Neural mechanisms underlying respiratory rhythm generation in the lamprey.

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ARTICLE IN PRESS

RESPNB-2373; No. of Pages 10

Respiratory Physiology & Neurobiology xxx (2014) xxx–xxx

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

Neural mechanisms underlying respiratory rhythm generation in the lamprey Fulvia Bongianni ∗ , Donatella Mutolo, Elenia Cinelli, Tito Pantaleo Dipartimento di Medicina Sperimentale e Clinica, Sezione Scienze Fisiologiche, Università degli Studi di Firenze, Viale G.B. Morgagni 63, 50134 Firenze, Italy

a r t i c l e

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Article history: Accepted 5 September 2014 Available online xxx Keywords: Respiratory rhythm generation Control of breathing Evolution of breathing GABAA and glycine receptors Glutamatergic transmission

a b s t r a c t 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. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The vertebrate nervous system is organized in a similar way throughout vertebrate phylogenesis, although the level of complexity increases. Lampreys are jawless vertebrates known as cyclostomes. They have changed comparatively little during evolution, and became separated from the main vertebrate line 560 million years ago (Kumar and Hedges, 1998). The lamprey central nervous system can be regarded as a vertebrate prototype, with the experimental advantage that it has fewer neurons than higher vertebrates and can be maintained in vitro. Studies on neuronal networks of the lamprey proved to be highly useful to provide insights into the basic mechanisms of central pattern generators (CPGs) of rhythmic activities, such as locomotion and respiration (Grillner, 2003, 2006). The lamprey model has been used for many years to identify the cellular mechanisms involved in the generation and control of locomotion in extreme detail and, more recently, to investigate the neural mechanisms underlying respiratory rhythm generation. The basic features of the neural organization as well as those of rhythmogenic networks have been conserved throughout vertebrate evolution (Grillner, 2003; Mutolo et al., 2007, 2010;

∗ Corresponding author. Tel.: +39 055 2751608; fax: +39 055 4379506. E-mail address: fulvia.bongianni@unifi.it (F. Bongianni).

Robertson et al., 2007; Kinkead, 2009; Ericsson et al., 2011, 2013; Stephenson-Jones et al., 2011, 2012a, 2012b; Cinelli et al., 2013). This review examines the main characteristics of the lamprey respiratory network and describes recent results concerning respiratory rhythm generation and the relevant role of some neurotransmitters and neuromodulators. We also consider the characteristics of respiratory CPGs during vertebrate evolution and possible evolutionary trends in respiratory rhythm generation.

2. General features of the lamprey respiratory system In the adult lamprey, breathing is produced by synchronous contractions of the branchial muscles that force water out of the gill openings; the inhalation phase is passive and is produced by the elastic recoil of cartilaginous baskets surrounding the gill sacs (Rovainen, 1977, 1979). The isolated brainstem of the adult lamprey spontaneously generates respiratory neuronal activity in vitro; this activity closely resembles that underlying the respiratory behavior of intact animals and persists after transections of the brain at both the obex and isthmus level (Rovainen, 1977, 1983; Thompson, 1985; Russell, 1986). Thus, both the neural network responsible for respiratory rhythm generation and respiratory motoneurons are located within the brainstem. The results obtained in this preparation contribute to improve current knowledge on the synaptic transmission within the respiratory network of the lamprey and,

http://dx.doi.org/10.1016/j.resp.2014.09.003 1569-9048/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Bongianni, F., et al., Neural mechanisms underlying respiratory rhythm generation in the lamprey. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.09.003

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ARTICLE IN PRESS F. Bongianni et al. / Respiratory Physiology & Neurobiology xxx (2014) xxx–xxx

Fig. 1. Localization of the pTRG. (A) Schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon showing the levels of the coronal sections illustrated in B and C (arrows) and the location of the pTRG (pink area). (B) Photomicrograph of a transverse section of the rostral rhombencephalon showing the location of an horseradish peroxidase injection into the pTRG. (C) Photomicrograph of a transverse sections of the rhombencephalon showing the location of fluorescent beads microinjected into the pTRG (green). ARRN, anterior rhombencephalic reticular nucleus; I1 , isthmic Müller cell; nVm, motor root of the trigeminal nerve; nVs, sensory root of the trigeminal nerve; pTRG, paratrigeminal respiratory group; SL, sulcus limitans of His. V, trigeminal motor nucleus; VII, facial motor nucleus; IX, glossopharyngeal motor nucleus; X, vagal motor nucleus. B and C adapted from Mutolo et al. (2007) and Cinelli et al. (2014), respectively.

