The neuronal determinants of respiratory rhythm

Progress in NeurobiologyVol. 27, pp. 101 to 182, 1986

0301-0082/86/$0.00+0.50 1986 Pergamon Journals Lid

Printed in Great Britain,

THE NEURONAL

DETERMINANTS RHYTHM

OF RESPIRATORY

SUSAN LONG* a n d JAMES DU~rN

Department of Physiology, University of Toronto, Toronto, Ontario MSS lAB, Canada (Received 8 January 1986)

Contents Abbreviations I. Introduction and historical background 2. Respiratory neurons 2.1. Ventrolateral nucleus tractus solitarius respiratory neurons 2.1.1. 'Late-peak' inspiratory neurons 2.1.1.1. Location and morphology 2.1.1.2. Patterns of activity 2.1.1.3. Functional interrelations 2.1.1.4. Afferents 2.1.1.5. Projections and synaptic connections 2.1.1.6. Summary 2.2. Nucleus retroambigualis respiratory neurons 2.2.1. Rostral nucleus retroambigualis 2.2.1.1. 'Late-peak' inspiratory neurons 2.2.1.2. 'Early-burst' inspiratory neurons 2.2.1.3. 'Post-inspiratory' neurons 2.2.2. Caudal nucleus retroambigualis 2.2.2.1. 'Late-peak' expiratory neurons 2.3. Botzinger complex respiratory neurons 2.3.1. 'Late-peak' expiratory neurons 2.3.1.1. Location and morphology 2.3.1.2. Patterns of activity 2.3.1.3. Afferents 2.3.1.4. Projections and synaptic connections 2.3.1.5. Summary 2.4. Upper cervical respiratory neurons 2.4.1. Upper cervical inspiratory neurons 2.4.1.1. Location and morphology 2.4.1.2, Patterns of activity 2.4.1.3, Afferents 2.4.1.4. Projections and synaptic connections 2.4.1.5. Summary 3. Conclusion Acknowledgements References

101 101 107 107 107 107 109 112 113 121 125 126 126 126 142 146 150 150 162 162 162 162 163 164 167 168 168 168 169 169 170 171 171 174 174

Abbreviations vl-NTS--ventrolateral nucleus tractus solitarius; NRA--nucleus retroambigualis; r-NRA--rostral NRA; c-NRA---caudal NRA; BOT--Botzinger complex; NA--nucleus ambiguus; BS--bulbospinal; NAA--nonantidromically-activated; EPSP--cxcitatory postsynaptic potential; IPSP--inhibitory postsynaptic potential; PSP--postsynaptic potential; R~--alpha type inspiratory neuron; Rr--beta type inspiratory neuron; H R P - horseradish peroxidase; STA--spike-triggered averaging.

1. Introduction and Historical Background In the last 100 years, our concept of the organization and function of the brain stem mechanisms responsible for respiratory rhythm generation has been one of increasing complexity. The early ablation and sectioning experiments (LeGallois, 1812; Flourens, * Present address: Susan Long, M.Sc., Department of Respiratory Medicine, West Park Hospital, 82 Buttonwood Avenue, Toronto, Ontario M6M 2J5, Canada. J.PN. 27/2--&

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1842; Markwald, 1888) localized the rhythm generator to the lower brain stem and, in particular, the medulla. Then, during the period between 1880 and 1930, anatomists and comparative neurologists provided accurate descriptions of the locations, organization, and pathways of the brain stem nuclei (Ramon y Cajal, 1909; Herrick, 1922). Transections at various levels of the neuraxis demonstrated that the respiratory rhythm generator resides in the lower brain stem (pons and medulla) (Lumsden, 1922a, b; Pitts et al., 1939a, b, c; Pitts, 1946) and resulted in the assignment of respiratory functions to various regions of the pontomedullary complex. The eupneic pattern of breathing was preserved after transection at any level rostral to the caudal edge of the inferior colliculus (Lumsden, 1923a, b; Wang et al., 1957). Although stimulation of suprapontine structures in an intact preparation can affect the rate and depth of breathing, e.g. Kabat, 1936 (see review by Hockman and Duffin, 1973), these structures are not thought to be directly involved in the generation of respiratory rhythm. These studies formed the basis for the traditional multiple centre view of respiratory rhythm generation (Lumsden, 1923a, b; Pitts et al., 1939a, b, c; Pitts, 1946), which has been used as the basis for much of the research of subsequent years. For the past 30 years, respiratory neurons have been intensively studied using extra- and intracellular recordings of neuronal activity; histological techniques such as horseradish peroxidase (HRP) and intracellular dyes; classical electrophysiological techniques such as microstimulation-response and antidromic mapping; as well as the newer neurophysiological methods such as cross-correlation histograms, antidromic latency measures, and spike-triggered averaging (STA) of intracellular potentials. The common goal of all such studies has been to explain the generation of respiratory rhythm and to determine how sensory and other inputs to the lower brain stem are processed to produce a respiratory motor response. To ascertain which neurons are involved in the genesis of respiratory rhythm, microelectrode explorations have been made in different regions of the lower brain stem of the cat. Most studies have been done in the cat so that, unless otherwise stated, all of the data cited in this review were obtained from this animal. These recordings have revealed the presence of different types of respiratory neurons. Consequently, units that discharge solely during inspiration (defined according to the augmenting portion of the phrenic nerve or diaphragmatic discharge) or during expiration (the silent phase in phrenic neurogram or diaphragmatic electromyogram recordings) are classified as inspiratory or expiratory neurons, respectively. Those neurons which fire during only one phase of respiration but whose peak-firing frequency occurs at a particular point in the phase are subcategorized according to the time of peak-firing in relation to the onset of the specific respiratory phase. For example, 'early-burst' inspiratory neurons fire maximally at the onset of the inspiratory phase; and 'late-peak' inspiratory neurons have an augmenting discharge frequency with the peak frequency not occurring until late in the inspiratory phase. More recently, a third type of neuron whose discharge coincides with the period of declining discharge in the phrenic nerve or diaphragmatic discharge has been classified as a 'post-inspiratory' neuron, and with respect to the respiratory cycle, this period of declining inspiratory muscle activity has been labeled as the post-inspiratory phase (Richter, 1982a) or as the stage I expiratory phase (Richter, 1982b; Richter and Ballantyne, 1983). In the latter instance, the expiratory phase (the silent period of the phrenic neurogram) was classified as Stage II expiration (Richter, 1982b; Richter and Ballantyne, 1983). For neurons which display a discharge pattern which cannot be associated solely with one phase of the respiratory cycle but which is one of increased discharge during inspiration and a decreased discharge during expiration or vice versa, the term 'phase-spanning' has been utilized. Such respiratory neurons have been predominantly associated with pontine regions of the brain stem. A large number of respiratory neurons, many of which are phase-spanning types, can be found in the dorsolateral, rostral pons of vagotomized, spinally transected (C7), unanaesthetized cats (Bertrand and Hugelin, 1971; Bertrand et al., 1973, 1974). However, the number of neurons which exhibit a respiratory rhythm may be markedly reduced if

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the vagi are left intact (Feldman et al., 1976). Recordings from awake cats with vagi intact nevertheless show pontine neurons with a respiratory rhythm (Sieck and Harper, 1980, 1981). Destruction of this region, classically identified as the pneumotaxic centre (Lumsden, 1923a, b), produces a slower, deeper pattern of breathing in anaesthetized animals (Bertrand and Hugelin, 1971; Bertrand et al., 1974; Feldman and Gautier, 1976; Knox and King, 1976; von Euler et al., 1976). With bilateral vagotomy, the pattern of breathing becomes apneustic (inspiratory gasping). However, chronic preparations with bilateral vagotomy and pneumotaxic centre lesions which breathe apneustically when anaesthetized, eventually return to an eupneic pattern of breathing when awake (St. John et al., 1972). These observations tend to discount the possibility that these rostral pontine neurons are the primary source of respiratory rhythm generation. Two anatomically designated nuclei have been recognized in the pons; the nucleus parabrachialis medialis and the more laterally located Kolliker-Fuse nucleus. The latter nucleus has a predominance of phasically discharging inspiratory neurons (Cohen, 1958; Takagi and Nakayama, 1958), while the former contains both inspiratory and expiratory neurons as well as phase-spanning neurons, and many of these have an underlying tonic discharge. Extensive neural pathways exist between these pontine nuclei and the major medullary respiratory nuclei (to be detailed in later sections) (Kalia, 1977; Denavit-Saubie and Riche, 1977; King, 1980; St. John, 1982). Coupled with the knowledge that electrical stimulation of these regions can provoke a premature termination of the current respiratory phase (changing inspiration to expiration and vice versa) (Feldman and Gautier, 1976; von Euler and Trippenbach, 1976; Cohen, 1971), these observations have led to the hypothesis that these rostral pontine respiratory nuclei may act "to finely tune the pattern generator" (Mitchell and Berger, 1981). The postulation of an apneustic centre in the caudal pons, which would facilitate inspiration and promote apneusis if released from rostral pontine and vagal afferent influences, was originally proposed to explain the results of early transection experiments (Wang et al., 1957; Hukuhara, 1973). However, only one report (Cohen and Wang, 1959) of respiratory neurons in the caudal pons has been published, despite numerous microelectrode explorations (Hukuhara et al., 1954; Achard and Bucher, 1954; Ngai and Wang, 1957; Kahn and Wang, 1966; St. John and Wang, 1977). Sears (1977) has suggested that apneusis is a functional state of the system involving the reticular activating system rather than the action of an apneustic centre. Earlier work by Andersen and Sears (1970) had led to the view that the caudal pons was the origin of a reticulospinal pathway involved in biasing inspiratory motoneurons. This concept is supported by the response of vagotomized cats to chronic nucleus parabrachialis and Kolliker-Fuse nucleus lesions in the awake and anaesthetized state (St. John et al., 1972); their awake eupneic breathing pattern was converted to one of apneusis when they were anaesthetized. Similarly, in the case of partial, rostral pontine lesions, an apneustic breathing pattern can be converted to one of eupnea by an increase in body temperature (von Euler et al., 1976). Hence, the facilitation of inspiration and the production of apneusis would appear to be a function of the activity of the reticular activating system and separate from the basic respiratory rhythm production. Notwithstanding the profound effects that these pontine nuclei (St. John and Bianchi, 1983), as well as higher structures such as the hippocampus (Duttin and Hockman, 1972), can have on the pattern of ventilation, it is thought that the generation of respiratory rhythm occurs in the medulla, since cats with high medullary transections continue to breathe rhythmically, albeit with an altered pattern (Lumsden, 1923b; Pitts, 1946; Wang et al., 1957). Consequently, the respiratory neurons of the medulla have been the subject of intense scrutiny. During the past 50 years, respiratory neuronal activity has been associated with the dorsomedial and the ventrolateral aspects of the medulla (Gesell et al., 1936; Gesell, 1940; Amoroso et al., 1951; Achard and Bucher, 1954; von Baumgarten et al., 1957; Batsel,

