Noeud Vital and the Respiratory Centers.

We now recognize that the main breathing generator resides principally in the medulla oblongata. Vivisectionists-specifically, Julien Legallois-discovered "the respiratory center." Cutting through the brainstem stops respiration but not if the medulla remains intact and the brain is sliced in successive portions. Pierre Flourens localized surgical ablation experiments further identified a 1-mm area in the medulla, which he called vital knot or node (noeud vital). Detailed characterization had to wait until the 1920s, when Lumsden carried out more specific transection experiments to improve morphological differentiation of the respiratory center into inspiratory and expiratory divisions.

Serotonergic projections to the ventral respiratory column from raphe nuclei in rats.

The ventral respiratory column (VRC) generates rhythmical respiration and is divided into four compartments: the Bötzinger complex (BC), pre-Bötzinger complex (PBC), rostral ventral respiratory group (rVRG), and caudal ventral respiratory group (cVRG). Serotonergic nerve fibers are densely distributed in the rostral to caudal VRC and serotonin would be one of the important modulators for the respiratory control in the VRC. In the present study, to elucidate detailed distribution of serotonergic neurons in raphe nuclei projecting to the various rostrocaudal levels of VRC, we performed combination of retrograde tracing technique by cholera toxin B subunit (CTB) with immunohistochemistry for tryptophan hydroxylase 2 (TPH2). The double-immunoreactive neurons with CTB and TPH2 were distributed in the both rostral and caudal raphe nuclei, i.e. dorsal raphe nucleus, raphe magnus nucleus, gigantocellular reticular nucleus alpha and ventral parts, lateral paragigantocellular nucleus, parapyramidal area, raphe obscurus nucleus, and raphe pallidus nucleus. The distributions of double-immunoreactive neurons were similar among injection groups of BC, PBC, anterior rVRG, and posterior rVRG/cVRG. In conclusion, serotonergic neurons in both rostral and caudal raphe nuclei projected throughout the VRC and these serotonergic projections may contribute to respiratory responses to various environmental and vital changes.

A novel excitatory network for the control of breathing.

Breathing must be tightly coordinated with other behaviours such as vocalization, swallowing, and coughing. These behaviours occur after inspiration, during a respiratory phase termed postinspiration. Failure to coordinate postinspiration with inspiration can result in aspiration pneumonia, the leading cause of death in Alzheimer's disease, Parkinson's disease, dementia, and other neurodegenerative diseases. Here we describe an excitatory network that generates the neuronal correlate of postinspiratory activity in mice. Glutamatergic-cholinergic neurons form the basis of this network, and GABA (γ-aminobutyric acid)-mediated inhibition establishes the timing and coordination relative to inspiration. We refer to this network as the postinspiratory complex (PiCo). The PiCo has autonomous rhythm-generating properties and is necessary and sufficient for postinspiratory activity in vivo.The PiCo also shows distinct responses to neuromodulators when compared to other excitatory brainstem networks. On the basis of the discovery of the PiCo, we propose that each of the three phases of breathing is generated by a distinct excitatory network: the pre-Bötzinger complex, which has been linked to inspiration; the PiCo, as described here for the neuronal control of postinspiration; and the lateral parafacial region (pF(L)), which has been associated with active expiration, a respiratory phase that is recruited during high metabolic demand.

Neural mechanisms underlying respiratory rhythm generation in the lamprey.

The isolated brainstem of the adult lamprey spontaneously generates respiratory activity. The paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator, has been anatomically and functionally characterized. It is sensitive to opioids, neurokinins and acetylcholine. Excitatory amino acids, but not GABA and glycine, play a crucial role in the respiratory rhythmogenesis. These results are corroborated by immunohistochemical data. While only GABA exerts an important modulatory control on the pTRG, both GABA and glycine markedly influence the respiratory frequency via neurons projecting from the vagal motoneuron region to the pTRG. Noticeably, the removal of GABAergic transmission within the pTRG causes the resumption of rhythmic activity during apnea induced by blockade of glutamatergic transmission. The same result is obtained by microinjections of substance P or nicotine into the pTRG during apnea. The results prompted us to present some considerations on the phylogenesis of respiratory pattern generation. They may also encourage comparative studies on the basic mechanisms underlying respiratory rhythmogenesis of vertebrates.

Involvement of the avian song system in reproductive behaviour.

