The brainstem respiratory network: An overview of a half century of research

Respiratory Physiology & Neurobiology 168 (2009) 4–12

Contents lists available at ScienceDirect

Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol

Frontiers review

The brainstem respiratory network: An overview of a half century of research Armand L. Bianchi, Christian Gestreau ∗ CRN2M, Département PNV, MP3-Respiration, Université Aix-Marseille II & III, Marseille, France

a r t i c l e

i n f o

Article history: Accepted 22 April 2009 Keywords: Brainstem respiratory network Central pattern generator Experimental models Non-respiratory behaviors Network reconfiguration

a b s t r a c t 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. © 2009 Elsevier B.V. All rights reserved.

1. Introduction During the second half of the 20th century the concept of a respiratory center has progressively evolved from an undefined respiratory neuronal system as part of the brainstem reticular formation to the modern concept of a central pattern generator (CPG). During this period various respiratory neuronal groups were recognized within the brainstem each of them having a well defined function in producing the respiratory drive to the motor outputs and breathing muscles. Those looking for the real respiratory neurons within the brainstem might keep in mind that for O2 up take and CO2 release, air breathing animals and specifically mammals have developed neuronal groups which must coordinate the activities of two essential parts of the respiratory system: one is devoted to create air aspiration and air expulsion, the pump muscles, and the other for regulating air flow, the valve muscles (Fig. 1). In addition, they might be aware that the respiratory muscles are involved in several functions other than respiration. In these circumstances the respiratory CPG must adapt to a new behavioral situation quickly and to do this undergo rapid reconfiguration. The CPG can be seen as an

∗ Corresponding author at: CRN2M, Département de Physiologie Neurovégétative (PNV), Equipe MP3-Respiration, Université Paul Cézanne, Campus Saint-Jérôme, Case 362, Avenue Escadrille Normandie-Niemen, 13397 Marseille cedex 20, France. Tel.: +33 491288451; fax: +33 4912888885. E-mail address: [email protected] (C. Gestreau). 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.04.019

dynamic assembly of neurons able to produce a rhythmic discharge that can easily adapted to generate different breathing patterns, and to contribute almost entirely or only partially to non-respiratory behaviors such as coughing, swallowing or vomiting. 2. Discharge activities of the respiratory motor outputs versus spike discharge activities of brainstem respiratory neurons The sequence of breathing is composed of three essential phases as defined by Diethelm Richter (1982), i.e., inspiration (I), post-inspiration (or early expiration, E1) and late expiration (E2). In eupnea, the main inspiratory pump muscles, the diaphragm, and external intercostal muscles with their respective nerves develop an augmenting inspiratory activity, which is prolonged by a declining activity during early expiration. The expiratory outputs (abdominal muscles and internal intercostal muscles) are active in late expiration, although the phase of expiration can be passive in quiet breathing so that no discharge is observed in these motor outputs (Fig. 1). It is also important to consider the patterns of activity of the nerves driving the valve muscles since their motoneurons are included within the brainstem together with the respiratory neurons of the CPG (Fig. 1A and C). Their axons are distributed within several cranial nerves. The vagus nerve and its branch the recurrent laryngeal nerve (RLN) contain motoneurons active in inspiration (for abduction of the glottis) and motoneurons active in early expiration (for adduction of the glottis that reduce the airflow at this

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

5

Fig. 1. (A) Dorsal view of the brainstem showing locations of respiratory neurons in dorsal respiratory group (DRG) and ventral respiratory column (VRC), their axonal projections, and cranial nerves to the valve muscles. Respiratory cycle progresses in three phases: I inspiration; E1 post-inspiration or early expiration; E2 late expiration. (B) Six basic patterns of action potentials recorded in brainstem DRG and VRC. Discharge activities in cranial outputs (C) and spinal respiratory outputs (D). The data were obtained from several decerebrate cats. Time duration of respiratory cycles has been matched using computer software. Abbreviations: Pre-I, pre-inspiratory; e-I; early inspiratory; I aug, inspiratory augmenting; Late I, late inspiratory; Post I, post-inspiratory; E aug, expiratory augmenting; Phr, phrenic nerve; Abd, L1 lumbar nerve branch; RLN, recurrent laryngeal nerve; SLN, superior laryngeal nerve; VII, facial nerve; IX, glossopharyngeal nerve; X, vagus nerve; PH-X: pharyngeal branch of the X.

period). These changes in upper airway resistance associated with the respiratory cycle are determinant for ventilation. Indeed, the negative pressure generated by contraction of diaphragm and external intercostal muscles in inspiration must be counteracted by a decrease in upper airway resistance to prevent the risk of obstructive apnea. Conversely, an increase in upper airway resistance in early expiration allows a smooth transition between inspiration and expiration. It decreases the speed of the expiratory airflow, preventing therefore alveolar collapse. Other brainstem motoneurons innervating the oro-pharyngeal region and their respective nerves are active during the respiratory cycle. The glossopharyngeal nerve (IX) is responsible for pharyngeal dilatation in inspiration, the pharyngeal branch of the vagus nerve (PH-X) for pharyngeal constriction in expiration, and the hypoglossal nerve (XII) for tongue protrusion in inspiration (mainly via contraction of the genioglossus muscle) (Fig. 1A and C). Both pharyngeal dilatator and tongue protrusor muscles contribute to upper airway patency, i.e. they maintain an open airspace for effective breathing and prevent obstructive apneas (Chan et al., 2006). However, recordings of genioglossus muscle activity or XII activity in conscious awake rats reported little phasic respiratory-related tongue movements or XII discharges as compared with sleep states or anesthesia (Jelev et al., 2001; Roda et al., 2004; Chan et al., 2006; Besnard et al., 2007). By contrast, robust inspiratory-related XII discharges are more often observed in decerebrate or anesthetized animals with vagotomy (Grélot et al., 1989; Kubin et al., 1992) than when the vagi are left intact (for example Gestreau et al., 1996, 2000). These observations suggest that changes in arterial PCO2 and/or vagal afferent inputs contribute significantly to phasic respiratory activity of tongue muscles. Many other oro-pharyngeal muscles also active during respiration are innervated by the facial nerve and branches of the fifth nerve (not shown in Fig. 1).

Extracellular unitary recordings from the respiratory neurons in the brainstem were largely developed during the second half of the 20th century. In most studies of this type, the major concerns were the description of the discharge patterns of the respiratory neurons and their anatomical location (Batsel, 1964). The identification of neurons was performed by comparison of their patterns of discharge with those recorded from the peripheral nerves, i.e., augmenting or decrementing discharge as in phrenic or laryngeal nerves, respectively (Fig. 1B–D). On this basis, an important contribution was made by Morton Cohen (1970). He induced changes in respiratory rhythmicity and compared the changes in activity of individual respiratory brainstem neurons with the changes of the overall respiratory cycle, as indicated by the phrenic nerve discharge. He used three major experimental variables, CO2 tension, lung inflation and brainstem stimulation. Based on the responses of the various classes of neurons, the functional role of such neurons within the central respiratory network could be hypothesized. 3. Antidromic stimulation: a tool for functional identification of brainstem respiratory neurons Another step was reached when additional characterization of the respiratory neurons was obtained by using antidromic stimulation of their axonal pathway. In the mid sixties such a method of identification was used to identify the spinal axons of inspiratory neurons located in the nucleus of the solitary tract (NTS; Nakayama and Baumgarten, 1964). Subsequent studies identified the spinal axons of respiratory neurons of the ventro-lateral medulla (Merrill, 1970). Extensive extracellular recording studies of brainstem respiratory neurons during systematic microstimulation of the either the