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MEOULLA

C1

~-~

C5

~

,

T4

..........

x3~

FIG. 1. A sectional view of the medulla and spinal cord, illustrating the location of respiratory neurons, in the Botzinger complex (BOT), the rostral nucleus retroambigualis (r-NRA), the ventrolateral portion of the nucleus tractus solitarius (vI-NTS), the caudal nucleus retroambigualis (c-NRA), the upper cervical region (CIN), the phrenic nucleus (Ph Mn) and the intercostal nucleus (Int Mn). The numerals above the medullary sections indicate the position of the section in mm with respect to the obex.

1964). While subsequent anatomical and electrophysiological studies of these two medullary regions have more specifically identified the locations and types of respiratory neurons, the extent of respiratory neuronal activity within the medulla has been enlarged by the recent identification of other concentrations of neurons which appear to be part of the respiratory system. To facilitate the examination of these respiratory neurons and their relationship to the generation of periodic breathing movements, it is first necessary to define the anatomy and physiology of the various neuronal populations. For those interested in a more detailed review of the historical background, the reviews by Wyman (1977), Cohen (1979), and Mitchell and Berger (1981) are recommended. Von Baumgarten and colleagues (1957) first identified a concentration of inspiratory neurons in the dorsomedial aspect of the medulla, associated with the nucleus tractus solitarius (NTS). Occasionally referred to as the dorsal respiratory group (DRG), this population of 'late-peak' inspiratory neurons (Berger, 1977; Lipski et al., 1979; Graham and Duffin, 1982, 1985; Merrill et al., 1983; Averill et al., 1984; Berger et al., 1984) has been anatomically located in the ventrolateral portion of the nucleus tractus solitarius (vl-NTS) (von Baumgarten et al., 1957) (see Figure 1). Two types of vI-NTS inspiratory neurons (R~ and Rp) have been described (von Baumgarten and Kanzow, 1958) on the basis of their response to lung volume manipulation. While lung inflation appears to inhibit the R~ cells, it excites the Ra cells via a monosynaptic excitatory connection between Ra cells and the slowly adapting pulmonary stretch receptors (Averill et al., 1984). Functionally, both the R= and Ra vI-NTS inspiratory neurons have been identified as a source of monosynaptic excitatory drive to the spinal inspiratory motoneurons (phrenic and external intercostal) (Fedorko et al., 1983; Lipski et al., 1983; Lipski and Duffin, 1985). [Note: to reflect the inspiratory discharge pattern of R~ and R~ neurons, Mitchell and Berger (1975) suggested the terminology I~ and Ia, respectively; both sets of terminology are used in the literature.] Recent studies have also recorded expiratory activity within the vicinity of the vI-NTS (von Euler et al., 1973a; Berger, 1977; Feldman and Cohen, 1978), however, it appears to have been made from axons of the expiratory neurons of the Botzinger complex and from the expiratory laryngeal motoneurons of the nucleus ambiguus (NA) (for a detailed explanation, see Richter et al., 1979a, p. 254; Merrill et al., 1983, pp. 48-49). In addition, within the vI-NTS and the medial NTS, the existence of a group of pump or P-cells which discharge as a consequence of monosynaptic excitatory input from pulmonary stretch receptors, irrespective of the timing of this excitatory input during the respiratory cycle,

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has been demonstrated (Berger, 1977; Averill et al., 1984; Cohen and Feldman, 1984; Donoghue et al., 1985). Unlike the R~ and Rp cells, these cells do not appear to be actively inhibited during the expiratory phase, as judged by their lung inflation response during that phase (Cohen and Feldman, 1984), or to have projections to the spinal cord (Berger, 1977). A further indication that the 'P' cells are a distinct population in the vI-NTS is their more dorsal location compared with that of R~ and Ra cells (Cohen and Feldman, 1984). Hence, these cells appear to be interneurons which are involved in the transmission of pulmonary stretch receptor information but their role at present is not understood (see for further details: Berger, 1977; Lipski et al., 1979; Bowden and Duffin, 1980; Averill et al., 1984; Feldman and Cohen, 1984; Donaghue et al., 1985). In the ventrolateral aspect of the medulla, a concentration of respiratory neurons, associated with the NA were originally described by Achard and Bucher (1954) and Batsel (1964). The studies of Merrill (1970, 1974a) and Bianchi (1971, 1974) subsequently identified a column of respiratory neurons slightly lateral to the NA in the rostral medulla but it also extends caudally to approximately C~. Although only the portion caudal to the obex corresponded with the nucleus retroambigualis (NRA), as described by Olszewski and Baxter (1954), Merrill (1970) utilized the term NRA to refer to the entire column of respiratory neurons. In addition to this anatomical difference, these two respiratory nuclei (NA and NRA) have been distinguished from one another on a functional basis; the cells of the NA are predominantly glossopharyngeal and vagal motoneurons (Lawn, 1966a, b) while those of the NRA are primarily pre-motor neurons and propriobulbar neurons (Merrill, 1970, 1974a; Bianchi, 1971; Richter, 1982b). As the respiratory motoneurons of the NA are suppressed during deep pentobarbital anaesthesia (Merrill, 1974a, 1979), the persistence of a eupneic respiratory pattern in this state strongly suggests that these cells are not essential for the central production of periodic respiratory movements (Merrill, 1981). Consequently, within this ventrolateral group (sometimes referred to in the literature as the ventral respiratory grouly--VRG), the respiratory neurons of the NA are neglected in the present discussion. Rostral to the level of the obex, the NRA will be referred to as the rostral NRA (r-NRA) (Fig. 1) and it contains three different respiratory neuron populations. One population has a 'late-peak' inspiratory discharge which is quite similar to that of the vI-NTS. While a large proportion of these 'late-peak' cells are bulbospinal (BS) and are likely a source of drive to inspiratory motoneurons (Hilaire and Monteau, 1976; Fedorko et ai., 1983; Feldman and Speck, 1983a; Lipski and Merrill, 1983; Sears et al., 1985), it appears that other 'late-peak' cells have only intra-medullary projections (Kreuter et al., 1977; Richter, 1982b; Ballantyne and Richter, 1984); the role of the latter neurons is not presently known. The other two respiratory populations, the 'early-burst' inspiratory neurons and the 'post-inspiratory' neurons, also appear to be propriobulbar (Bianchi, 1971, 1974; Merrill, 1972b, 1974a; Richter and Ballantyne, 1981, 1983; Ballantyne and Richter, 1982). The 'early-burst' cells are purported to be a source of inhibition to other medullary respiratory neurons, thereby either preventing their discharge during the inspiratory phase (i.e. inhibition of expiratory and 'post-inspiratory' neurons) (Merrill, 1972b, 1974a, 1979; Richter and Ballantyne, 1981, 1983; Richter, 1982b) or modifying their discharge pattern during their active phase (i.e. inhibition of some 'late-peak' inspiratory neurons) (Ballantyne and Richter, 1984). Similarly, the 'post-inspiratory' neurons are believed to be a source of inhibition to other medullary respiratory neurons (i.e. expiratory and 'early-burst' inspiratory neurons) during the post-inspiratory phase as well as a source of excitation to the 'late-peak' inspiratory neurons of both the vI-NTS and the r-NRA (Richter and Ballantyne, 1981, 1983; Ballantyne and Richter, 1982, 1985; Richter, 1982b). Hence, these latter two types of propriobulbar neurons in the r-NRA appear to play an integral role in the determination of the discharge patterns characteristic of the medullary respiratory neurons. Within the nucleus retroambigualis caudal to the level of the obex (c-NRA) (Fig. 1) is a population of primarily 'late-peak' expiratory BS neurons which is thought to be a source of excitatory drive to the spinal expiratory motoneurons (i.e. internal intercostal and