The song system of songbirds consists of an interconnected set of forebrain nuclei that has traditionally been regarded as dedicated to the learning and production of song. Here, however, we suggest that the song system could also influence muscles used in reproductive behaviour, such as the cloacal sphincter muscle. We show that the same medullary nucleus, retroambigualis (RAm), that projects upon spinal motoneurons innervating expiratory muscles (which provide the pressure head for vocalization) and upon vocal motoneurons for respiratory-vocal coordination also projects upon cloacal motoneurons. Furthermore, RAm neurons projecting to sacral spinal levels were shown to receive direct projections from nucleus robustus arcopallialis (RA) of the forebrain song system. Thus, by indicating a possible disynaptic relationship between RA and motoneurons innervating the reproductive organ, in both males and females, these results potentially extend the role of the song system to include consummatory as well as appetitive aspects of reproductive behaviour.

Anatomical and functional pathways of rhythmogenic inspiratory premotor information flow originating in the pre-Bötzinger complex in the rat medulla.

The pre-Bötzinger complex (preBötC) of the ventrolateral medulla is the kernel for inspiratory rhythm generation. However, it is not fully understood how inspiratory neural activity is generated in the preBötC and propagates to other medullary regions. We analyzed the detailed anatomical connectivity to and from the preBötC and functional aspects of the inspiratory information propagation from the preBötC on the transverse plane of the medulla oblongata. Tract-tracing with immunohistochemistry in young adult rats demonstrated that neurokinin-1 receptor- and somatostatin-immunoreactive neurons in the preBötC, which could be involved in respiratory rhythmogenesis, are embedded in the plexus of axons originating in the contralateral preBötC. By voltage-imaging in rhythmically active slices of neonatal rats, we analyzed origination and propagation of inspiratory neural activity as depolarizing wave dynamics on the entire transverse plane as well as within the preBötC. Novel combination of pharmacological blockade of glutamatergic transmission and mathematical subtraction of the video images under blockade from the control images enabled to extract glutamatergic signal propagations. By ultra-high-speed voltage-imaging we first demonstrated the inter-preBötC conduction process of inspiratory action potentials. Intra-preBötC imaging with high spatiotemporal resolution during a single spontaneous inspiratory cycle unveiled deterministic nonlinearities, i.e., chaos, in the population recruitment. Collectively, we comprehensively elucidated the anatomical pathways to and from the preBötC and dynamics of inspiratory neural information propagation: (1) From the preBötC in one side to the contralateral preBötC, which would synchronize the bilateral rhythmogenic kernels, (2) from the preBötC directly to the bilateral hypoglossal premotor and motor areas as well as to the nuclei tractus solitarius, and (3) from the hypoglossal premotor areas toward the hypoglossal motor nuclei. The coincidence of identified anatomical and functional connectivity between the preBötC and other regions in adult and neonatal rats, respectively, indicates that this fundamental connectivity is already well developed at the time of birth.

Brainstem respiratory networks: building blocks and microcircuits.

Breathing movements in mammals are driven by rhythmic neural activity generated within spatially and functionally organized brainstem neural circuits comprising the respiratory central pattern generator (CPG). This rhythmic activity provides homeostatic regulation of gases in blood and tissues and integrates breathing with other motor acts. We review new insights into the spatial-functional organization of key neural microcircuits of this CPG from recent multidisciplinary experimental and computational studies. The emerging view is that the microcircuit organization within the CPG allows the generation of multiple rhythmic breathing patterns and adaptive switching between them, depending on physiological or pathophysiological conditions. These insights open the possibility for site- and mechanism-specific interventions to treat various disorders of the neural control of breathing.

Chapter 3--networks within networks: the neuronal control of breathing.

Breathing emerges through complex network interactions involving neurons distributed throughout the nervous system. The respiratory rhythm generating network is composed of micro networks functioning within larger networks to generate distinct rhythms and patterns that characterize breathing. The pre-Bötzinger complex, a rhythm generating network located within the ventrolateral medulla assumes a core function without which respiratory rhythm generation and breathing cease altogether. It contains subnetworks with distinct synaptic and intrinsic membrane properties that give rise to different types of respiratory rhythmic activities including eupneic, sigh, and gasping activities. While critical aspects of these rhythmic activities are preserved when isolated in in vitro preparations, the pre-Bötzinger complex functions in the behaving animal as part of a larger network that receives important inputs from areas such as the pons and parafacial nucleus. The respiratory network is also an integrator of modulatory and sensory inputs that imbue the network with the important ability to adapt to changes in the behavioral, metabolic, and developmental conditions of the organism. This review summarizes our current understanding of these interactions and relates the emerging concepts to insights gained in other rhythm generating networks.

Chapter 14--looking forward to breathing.