possibly, to obtain new hints for further investigations on the basic neural mechanisms operating in the respiratory network of higher vertebrates, including mammals. The vast majority of respiratory motoneurons are located in the facial, glossopharyngeal and, especially, vagal nuclei, while the neural aggregate responsible for respiratory rhythm generation appears to be located in a region rostrolateral to the trigeminal motor nucleus (Rovainen, 1977, 1979, 1983, 1985; Thompson, 1985; Russell, 1986; Bongianni et al., 1999, 2002, 2006; Guimond et al., 2003; Martel et al., 2007; Mutolo et al., 2007). Mutolo et al. (2007) reported that opioids have a modulatory role in the respiratory network and, in particular, that microinjections of the ␮-opioid receptor agonist DAMGO at sites rostrolateral to the trigeminal motor nucleus abolish the respiratory rhythm. These apneic responses support the hypothesis that this specific opioid-sensitive region likely has a pivotal role in respiratory rhythmogenesis. Mutolo et al. (2007) proposed to name this area the paratrigeminal respiratory group (pTRG). The results on the depressant effects of opioids on the lamprey respiratory activity also imply that the inhibitory role of opioids on respiration is present at an early stage of vertebrate evolution. Respirationrelated neurons with different firing patterns are present in the pTRG (Mutolo et al., 2007, 2010), thus corroborating our hypothesis on the involvement of the pTRG in respiratory rhythm generation. The different discharge patterns encountered in the pTRG may suggest different neuronal functions, but at present any attempt to ascribe a specific role to each type of neurons is only speculative.

3. Anatomical and functional characterization of the pTRG An anatomical and functional characterization of the pTRG region has been recently provided (Cinelli et al., 2013, 2014).

By retrograde labeling, we found neurons located in the isthmic periventricular cell layer with axonal projections to the vagal motoneuron region. Projecting neurons can be easily identified by anatomical landmarks, i.e. they are located in a dorsal aspect of the anterior rhombencephalic reticular nucleus, at the level of the isthmic Müller cell I1 , close to the sulcus limitans of His. This region corresponds closely to the pTRG as defined in our previous studies (Mutolo et al., 2007, 2010, 2011). Neurons located in the pTRG project to the ipsilateral and contralateral vagal motor nucleus as well as to the contralateral pTRG (Rovainen, 1985; Thompson, 1985; Russell, 1986; Gariépy et al., 2012; Cinelli et al., 2013). The results obtained with microinjections of several neuroactive drugs, such as DAMGO, substance P (SP), acetylcholine (ACh), glutamate or GABA receptor agonists and antagonists, exactly into this region (Mutolo et al., 2007, 2010, 2011; Cinelli et al., 2013, 2014) help to identify and characterize the pTRG and to support the notion that it corresponds to the respiratory CPG (see below). A schematic representation of a dorsal view of the lamprey mesencephalon/rhombencephalon showing the respiration-related areas along with photomicrographs of transverse sections illustrating the localization of the pTRG is reported in Fig. 1.

4. Glutamatergic mechanisms in the respiratory rhythmogenesis Endogenously released excitatory amino acids play a crucial role in the lamprey respiratory rhythmogenesis acting on ionotropic receptors (Bongianni et al., 1999) and exert a modulatory role on respiratory activity via metabotropic receptors (Bongianni et al., 2002). The suppression of respiratory activity caused by bath application of ionotropic glutamate receptor antagonists (Bongianni et al., 1999; Mutolo et al., 2011) is mimicked by microinjections


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Fig. 2. Examples of respiratory responses evoked by blockade of ionotropic glutamate receptors and by blockade or activation of the pTRG region. (A) Suppression of respiratory rhythmic activity ∼20 min after bath application of a mixture of 20 ␮M CNQX and 100 ␮M D-AP5. A schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon in the perfused recording chamber is shown. (B) Suppression of respiratory rhythmic activity ∼ 1 min after a unilateral microinjection of a mixture of 1 mM CNQX and 5 mM D-AP5 into the pTRG region. (C) Increases in respiratory frequency and peak vagal activity ∼2 min after a unilateral microinjection of a mixture of 1 mM AMPA and 2 mM NMDA into the pTRG. The site where drugs were microinjected (pink area) is shown on a schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon. VA, raw vagal nerve activity; IVA, integrated vagal nerve activity. Modified from Cinelli et al. (2013).