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abdominal) (Kirkwood and Sears, 1973; Lipski and Merrill, 1983; Sears et al., 1985). A second population of 'late-peak' expiratory neurons, the Botzinger complex (BOT), has recently been identified at the extreme rostral part of the NA-NRA, in the vicinity of the retrofacial nucleus (Kalia et al., 1979; Lipski and Merrill, 1980) (Fig. 1). Electrophysiological studies indicate that these cells are a source of strong monosynaptic inhibition of the 'late-peak' inspiratory neurons of both the vI-NTS and the r-NRA as well as the phrenic motoneurons during the expiratory phase (Merrill et al., 1983; Merrill and Fedorko, 1984; Fedorko and Merrill, 1984b). While it has been reported that the BOT also contains other types of respiratory neurons (i.e. 'late-peak' inspiratory, 'early-burst' inspiratory neurons), it is not clear as to whether these other types of neurons were actually recorded from the rostral limits of the NA-NRA regions or from the BOT (see BOT-Section 2.3.1.2 for further details). For the purposes of this review, both the c°NRA and BOT respiratory neuron populations will be considered to contain 'late-peak' expiratory neurons. While the brain stem genesis of respiratory rhythm appears to be independent of spinal mechanisms, other recent experiments suggest that the isolated spinal cord (Cz or C2 level transection--Coglianese et al., 1977; C~ level transection--Aoki et al., 1978, 1980, 1983a) is capable of generating spontaneous respiratory activity. In support of this view of a 'spinal' respiratory rhythm generator, Viala and colleagues (Viala and Vida, 1978; Viala et al., 1979; Viala and Freton, 1983) have reported a pharmacologic activation (nialamide and DOPA) of rhythmic discharge in the phrenic nerves of rabbits with high spinal transections (C2-C3 segments). However, the actual existence and the location of this respiratory rhythm generator in the spinal cord remain in question for the following reasons. Firstly, the respiratory pattern generator described by Viala's laboratory (Viala and Vidal, 1978; Viala et al., 1979; Viala and Freton, 1983) was only present in rabbits with high spinal transections (C2-C3 levels) following pharmacologic activation. Secondly, it is doubtful that the spinal respiratory rhythm generator described by both Aoki's laboratory (Aoki et al., 1978, 1980, 1983a) and Viala's laboratory (Viala and Vidal, 1978; Viala et al., 1979; Viala and Freton, 1983) are one and the same. The 'spinal' respiratory rhythm generator described by Aoki and colleagues has been localized within the Ct and C2 segments since spontaneous respiratory rhythm does not occur in animals spinalized at the C3 level (Aoki et al., 1980); in Viala's preparation, their pharmacologically induced rhythm generator has also been localized to the cervical cord but caudal to the C 3 segment (Viala and Freton, unpublished data, as cited by Viala and Freton, 1983). Thirdly, Webber and Pleschka (1983, 1984) were unable to detect any rhythmic activity in the phrenic nerve of decerebrate rabbits with high cervical cord cold blockade (C2 segment); the cold blockade technique was selected over cord transection so as to minimize surgical trauma to the cord. Fourthly, St. John and co-workers (1981) were unable to detect any respiratory activity in the phrenic nerve of their cats transected at the C~ level. However, if the 'spinal' respiratory rhythm generator is located in the C1~C2segments as suggested by Aoki et al. (1980, 1982, 1983a), then this rhythm generator may have been inactivated in the cold blockade study (Webber and Pleschka, 1983, 1984) and destroyed by the spinal transection technique used by St. John and co-workers (1981) (upon completion of the C~ transection, a section of the spinal cord approximately 3 mm in length was removed). Hence, it appears that further experimentation is required to verify the existence of such a 'spinal' respiratory rhythm generator and, if so, to identify its location within the cervical cord. In an attempt to explain the neural mechanisms underlying this purported 'spinal' respiration, Aoki and colleagues (1980, 1982, 1983a) explored the upper cervical spinal segments of nonspinalized cats and made extracellular and intracellular recordings from inspiratory neurons which they discovered there (Fig. 1). While these investigators speculate that these neurons may be responsible for the generation of 'spinal' respiratory rhythm, the data available to date do not prove or disprove this hypothesis (Aoki et al., 1980, 1982, 1983a, b, 1984; Lipski and Duffin, 1986). The following review of literature will focus primarily on the current knowledge of the

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medullary and upper cervical respiratory neurons involved in the generation of the rhythmic drive to respiratory motoneurons and, in particular, on their known membrane and network properties. For a review of respiratory neurophysiology in general see Cohen (1979), Mitchell and Berger (1981) and von Euler (1983). To facilitate the examination of this catalog of respiratory neurons, each population will be reviewed in relation to their location and morphology; patterns of activity; functional interrelations; afferents; and projections and synaptic connections. 2. Respiratory Neurons 2.1. VENTROLATERAL NUCLEUS TRACTUS SOLITARIUS RESPIRATORY NEURONS 2.1.1. ' L a t e - p e a k '

inspiratory neurons

2.1.1.1. Location and m o r p h o l o g y

Following the original demonstration of the R~ and Ra inspiratory neurons in the proximity of the NTS by yon Baumgarten and colleagues (1957), numerous investigators (yon Baumgarten and Kanzow, 1958; Nelson, 1959; yon Baumgarten et al., 1960; Porter, 1963; Nakayama and yon Baumgarten, 1964; Batsel, 1964, 1965; Bianchi, 1971, 1974; yon Euler et al., 1973a; Cohen et al., 1974; Merrill, 1974a, 1975; Mitchell and Herbert, 1974b; Bianchi and Barillot, 1975; Feldman et al., 1976; Berger, 1977; Lipski et al., 1977; Taylor et al., 1978; Barillot and Bianchi, 1979; Bowden and Duffin, 1980; Graham and Duffin, 1982, 1985; Merrill et al., 1983; Berger et al., 1984; Cohen and Feldman, 1984) have confirmed that both the R~ and Ra 'late-peak' inspiratory neurons are located in densely packed, bilateral aggregations, parallel to the neuraxis, in association with the ventrolateral subnucleus of the nucleus tractus solitarius. This subnucleus is situated 2-3 mm lateral to the midline, extending 2-3 mm rostral from the level of the obex and lying at a depth of 0.6-2.6 mm below the dorsal surface. Recently, respiratory activity has also been recorded in the medial part of the NTS, medial to the sulcus intermediolateralis (Pantaleo and Corda, 1984; Donoghue et al., 1985; Kubin et al., 1985). These extraceUular recordings, purportedly made from neurons, demonstrated various activity patterns: inspiratory (R~ and Ra types--defined by their responses to lung inflation), expiratory, 'pump' type (defined by activity present solely in response to lung inflation), and post-inspiratory. However, as the details for most of these observed activities have been presented in abstract form only (Pantaleo and Corda, 1984; Kubin et al., 1985), no electrophysiological data were made available to support the contention that the recordings were from neurons and not from the passing axons of respiratory cells. As has been pointed out by Merrill et al. (1983), differentiation between axonal and cell body recordings can be extremely difficult in some situations (i.e. the inflections on the rising phases of extracellularly recorded spikes which are associated with neuronal recordings may be present in the instance of blocked proximal nodes of Ranvier). In particular, as some of the R~ Ra-type activities recorded in the medial NTS had spinal axonal projections, the knowledge that the axons of R~ and R B vI-NTS cells course medially, crossing the midline rostral to the cell bodies before descending into the spinal cord (Bianchi, 1971; yon Euler et al., 1973b; Cohen et al., 1974; Merrill, 1974a; Berger, 1977; Lipski et al., 1979; Berger et al., 1983, 1984), suggests the medial NTS P~ and Ra activities could have been recorded from the passing axons of R~ and Ra vl-NTS inspiratory neurons. While Donoghue and colleagues (1985) did record intracellularly from pump-type cells in the medial division of the NTS, their neuronal discharge response characteristics (i.e. excited by lung inflation irrespective of the respiratory phase; absence of discharge during a no-inflation inspiratory phase) is not supportive of their classification as other than peripheral afferent respiratory neurons. The earliest morphological study for the 'late-peak' inspiratory neurons of the vI-NTS (yon Euler et al., 1973a), labeled physiologically identified cells (n = 10) with Procion yellow. Since this study, which provided a limited description of the extent of dendrites and axons, Berger and colleagues (1984), utilizing the enzyme horseradish peroxidase

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(HRP), have provided a more detailed morphological description of these neurons (n = 11), their dendritic processes and their initial axonal trajectories. In the transverse plane, the Procion yellow study (von Euler et aL, 1973a) demonstrated an average somal diameter of 30/~m (range, 20-60 #m), while the more recent study (Berger et al., 1984) observed a similar average somal diameter of 30.4/~m (range of minor and major somal diameters of 13.5-45.3/~m, respectively), with the major somal diameters oriented in a mediolateral direction. The average minor and major somal diameters and the surface area of transversely sectioned cells were 20.2/tm, 40.7 #m, and 2.55 pm 2, respectively. Furthermore, the mean somal diameter in the horizontal plane was 38.2 #m (Berger et al., 1984). In comparison to an average of four primary dendrites per cell (yon Euler et al., 1973a), the HRP study (Berger et aL, 1984) demonstrated an average of 6.2 primary dendrites (range, 4-10) which frequently projected rostrally or caudally up to 1 mm from the soma. Primary dendrites generally remained parallel and ventral to the tractus solitarius, and appeared to be preferentially oriented in the horizontal plane, with the emergence of profuse dendritic branching along their course. In the transverse plane, in contrast to the dorsomedial and ventrolateral dendritic orientation described by von Euler and colleagues (1973a), Berger and colleagues (1984) found a predominantly medial and lateral orientation, but to some extent a dorsal and ventral one. Further, the latter investigators observed that, in general, the dendrites had numerous spines and appendages. Overall, the similarity of the organization of the dendritic trees for the labeled vI-NTS cells together with the proximity of these labeled cells to unidentified, counter-stained neurons of similar or larger somal diameter (i.e. likely to be inspiratory) led Berger and colleagues (1984) to suggest that an overlap of their dendritic trees might provide for local neuronal synaptic interactions or indirect coupling of neuronal activity as a consequence of extracellular field potentials and/or alterations in the extracellular ionic environment (Kreuter et al., 1977; Richter et al., 1977). The axons were found to originate from either the cell body (n = 3) or a large primary dendrite close to the cell body (n -- 4). The mean diameter of the initial segment and of the average myelinated portion of the axon (three point determination) was 1.73 _+0.26 #m (S.D.) and 3.23 _+0.69/~m (S.D.), respectively (Berger et al., 1984). Although the initial trajectories of the axons were variable (i.e. both dorsal and ventral departures from the soma occurred), thereafter the axons always coursed in a ventral direction away from the cell bodies before turning medially and crossihg the midline of the medulla rostral to the cell bodies (Berger et al., 1984). In addition, for 3/7 neurons, the axon bifurcated in the ipsilateral medulla with one branch entering the contralateral medulla and the other remaining ipsilateral and projecting caudally. Axons could be traced within the medulla for several millimeters (in one instance, up to 8 mm) (Berger et aL, 1984). The possible destinations of these anatomically traced axons will be discussed in the Section 2.1.1.5. As this sample of HRP labeled cells exhibited a similarity in their somal and axonal morphology as well as in the extent and orientation of their dendritic trees, Berger and colleagues (1984) suggested that the 'late-peak' inspiratory neurons of the vl-NTS appear to be morphologically homogeneous, but that, based on observation of both stained and similar-sized counter-stained cells, these cells are few in number. An additional HRP study which physiologically identified the inspiratory cells of the vl-NTS as either R~ or R~ (Berger et al., 1983, 1985) not only supports the previous description of the somal, axonal and dendritic morphology of these inspiratory cells but also demonstrates that the R~ (n = 7) and the Rp (n = 7) neurons do not constitute two morphologically distinct types of neurons (see Berger et al., 1983, 1985 for details). Berger and colleagues (1983, 1985) therefore conclude that the different responses of the R~ and R~ neurons to lung inflation must be a result of differences in their afferent connectivity (see Section 2. I. 1.4 for details). Neurons in the vI-NTS having spinal axonal projections to the immediate vicinity of the respiratory motoneurons (i.e. motoneurons supplying the diaphragm, intercostals and abdominals) have been labeled using the technique of retrograde transport of HRP (Rikard-Bell et al., 1984, 1985). While the discharge behaviour of these labeled BS neurons in the vl-NTS is not known, the demonstration that 'late-peak' inspiratory vI-NTS neurons

