth under a very wide range of metabolic demands and behavioral states. The quest to find where within the CNS this life-long, repetitive respiratory rhythm is generated has an extensive history. This includes the writings of Galen, the physician to the gladiators, who noted that a breathing rhythm continues as long as nervous tissue above the lower neck region is intact. Animal experimentation in the 1790s demonstrated that a breathing rhythm persisted after removal of the cerebrum and cerebellum in rabbit preparations. Refinement of this approach in the early 1800s, also using the rabbit model, narrowed the critical region to the medulla and lower pons. A few decades later, Flourens reported persistent breathing movements of the mouth with only a limited portion of the medulla remaining intact. He referred to this medullary 45 Respiration 1425 region as the “noeud vitale.” The fact that each half of the brainstem could independently generate respiratory rhythms was shown in 1880 after midsagittal division of the rabbit medulla. Experiments involving sectioning of the anesthetized cat brainstem at several levels generated data suggesting that the pons plays an important role in the generation of a normal respiratory rhythm. The importance of the pons was challenged during that era, and it was concluded that the basic respiratory rhythm is indeed generated in the medulla, and the pons provides important conditioning input. The late nineteenth century saw the advent of electrical stimulation as an approach to investigate brainstem respiratory centers. This approach was not particularly useful for identifying a discrete region controlling rhythm generation; however, it did reveal that there are multiple regions in the brainstem that influence respiratory rate and pattern when stimulated. Ramon y Cajal, who made early contributions to many concepts in this textbook, contributed an anatomical perspective to the question. He examined the efferent and afferent connections of respiratory-related nerves in rats and cats and came up with a network model of respiratory pattern generation between various brainstem nuclei. Variations on that network model were in vogue for many years prior to the series of experiments described in the “Discovery of the RespiratoryRhythm-Generating Center” section below. In hindsight, given the current models of respiratory rhythmogenesis, it is remarkable how insightful those early investigators were based on data from relatively rudimentary experimental approaches. The respiratory network that produces and controls breathing can be divided into 5 main components as diagrammed in Fig. 45.1: (1) rhythm-generating networks that produce the basic neural oscillation that underlies the rhythmic act of breathing; (2) pattern forming systems that translate the basic oscillation into a coordinated pattern of activity in the various motoneuron pools innervating the respiratory muscles; (3) respiratory muscles, which comprise pump, airway, and accessory muscles that generate inspiratory and expiratory airflow; (4) regulatory elements that respond to and process chemosensory and mechanosensory information from the CNS and periphery; and (5) integrative components that coordinate respiratory movements with other movements such as locomotion, speech, chewing, and swallowing. Each of these topics will be discussed in the following sections, and relevant historical context is provided within. Respiratory Rhythm Is Generated within the Brainstem Discovery of the Respiratory-Rhythm-Generating Center As outlined above, the pons and medulla have been a long-standing focus of investigation. There are three main interconnected regions of the brainstem that contain concentrated populations of respiratory neurons classified based on the phase in which they are active (e.g., inspiratory, expiratory, phase-spanning) and their firing patterns (e.g., decrementing or augmenting). The pontine respiratory group (PRG) is located within the dorsolateral pons and includes the parabrachial 1426 J.J. Greer and G.D. Funk Fig. 45.1 The respiratory control network can be divided into five main components: (1) a rhythmgenerating network that produces the basic oscillation; (2) a pattern-forming system that produces a coordinated pattern of activity in the respiratory muscles; (3) respiratory muscles; (4) mechanosensory and chemosensory regulatory elements; and (5) integrative components coordinate breathing with other behaviours and K€ olliker-Fuse nuclei (Fig. 45.2). The dorsal respiratory group (DRG) is located within the nucleus of the solitary tract (NTS). The ventral respiratory column (VRC) is a column of ventrally located medullary neurons extending from the facial nucleus to the spinal cord. A series of systematic studies addressed the specific question of which of these neuronal populations are essential for generating the basic respiratory rhythm. Following decades of combining neuronal recordings with stimulation, lesioning, cooling, and ablations within the brainstem of anesthetized or decerebrate animal models, a major advance resulted from experiments utilizing an in vitro brainstemspinal cord preparation isolated from neonatal rats (Fig. 45.3). Remarkably, the 45 Respiration 1427 Fig. 45.2 Respiratory-related regions of the brainstem of the rat shown in horizontal (a) and sagittal (b) views. Respiratory-related regions comprise a nearly continuous column in the lateral brainstem. The boundaries depicted between the various brainstem compartments reflect functional distinctions between adjacent regions relative to their impact on breathing. Abbreviations: 5n trigeminal nerve, 7 facial nucleus, 7n facial nerve, A5 A5 noradrenergic neuronal group, AmbC compact part of nucleus ambiguus, AP area postrema, B€ otC B€ otzinger complex, cVRG caudal division of ventral respiratory group, DRG dorsal respiratory group, I5 intertrigeminal area, icp inferior cerebellar peduncle, KF K€ olliker-Fuse nucleus, LPBr lateral parabrachial region, LRt lateral reticular nucleus, mcp medial cerebellar peduncle, Mo5 motor nucleus of the trigeminal nerve, MPBr medial parabrachial region, NTS nucleus of the solitary tract, pFRG parafacial respiratory group, Pn basilar pontine nuclei, preB€ otC pre-B€ otzinger complex, PRG pontine respiratory group, RTN retrotrapezoid nucleus, rVRG rostral division of ventral respiratory group, scp superior cerebellar peduncle, SO superior olive, sol solitary tract, SolC commissural subdivision of the nucleus of the solitary tract, SolVL ventrolateral subdivision of the nucleus of the solitary tract, sp5 spinal trigeminal tract, vlPons ventrolateral pontine region, VRC ventral respiratory column of the medulla, VRG ventral respiratory group (Adapted with permission from Alheid and McCrimmon (2008) Respir Physiol Neurobiol. 64:3–11) 1428 a J.J. Greer and G.D. Funk b X IX c1 5 sec c5 T2 T8 5 sec Fig. 45.3 In vitro brainstem spinal cord preparation isolated from newborn rats. Photomicrographs of (a) in vitro brainstem-spinal cord preparation with recording suction electrodes. (b) Spontaneous respiratory discharge generated by brainstem spinal cord preparation. Distribution of spontaneous motor activity is illustrated by recordings of cranial (IX, X) and spinal motoneuron discharge (C1, C4, C5, T2, and T8) on ventral roots (Adapted with permission from Smith et al. (1991) J Neurophysiol. 64:1149–1169) preparation, when bathed in an oxygenated modified Krebs solution, spontaneously generates robust rhythmic respiratory-related activity for several hours. The activity can be readily monitored by simply watching the rhythmic rib cage movements if the rib cage is left intact. More precise monitoring is made by recording rhythmic neuronal discharges from axons contained in ventral roots and cranial nerves that innervate the diaphragm, respiratory rib cage, and upper airway musculature (Fig. 45.3). This experimental model provided the opportunity to carefully dissect away various parts of the brainstem respiratory groups while monitoring for perturbations of the respiratory rhythm. Removal of the PRG and DRG did not significantly alter rhythmic respiratory activity. This left the VRC as the likely candidate for containing the neuronal populations generating the basic rhythm. The VRC is a long column of cells that extends the rostrocaudal length of the medulla and is comprised of several groups of neurons based on anatomical and functional criteria. Specifically, from rostral to caudal, there are the parafacial respiratory group/retrotrapezoid nucleus complex (pFRG/RTN), the B€ otzinger complex, the pre-B€otzinger complex (preB€otC), the rostral ventral respiratory group (rVRG), and the caudal ventral respiratory group (cVRG). The sequential sectioning of fine slices of tissue along the rostrocaudal extent 45 Respiration 1429 Fig. 45.4 Medullary slice preparation containing the preB€ otC complex generates spontaneous respiratory rhythm. Left panel – imaging of preB€ otC neuronal activity in a medullary slice preparation using a calcium-sensitive dye. Center panel – schematic of medullary slice that contains the preB€ otC and hypoglossal (XII) motoneurons. Right panel – whole-cell patch recording of a preB€ otC inspiratory neuron that fires action potentials in approximate synchrony with the inspiratory motor discharge recorded from the hypoglossal (XII) nerve root of medullary slice preparation (Adapted with permission from Thoby-Brisson et al. (2005) J Neurosci. 25:4307–4318) of the VRC revealed that the well-delineated region of the preB€otC was the source of the basic inspiratory rhythm. In fact, a thin medullary slice of tissue containing the preB€ otC region, isolated from the remainder of the CNS, will generate robust inspiratory neuronal activity in vitro that can be monitored via electrophysiological recordings or imaging using voltage- or calcium-dependent dyes (Fig. 45.4). The critical role of the preB€ otC was further demonstrated in the intact nervous system of adult rodents by Feldman and colleagues. The first strategy was to determine if rhythmogenic preB€otC neurons could be differentiated from surrounding cells by the types of neurotransmitters and receptors they express. It became apparent that many preB€otC neurons expressed the neurokinin 1 receptor (NK1R), the neurotransmitter somatostatin, and transporters identifying them as glutamate releasing neurons. The NK1R expression was utilized for the second strategic study. Normally, the neurotransmitter substance P (SubP) released at a synapse binds to NK1R and is then internalized while bound to the receptor. In this series of experiments, the plant toxin saporin was attached to SubP and applied to the preB€ otC region. This resulted in the internalization of the toxin and subsequent perturbation of protein synthesis and cell death. The selective killing of preB€otC neurons resulted in a marked destabilization of breathing rhythm. The third strategic study utilized the somatostatin expression of preB€ otC neurons. Experiments were performed to induce the expression of the Drosophila allatostatin receptor in somatostatinexpressing preB€ otC neurons. The allatostatin receptor, when expressed in neurons, is coupled to GIRK channels (inward-rectifying K+ channels). However, there are no natural ligands for the allatostatin receptor in the mammalian CNS, and thus, neuronal function is not affected. In this study, exogenous allatostatin was administered to activate the receptors which induced a hyperpolarization of the cell membrane due to the increase in potassium conductance. The ensuing silencing of preB€otC neurons led to the rapid suppression of rhythmic inspiratory drive that was reversible upon the removal of allatostatin. 1430 J.J. Greer and G.D. Funk Fig. 45.5 Two regions of the VRC that generate rhythmic respiratory-related activity. Imaging of respiratory activity in the pFRG/RTN and preB€otC regions detected in the brainstem of a newborn rat in vitro preparation using a voltage-sensitive dye (Adapted with permission from Oku Y et al. (2007) J Physiol. 