6

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

spinal cord or vagus nerve for inducing antidromic invasion was performed (Bianchi, 1969, 1971, 1974). These studies made it possible to recognize three anatomical and functional categories of brainstem respiratory neurons: • Bulbo-spinal neurons sending their axons down to the spinal cord. • Propriobubar neurons playing the role of an interneuronal brainstem network. • Laryngeal motoneurons sending their axons down to the vagus (laryngeal) nerve. Characterization of propriobulbar neurons was made on the basis of a negative finding, i.e. the lack of antidromic activation following stimulation of the vagus nerve or the spinal cord. Thus, this test does not constitute a definitive evidence for the existence of such neurons, although results obtained after viral transfection (Dobbins and Feldman, 1995) and cross-correlation analyses (Graham and Duffin, 1985; Duffin et al., 2000) confirm their existence. In associating the discharge patterns of the medullary respiratory neurons with their axonal projections (Fig. 1A and B) it was observed that (Bianchi, 1974): • Pre-inspiratory (Pre-I) and early-inspiratory (e-I) discharging neurons are mainly propriobulbar interneurons. • Augmenting inspiratory and late discharging inspiratory neurons are mainly bulbo-spinal neurons or pre-motoneurons (but not exclusively, a few can be propriobulbar). • Augmenting expiratory neurons are mainly bulbo-spinal in the caudal medulla but propriobulbar in the rostral medulla (see next Section 4). • Post-inspiratory neurons are often laryngeal motoneurons but some of them are propriobulbar, including medullary laryngeal pre-motoneurons (Baekey et al., 2001). Inspiratory augmenting neurons could be identified as propriobulbar, and in some examples as motoneurons, although the latter exhibited slight changes in discharge patterns. 4. Locations of respiratory neurons and their axonal projections The functional characterization of the brainstem respiratory neurons described above is also linked to their anatomical location in either the dorsal respiratory group (DRG) in the NTS region, or the ventral respiratory column (VRC) in the ventro-lateral medulla including the nucleus ambiguus (NA; Fig. 1A and B). • The DRG contains mainly inspiratory bulbo-spinal neurons in cats but not in rats (Duffin et al., 2000), and this includes phrenic premotoneurons. • The VRC contains both bulbo-spinal and propriobulbar neurons in close vicinity with the motoneurons of the NA in cats, as well as in rats. • Cranial (laryngeal and pharyngeal) motoneurons of the brainstem constitute the NA, the rostral border of which extends towards the facial motor nucleus. They all innervate valve muscles and exhibit respiratory activity. This column of motoneurons can be considered as belonging anatomically to the VRC since bulbo-spinal neurons and laryngeal or pharyngeal neurons can be found in close proximity. The axonal projections of respiratory neurons of the rostral VRC named retrofacial nucleus target the VRC or DRG (Merrill, 1974; Richter et al., 1979; Bianchi and Barillot, 1982; Grélot et al., 1988). In addition, another important respiratory region is located in the

dorso-lateral pons, and has been named the pontine respiratory group (PRG). Using the antidromic method, it has been shown that reciprocal connections exist between the PRG and the medullary respiratory regions (Bianchi and St John, 1981, 1982). More recent in vivo and in situ experiments in rats have identified a precise role for the Kölliker–Fuse nucleus (KF). This PRG nucleus controls the duration of the respiratory cycle and, more specifically, the postinspiratory phase (Dutschmann and Herbert, 2006; Smith et al., 2007). It is also involved in apneic responses associated with the diving reflex which is accompanied by prolonged activation of postinspiration (Dutschmann and Herbert, 1996; Chamberlin and Saper, 1998). Importantly, the KF nucleus differentially controls pump versus respiratory valve muscle contraction during respiratory and non-respiratory functions (Gestreau et al., 2005, 2008; Dutschmann et al., 2007). Indeed, glutamate-induced stimulation of the intermediate KF triggers a long lasting suppression of phasic inspiratory XII nerve discharge with minimal change in phrenic nerve activity (see Fig. 5 in Gestreau et al., 2005). By contrast, lesion of the same region resulted in an increase in XII nerve discharge associated not only with inspiration, but also with pharyngeal swallowing (see Fig. 7 in Gestreau et al., 2005). Recent data also indicate that injection of the excitatory peptide orexin in the intermediate KF enhances pre-inspiratory activity of the XII nerve (Dutschmann et al., 2007). Since a lack of orexin is associated with exaggerated sleep apneas, a sleep state during which a decrease in tongue muscle activity occurs (Gestreau et al., 2008), we suggest that the KF nucleus is an important determinant in airway patency during sleep–wake transitions. The reported changes in activity of tongue muscles is likely mediated by hypoglossal premotor neurons located at the ventral border of the KF that can control both tongue protusors and retrusors (Roda et al., 2004; Gestreau et al., 2005). Thus, together with the demonstration that the KF nucleus can influence post-inspiratory activity of laryngeal motoneurons (Dutschmann and Herbert, 2006), the data indicated above suggest that the KF contributes to coordination of respiratory muscles during respiratory and non-respiratory behaviors. 5. Synaptic connections Another important step was to determine the nature of the synaptic connections between respiratory brainstem neurons. This was inferred from intracellular recording and confirmed by crosscorrelation and spike-triggered averaging. Stable intracellular recordings of respiratory neurons were obtained during the last decades of the 20th century providing information on the time course and intensity of rhythmic depolarization and repolarization of respiratory neurons in anesthetized animals (Ballantyne and Richter, 1984, 1986). By examination of membrane potential trajectories during the respiratory phases it was inferred that reciprocal synaptic inhibition exists between I augmenting and E augmenting neurons. Similarly the membrane potential trajectories of Early I and Post I neurons indicated a possible reciprocal inhibitory influence also existed between these two neuronal groups (see details in Ballantyne and Richter, 1984). Cross-correlation studies and spike-triggered averaging were also used to investigate and confirm the nature of synaptic connections between respiratory neurons as inferred from intracellular recordings (Graham and Duffin, 1981; Madden and Remmers, 1982; Merrill et al., 1983; Feldman and Speck, 1983; Hilaire et al., 1984; Graham and Duffin, 1985; Duffin et al., 2000). 6. Model for respiratory rhythm generation Close examination of these connections allowed a model of respiratory rhythm generation to be developed (Bianchi et al., 1995). In this model, the inhibitory connections are considered as the main