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monosynaptically drive the spinal inspiratory motoneurons (see Section 2.1.1.5 for details) strongly suggests that these retrogradely labeled cells belong to the 'late-peak' inspiratory population. This contention is supported by the morphological similarities between the inspiratory cells labeled by Berger and colleagues (1983, 1984, 1985) and the retrogradely labeled cells, at least, from the thoracic injection sites (upper, T3-T4; lower, Ts-T9). In the transverse plane, the mean somal diameters relative to the upper and lower thoracic injection sites were 28.6/tm (range, 16.8-37.2#m) and 31.5#m (range, 19-43.4pm), respectively; these values are comparable to those reported by Berger et al. (1983, 1984, 1985). A further similarity involved the orientation of the retrogradely labeled dendrites in the transverse plane though the number of labeled primary dendrites was fewer (up to 3 only). The latter finding of few dendritic trunks could be due to the utilization of only transverse sections and/or less extensive filling of the neuronal processes with the retrograde technique (Rikard-Bell et al., 1985). If, indeed, the cells labeled in the vl-NTS following thoracic injection are inspiratory cells, then the dorsoventral separation of the ceils labeled from the upper and lower thoracic motoneuron regions, respectively, is suggestive of a somatotopic organization of the vl-NTS (Rikard-Bell et al., 1985). In contrast, the vl-NTS cells retrogradely labeled following injections into the phrenic nucleus (Rikard-Bell et al., 1984) were significantly smaller (18.3 _ 3.8/tin (S.D.); range 10.7-32.9/~m) than either the cells retrogradely labeled from the thoracic cord injections (Rikard-Bell et al., 1985) or the inspiratory neurons identified by intracellular injection of Procion yellow (30/tm; von Euler et al., 1973a) and HRP (30.4/~m, Berger et al., 1984), in the transverse plane, The failure of the HRP injections into the phrenic nucleus to label cells with morphological characteristics similar to the BS inspiratory cells is surprising since they have been shown to have axonal collaterals which arborize in the phrenic nucleus (Merrill, 1979) and monosynaptic connections with phrenic motoneurons (Fedorko et al., 1983; Lipski et al., 1983). 2.1.1.2. Patterns o f activity

Extracellular recordings of the 'late-peak' inspiratory neurons (R~ and Ra types) of the vI-NTS have demonstrated an augmenting discharge pattern (i.e. von Baumgarten et al., 1957; Batsel, 1964; Bianchi, 1971, 1974; Hilaire and Monteau, 1975; Taylor et al., 1978; Baker and Remmers, 1980; Graham and Duffin, 1982; Feldman and Speck, 1983a; Lipski et al., 1983; Averill et al., 1984). Characterized by a wide range of recruitment times (Bianchi, 1971, 1974; Hilaire and Monteau, 1975; Taylor et al., 1978; Baker and Remmers, 1980; Lipski et al., 1983), these cells display discharge onsets ranging from 20-30% of the inspiratory time preceding the phase transition from expiration to inspiration through to the remaining 20% of the inspiratory phase (defined by phrenic neurogram or diaphragmatic myogram discharge). However, it appears that only a few of the 'late-peak' cells have onset times which precede the phrenic nerve onset (Lipski et al., 1983; Cohen and Feldman, 1984). Recently, specific examination of the discharge patterns of individual R~ and Ra types (Lipski et al., 1983; AveriU et al., 1984; Cohen and Feldman, 1984) has shown that the delay from the beginning of the inspiratory phase to the onset of cell discharge was generally longer for Ra (n = 17) than for R~ (n = 18) cells. More specifically, most Ra cells started to discharge in the latter two-thirds of the inspiratory phase, while most R~ cells did so early in the phase. Lipski and colleagues (1983) found that these discharge onsets appeared to be unimodally distributed for both cell types during both control respiratory cycles and 'no inflation' tests (see Fig. 3, Lipski et al., 1983). However, the results of other investigators (Cohen and Feldman, 1977; Baker and Remmers, 1980; Marino et al., 1981; Averill et aL, 1984; Cohen and Feldman, 1984) suggest the classification of Ra neurons into two categories based on discharge onset: early and late R Btypes. As a further distinction between R~ and R~ types, Averill and colleagues (1984) found that the peak discharge frequency of Ra neurons was frequently delayed in relation to that of the R~ cells, and usually occurred at the onset of the phrenic post-inspiratory activity. Since this difference between R~ and R e peak discharge frequencies was not observed during 'no-inflation' tests,

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it appears that it is likely due to the slowly adapting pulmonary stretch receptor input to the R e neurons. Based on the distribution of the discharge peaks of Ra cells, Lipski and colleagues (1983) have reflected on the possible involvement of these cells in inspiration termination. From their observations that the peak discharge of R~ cells lags the peak of phrenic rootlet discharge, during a control breath, (likely as a consequence of the lag in lung inflation), and occurs in the period when discharge of R~ cells start to decrease, they suggest that R e cells might be involved in inhibition of medullary inspiratory activity; this role would be in addition to their role in generating the post-inspiratory ('post-ramp') phrenic activity. Alternatively, the fact that, during 'no inflation', both R~ and RI~ types appear to reach their discharge peak at approximately the same time led Lipski et al. (1983) to suggest that R~ types are switched off by a third source. Hence, the specific role of Ra cells at the medullary level remains unclear. Without differentiating between R~ and R e types, Taylor and colleagues (1978) observed that the gradually increasing output from the vI-NTS inspiratory group does not result so much from increasing discharge frequencies in individual cells, but from graded recruitment of further cells. From the results of Lipski and colleagues (1983), the Ra cells appear to be more involved in this graded recruitment, particularly during the latter half of the inspiratory phase. Moreover, as later recruited cells do not necessarily terminate their discharge before earlier recruited ones, the basis of recruitment of vI-NTS inspiratory cells does not appear to be simply a threshold type. While the incompatibility of these results with threshold recruitment led Taylor and co-workers (1978) to conclude that these inspiratory neurons are controlled by input drives which are specific to individual neurons rather than homogeneously shared, the recent intracellular study of PSPs in these neurons by Ballantyne and Richter (1984) suggests that this recruitment phenomenon is more likely a result of complex excitatory postsynaptic potential--inhibitory postsynaptic potential (EPSP-IPSP) interactions during the inspiratory phase (see below). Intracellular examination of the periodic activity of these cells has involved analysis of the membrane potential trajectory during the respiratory cycle and experimental reversal of IPSPs; the latter occurs when chloride ions enter the cell either by diffusion (due to electrode penetration of the membrane) or by injection (due to passage of chloride or a negative current through the electrode). That the characteristic burst of action potentials of increasing frequency during the inspiratory phase are evoked by an incrementing pattern of EPSPs was shown by the first intracellular recordings of the inspiratory neurons of the vl-NTS (von Baumgarten et al., 1960; Salmoiraghi and von Baumgarten, 1961). The presumption that the subsequent repolarization of these cells during the expiratory phase was a consequence of synaptic inhibition (IPSPs) was eventually established by chloride reversal of the hyperpolarization (Mitchell and Herbert, 1974b; Richter et al., 1979a; Richter and Ballantyne, 1981; Richter, 1982a, b; Merrill et al., 1983; Ballantyne and Richter, 1984). More recent, detailed intracellular work from the laboratory of Richter (Richter, 1980, 1981, 1982a, b; Richter and Ballantyne, 1981; Ballantyne and Richter, 1982, 1984) has revealed more complex postsynaptic patterns in these 'late-peak' inspiratory neurons, particularly during their active phase. In addition to the incrementing pattern of EPSPs received during the inspiratory phase, there is evidence for two different patterns of inhibitory inputs to these cells though rarely do the two inhibitory patterns co-exist in the same inspiratory neurons, at least within the tested range of experimental conditions (Ballantyne and Richter, 1984). In the instance of an early inspiratory inhibitory pattern, a declining pattern of IPSPs during early inspiration interact with the 'throughout' incrementing EPSP pattern. The second pattern (late inspiratory inhibition) is characterized by the same 'throughout' incrementing EPSP pattern interaction with an incrementing IPSP pattern late in the inspiratory phase (Richter, 1980, 1981, 1982a, b; Richter and Ballantyne, 1981; Ballantyne and Richter, 1982, 1984). Chloride reversal of the inspiratory IPSPs in R~ (n = 12) and R/~ (n = 5) inspiratory neurons (Ballantyne and Richter, 1984) demonstrated early inspiratory inhibition for 6 R~

