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Reflections on respiratory rhythm generation
Progress in Brain Research, Vol. 143 ISSN 0079-6123 Copyright ß 2004 Elsevier BV. All rights reserved
CHAPTER 7
Reflections on respiratory rhythm generation Kazuhisa Ezure* Department of Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan
Abstract: Knowledge about neuronal mechanisms that control respiration is being advanced rapidly by studies that make use of both mature in vivo animals and in vitro neonates. The available data suggest that particular types of neurons within selected networks of the ventrolateral medulla are essential for respiratory rhythm generation. There are many uncertainties, however, about the correspondence between neurons identified by the above two approaches, because there are virtually no studies that have combined them. In this chapter, I propose a hypothesis that shows how neonatal respiratory neurons, with either retained or modified intrinsic cellular properties, develop into mature, wellcharacterized respiratory neurons located in medullary areas called the Bo¨tzinger and pre-Bo¨tzinger complex. Currently, the most plausible models of respiratory rhythmogenesis are hybrid ones that include both intrinsic cellular and network properties.
Introduction
recording from whole amounts of portions of brainstem-spinal cord or thin brainstem slices in neonates (Suzue, 1984; Onimaru and Homma, 1987; Smith et al., 1990). These much-more-reduced in vitro preparations allow precise control of the extracellular and intracellular environment of single neurons (e.g., by using patch-clamp electrodes), and have thereby revealed many microproperties of the respiratory system at the cellular and subcellular level (e.g., Kudo et al. and Nakamura et al., Chapters 5 and 9 of this volume). The combined data from mature in vivo and neonatal in vitro preparations now suggest that particular types of neurons within particular parts of the ventrolateral medulla and their networks are essential for respiratory rhythm generation (see also Kirkwood and Ford, Chapter 10 of this volume). There are incongruities, however, in the knowledge obtained by the use of these two approaches. Because there are virtually no studies that have combined them, there is much current uncertainty about information obtained from neonatal in vitro
Respiratory movement, ventilation of the lungs with air, is characterized by its automatic rhythmicity and finely regulated contraction of respiration-related muscles that accommodate moment-to-moment chemical needs and mechanical conditions. The basic question of how the automatic rhythmicity is produced by the CNS has attracted much attention from physiologists. Recently, this field has made substantial progress using two different but complementary approaches. One is the in vivo approach on surgically reduced mature whole animals, whose use, together with refined electrophysiological and neuroanatomical methods at the level of single neurons, has greatly increased our knowledge about the overall respiratory system and its network mechanisms (Feldman, 1986; Bianchi et al., 1995; Ezure, 1996). The other is the in vitro approach, which involves *Corresponding author: Tel.: þ 81-42-325-3881; Fax: þ 81-42-321-8678; E-mail: [email protected]
67 DOI: 10.1016/S0079-6123(03)43007-0
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preparations versus mature in vivo preparations. The purpose of this chapter is to address this issue, with special emphasis on the genesis of the respiratory rhythm.
Key questions In vivo studies using adult mammals have identified a wide variety of respiratory neurons (defined below), their efferent and afferent properties, and their interconnections (Cohen, 1979; Feldman, 1986; Ezure, 1990; Bianchi et al., 1995). In the brainstem’s network of respiratory neurons, excitatory and inhibitory synaptic connections play a crucial role in the generation of the respiratory rhythm. In particular, glycinergic and GABAergic inhibitory mechanisms are essential in adult animals (Hayashi and Lipski, 1992; Paton and Richter, 1995; Pierrefiche et al., 1998). On the other hand, in vitro studies on largely neonatal rats have revealed pacemakerlike neurons. These, together with their excitatory (not inhibitory) interconnections, have been put forward as the fundamental kernel for respiratory rhythm generation (Onimaru and Homma, 1987; Ballanyi et al., 1999; Gray et al., 1999; Johnson et al., 2001). To what extent are the mechanisms of respiratory rhythm generation inferred in neonatal animals comparable with those inferred in mature mammals? To answer this question, it is first necessary to determine how pacemaker-like neurons found in neonatal in vitro preparations correspond to or develop into neurons identified in mature in vivo animals. Next, resolution is required of the longstanding issue: the relative importance for rhythm generation of network versus pacemaker properties. Which of the two is essential for the rhythm, or are both critical?
Subtle aspects of respiratory center terminology Various neurons in the central nervous system fire in synchrony with respiration and are called respirationrelated neurons, or simply, respiratory neurons. Of these, the ones primarily responsible for respiratory
control constitute the so-called ‘respiratory center’. It is composed of three major cell groups in the medulla: the dorsal respiratory group (DRG), the ventral respiratory group (VRG) and the Bo¨tzinger complex (BOT). These groups, and other cells of the overall respiratory center, include neurons with different functions. Some excite or inhibit other respiratory cells. Others are bulbospinal neurons that transmit respiratory activity to the spinal cord. Still others are motoneurons that innervate upper airway muscles. A variety of other neurons exchange information between the respiratory center and other neuronal centers and networks. Finally, chemoreceptor neurons, which may or may not have a respiratory rhythm, help sustain the activity of rhythm-generating neurons. These neurons, as a whole, comprise the respiratory center whose overall operation is to produce automatic rhythmicity, receive chemical and mechanical information, and integrate this information for the subsequent generation of motor outputs that result in optimal gas exchange within the lungs. Some terms require careful definition. For example, ‘rostral ventrolateral medulla (RVLM)’ was originally used to demarcate cardiovascularrelated neurons (Ross et al., 1984). Anatomically, it refers to the medullary region located immediately caudal to the facial nucleus, extending from the compact formation of the nucleus ambiguus to the ventral medullary surface. The BOT, which is functionally defined as the region containing E-AUG (see the next section) neurons (Merrill et al., 1983), is included within the RVLM. Thus, the RVLM includes both the BOT and the region containing cardiovascular presympathetic neurons (Kanjhan et al., 1995). The pre-Bo¨tzinger complex (pre-BOT) is a region of respiratory neurons caudal to the BOT (Smith et al., 1991). Its boundaries are reasonably well defined, albeit not with absolute precision (Pilowsky and Feldman, 2001). Anatomically, the pre-BOT is reported to be situated caudal to the compact formation of the nucleus ambiguus, i.e., just caudal to the RVLM (Johnson et al., 1994). Functionally, it corresponds to the transitional zone between the BOT and the rostral VRG (Ezure, 1990, 1996; Connelly et al., 1992; Johnson et al., 1994). In this chapter, the term pre-BOT/RVLM refers collectively to the RVLM and the pre-BOT region.