I provide a personal view of the developments since ~1986 that underlie the contemporary view(s) about how the rhythm of breathing is generated and how the pattern of breathing is modulated. Two sites in the mammalian brainstem are likely to participate in respiratory rhythm generation: the preBötzinger Complex (preBötC), first described and intensely investigated since 1990, plays a well-documented essential role in normal breathing in mammals of all ages and may be principally involved in controlling inspiratory motor activity, and the retrotrapezoid/parafacial respiratory group (RTN/pFRG) that appears to play at least a modulatory role in neonatal and juvenile rodents and may be a conditional oscillator that controls active expiration.

Central respiratory chemoreception.

By definition central respiratory chemoreceptors (CRCs) are cells that are sensitive to changes in brain PCO(2) or pH and contribute to the stimulation of breathing elicited by hypercapnia or metabolic acidosis. CO(2) most likely works by lowering pH. The pertinent proton receptors have not been identified and may be ion channels. CRCs are probably neurons but may also include acid-sensitive glia and vascular cells that communicate with neurons via paracrine mechanisms. Retrotrapezoid nucleus (RTN) neurons are the most completely characterized CRCs. Their high sensitivity to CO(2) in vivo presumably relies on their intrinsic acid sensitivity, excitatory inputs from the carotid bodies and brain regions such as raphe and hypothalamus, and facilitating influences from neighboring astrocytes. RTN neurons are necessary for the respiratory network to respond to CO(2) during the perinatal period and under anesthesia. In conscious adults, RTN neurons contribute to an unknown degree to the pH-dependent regulation of breathing rate, inspiratory, and expiratory activity. The abnormal prenatal development of RTN neurons probably contributes to the congenital central hypoventilation syndrome. Other CRCs presumably exist, but the supportive evidence is less complete. The proposed locations of these CRCs are the medullary raphe, the nucleus tractus solitarius, the ventrolateral medulla, the fastigial nucleus, and the hypothalamus. Several wake-promoting systems (serotonergic and catecholaminergic neurons, orexinergic neurons) are also putative CRCs. Their contribution to central respiratory chemoreception may be behavior dependent or vary according to the state of vigilance.

Respiratory responses induced by blockades of GABA and glycine receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit.

The respiratory role of GABA(A), GABA(B) and glycine receptors within the Bötzinger complex (BötC) and the pre-Bötzinger complex (preBötC) was investigated in alpha-chloralose-urethane anesthetized, vagotomized, paralysed and artificially ventilated rabbits by using bilateral microinjections (30-50 nl) of GABA and glycine receptor agonists and antagonists. GABA(A) receptor blockade by bicuculline (5mM) or gabazine (2mM) within the BötC induced strong depression of respiratory activity up to apnea. The latter was reversed by hypercapnia. Glycine receptor blockade by strychnine (5mM) within the BötC decreased the frequency and amplitude of phrenic bursts. Bicuculline microinjections into the preBötC caused decreases in respiratory frequency and the appearance of two alternating different levels of peak phrenic activity. Strychnine microinjections into the preBötC increased respiratory frequency and decreased peak phrenic amplitude. GABA(A), but not glycine receptor antagonism within the preBötC restored respiratory rhythmicity during apnea due to bicuculline or gabazine applied to the BötC. GABA(B) receptor blockade by CGP-35348 (50mM) within the BötC and the preBötC did not affect baseline respiratory activity, though microinjections of the GABA(B) receptor agonist baclofen (1mM) into the same regions altered respiratory activity. The results show that only GABA(A) and glycine receptors within the BötC and the preBötC mediate a potent control on both the intensity and frequency of inspiratory activity during eupneic breathing. This study is the first to provide evidence that these inhibitory receptors have a respiratory function within the BötC.

Retrotrapezoid nucleus and parafacial respiratory group.

The rat retrotrapezoid nucleus (RTN) contains about 2000 Phox2b-expressing glutamatergic neurons (ccRTN neurons; 800 in mice) with a well-understood developmental lineage. ccRTN neuron development fails in mice carrying a Phox2b mutation commonly present in the congenital central hypoventilation syndrome. In adulthood, ccRTN neurons regulate the breathing rate and intensity, and may regulate active expiration along with other neighboring respiratory neurons. Prenatally, ccRTN neurons form an autonomous oscillator (embryonic parafacial group, e-pF) that activates and possibly paces inspiration. The pacemaker properties of the ccRTN neurons probably vanish after birth to be replaced by synaptic drives. The neonatal parafacial respiratory group (pfRG) may represent a transitional phase during which ccRTN neurons lose their group pacemaker properties. ccRTN neurons are activated by acidification via an intrinsic mechanism or via ATP released by glia. In summary, throughout life, ccRTN neurons seem to be a critical hub for the regulation of CO(2) via breathing.

Study of the human hypoglossal nucleus: normal development and morpho-functional alterations in sudden unexplained late fetal and infant death.