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and 2 Ra neurons while late inspiratory inhibition occurred in the remaining 9 R~ and R a cells. These postsynaptic patterns suggest that both the R~ and R a neurons should have two types of discharge patterns; that is, an early onset discharge (late inspiratory inhibition) pattern and a late onset discharge (early inspiratory inhibition) pattern. While recent extracellular recordings of vI-NTS inspiratory neurons have confirmed the existence of such early and late onset discharge patterns for Ra neurons (Lipski et al., 1983; Averill et al., 1984), the onset of discharge for R~ neurons (Lipski et al., 1983) generally occurs near the beginning of the inspiratory phase (Lipski et al., 1983; Averill et al., 1984). It therefore seems unlikely that the majority of R~ (n = 37) cells are subject to this early inspiratory inhibition, although Ballantyne and Richter's results suggest that early and late inhibition of R~ vI-NTS cells occur with similar frequency (i.e. 50%, n = 12). Regardless of the frequency of occurrence of early inspiratory inhibition in the R~ or, for that matter, the Ra cells, pulmonary stretch receptor inputs do not appear to play a role in the determination of the discharge onset, as shown by the similarity of discharge onsets for control and 'no inflation' inspiratory cycles (Lipski et al., 1983). Detailed examination of the inspiratory inhibitory synapses to the 'late-peak' inspiratory neurons suggests that the early inspiratory IPSPs arise at distal dendritic sites as shown by the features of the IPSP reversal: the long time course of the chloride injection/diffusion required for reversal and the minimal or absent change in the neuron's input conductance (Ballantyne and Richter, 1984). In contrast, the late inspiratory IPSPs appear to arise at or close to the cell soma; this is suggested by the relatively shorter time course of IPSP reversal following either chloride injection or diffusion into the cells and the associated increase in neuronal input conductance (Richter et al., 1979a, b; Richter, 1982; Ballantyne and Richter, 1984). In contrast to the complexity of the postsynaptic potentials (PSPs) recorded in these 'late-peak' inspiratory neurons during the inspiratory phase, the pattern of synaptic activity during the remainder of the respiratory cycle is relatively uncomplicated. The transition to the post-inspiratory phase (the period of declining discharge in the phrenic nerve) is marked by a rapid depolarization and a decrementing pattern of EPSPs, thereby producing a decrementing neuronal discharge (Richter, 1981; Richter and Ballantyne, 1981; Richter, 1982a, b). The onset of the expiratory phase is coincident with the cessation of their declining pattern of discharge and the onset of the incrementing pattern of postsynaptic inhibition (Mitchell and Herbert, 1974b; Richter et al., 1979a; Richter and Ballantyne, 1981; Richter, 1982a, b; Ballantyne and Richter, 1984). Similar to the late inspiratory IPSPs, these expiratory IPSPs appear to arise at or close to the cell soma as suggested by the time course of the IPSP reversal and the associated change in neuronal input conductance (Richter et al., 1979a, b; Richter, 1982; Ballantyne and Richter, 1984). With the exception of the inhibitory postsynaptic pattern during the expiratory phase, the sources of input which produce these varying synaptic patterns have yet to be conclusively revealed. However, intracellular evaluation of the patterns of postsynaptic potentials in some of the other types of respiratory neurons have identified correlations in the time-intensity profile of the discharge patterns, suggestive of neuronal connectivity. These known and potential sources of input will be examined in subsequent sections. Recently, Acker and Richter (1985) have demonstrated the occurrence of fluctuations in the ionic activities in the extracellular vicinity of these vl-NTS neurons during the respiratory cycle. For the R~ neurons (R~ cells were not examined), the extracellular activities of potassium and calcium were found to increase and decrease, respectively, during the inspiratory phase; the directions of these fluctuations were reversed during the expiratory phase. These rhythmic changes in the ionic environment appear to be related either to the extracellular oxygen tension or to peripheral chemoreceptor activity since hypoxia or asphyxia accentuated both the increase in extracellular potassium activity and the decrease in the extracellular calcium activity and was associated with both an increase in the frequency of the R e neuron discharge and the strength of the phrenic nerve discharge. These results, together with the finding that changes in the ionic environment of neurons

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in various species can influence their intrinsic membrane properties and their response to synaptic inputs (see Neher and Lux, 1973; Richter et al., 1978; Adams et al., 1982), led Acker and Richter (1985) to conclude that various membrane conductances and transport systems are active in the determination of the postsynaptic responses in, at least, the R B neurons of the vI-NTS and suggest that synaptic interactions alone do not explain the rhythmic pattern of discharge in the medullary respiratory neurons. Such involvement of other mechanisms (i.e. intrinsic membrane properties) in the determination of rhythmic respiratory discharge will receive further attention in the 'Patterns of Activity' Sections for the other categories of respiratory neurons. 2.1.1.3. Functional interrelations

Discussion of the patterns of activity of the 'late-peak' inspiratory neurons of the vI-NTS has identified the synchronization of their phasic neuronal discharge on a long time scale (seconds) and their prominent postsynaptic potentials. Both these features strongly suggest that synaptic connectivity amongst the inspiratory neurons is an essential feature in the central production of respiration. Such connectivity between any pair of neurons can be demonstrated by correlation of their discharge patterns on a millisecond time scale (Perkel et al., 1967a, b; Moore et al., 1970; Kirkwood, 1979; Knox, 1981). In addition, correlation can occur if two neurons share a common input from a third neuron. As previously discussed, the apparent extensive overlap of the dendritic trees of neighbouring 'late-peak' inspiratory neurons suggests that direct interneuronal interactions are anatomically possible (Berger et al., 1983, 1984, 1985). Furthermore, short term synchronization of discharge is more likely for neighbouring neurons which lie within the axonal arborization field of an afferent neuron thereby sharing a common input. The earliest cross-correlation study of near neighbouring inspiratory neurons of this nucleus (R, and R~ classifications were not made) (Feldman et al., 1980) reported short-term synchrony in their discharges, however, these results were based upon single electrode techniques where false positive correlations are easily obtained. Using pairs of microelectrodes with small electrode spacings (100-350#m), Graham and Duffin (1982) demonstrated significant short time scale correlations for all R T R ~ pairs (n = 9), and in approximately 50% of the R~-R~ pairs (n = 7) and R~-R~ pairs (n = 6); however, the bulbospinal character of these neurons was not verified. Whether these correlations are due to common excitation or to complex synaptic interactions could not be distinguished although common vagal afferent input does not appear to be responsible since bilateral vagotomy had no appreciable effect on these short-term correlations. In addition, Madden and Remmers (1982) demonstrated short-time scale correlations in the discharge of neighbouring 'late-peak' inspiratory neuronal pairs (typical electrode tip separation, 250/~m) but also failed to investigate the presence or absence of spinal axonal projections for these pairs, While the patterns of positive correlations for R~-R~ pairs (50%, n = 22) and for R~-R~ pairs (47%, n = 38) were in close agreement with those of Graham and Duffin (1982), Madden and Returners (1982) reported few (20%, n = 14) positive cross-correlations among R~-R~ near neighbouring pairs. The latter difference in findings is likely due to differences in experimental preparations and in neuron type classification. Recent cross-correlation results of Hilaire and colleagues (1984) showed that 37.5% (15/40) of inspiratory BS neuron pairs showed short-time scale correlations, while mixed pairs of inspiratory neurons (one BS cell and one non-antidromically-activated (NAA) cell from either the cord or the ipsilateral vagus nerve which they called 'propriobulbar') were less frequently correlated (17%, 4/23). No correlations were demonstrated between pairs of inspiratory 'propriobulbar' neurons (n = 5). However, as this study neither identified the proximity of the neuronal pairs (recorded using two independent microdrives) nor did it classify the neurons as R~ and R B types, it is difficult to make further comparison with the two preceding studies (Graham and Duffin, 1982; Madden and Remmers, 1982). On the other hand, their contention that a higher incidence of correlation occurred between BS neuronal pairs than between BS-'propriobulbar' pairs (Hilaire et al., 1984) is supported

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by that of Feldman and Speck (1983a), who reported that short-term synchronization of discharge occurred more frequently for pairs (41%, n = 17) in which both neurons (R~, Ra types unidentified) were correlated to the phrenic discharge (identified by crosscorrelation of unit and phrenic nerve discharges). Feldman and Speck (1983a) also demonstrated that the correlated inspiratory vI-NTS neuron pairs (24.3%, n = 70) did not appear to vary in probability or strength of short-term synchrony for electrode spacings up to 2000 pm, a finding at odds with the majority of cross-correlation studies. Overall, the results of these studies show that short-term synchronization of discharge occurs for both homogeneous (R~R~ pairs, Rv-R p pairs) and heterogeneous (R~Ra pairs) inspiratory neurons in the vl-NTS. However, for all of these studies (Feldman et al., 1980; Graham and Duffin, 1982; Madden and Remmers, 1982; Feldman and Speck, 1983a; Hilaire et al., 1984), the shapes of the cross-correlation peaks and their locations relative to time zero do not unequivocally differentiate between common synaptic input to these neurons and interaction between them (but see Feldman and Speck, 1983a and Hilaire et al., 1984). Recently, in an attempt to distinguish between these synaptic possibilities, Graham and Dutiin (1985) performed further cross-correlation studies combined with spinal antidromic activation of one neuron of positively cross-correlated near neighbouring inspiratory pairs in the vI-NTS (for details of the technique, see Graham and Duffin, 1985). For 9 pairs of positively correlated inspiratory neurons (3 pairs of R~-R~, 3 pairs of R f R ~ , and 3 pairs of R~-R~) tested with this technique, 2 pairs of BS neurons showed evidence of interneuronal interaction (R~ exciting R~, Rp exciting R~). Due to the short interaction latencies for these two neuronal pairs (i.e. 0.6 msec and 0.2 msec), the extensive overlap of 'late-peak' inspiratory dendritic trees (Berger et al., 1983, 1984, 1985), and the apparent absence of short-axon collaterals, Graham and Duffin (1985) suggested that the interactions were mediated by dendrodendritic or electrotonic synapses. For most of the remaining 7 pairs (all 3 Ra-R~ pairs), the failure of neuronal interactions to account for the short-time scale correlations indicates that a common synaptic excitation was responsible for the discharge synchronization (Graham and Duffin, 1985). In summary, the 'late-peak' inspiratory neurons of the vI-NTS demonstrate short-term synchronization of firing of their discharge (Graham and Dutfin, 1982; Madden and Remmers, 1982; Feldman and Speck, 1983a; Hilaire et al., 1984; Graham and Duffin, 1985). In particular, the R~ neurons have a high incidence of synchronization which appears to be due to common input (Graham and Duffin, 1985). While the incidence of synchronization for R~-R~ types and R~-Ra types appears to be lower, preliminary results have indicated the existence of interneuronal interaction for one pair of each type. Although some studies have suggested the existence of nonbulbospinal, nonvagal inspiratory neurons of the vI-NTS (Ballantyne and Richter, 1984; Hilaire et al., 1984) and that the BS neurons are more likely to show short-term synchronization of discharge, recent morphological and cross-correlation studies (Berger et al., 1983, 1984, 1985; Graham and Duffin, 1985) do not support either of these contentions. Overall, such short-term synchronization of these BS neurons via the mechanisms of common input as well as neuronal interaction suggests that the manner in which the population as a whole is synchronized is quite complex. 2.1.1.4. Afferents