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In vivo studies reveal network properties Respiratory neurons that fire during the inspiratory (I) phase of breathing are called I neurons, whereas those that fire during the expiratory (E) phase are termed E neurons. Phase-spanning neurons fire in both phases (Cohen, 1979). In both phases, respiratory neurons exhibit characteristic firing patterns, e.g., there are augmenting (AUG), decrementing (DEC) and constant (CON) types of discharge (Ezure, 1990; Duffin et al., 1995). A large number of neurons in the respiratory center are motoneurons that supply upper airway muscles. They do not contribute to rhythm generation, however. Some bulbospinal neurons, such as the E-AUG neurons of the caudal VRG, have no medullary collaterals. Therefore, they, too, do not contribute to rhythm generation (Arita et al., 1987). On the other hand, many other of the respiratory neurons identified to date have medullary projections and can indeed contribute to rhythm generation. For example, bulbospinal I-AUG neurons of the DRG and VRG excite not only spinal I neurons but also medullary I neurons via their medullary axon collaterals (Ezure and Manabe, 1989a). E-AUG neurons of the BOT, whose distribution actually defines this region, arborize extensively within the respiratory center and maintain the E phase by widely inhibiting I neurons (Merrill et al., 1983; Otake et al., 1987; Ezure, 1990; Jiang and Lipski, 1990; Bryant et al., 1993). E-DEC, I-CON and I-DEC neurons of the BOT and VRG are propriobulbar neurons with extensive medullary projections (Ezure et al., 1989b; Otake et al., 1990). E-DEC neurons contribute to the transition from the I to E phase by inhibiting I neurons (Ezure and Manabe, 1988). This transition may be assisted by the firing of I/E ‘phase-spanning’ neurons under some conditions, albeit this function has not yet been established unequivocally. I-DEC neurons begin firing at the transition from the E to I phase, and they inhibit not only E but also I neurons (Ezure, 1990). I-CON neurons exhibit a burst of discharge in the I phase, and make extensive excitatory connections with various types of I neuron (Ezure et al., 1989b). Of the above, I-CON, I-AUG, I-DEC and E-AUG (of the BOT) neurons are of particular
interest in relation to neurons studied in neonatal in vitro preparations. Those are located within the pre-BOT/RVLM region, where they span the BOT and rostral part of the VRG.
In vitro analysis of the RVLM/pre-BOT The pre-BOT of neonates was proposed by Feldman and colleagues to contain a rhythm-generating kernel (Smith et al., 1991; Rekling and Feldman, 1998). Alternatively, Onimaru and colleagues stress that a more rostral region, the RVLM, contains such a kernel (Onimaru et al., 1997; Ballanyi et al., 1999). In both of these in-vitro-derived hypotheses, the rhythm-generating kernel comprises a network of synaptically coupled excitatory neurons that possess pacemaker-like properties. Their hypotheses share four findings. (1) In the pre-BOT/RVLM of neonatal rats, neurons possessing some properties of pacemaker-like activity have been identified by two independent groups (Onimaru and Homma, 1987; Smith et al., 1991). (2) A medullary slice in either the transverse or sagittal plane can produce a respiratory rhythm if it includes the pre-BOT region (Smith et al., 1991; Paton et al., 1994). (3) Rhythmic activity persists even after blockage of inhibitory synaptic transmission (Feldman and Smith, 1989; Onimaru et al., 1990), thereby indicating that glycinergic and/ or GABAergic inhibition is not necessary for basic rhythm generation. (4) Chemical and mechanical manipulation of the pre-BOT/RVLM region strongly modulates and can even abolish the respiratory rhythm (Smith et al., 1991; Funk et al., 1993; Gray et al., 1999). The RVLM pacemaker-like neurons identified by Onimaru and colleagues are called ‘pre-I neurons’. They start firing before the I phase, receive inhibition during this phase, and fire again after it (post-I phase), i.e., they exhibit characteristic biphasic E activity. Some (20%) of them do not receive inhibition in the I phase. When their synaptic connections are blocked by a low Ca2 þ /high Mg2 þ bathing medium, these neurons still continue to discharge in bursts. On the other hand, the pacemaker-like neurons found by Feldman and colleagues within the pre-BOT are called conditional busters. They exhibit bursting activity during blockage of
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synaptic transmission when their membrane potential voltage is within a selected range. Many I neurons exhibit this property in in vitro preparations (Rekling and Feldman 1998; Butera et al., 1999). Pre-I neurons of the RVLM also display conditional bursting (Ballanyi et al., 1999). Although the two pacemaker types are clearly different in properties and location, motor output is dependent on their activity. The two regions seem to produce rhythmic activity either independently or jointly (Mellen and Feldman, 2001). Thus, a substantial amount of evidence supports the conclusion that at least in reduced neonatal preparations, a kernel for rhythm generation comprises pacemaker-like neurons and their excitatory connections in the pre-BOT/RVLM region.