This study evaluated the development and the involvement in sudden perinatal and infant death of the medullary hypoglossal nucleus, a nucleus that, besides to coordinate swallowing, chewing and vocalization, takes part in inspiration. Through histological, morphometrical and immunohistochemical methods in 65 cases of perinatal and infant victims (29 stillbirths, 7 newborns and 29 infants), who died of both unknown and known cause, the authors observed developmental anomalies of the hypoglossal nucleus (HGN) in high percentage of sudden unexplained fetal and infant deaths. In particular, HGN hypoplasia, hyperplasia, positive expression of somatostatin and absence of interneurons were frequently found particularly in infant deaths, with a significant correlation with maternal smoking.

Fluorescence imaging of active respiratory networks.

Breathing in mammals is controlled by neural networks in the brainstem such as the pre-Bötzinger complex (preBötC) and the parafacial respiratory group (pFRG). Exploring these rhythmogenic networks and their interactions is greatly facilitated by live fluorescence imaging that enables analysis of (i) spatiotemporal patterns of respiratory (population) activities, (ii) (sub)cellular signaling in identified respiratory neurons, and (iii) membrane properties of respiratory neurons that are fluorescence-tagged for characteristic markers. Transversal medullary slices containing the preBötC and "en bloc" brainstem-spinal cord preparations with a functional preBötC/pFRG dual respiratory center which interacts, e.g., with pontine structures, are used for respiratory imaging in perinatal rodents. Imaging of less reduced (mature) respiratory networks is feasible in arterially-perfused "working-heart-brainstem" preparations from rodents. In these in situ models, imaging with voltage and Ca2+ sensitive dyes is established for assessment of respiratory (population) activities. Here, we summarize findings from diverse live imaging approaches in these models and point out potential pitfalls and future perspectives of respiratory-related optical recording.

The brainstem respiratory network: an overview of a half century of research.

This review aims at summarizing the work performed over 40 years by Professor Armand Bianchi and the research team he directed, which was devoted to the study of the central respiratory network. The major steps towards the understanding of this complex network will be presented together with methodological considerations. This includes the sequential progress that was made in the identification and characterization of respiratory neurons as deduced from inferences gleaned from intracellular recordings, which revealed putative synaptic connections within the respiratory network. Also reviewed is a comparison of in vivo versus in vitro approaches. The search for the "real" respiratory neurons must consider that those neurons are redundantly represented within the brainstem and express a wide variety of patterns. The last part of this review focuses on the concept that the brainstem respiratory circuitry forms part of a multifunctional network subserving both respiration and non-respiratory motor behaviors. Numerous data provide evidence that the respiratory network operates as a dynamic assembly of neurons, some of which can belong to several networks involved in the coordination of respiratory muscles during functions that include coughing, swallowing and vomiting.

5-HT2A receptors are concentrated in regions of the human infant medulla involved in respiratory and autonomic control.

The serotonergic (5-HT) system in the human medulla oblongata is well-recognized to play an important role in the regulation of respiratory and autonomic function. In this study, using both immunocytochemistry (n=5) and tissue section autoradiography with the radioligand (125)I-1-(2,5-dimethoxy-4-iodo-phenyl)2-aminopropane (n=7), we examine the normative development and distribution of the 5-HT(2A) receptor in the human medulla during the last part of gestation and first postnatal year when dramatic changes are known to occur in respiratory and autonomic control, in part mediated by the 5-HT(2A) receptor. High 5-HT(2A) receptor binding was observed in the dorsal motor nucleus of the vagus (preganglionic parasympathetic output) and hypoglossal nucleus (airway patency); intermediate binding was present in the nucleus of the solitary tract (visceral sensory input), gigantocellularis, intermediate reticular zone, and paragigantocellularis lateralis. Negligible binding was present in the raphé obscurus and arcuate nucleus. The pattern of 5-HT(2A) immunoreactivity paralleled that of binding density. By 15 gestational weeks, the relative distribution of the 5-HT(2A) receptor was similar to that in infancy. In all nuclei sampled, 5-HT(2A) receptor binding increased with age, with significant increases in the hypoglossal nucleus (p=0.027), principal inferior olive (p=0.044), and medial accessory olive (0.038). Thus, 5-HT(2A) receptors are concentrated in regions involved in autonomic and respiratory control in the human infant medulla, and their developmental profile changes over the first year of life in the hypoglossal nucleus critical to airway patency and the inferior olivary complex essential to cerebellar function.

Avian nucleus retroambigualis: cell types and projections to other respiratory-vocal nuclei in the brain of the zebra finch (Taeniopygia guttata).