15;585:175–186) The data from in vitro and in vivo rodent studies was subsequently extended to the examination of human tissue. A concentrated cluster of NK1R-positive neurons were found in a region of the human brain that correlates well with the rodent preB€ otC. In addition, autopsy studies from patients with the neurodegenerative disorder, multiple system atrophy, demonstrated a significant loss of NK1R expressing neurons in the region of the preB€otC. This is consistent with the severe respiratory disorders, including central sleep apnea, associated with the disease. The model of rhythmogenesis subsequently has become more complicated with evidence suggesting that a second site contributes to the rhythm of breathing. Studies utilizing imaging (Fig. 45.5) and recording of respiratory neuronal activity demonstrate rhythmic activity in the rostral region of the VRC adjacent to the facial nucleus, referred to as the pFRG/RTN (shown in Fig. 45.2). The pFRG/RTN rhythmic activity starts slightly prior to preB€otC discharge and persists when the preB€ otC rhythm has been blocked pharmacologically with opiates. Lesioning experiments have demonstrated synaptic connections between the two nuclei, and removal of input from pFRG/RTN to the preB€otC can modulate the frequency of inspiratory rhythm. More strikingly, the removal of pFRG/RTN input results in the complete loss of active expiratory activity (Fig. 45.6). Further, stimulation of the pFRG/RTN area leads to the expression of previously subthreshold expiratory phase activity and a resetting of the respiratory rhythm. The current proposal is that the preB€otC generates inspiratory rhythm and the pFRG/RTN generates expiratory activity during forceful, active exhalation and that these two oscillators are coupled. There is further speculation that a phylogenic relationship exists between these two coupled mammalian oscillators and the rhythm generators in the developing amphibian brainstem that independently, although in a coordinated manner, regulate lung and buccal respiratory patterns. 45 Respiration 1431 Fig. 45.6 Functional data showing differential roles of pFRG/RTN and preB€ otC in control of active expiration and inspiration. (a) Transection rostral to the facial nucleus, that leaves the pFRG/RTN and preB€ otC intact, does not affect breathing pattern. (b) Transection at the caudal end of the facial nucleus, to remove input from the pFRG/RTN, but not the preB€ otC, eliminates active expiratory EMGABD bursts while maintaining robust inspiratory activity. (c). Schematic showing the levels of transections with accompanying histological verification of location. V motor trigeminal nucleus, VII facial nucleus, LRN lateral reticular nucleus, preB€ otC pre-B€ otzinger complex, B€ otC B€otzinger complex, pFRG parafacial respiratory group, RTN retrotrapezoid nucleus, SO superior olivary complex, Ac ambiguus nucleus compact (Adapted with permission from Janczewski and Feldman (2006) J Physiol. 15;570:407–420) Neuronal and Network Properties Underlying Respiratory Rhythmogenesis The question of how neurons are organized and function within the preB€otC to generate inspiratory rhythmogenesis has not been fully answered. Early models proposed that respiratory rhythm emerged as the result of synaptic interactions between distinct populations of neurons that formed a network within the brainstem. A central component of those models was that inhibitory neurotransmission was necessary for the phase transition between inspiration and expiration. This concept was tested by blocking multiple types of inhibitory neurotransmission. While the motor pattern is markedly altered under those conditions, the basic respiratory rhythm persists. This led to the establishment and testing of the hypothesis that rhythm was 1432 J.J. Greer and G.D. Funk generated by the action of neurons with pacemaker properties, similar to what is present in a number of invertebrate rhythmic neuronal systems. Indeed, whole-cell intracellular recording studies identified two classes of respiratory neurons with bursting-pacemaker properties within a subpopulation of preB€otC neurons in medullary slice preparations. One class depends on a persistent Na+ current (INaP) and the other on Ca2+ and/or Ca2+-activated nonselective and voltage-insensitive cation (ICAN) currents for their bursting activity. However, rhythmic inspiratory activity can persist under some conditions when both types of bursting-pacemaker neurons are pharmacologically blocked. This has led to the group-pacemaker hypothesis. In this model, the emerging respiratory rhythm depends on recurrent glutamatergic excitatory synaptic inputs between many preB€ otC neurons. In addition, the INaP and ICAN that promote the bursting properties act to amplify the synaptic depolarizations and are important for the full inspiratory drive potential characteristic of preB€otC neurons. The preB€ otC and pFRG/RTN in the intact behaving organism function within a much broader neural system. The generation of completely normal or eupneic breathing requires inputs from many other central and peripheral network elements. Synaptic inputs (both excitatory and inhibitory) from the PRG and DRG are particularly important for respiratory sensorimotor integration and the shaping of the various phases of inspiratory and expiratory motor patterns. There are inputs from other brainstem regions and higher centers that regulate respiratory output and reconfigure the rhythmogenic activity to accommodate behaviors such as suckling, swallowing, sniffing, chewing, coughing, vomiting, and vocalization. The modification of rhythmic preB€ otC output for the generation of gasping is particularly important for autoresuscitation and survival during extreme tissue hypoxia. Neuromodulation of Central Respiratory Rhythmogenesis Which neurotransmitter is primarily essential for generating the basic respiratory rhythm within the preB€ otC? The answer is glutamate. Among the various types of glutamate receptors, it is the block of AMPA [2-amino-3-(5-methyl-3-oxo-1,2oxazol-4-yl)propanoic acid] receptors alone that causes a dose-dependent decrease and ultimate cessation of respiratory frequency. The inspiratory drive potential generated via activation of AMPA receptors is further enhanced by activation of NMDA and group I metabotropic glutamate receptors. In addition, preB€otC neurons receive multiple modulatory inputs from many areas outside and within the vicinity of the preB€ otC. The neurotransmitters acetylcholine, norepinephrine, histamine, serotonin (5-HT), dopamine, adenosine triphosphate (ATP), SubP, and cholecystokinin (CCK), often by converging onto similar second messenger systems, can increase frequency, regularity, and amplitude of respiratory activity. The medullary raphe nuclei are an important source of neuromodulatory input regulating respiratory rhythmogenesis. The three major neurotransmitters released from the raphe complex, 5-HT, thyrotropin-releasing hormone (TRH), and SubP, all have excitatory actions on respiratory rhythmogenesis. The noradrenergic 45 Respiration 1433 control of respiratory frequency via synaptic inputs from neurons belonging to the locus coeruleus and the pontine A5 and A6 nuclei is variable with the predominant excitatory versus inhibitory action being determined by the relative activation of medullary a1 (excitatory) and a2 (inhibitory) receptors. The neurotransmitters g-aminobutyric acid (GABA) and glycine, the principal mediators of fast chloride-mediated inhibitory transmission in the mammalian CNS, modulate respiratory rhythmogenesis and the patterning of motor output. In addition, GABAA receptor-mediated control of respiratory activity is markedly enhanced by neurosteroids released during stress induced by hypoxia, asphyxia, parturition, ethanol exposure, and infection. Opiates, used widely as analgesics to treat acute and chronic pain, are powerful respiratory depressants. Balancing the trade-off between analgesia and sedation and respiratory depression is one of the major clinical challenges of anesthesiology, pain, and intensive care medicine. A significant component of the suppression of respiratory rhythmogenesis results from activation of m-opiate receptors specifically within the preB€ otC. The mechanism of action includes presynaptic mediated suppression of glutamate release and postsynaptic hyperpolarization of rhythmogenic preB€ otC neurons. There are also indirect contributions to opioidinduced respiratory depression from other neuronal regions, including cortical inputs, chemoreceptors, and NTS. Similarly, anesthetics, many of which act via activation of GABAA receptors, inhibit preB€ otC neuronal function and thus respiratory rhythmogenesis. The activation of somatostatin receptors has potent inhibitory effects on respiratory rhythm generation. Acetylcholine has excitatory actions via activation of nicotinic (nACh) and M3 muscarinic (mACh) receptors, while M2 receptor activation is inhibitory to respiratory rhythm. The purinergic control of respiratory frequency is complex, with ATP being excitatory and its degradation product adenosine being inhibitory. Breathing Movements Are Generated by a Pattern Generator That Underlies the Coordinated Recruitment of Rib Cage, Abdominal and Upper Airway Muscles The respiratory rhythmogenic drive generated in the brainstem is transmitted via networks of interneurons and premotoneurons to several respiratory motoneuron populations. At each synapse leading up to the neuromuscular junction, synaptic and intrinsic membrane properties interact to produce the muscle-specific patterns of respiratory activation. This includes neurons controlling rib cage, abdominal and upper airway musculature. Respiratory pattern generating neurons, motoneurons, and the muscles they innervate are typically active either during the inspiratory (inhalation) or expiratory (exhalation) phase of the breathing cycle. The expiratory phase often displays two distinct components. Some respiratory neurons have a higher intensity of activation during the early stage of expiration (stage I or postinspiratory), for example, neurons controlling upper airway muscles that aid in 1434 J.J. Greer and G.D. Funk slowing expiratory flow rate, while other neurons have the predominance of their activity during the late (or stage II) phase of expiration, for example, those controlling the internal intercostal muscles. Respiratory Muscles Controlling the Rib Cage and Abdomen Air flow in and out of the lungs is regulated by the mechanical action of the rib cage moving in axial and radial directions. Inspiration is an active phase controlled by the contraction of the diaphragm, levator costae, scalene, and parasternal and external intercostals muscles (i.e., pump muscles). In addition, the sternocleidomastoid, pectoralis, serratus anterior, and trapezius muscles are recruited during forceful inspiration (i.e., accessory muscles). Expiration is largely passive. During forced expiration, for instance during strenuous exercise, the rib cage movements are also controlled by abdominal and internal intercostal muscles (Fig. 45.7). Respiratory Spinal Motoneurons Phrenic motoneurons innervate the diaphragm muscle. They receive their phasic inspiratory drive from axons that descend from the medulla to the cervical spinal cord. Specifically, the descending medullary neurons are located in the DRG and rVRG. The majority of connections from the medulla to phrenic motoneurons are monosynaptic. Glutamate is the main neurotransmitter mediating the transmission of inspiratory drive from medullary neurons to phrenic motoneurons, largely via AMPA receptors. There are also descending inhibitory synaptic inputs from the medulla onto phrenic motoneurons. The primary inhibitory sources arise from brainstem neurons located in the cVRG and B€ otC. The inhibitory drive during the expiratory phase ensures that the diaphragm does not contract during exhalation. Interestingly, there is also inhibitory drive to phrenic motoneurons during the inspiratory phase. It is the balance of inhibitory versus opposing excitatory synaptic drive that determines the activity of phrenic motoneurons and diaphragm contraction. This “push-pull” balance of excitation and inhibition is a strategy widely used by the CNS to further enhance the potential for regulating motoneuron excitability. In addition to the fast excitatory and inhibitory inspiratory drives mediated by the amino acid transmitters, synaptic endings converge onto the phrenic motoneuron pool that contain a multitude of neurotransmitters, including 5-HT, TRH, norepinephrine, SubP, met-enkephalin, cholecystokinin, galanin, neuropeptide Y, and adenosine. The neuromodulators regulate phrenic motoneuron excitability on a slower time scale relative to the amino acid transmitters. In addition, the activity of these modulatory neurons often varies between states (e.g., sleep-wake cycling). Internal and external intercostals, as well as triangularis sterni and parasternal motoneurons, are located in the ventromedial region of the spinal cord that corresponds to the intercostal space in which the muscle is located. Inspiratory drive 45 Respiration 1435 Sternocleidomastoid Scalene Parasternal Serratus anterior Internal