Some ideas on the missing link Figure 1 provides a schematic representation of the ideas presented in the following sections.
Correspondence between neurons studied in in vivo versus in vitro preparations Currently, there is uniform consensus that the preBOT/RVLM is essential for rhythm generation, as shown in both in vivo and in vitro studies. In vivo studies on adult cats have revealed critical excitatory and inhibitory respiratory neurons in this region. First, I-CON and I-DEC neurons, which are fundamental excitatory and inhibitory neurons, are
Fig. 1. Hypothetical correspondence between neurons identified in neonatal in vitro versus mature in vivo preparations. Studies in both preparations agree that respiration-related neurons in the pre-BOT/RVLM region and their interconnections are essential for rhythm generation. Many of the neonatal respiratory neurons have pacemaker or conditional bursting properties. These properties together with excitatory connections may produce the basic respiratory rhythm, with the inspiratory and the expiratory phases differentiated by mutual inhibition between the respective neuron groups. Neonatal respiratory neurons, while either retaining or modifying their intrinsic cellular properties, may develop into mature respiratory neurons. These are proposed to correspond to well-characterized respiratory neurons of the mature pre-BOT/RVLM. Neurons with filled and open ellipsoids are inhibitory and excitatory, respectively.
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concentrated in the region caudal to BOT and rostral to the rVRG. This region presumably corresponds to the pre-BOT (Ezure, 1990, 1996; Connelly et al., 1992). Second, E-AUG and E-DEC neurons, which are fundamental inhibitory neurons, are distributed in the BOT, and a part of the RVLM. It is parsimonious to propose that these neurons are the mature equivalent of pre-BOT/RVLM neurons found in neonatal animals. The author further proposes that I-CON and I-DEC neurons found in mature in vivo preparations correspond to the pre-BOT pacemaker-like neurons found in neonates. I-CON (rather than I-DEC) neurons are more likely to make excitatory connections in neonates. They have strong and widely distributed excitatory connections in adult cats, and are virtually the only known excitatory neurons in the pre-BOT region. Excitatory synaptic connections between the I-CON neurons, themselves, are feasible, because they project extensively to both the pre-BOT and several other medullary regions (Otake et al., 1990). Although I-AUG neurons make excitatory synaptic connections with I neurons (Ezure and Manabe, 1989a), the former are not pre-BOT neurons in the strict sense of the definition. Thus, the equivalent of the I-CON neuron in both mature rat and cat is likely to correspond to a pacemaker neuron with excitatory connections in the neonatal rat. Furthermore, the I-DEC neuron, which has inhibitory connections and is thereby not found in excitatory networks, may also be the equivalent of a pacemaker-like neuron in neonatal preparations. On the other hand, Feldman and colleagues hypothesize that the E–I phase-spanning neurons found in the pre-BOT region of adult (and neonatal) rodents have pacemaker properties and contribute primarily to rhythm generation (Smith et al., 1990; Funk and Feldman, 1995; Sun et al., 1998; Pilowsky and Feldman, 2001; Wang et al., 2001). These neurons have discharge that begins in the late E phase and continues into the I phase. They are also called ‘pre-I neurons’. Here they are called ‘pre-I–I neurons’ to discriminate them from Onimaru’s pre-I neurons of the RVLM. These pre-I–I neurons are reported to comprise a conspicuous population in the pre-BOT region in rodents (Paton, 1997; Sun et al., 1998; Wang et al., 2001). They are found also in adult cats (Connelly et al., 1992; Schwarzacher et al., 1995),
albeit in a smaller-sized population. It remains unclear whether pre-I–I neurons constitute a special group and whether they are equivalent to I-CON and I-DEC neurons of cats. Indeed, most I-CON and I-DEC neurons and some I-AUG neurons in the cat initiate their firing before the onset of the I phase, and thereby exhibit more-or-less preinspiratory firing. It is also not currently known if pre-I–I neurons have excitatory or inhibitory connections, or both. Such a determination is eagerly awaited (Wang et al., 2001). The final hypothesis is that the pre-I neurons of the neonatal RVLM, biphasic E neurons, develop into E-AUG or E-DEC neurons of the mature BOT. At a first glance, this hypothesis may seem farfetched, but it is plausible for several reasons. One of the most characteristic properties of neonatal pacemaker-like neurons is the synaptic inhibition they receive in the I phase. Therefore, they should almost certainly behave like E neurons in mature animals. Next, by definition, the region of these pre-I neurons in the RVLM is the BOT. Although some cardiovascular-related neurons of the RVLM have a respiratory rhythm in mature preparations (Ha¨bler et al., 1994; Sun, 1995), such a rhythm is faint, and it is unlikely that such cells correspond to pre-I neurons. Therefore, only E-AUG and E-DEC neurons in the adult RVLM correspond to neonatal pre-I neurons. Furthermore, the inhibition of pre-I neurons in the I phase has an equivalent in connectivity observed in adult cats (Ezure, 1990), i.e., I-DEC neurons that are located slightly caudal to the RVLM and inhibit E-AUG neurons. In summary, immature biphasic E neurons (pre-I neurons of the neonatal RVLM) are potentially analogous to mature E-AUG (and/or E-DEC) neurons, which can indeed exhibit biphasic E firing under some conditions. About this hypothesis, however, there are two reservations. First, E-AUG neurons already exist in immature preparations (Smith et al., 1990; Arata et al., 1998). However, it is not certain whether they are interneurons or cranial motoneurons. It is assumed that these neurons may not necessarily develop into mature E-AUG neurons, since the former are possibly inhibited by the pre-I neurons (Arata et al., 1998) that change their own firing during development. Second, some pre-I neurons seem excitatory (Onimaru et al., 1997) and
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may not correspond to mature E-AUG neurons, which are inhibitory. A group of pre-I neurons that are not inhibited at the I phase are I neurons from the beginning and it is assumed that they may develop into mature I neurons, such as I-CON neurons. If biphasic type pre-I neurons are excitatory, their corresponding neurons in mature animals cannot be specified. Here, it should be emphasized that any expiratory neurons that excite E-AUG neurons of the mature BOT have not yet been identified. Such excitatory neurons may not exist. E-AUG (and/or E-DEC) neurons of the BOT may receive tonic (possibly chemical) drives that modulate their intrinsic membrane properties as well as inhibitory inputs from I neurons. That is, these BOT neurons are called E neurons because they are inhibited by the other group of neurons that are called I neurons (see the next section for related discussion).
