Parafacial Respiratory Group Research Articles

Retrotrapezoid nucleus, respiratory chemosensitivity and breathing automaticity.

Breathing automaticity and CO(2) regulation are inseparable neural processes. The retrotrapezoid nucleus (RTN), a group of glutamatergic neurons that express the transcription factor Phox2b, may be a crucial nodal point through which breathing automaticity is regulated to maintain CO(2) constant. This review updates the analysis presented in prior publications. Additional evidence that RTN neurons have central respiratory chemoreceptor properties is presented, but this is only one of many factors that determine their activity. The RTN is also regulated by powerful inputs from the carotid bodies and, at least in the adult, by many other synaptic inputs. We also analyze how RTN neurons may control the activity of the downstream central respiratory pattern generator. Specifically, we review the evidence which suggests that RTN neurons (a) innervate the entire ventral respiratory column and (b) control both inspiration and expiration. Finally, we argue that the RTN neurons are the adult form of the parafacial respiratory group in neonate rats.

CO2-Sensitive Preinspiratory Neurons of the Parafacial Respiratory Group Express Phox2b in the Neonatal Rat

Phox2b protein is a specific marker for neurons in the parafacial region of the ventral medulla, which are proposed to play a role in central chemoreception and postnatal survival. Mutations of PHOX2B cause congenital central hypoventilation syndrome. However, there have been no reports concerning electrophysiological characteristics of these Phox2b-expressing neurons in the parafacial region of the neonate immediately after birth. This region overlaps with the parafacial respiratory group (pFRG) composed predominantly of preinspiratory (Pre-I) neurons that are involved in respiratory rhythm generation. We studied (1) whether pFRG neurons are Phox2b immunoreactive or not and (2) whether they show intrinsic CO(2) chemosensitivity. We found that most pFRG/Pre-I neurons were Phox2b immunoreactive and depolarized upon increase in CO(2) concentration under condition of action potential-dependent synaptic transmission blockade by tetrodotoxin. We also confirmed that these pFRG neurons expressed neurokinin-1 receptor. They were tyrosine hydroxylase negative and presumed to be glutamatergic. Our findings suggest that Phox2b-expressing parafacial neurons play a role in respiratory rhythm generation as well as central chemoreception and thus are essential for postnatal survival.

Pacemakers handshake synchronization mechanism of mammalian respiratory rhythmogenesis

Inspiratory and expiratory rhythms in mammals are thought to be generated by pacemaker-like neurons in 2 discrete brainstem regions: pre-Bötzinger complex (preBötC) and parafacial respiratory group (pFRG). How these putative pacemakers or pacemaker networks may interact to set the overall respiratory rhythm in synchrony remains unclear. Here, we show that a pacemakers 2-way "handshake" process comprising pFRG excitation of the preBötC, followed by reverse inhibition and postinhibitory rebound (PIR) excitation of the pFRG and postinspiratory feedback inhibition of the preBötC, can provide a phase-locked mechanism that sequentially resets and, hence, synchronizes the inspiratory and expiratory rhythms in neonates. The order of this handshake sequence and its progression vary depending on the relative excitabilities of the preBötC vs. the pFRG and resultant modulations of the PIR in various excited and depressed states, leading to complex inspiratory and expiratory phase-resetting behaviors in neonates and adults. This parsimonious model of pacemakers synchronization and mutual entrainment replicates key experimental data in vitro and in vivo that delineate the developmental changes in respiratory rhythm from neonates to maturity, elucidating their underlying mechanisms and suggesting hypotheses for further experimental testing. Such a pacemakers handshake process with conjugate excitation-inhibition and PIR provides a reinforcing and evolutionarily advantageous fail-safe mechanism for respiratory rhythmogenesis in mammals.