intercostal External Intercostal Diaphragm Rectus abdominis INHALATION EXHALATION Fig. 45.7 Respiratory muscles innervated by spinal respiratory motoneurons that are recruited during inhalation and exhalation. Levator costae (occupying most rostral regions of each intercostal space), triangularis sterni (underlie parasternal muscles), and other abdominal muscles besides rectus abdominis not shown (Copyright 2004 Pearson Education, Inc. publishing as Benjamin Cummings) transmission to intercostal respiratory motoneurons arises from the rVRG and DRG. Expiratory drive arises from the cVRG. Unlike phrenic motoneurons, there seems to be very little monosynaptic input to intercostal respiratory motoneurons from the medulla. Rather, there is a segmental network of interneurons that transmit reciprocal inhibition between inspiratory and expiratory intercostals motoneurons. The neurochemical control of respiratory motoneurons controlling intercostal musculature has not been studied in detail, but AMPA receptor activation seems to be an important contributor. Abdominal muscles are primarily innervated by motoneurons located in the lower thoracic and upper lumbar spinal cord. Premotoneurons supplying abdominal motoneurons are localized in the cVRG. Contraction of the rectus abdominis, oblique abdominis, and transversus abdominis muscles increases intra-abdominal pressure that causes upward displacement of the diaphragm muscle during forceful expiration. Control of the Upper Airway In order to maintain adequate ventilation, the upper airway must remain open across different postural positions and sleep-wake cycles. This is accomplished by contraction of upper airway muscles, such as the genioglossus (tongue) and the hyoid 1436 J.J. Greer and G.D. Funk muscles. The airway muscles are typically activated prior to the main inspiratory pump muscle, the diaphragm, and this decreases airway resistance to facilitate airflow generated by the downward contraction of the diaphragm. The respiratory modulation of upper airway muscles is superimposed on a background of tonic activation. This is important for setting baseline airway size and stiffness necessary for counteracting the subatmospheric collapsing pressures generated during inspiration. Glutamate, acting largely via AMPA receptors, is an important component of basic inspiratory drive transmission to hypoglossal motoneurons. Suppression of upper airway muscle activation during sleep can result in episodes of decreased muscle tone that leaves the upper airspace vulnerable to collapse (Obstructive Sleep Apnea discussed below). Spinal Cord Injury and Breathing The relative importance of each population of respiratory motoneurons for breathing movements is clearly evident from the effects of spinal cord injury. With a spinal cord injury at the level of C4 and higher, all of the major muscles controlling rib cage expansion (diaphragm, external intercostal, and parasternal) are paralyzed. Patients will require the use of a mechanical ventilator except for short durations where a combination of the recruitment of neck inspiratory accessory muscles (sternocleidomastoid and scalenus) in combination with glossopharyngeal breathing can produce adequate ventilation. Glossopharyngeal (or frog) breathing uses the muscles of the tongue and the throat to force air into the lungs through repetitious cycles of 6–10 gulps of air followed by exhalation. Injuries between C4 and T6 leave the diaphragm functioning, but the loss of intercostal activity results in the patient experiencing a sensation of breathlessness (dyspnea) despite having adequate gas exchange. Injuries between T6 and T12 do not normally affect quiet breathing; however, the ability to cough is impaired. Normal breathing and cough reflexes are preserved with injuries below T12. Respiratory Rate and Pattern Are Regulated by Chemoreceptive and Mechanoreceptive Feedback Chemoreception The respiratory system works in conjunction with the cardiovascular system to provide oxygen to cells for cellular metabolism, remove the waste product CO2 and maintain proper pH levels. For the system to work efficiently, feedback from sensors that constantly monitor levels of O2, CO2, and pH in the blood and brain is necessary. There are two main groups of chemoreceptors that perform this function. The peripheral chemoreceptors detect changes in these variables in the blood, while central chemoreceptors monitor the CNS. When the chemoreceptors detect that O2, CO2, and/or pH levels deviate from normal baseline levels, they 45 Respiration 1437 transmit nerve signals through a feedback loop to induce compensatory changes in the depth and/or rate of breathing. Thus, chemoreceptors are key to the homeostatic control function of the respiratory network. Hypoxia Breathing is typically described in terms of the number of breaths taken per minute (frequency) and the size or depth of each breath (tidal volume). A 70-kg person at rest typically has a tidal volume of 500 ml per breath and takes 12 breaths per minute. The product of the two variables is referred to as the minute ventilation (V_ E). Measurement of blood gases is reported in partial pressures. For example, the measurement of oxygen and carbon dioxide in the arterial blood is reported as PaO2 and PaCO2, respectively. The relationship between V_ E and the partial pressure of oxygen in the alveoli (PAO2) is shown in Fig. 45.8 (in healthy individuals, PAO2 is a reasonable estimate of arterial PaO2). This relationship is measured in a laboratory setting by monitoring a subject’s ventilation when they breathe air containing different levels of oxygen. Under normal conditions, PaO2 drops when a subject does not breathe sufficiently to match metabolic demand. This drop in PO2 is detected, the neuronal feedback loop is activated, and ventilation increases, restoring PaO2. Experimental control over the O2 level of the inspired air “short-circuits” the normal feedback loop because no matter how much subjects increase ventilation, they will not be able to restore PaO2. The resultant relationship describes the potency of hypoxia as a source of respiratory drive. The experiment is performed under two conditions. In the first, the only manipulation is to alter the level of hypoxia and monitor ventilation. The resultant relationship is shown in Fig. 45.8 (blue curve). There are two main points. At resting levels of PO2 (near 100 mmHg), the relationship is quite flat, indicating that the system is not very sensitive to changes in PO2 in this range. However, as hypoxia becomes increasingly severe near PaO2 levels of 60 mmHg, the sensitivity increases, as indicated by the progressive increase in the slope of the V_ E/PO2 relationship. In the second experimental condition, CO2 is added to the inspired air to prevent the fall in CO2 that results from the increased ventilation (Fig. 45.8, red curve). This curve reveals the true sensitivity of ventilation to hypoxia. A key feature of the hypoxic ventilatory response is that it is biphasic (Fig. 45.9). In most adult mammals, it is characterized by an initial increase in ventilation that peaks within the first 1–2 min and then decreases over the next 2–5 min to a level that remains above control. This secondary decrease in ventilation, referred to as hypoxic ventilatory depression, is more pronounced in newborn and premature mammals such that ventilation can actually fall below baseline levels (as shown for an 8-day-old infant in Fig. 45.9). Part of this decrease in ventilation is due to the fact that hypoxia actually depresses metabolism. The reduced metabolism is an appropriate adaptive response to reduce the use of oxygen that is in short supply. However, the inhibition of respiratory networks is clearly not adaptive as it does not meet the demand for bringing in more oxygen via increased ventilation. This disadvantageous suppression of ventilation may be due to the hypoxic-induced release of inhibitory neuromodulators within the brainstem respiratory circuitry, including adenosine, taurine, neurosteroids, opioids, prostaglandins, and GABA. 1438 60 PCO2 held constant at 42.6 mmHg 40 Minute Ventilation Fig. 45.8 Ventilatory response to hypoxia of a human subject. The red curve is the ventilatory response when PCO2 is kept constant by adding CO2 to the inspired gas. The blue curve is the response when PCO2 was not maintained constant. In this case, as ventilation increases in response to the decrease in PO2, PCO2 continues to decrease. The ventilatory response to hypoxia is therefore blunted because the CO2 drive to breathe is constantly falling and offsetting the increased hypoxic drive. The numbers next to the blue curve represent the PCO2 measured at each point (Adapted with permission from Loeschcke and Gertz (1958) Pfleugers Archiv. 267:460–477) J.J. Greer and G.D. Funk 20 30 37 PCO2 allowed to decrease 0 20 60 39 100 140 PO2 (mmHg) Hypercapnia Feedback signals in response to an increase in the levels of CO2 (hypercapnia) cause an increase in ventilation. This respiratory response to hypercapnia is attributed 20% to peripheral and 80% to central chemoreceptors. The relationship between V_ E and PaCO2, measured in a similar manner to the hypoxic sensitivity, is shown in Fig. 45.10 at three different levels of PaO2. At normal levels of oxygen (normoxia), the relationship is characterized by a hockey-stick-shaped curve with two key features. First, near baseline levels of CO2, the relationship is linear with a significant slope meaning that even small changes from baseline will evoke a significant compensatory ventilatory response. This differs markedly from the V_ E/PO2 relationship and illustrates that under normal conditions, CO2/pH is the primary regulated variable. Second, ventilation decreases linearly with PCO2 until 26 mmHg when the relationship becomes flat (the blade of the hockey stick). Over this range, CO2 is so low that it no longer provides a chemical drive to breathe. The level of ventilation observed here is determined by PaO2. The relationship between V_ E and CO2 when there is also hypoxia is similar to that in normoxia with two noteworthy differences. First, the slope of the V_ E/PaCO2 relationship is steeper, indicating a positive nonlinear interaction between 45 Respiration 1439 8 days old 18 days old +40 +20 % Change in Ventilation 0 −20 −40 O2(%) 21 15 Hypoxia 21 Hypoxia 15 5 min Fig. 45.9 Ventilatory responses to mild (15%) hypoxia in premature infants. In the youngest group, an initial increase in ventilation is followed by a respiratory depression in which ventilation falls below baseline during the hypoxic exposure and often remains below baseline after returning to normoxia. In older infants, the ventilatory increase is more sustained, remaining above baseline throughout the period of hypoxia (Adapted with permission from Waters and Gozal (2003) Respir Physiol & Neurobiol. 136:115–129) the O2 and CO2 respiratory drives (i.e., there is a larger change in ventilation for the same change in PCO2). Second, the flat “blade” component of the hockey stick is still present, but it occurs at a higher level of V_ E, and the inflection point in the stick is at lower levels of PaCO2, reflecting the higher O2 drive in this condition. In hyperoxia (curve 3), the slope of the linear V_ E/PaCO2 relationship is reduced from that in hypoxia and normoxia, again indicative of the interaction between CO2 and O2 drives. In addition, the relationship is linear throughout. When PaCO2 falls to <28 mmHg, the relationship intersects with the x-axis; ventilation falls to zero. This illustrates a very important feature of the central respiratory network; while capable of generating a rhythm when isolated from all peripheral inputs in vitro, it requires a baseline level of tonic drive to operate. Chemoreceptor input is a major source of this tonic drive in vivo; if blood PaO2 is maintained and PCO2 is reduced, central respiratory rhythm and breathing will cease. Peripheral Chemoreceptors In mammals, the main group of peripheral chemoreceptors is located bilaterally in the carotid body where the common carotid artery bifurcates into the internal and external carotid arteries (Fig. 45.11a, b). The carotid bodies are small sensory organs, but per gram of tissue, they are the most highly perfused structure in the body. This along with their position between the heart and the CNS places them ideally as monitors of blood en route to the brain. In addition, there are contributions from chemosensing aortic bodies that are exposed to blood flowing out of the heart through the aortic arch to the remainder of the body. Even at rest, the ongoing feedback from the peripheral chemoreceptors contributes approximately 20% to the ventilatory drive. This has been demonstrated by the experimental manipulation of briefly having the subject breathe 100% O2 which momentarily silences the feedback from peripheral chemoreceptors. 