Network versus intrinsic cellular properties It may not be meaningful to ask which is essential for the rhythm generation—network or pacemaker properties. Evidence obtained from both mature in vivo and neonatal in vitro preparations has shown that rhythm generation requires synaptic connections between relevant neurons. Without excitatory synaptic connections, synchronized motor activity cannot be produced in neonatal animals, even if each pacemaker neuron in the circuit maintains its own independent rhythmicity. One major difference is that inhibitory mechanisms are absolutely essential for rhythm generation in mature but not neonatal preparations. In the latter, inhibitory connections are nonetheless important. Although their blockage does not completely stop the primary rhythm, it does perturb both qualitative and quantitative features of the overall rhythmicity (Funk and Feldman, 1995; Ballanyi et al., 1999). For example, Iizuka (1999) showed that after such blockage, the basic alternation between I and E activity disappeared, and both activities synchronized. His work had the important implication that primary respiratory activity is neither inspiratory nor expiratory. Rather, the two phases are differentiated only by the presence of reciprocal inhibition. It is evident, therefore, that both excitatory and inhibitory connections are
essential for eupnea in both mature and neonatal animals. Admittedly, synaptic inhibition seems more significant for rhythm generation in mature versus neonatal animals. The fact that even 22-day-old mice can produce a respiratory rhythm after blocking glycinergic inhibition (Ramirez et al., 1996) suggests, however, that the role of inhibitory connections is not so qualitatively different in the two preparations. A more appropriate question is whether the pacemaker mechanisms that are essential for the neonatal rhythm are also involved in the mature rhythm. The author is that intrinsic cellular properties, which may not necessarily be called pacemaker properties, are crucially involved in rhythm generation in adult animals. This idea is in keeping with various current hybrid models that combine active membrane properties like conditional bursting with network circuitry into the rhythm-generating kernel (Bianchi et al., 1995; Funk and Feldman, 1995; Ramirez and Richter, 1996; Butera et al., 1999). It does not seem, however, that autonomic rhythmicity can be produced by respiratory neurons, simply on the basis of their passive and active membrane properties and their excitatory and inhibitory synaptic connections. For example, it is quite difficult to interpret the firing patterns of respiratory neurons such as I-DEC and I-CON neurons without taking into account their intrinsic cellular properties. There seem to be no respiratory neurons whose synaptic connections to I-DEC neurons alone make their firing pattern decremental. It is also hard to account for the sudden start and stop of I-CON neuron firing solely on the basis of their synaptic bombardment. Therefore, it is reasonable to propose that the intrinsic and neuromodulated ionic mechanisms of I-CON and I-DEC neurons play a crucial role during generation of the respiratory rhythm.
Concluding thoughts Knowledge about neuronal mechanisms of respiration is rapidly increasing by studies that use both mature in vivo animals and in vitro neonates. Although there are missing links between the two approaches, respiratory physiologists are well positioned to model the mechanisms of respiratory
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rhythm generation. The available data suggest that the neuronal mechanisms for respiratory rhythm generation may be basically similar in both neonates and adults. Currently, hybrid models of respiratory rhythmogenesis that include both intrinsic cellular properties and network properties seem the most plausible ones. Pacemaker-like neurons in neonates may change throughout development, but there is indication that they still exist and function in the mature pre-BOT/RVLM region. In other words, respiratory neurons of adults give indication of their neonatal ancestry. Respiratory neurons in the preBOT/RVLM region with intrinsic properties like spike-frequency adaptation and neuromodulated ionic properties like conditional bursting, together with their excitatory and inhibitory interconnections, may be primarily responsible for the respiratory rhythm.