The homeodomain protein Dbx1 specifies a vital population of respiratory interneurons in the mouse

Recent advances in the neurobiology of breathing indicate that the Respiratory Rhythm Generator (RRG) relies on the coupling of two distinct oscillators ventrally located in the brainstem: the para-facial respiratory group (pFRG), comprising pre-inspiratory neurons, and the PreBötzinger complex (PreBötC) playing a dominant role in pacing inspiration. Although vital for life ex utero, the prenatal development of the RRG remains poorly understood. Particularly little is known about the origin in the neural tube of interneurons forming these oscillators. We are addressing this issue considering homeodomain proteins defining distinct progenitor domains along the dorsal ventral (DV) axis of the neural tube. We here report on the role of Dbx1, expressed in the domain of neural progenitors giving rise to the V0 class of interneurons in the mouse. Electrophysiological recordings performed on en bloc and preBötC slice preparations from Dbx1 null (Dbx1LacZ/LacZ) embryos show that the preBötC, but not the pFRG, fails to become active. At birth, Dbx1 null neonates immediately die from a complete failure to breathe. Anatomical analysis in Dbx1LacZ/+ embryos revealed the presence of beta-Gal expressing cells in the preBötC but not the pFRG area. Optical recording of these cells in the Dbx1GFP/GFP line using the calcium indicator Calcium-green-1 AM ascertained their rhythmic status. The majority of beta-Gal cells in the preBötC expressed the mRNA for the type 2 vesicular glutamate transporter (vGlut2) suggesting their role in excitatory synaptic processing. Indeed, the absence of rhythmic activity in Dbx1LacZ/LacZ embryos was associated to a massive loss of vGlut2 expressing cells in the preBötC Glutamatergic synaptic transmission (i) within the preBötC on one side and (ii) over the midline across the left and right preBötC was found impaired in the mutant. We conclude that Dbx1-derived V0 interneurons constitute an obligatory component of the preBötC oscillator.

Hypoplasia of the Parafacial/Facial Complex: A Very Frequent Finding in Sudden Unexplained Fetal Death

AIMS - To define firstly in man the localization and the anatomical boundaries of the parafacial respiratory group in the brainstem. Thereafter, to determine whether this center, given its essential role in the respiratory rhythm- generating circuit, showed abnormalities in sudden unexplained perinatal and infant deaths, like other nuclei and/or struc- tures of the brainstem and cerebellum checking vital functions, that we have previously reported. METHODS - In 67 brains collected from 29 stillbirths, 9 newborns and 29 infants, died of both known and unknown cause, an in-depth histological examination of the autonomic nervous system was made, according to the protocol rou- tinely followed by the Institute of Pathology, University of Milan. In particular we analyzed the parafacial and facial nu- clei in serial sections of caudal pons. RESULTS - We firstly identified and defined the normal structure of the parafacial/facial complex in control cases. Be- sides we diagnosed the hypoplasia of these nuclei in 75% of sudden unexplained fetal deaths and never after birth. CONCLUSIONS - We formulate the hypothesis that the hypoplasia of the parafacial/facial complex is a specific marker of unexplained stillbirth, and that the normal development of this complex is essential for extra-uterine life.

Reconfiguration of respiratory-related population activity in a rostrally tilted transversal slice preparation following blockade of inhibitory neurotransmission in neonatal rats

Recent studies showed that respiratory rhythm generation depends on oscillators located in the pre-Bötzinger complex (pre-BötC) and the parafacial respiratory group (pFRG). To study inhibitory synaptic interactions between these two oscillators, we developed a rostrally tilted transversal slice preparation, which preserves these regions. The onset of rhythmic mass activity in the retrotrapezoid nucleus (RTN)/pFRG preceded that of the pre-BötC. Blockade of glycinergic and gamma-aminobutyric acidic inhibition synchronized pre-BötC and RTN/pFRG activity and significantly increased pre-BötC burst frequency, amplitude, and duration. Population imaging revealed recruitment of inspiratory-like neurones, while expiratory-like neurones lost their phasic activity. The reconfiguration after disinhibition reveals: (1) synaptic inhibition of the pre-BötC arising from the RTN/pFRG, (2) excitatory drive from the RTN/pFRG that triggers the pre-BötC burst. Our findings support the view that these synaptic interactions in vitro relate to the initiation of the inspiratory phase or to the steering of the expiratory-inspiratory phase transition in vivo.