1440 60 hypoxia normoxia 40 Minute Ventilation Fig. 45.10 Ventilatory response of a human subject to hypercapnia at three levels of PO2: hypoxia, normoxia, and hyperoxia. The response to PCO2 is linear at each level of PO2, but the slope of the relationship increases with increasing hypoxia (Adapted with permission from Nielsen and Smith (1952) Acta Physiologica Scand. 24:293–313) J.J. Greer and G.D. Funk 20 hyperoxia 0 20 40 30 PCO2 (mmHg) 50 Carotid bodies contain two main types of cells, neuron-like glomus (type I) cells and glia-like sustentacular (type II) cells (Fig. 45.11c). Sustentacular (derived from the Latin word for “support”) refers to cells that provide a supportive function, similar to those of astrocytes in the CNS. However, just as astrocytes in the CNS are emerging as important participants in information processing, involvement of sustentacular cells in signal processing in the carotid body is under intense investigation. Glomus type I cells are the actual sensory cells, detecting primarily changes in PO2 but also PCO2/pH. They have a fine granular appearance due to their high content of neurotransmitter-containing vesicles. The molecular mechanisms underlying hypoxia sensing are not completely known but appear to involve closure of a K+ (TASK) channel that is normally open at rest (most likely an acid-sensitive two-pore domain K+ [TASK] channel) (Fig. 45.11c). These channels are very sensitive to inhibition of mitochondrial energy metabolism. They close in response to hypoxia via unknown mechanisms and are the major contributor to the hypoxiaevoked depolarization of glomus type I cells. Elevated CO2 and the resultant decrease in pH directly inhibit these acid-sensitive K+ channels causing the CO2-dependent depolarization of glomus type I cells (Fig. 45.11c). Regardless of the stimulus, glomus type I cell depolarization opens voltage-gated Ca2+ channels, leading to Ca2+ influx which triggers vesicle fusion with the cell membrane and 45 Respiration 1441 Fig. 45.11 Carotid body chemoreceptors: location, transduction mechanisms, and central projections. The carotid bodies are located bilaterally in the neck (a) at the bifurcation of the common carotid artery into the internal and external carotid arteries. (b) Enlarged view of the box in (a) showing the carotid body and its afferent innervation via the carotid sinus nerve. (c) Enlarged view of a cross section through the carotid body (at the blue line in b) showing arrangement of glomus cells (type I; the sensor), sustentacular cells (type II; glia-like), capillaries, and carotid sinus nerve endings. Signal transduction pathways for conversion of increased CO2 (upper glomus cell [steps i-vii] and decreased O2 (lower glomus cell [steps 1–7]) into transmitter release and action potential generation in carotid sinus nerve are depicted. (d) Central projections of carotid sinus nerve afferent fibers in the medulla. The first-order excitatory synapse is in the NTS. These neurons project to the pontine respiratory group (PRG) and chemosensitive neurons in the pFRG/RTN which in turn send excitatory projections to the preB€ otC and possibly other regions of the VRC neurotransmitter release in proportion to the magnitude of the hypoxic (or hypercapnic) stimulus. Glomus cell vesicles contain a host of neurotransmitters, but it is primarily ATP and acetylcholine that excite afferent terminals of the carotid sinus nerve (a branch of glossopharyngeal nerve) by activating P2X (ATP-gated ion channels) and acetylcholine receptors, respectively. Impulses conduct centrally 1442 J.J. Greer and G.D. Funk and make excitatory synapses in the caudal NTS (Fig. 45.11d, see also Fig. 45.2). The NTS is a major integration site for visceral sensory information from the pharynx, larynx, respiratory tracts, heart, and large blood vessels coming through the vagus (X), glossopharyngeal (IX), and facial (VII) nerves. From this first-order synapse in the NTS, second-order neurons synapse in the RTN (important central chemoreceptive site discussed below) and pontine respiratory groups, which in turn project to the VRC and hypothalamus to bring about changes in ventilation. Central Chemoreceptors Chemosensitive sites: CO2 diffuses rapidly from blood vessels across the bloodbrain barrier. Thus, elevations in PaCO2 rapidly appear in the poorly buffered extracellular and cerebrospinal fluid of the brain. Central chemoreception refers specifically to the feedback process whereby changes in brain CO2 (or pH) evoke the changes in ventilation that maintain arterial CO2 (or pH) near steady-state levels. The dominant theory is that CO2, working indirectly through its effect on pH, stimulates breathing by activating different types of acid-sensitive CNS neurons located at multiple sites. Initial attention focused on the ventral medullary surface following the observation of dramatic increases in ventilation in response to local acidification of two regions on the ventral surface of the medulla. Named after the investigators that described them in the 1960s and 1970s, Mitchell’s area is in the rostral medulla (approximately under the facial nucleus) and corresponds approximately with the RTN, while Loeschcke’s area is in the caudal medulla. Subsequent to those pioneering studies, a variety of areas that evoke increases in ventilation upon local acidification have been discovered. Putative chemosensory sites in the brainstem include the caudal NTS and adjoining dorsal motor nucleus of the vagus, rostroventrolateral medulla, preB€otC, medullary raphe, locus coeruleus, and RTN. The relative importance of the different regions may change with sleep-wake state, but, in general, there seems to be a modest increase in ventilation of 20% in response to acidification of each individual region. If multiple regions are activated together, the overall change in ventilation reflects the additive effects from each region. Those observations led to the view that central chemosensitivity derives from the independent actions of these multiple sites that then converge on the central respiratory controller to produce an integrated respiratory response (Fig. 45.12a). An alternate emerging view is that the RTN is a particularly important site of central CO2 chemosensitivity and that it extends excitatory glutamatergic projections to the preB€ otC to alter ventilation. This view further proposes that the RTN acts as an integration center for chemosensory information coming from the periphery and other sites in the CNS (see Fig. 45.12b). This proposed model is based on several observations. The RTN, which contains the most thoroughly characterized group of central chemosensitive neurons, receives axonal projections from other putative central chemosensitive regions. Experimentalists have selectively lesioned RTN neurons in adult rats and noted a marked decrease in CO2 sensitivity. Further, a genetically engineered mouse model that is lacking the Phox2b transcription factor is born without a functioning RTN (i.e., Phox2b is critical for RTN 45 Respiration 1443 Fig. 45.12 Hypothesized configurations of multiple chemosensory sites underlying central chemoreception. (a) The prevailing view is that multiple sites converge on the central respiratory controller (preB€otC/VRC) to produce an integrated CO2/pH respiratory response. (b) An emerging view is that the RTN is a key chemosensory site, but also the central integration site of chemosensory information arising from the other sites (including peripheral chemoreceptors) that then influences the respiratory controller. (c) A third possibility represents of hybrid of (a) and (b) in which only some regions feed through the RTN. Abbreviations: NTS nucleus tractus solitarius, RTN retrotrapezoid nucleus, RVLM rostroventrolateral medulla, VRC ventral respiratory column formation). Those mice are capable of generating a respiratory rhythm but lack a central CO2 response. In addition, in one of “mother nature’s” experiments, patients with the rare disorder called congenital central hypoventilation syndrome (discussed further in Genetics and Breathing Disorders) also lack Phox2b neurons and central chemosensitivity. A third model states that there is a hybrid organization where only some chemosensitive regions feed through the RTN and others work independently to control ventilation (Fig. 45.12c). 1444 J.J. Greer and G.D. Funk Molecular mechanism of CO2/pH sensing: How do chemosensitive neurons actually “sense” changes in pH? This basic issue has not been fully resolved, but acid-sensitive ion channels in the membranes of chemosensitive neurons are currently hypothesized as the proton sensors. Numerous candidate channels have been described and their pH sensitivity characterized in significant detail. Despite this, no single ion channel has been shown to directly contribute to central respiratory chemosensitivity. The search is confounded by the fact that the chemosensitivity of individual neurons may depend on multiple acid-sensitive ion channels. Also, the chemosensitivity of the different classes of chemosensitive neurons (e.g., RTN vs. locus coeruleus vs. raphe neurons) may depend on a different complement of acidsensitive ion channels. Finally, neurons that are not involved in respiratory chemosensation may also express acid-sensitive channels. A probable list of candidate acid-sensitive channels includes the following: specific subtypes of acid-sensitive two-pore domain K+ (TASK) channels (close with decreased pH); inwardly rectifying K+ channels (Kir1.1, Kir4.1-Kir5.1, close with decreased pH); acid-sensitive ion channels that open in response to decreased pH and permeate both Na+ and Ca2+; and ATP-gated P2X2 receptors. However, knockout mice lacking each of these candidate genes have essentially normal ventilatory responses to normoxic hypercapnia. Astrocytes: Chemosensory cells may not be limited to neurons. Within the RTN, astrocytes are emerging as important contributors to central chemoreception. They release ATP in response to decreases in pH via Ca2+-dependent, exocytotic release and possibly also via Ca2+-independent, CO2-sensitive gating of hemichannels (half of a gap junction that can open independently) (Fig. 45.13). The released ATP excites local chemosensitive RTN neurons and may account for 20% of the overall CO2 ventilatory response of the RTN. In summary, central chemosensitivity likely derives from multiple chemoreceptive cells and multiple chemosensory mechanisms. Mechanoreception A key component of any motor control system is a sensory network that provides rapid feedback about the status of the limbs, joints, and muscles to allow for necessary adjustments. Note that speed is critical since the afferent feedback is relevant for modification of the behavior as it is happening. For example, in locomotion, information from muscle spindle afferents provides critical information about muscle length and limb position, Golgi tendon organs provide information about muscle tension, and cutaneous afferents provide information about interactions between the limbs/body and the immediate environment. Similarly, the respiratory network receives breath-by-breath information about the mechanical status of respiratory muscles, airways, lungs, and thorax, to achieve efficient breathing. Unlike the locomotor system, however, very little of the afferent information comes from the muscles per se. The main source of mechanoreceptive feedback derives from visceral afferents from the lung, chest wall, and airways. These reflexes can be classified as regulatory or protective. 