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

type of connections between the different categories of brainstem respiratory neurons. These connections were assumed on the basis of responses to several manipulations or measurements during intracellular recordings. These include transfer of chloride from the micropipette into the cell by a negative current injection, thereby inducing reversal of inhibitory post-synaptic potentials. Also, input resistance was inferred by changes in voltage responses of neurons to current pulses (for example Bianchi et al., 1988). In addition, putative neurotransmitters involved in respiratory drive potentials were determined by iontophoresic applications (for example Pierrefiche et al., 1991). As regards rhythm generation, the proposed model of the respiratory CPG corresponds to the three phases (I, E1, E2) oscillator, as initially described by Richter (1982). It is important to note that the pre-motoneurons or bulbo-spinal neurons are considered as part of the respiratory CPG since they are involved in the processes of re-excitation and ramp excitation. The later processes are indeed considered as crucial for pattern generation. As already stated in a former review (Bianchi et al., 1995), respiratory pattern and rhythm generation are established by both excitatory and inhibitory synaptic inputs which transform short-lasting events like action potentials into longer processes that determine the three phases of the respiratory motor sequence. Thus, any alteration of the brainstem respiratory network such as transections (Smith et al., 2007) or blockade of synaptic transmission (Paton and Richter, 1995; Pierrefiche et al., 1998; Funke et al., 2008) induces drastic changes in both pattern and rhythm generation, resulting in inspiratory-like bursts before complete rhythm extinction. 7. Contribution of in vitro preparations Isolated brainstem spinal cord preparations (Suzue, 1984; Onimaru and Homma, 1987; Hilaire et al., 1989) as well as brainstem slices (Champagnat et al., 1983; Dekin and Getting, 1984; Smith et al., 1991) contain neurons with intrinsic oscillatory properties located in respiratory regions. All these properties revealed in vitro must play a key role in respiratory pattern and rhythm generation, and must be recognized to develop any model of CPG (Bianchi et al., 1995; Rybak et al., 2004). Unfortunately, most of these properties, such as ionic membrane conductances, intrinsic oscillatory properties, and various sorts of ionic currents have been demonstrated in vitro but their existence and function in vivo remains to be demonstrated. Hence, it is difficult to determine whether the intrinsic membrane properties expressed by neurons recorded in vitro are compatible with those of neurons recorded in vivo. In addition, in such in vitro preparations oxygenation of the tissue is very limited since superfusion provides oxygen to a level of 200 ␮m below the surface only (Paton et al., 1994), hence reducing the physiological viability of many respiratory cells (Brockhaus et al., 1993). The role of hypoxia is of importance in determining the dramatic change in the respiratory pattern generation, which occurs in these circumstances. Indeed, using the in situ perfused brainstem preparation, Paton has demonstrated the fundamental role of hypoxia in shaping the respiratory pattern (Paton, 1996; Paton et al., 2006). Regarding in vivo experiments, it was possible to manipulate the circulatory process to the brainstem of anesthetized, ventilated cats. For example, apneusis was obtained after common carotid occlusion followed by the ligature of the ventral basilary artery (Fig. 2A). This apneusis likely resulted from poor oxygenation (hypoxic conditions) of the brainstem tissue due to the reduced blood flow. This hypothesis was further confirmed by intravenous infusion of ephedrine, an amine inducing a high level of blood pressure used to restore blood flow to the brainstem. Indeed, the animal was able to recover from apneusis to eupnea in parallel with the increase in BP with ephedrine infusions (Fig. 2B and C). Gasping was obtained following vagotomy and disconnection of the pons from the medulla as it was described a long time ago by

7

Fig. 2. Effect of changes in blood circulatory process in the brainstem onto breathing patterns in anesthetized cat with occluded common carotids. (A) Eupneic breathing (A1) before ligature of basilar artery (arrow head at blood pressure recording in A1). Location of ligature is indicated by arrow in semi-schematic drawing of the brainstem (A2). (B) Apneusis occurred 1 min 40 after ligature of basilar artery. (C) Eupneic breathing pattern was restored when blood pressure was enhanced by Ephedrine i.v. injection (arrow).  Time lag between B and C traces, 20 s. Abbreviations: Phr, phrenic nerve activity; Phr, integrated phrenic nerve activity; BP, blood pressure; TP, tracheal pressure.

Lumsden (1923). Hence, gasping appears as a process of autoresuscitation. In neonate and adult animals in vivo gasping was induced by exposure to anoxia (Wang et al., 1996; St John, 1997). The same holds true in juvenile animals studied in situ, where the perfusion can be precisely controlled (St John and Paton, 2000; Paton et al., 2006). Thus, the presence of hypoxia or even anoxia should be carefully considered when choosing the type of experimental preparation to be used for studying groups of neurons involved in respiratory rhythm generation. As an alternative to in vitro preparations or intact animals, the arterially perfused brainstem preparation (Paton, 1996) seems to be the best approach for future studies of brainstem neuronal populations controlling breathing. 8. Non-respiratory functions of the respiratory system 8.1. Breathing is at the crossroads of autonomic and somatic functions The respiratory pump and valve muscles are also involved in many non-respiratory functions, which in some circumstances are opposite to ventilation. In these circumstances they contract independently of ventilation, which means that the respiratory CPG has to be reconfigured. This is the case in postural control, phonation, protective reflexes of the upper airways (sneezing, swallowing, coughing), and expulsive maneuvers (defecation, parturition, micturition, vomiting). The postural function of the respiratory muscles is of importance in air breathing animals since individuals can continue to breathe in any body position (Duron, 1973; Duron and Rose, 1997). Ventilatory muscles are critically involved during phonation, but expiratory processes must dominate during this behavior up to a certain limit of O2 –CO2 equilibrium (Plum, 1970; Sears, 1971). Another important function of the respiratory muscles of the oro-pharyngeal and laryngeal region is to protect the upper airway from aspiration of food or foreign particles. The CPG for breathing is actually at a crossroads of autonomic and somatic functions. It is able to ensure homeostasis and maintains life by constant re-oxygenation of blood, which in cooperation

8

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

Fig. 3. Discharge activities in phrenic (Phr), abdominal (Abd), and hypoglossal nerve (XII) during respiratory and non-respiratory behaviors. (A) Breathing. (B) swallowing (sw, stars) and coughing (cg, arrows) induced by electrical stimulation of superior laryngeal nerve (Sln stim.). (C) Vagal-induced vomiting (X stim.).

with the circulatory system ensures tissues remain viable. Dynamic interactions among populations of neurons may be necessary not only for producing different breathing patterns, but also in providing the appropriate response to various kinds of autonomic and somatic inputs. Afferents from both visceral and somatic receptors converge on most brainstem respiratory neurons. Indeed, the respiratory CPG receive sensory inputs (respiratory and non-respiratory) via both the vagus and glossophrayngeal nerves, which can trigger protective and expulsive reflexes. In addition, different neurons involved in the generation of brainstem respiratory outputs also receive inputs from the forebrain. For example, the phrenic, intercostal, and laryngeal recurrent nerves are activated at short latency by forebrain stimulation (Bassal and Bianchi, 1981a, 1981b). Indeed, following acute forebrain stimulation (sensory and motor areas of the orbitary gyrus), bulbo-spinal neurons could be activated at short latency whereas propriobulbar neurons were inhibited (Bassal et al., 1981). Hence, the forebrain directly drives the motoneurons of the respiratory system but also the premotor neurons, namely the bulbo-spinal neurons, while the respiratory interneurons of respiratory CPG are inhibited. 8.2. Experimental approaches In our laboratory, we have more specifically studied reconfiguration of the respiratory network during ingestive or expulsive behaviors, such as swallowing and coughing triggered by superior laryngeal nerve (SLN) stimulation, as well as vomiting elicited by vagal stimulation or injection of various emetic drugs (Fig. 3). These studies were greatly motivated by the discovery of multifunctional networks in invertebrates, such as in the stomatogastric system of crustaceans (Marder, 1988; Meyrand et al., 1991; Weimann et al., 1991). Our experiments tested the hypothesis that the mammalian brainstem respiratory network has the same property as multifunctional circuits of invertebrates, i.e. that the same neuron has the ability to switch from one network to another. The experimental approaches consisted of recording activities of respiratory nerves and neurons in fictive situations, since the animals were decerebrate or anesthetized, paralyzed and ventilated artificially. The specific objectives were to provide evidence for reconfiguration of the respiratory CPG when other brainstem CPGs operate to control the same muscle targets during functions other than breathing. In the following sections, we will summarize the main neurological features of a variety of fictive behaviors and explain why the experimental protocols used were suitable for studying reconfiguration of the respiratory network. Control of fictive breathing was performed by recording the phrenic nerve and a lumbar nerve rootlet to abdominal mus-