(a) Brain stem, cortex and spinal cord. Subsequent to the initial discovery of inspiratory neurons in the vI-NTS by von Baumgarten and colleagues (1957), progress in the identification of afferent connections, particularly from the medullary respiratory neurons, to the vI-NTS group has been slow. Intensive antidromic mapping (Merrill, 1974a, 1979) and neuroanatomical studies (Kalia et al., 1979; Bystrzycka, 1980; Kalia et al., 1981) have failed to demonstrate any evidence of projection of the 'late-peak' inspiratory r-NRA neurons, the 'early-burst' inspiratory r-NRA neurons, or the expiratory neurons of the c-NRA to the vI-NTS region. As the 'post-inspiratory' neurons are also located in the r-NRA, presumably the failure of neuroanatomical studies (Kalia et al., 1979; Bystrzycka,

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1980) to demonstrate any projections originating from the r-NRA region suggests that they, too, do not send collaterals to the vl-NTS, though such a projection has not been electrophysiologically investigated. Despite the similarities of the time course and pattern of EPSPs in the 'late-peak' inspiratory neurons of both the vI-NTS and the r-NRA, the apparent absence of r-NRA projections to the vl-NTS together with the absence of short-time scale correlation of discharge between 'late-peak' inspiratory neurons of the 2 groups (n = 20 pairs) (Feldman et al., 1980) suggest that vi-NTS neurons are not synaptically acted on by the 'late-peak' inspiratory r-NRA group. Similar comparison of the time course and pattern of IPSPs in those 'late-peak' vl-NTS inspiratory neurons which receive 'early-inspiratory' inhibition (Ballantyne and Richter, 1984) to the time course and discharge pattern of 'early-burst' inspiratory r-NRA cells (Merrill, 1974a) suggests that the latter cell type is inhibitory to the former (Ballantyne and Richter, 1984; Ballantyne and Richter, 1985). However, the absence of projections from the 'early-burst' cells to the vl-NTS region does not lend support to this hypothesis. Examination of the time-intensity profile of IPSPs in the 'late-peak' inspiratory vl-NTS neurons during the post-inspiratory phase demonstrates a close parallel to the abrupt onset, decrementing discharge of 'post-inspiratory' neurons located in the r-NRA. As for the hypothesis of an inhibitory pathway from 'early-burst' inspiratory cells to the vl-NTS, the 'post-inspiratory' r-NRA neuron appears to be a plausible candidate for the postinspiratory phase inhibition of the vI-NTS inspiratory cells (Ballantyne and Richter, 1985). However, the absence of proof of projections, at least on an anatomical basis, from the 'post-inspiratory' neurons to the vI-NTS is not supportive of such a connection. While the parallel in the time-intensity profile of the incrementing EPSPs in the c-NRA expiratory neurons with that of the incrementing IPSPs in the vl-NTS inspiratory neurons might suggest an inhibitory connection between these expiratory and inspiratory populations, this appears unlikely for two reasons. Neuroanatomical (Kalia et al., 1979; Bystrzycka, 1980; Kalia et al., 1981) and electrophysiological (Merrill, 1974a, 1979; Merrill et al., 1983) studies have failed to demonstrate the relevant axonal projections to the vI-NTS. Second, spike-triggered averaging of post-synaptic noise in 'late-peak' inspiratory vl-NTS neurons failed to reveal any synaptic connections from c-NRA expiratory cells (Merrill et al., 1983). Recent neuroanatomical and electrophysiological studies (Kalia et al., 1979; Bystrzycka, 1980; Lipski and Merrill, 1980; Bianchi and Barillot, 1982; Fedorko and Merrill, 1984a), however, have identified projections from the expiratory neurons in the Botzinger complex to the ipsi- and contralateral vl-NTS inspiratory regions. Moreover, Merrill and colleagues (1983) have provided direct evidence, in the form of intraceUular spike-triggered unitary IPSPs, for monosynaptic inhibition of both R~ and Ra inspiratory vI-NTS neurons, at least contralaterally, by the BOT expiratory neurons. In addition to the BOT expiratory projections, other brain stem sites have been neuroanatomically demonstrated to send axonal projections to the vI-NTS region. The retrograde transport of HRP (Bystrzycka, 1980) identified projections arising from: (1) the contralateral nucleus paragigantiocellularis dorsalis; (2) the ipsilateral medial and lateral parabrachial nuclei as well as the ipsilateral Kolliker-Fuse nucleus (this pontine projection is quite weak); and (3) the contralateral vI-NTS region. Recent electrophysiological work by Bianchi and St. John (1982) has shown that a limited number of respiratory neurons in the vicinity of the nucleus parabrachialis medialis and Kolliker-Fuse nucleus project to the ipsi- and contralateral vI-NTS inspiratory region; the largest proportion of these pontobulbar neurons had phase-spanning patterns of activity. In the same regions of the pons, a number of non-respiratory neurons were also found to project to the vl-NTS region. Whether any of these respiratory as well as non-respiratory cells have synaptic influence on the vl-NTS inspiratory neurons is not known at present. As for the anatomically demonstrated projections from the ipsilateral to the contralateral vI-NTS region, the existence of short-time scale correlation for only 5% of ipsilateral-contralateral vI-NTS inspiratory pairs (n = 20) (Feldman et al., 1980) does not

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support the existence of synaptic input from inspiratory cells of the vI-NTS to those of the contralateral vI-NTS. Hence, these crossed projections might have originated from non-respiratory vI-NTS cells or resulted from the uptake of H R P by passing axons. Evidence for projections to the NTS from more rostral sites is limited. While cells in the cerebral cortex (Brodal et al., 1956) have been neuroanatomically shown to project to the NTS, afferent fibre termination does not appear to occur in the ventrolateral subnucleus. Similarly, neuroanatomically identified projections from the spinal cord to the NTS do not appear to send terminal branches to the vI-NTS (Torvik, 1956). Recent electrophysiological work, however, suggests that the phrenic nerve afferents project to the lateral reticular nucleus (Macron et al., 1985); external cuneate nucleus (Speck et al., 1985); the NA (Macron et al., 1985; Speck et al., 1985); and perhaps, the N R A (identified only as the ventral respiratory group, Speck et al., 1985), as demonstrated by the recording of responses evoked by electrical stimulation of the central cut end of the phrenic nerve. Such stimulation was found to evoke activity in the ipsilateral inspiratory neurons (presumably R~ and Ra) of the vI-NTS at latencies of 5-15 msec, while a marked transient reduction in the motor discharge of the contralateral phrenic nerve occurred at an onset latency of 7-10 msec. Furthermore, the results of other electrical stimulation studies have suggested that the proprioceptive afferents (Group I) of intercostal (internal and external) and abdominal muscles as well as thoracic wall cutaneous receptors also influence the discharge of the vI-NTS inspiratory cells (R~ and Ra), although the effect appears to be inhibitory (i.e. terminates ongoing activity) and not excitatory as was shown for the phrenic afferents (Shannon, 1980; Shannon and Freeman, 1981; Bolser et al., 1983, 1984; Shannon et al., 1985a, b). Through selective excitation of the Group Ia (muscle spindle endings) and Group Ib (golgi tendon organs), using muscle vibration and muscle contraction, elicited by a ventral root stimulation, respectively, Bolser and colleagues (Bolser et al., 1983, 1984; Shannon et al., 1985b) concluded that it is increased golgi tendon organ activity which reduces vl-NTS inspiratory neuron (also r-NRA 'late-peak' cells) and phrenic nerve activity; activation of muscle spindle endings appears to have minimal or no effect on the vl-NTS cells. Presumably the effects of Group Iavs Ib afferents of the abdominal muscles are similar to those of the intercostal afferents, though they were not selectively tested. In turn, since generalized stimulation of intercostal Group I proprioceptive afferents also evoked activity in neurons in close proximity to spontaneously active vl-NTS inspiratory cells, Shannon and Lindsey (1983) suggest that these cells function as interneurons in the inhibitory respiratory muscle Group Ib afferent pathway to the vI-NTS inspiratory cells. Following such demonstrations that electrical activation of respiratory muscle proprioceptive afferents influences the vl-NTS inspiratory neuron discharge, Shannon and colleagues (1985a) have recently reported that inspiratory duration is prolonged by tracheal occlusion in vagotomized cats; however, the rate of rise as well as the peak frequency of discharge remained unchanged. Such prolongation of the duration of neuronal activity is likely due to phrenic and inspiratory intercostal afferents since this response was abolished following both cervical ( C 3 - C 7 ) and thoracic (TI-Tg) dorsal rhizotomies, but is still present with only cervical or thoracic dorsal roots intact. (b) Cranial nerves and central chemoreceptors. By virtue of the location of these 'late-peak' inspiratory neurons within a subnucleus of the NTS, the major projection site of the afferents of the IXth and Xth cranial nerves, and their branches (Cottle, 1964; Berger, 1979; Kalia and Mesulam, 1980a, b; Kalia and Welles, 1980; Panneton and Loewy, 1980; Ciriello et aL, 1981a, b; Davies and Kalia, 1981; Donoghue et al., 1982a, b; Nomura and Mizuno, 1982; Donoghue et al., 1984; Kubin and Davies, 1985; Spyer, 1985), it would appear likely that these neurons play a role in the processing and integration of viscerosensory input at the premotor respiratory neuron level. The earliest electrophysiological suggestion of such a role for the vI-NTS inspiratory cells arose from a study involving lung volume manipulation (von Baumgarten and Kanzow, 1958). That some vI-NTS cells (Ra type) could be excited by lung inflation in