45 Respiration 1445 Fig. 45.13 Schematic illustration of the hypothetical mechanism(s) underlying the contribution of chemosensitive RTN neurons and astrocytes to central chemoreception. Elevated CO2 in the blood diffuses across the blood vessel/capillary wall, increasing CO2 and H+ in the extracellular space surrounding neurons and astrocytes (1). Astrocytes near the ventral medullary surface including those in the glia limitans respond in two ways. Depicted in the middle astrocyte, elevated CO2 (intracellular or extracellular) may evoke release of ATP through CO2-sensitive Cx26 hemichannels (i.e., Cx26 hemichannels act as the CO2 sensor) (2). Depicted in the right astrocyte, CO2 or H+ causes intracellular Ca2+ release (3) and Ca2+-dependent, exocytotic release of ATP (4). ATP released via one or both of these mechanisms excites chemosensitive RTN neurons through a P2Y (5), G-protein-coupled receptor-dependent mechanism that either modulates an unknown membrane conductance (6) or the acid-sensitive ion channels directly (7). RTN neurons are also directly sensitive to intra- or extracellular acidification; the H+ sensor may be K+ channels that are open at rest and close in response to increased H+ (8). Increased output from the RTN to the ventral respiratory column (VRC) (9) causes ventilation to increase. NOTE: ATP-dependent processes mediate 20% of the central chemosensory response. The remainder of the response reflects direct activation of RTN and other chemosensory neurons Regulatory reflexes are important in controlling both the rate and depth of normal breathing on a cycle-by-cycle basis. They are mediated by receptors that provide information relevant to the ongoing breathing (i.e., lung inflation, pressure in the trachea, chest wall/body position, etc.) and primarily change the pattern of respiratory motor output rather than the level of ventilation. The main class of regulatory reflexes is the Breuer-Hering respiratory reflexes that are mediated by slowly adapting receptors (SARs) located primarily in the smooth muscle of the airways. These receptors have a background level of tonic discharge 1446 J.J. Greer and G.D. Funk that increases with lung inflation/airway distension. They are referred to as SARs because their discharge decays slowly in response to a constant, sustained stimulus. SAR signals travel in rapidly conducting myelinated axons of the vagus (X) nerve to the first-order excitatory synapse on pump cells (P cells) in the NTS. Second-order synapses are on specific types of inspiratory and expiratory neurons in the VRC. The main impact of SAR feedback on basal breathing pattern is immediately apparent when input from the vagus nerve is removed. In the majority of mammals, this results in a very slow breathing frequency with each breath being of large amplitude (Fig. 45.14a). Surprisingly, in humans after heart-lung transplant, breathing is normal during relaxed wakefulness and sleep despite the absence of a control loop involving pulmonary mechanoreceptors. This suggests that vagal feedback is not as critical in adult humans as in other species. The two main Breuer-Hering reflexes are the inspiratory terminating reflex and the expiratory prolonging reflex. Both are mediated by SARs, but the evoked reflex is dependent on the phase of the respiratory cycle in which the afferent information arrives. The inspiratory terminating reflex is important in the breath-to-breath control of inspiratory duration (Fig. 45.14b). As the lungs inflate during inspiration, SAR activity increases, exciting P cells, which in turn excite neurons in the VRC that discharge late in the inspiratory phase. These “late-inspiratory neurons” terminate inspiration by inhibiting other inspiratory neurons (Fig. 45.15a). During expiration, SAR activity typically decreases with lung volume. However, if there is a sudden increase in SAR activity, or a slowing in the normal decrease in SAR activity that accompanies expiration, the elevated activity of P cells excites VRC neurons that show decrementing patterns of discharge during expiration. These “decrementing E cells” inhibit a type of inspiratory neuron important in terminating expiration and initiating inspiration. Their inhibition therefore delays the onset of the next inspiration (Fig. 45.15b). The hypothesized function is that if something limits expiratory airflow, this reflex extends the expiratory period to allow normal emptying of the lung. Proprioceptors in the diaphragm with afferent, myelinated axons in the phrenic nerve are sparse compared to most skeletal muscles but appear to operate in a manner analogous to that of the SARs, that is, their activation inhibits inspiratory neurons and shortens inspiration. Protective reflexes are mediated by receptors that have a low level of tonic discharge and become active only in unusual situations. These receptors are often chemoreceptors, supplied primarily by nonmyelinated fibers. In the upper airway (larynx), multimodal, nonmyelinated C fibers sensitive to temperature and irritants convey information centrally via the recurrent laryngeal or superior laryngeal nerves (branches of vagus nerve) and evoke cough, vocal cord adduction, and defensive changes in breathing pattern. Chemosensitive C fibers with axons in the vagus nerve are also present throughout the lower respiratory tract from the trachea to the distal portions of the bronchial tree. Stimulation of these neuron endings in the airways and alveolar wall by chemical irritants and edema (fluid in the lungs) evokes a brief apnea followed by rapid shallow breathing. The apnea is achieved through inhibition of central inspiratory and expiratory neurons. It is protective in that it prevents further inhalation of the offending compound. 45 Respiration 1447 Fig. 45.14 Breathing pattern in the presence of afferent feedback from slowly adapting receptors is faster and of smaller amplitude due to the SAR-mediated Breuer-Hering inspiratory termination reflex. (a) Recording of integrated motor activity from the phrenic nerve innervating the diaphragm in the presence of SAR feedback is faster and of smaller burst amplitude than in the absence of feedback (Modified with permission from Felman and Gauthier (1976) J Neurophysiol. 39:31–44). (b) Recordings of integrated nerve activity from the vagus nerve (mainly from SARs) and the motor activity from the phrenic nerve. In the absence of SAR feedback, inspiratory duration indicated by phrenic nerve activity reflects the intrinsic period of the rhythm generator (left panel). Normal incrementing discharge of SARs terminates inspiratory such that the inspiratory period in the presence of feedback is shorter than in its absence (right panel). (Traces from left are superimposed as dashed lines in the right panel to facilitate comparison of inspiratory period in the presence and absence of SAR feedback) (Redrawn from von Euler (1986) Handbook of Physiology, Sect. 3, The Respiratory System, Vol. II Control of Breathing. p. 1–68. Unpublished observations by C. von Euler and T. Trippenbach. Am Physiol Soc, used with permission) Rapidly adapting receptors, which have also been referred to as irritant receptors, are located in the epithelial and subepithelial layers of the mucosa in the lower respiratory tract and send myelinated axons centrally via the vagus nerve. They were first identified by their rapidly adapting response to rapid lung inflation. In addition to rapid inflation, RARs respond to large lung inflation or deflation, airborne irritants, and lung edema. Their activation is associated with a rapid burst of activity in phrenic motoneurons. They initiate multiple reflexes depending on the stimulus and location. Activation of RARs near the tracheal bifurcation by inhaled irritants elicits cough, a protective reflex that clears the airway. RARs can elicit rapid breathing and are also sensitive to changes in lung mechanics. During quiet resting breathing, lung compliance decreases by as much as 30% (i.e., it becomes stiffer). As lung stiffness increases, RARs discharge high-frequency bursts of action potentials at the peak of each inspiration. At some critical level of activity, the RARs trigger a sigh, or augmented breath, which restores lung volume and compliance to optimum values. 1448 J.J. Greer and G.D. Funk a b NA NTS Exp. Exp. neuron neuron IV Ventricle VRC Area Area Postrema Postrema Insp. Insp. term term neuron neuron Insp. Insp. neuron neuron Pump Pump cell cell Insp. Insp. preMN preMN excitatory excitatory inhibitory inhibitory To To Insp. Insp. MNs MNs SAR SAR Feedback Feedback c Lung Lung inflation inflation during during expiration expiration excites excites expiratory expiratory neurons, neurons, prolonging prolonging expiration expiration ∫Phrenic ∫Phrenic Nerve Nerve –10 –10 mV mV Mem. Mem. Potential Potential Exp. Exp. neuron neuron –60 –60 mV mV Tracheal Tracheal Pressure Pressure d 10 10 cm cm H H22O O Lung Lung inflation inflation 22 sec sec Lung Lung inflation inflation during during expiration expiration inhibits inhibits inspiratory inspiratory neurons, neurons, prolonging prolonging expiration expiration –40 –40 mV mV Mem. Mem. Potential Potential Insp. Insp. neuron neuron –60 –60 mV mV ∫Phrenic ∫Phrenic Nerve Nerve Tracheal Tracheal Pressure Pressure 10 10 cm cm H H2200 Lung Lung inflation inflation 22 sec sec Fig. 45.15 Breuer-Hering reflex pathways. (a) Dorsal view of rat brainstem showing respiratoryrelated nuclei (from Fig. 45.3) and hypothesized pathways underlying the Breuer-Hering inspiratory termination and expiratory prolonging reflexes. SARs in the large airways activate pump cells in the NTS. Activation of pump cells during inspiration excites inspiratory terminating neurons in the VRG. Activation of pump cells during expiration causes subsequent excitation of expiratory neurons that in turn inhibit inspiratory neurons necessary for initiating and maintaining inspiration; the result is expiratory prolongation. (b) Image of an expiratory and inspiratory neuron labeled with fluorescent markers while their respiratory-related changes in membrane potential (shown in the traces in panels c and d) were recorded with an intracellular electrode. (c) Membrane potential recording of an expiratory neuron like that shown in (b) along with inspiratory activity from the phrenic nerve. A recording of tracheal pressure shows that when the lung is inflated during expiration, the expiratory neurons discharge for much longer than during a normal breath, that is, expiration is prolonged. It is hypothesized that the increased activity of this type of expiratory cell is responsible for the inhibition of the inspiratory neuron evoked in (d) by lung inflation during expiration (Adapted with permission from Hayashi, Coles and McCrimmon (1996) J Neurosci. 16(20):6526–6536) 45 Respiration 1449 Respiratory Network Activity Is Integrated with Networks Controlling Sleep, Exercise, and Locomotion Sleep Sleep is a remarkably complex behavior that occupies approximately one third of our lives. It is virtually ubiquitous among animals - even fruit flies exhibit periods of inactivity akin to sleep. The physiological function of sleep is not fully understood; however, its necessity is obvious from the severely impaired mental, physical, and emotional function after even 24 h of sleep deprivation. Sleep can be divided into two main substates: non-REM (also referred to as slow wave or quiet sleep) and REM (also referred to as active, paradoxical, or dreaming sleep). NonREM sleep is further divided into four stages (I–IV) that we progress through sequentially each night to the deepest stage IV sleep. Cortical EEG activity in non-REM is easily distinguished from that in wakefulness by the emergence of slow, synchronous oscillations in brain activity. Periods of non-REM alternate with REM sleep, which in terms of brain activity is much more like wakefulness. Sleep networks interact significantly with motor and homeostatic control systems. Most significant in the context of respiratory control is that during REM sleep, most skeletal muscles show dramatic reductions in excitability. The muscle atonia of REM sleep is a signature feature of this state. The condition known as REM behavior disorder exemplifies the advantage of remaining motionless during REM. Those patients lack REM-related atonia and physically act out their dreams that can involve kicking, screaming, punching, and jumping out of bed. At the same time, paralysis of all respiratory muscles during sleep presents its own unique set of problems. Not surprisingly, there is differential sensitivity of respiratory muscles to this atonia. Phrenic motoneurons driving the main inspiratory pump muscle, the diaphragm, are least affected by pathways underlying REM sleep atonia. Motoneurons controlling airway muscles, in contrast, are like most other skeletal muscles and show dramatically reduced excitability in REM sleep. While this is not a concern for most animals, the highly compliant airway of humans, which evolved in association with the development of speech, predisposes some individuals with anatomically compromised airways to sleep-disordered breathing (see below; also Obstructive Sleep Apnea). Exercise Elite athletes can increase ventilation 30–40-fold to support maximum exercise. Ventilation levels >200 L/min have been recorded in elite cyclists, which compares with 5 L/min at rest. The respiratory network must control both movement and homeostasis to achieve this level of dynamic regulation (Fig. 45.16). The ventilatory response