Abbreviations BOT CNS DRG E E-AUG E-DEC I I-CON I-DEC pre-BOT RVLM VRG
Bo¨tzinger complex central nervous system dorsal respiratory group expiratory augmenting expiratory decrementing expiratory inspiratory constant inspiratory decrementing inspiratory pre-Bo¨tzinger complex rostral ventrolateral medulla ventral respiratory group
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CHAPTER 7
Reflections on respiratory rhythm generation Kazuhisa Ezure* Department of Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan
Abstract: Knowledge about neuronal mechanisms that control respiration is being advanced rapidly by studies that make use of both mature in vivo animals and in vitro neonates. The available data suggest that particular types of neurons within selected networks of the ventrolateral medulla are essential for respiratory rhythm generation. There are many uncertainties, however, about the correspondence between neurons identified by the above two approaches, because there are virtually no studies that have combined them. In this chapter, I propose a hypothesis that shows how neonatal respiratory neurons, with either retained or modified intrinsic cellular properties, develop into mature, wellcharacterized respiratory neurons located in medullary areas called the Bo¨tzinger and pre-Bo¨tzinger complex. Currently, the most plausible models of respiratory rhythmogenesis are hybrid ones that include both intrinsic cellular and network properties.
Introduction
recording from whole amounts of portions of brainstem-spinal cord or thin brainstem slices in neonates (Suzue, 1984; Onimaru and Homma, 1987; Smith et al., 1990). These much-more-reduced in vitro preparations allow precise control of the extracellular and intracellular environment of single neurons (e.g., by using patch-clamp electrodes), and have thereby revealed many microproperties of the respiratory system at the cellular and subcellular level (e.g., Kudo et al. and Nakamura et al., Chapters 5 and 9 of this volume). The combined data from mature in vivo and neonatal in vitro preparations now suggest that particular types of neurons within particular parts of the ventrolateral medulla and their networks are essential for respiratory rhythm generation (see also Kirkwood and Ford, Chapter 10 of this volume). There are incongruities, however, in the knowledge obtained by the use of these two approaches. Because there are virtually no studies that have combined them, there is much current uncertainty about information obtained from neonatal in vitro
Respiratory movement, ventilation of the lungs with air, is characterized by its automatic rhythmicity and finely regulated contraction of respiration-related muscles that accommodate moment-to-moment chemical needs and mechanical conditions. The basic question of how the automatic rhythmicity is produced by the CNS has attracted much attention from physiologists. Recently, this field has made substantial progress using two different but complementary approaches. One is the in vivo approach on surgically reduced mature whole animals, whose use, together with refined electrophysiological and neuroanatomical methods at the level of single neurons, has greatly increased our knowledge about the overall respiratory system and its network mechanisms (Feldman, 1986; Bianchi et al., 1995; Ezure, 1996). The other is the in vitro approach, which involves *Corresponding author: Tel.: þ 81-42-325-3881; Fax: þ 81-42-321-8678; E-mail: [email protected]
67 DOI: 10.1016/S0079-6123(03)43007-0
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preparations versus mature in vivo preparations. The purpose of this chapter is to address this issue, with special emphasis on the genesis of the respiratory rhythm.
Key questions In vivo studies using adult mammals have identified a wide variety of respiratory neurons (defined below), their efferent and afferent properties, and their interconnections (Cohen, 1979; Feldman, 1986; Ezure, 1990; Bianchi et al., 1995). In the brainstem’s network of respiratory neurons, excitatory and inhibitory synaptic connections play a crucial role in the generation of the respiratory rhythm. In particular, glycinergic and GABAergic inhibitory mechanisms are essential in adult animals (Hayashi and Lipski, 1992; Paton and Richter, 1995; Pierrefiche et al., 1998). On the other hand, in vitro studies on largely neonatal rats have revealed pacemakerlike neurons. These, together with their excitatory (not inhibitory) interconnections, have been put forward as the fundamental kernel for respiratory rhythm generation (Onimaru and Homma, 1987; Ballanyi et al., 1999; Gray et al., 1999; Johnson et al., 2001). To what extent are the mechanisms of respiratory rhythm generation inferred in neonatal animals comparable with those inferred in mature mammals? To answer this question, it is first necessary to determine how pacemaker-like neurons found in neonatal in vitro preparations correspond to or develop into neurons identified in mature in vivo animals. Next, resolution is required of the longstanding issue: the relative importance for rhythm generation of network versus pacemaker properties. Which of the two is essential for the rhythm, or are both critical?
Subtle aspects of respiratory center terminology Various neurons in the central nervous system fire in synchrony with respiration and are called respirationrelated neurons, or simply, respiratory neurons. Of these, the ones primarily responsible for respiratory
control constitute the so-called ‘respiratory center’. It is composed of three major cell groups in the medulla: the dorsal respiratory group (DRG), the ventral respiratory group (VRG) and the Bo¨tzinger complex (BOT). These groups, and other cells of the overall respiratory center, include neurons with different functions. Some excite or inhibit other respiratory cells. Others are bulbospinal neurons that transmit respiratory activity to the spinal cord. Still others are motoneurons that innervate upper airway muscles. A variety of other neurons exchange information between the respiratory center and other neuronal centers and networks. Finally, chemoreceptor neurons, which may or may not have a respiratory rhythm, help sustain the activity of rhythm-generating neurons. These neurons, as a whole, comprise the respiratory center whose overall operation is to produce automatic rhythmicity, receive chemical and mechanical information, and integrate this information for the subsequent generation of motor outputs that result in optimal gas exchange within the lungs. Some terms require careful definition. For example, ‘rostral ventrolateral medulla (RVLM)’ was originally used to demarcate cardiovascularrelated neurons (Ross et al., 1984). Anatomically, it refers to the medullary region located immediately caudal to the facial nucleus, extending from the compact formation of the nucleus ambiguus to the ventral medullary surface. The BOT, which is functionally defined as the region containing E-AUG (see the next section) neurons (Merrill et al., 1983), is included within the RVLM. Thus, the RVLM includes both the BOT and the region containing cardiovascular presympathetic neurons (Kanjhan et al., 1995). The pre-Bo¨tzinger complex (pre-BOT) is a region of respiratory neurons caudal to the BOT (Smith et al., 1991). Its boundaries are reasonably well defined, albeit not with absolute precision (Pilowsky and Feldman, 2001). Anatomically, the pre-BOT is reported to be situated caudal to the compact formation of the nucleus ambiguus, i.e., just caudal to the RVLM (Johnson et al., 1994). Functionally, it corresponds to the transitional zone between the BOT and the rostral VRG (Ezure, 1990, 1996; Connelly et al., 1992; Johnson et al., 1994). In this chapter, the term pre-BOT/RVLM refers collectively to the RVLM and the pre-BOT region.