of these drugs into the pTRG (Martel et al., 2007; Cinelli et al., 2013, 2014), thus supporting the hypothesis that this region is crucial for respiratory rhythm generation. Examples of the apnea caused by ionotropic glutamate receptor blockade are reported in Fig. 2A and B. In agreement with this finding and with the view that changes in respiratory frequency are due to an action on the central mechanisms generating the respiratory rhythm (Gray et al., 1999; Feldman and Del Negro, 2006; Bongianni et al., 2008), we have shown that microinjections of glutamate agonists into the pTRG cause a marked increase in respiratory frequency associated with increases in peak amplitude and duration of vagal bursts (Cinelli et al., 2013). Fig. 2C shows increases in respiratory frequency following a microinjection of glutamate receptor agonists into the pTRG. Recently, evidence has been provided that pTRG neurons, retrogradely labeled from the vagal motoneuron region, are immunoreactive for glutamate, thus showing that glutamatergic transmission mediates the excitatory input to vagal motoneurons (Fig. 3). In the pTRG region there are also glutamate-expressing cells, not retrogradely labeled, that may represent interneurons of the respiratory CPG (Cinelli et al., 2013). Accordingly, a recent study reported the presence of numerous glutamatergic neurons in the isthmic region of the lamprey where we ˜ et al., have identified retrogradely labeled neurons (Villar-Cervino 2012). 5. GABAergic and glycinergic mechanisms in the respiratory network Both GABA- and glycine-mediated inhibition are not essential for respiratory rhythm generation (Rovainen, 1983; Bongianni et al., 2006), but may represent mechanisms suitable to regulate

the excitability level of the respiratory network (Mutolo et al., 2011; Cinelli et al., 2014). Blockade of GABAA and/or glycine receptors by bath application of the appropriate antagonists causes potent excitatory effects on respiration by increasing the frequency and amplitude of vagal bursts. On the other hand, GABAB receptor antagonists applied to the bath induce slight decreases in the respiratory frequency (Bongianni et al., 2006). Antagonist microinjections into the pTRG revealed that an important modulatory control is exerted at that level by GABA acting on GABAA and GABAB receptors, while a glycinergic modulation is lacking (Cinelli et al., 2014). GABAB antagonism within the pTRG induces only modest decreases in respiratory frequency probably due to a presynaptic mechanism. The blockade of GABAA receptors increases respiratory network excitability apparently by acting on the mechanisms generating respiratory bursts within the pTRG with little influence on the interburst period. These outcomes suggest that GABA receptors may conceivably contribute to an inhibitory control of the excitability of pTRG neurons and to generate a more regular respiratory rhythm. Consistent with our interpretation, an increase in neuronal excitability of the preBötzinger complex (preBötC), the proposed mammalian respiratory CPG, has been observed following a blockade of GABAA receptors both in in vitro and in vivo preparations (Kam et al., 2013). Furthermore, during the apnea caused by bath application of ionotropic glutamate receptor antagonists, the blockade of GABAA , but not glycine receptors within the pTRG causes the resumption of rhythmic activity (Cinelli et al., 2014), thus underlining the prominent role of GABAergic mechanisms within the pTRG. These effects are illustrated in Fig. 4A and B. The inhibitory control of pTRG neurons is also emphasized by the prolonged apnea caused by the GABAA agonist muscimol microinjected into the pTRG (Fig. 4C). Retrogradely labeled neurons within the pTRG are




Fig. 3. Distribution of glutamate immunoreactivity in the pTRG. (A) Schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon showing the site of a Neurobiotin injection into the vagal nucleus (green) and the location of retrogradely labeled neurons within the pTRG (green circles). (B) Photomicrograph of a transverse section at the level of the rostral rhombencephalon showing retrogradely labeled neurons (merged, Neurobiotin green + glutamate immunoreactivity red signals) in the pTRG region after a Neurobiotin injection into the vagal motoneuron pool. (C) Photomicrographs at a higher magnification of the portion of the transverse section indicated by the white rectangle in B showing retrogradely labeled neurons, glutamate immunoreactivity and merged image at the level of the pTRG region. Retrogradely labeled neurons displaying immunoreactivity for glutamate are indicated by white arrows. ARRN, anterior rhombencephalic reticular nucleus; I1 , isthmic Müller cell; pTRG, paratrigeminal respiratory group; V, trigeminal motor nucleus; VII, facial motor nucleus; IX, glossopharyngeal motor nucleus; X, vagal motor nucleus. Scale bars: B, 200 ␮m; C, 100 ␮m. Modified from Cinelli et al. (2013).