to moderate exercise follows a well-characterized time course that comprises three distinct phases (Fig. 45.17). At the onset of exercise, breathing increases rapidly (phase I), that is, within 10–15 s or often with the first 1450 J.J. Greer and G.D. Funk Fig. 45.16 Central respiratory networks control both movement and homeostasis, two of the major controlling functions of the brain breath. Minute ventilation and metabolic rate then increase gradually, in parallel over several minutes (phase II) to reach a steady state (phase III). The same phases are repeated in the same order when exercise stops, with a rapid phase I decrease in ventilation followed by a gradual decrease in ventilation and metabolic rate back to resting levels. The mechanisms that increase breathing in proportion to the increase in metabolic rate remain one of the most controversial issues in Physiology. It is a simple question. What is the trigger to increase ventilation when we exercise? The most intuitive answer is the decrease in oxygen and buildup of CO2 that result from increased activity. However, in virtually all animals tested, the ventilatory control system is so efficient in increasing ventilation during exercise that PaO2 actually increases and PaCO2 decreases. In other words, there is no blood-borne (humoral) chemical stimulus necessary to evoke the increase in ventilation. If humoral signals do not cause the initial ventilatory increase, what does? Valuable insight comes from the kinetics of the three-phase ventilatory response. The rapid phase I increase in ventilation occurs before any effect of increased muscle metabolism can alter blood gases and circulate to the chemoreceptors. This suggested to pioneers in the field in the late 1800s that neural (or neurogenic) signals in the form of feedback signals from the working muscles or feed-forward signals from locomotor centers in the brain to the respiratory networks may be important contributors. Hypothesized mechanisms have changed little since this very early work of Krogh and Lindhard. However, it now appears that the ventilatory response to exercise reflects coordinated interaction of neurohumoral and multiple neurogenic mechanisms. What remains is to determine the relative roles of these mechanisms in each of the different phases. 45 Respiration 1451 Fig. 45.17 Time course of the ventilatory response during moderate exercise and recovery. Phase I represents the rapid initial increase in ventilation that occurs at the transition from rest to exercise. Phase II represents the gradual increase into steady-state levels of ventilation that are achieved in phase III (Adapted from Wasserman, Whipp and Casaburi (1986) Handbook of Physiology, Sect. 3, The Respiratory System, Vol. II Control of Breathing. p. 595–619. Am Physiol Soc, used with permission) The phase I increase in ventilation is neurogenic in origin. An extensive body of creative experimentation has explored the contribution of various feedback signals to the phase I increase in ventilation. Activation of type I and II afferent fibers, large diameter myelinated fibers that carry information from muscle spindles and Golgi tendon organs, produce only minor increases in ventilation. Thus, these afferents are not considered as a major contributor; however, they may provide mechanoreceptive information about the timing of the locomotor cycle important for coordinating respiration with locomotor rhythm (see Locomotor-Respiratory Coordination). Type III, or small-diameter myelinated, fibers transmit information from free nerve endings in the muscle. Type IV, or nonmyelinated, fibers are chemosensitive and respond to local changes in K+, pH, etc. Approximately half of the type III and IV fibers respond to ergoreceptive stimuli (nonnoxious stimuli related to muscle contraction and/or metabolic products of muscle work); these ergoreceptive fibers are significant contributors to the rapid onset phase I increase in ventilation. Feed-forward neurogenic mechanisms from locomotor networks also contribute to the phase I increase in ventilation during exercise. Almost 100 years after it was originally hypothesized, investigators finally demonstrated in 1983 that parallel drives from higher centers that activate locomotor networks also activate respiratory centers and contribute to the ventilatory response to exercise. They stimulated a midbrain locomotor-inducing nucleus, the mesencephalic locomotor region, in decerebrate animals to activate locomotion. Rapid, parallel increases in ventilation were noted. When stimulation intensity increased, locomotor activity, metabolic rate, and ventilatory activity all increased in parallel. Under these conditions, as in intact animals, the increase in ventilation could reflect feedback or feed-forward processes. In the critical experiment, the animals were paralyzed and locomotor centers were again stimulated. In this case, in the complete 1452 J.J. Greer and G.D. Funk absence of all afferent feedback, the activity of central locomotor and respiratory networks still increased in parallel. Similar feed-forward mechanisms operate in humans. When conscious, but completely paralyzed subjects were asked to perform different types of exercise (handgrip, ankle dorsiflexion, contraction of all limb muscles), phase I increases in heart rate and blood pressure occurred, despite complete lack of any muscular contraction. In other words, despite the fact that not a single muscle had contracted, the mere central command to “exercise” was sufficient to evoke the initial response. Phases II and III of the ventilatory response are under neurogenic and neurohumoral control. Neurogenic feed-forward and feedback mechanisms continue in phases II and III. In addition, chemoreceptor mechanisms are implicated in these later phases. The delayed onset of phase II (10–15 s), which coincides roughly with delay for blood-borne transfer of chemical signals from exercising muscles to putative chemoreceptor sites, suggests a significant neurohumoral (blood-borne) contribution. In addition, the time course of the phase II increase in ventilation is sluggish in the absence of arterial chemoreceptors. Nevertheless, the precise nature of the blood-borne signal is uncertain because PO2, PCO2, and pH change little in submaximal exercise. Possibilities include catecholamines (i.e., epinephrine), O2, CO2, K+, or oscillations in pH, PCO2, and PO2. Thus, while feedback and feedforward mechanisms give an approximately proportional increase in ventilation and metabolic rate, peripheral chemoreceptors (with perhaps a small contribution from central chemoreceptors) appear to ensure precise matching. This interaction between neurogenic and neurohumoral mechanisms continues into phase III. Coordination of Breathing with Other Behaviors Respiratory muscles serve multiple functions outside of breathing. In some cases, the competing demands cannot be met simultaneously. The tongue, for example, is important in breathing (maintaining an open airway), suckling, swallowing, and speech. Breathing is not compatible with most of those behaviors; so only one can be expressed at a time. Breathing is stopped during swallowing via a reflex from sensory afferents in the upper airway. Similarly, breathing is stopped during the Valsalva maneuver which requires that the glottis is closed and thoracic and abdominal muscles are contracted to increase intrathoracic and intra-abdominal pressure. In contrast, speech is well coordinated with breathing as it occurs during the expiratory phase. Many of the muscles used in respiration also have postural or locomotor roles. Coordination between locomotor and respiratory rhythms is observed in virtually all vertebrate phyla. Quadrupeds from the gerbil to the rhinoceros take one breath per stride during galloping, with the breath always occurring at the same part of the locomotor cycle. Similarly, in some birds during flight, inspiration is synchronized 1:1 with the upstroke of the wings. Similarly, the number of strides taken per breath by trained human runners typically shows an integer relationship. A 2:1 ratio 45 Respiration 1453 appears to be preferred, but it is highly variable (4:1, 3:1, 2:1, 1:1, 5:2, and 3:2), both between and within subjects. This variability indicates the coordination is not simply due to an obligatory mechanical interaction. Coordination of locomotor and respiratory rhythms is achieved through a combination of feedback and feed-forward neural mechanisms. Afferent feedback, most likely from type I and II afferent fibers, entrains respiratory rhythm to the locomotor cycle via a pathway that involves first the pontine parabrachial respiratory group and subsequent inhibition of inspiratory neurons in the VRC. Additional entrainment arises from respiratory mechanoreceptors (SARs, RARs) that detect cyclic changes in thoracic pressure and volume. Afferent feedback, however, is not necessary. When “flight” is activated in birds (Canada geese) after complete muscular paralysis, the relationship between the neural outputs of the locomotor and respiratory networks to their respective muscles is synchronized (1:1). During free flight, it is primarily 3:1 but varies to 2:1 and 4:1. Thus, while the locomotor and respiratory networks interact centrally to produce coordinated outputs, under the labile conditions of actual locomotion, feedback from limb and respiratory mechanoreceptors is likely critical in altering respiratory timing to adjust the pattern of coordination. Functionally, such coordination is hypothesized to reduce the work of breathing by reducing the mechanical interference between locomotion and respiration and transferring part of the work of breathing from respiratory to locomotor muscles. For example, by acting as a bellows, the action of bending and extending the trunk during galloping in the horse may relieve the work of the diaphragm. Similarly, inertial oscillations of the viscera in hopping wallabies and trotting dogs may act as a piston mechanism that assists in producing respiratory airflow. One of the most intriguing examples of coordination between multiple motor networks is in insectivorous microchiropteran bats that use extremely highfrequency sound pulses to provide information about the location of airborne prey. Breathing and wingbeat are synchronized, with expiration occurring during downstroke. Vocalization is also synchronized such that the high-frequency echolocation pulses, which require extremely high rates of expiratory airflow, are only emitted during the downstroke of the wingbeat. The energetic demands of echolocation are extremely high for these bats at rest, but it comes at virtually no extra cost during flight due to this precise coordination of activities in these three networks. The Respiratory Control Network Is Highly Plastic Perinatal Environment Research into the developmental origins of health and disease has established that the environment in utero and during the postnatal period can have long-term effects on physiological processes. This includes the respiratory system that exhibits considerable plasticity in response to perinatal experiences. The effects 1454 J.J. Greer and G.D. Funk of hypoxia are a clear example. In utero hypoxia can lead to longer-term modifications of respiratory control that extend into the newborn period causing persistent hyperventilation and an exaggerated response to hypoxia. Repeated bouts of hypoxia in the immediate newborn period, such as those that occur with apnea of prematurity, can lead to a long-term increase in baseline ventilation, significantly heightened sensitivity to opiate-induced respiratory depression, and a decreased ventilatory response to hypoxia. Exposure to drugs in utero is another example, with nicotine being the best studied. Nicotine exposure in utero leads to the suppression of fetal breathing movements necessary for proper lung development. Longer-term effects that appear in the newborn period include reduced baseline ventilation, a more unstable breathing pattern, increased bouts of apnea, a diminished capacity for autoresuscitation following severe hypoxic exposure (e.g., face in pillow), and increased risk for sudden infant death syndrome (SIDS, discussed below). Plasticity in the Adult System It is only within the last 15 years that the conventional view of respiratory control networks as rigid, largely immutable structures has been replaced with the current view that recognizes the enormous capacity of the mature respiratory control system to undergo plasticity. For example, when goats and humans undergo repeated exercise trials (20–70) with added dead space (i.e., they breathe through a tube), the ventilatory response to exercise is enhanced. The enhanced response persists in subsequent baseline exercise trials after the dead space is removed. This persistent enhancement of the ventilatory response is referred to as longterm facilitation and is a form of motor learning or neuroplasticity. Long-term modulation of the ventilatory response to exercise in human has also been observed in response to repeated pairings of exercise with increased inspired CO2 (high number of pairing appears critical) and with inspiratory resistive loading (without an elevated CO2 stimulus). This latter finding is significant because it suggests that long-term modulation of ventilatory control is not limited to conditions associated with long-term disruptions in blood-gas homeostasis; it may extend to conditions in which respiratory system mechanics are altered for prolonged periods. In other words, respiratory plasticity (long-term modulation) may help individuals adapt and maintain appropriate respiratory responses to mechanical challenges that arise through weight gain, aging, and lung disease. The field of respiratory plasticity has advanced enormously over the last decade. Many of the intracellular signaling pathways responsible for the adaptive changes in the healthy nervous system of animal models of respiratory plasticity are being resolved. There is considerable excitement about the potential of exploiting these mechanisms to address clinical conditions, including traumatic injury (including spinal cord injury), obstructive sleep apnea, amyotrophic lateral sclerosis, and other neurodegenerative diseases in which respiration is compromised. 