Anoxic persistence of lumbar respiratory bursts and block of lumbar locomotion in newborn rat brainstem–spinal cords

The tolerance of breathing in neonates to oxygen depletion is reflected by persistence of inspiratory-related motor output during sustained anoxia in newborn rat brainstem preparations. It is not known whether lumbar motor networks innervating expiratory abdominal muscles are, in contrast, inhibited by anoxia similar to locomotor networks in neonatal mouse lumbar cords. To test this, we recorded inspiratory-related cervical/hypoglossal plus pre/postinspiratory lumbar/facial nerve activities and, sometimes simultaneously, locomotor rhythms in newborn rat brainstem-spinal cords. Chemical anoxia slowed 1 : 1-coupled cervical and lumbar respiratory rhythms and induced cervical burst doublets associated with depressed preinspiratory and augmented postinspiratory lumbar activities. Similarly, anoxia evoked repetitive hypoglossal bursts and shifted facial activity toward augmented postinspiratory bursting in medullas without spinal cord. Selective lumbar anoxia augmented pre/postinspiratory lumbar bursting without slowing the rhythm. This suggests a medullary origin of both anoxic inspiratory double bursts and preinspiratory depression, but a mixed medullary/lumbar origin of boosted postinspiratory lumbar activity. Lumbar respiratory rhythm is likely to be generated by the parafacial respiratory group expiratory centre as indicated by lack of normoxic and anoxic bursting following brainstem transection between the facial motonucleus and the more caudal pre-Bötzinger complex inspiratory centre. Opposed to sustained respiratory activities, anoxia reversibly abolished non-rhythmic spinal discharges and electrically or chemically evoked lumbar locomotor activities, followed by pronounced postanoxic spinal hyperexcitability. We hypothesize that (i) the anoxia tolerance of neonatal breathing includes pFRG-driven lumbar expiratory networks, (ii) the anoxic respiratory pattern transformation is due to disturbed inspiratory-expiratory centre interactions, and (iii) postanoxic lumbar hyperexcitability contributes to spasticity in cerebral palsy.

Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla

Two putative respiratory rhythm generators (RRGs), the para-facial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC), have been identified in the neonatal rodent brainstem. To elucidate their functional roles during the neonatal period, we evaluated developmental changes of these RRGs by optical imaging using a voltage-sensitive dye. Optical signals, recorded from the ventral medulla of brainstem-spinal cord preparations of neonatal (P0-P4) rats (n = 44), were analysed by a cross correlation method. With development during the first few postnatal days, the respiratory-related activity in the pFRG reduced and shifted from a preinspiratory (P0-P1) to an inspiratory (P2-P4) pattern, whereas preBötC activity remained unchanged. The mu-opioid agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) augmented preinspiratory activity in the pFRG, while the mu-opioid antagonist naloxone induced changes in spatiotemporal activation profiles that closely mimicked the developmental changes. These results are consistent with the recently proposed hypothesis by Janczewski and Feldman that the pFRG is activated to compensate for the depression of the preBötC by perinatal opiate surge. We conclude that significant reorganization of the respiratory neuronal network, characterized by a reduction of preinspiratory activity in the pFRG, occurs at P1-P2 in rats. The changes in spatiotemporal activation profiles of the pFRG neurones may reflect changes in the mode of coupling of the two respiratory rhythm generators.

Distinct roles of Hoxa2 and Krox20in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening

BackgroundLittle is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of Hoxa2, the only Hox gene expressed up to r2, and of Krox20, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of Hoxa2 by Krox20 in r3.ResultsWe found that Hoxa2 mutants displayed an impaired oro-buccal reflex, similarly to Krox20 mutants. In contrast, while Krox20 is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency, Hoxa2 inactivation did not affect neonatal breathing frequency. Instead, we found that Hoxa2-/- but not Krox20-/- mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of Hoxa2 expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory.ConclusionThus, inspiratory shaping and respiratory frequency are under the control of distinct Hox-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.