cles (Fig. 3A). Fictive coughing and swallowing were elicited by SLN stimulation. Typically, repetitive episodes of coughing were obtained at low frequency (4–8 Hz) whereas higher frequency (30–40 Hz) preferentially elicited repetitive swallowing. But, both coughing and swallowing could be obtained at any frequency (Fig. 3B; see also Gestreau et al., 1996). Fictive coughing (arrows in Fig. 3B) is characterized by an enhanced phrenic nerve discharge (the inspiratory phase), a large and concomitant discharge in the recurrent laryngeal and lumbar nerves (the compressive phase), followed by a prolonged lumbar nerve discharge (the explusive phase). These discharge patterns on respiratory nerves or motoneurons have been described in previous studies (Bolser, 1991; Grélot and Milano, 1991; Bianchi et al., 1992; Grélot et al., 1992; Grélot and Miller, 1997; Milano et al., 1992; Gestreau et al., 1996, 2000; Roda et al., 2002). Fictive swallowing consists in enhanced discharge activities from both the hypoglossal nerve (XII) and the pharyngeal branch of the vagus nerve (Ph-X), while both phrenic and lumbar nerves are almost completely silent (asterisks in Fig. 3B; Grélot and Bianchi, 1992; Gestreau et al., 1996, 2000; Roda et al., 2002). Careful examination of the phrenic nerve discharge at the onset of swallowing shows a small and variable burst of activity, called “phrenic breakthrough” (Jodkowski et al., 1988), although no diaphragmatic contraction occurs in real swallowing. We also studied the role of respiratory outputs and central neurons during fictive vomiting obtained by repetitive stimulation of the upper diaphragmatic vagus nerve below the recurrent laryngeal nerve bifurcation (Grélot and Bianchi, 1992; Grélot et al., 1992, 1993, 1996). Fictive vomiting is characterized by the rapid onset of very large, repetitive and synchronous discharges in phrenic and abdominal nerves (namely retching). This corresponds to a drastic change in the discharge patterns of these two nerves, which is clearly opposite to the normal pattern in fictive breathing characterized by alternate phrenic and lumbar discharges. During vomiting, the diaphragm and abdominal muscles are transiently not used for ventilation. They act as compressive and expulsive muscles since there is a glottic closure during retching allowing abdominal pressure to rise thereby applying extra-mural pressure on the gastro-intestinal contents. At the end of the last retching phase, phrenic nerve activity resumes whereas the lumbar nerve discharge is prolonged, corresponding to the expulsive phase of vomiting (Fig. 3C). It is important to note that the discharge patterns recorded in vivo from cranial and spinal nerves during the fictive behaviors presented above are almost similar to those recorded during real behaviors (Grélot and Milano, 1991; Bolser, 1991; Dick et al., 1993; Jean, 2001). This holds true although electrical stimulation used to trigger these functions does clearly not correspond to physiological stimuli. One important characteristic of brainstem CPGs is that they are able to program the motor sequence almost entirely without sensory afferent feedback. The reflexive nature of swallowing, coughing or vomiting seems to be poorly dependent on the rate and temporal codes carried by afferent fibers. This is particularly evident for the swallowing CPG, since simple pharmacological injection of glutamate into the NTS can trigger the buccopharyngeal stage of swallowing (Bieger and Neuhuber, 2006). 8.3. Discharge patterns of brainstem respiratory neurons during non-respiratory motor activities Changes in membrane potential trajectories and discharge patterns of brainstem respiratory neurons including cranial and spinal motoneurons and interneurons have been studied during fictive coughing, swallowing and vomiting (Gestreau et al., 1996, 2000; Grélot et al., 1996; Roda et al., 2002). During the inspiratory phase of coughing, the augmenting inspiratory bulbo-spinal and propriobulbar neurons of both DRG and VRC are strongly activated.

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

Thus, the enhanced phrenic nerve discharge associated with the inspiratory phase of coughing is mediated by the inspiratory elements of the respiratory CPG. The control of the glottis during coughing also depends on neurons of the respiratory network. Indeed, inspiratory laryngeal motor and premotor neurons control the inspiratory phase of coughing associated with an abduction of the glottis. The compressive phase of cough is mediated by the same expiratory motor and premotor neurons as those involved in breathing (Gestreau et al., 1996, 2000; Shiba et al., 1999; Baekey et al., 2001). In addition, the expiratory bulbo-spinal and propriobulbar neurons that are normally active during late expiration have been found to discharge with a prolonged decrementing discharge during both the compressive and expiratory phases of coughing. Therefore, muscle contractions associated with these phases of coughing depend on the expiratory elements of the respiratory CPG. Since most of the neuronal elements of the respiratory CPG are involved during coughing, this strongly suggests that a large part of the respiratory CPG reconfigures during coughing. Other neurons located in medullary regions that are not considered as respiratory per se have also been suggested to contribute to coughing (for details, see Gestreau et al., 1997; Jakus et al., 2000). A large number (>80%) of DRG inspiratory bulbo-spinal neurons, presumed to be phrenic premotor neurons, have been found to be activated briefly during the buccopharyngeal stage of swallowing (Gestreau et al., 1996). Similarly, results from the same study suggest that DRG inspiratory propriobulbar neurons are also involved in swallowing. About half of the VRC inspiratory augmenting (I Aug) and decrementing (I Dec) neurons exhibit a brief discharge during swallowing, i.e. during the first half of the hypoglossal nerve discharge (Oku et al., 1994). These striking results contrast markedly with the lack of respiratory movement of the diaphragm during swallowing. As suggested by Oku and collaborators (1994), theses discharges may be related to brief phrenic bursts which could occur during the bucco-pharyngeal stages of swallowing. Alternatively, since DRG inspiratory bulbo-spinal neurons have been found to possess bifurcated axons to the phrenic and hypoglossal motor nuclei (Ono et al., 1994), the question remains as to whether the discharge activity of the respiratory DRG neurons could participate to swallowing-related movements of the tongue. The discharge activities and membrane potential trajectory changes of several groups of respiratory neurons have also been recorded during vomiting. We have shown that the inspiratory bulbo-spinal (I Aug) neurons of both DRG and VRC are strongly inhibited during periods of phrenic nerve activity associated with vomiting (Bianchi and Grélot, 1989). Indeed, a large hyperpolarization develops during the retching phase of vomiting. This inhibition is chloride-dependent, as shown by its reversal on membrane hyperpolarization (Bianchi and Grélot, 1989). Between retches, these bulbo-spinal I Aug neurons are transiently depolarized due to post-inhibitory rebounds. Reconfiguration of the respiratory network during vomiting may also be illustrated by changes in activity of expiratory neurons. The augmenting expiratory neurons of the caudal VRC are active during the retching phases, and thus fire appropriately to activate expiratory motoneurons in vomiting, which leads to abdominal muscle contractions. By contrast, the expiratory augmenting neurons of rostral VRC (the Bötzinger complex) are active between retches, and inhibited during these phases of vomiting. Thus, opposite changes in phase relationships between neurons and respiratory motor discharges occur between breathing and vomiting. Based on these changes, a model of reconfiguration of the respiratory CPG during vomiting has been proposed (Grélot and Miller, 1997). In this model, the pathways driving the phrenic and expiratory motoneurons during vomiting are different from those conveying the central respiratory drive. These pathways may originate from non-respiratory neurons that are recruited and form the vomiting CPG transiently, but these neurons remain to be identified.