45 Respiration 1455 Respiratory Pathologies Result from Abnormal Neural Control Mechanisms Apnea, the term comes from the Greek word meaning “without wind.” Sleep apnea is a disorder characterized by abnormal pauses in breathing during sleep. There are three forms of sleep apnea: central, obstructive, and complex or mixed sleep apnea (i.e., a combination of central and obstructive). Sleep-disordered breathing describes a group of disorders characterized by abnormalities of respiratory pattern (pauses in breathing) during sleep. One of the most common and most serious of these conditions is obstructive sleep apnea (OSA; Fig. 45.18b). It affects upwards of 10% of adult males (5% of females) and is characterized by repeated episodes (hundreds/night) of airway obstruction, apnea, decreased blood O2, elevated CO2, blood pressure swings, and sudden arousals. This nightly series of harmful metabolic insults often lead to cardiovascular disease including angina, myocardial infarction, stroke, left ventricular dysfunction, and sustained daytime hypertension. Further, the loss of quality sleep results in a very marked increase in motor vehicle and occupational accidents. Mechanistically, it is important to emphasize that OSA is specific to sleep. Even patients with the most severe cases breathe normally when awake. The neurotransmitters that provide modulation of hypoglossal motoneuron activity, and presumably the activity of other airway motoneurons, change dynamically across sleep-wake states. The excitatory drive to upper airway motoneurons from medullary raphé neurons (release 5-HT, TRH, and SubP) and the locus coeruleus complex (release noradrenalin) declines from wakefulness to non-REM sleep, with minimal firing in REM. This loss of excitatory input during sleep is referred to as disfacilitation. In addition, there is active inhibition of upper airway motoneurons during sleep. Specifically, activation of a subset of cholinergic neurons involved in the generation of REM sleep causes net decreases in respiratory-related motoneuron activity, with muscarinic receptor-mediated suppression predominating over nicotinic excitation. Further active inhibition arises from the release of glycine from the nucleus pontis oralis that is activated during REM sleep. The relative importance of disfacilitation versus active inhibition in upper airway atonia during sleep and the potential role of other neurotransmitter systems are not fully understood. This, in part, explains the current lack of success in developing an effective pharmacological therapy for OSA. Central apnea occurs when there is a lack of respiratory drive from brainstem regions to pump and airway muscles (Fig. 45.18a). Conditions that can cause or lead to central sleep apnea include bulbar poliomyelitis, encephalitis affecting the brainstem, neurodegenerative illnesses such as Parkinson’s disease and multiple system atrophy, stroke affecting the brainstem, congestive heart failure, and use of certain medications such as narcotic-containing analgesics. Cheyne-Stokes respiration, whose description is attributed to the Irish physicians John Cheyne and George Stokes in the nineteenth century, is a specific pattern of breathing characterized by progressively deeper (increased tidal volume) and sometimes faster breathing followed by central apnea (Fig. 45.18c). The pattern 1456 J.J. Greer and G.D. Funk Fig. 45.18 Pathological patterns of breathing. (a) Central apnea is characterized by lack of central drive to respiratory muscles and thus loss of rib cage movement and air flow. (b) Obstructive apnea is associated with activation of respiratory rib cage and muscles as evident from movements of the thorax and abdomen, but there is reduced or no airflow due to obstruction of the airway. (c) Cheyne-Stokes respiration is characterized by repeated waxing and waning of progressively deeper breaths followed by apnea. (d) Left panel shows normal (eupnea) breathing R pattern as detected by integrated ( PN) and raw phrenic nerve recording (PN). Right panel shows experimentally induced apneusitic breathing pattern induced in anesthetized animal model after pontine lesion and cutting of the vagus nerve (Adapted with permission from Haji et al., Neurosci Res. 1998, 32:323–331) repeats with each cycle typically lasting 0.5–2 min and can occur during sleep and wakefulness, although more common during sleep. This pattern of breathing is thought to be caused by altered chemosensitivity to hypoxia and hypercapnia, together with a prolonged circulatory time, and is observed in association with heart failure and brainstem injury caused by stroke, tumors, or toxins. CheyneStokes respiration is also present in healthy individuals in the hypoxic environment of very high altitudes. Apneustic breathing (or apneusis – derived from the Greek word for breathholding) is characterized by a very prolonged inspiratory effort (Fig. 45.18d). In animal models, it is observed after concurrent removal of network elements that normally regulate the termination of inspiration, that is, SAR mechanosensory feedback from the vagus nerve and pontine respiratory groups. However, in the vast majority of human cases, vagal input is intact and trauma or stroke in the pons or medulla is the primary cause of apneusis. 45 Respiration 1457 Apnea of prematurity is defined as cessation of breathing by a premature infant that lasts for more than 15 seconds and is accompanied by low blood oxygen levels (hypoxemia) and lowered heart rate (bradycardia). Apnea of prematurity, which can be obstructive, central, and mixed, occurs to varying degrees in more than 85% of infants who are born at less than 34 weeks of gestation (full term is considered between 37 and 42 weeks). The incidence of apneas in premature infants is most common during REM sleep. Aside from conditions such as congenital abnormalities (e.g., cervicofacial malformations, laryngomalacia, vocal cord paralysis), there are two primary mechanisms involved in mixed/obstructive apneas in the newborn. The most frequently recognized mechanism is passive upper airway collapse during inspiration, which can occur under conditions such as flexion of the neck, nose obstruction, or lack of upper airway muscle tone. In addition, there can be active closure of the airway as part of the laryngeal chemoreflexes triggered by stimulation of laryngeal mucosal receptors in response to salivary or respiratory secretions, gastroesophageal reflux, and during feeding. Methylxanthines (theophylline and caffeine) are effective treatments for apnea of prematurity. Administration results in improved respiratory drive and reduced need for ventilation and diminishes the likelihood of chronic lung disease, cerebral palsy, and cognitive delay. Mechanisms of action are thought to include blockade of adenosine receptors that act through both peripheral and central effects and the additional increase in respiratory neuronal excitability associated with an elevation of cAMP levels. The currently recognized unwanted side effects include increased heart rate (tachycardia) and disruption of sleep-state architecture. The apneas typically subside as the infant matures during the first several postnatal weeks. It should be made clear that sudden infant death syndrome (SIDS), defined clinically as a sudden death of an infant that is unexpected by medical history and remains unexplained after postmortem investigation, is not correlated with apnea of prematurity. In fact, few infants die of SIDS under 1 month of age; rather, the incidence peaks between 2 and 4 months and then declines again after 5 months. The underlying cause(s) is/are unknown, but failed autoresuscitation from hypoxicinduced apnea sleep has been postulated to be involved in the sequence of events leading to SIDS. Major risk factors for SIDS include prone sleeping, maternal smoking, recent infection, and prematurity. The decline in the incidence of SIDS with the adaptation of positioning sleeping infants on their back or side has been very significant. Presumably, this has led to a decrease in the obstruction of breathing that can trigger the fatal hypoxic event in susceptible infants. Genetics and breathing disorders: In human infants, dysfunction of the neural control of breathing is seen in a variety of genetic diseases that include congenital central hypoventilation syndrome (CCHS), Rett syndrome, and Prader-Willi syndrome. CCHS is a rare condition typically caused by a mutation of the PHOX2B gene that encodes a transcription factor expressed in central and peripheral neurons involved in respiratory chemoreception, including those in the pFRG/ RTN. CCHS is characterized by adequate (although still compromised) ventilation while the patient is awake and by hypoventilation and apnea during sleep that often requires intervention with mechanical ventilation. CCHS was previously referred to 1458 J.J. Greer and G.D. Funk as Ondine’s curse in reference to the German folktale of Ondine, a water nymph who cursed her unfaithful husband. “As long as you are awake, you shall have your breath, but should you ever fall asleep, then that breath will be taken from you and you will die!” Rett syndrome results from a mutation in the X-linked gene encoding the transcription factor methyl-CpG-binding protein 2 (MeCP2) in neurons. Children with Rett syndrome typically appear normal until 6–18 months of life, when there is a prolonged period of regression characterized by a loss of communication and motor skills, onset of stereotyped hand movements, gait disturbances, and irregular breathing patterns that include periods of hyperventilation, breath-holding, and lifethreatening apneas. Unlike many CNS disorders of breathing which are specific or exaggerated in sleep, the breathing abnormalities are most prominent during wakefulness. This suggests an abnormality in regulatory inputs to the respiratory rhythm generating center that are most prominent when awake (e.g., associated with stress). Prader-Willi syndrome is a genetic disorder in which genes located on a specific region of chromosome 15 (q11-13) are deleted or unexpressed on the paternal chromosome. Prader-Willi syndrome patients typically have some degree of learning disabilities, low muscle tone, short stature if not treated with growth hormone, incomplete sexual development, and a chronic feeling of hunger that can lead to marked obesity. In addition there is an abnormally high incidence of central and obstructive apneas. Each of the above mentioned genetic conditions has provided insights into transcriptional control of neurons that modulate breathing. However, none of the genes underlying the respiratory phenotypes are expressed in the preB€otC per se. Recent insight into the transcriptional control of preB€otC development has been made with the observation that a deletion of the gene encoding the transcription factor Dbx1 (developing brain homeobox protein 1) in mice results in the loss of glutamatergic preB€ otC neurons and inspiratory