lacking GABA, but are surrounded by GABA-immunoreactive structures (Fig. 4D). Interestingly, increases in respiratory frequency caused by bath application of bicuculline or strychnine (Rovainen, 1983; Bongianni et al., 2006) are mimicked by microinjections of these drugs into the vagal motoneuron region (Cinelli et al., 2014). The effects caused by blockade of GABAA and glycine receptors within the vagal motoneuron region are reported in Fig. 5. Evidence has been provided that neurons within this region receive GABAergic and glycinergic inputs and are involved in the regulation of respiratory frequency via ascending excitatory projections to the pTRG (Cinelli et al., 2014). Projecting neurons are retrogradely labeled by injections of Neurobiotin into the pTRG. Preliminary results show that these neurons display glutamate immunoreactivity (unpublished data). Of note, bicuculline applied to the vagal motoneuron region during apnea caused by ionotropic glutamate receptor blockade does not restore the respiratory rhythm, thus suggesting that this region does not possess the rhythmogenic properties hypothesized by previous studies (see e.g. Kawasaki, 1979, 1984; Thompson, 1985, 1990; Martel et al., 2007). However, the functional role of GABAergic and glycinergic inputs to neurons located in the vagal motoneuron region remains unclear. 6. Respiratory role of neurokinins and acetylcholine Neurokinins (NKs) have an important modulatory role in the lamprey respiratory network (Mutolo et al., 2010). Microinjections of SP as well as NK1 , NK2 and NK3 receptor agonists into the pTRG increase the frequency and amplitude of vagal bursts. Furthermore, SP microinjections into the pTRG (Fig. 6A) restore rhythmic respiratory activity during apnea induced by bath application of riluzole and flufenamic acid used to block the burst-promoting currents, i.e. the persistent Na+ current (INaP ) and the Ca2+ -activated nonspecific cation current (ICAN ), respectively. The rhythmogenic role of SP is also confirmed by recent findings (Cinelli et al., 2013) showing that the respiratory rhythm can be re-established by SP microinjected into the pTRG during apnea caused by a blockade of ionotropic glutamate receptors within this region (Fig. 6B). The

presence of an intense SP-immunoreactivity in close proximity to pTRG neurons is consistent with these findings (Fig. 6C). ACh plays an important excitatory role on respiration under basal conditions and is also capable per se of maintaining rhythmic respiratory activity when both fast excitatory and inhibitory neurotransmission are impaired. Both these effects are achieved through an action on ␣7 nicotinic ACh receptors of pTRG neurons (Mutolo et al., 2011). Activation of these receptors by nicotine increases respiratory frequency, while their blockade with D-tubocurarine or ␣-bungarotoxin reduces respiratory frequency and increases the duration of vagal bursts. Combined histological and functional findings strongly support the hypothesis that pTRG neurons expressing ␣7 nicotinic ACh receptors may have a rhythmogenic role (Mutolo et al., 2011; Cinelli et al., 2013). During blockade of both fast excitatory and inhibitory neurotransmission, the respiratory rhythmic activity persists, although at reduced frequency, and is suppressed by blockade of pTRG ␣7 nicotinic ACh receptors (Fig. 7A). Furthermore, during the apnea induced by the blockade of ionotropic glutamate receptors within the pTRG, microinjections of nicotine into the same region restore rhythmic respiratory activity (Fig. 7B). It is noteworthy that immunohistochemical experiments revealed the presence of ␣-bungarotoxin binding sites (indicating nicotinic receptors) throughout the pTRG area and particularly on the soma of retrogradely labeled neurons projecting to the vagal motoneuron region (Fig. 7C). In agreement with previous findings (Pombal et al., 2001; Le Ray et al., 2003), we have also provided evidence that cholinergic neurons are close to and intermingled with retrogradely labeled pTRG neurons (Cinelli et al., 2013). Together, these findings identify a novel cholinergic modulatory and possibly subsidiary rhythmogenic mechanism within the lamprey respiratory network and motivate further studies on the respiratory role of cholinergic receptors in different animal species. The findings on the resumption of respiratory rhythm following SP or nicotine microinjections into the pTRG fit the “group-pacemaker” hypothesis proposed for respiratory rhythm generation in mammals (Del Negro et al., 2005; Feldman and Del Negro, 2006). It was found that a blockade of the burst-promoting currents eliminates the respiratory rhythm that, however, could