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In vivo studies reveal network properties Respiratory neurons that fire during the inspiratory (I) phase of breathing are called I neurons, whereas those that fire during the expiratory (E) phase are termed E neurons. Phase-spanning neurons fire in both phases (Cohen, 1979). In both phases, respiratory neurons exhibit characteristic firing patterns, e.g., there are augmenting (AUG), decrementing (DEC) and constant (CON) types of discharge (Ezure, 1990; Duffin et al., 1995). A large number of neurons in the respiratory center are motoneurons that supply upper airway muscles. They do not contribute to rhythm generation, however. Some bulbospinal neurons, such as the E-AUG neurons of the caudal VRG, have no medullary collaterals. Therefore, they, too, do not contribute to rhythm generation (Arita et al., 1987). On the other hand, many other of the respiratory neurons identified to date have medullary projections and can indeed contribute to rhythm generation. For example, bulbospinal I-AUG neurons of the DRG and VRG excite not only spinal I neurons but also medullary I neurons via their medullary axon collaterals (Ezure and Manabe, 1989a). E-AUG neurons of the BOT, whose distribution actually defines this region, arborize extensively within the respiratory center and maintain the E phase by widely inhibiting I neurons (Merrill et al., 1983; Otake et al., 1987; Ezure, 1990; Jiang and Lipski, 1990; Bryant et al., 1993). E-DEC, I-CON and I-DEC neurons of the BOT and VRG are propriobulbar neurons with extensive medullary projections (Ezure et al., 1989b; Otake et al., 1990). E-DEC neurons contribute to the transition from the I to E phase by inhibiting I neurons (Ezure and Manabe, 1988). This transition may be assisted by the firing of I/E ‘phase-spanning’ neurons under some conditions, albeit this function has not yet been established unequivocally. I-DEC neurons begin firing at the transition from the E to I phase, and they inhibit not only E but also I neurons (Ezure, 1990). I-CON neurons exhibit a burst of discharge in the I phase, and make extensive excitatory connections with various types of I neuron (Ezure et al., 1989b). Of the above, I-CON, I-AUG, I-DEC and E-AUG (of the BOT) neurons are of particular
interest in relation to neurons studied in neonatal in vitro preparations. Those are located within the pre-BOT/RVLM region, where they span the BOT and rostral part of the VRG.
In vitro analysis of the RVLM/pre-BOT The pre-BOT of neonates was proposed by Feldman and colleagues to contain a rhythm-generating kernel (Smith et al., 1991; Rekling and Feldman, 1998). Alternatively, Onimaru and colleagues stress that a more rostral region, the RVLM, contains such a kernel (Onimaru et al., 1997; Ballanyi et al., 1999). In both of these in-vitro-derived hypotheses, the rhythm-generating kernel comprises a network of synaptically coupled excitatory neurons that possess pacemaker-like properties. Their hypotheses share four findings. (1) In the pre-BOT/RVLM of neonatal rats, neurons possessing some properties of pacemaker-like activity have been identified by two independent groups (Onimaru and Homma, 1987; Smith et al., 1991). (2) A medullary slice in either the transverse or sagittal plane can produce a respiratory rhythm if it includes the pre-BOT region (Smith et al., 1991; Paton et al., 1994). (3) Rhythmic activity persists even after blockage of inhibitory synaptic transmission (Feldman and Smith, 1989; Onimaru et al., 1990), thereby indicating that glycinergic and/ or GABAergic inhibition is not necessary for basic rhythm generation. (4) Chemical and mechanical manipulation of the pre-BOT/RVLM region strongly modulates and can even abolish the respiratory rhythm (Smith et al., 1991; Funk et al., 1993; Gray et al., 1999). The RVLM pacemaker-like neurons identified by Onimaru and colleagues are called ‘pre-I neurons’. They start firing before the I phase, receive inhibition during this phase, and fire again after it (post-I phase), i.e., they exhibit characteristic biphasic E activity. Some (20%) of them do not receive inhibition in the I phase. When their synaptic connections are blocked by a low Ca2 þ /high Mg2 þ bathing medium, these neurons still continue to discharge in bursts. On the other hand, the pacemaker-like neurons found by Feldman and colleagues within the pre-BOT are called conditional busters. They exhibit bursting activity during blockage of
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synaptic transmission when their membrane potential voltage is within a selected range. Many I neurons exhibit this property in in vitro preparations (Rekling and Feldman 1998; Butera et al., 1999). Pre-I neurons of the RVLM also display conditional bursting (Ballanyi et al., 1999). Although the two pacemaker types are clearly different in properties and location, motor output is dependent on their activity. The two regions seem to produce rhythmic activity either independently or jointly (Mellen and Feldman, 2001). Thus, a substantial amount of evidence supports the conclusion that at least in reduced neonatal preparations, a kernel for rhythm generation comprises pacemaker-like neurons and their excitatory connections in the pre-BOT/RVLM region.