9

The role of pontine neurons has been investigated in swallowing (see refs in Jean, 2001; Gestreau et al., 2005) and coughing (Gestreau et al., 1997; Poliacek et al., 2004, 2005; Shannon et al., 2004). To our knowledge, no study has assigned a role of PRG in vomiting. A primary sensory relay for laryngeal ascending projections has been demonstrated in a pontine region dorsal to the Vth motor nucleus in sheep (Car et al., 1975). It has been reported that pontine neurons are synaptically activated by laryngeal stimulation and spontaneously activated during swallowing, nevertheless their activity is abolished after paralysis of the animals (Jean, 2001). Although we suggested that KF neurons are most likely involved in the control of motor output during breathing and swallowing in decerebrate and spontaneously breathing rats (Gestreau et al., 2005), their discharge patterns as well as the peripheral versus central origin of their discharge remain to be determined. Several anatomical studies based on either c-Fos expression (Gestreau et al., 1997; Jakus et al., 2008) or kainic acid inactivation (Poliacek et al., 2004, 2005) have suggested that pontine neurons are involved in coughing. In addition, the discharge patterns of pontine respiratory as well as non-respiratory neurons are altered during coughing (Shannon et al., 2004). In particular, most respiratory-modulated neurons including I-Dec, E-Dec and E-Aug display appropriate changes in their firing rates during inspiratory or expiratory phases of coughing. Altogether, these results suggest an involvement of the PRG in the cough motor pattern although non-respiratory pontine neurons could be also involved (Poliacek et al., 2005). 8.4. Possible mechanisms involved in reconfiguration processes At present, the mechanisms responsible for switching from respiration to other antagonistic behaviors are not known. Due to the complexity of the brainstem circuitry and intrinsic organization of CPGs for breathing, swallowing, coughing and vomiting, progresses in this field are rendered extremely difficult. However, future experiments will hopefully solve several challenging questions. For example, how does the stimulation at a fixed frequency of the same afferent pathway, e.g. SLN 10 Hz, can trigger pharyngeal swallowing and/or coughing? Despite the observation that swallowing occurs only at respiratory phase transitions (Dick et al., 1993), what is the neuronal substrate for interaction between breathing and swallowing? Is there a dynamical reorganization of the strength of connectivity among respiratory neurons during reconfiguration? What roles play the intrinsic membrane properties of neurons? In the following section, we will only consider some cellular mechanisms that may be involved in a process of selection/exclusion of given neurons in multiple functions. Various mechanisms of pre-synaptic inhibition may contribute to reconfiguration at the level of the NTS, a gateway for many primary afferents from visceral sensory receptors (Gatti et al., 1995; Bailey et al., 2006; Kline et al., 2009). We speculate that similar cellular mechanisms can operate in various subdivisions of the NTS allowing a variety of autonomic reflexes. In most cases, the net NTS output is determined by a balance of excitatory glutamatergic and inhibitory GABAergic inputs. For example, it has been shown that glutamate released from the primary central afferent terminals can spill over pre-synaptic metabotropic glutamate receptors on GABA interneurons to suppress GABA release at the secondorder baroreceptor neurons (Chen and Bonham, 2005). Also, GABA agonist or antagonist microinjections in the NTS can suppress or enhance swallowing, respectively (Wang and Bieger, 1991; Bieger and Neuhuber, 2006). In a different context, modulation of swallowing reflex by orofacial pain has been shown to also depend on GABAergic neurons including those of the NTS (Tsujimura et al., 2009). Thus, endogenous glutamate released by stimulation of peripheral afferent inputs may reduce GABA transmission and

10

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

increase the excitability of NTS neurons involved in reconfiguration. Recently, it has been suggested that glutamate spillover at immature NTS synapses may contribute to synaptic processing (Balland et al., 2008), but whether this holds true in interacting CPGs of adult animals remains to be determined. At the level of the lateral and medial medullary reticular formation, microinjections of drugs acting on GABAergic and glutamatergic synapses have also been shown to alter one or several behaviors (Chen and Travers, 2003; Harada et al., 2005). For example, activation of GABAA receptors (Chen et al., 2001), or selective blockade of NMDA or AMPA/kainate receptors (Chen and Travers, 2003) in the lateral reticular formation reversibly suppressed swallowing. Interestingly, similar pharmacological manipulations were able to switch ingestion to a rejection behavior (Chen and Travers, 2003). Injection within the parvocellular reticular formation of bicuculline, a GABAA receptor antagonist, resulted in facilitation of swallowing and also generated coughing (Harada et al., 2005). Therefore, it has been postulated that GABAergic system would play an essential role for switching swallowing and coughing depending on the nature of sensory inputs to the medullary reticular formation through the NTS (Harada et al., 2005). Neuromodulatory influences upon CPGs may enable behavioral flexibility. As demonstrated in the crustacean stomatogastric system, one neuromodulatory neuron may exert several and opposite target-specific effects, such as transient hyperpolarization of some neurons and long lasting-depolarization of others (Faumont et al., 2005). This kind of dual effects also exists in the mammalian brainstem, and the resulting alteration of firing may be responsible for different circuit configurations and interactions (for review see Dickinson, 2006). 9. Concluding remarks To conclude, we provided evidence that some neuronal elements that belong to the respiratory CPG are shared by different operating networks pertaining to distinctive behaviors. As opposed to the respiratory motor sequence that remains spontaneously active throughout life, non-respiratory behaviors need to be triggered by specific afferent inputs. Also, once triggered, these non-respiratory behaviors occur as stereotyped motor sequences which do not require afferent feedback. However, all of these centrally programmed motor sequences can be modulated by afferent inputs depending on physiological conditions. Fig. 4 summarizes the extent of overlap between the CPGs for breathing, coughing, swallowing and vomiting which are responsible for antagonistic functions requiring entirely or partially the same motor outputs. This concept of multifunctional pattern-generating circuits has been clearly demonstrated in invertebrates and is well accepted in mammals (Dickinson, 2006; Briggman and Kristan, 2008). It has been proposed that coughing mainly results from the reconfiguration of the respiratory network since most respiratory neurons are involved during both breathing and coughing, including changes in membrane potential trajectories, altered firing rates and patterns of activity (Gestreau et al., 1996; Grélot et al., 1996; Baekey et al., 2001; Shannon et al., 2004). Interestingly, a network model based on data from in vivo experiments, and the established connectivity between the main respiratory groups, was shown to reproduce both the characteristic changes in neuronal and motor patterns observed in vivo during transition from breathing to coughing (Rybak et al., 2008). Other data from in vivo experiments obtained in our laboratory (Grélot and Bianchi, 1996) and elsewhere (Nakazawa et al., 1999), have suggested that less brainstem respiratory neurons are involved in swallowing and vomiting than in coughing. Although vomiting requires the contraction of many respiratory muscles whereas swallowing only recruits the activation of respiratory

Fig. 4. Schematic representation of the dynamic interactions between the respiratory, coughing, swallowing and vomiting CPGs. Afferent or central inputs impinge on the respiratory CPG to trigger protective and expulsive reflexes. Most respiratory neurons are involved during both breathing and coughing, explaining a large overlap of both CPGs. Swallowing and vomiting mainly depend on separate CPGs, i.e. elements outside the respiratory CPG. However, some respiratory neurons (dashed areas) also serve these non-respiratory behaviors, explaining the partial overlaps shown between CPGs.