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Fig. 4. Role of GABAA receptors in the resumption of respiratory activity during blockade of ionotropic glutamate receptors. (A) Bath application of 20 ␮M CNQX and 100 ␮M D-AP5 abolished the respiratory rhythm that was restored by 10 ␮M bicuculline (Bic) added to the bath. A schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon in the perfused recording chamber is shown. (B) Bilateral microinjections of 1 mM Bic into the pTRG restored the respiratory rhythm during apnea caused by bath application of 20 ␮M CNQX and 100 ␮M D-AP5. The sites where Bic was microinjected (pink areas) are shown on a schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon. (C) Suppression of respiratory rhythmic activity ∼1 min after a unilateral microinjection of 0.2 mM muscimol (Mus) into the pTRG region. (D) Photomicrograph of a transverse section from the isthmic region at the level of the pTRG (left panel) showing retrogradely labeled neurons (green) after bilateral injections of Neurobiotin into the vagal motoneuron pools. GABA immunoreactivity is shown in red (scale bar 200 ␮m). A photomicrograph at a higher magnification of the portion of the transverse section (white box) showing retrogradely labeled neurons within the pTRG surrounded by GABA-immunoreactive structures is reported in the right panel (scale bar 25 ␮), ARRN, anterior rhombencephalic reticular nucleus; I1 , isthmic Müller cell; pTRG, paratrigeminal respiratory group; V, trigeminal motor nucleus; VII, facial motor nucleus; IX, glossopharyngeal motor nucleus; X, vagal motor nucleus; VA, raw vagal nerve activity; IVA, integrated vagal nerve activity. Modified from Cinelli et al. (2014).

be restored by increasing network excitability by exogenous excitatory agents (Del Negro et al., 2005; Feldman and Del Negro, 2006). The resumption of respiratory rhythmic activity was suggested to result from synaptic glutamatergic interconnections that combine with the intrinsic membrane properties of neurons without the involvement of pacemaker neurons. However, how this could have occurred in our preparations during ionotropic glutamate receptor blockade within the pTRG is at present only matter of speculation and the reasons underlying respiratory rhythm resumption in the lamprey remain unclear (see Cinelli et al., 2013). An involvement of

metabotropic glutamate receptors seems unlikely since their blockade during a concomitant removal of fast synaptic excitatory and inhibitory transmission did not produce any change in respiration (Mutolo et al., 2011). SP effects may be due to an increase in an ˜ and Ramirez, 2004; Ben ICAN -dependent bursting mechanism (Pena Mabrouk and Tryba, 2010). On the other hand, nicotine could have produced the resumption of rhythmic activity by increasing the excitability of pTRG neurons through a Ca2+ -dependent mechanism (Albuquerque et al., 2009). Finally, we can hypothesize a role of gap junctions in respiratory rhythm resumption, although at present




Fig. 5. Respiratory role of GABAA and glycine receptors within the vagal motor nucleus. (A) Marked increases in respiratory frequency ∼4 min after a unilateral microinjection of 1 mM bicuculline (Bic) into the region of vagal motoneurons (MN region). (B) Marked increases in respiratory frequency ∼3 min after a unilateral microinjection of 1 mM strychnine (Stryc) into the vagal MN region. Sites where Bic or Stryc microinjections (blue area) were performed are shown on a schematic illustration of a dorsal view of the lamprey mesencephalon/rhombencephalon. V, trigeminal motor nucleus; VII, facial motor nucleus; IX, glossopharyngeal motor nucleus; X, vagal motor nucleus; VA, raw vagal nerve activity; IVA, integrated vagal nerve activity. Modified from Cinelli et al. (2014).