Some ideas on the missing link Figure 1 provides a schematic representation of the ideas presented in the following sections.
Correspondence between neurons studied in in vivo versus in vitro preparations Currently, there is uniform consensus that the preBOT/RVLM is essential for rhythm generation, as shown in both in vivo and in vitro studies. In vivo studies on adult cats have revealed critical excitatory and inhibitory respiratory neurons in this region. First, I-CON and I-DEC neurons, which are fundamental excitatory and inhibitory neurons, are
Fig. 1. Hypothetical correspondence between neurons identified in neonatal in vitro versus mature in vivo preparations. Studies in both preparations agree that respiration-related neurons in the pre-BOT/RVLM region and their interconnections are essential for rhythm generation. Many of the neonatal respiratory neurons have pacemaker or conditional bursting properties. These properties together with excitatory connections may produce the basic respiratory rhythm, with the inspiratory and the expiratory phases differentiated by mutual inhibition between the respective neuron groups. Neonatal respiratory neurons, while either retaining or modifying their intrinsic cellular properties, may develop into mature respiratory neurons. These are proposed to correspond to well-characterized respiratory neurons of the mature pre-BOT/RVLM. Neurons with filled and open ellipsoids are inhibitory and excitatory, respectively.
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concentrated in the region caudal to BOT and rostral to the rVRG. This region presumably corresponds to the pre-BOT (Ezure, 1990, 1996; Connelly et al., 1992). Second, E-AUG and E-DEC neurons, which are fundamental inhibitory neurons, are distributed in the BOT, and a part of the RVLM. It is parsimonious to propose that these neurons are the mature equivalent of pre-BOT/RVLM neurons found in neonatal animals. The author further proposes that I-CON and I-DEC neurons found in mature in vivo preparations correspond to the pre-BOT pacemaker-like neurons found in neonates. I-CON (rather than I-DEC) neurons are more likely to make excitatory connections in neonates. They have strong and widely distributed excitatory connections in adult cats, and are virtually the only known excitatory neurons in the pre-BOT region. Excitatory synaptic connections between the I-CON neurons, themselves, are feasible, because they project extensively to both the pre-BOT and several other medullary regions (Otake et al., 1990). Although I-AUG neurons make excitatory synaptic connections with I neurons (Ezure and Manabe, 1989a), the former are not pre-BOT neurons in the strict sense of the definition. Thus, the equivalent of the I-CON neuron in both mature rat and cat is likely to correspond to a pacemaker neuron with excitatory connections in the neonatal rat. Furthermore, the I-DEC neuron, which has inhibitory connections and is thereby not found in excitatory networks, may also be the equivalent of a pacemaker-like neuron in neonatal preparations. On the other hand, Feldman and colleagues hypothesize that the E–I phase-spanning neurons found in the pre-BOT region of adult (and neonatal) rodents have pacemaker properties and contribute primarily to rhythm generation (Smith et al., 1990; Funk and Feldman, 1995; Sun et al., 1998; Pilowsky and Feldman, 2001; Wang et al., 2001). These neurons have discharge that begins in the late E phase and continues into the I phase. They are also called ‘pre-I neurons’. Here they are called ‘pre-I–I neurons’ to discriminate them from Onimaru’s pre-I neurons of the RVLM. These pre-I–I neurons are reported to comprise a conspicuous population in the pre-BOT region in rodents (Paton, 1997; Sun et al., 1998; Wang et al., 2001). They are found also in adult cats (Connelly et al., 1992; Schwarzacher et al., 1995),
albeit in a smaller-sized population. It remains unclear whether pre-I–I neurons constitute a special group and whether they are equivalent to I-CON and I-DEC neurons of cats. Indeed, most I-CON and I-DEC neurons and some I-AUG neurons in the cat initiate their firing before the onset of the I phase, and thereby exhibit more-or-less preinspiratory firing. It is also not currently known if pre-I–I neurons have excitatory or inhibitory connections, or both. Such a determination is eagerly awaited (Wang et al., 2001). The final hypothesis is that the pre-I neurons of the neonatal RVLM, biphasic E neurons, develop into E-AUG or E-DEC neurons of the mature BOT. At a first glance, this hypothesis may seem farfetched, but it is plausible for several reasons. One of the most characteristic properties of neonatal pacemaker-like neurons is the synaptic inhibition they receive in the I phase. Therefore, they should almost certainly behave like E neurons in mature animals. Next, by definition, the region of these pre-I neurons in the RVLM is the BOT. Although some cardiovascular-related neurons of the RVLM have a respiratory rhythm in mature preparations (Ha¨bler et al., 1994; Sun, 1995), such a rhythm is faint, and it is unlikely that such cells correspond to pre-I neurons. Therefore, only E-AUG and E-DEC neurons in the adult RVLM correspond to neonatal pre-I neurons. Furthermore, the inhibition of pre-I neurons in the I phase has an equivalent in connectivity observed in adult cats (Ezure, 1990), i.e., I-DEC neurons that are located slightly caudal to the RVLM and inhibit E-AUG neurons. In summary, immature biphasic E neurons (pre-I neurons of the neonatal RVLM) are potentially analogous to mature E-AUG (and/or E-DEC) neurons, which can indeed exhibit biphasic E firing under some conditions. About this hypothesis, however, there are two reservations. First, E-AUG neurons already exist in immature preparations (Smith et al., 1990; Arata et al., 1998). However, it is not certain whether they are interneurons or cranial motoneurons. It is assumed that these neurons may not necessarily develop into mature E-AUG neurons, since the former are possibly inhibited by the pre-I neurons (Arata et al., 1998) that change their own firing during development. Second, some pre-I neurons seem excitatory (Onimaru et al., 1997) and
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may not correspond to mature E-AUG neurons, which are inhibitory. A group of pre-I neurons that are not inhibited at the I phase are I neurons from the beginning and it is assumed that they may develop into mature I neurons, such as I-CON neurons. If biphasic type pre-I neurons are excitatory, their corresponding neurons in mature animals cannot be specified. Here, it should be emphasized that any expiratory neurons that excite E-AUG neurons of the mature BOT have not yet been identified. Such excitatory neurons may not exist. E-AUG (and/or E-DEC) neurons of the BOT may receive tonic (possibly chemical) drives that modulate their intrinsic membrane properties as well as inhibitory inputs from I neurons. That is, these BOT neurons are called E neurons because they are inhibited by the other group of neurons that are called I neurons (see the next section for related discussion).