valve muscles, both motor activities depend on separate CPGs. Thus, the so-called respiratory muscles are multifunctional muscle groups (Briggman and Kristan, 2008) since they are used during non-respiratory behaviors. However, different types of dynamic reorganization among brainstem networks must exist to coordinate respiratory muscles that are appropriate for the motor behavior being expressed. References Baekey, D.M., Morris, K.F., Gestreau, C., Li, Z., Lindsey, B.G., Shannon, R., 2001. Medullary respiratory neurones and control of laryngeal motoneurones during fictive eupnoea and cough in the cat. J. Physiol. (Lond.). 534, 565–581. Bailey, T.W., Jin, Y.H., Doyle, M.W., Smith, S.M., Andresen, M.C., 2006. Vasopressin inhibits glutamate release via two distinct modes in the brainstem. J. Neurosci. 26, 6131–6142. Balland, B., Lachamp, P., Kessler, J.P., Tell, F., 2008. Silent synapses in developing rat nucleus tractus solitarii have AMPA receptors. J. Neurosci. 28, 4624–4634. Ballantyne, D., Richter, D.W., 1986. The non-uniform character of expiratory synaptic activity in expiratory bulbospinal neurones of the cat. J. Physiol. (Lond.) 370, 433–456. Ballantyne, D., Richter, D.W., 1984. Post-synaptic inhibition of bulbar inspiratory neurones in the cat. J. Physiol. (Lond.) 348, 67–87. Bassal, M., Bianchi, A.L., 1981a. Effets de la stimulation des structures nerveuses centrales sur les activités respiratoires efférentes chez le chat. I. Réponses à la stimulation corticale. J. Physiol. (Paris) 77, 741–757. Bassal, M., Bianchi, A.L., 1981b. Effets de la stimulation des structures nerveuses centrales sur les activités respiratoires efférentes chez le chat. II. Réponses à la stimulation sous-corticale. J. Physiol. (Paris) 77, 759–777. Bassal, M., Bianchi, A.L., Dussardier, M., 1981. Effets de la stimulation des structures nerveuses centrales sur l’activité des neurones respiratoires chez le chat. J. Physiol. (Paris) 77, 779–795. Batsel, H.L., 1964. Localization of bulbar respiratory center by microelectrode sounding. Exp. Neurol. 9, 410–426. Besnard, S., Massé, F., Verdaguer, M., Cappelin, B., Meurice, J.C., Gestreau, C., 2007. Time- and dose-related effects of three 5-HT receptor ligands on the genioglossus activity in anesthetized and conscious rats. Sleep Breath. 11, 275–284. Bianchi, A.L., 1969. Activation antidromique des neurones respiratoires bulbaires. J. Physiol. (Paris) 61 (Suppl. 1), 91. Bianchi, A.L., 1971. Localisation et étude des neurones respiratoires bulbaires. Mise en jeu antidromique par stimulation spinale ou vagale. J. Physiol. (Paris) 63, 5–40. Bianchi, A.L., 1974. Modalités de décharge et propriétés anatamo-fonctionelles des neurones respiratoires bulbaires. J. Physiol. (Paris) 68, 555–587. Bianchi, A.L., St John, W.M., 1981. Pontile axonal projections of medullary respiratory neurons. Respir. Physiol. 45, 167–183.

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12 Bianchi, A.L., Barillot, J.C., 1982. Respiratory neurons in the region of the retrofacial nucleus: pontile, medullary, spinal and vagal projections. Neurosci. Lett. 31, 277–282. Bianchi, A.L., St John, W.M., 1982. Medullary axonal projections of respiratory neurons of pontile pneumotaxic center. Respir. Physiol. 48, 357–373. Bianchi, A.L., Grélot, L., Iscoe, S., Remmers, J.E., 1988. Electrophysiological properties of respiratory neurones in the cat: an intracellular study. J. Physiol. (Lond.) 407, 293–310. Bianchi, A.L., Grélot, L., 1989. Converse motor output of inspiratory bulbospinal premotoneurones during vomiting. Neurosci. Lett. 104, 298–302. Bianchi, A.L., Grélot, L., Miller, A.D., King, G.L., 1992. Mechanisms and Control of Emesis. INSERM/John Libbey Eurotext Ltd., Paris. Bianchi, A.L., Denavit-Saubié, M., Champagnat, J., 1995. Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75, 1–46. Bieger, D., Neuhuber, W.L., 2006. Neural circuits and mediators regulating swallowing in the brainstem. Gastro-Intestinal Motility, online: 16-5-2006. Bolser, D.C., 1991. Fictive cough in the cat. J. Appl. Physiol. 71, 2325–2331. Briggman, K.L., Kristan, W.B., 2008. Multifunctional pattern-generating circuits. Annu. Rev. Neurosci. 31, 271–294. Brockhaus, J., Ballanyi, K., Smith, J.C., Richter, D.W., 1993. Microenvironment of respiratory neurons in the in vitro brainstem-spinal cord of neonatal rats. J. Physiol. (Lond.) 462, 421–445. Car, A., Jean, A., Roman, C., 1975. A pontine primary relay for ascending projections of the superior laryngeal nerve. Exp. Brain Res. 22, 197–210. Chamberlin, N.L., Saper, C.B., 1998. Brainstem neural network mediating apneic reflexes in the rat. J. Neurosci. 18, 371–387. Champagnat, J., Denavit-Saubié, M., Siggins, G.R., 1983. Rhythmic neuronal activities in the nucleus of the tractus solitarius isolated in vitro. Brain Res. 280, 155–159. Chan, E., Steenland, H.W., Liu, H., Horner, R.L., 2006. Endogenous excitatory drive modulating respiratory muscle activity across sleep–wake states. Am. J. Respir. Crit Care Med. 174, 1264–1273. Chen, C.Y., Bonham, A.C., 2005. Glutamate suppresses GABA release via presynaptic metabotropic glutamate receptors at baroreceptor neurones in rats. J. Physiol. (Lond.) 562, 535–551. Chen, Z., Travers, J.B., 2003. Inactivation of amino acid receptors in medullary reticular formation modulates and suppresses ingestion and rejection responses in the awake rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285, 68–83. Chen, Z., Travers, S.P., Travers, J.B., 2001. Muscimol infusions in the brain stem reticular formation reversibly block ingestion in the awake rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, 1085–1094. Cohen, M.I., 1970. How respiratory rhythm originates: evidence from discharge patterns of brainstem respiratory neurons. In: Porter, R. (Ed.), Ciba Foundation Hering-Breuer Centenary Symposium. Breathing, Churchill, J.&A., London, pp. 125–157. Dekin, M.S., Getting, P.A., 1984. Firing pattern of neurons in the nucleus tractus solitarius: modulation by membrane hyperpolarization. Brain Res. 324, 180–184. Dick, T.E., Oku, Y., Romaniuk, J.R., Cherniack, N.S., 1993. Interaction between central pattern generators for breathing and swallowing in the cat. J. Physiol. (Lond.) 465, 715–730. Dickinson, P.S., 2006. Neuromodulation of central pattern generators in invertebrates and vertebrates. Cur. Opin. Neurobiol. 16, 604–614. Dobbins, E.G., Feldman, J.L., 1995. Differential innervation of protruder and retractor muscles of the tongue in rat. J. Comp. Neurol. 357, 376–394. Duffin, J., Tian, G.-F., Peever, J.H., 2000. Functional synaptic connections among respiratory neurons. Resp. Physiol. Neurobiol. 122, 237–246. Duron, B., 1973. Postural and ventilatory functions of intercostal muscles. Acta Neurobiol. Exp. (Wars.) 33, 355–380. Duron, B., Rose, D., 1997. The intercostal muscles. In: Miller, A.D., Bianchi, A.L., Bishop, B.P. (Eds.), Neural Control of the Respiratory Muscles. CRC Press, Boca Raton, pp. 21–33. Dutschmann, M., Herbert, H., 1996. The Kölliker Fuse nucleus mediates the trigeminally induced apnoea in the rat. Neuroreport 7, 1432–1436. Dutschmann, M., Herbert, H., 2006. The Kölliker Fuse nucleus gates the postinspiratory phase of the respiratory cycle to control inspiratory off-switch and upper airway resistance. Eur. J. Neurosci. 24, 1071–1084. Dutschmann, M., Kron, M., Morschel, M., Gestreau, C., 2007. Activation of Orexin B receptors in the pontine Kölliker–Fuse nucleus modulates preinspiratory hypoglossal motor activity in rat. Respir. Physiol. Neurobiol. 159, 232–235. Faumont, S., Combes, D., Meyrand, P., Simmers, J., 2005. Reconfiguration of multiple motor networks by short- and long-term actions of an identified modulatory neuron. Eur. J. Neurosci. 22, 2489–2502. Feldman, J.L., Speck, D.F., 1983. Interactions among inspiratory neurons in dorsal and ventral respiratory groups in cat medulla. J. Neurophysiol. 49, 472–490. Funke, F., Müller, M., Dutschmann, M., 2008. Reconfiguration of respiratory-related population activity in a rostrally tilted transversal slice preparation following blockade of inhibitory neurotransmission in neonatal rats. Pflugers Arch. 457, 185–195. Gatti, P.J., Shirahata, M., Johnson, T.A., Massari, V.J., 1995. Synaptic interactions of substance P immunoreactive nerve terminals in the baro- and chemoreceptor reflexes of the cat. Brain Res. 693, 133–147. Gestreau, C., Milano, S., Bianchi, A.L., Grélot, L., 1996. Activity of dorsal respiratory group inspiratory neurons during laryngeal-induced fictive coughing and swallowing in decerebrate cats. Exp. Brain Res. 108, 247–256.