no information is available on their presence and function in the lamprey respiratory network. 7. Evolutionary conserved characteristics of the respiratory CPG The most important findings on the connectivity within the respiratory network of the lamprey and relevant neurotransmitter influences are schematically illustrated in Fig. 8. We believe that the pTRG has a crucial role in respiratory rhythm generation similar to that attributed to the preBötC in mammals (Feldman and Del Negro, 2006; Smith et al., 1991). Like the pTRG, the preBötC contains predominantly glutamatergic neurons that express NK1 receptors and are specifically sensitive to opioids and SP (Gray et al., 1999, 2001; Guyenet et al., 2002; Feldman and Del Negro, 2006; Feldman et al., 2013). A possible difference is that the lamprey pTRG is located in the rostral rhombencephalon/isthmic region corresponding to the rostral pons, while the preBötC is located in the medulla. The pontine respiratory group and especially the Kölliker-Fuse nucleus have important respiratory functions, such as the regulation of the inspiratory–expiratory phase transition and the genesis of the postinspiratory activity (Dutschmann and Dick, 2012; Bautista and Dutschmann, 2014; Poon and Song, 2014 also for further Refs.). However, available data do not support the notion that they display functional characteristics similar to those observed in the pTRG and in the preBötC. As to the inhibitory control of respiration, the respiratory rhythmogenesis persists after a blockade of synaptic inhibition, not only in neonatal rodent preparations (reviewed in Feldman et al., 2013), but also in the adult lamprey (Rovainen, 1983; Bongianni et al., 2006; Cinelli et al., 2014) and in the adult turtle (Johnson et al., 2002, 2007). In addition, a blockade of synaptic inhibition in the pre-metamorphic tadpole abolishes fictive gill ventilation, but not lung ventilation (Galante et al., 1996; Broch et al., 2002). These findings are consistent, at least to some extent, with recent results in adult mammals (e.g. Bongianni et al., 2010; Feldman et al., 2013; Janczewski et al., 2013; Kam et al., 2013 also for further Refs.). Intriguing proposals on the homology between oscillators in mammals and lower vertebrates have been advanced despite the

insufficiency of available supporting data (Wilson et al., 2006). In agreement with Kinkead (2009), we believe that the lamprey pTRG displays a high homology not only with the mammalian preBötC, but also with the neural mechanisms generating lung ventilation in amphibians (Wilson et al., 2002; Vasilakos et al., 2005; Chen and Hedrick, 2008; Kottick et al., 2013) and turtles (Johnson et al., 2002, 2007), rather than with those that generate gill respiration in tadpoles (Galante et al., 1996; Broch et al., 2002). All these different oscillators have as a possible underlying rhythm generating mechanism the “group-pacemaker” model (Del Negro et al., 2005; Feldman and Del Negro, 2006). In addition, they display opioid sensitivity and, at least in frogs and mammals, SP sensitivity. Admittedly, in the lamprey the active phase is expiration, thus the pTRG could more appropriately correspond to the retrotrapezoid nucleus/parafacial respiratory group, i.e. the hypothesized rostral expiratory oscillator of mammals (see e.g. Onimaru et al., 2009; Thoby-Brisson et al., 2009; Guyenet and Mulkey, 2010; Feldman et al., 2013; Smith et al., 2013). This oscillator may display burst activity involving endogenous INaP -dependent properties, as it occurs in the preBötC (Fortin and Thoby-Brisson, 2009; ThobyBrisson et al., 2009; Molkov et al., 2010). In addition, it contains glutamatergic neurons that express NK1 receptors, but it is not sensitive to opioids (Mulkey et al., 2004; Onimaru et al., 2008; Takakura et al., 2008; Lazarenko et al., 2009; Thoby-Brisson et al., 2009; for reviews, see Feldman et al., 2013; Guyenet and Mulkey, 2010). In conclusion, similarly to other neurophysiological features (Ericsson et al., 2011, 2013; Stephenson-Jones et al., 2011, 2012a, 2012b), the basic oscillatory and neuromodulatory mechanisms of the respiratory network seem to be highly evolutionary conserved regardless of their location and their inspiratory or expiratory function. 8. Considerations on the evolutionary trends in respiratory rhythm generation across the vertebrate classes The finding that the respiratory rhythm generator in the lamprey, and possibly also in jawed fishes, is localized within the reticular formation close to the trigeminal nucleus is not surprising. In fact, the evolutionary origin of respiratory mechanisms in vertebrates is from structures and pumps initially associated with