Network versus intrinsic cellular properties It may not be meaningful to ask which is essential for the rhythm generation—network or pacemaker properties. Evidence obtained from both mature in vivo and neonatal in vitro preparations has shown that rhythm generation requires synaptic connections between relevant neurons. Without excitatory synaptic connections, synchronized motor activity cannot be produced in neonatal animals, even if each pacemaker neuron in the circuit maintains its own independent rhythmicity. One major difference is that inhibitory mechanisms are absolutely essential for rhythm generation in mature but not neonatal preparations. In the latter, inhibitory connections are nonetheless important. Although their blockage does not completely stop the primary rhythm, it does perturb both qualitative and quantitative features of the overall rhythmicity (Funk and Feldman, 1995; Ballanyi et al., 1999). For example, Iizuka (1999) showed that after such blockage, the basic alternation between I and E activity disappeared, and both activities synchronized. His work had the important implication that primary respiratory activity is neither inspiratory nor expiratory. Rather, the two phases are differentiated only by the presence of reciprocal inhibition. It is evident, therefore, that both excitatory and inhibitory connections are
essential for eupnea in both mature and neonatal animals. Admittedly, synaptic inhibition seems more significant for rhythm generation in mature versus neonatal animals. The fact that even 22-day-old mice can produce a respiratory rhythm after blocking glycinergic inhibition (Ramirez et al., 1996) suggests, however, that the role of inhibitory connections is not so qualitatively different in the two preparations. A more appropriate question is whether the pacemaker mechanisms that are essential for the neonatal rhythm are also involved in the mature rhythm. The author is that intrinsic cellular properties, which may not necessarily be called pacemaker properties, are crucially involved in rhythm generation in adult animals. This idea is in keeping with various current hybrid models that combine active membrane properties like conditional bursting with network circuitry into the rhythm-generating kernel (Bianchi et al., 1995; Funk and Feldman, 1995; Ramirez and Richter, 1996; Butera et al., 1999). It does not seem, however, that autonomic rhythmicity can be produced by respiratory neurons, simply on the basis of their passive and active membrane properties and their excitatory and inhibitory synaptic connections. For example, it is quite difficult to interpret the firing patterns of respiratory neurons such as I-DEC and I-CON neurons without taking into account their intrinsic cellular properties. There seem to be no respiratory neurons whose synaptic connections to I-DEC neurons alone make their firing pattern decremental. It is also hard to account for the sudden start and stop of I-CON neuron firing solely on the basis of their synaptic bombardment. Therefore, it is reasonable to propose that the intrinsic and neuromodulated ionic mechanisms of I-CON and I-DEC neurons play a crucial role during generation of the respiratory rhythm.
Concluding thoughts Knowledge about neuronal mechanisms of respiration is rapidly increasing by studies that use both mature in vivo animals and in vitro neonates. Although there are missing links between the two approaches, respiratory physiologists are well positioned to model the mechanisms of respiratory
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rhythm generation. The available data suggest that the neuronal mechanisms for respiratory rhythm generation may be basically similar in both neonates and adults. Currently, hybrid models of respiratory rhythmogenesis that include both intrinsic cellular properties and network properties seem the most plausible ones. Pacemaker-like neurons in neonates may change throughout development, but there is indication that they still exist and function in the mature pre-BOT/RVLM region. In other words, respiratory neurons of adults give indication of their neonatal ancestry. Respiratory neurons in the preBOT/RVLM region with intrinsic properties like spike-frequency adaptation and neuromodulated ionic properties like conditional bursting, together with their excitatory and inhibitory interconnections, may be primarily responsible for the respiratory rhythm.
Abbreviations BOT CNS DRG E E-AUG E-DEC I I-CON I-DEC pre-BOT RVLM VRG
Bo¨tzinger complex central nervous system dorsal respiratory group expiratory augmenting expiratory decrementing expiratory inspiratory constant inspiratory decrementing inspiratory pre-Bo¨tzinger complex rostral ventrolateral medulla ventral respiratory group
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