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inspiration as well as during the silent period of phrenic nerve discharge (i.e. expiration) led von Baumgarten and Kanzow 0958) to suggest that R~ cells were brainstem interneurons located in the afferent portion of the pathway which mediates the HeringBreuer reflex (inspiratory-inhibiting lung-inflation reflex). Taken with the earlier demonstration that slowly adapting pulmonary vagal stretch receptors are responsible for this reflex (Larrabee and Knowlton, 1946; Knowlton and Larrabee, 1946), it would seem that R~ cells are synaptically excited by pulmonary stretch receptors. As the time-intensity profiles of the discharge of R~ neurons paralleled that of the phrenic nerve activity, regardless of the applied lung inflation manoeuvres, yon Baumgarten and Kanzow (1958) further suggested that the R~ group drive the phrenic motoneurons (i.e. pre-motor function) but are inhibited by R e neurons during the inspiratory phase. Numerous subsequent electrophysiological studies have also identified these two types of vl-NTS inspiratory cells (R~ and Re), utilizing lung inflation manoeuvres primarily during expiration (the method introduced by yon Baumgarten and Kanzow in 1958) (Cohen, 1969; Bianchi and Barillot, 1971, 1975; Berger, 1977; Lipski et al., 1979, 1983; Bowden and Duffin, 1980; Graham and Duffin, 1982, 1985; Cohen and Feldman, 1984); cervical vagal nerve stimulation (von Euler et al., 1973a; Sessle et al., 1978; Richter et al., 1979a, b); and, most recently, 'no-inflation' tests during the inspiratory phase (von Euler et al., 1973a; Cohen and Feldman, 1977; Cohen, 1979; Baker and Remmers, 1980; Marino et al., 1981; Lipski et al., 1983; Averill et al., 1984; Cohen and Feldman, 1984; Dick and Berger, 1985; Iscoe and Long, 1985). This latter test involves the omission of inspiratory lung inflation for some respiratory cycles in artificially ventilated animals for which lung inflation is triggered or controlled by the phrenic nerve activity; no-inflation cycles thereby eliminate phasic pulmonary stretch receptor input to the vl-NTS inspiratory cells. Alternatively, in spontaneously breathing animals, Duffin and colleagues (Bowden and Duffin, 1980; Graham and Duffin, 1982, 1985) have performed a 'no-inflation' test during inspiration by an occlusion of the airway which was controlled by the diaphragmatic EMG activity. In either type of preparation, an excitatory effect of pulmonary stretch receptors was indicated by reduction of a neuron's discharge during the 'no-inflation' inspiration. As these three techniques differ in their abilities to selectively test the influence of pulmonary stretch receptor input on the vl-NTS cells (i.e. cervical vagal nerve stimulation involves other vagal inputs), published data on R,-R Bneurons may be associated with an overestimation of the proportion of Re types when identification is based on cervical vagus nerve stimulation, but with mistaken identification of R e neurons as R, types when lung inflation testing is only applied during the expiratory phase, in the latter instance, the demonstrations that 'late-peak' inspiratory neurons are actively inhibited during the expiratory phase (Richter et al., 1979a; Merrill et al., 1983; Ballantyne and Richter, 1984) and that such IPSPs can partially shunt excitatory synaptic current (Camerer et al., 1979) suggest, at least some, R e neurons would not be brought to discharge threshold despite the receipt of excitatory input from pulmonary stretch receptors (Lipski et al., 1983). Furthermore, several studies (Cohen, 1979; Bowden and Duffin, 1980; Cohen and Feldman, 1980, 1984; Lipski et al., 1983) have recently shown the 'no-inflation' test during inspiration to be more sensitive than the lung inflation test, particularly when performed in expiration, for the detection of pulmonary stretch receptor input to R~ cells (see for further details, Lipski et al., 1983). However, both these testing procedures are still subject to incorrect identification of R~ as R~ cells when lung inflations are started from a particularly low functional residual capacity [i.e. animals which are paralyzed, thoracotomized, and artificially ventilated with no expiratory threshold load (Lipski et al., 1979)]. In this instance, some R e cells may have received pulmonary stretch receptor input but insufficient either to cause a change in discharge pattern (i.e. inflation vs no-inflation during inspiration) or to induce neuronal discharge (i.e. lung inflation during expiration). On the other hand, Averill and colleagues (1984) observed that R~ neurons could be consistently identified by their response to electrical stimulation of the ipsilateral vagus at stimulus intensities just suprathreshold for activating pulmonary stretch receptors. Based on guidelines set forth by Cohen and Feldman (Cohen and Feldman, 1977, 1980;

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Cohen, 1979; but see Cohen and Feldman, 1984), the classification of R~ neurons from the 'no-inflation' test is made if their discharge during 'no-inflation' cycles compared to that during lung inflation satisfy the following criteria: (1) the onset of discharge is delayed relative to onset of phrenic nerve activity (or, more delayed for those cells whose lung inflation discharge onsets do not precede the phrenic nerve onset); (2) the rate of discharge augmentation is reduced (i.e. decreased slope for a cycle-triggered histogram); and (3) the peak discharge frequency is decreased. However, Lipski and colleagues (1983) have observed that the third criterion, the change of peak frequency, both absolute and relative to peak phrenic nerve discharge, is variable and therefore should not be used to identify R, neurons (for detailed explanation see Lipski et al., 1983). A further problem in classification arises if the 'no-inflation' inspiration is associated with a decrease in the rate of augmentation of both neuron and phrenic nerve discharge as the decreased neuron discharge could be due to decreased central inspiratory drive and/or removal of pulmonary stretch receptor input (Lipski et al., 1983). In addition to the physiological evidence from lung inflation tests that pulmonary vagal afferents synaptically act on the vI-NTS inspiratory neurons, a recent neuroanatomical study (Kalia and Mesulam, 1980b) suggests that pulmonary afferents project to the NTS, with pulmonary afferent terminations in the medial, and to some extent the ventrolateral, NTS. Furthermore, both antidromic mapping of single pulmonary stretch receptor afferents (Donoghue et al., 1982b) and spike-triggered averaging of extracellular field potentials of pulmonary stretch receptor afferents (Berger and Averill, 1983) have demonstrated that functionally identified slowly adapting pulmonary stretch receptor afferents do project to and terminate in the ipsilateral vl-NTS region as well as the medial and dorsolateral subnuclei of NTS. The proportion of afferents terminating in the vI-NTS, however, is small compared to that of other regions in the NTS. These demonstrations of pulmonary stretch receptor afferent termination in the vI-NTS, together with the short average difference in the latencies to excitation of pulmonary stretch receptor afferents in the vl-NTS and to the onset of discharge in R, neurons during ipsilateral vagal nerve stimulation (yon Euler et al., 1973a; Richter et al., 1979b; Backman et al., 1984) are suggestive of monosynaptic excitatory connections between pulmonary stretch receptor afferents and R, neurons. This connection to both early and late onset R~ neurons has recently been confirmed by cross-correlation analysis (20%, n = 44) (Averill et al., 1984) and by intraceUular spike-triggered averaging (Backman et al., 1984; Berger et al., 1985). This finding is further substantiated by examination of the latency differences of evoked action potentials recorded centrally from pulmonary stretch receptor afferents and of EPSPs from R~ neurons during electrical stimulation of the vagus nerve; the latencies were too small to allow for more than one intercalated synapse (Backman et al., 1984). In contrast, no evidence was obtained demonstrating synaptic connectivity between pulmonary stretch receptor afferents and R~ neurons (Averill et al., 1984; Backman et al., 1984; Berger et al., 1985). This evidence for synaptic connectivity between pulmonary stretch receptor afferents and Rp, but not R~, neurons, taken with the demonstrations that the R~ and R~ neurons are morphologically indistinct (Berger et al., 1983, 1984, 1985) and that they both monosynaptically excite phrenic and inspiratory intercostal motoneurons (Fedorko et al., 1983; Lipski et al., 1983; Lipski and Duflin, 1985), strongly suggest that the differing responses of R~ and R~ neurons to lung inflation arise only from differences in their afferent connectivity. In fact, since the categorization of these neurons on the basis of their responses to lung inflation tests is not always clear (Lipski et al., 1983; Averill et al., 1984; Cohen and Feldman, 1984), it appears that the vI-NTS neurons exhibit a continuum of behaviour with respect to pulmonary stretch receptor input (see Cohen and Feldman, 1984). Moreover, such new information concerning the afferent and efferent connections of R~ cells provides evidence for a disynaptic excitatory pathway between slowly adapting pulmonary stretch receptors and the inspiratory motoneurons via activation of R~ neurons. These results are, however, in conflict with the classical view (yon Baumgarten and Kanzow, 1958) that the output from R~ neurons is inhibitory to the 'pre-motor' group, J.P.N 27/2--B