11

Gestreau, C., Bianchi, A.L., Grélot, L., 1997. Differential brainstem Fos-like immunoreactivity after laryngeal-induced coughing and its reduction by codeine. J. Neurosci. 17, 9340–9352. Gestreau, C., Grélot, L., Bianchi, A.L., 2000. Activity of respiratory laryngeal motoneurons during fictive coughing and swallowing. Exp. Brain Res. 130, 27–34. Gestreau, C., Dutschmann, M., Obled, S., Bianchi, A.L., 2005. Activation of XII motoneurons and premotor neurons during various oropharyngeal behaviors. Respir. Physiol. Neurobiol. 147, 159–176. Gestreau, C., Bévengut, M., Dutschmann, M., 2008. The dual role of orexin/hypocretin system in modulating wakefuleness and respiratory drive. Curr. Opin. Pulm. Med. 14, 512–518. Graham, K., Duffin, J., 1981. Cross correlation of medullary expiratory neurons in the cat. Exp. Neurol. 73, 451–464. Graham, K., Duffin, J., 1985. Short-latency interactions among dorsomedial medullary inspiratory neurons in the cat. Exp. Neurol. 88, 726–741. Grélot, L., Bianchi, A.L., Iscoe, S., Remmers, J.E., 1988. Expiratory neurones of the rostral medulla: anatomical and functional correlates. Neurosci. Lett. 50, 23–40. Grélot, L., Barillot, J.C., Bianchi, A.L., 1989. Pharyngeal motoneurones: respiratoryrelated activity and responses to laryngeal afferents in the decerebrate cat. Exp. Brain Res. 78, 336–344. Grélot, L., Milano, S., 1991. Diaphragmatic and abdominal muscle activity during coughing in the decerebrate cat. Neuroreport 2, 165–168. Grélot, L., Bianchi, A.L., 1992. Activities of cranial and spinal respiratory-related motoneurons during vomiting. In: Bianchi, A.L., Grélot, L., Miller, A.D., King, G. (Eds.), Mechanisms and Control of Emesis. INSERM/John Libbey Eurotext Ltd., Paris, pp. 29–39. Grélot, L., Milano, S., Portillo, F., Miller, A.D., Bianchi, A.L., 1992. Membrane potential changes of phrenic motoneurons during fictive vomiting, coughing, and swallowing in the decerebrate cat. J. Neurophysiol. 68, 2110–2119. Grélot, L., Milano, S., Miller, A.D., Portillo, F., 1993. Respiratory interneurons of the lower cervical (C4-C5) cord: membrane potential changes during coughing, vomiting, and swallowing in the decerebrate cat. Pflüg. Arch. 425, 313–320. Grélot, L., Bianchi, A.L., 1996. Multifunctional medullary respiratory neurons. In: Miller, A.D., Bianchi, A.L., Bishop, B.P. (Eds.), Neural Control of the Respiratory Muscles. CRC Press, Boca Raton, FL, pp. 299–306. Grélot, L., Milano, S., Gestreau, C., Bianchi, A.L., 1996. Are medullary respiratory neurones multipurpose neurones? In: Bostock, H., Kirkwood, P.A., Pullen, A.H. (Eds.), The Neurobiology of Disease: Contributions from Neuroscience to Clinical Neurology. Cambridge University Press, pp. 299–308. Grélot, L., Miller, A.D., 1997. Neural Control of Respiratory Muscles activation during Vomiting. In: Miller, A.D., Bianchi, A.L., Bishop, B.P. (Eds.), Neural Control of the Respiratory Muscles. CRC Press, Boca Raton, FL, pp. 239–248. Harada, H., Takakusaki, K., Kita, S., Matsuda, M., Nonaka, S., Sakamoto, T., 2005. Effects of injecting GABAergic agents into the medullary reticular formation upon swallowing induced by the superior laryngeal nerve stimulation in decerebrate cats. Neurosci. Res. 51, 395–404. Hilaire, G., Monteau, R., Bianchi, A.L., 1984. A cross-correlation study of interactions among respiratory neurons of dorsal, ventral and retrofacial groups in cat medulla. Brain Res. 302, 19–31. Hilaire, G., Monteau, R., Errchidi, S., 1989. Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat. Brain Res. 485, 325–332. Jakus, J., Stransky, A., Poliacek, I., Barani, H., Bosel’ova, L., 2000. Kainic acid lesions to the lateral tegmental field of medulla: effects on cough, expiration and aspiration reflexes in anesthetized cats. Physiol. Res. 49, 387–398. Jakus, J., Poliacek, I., Halasova, E., Murin, P., Knocikova, J., Tomori, Z., Bolser, D.C., 2008. Brainstem circuitry of tracheal-bronchial cough: c-fos study in anesthetized cats. Respir. Physiol. Neurobiol. 160, 289–300. Jean, A., 2001. Brain stem control of swallowing: neuronal network and cellular mechanisms. Physiol. Rev. 81, 929–969. Jelev, A., Sood, S., Liu, H., Nolan, P., Horner, R.L., 2001. Microdialysis perfusion of 5-HT into hypoglossal motor nucleus differentially modulates genioglossus activity across natural sleep–wake states in rats. J. Physiol. 532,467–481. Jodkowski, J.S., Viana, F., Dick, T.E., Berger, A.J., 1988. Repetitive firing properties of phrenic motoneurons in the cat. J. Neurophysiol. 60, 687–702. Kline, D.D., Hendricks, G., Hermann, G.E., Rogers, R.C., Kunze, D.L., 2009. Dopamine inhibits N-type channels in visceral afferents to reduce synaptic transmitter release under normoxic and chronic intermittent hypoxic conditions. J. Neurophysiol. 101, 2270–2278. Kubin, L., Tojima, H., Davies, R.O., Pack, A.I., 1992. Serotonergic excitatory drive to hypoglossal motoneurons in the decerebrate cat. Neurosci. Lett. 139, 243–248. Lumsden, T., 1923. Observations on the respiratory centres. J. Physiol. (Lond.) 57, 354–367. Madden, K.P., Remmers, J.E., 1982. Short time scale correlations between spike activity of neighboring respiratory neurons of nucleus tractus solitarius. J. Neurophysiol. 48, 749–760. Marder, E., 1988. Modulating a neuronal network. Nature 335, 1–2. Merrill, E.G., 1970. The lateral respiratory neurons of the medulla: their association with nucleus ambiguus, nucleus retroambigualis, the spinal accessory nucleus and the spinal cord. Brain Res. 24, 11–28. Merrill, E.G., 1974. Finding a respiration function for the medullary respiratory neurons. In: Bellairs, R., Gray, E.G. (Eds.), Essay on the Nervous System. Clarendon Press, Oxford, pp. 451–486. Merrill, E.G., Lipski, J., Kubin, L., Fedorko, L., 1983. Origin of the expiratory inhibition of nucleus tractus solitarius inspiratory neurones. Brain Res. 263, 43–50.