rhythmogenesis. Further, expression of the Robo3 (roundabout homolog 3) gene is necessary for guiding the axons that cross the midline and ensure bilateral synchronous activity of preB€otC neurons. Summary of Major Concepts An understanding of the neural control of breathing starts with an appreciation of the source of basic respiratory rhythmic drive within the ventrolateral medulla. A network of neurons within the preB€ otC is necessary for the generation of the primary active phase of breathing, inspiration. The current hypothesis is that a second rhythmogenic circuit within the pFRG/RTN is important for generating the expiratory phase of activity during forceful breathing (i.e., active expiration). However, the final pattern of rhythmic drive that emerges and coordinates the activation of the rib cage and abdominal and upper airway musculature to produce normal (eupneic) breathing in intact mammals is ultimately determined by complex synaptic interactions between those rhythmogenic centers and other parts of the VRC, as well as the DRG and PRG. Further, similar to all motor systems, there is 45 Respiration 1459 a continuous feedback of afferent signals that tune the respiratory neural output. In the specific case of respiration, the concentration (or partial pressure) of oxygen and carbon dioxide and pH levels in blood, tissue, and the brain are the key components being regulated, and thus, feedback on their levels is particularly critical for the homeostatic control of respiratory network activity. This feedback arises from peripheral and central sources. Peripheral chemosensory information regarding plasma oxygen and carbon dioxide levels arises primarily from the aortic and carotid bodies. Centrally, there are multiple brainstem regions that contain neurons (and astrocytes) sensitive to pH that ultimately converge directly or indirectly on the central respiratory controller. Mechanoreceptive feedback from the lung, airways, and respiratory muscles further regulates breathing rate and pattern. During exercise, there is a very efficient neural feed-forward system in place whereby motor centers responsible for driving locomotor activity send parallel signals to respiratory networks and concomitantly respiratory activity. Afferent feedback from working muscles contributes to this neural drive and, in combination with chemoreceptor feedback, provides a precise matching of ventilation and metabolic rate. In addition, the respiratory control system is not hardwired and immutable. It can undergo long-term adaptive changes in response to environmental, physiological, and disease challenges. Thus, in general, the respiratory system is extremely robust, but also highly adaptable, operating efficiently in a seamless manner to meet a very wide range of metabolic and behavioral demands. However, there are certain developmental, state-dependent, and disease-related conditions where there are impairments in respiratory rhythmogenesis, afferent feedback, and/or drive to respiratory muscles. This is exemplified by the instability in respiratory rhythm and inability to keep the airways open in prematurely born infants. In adults, the trade-off of having a compliant upper airway necessary for speech has the downside of a susceptibility to airway obstruction during certain sleep states. Neuronal damage or deficiencies in key transcriptional control mechanisms, particularly within the brainstem, can affect chemoreception or the proper balance of synaptic drive from multiple brainstem regions and neuromodulators necessary for stable rhythmogenesis. Outlook In the context of understanding the organization and function of mammalian motor systems, the respiratory field is at a relatively advanced stage (e.g., compared to the locomotor system). However, there remain significant deficits in our understanding of some very fundamental mechanisms, including the following. Rhythmogenesis: The discovery of the preB€ otC was a seminal advancement in the field of respiratory neural control. The challenge going forward will be to determine the cellular and synaptic mechanisms within this region that are responsible for rhythm generation. There are sufficient data to provide a foundation for the group-pacemaker hypothesis, but it remains to be rigorously tested. Further, it is important to note that much of the cellular data on which current models of how 1460 J.J. Greer and G.D. Funk the respiratory rhythm is generated are derived from in vitro experiments using reduced perinatal rodent preparations. A thorough investigation of respiratory neuronal properties within the preB€ otC in intact models is lacking. Further, the underlying mechanisms may vary under different behavioral states and with age and disease. The proposal that the pFRG/RTN is a second oscillator responsible for the expiratory phase of breathing requires further verification. Very little is known about how rhythmogenesis is generated within the pFRG/RTN and how it couples with the preB€ otC. There are also questions regarding the relative role of the two oscillators at different developmental stages (i.e., the pFRG/RTN may play a more important role during the immediate newborn period) and behavioral states. How the respiratory activity within the PRG and DRG interacts and modulates preB€otC and pFRG/RTN rhythmic activity is not well understood. These regions are clearly essential in producing a fully evolved pattern of normal breathing. Application of novel methods such as optogenetics that allow neurons to be switched on and off in milliseconds using light could greatly facilitate a deeper understanding of the importance of these various brainstem nuclei. Neurochemical control: There are a multitude of neuromodulators that regulate breathing over multiple time scales. In addition, the relative balance of excitatory versus inhibitory synaptic input to rhythm-generating centers and respiratory motoneurons fluctuates across age (e.g., apnea of prematurity) and sleep-wake states (e.g., OSA). Further, there is a diverse number of receptor subtypes for each neurotransmitter family, and they have yet to be delineated among various respiratory nuclei. Developing drug therapies for central respiratory disorders is a serious unmet medical need, and advances will depend on a much more comprehensive understanding of the neurochemical control of breathing. This may require, for instance, the further development of microdialysis approaches and novel methods for measuring neurotransmitter levels in the extracellular space of respiratory nuclei in behaving animal models. The role of astrocytes in the modulation of respiratory network activity is also of emerging interest. Astrocytes express many of the same transmitter receptors as neurons, respond to the activation of these receptors, and in turn release neuroactive compounds that modulate synaptic function. Our understanding of their role will accelerate as pharmacological, molecular, and imaging (including optogenetic) approaches are developed that permit their selective manipulation. Chemoreception: Central and peripheral chemoreceptors are very active areas of investigation due to their critical role in respiratory homeostasis and respiratory disorders. Yet, the relative importance of the RTN, raphe complex, locus coeruleus, and other brainstem regions for central chemoreception is unclear. The role of glia in the processes has only recently gained attention. The most basic mechanisms by which changes in hydrogen ion concentration are detected and translated into changes in neuronal excitability and subsequently transmitted to induce a change in ventilation remain unresolved. Similarly, the mechanisms by which peripheral chemoreceptors respond to changes in PaO2 and PaCO2 are only partially understood. 45 Respiration 1461 Development and genetics: From a developmental perspective, the timing of preB€ otC formation, at least in the rodent, is fairly well understood. However, little is known about the development of other respiratory brainstem nuclei. Further, the genetic control mechanisms underlying the development of preB€otC and other respiratory nuclei are a newly emerging field. The recent discovery of the critical role of the DBX1 gene for preB€ otC formation is an important foundation. The deficiencies underlying abnormal breathing in genetic disorders such as Rett syndrome, Prader-Willi syndrome, and CCHS will hopefully be advanced by investigations of genetically engineered mouse models that replicate some of the disease characteristics. Importantly, those models provide opportunities for evaluating therapeutic interventions. Breathing in the newborn: Infants born prematurely are prone to having their central respiratory drive shut down and their airways collapse while sleeping. It is unclear whether this is due to an immaturity of the respiratory control network or a lack of appropriate balance of excitatory/inhibitory synaptic drive. Exposure to a hypoxic in utero environment alters the response to hypoxia after birth. The mechanisms underlying this clinically problematic phenomenon are not understood. A main predisposing factor to SIDS is prenatal nicotine exposure. However, it is not clear how this exposure alters the neural networks controlling breathing. Further, how those changes impair the infant’s ability to autoresuscitate in the presence of a hypoxic event is unknown. If SIDS is related to a failure of the respiratory system to mature properly, why is the peak period of deaths delayed to the 1–3-month period; what is critical about the time window? Further Reading Alheid GF, McCrimmon DR (2008) The chemical neuroanatomy of breathing. Respir Physiol Neurobiol 164:3–11 Feldman JL, Del Negro CA (2006) Looking for inspiration: new perspectives on respiratory rhythm. Nat Rev Neurosci 7:232–242 Feldman JL, Gautier H (1976) Interaction of pulmonary afferents and pneumotaxic centre in control of respiratory pattern in cats. J Neurophysiol 39:31–44 Gray PA, Janczewski WA, Mellen N, McCrimmon DR, Feldman JL (2001) Normal breathing requires preB€otzinger complex neurokinin-1 receptor-expressing neurons. Nat Neurosci 4(9):927–930 Greer JJ, Funk GD, Ballanyi K (2006) Preparing for the first breath: prenatal maturation of respiratory neural control. J Physiol 570(Pt 3):437–444 Guyenet PG, Stornetta RL, Bayliss DA (2010) Central respiratory chemoreception. J Comp Neurol 518:3883–3906 Haji A et al (1998) NMDA receptor-mediated inspiratory off-switching in pneumotaxicdisconnected cats. Neurosci Res 32:323–331 Hayashi F, Coles SK, McCrimmon DR (1996) Respiratory neurons mediating the Breuer-Hering reflex prolongation of expiration in rat. J Neurosci 16(20):6526–6536 Horner RL (2009) Emerging principles and neural substrates underlying tonic sleep-statedependent influences on respiratory motor activity. Philos Trans R Soc Lond B Biol Sci 364(1529):2553–2564 1462 J.J. Greer and G.D. Funk Janczewski WA, Feldman JL (2006) Distinct rhythm generators for inspiration and expiration in the juvenile rat. J Physiol 570:407–420 Loeschcke HH, Gertz KH (1958) Einflub des 02-Druckes in der Einatmungsluft auf die Atemt€atigkeit des Menschen gepr€ uft unter Konstanthaltung des alveolaren C02-Druckes. Pfleugers Archiv 267:460–477 Mitchell GS, Johnson SM (2003) Neuroplasticity in respiratory motor control. J Appl Physiol 94(1):358–374 Nielsen M, Smith H (1952) Studies on the regulation of respiration in acute hypoxia; with a appendix on respiratory control during prolonged hypoxia. Acta Physiologica Scand 24:293–313 Nurse CA (2010) Neurotransmitter and neuromodulatory mechanisms at peripheral arterial chemoreceptors. Exp Physiol 95(6):657–667 Oku Y et al (2007) Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla. J Physiol 585:175–186 Ramirez JM, Tryba AK, Peña F (2004) Pacemaker neurons and neuronal networks: an integrative view. Curr Opin Neurobiol 14(6):665–674 Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL (1991) Pre-Botzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254:726–729 Smith JC, Abdala AP, Rybak IA, Paton JF (2009) Structural and functional architecture of respiratory networks in the mammalian brainstem. Philos Trans R Soc Lond B Biol Sci 364:2577–2587 Thoby-Brisson M et al (2005) Emergence of the pre-B€ otzinger respiratory rhythm generator in the mouse embryo. J Neurosci 25:4307–4318 Thoby-Brisson M, Karlén M, Wu N, Charnay P, Champagnat J, Fortin G (2009) Genetic identification of an embryonic parafacial oscillator coupling to the preB€ otzinger complex. Nat Neurosci 12:1028–1035 von Euler C (1986) Brain stem mechanisms for generation and control of breathing pattern. 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