Point:Counterpoint: Medullary pacemaker neurons are essential for both eupnea and gasping in mammals vs. medullary pacemaker neurons are essential for gasping, but not eupnea, in mammals

to the editor: Implicit in the commentaries concerning the possible role of medullary pacemakers in the neurogenesis of eupnea and gasping is one critical and recurring question: How are in vitro rhythms related to these classical patterns of automatic ventilatory activity? Suzue ([5][1]) considered

Functional Imaging Reveals Respiratory Network Activity During Hypoxic and Opioid Challenge in the Neonate Rat Tilted Sagittal Slab Preparation

In mammals, respiration-modulated networks are distributed rostrocaudally in the ventrolateral quadrant of the medulla. Recent studies have established that in neonate rodents, two spatially separate networks along this column-the parafacial respiratory group (pFRG) and the pre-Bötzinger complex (preBötC)-are hypothesized to be sufficient for respiratory rhythm generation, but little is known about the connectivity within or between these networks. To be able to observe how these networks interact, we have developed a neonate rat medullary tilted sagittal slab, which exposes one column of respiration-modulated neurons on its surface, permitting functional imaging with cellular resolution. Here we examined how respiratory networks responded to hypoxic challenge and opioid-induced depression. At the systems level, the sagittal slab was congruent with more intact preparations: hypoxic challenge led to a significant increase in respiratory period and inspiratory burst amplitude, consistent with gasping. At opioid concentrations sufficient to slow respiration, we observed periods at integer multiples of control, matching quantal slowing. Consistent with single-unit recordings in more intact preparations, respiratory networks were distributed bimodally along the rostrocaudal axis, with respiratory neurons concentrated at the caudal pole of the facial nucleus, and 350 microns caudally, at the level of the pFRG and the preBötC, respectively. Within these regions neurons active during hypoxia- and/or opioid-induced depression were ubiquitous and interdigitated. In particular, contrary to earlier reports, opiate-insensitive neurons were found at the level of the preBötC.

The pre-Bötzinger oscillator in the mouse embryo

Studies of the sites and mechanisms involved in mammalian respiratory rhythm generation point to two clusters of rhythmic neurons forming a coupled oscillator network within the brainstem. The location of these oscillators, the pre-Bötzinger complex (preBötC) at vagal level, and the para-facial respiratory group at facial level, probably result from regional patterning schemes specifying neural types in the hindbrain during embryogenesis. Here, we report evidence that the preBötC oscillator (i) is first active at embryonic stages, (ii) originates in the post-otic hindbrain neural tube and (iii) requires the glutamate vesicular transporter 2 for rhythm generation.

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal

The following letters are in response to the Point:Counterpoint series “The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal” that appears in this issue. To the Editor : Unique data from three of our past

The Last Word: Point:Counterpoint authors respond to commentaries on “The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal”

To the Editor : We hypothesize that respiratory rhythm originates from two interacting generators; the preBotzinger complex (preBotC) and the retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG). Some discussants view our hypothesis as too simplistic and proposed concepts of state-

Novel Data Supporting the Two Respiratory Rhythm Oscillator Hypothesis. Focus on “Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats”

Among the most rewarding moments in scientific discovery are when the pieces of a puzzle provided by different laboratories start to fall into place, forming a coherent and new view of the world. Several years ago, a consensus was emerging that respiratory rhythm in mammals arose from a single

Ontogeny of central rhythm generation in chicks and rodents

Recent studies help in understanding how the basic organization of brainstem neuronal circuits along the anterior-posterior (AP) axis is set by the Hox-dependent segmentation of the neural tube in vertebrate embryos. Neonatal respiratory abnormalities in Krox20(-/-), Hoxa1(-/-) and kreisler mutant mice indicate the vital role of a para-facial (Krox20-dependent, rhombomere 4-derived) respiratory group, that is distinct from the more caudal rhythm generator called Pre-Bötzinger complex. Embryological studies in the chick suggest homology and conservation of this Krox20-dependent induction of parafacial rhythms in birds and mammals. Calcium imaging in embryo indicate that rhythm generators may derive from different cell lineages within rhombomeres. In mice, the Pre-Bötzinger complex is found to be distinct from oscillators producing the earliest neuronal activity, a primordial low-frequency rhythm. In contrast, in chicks, maturation of the parafacial generator is tightly linked to the evolution of this primordial rhythm. It seems therefore that ontogeny of brainstem rhythm generation involves conserved processes specifying distinct AP domains in the neural tube, followed by diverse, lineage-specific regulations allowing the emergence of organized rhythm generators at a given AP level.

Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats

There are at least two respiration-related rhythm generators in the medulla: the pre-Bötzinger complex, which produces inspiratory (Insp) neuron bursts, and the parafacial respiratory group (pFRG), which produces predominantly preinspiratory (Pre-I) neuron bursts. The pFRG Pre-I neuron activity has not been correlated with motor neuron activity in slice or block preparations of rostral medulla. In this study, we attempted to detect pFRG Pre-I activity as motor output in the rostral medulla. We recorded respiratory activity of the facial nerve in the brain stem-spinal cord preparation of 0- to 2-day-old rats. Facial nerve activity consisted of preinspiratory, Insp, and postinspiratory activity. Pre- and postinspiratory activity corresponded well with membrane potential trajectories of Pre-I neurons in the rostral ventrolateral medulla. In response to perfusion of 1 microM DAMGO (a mu-opiate agonist), fourth cervical ventral root (C4) Insp activity was depressed and facial nerve activity continued to synchronize with Pre-I neuron bursts. After transverse sectioning between the levels of the pre-Bötzinger complex and the pFRG, C4 Insp activity recovered within 15 min, but facial nerve activity was inhibited. When DAMGO was applied, C4 Insp activity was inhibited, and rhythmic facial nerve activity recovered. Subsequent elevation of K+ concentration reinduced C4 activity, but facial nerve activity was inhibited. Whole cell recordings in the rostral block revealed the presence of putative Pre-I neurons, the activity of which was synchronized with facial nerve activity. These results show that the rostral medulla, not including the pre-Bötzinger complex, produces Pre-I-like rhythmic activity that can be monitored as facial nerve motor output in newborn rat in vitro preparations.

Regulation of Ventral Surface Chemoreceptors by the Central Respiratory Pattern Generator

The rat retrotrapezoid nucleus (RTN) contains neurons described as central chemoreceptors in the adult and respiratory rhythm-generating pacemakers in neonates [parafacial respiratory group (pfRG)]. Here we test the hypothesis that both RTN and pfRG neurons are intrinsically chemosensitive and tonically firing neurons whose respiratory rhythmicity is caused by a synaptic feedback from the central respiratory pattern generator (CPG). In halothane-anesthetized adults, RTN neurons were silent below 4.5% end-expiratory (e-exp) CO2. Their activity increased linearly (3.2 Hz/1% CO2) up to 6.5% (CPG threshold) and then more slowly to peak approximately 10 Hz at 10% CO2. Respiratory modulation of RTN neurons was absent below CPG threshold, gradually stronger beyond, and, like pfRG neurons, typically (42%) characterized by twin periods of reduced activity near phrenic inspiration. After CPG inactivation with kynurenate (KYN), RTN neurons discharged linearly as a function of e-exp CO2 (slope, +1.7 Hz/1% CO2) and arterial pH (threshold, 7.48; slope, 39 Hz/pH unit). In coronal brain slices (postnatal days 7-12), RTN chemosensitive neurons were silent at pH 7.55. Their activity increased linearly with acidification up to pH 7.2 (17 Hz/pH unit at 35 degrees C) and was always tonic. In conclusion, consistent with their postulated central chemoreceptor role, RTN/pfRG neurons encode pH linearly and discharge tonically when disconnected from the rest of the respiratory centers in vivo (KYN treatment) and in vitro. In vivo, RTN neurons receive respiratory synchronous inhibitory inputs that may serve as feedback and impart these neurons with their characteristic respiratory modulation.

Respiratory rhythm generation in the in vitro preparations: The role of the para-facial respiratory group

Respiratory rhythm generation in the in vitro preparations: The role of the para-facial respiratory group