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Fig. 6. Rhythmogenic role of substance P. (A) Bath coapplication of riluzole (RIL) and flufenamic acid (FFA) at 50 ␮M abolished the respiratory rhythm that was restarted by bilateral microinjections of 1 ␮M substance P (SP) into the pTRG. (B) Bilateral microinjections of 1 mM CNQX and 5 mM D-AP5 into the pTRG abolished the respiratory rhythm that was restored ∼1 min following bilateral microinjections of 1 ␮M SP into the same sites. (C) SP immunoreactivity within the pTRG. Confocal photomicrographs showing retrogradely labeled neurons (green signal) after injections of Neurobiotin into the vagal motoneuron pool, SP immunoreactivity (red signal) and merged image. Scale bar, 30 ␮m. VA, raw vagal nerve activity; IVA, integrated vagal nerve activity. Adapted from Cinelli et al. (2013).

feeding (Rovainen, 1996; Kardong, 2006; Kinkead, 2009; Milsom, 2010; Taylor et al., 2010). At this stage, the trigeminal motor mechanism plays a prominent role and is the first mover in the respiratory sequence that also involves other cranial motor nuclei. It is mainly responsible for velar pumping in the larval lamprey and for buccal pumping in jawed fishes (see e.g. Taylor et al., 2010). In addition, the velum of larval lampreys in the adult becomes a valve that allows breathing when the mouth is engaged in feeding behavior (Kinkead, 2009). Despite the fact that respiratory activity is produced by a tidal pump (adult lampreys) or by a suction/force pump driven by muscles innervated by branchiomeric and hypobranchial nerves (jawed fishes), the original generator of the respiratory rhythm may reside in the reticular formation close to the trigeminal nucleus and send drive projections to facial, glossopharyngeal and vagal motoneurons, thus maintaining a hierarchically dominant role. From an evolutionary point of view, we should recall that in air-breathing fishes, amphibians, reptiles, birds and mammals the respiratory activity changes progressively from a buccal/branchial ventilation

to a ventilation primarily driven by an aspiration pump (Kinkead, 2009; Milsom, 2010; Taylor et al., 2010). Despite the differences displayed by the different species in the pattern of conveying air and in the function of the respiratory muscles, evidence is accumulating that the respiratory rhythm generator within the reticular formation has shifted from a position close to the trigeminal nucleus, that has lost its primary pumping respiratory function, to a location close to the other cranial motor nuclei. These latter, along with spinal motor nuclei innervating the intercostal muscles and diaphragm, progressively acquire a prominent respiratory role. These changes in the location of the respiratory CPG obviously imply a caudal migration of the original rhythm generating mechanism or the development of a new respiratory oscillator or multiple oscillators, entrained to a large degree (Wilson et al., 2002, 2006; Vasilakos et al., 2005; Taylor et al., 2010; Kottick et al., 2013). The respiratory CPG of higher vertebrates and mammals remains placed in a cranial strategic position to drive close brainstem motoneurons that have to be engaged in advance and to send excitatory projections to lower respiratory muscles innervated




Fig. 7. Rhythmogenic role of acetylcholine. (A) Bath application of a cocktail solution containing 20 ␮M CNQX, 100 ␮M D-AP5, 10 ␮M bicuculline and 10 ␮M strychnine depressed respiratory activity that was completely abolished ∼5 min after bilateral microinjections of 2.5 ␮M ␣-bungarotoxin (␣BgTx) into the pTRG. (B) Bilateral microinjections of 1 mM CNQX and 5 mM D-AP5 into the pTRG abolished the respiratory rhythm that was restored ∼1 min following bilateral microinjections of 1 mM nicotine (Nic) into the same sites. (C) Distribution of ␣-bungarotoxin binding sites in the pTRG area. Photomicrographs showing retrogradely labeled neurons (red signal) following injections of Texas Red conjugated dextran into the region of vagal motoneurons, ␣-bungarotoxin binding sites (green signal) and merged image. Retrogradely labeled neurons (white arrows) are surrounded by ␣-bungarotoxin binding sites. Scale bar, 25 ␮m. VA, raw vagal nerve activity; IVA, integrated vagal nerve activity. Adapted from Cinelli et al. (2013).