12

A.L. Bianchi, C. Gestreau / Respiratory Physiology & Neurobiology 168 (2009) 4–12

Meyrand, P., Simmers, P., Moulins, M., 1991. Construction of a pattern generating circuit with neurons of different networks. Nature 351, 60–63. Milano, S., Grélot, L., Bianchi, A.L., Iscoe, S., 1992. Discharge patterns of phrenic motoneurons during fictive coughing and vomiting in decebrate cats. J. Appl. Physiol. 73, 1626–1636. Nakayama, S., Baumgarten, R.V., 1964. Localisierung absteigender Atmungsbahnem im Rückenmark der Katze mittels antidromer Reizung. Pflüg. Arch. 281, 231–244. Nakazawa, K., Umezaki, T., Zheng, Y., Miller, A.D., 1999. Behaviors of bulbar respiratory interneurons during fictive swallowing and vomiting. Otolaryngol. Head Neck Surg. 120, 412–418. Oku, Y., Tanaka, I., Ezure, K., 1994. Activity of bulbar respiratory neurons during fictive coughing and swallowing in the decerebrate cat. J. Physiol. (Lond.) 480, 309–324. Onimaru, H., Homma, I., 1987. Respiratory rhythm generator neurons in medulla of brainstem–spinal cord preparation from newborn rat. Brain Res. 403, 380–384. Ono, T., Ishiwata, Y., Inaba, N., Kuroda, T., Nakamura, Y., 1994. Hypoglossal premotor neurons with rhythmical inspiratory-related activity in the cat: localization and projection to the phrenic nucleus. Exp. Brain Res. 98, 1–12. Paton, J.F.R., 1996. A working heart-brainstem preparation of the mouse. J. Neurosci. Methods 65, 63–68. Paton, J.F.R., Ramirez, J.-M., Richter, D.W., 1994. Functionally intact in vitro preparation generating respiratory activity in neonatal and mature mammals. Pflugers Arch. 428, 250–260. Paton, J.F.R., Richter, D.W., 1995. Role of fast inhibitory synaptic mechanisms in respiratory rhythm generation in the maturing mouse. J. Physiol. (Lond.) 484, 505–521. Paton, J.F.R., Abdala, A.P.L., Koizumi, H., Smith, J.C., St.-John, W.M., 2006. Respiratory rhythm generation during gasping depends on persistent sodium current. Nat. Neurosci. 9, 311–313. Pierrefiche, O., Schmid, K., Foutz, A.S., Denavit-Saubie, M., 1991. Endogenous activation of NMDA and non-NMDA glutamate receptors on respiratory neurones in cat medulla. Neuropharmacology 30, 429–440. Pierrefiche, O., Schwarzacher, S.W., Bischoff, A.M., Richter, D.W., 1998. Blockade of synaptic inhibition within the pre-Bötzinger complex in the cat suppresses respiratory rhythm generation in vivo. J. Physiol. (Lond.) 509, 245–254. Plum, F., 1970. Neurological integration of behavioural and metabolic control of breathing. In: Porter, R. (Ed.), Breathing Hering–Breuer centenary symposium CIBA foundation. Churchill, London, pp. 159–181. Poliacek, I., Jakus, J., Stránsky, A., Baráni, H., Halasová, E., Tomori, Z., 2004. Cough, expiration and aspiration reflexes following kainic acid lesions to the pontine respiratory group in anesthetized cats. Physiol. Res. 53, 155–163. Poliacek, I., Stránsky, A., Szereda-Przestaszewska, M., Jakus, J., Baráni, H., Tomori, Z., Halasová, E., 2005. Cough and laryngeal muscle discharges in brainstem lesioned anaesthetized cats. Physiol. Res. 54, 645–654. Richter, D.W., Camerer, H., Meesmann, M., Roehrig, N., 1979. Studies on the synaptic interconnection between bulbar respiratory neurones of cats. Pflüg. Arch. 380 (3), 245–257.

Richter, D.W., 1982. Generation and maintenance of the respiratory rhythm. J. Exp. Biol. 100, 93–107. Roda, F., Gestreau, C., Bianchi, A.L., 2002. Discharge patterns of hypoglossal motoneurons during fictive breathing, coughing, and swallowing. J. Neurophysiol. 87, 1703–1711. Roda, F., Pio, J., Bianchi, A.L., Gestreau, C., 2004. Effects of anesthetics on hypoglossal nerve discharge and c-Fos expression in brainstem hypoglossal premotor neurons. J. Comp. Neurol. 468, 571–586. Rybak, I.A., Shevtsova, N.A., Paton, J.F., Dick, T.E., St-John, W.M., Mörschel, M., Dutschmann, M., 2004. Modeling the ponto-medullary respiratory network. Respir. Physiol. Neurobiol. 143, 307–319. Rybak, I.A., O’Connor, R., Ross, A., Shevtsova, N.A., Nuding, S.C., Segers, L.S., Shannon, R., Dick, T.E., Dunin-Barkowski, W.L., Orem, J.M., Solomon, I.C., Morris, K.F., Lindsey, B.G., 2008. Reconfiguration of the pontomedullary respiratory network: a computational modeling study with coordinated in vivo experiments. J. Neurophysiol. 100, 1770–1799. Sears, T.A., 1971. Breathing: a sensory-motor act. Sci. Basis of Med. Ann. Rev., 129–147. Shannon, R., Baekey, D.M., Morris, K.F., Nuding, S.C., Segers, L.S., Lindsey, B.G., 2004. Production of reflex cough by brainstem respiratory networks. Pulm. Pharmacol. Ther. 17, 369–376. Shiba, K., Satoh, I., Kobayashi, N., Hayashi, F., 1999. Multifunctional laryngeal motoneurons: an intracellular study in the cat. J. Neurosci. 19, 2717–2727. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W., Feldman, J.L., 1991. PreBötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726–729. Smith, J.C., Abdala, A.P., Koizumi, H., Rybak, I.A., Paton, J.F., 2007. Spatial and functional architecture of the mammalian brain stem respiratory network: a hierarchy of three oscillatory mechanisms. J. Neurophysiol. 98, 3370–3387. St John, W.M., 1997. Gasping. In: Miller, A.D., Bianchi, A.L., Bishop, B.P. (Eds.), Neural Control of the Respiratory Muscles. CRC Press Inc., pp. 195–202. St John, W.M., Paton, J.F.R., 2000. Characterizations of eupnea, apneusis and gasping in a perfused rat preparation. Respir. Physiol. 123, 201–213. Suzue, T., 1984. Respiratory rhythm generation in the “in vitro” brain stem spinal cord preparation of the neonatal rat. J. Physiol. (Lond.) 354, 173–183. Tsujimura, T., Kondo, M., Kitagawa, J., Tsuboi, Y., Saito, K., Tohara, H., Ueda, K., Sessle, B.J., Iwata, K., 2009. Involvement of ERK phosphorylation in brainstem neurons in modulation of swallowing reflex in rats. J. Physiol. 587, 805–817. Wang, W.T., Bieger, D., 1991. Role of solitarial GABAergic mechanisms in control of swallowing. Am. J. Physiol. Regul. Integr. Comp. Physiol. 261, 639–646. Wang, W.G., Fung, M.L., Darnall, R.A., St John, W.M., 1996. Characterizations and comparisons of eupnoea and gasping in neonatal rats. J. Physiol. (Lond.) 490, 277–292. Weimann, J.M., Meyrand, P., Marder, E., 1991. Neurons that form multiple pattern generators: identification and multiple activity patterns of gastric/pyloric neurons in the crab stomatogastric system. J. Neurophysiol. 65, 111–122.