In songbirds song production requires the intricate coordination of vocal and respiratory muscles under the executive influence of the telencephalon, as for speech in humans. In songbirds the site of this coordination is suspected to be the nucleus retroambigualis (RAm), because it contains premotor neurons projecting upon both vocal motoneurons and spinal motoneurons innervating expiratory muscles, and because it receives descending inputs from the telencephalic vocal control nucleus robustus archopallialis (RA). Here we used tract-tracing techniques to provide a more comprehensive account of the projections of RAm and to identify the different populations of RAm neurons. We found that RAm comprises diverse projection neuron types, including: 1) bulbospinal neurons that project, primarily contralaterally, upon expiratory motoneurons; 2) a separate group of neurons that project, primarily ipsilaterally, upon vocal motoneurons in the tracheosyringeal part of the hypoglossal nucleus (XIIts); 3) neurons that project throughout the ipsilateral and contralateral RAm; 4) another group that sends reciprocal, ascending projections to all the brainstem sources of afferents to RAm, namely, nucleus parambigualis, the ventrolateral nucleus of the rostral medulla, nucleus infra-olivarus superior, ventrolateral parabrachial nucleus, and dorsomedial nucleus of the intercollicular complex; and 5) a group of relatively large neurons that project their axons into the vagus nerve. Three morphological classes of RAm cells were identified by intracellular labeling, the dendritic arbors of which were confined to RAm, as defined by the terminal field of RA axons. Together the ascending and descending projections of RAm confirm its pivotal role in the mediation of respiratory-vocal control.

Background sodium current underlying respiratory rhythm regularity.

Rhythm-generating neural circuits underlying diverse behaviors such as locomotion, sleep states, digestion and respiration play critical roles in our lives. Irregularities in these rhythmic behaviors characterize disease states--thus, it is essential that we identify the ionic and/or cellular mechanisms that are necessary for triggering these rhythmic behaviors on a regular basis. Here, we examine which ionic conductances underlie regular or 'stable' respiratory activities, which are proposed to underlie eupnea, or normal quiet breathing. We used a mouse in vitro medullary slice preparation containing the rhythmogenic respiratory neural circuit, called the preBötzinger complex (preBötC), that underlies inspiratory respiratory activity. We varied either [K(+)](o) or [Na(+)](o), or blocked voltage-gated calcium channels, while recording from synaptically isolated respiratory pacemakers, and examined which of these manipulations resulted in their endogenous bursting becoming more irregular. Of these, lowering [Na(+)](o) increased the irregularity of endogenous bursting by synaptically isolated pacemakers. Lowering [Na(+)](o) also decreased the regularity of fictive eupneic activity generated by the ventral respiratory group (VRG) population and hypoglossal motor output. Voltage clamp data indicate that lowering [Na(+)](o), in a range that results in irregular population rhythm generation, decreased persistent sodium currents, but not transient sodium currents underlying action potentials. Our data suggest that background sodium currents play a major role in determining the regularity of the fictive eupneic respiratory rhythm.

Brain-derived neurotrophic factor enhances fetal respiratory rhythm frequency in the mouse preBötzinger complex in vitro.

Brain-derived neurotrophic factor (BDNF) is required during the prenatal period for normal development of the respiratory central command; however, the underlying mechanisms remain unknown. To approach this issue, the present study examined BDNF regulation of fetal respiratory rhythm generation in the preBötzinger complex (preBötC) of the mouse, using transverse brainstem slices obtained from prenatal day 16.5 animals. BDNF application (100 ng/mL, 15 min) increased the frequency of rhythmic population activity in the preBötC by 43%. This effect was not observed when preparations were exposed to nerve growth factor (100 ng/mL, 30 min) or pretreated with the tyrosine kinase inhibitor K252a (1 h, 200 nm), suggesting that BDNF regulation of preBötC activity requires activation of its cognate tyrosine receptor kinase, TrkB. Consistent with this finding, single-cell reverse transcription-polymerase chain reaction experiments showed that one third of the rhythmically active preBötC neurons analysed expressed TrkB mRNA. Moreover, 20% expressed BDNF mRNA, suggesting that the preBötC is both a target and a source of BDNF. At the network level, BDNF augmented activity of preBötC glutamatergic neurons and potentiated glutamatergic synaptic drives in respiratory neurons by 34%. At the cellular level, BDNF increased the activity frequency of endogenously bursting neurons by 53.3% but had no effect on basal membrane properties of respiratory follower neurons, including the Ih current. Our data indicate that BDNF signalling through TrkB can acutely modulate fetal respiratory rhythm in association with increased glutamatergic drive and bursting activity in the preBötC.