by spinal motoneurons. In fact, brainstem motoneurons are still recruited during respiration, but their main role changed and became that of maintaining stability and patency of the upper airways (e.g. Von Euler, 1986). Interestingly, trigeminal motoneurons display rhythmic respiratory activity in newborn rodents (e.g. Jacquin et al., 1999; Koizumi et al., 1999, 2002) and even in humans especially under particular conditions (Sauerland et al., 1981; StJohn and Bledsoe, 1985; Hollowell and Suratt, 1989; Hollowell et al., 1991). These findings may possibly account for the persistence of vestiges of the original trigeminal oscillator and for the high homology between the pTRG and the preBötC. It should be kept in mind that within the brainstem and spinal cord neural circuits capable of generating rhythmic motor behaviors develop in a segmental fashion (see Kinkead, 2009; Taylor et al., 2010). Each major group of respiratory motoneurons has been suggested to be coupled to its own rhythm generator (Champagnat and Fortin, 1997). This segmental configuration appears to be transient and reorganized over the course of the development to produce coordinated and effective movements (Kinkead, 2009). However, there is evidence of a resumption of a trigeminal rhythm following transection of the brainstem at the ponto-medullary junction as well

as after kainic acid lesions of the dorsal and ventral respiratory groups (St-John and Bledsoe, 1985). This could suggest the presence of a trigeminal oscillator in mammals under appropriate conditions. In our opinion, during the evolutive steps towards mammalian respiration there is a concomitant maturation of the respiratory network. The primordial respiratory trigeminal oscillator capable of generating a very simple respiratory pattern is progressively embedded into a complex distributed neural network subserving the generation of the breathing pattern in mammals (Smith et al., 2007, 2013). We believe that the main concern in the evolution of the neural control of breathing is not represented by the complexity of the breathing pattern that can be fairly complex also in lower vertebrates, but by other properties of respiration, such as rhythm stabilization, optimization of the energetic cost, integration with other non respiratory functions of respiratory muscles and adjustments to different behavioral and environmental conditions (Von Euler, 1986). Most of the presented considerations are speculative, nevertheless they may provide hints for further studies not only on the evolutionary trends in respiratory rhythm generation, but also on the control of




Fig. 8. Schematic drawing representing findings on the connectivity within the respiratory network and relevant neurotransmitter influences. The pTRG region is shown with its projections (pink) to ipsilateral and contralateral vagal motoneuron (red) regions and to the contralateral pTRG (Gariépy et al., 2012; Cinelli et al., 2013, 2014). Excitatory (yellow) and inhibitory (blue) influences on the pTRG region (Mutolo et al., 2007, 2010, 2011; Cinelli et al., 2013, 2014) and the vagal motoneuron region are illustrated. Glutamatergic (Glu) projections to the pTRG (green) from neurons located in the vagal area have also been reported. ACh, acetylcholine; GABA, ␥-aminobutyric acid; Gly, glycine; pTRG, paratrigeminal respiratory group region; SP, substance P; X, vagal motoneuron region. Modified from Cinelli et al. (2014).

breathing in mammals both under physiological and pathological conditions. Acknowledgments This study was supported by grants from the Ministry of Education, University, and Research of Italy and the A. Menarini United Pharmaceutical Industries. E.C. is supported by a Postdoctoral Fellowship from Regione Toscana and Menarini United Pharmaceutical Industries. References Albuquerque, E.X., Pereira, E.F., Alkondon, M., Rogers, S.W., 2009. Mammalian nicotinic acetylcholine receptors: from structure to function. Physiol. Rev. 89, 73–120. Bautista, T.G., Dutschmann, M., 2014. Inhibition of the pontine Kolliker-Fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat. Neuroscience 267, 22–29. Ben Mabrouk, F., Tryba, A.K., 2010. Substance P modulation of TRPC3/7 channels improves respiratory rhythm regularity and ICAN-dependent pacemaker activity. Eur. J. Neurosci. 31, 1219–1232. Bongianni, F., Deliagina, T.G., Grillner, S., 1999. Role of glutamate receptor subtypes in the lamprey respiratory network. Brain Res. 826, 298–302. Bongianni, F., Mutolo, D., Carfi, M., Pantaleo, T., 2002. Group I and II metabotropic glutamate receptors modulate respiratory activity in the lamprey. Eur. J. Neurosci. 16, 454–460. Bongianni, F., Mutolo, D., Cinelli, E., Pantaleo, T., 2008. Neurokinin receptor modulation of respiratory activity in the rabbit. Eur. J. Neurosci. 27, 3233–3243. Bongianni, F., Mutolo, D., Cinelli, E., Pantaleo, T., 2010. Respiratory responses induced by blockades of GABA and glycine receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit. Brain Res. 1344, 134–147. Bongianni, F., Mutolo, D., Nardone, F., Pantaleo, T., 2006. GABAergic and glycinergic inhibitory mechanisms in the lamprey respiratory control. Brain Res. 1090, 134–145.

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