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that is, the R, neurons; furthermore, the existence of an inhibitory connection from Ra to R, neurons also appears to be unlikely for the following reasons. The results of cross-correlation studies of R, and R¢ cells (Graham and Duffin, 1982, 1985) do not support this view of an inhibitory role for R~ neurons and, moreover, excitation of an R~ neuron by an R~ neuron was demonstrated for one neuronal pair. Second, the demonstration by Lipski and colleagues (1979) that the activation of R~ neurons with lung inflation during expiration also resulted in a concomitant depolarization of R, neurons (68, n = 28) (as measured by a decrease in antidromic latency) is also in disagreement with such an inhibitory action of Ra on R, neurons. Davies and colleagues (1983) have also reported that decreases in the antidromic latency of R, neurons occur in response to lung inflation applied during expiration (and maintained throughout the phase) and in a pattern which differs from that of R~ cells. In view of the demonstration of this disynaptic excitatory pathway from pulmonary stretch receptors to inspiratory motoneurons with Ro cells as the interposed 'interneuron' in the pathway, it is difficult to interpret some recent experimental observations. As it is assumed that Ra activity is decreased by 'no-inflation' elimination of pulmonary stretch receptor input, the augmentation of phrenic nerve activity during such a test, observed most frequently in deeply anaesthetized cats, remains unexplained (Lipski et al., 1983). Lipski and colleagues (1983) suggest that increased activity in other pre-motor neurons (or, other afferent pathways) must be responsible for such increased phrenic activity. Also unexplained is the maintenance of inspiratory duration during maintained elevations in functional residual capacity (i.e. 10 min) despite the demonstration that the increased pulmonary stretch receptor activity only partially adapts (Finkler and Iscoe, 1984). However, Iscoe and Long (1985) found that R~ cells (n = 4) showed no overall change in either peak frequency of discharge or time of discharge onset during inflations from an elevated end-expiratory lung volume, thereby suggesting that Ra cells habituate to the elevated pulmonary stretch receptor activity within the period of elevated lung inflation (i.e. 5-10min). Alternatively, Berger and colleagues (1985) have suggested that such lung-volume dependent reflexes may be mediated not by Ra cells but by the vl-NTS pump-cells and/or other relay sites in the NTS region where the majority of the pulmonary stretch receptor afferents terminate. In contrast to the two response profiles of vl-NTS neurons to pulmonary stretch receptor input (i.e. lung inflation vs 'no-inflation' inspiration), the performance, during expiration, of test lung inflations with volumes large enough to evoke the gasp response (i.e. Head's paradoxical reflex) (Larrabee and Knowlton, 1946; Widdicombe, 1954) is associated with excitation of both R, and R~ neurons as well as a 'paradoxical' discharge in the phrenic nerve/diaphragm (Berger, 1977; Lipski et al., 1979; Bowden and Duffin, 1980; Lipski et al., 1983). As such lung hyperinflation is thought to activate rapidly adapting pulmonary receptors (Knowlton and Larrabee, 1946; Larrabee and Knowlton, 1946; Sellick and Widdicombe, 1970), it is possible that their central afferents mediate the excitation of both R, and R B neurons. While neuroanatomical studies (Cottle, 1964; Kalia and Mesulam, 1980a) have demonstrated that vagal visceral afferents terminate principally in the NTS, the projection patterns of functionally identified rapidly adapting pulmonary receptors and their sites of termination within the NTS subnuclei has only recently been determined, using the antidromic mapping technique (Kubin and Davies, 1984, 1985). The majority of the tested afferents (n = 11) had a primarily ipsilateral projection to the ventral portion of the medial part of the caudal NTS with at least one major branch coursing to the contralateral medial part of the caudal NTS. Some branches of these afferents did project to the lateral NTS and, to a lesser extent, the vI-NTS. These results suggest that the rapidly adapting pulmonary receptors are likely to exert any synaptic effects on vi-NTS inspiratory neurons via second order neurons, presumably located at the termination site of their afferent fibres. Hence, the central processing for rapidly adapting pulmonary receptor input appears to be quite different from that demonstrated for slowly adapting pulmonary stretch receptor input.

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The vI-NTS inspiratory cells also appear to be involved in the processing of superior laryngeal afferent input since electrical stimulation of this nerve elicits a short latency excitation of ipsilateral vI-NTS inspiratory cells as well as the phrenic nerve (Berger, 1977; Sessle et al., 1978; Iscoe et al., 1979; Cohen and Donnelly, 1982; Donnelly et al., 1983). This observation, together with the bulbospinal character of vl-NTS inspiratory neurons, led Berger (1977) to suggest that the phrenic nerve excitation involves a disynaptic pathway via these inspiratory cells. More recently, such short latency excitation has been demonstrated for both early and late Ra neurons, and for late R~ neurons, but not for early R~ cells, following ipsilateral superior laryngeal nerve stimulation (Donnelly et al., 1983). However, as this short-latency excitation was followed by period of silence (i.e. inhibition) in the phrenic activity as well as in both R, and Ra cells, Donnelly and colleagues (1983) concluded that all types of vI-NTS inspiratory neurons are influenced by superior laryngeal afferents, but early R~ cells are least affected. These results, taken with the demonstration that uni- or bilateral vl-NTS destruction resulted in attenuation or abolition of the short-latency phrenic excitation from superior laryngeal stimulation, are consistent with Berger's original proposal for the mediation of the response via, at least, the Rp neurons. As yet, however, the precise regions of the NTS innervated by the superior laryngeal afferents are unresolved. As previously mentioned, the NTS is also the major projection site of the afferents of the IXth cranial nerve (Cottle, 1964; Ciriello et al., 1981b; Nomura and Mizuno, 1982). Of its three main peripheral branches, the carotid sinus nerve, with its carotid body chemoreceptor afferent fibres, is of particular interest with respect to respiratory control. Recent studies utilizing the transganglionic transport of H R P (Berger, 1979a; Panneton and Loewy, 1980; Ciriello et al., 1981a; Davies and Kalia, 1981; Nomura and Mizuno, 1982) have shown that the afferent fibres of the carotid sinus nerve (both chemoreceptor and baroreceptor afferents) terminate principally in several subnuclei of the ipsilateral NTS (with a less intense contralateral projection): the dorsal aspect of the medial NTS, the lateral NTS, and the commissural NTS (but see Davies and Kalia, 1981). Terminations within the vl-NTS were found to be much less intense. A subsequent antidromic mapping study (Donoghue et al., 1984) has determined that the central projections of individual physiologically identified chemoreceptor afferents (n = 13) have a preference for extensive terminations in the dorsomedial and medial NTS (ipsilateral) and the ipsi- and contralateral commissural NTS; a sparse projection to the lateral NTS occurred in a few cases. These results, taken with their inability to antidromically activate these afferents from the vI-NTS, suggest that the medial NTS, and not the vI-NTS, region is the primary relay site for chemoreceptor afferent input (Spyer, 1985). This is, however, at odds with the demonstration of monosynaptic excitatory connections between carotid body chemoreceptors and inspiratory BS neurons of the ipsilateral vI-NTS, using cross-correlation analysis (Kirkwood et al., 1979). As no estimates for the frequency of this connection were given, it may be that chemoreceptor afferents have a sparse projection to the vI-NTS region resulting in only occasional synaptic action on the inspiratory neurons. Alternatively, Donoghue and colleagues (1984) suggest that such interaction between the medially located chemoreceptor afferents and vl-NTS inspiratory cells might occur via axodendritic synapses, based on a personal communication from D. W. Richter that these inspiratory neurons have extensive dendritic arborizations passing far from the vl-NTS into other NTS subnuclei and beyond, as revealed by HRP labeling. This latter observation, however, is not consistent with the recent observation, also using H R P labeling, (Berger et al., 1984) that the dendritic trees of vI-NTS inspiratory neurons do not enter the NTS but remain almost exclusively ventral to this structure. Hence, whether input from the carotid body chemoreceptors to the vI-NTS inspiratory neurons is processed directly or via an interneuron is presently unresolved. Irrespective of the processing mechanism for this chemoreceptor input, numerous studies have reported enhanced inspiratory discharge of these vI-NTS neurons as a consequence of carotid body chemoreceptor stimulation (Davies and Edwards, 1975; Lipski et al., 1977; St. John, 1981; Laubie et al., 1983). However, the effect of such

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chemoreceptor stimulation on these cells was actually found to vary in phase with the respiratory cycle in that they are excited by the delivery of phasic stimuli in the inspiratory phase, but are mainly inhibited when stimuli are applied during the expiratory phase (Lipski et al., 1977). As the excitability of carotid sinus nerve afferent terminals shows no respiratory variation (Jordan et al., 1981), the mechanism responsible for such respiratory modulation does not appear to be presynaptic inhibition of chemoreceptor afferent fibres. Alternatively, the recent demonstration that BOT expiratory neurons project to the vI-NTS and are excited by stimulation of carotid chemoreceptors (close arterial injections of CO2 equilibrated saline) during the expiratory phase (Lipski et al., 1984) suggests that the carotid chemoreceptor inhibition of the vI-NTS inspiratory cells during expiration is evoked indirectly, through excitation of the BOT expiratory cells which, in turn, inhibit the vI-NTS cells. The demonstrated monosynaptic inhibitory connection from BOT expiratory neurons to the vl-NTS inspiratory group (R~ and R~) (Merrill et al., 1983) lends further support to this latter interpretation. Such a role for the BOT expiratory cells would seem likely as they also have been shown to inhibit the phrenic motoneurons during the expiratory phase. Thus, in the inspiratory phase, carotid chemoreceptor stimulation results in direct excitation (though not necessarily monosynaptic) of the vl-NTS inspiratory neurons, while in the expiratory phase, such stimulation inhibits these neurons indirectly, probably via the BOT expiratory pathway. Comparison of the discharge of the vI-NTS inspiratory cells (n = 23) during equivalent elevations of ventilatory activity (i.e. peak integrated phrenic nerve activity) from pharmacological stimulation of the carotid chemoreceptors and from hyperoxic hypercapnia has shown similar augmentations of activity for most vl-NTS cells (St. John, 1981). As the ventilatory changes which result during hyperoxic hypercapnia have been attributed primarily to the action of central chemoreceptors (Sorensen, 1971; Berkenbosch et al., 1979), St. John suggests that the vI-NTS inspiratory neurons receive equivalent excitatory inputs from both the peripheral and central chemoreceptors. Moreover, the finding that hyperoxic hypercapnia was also associated with increases in activity of both the inspiratory and the expiratory NRA neurons is consistent with the earlier conclusion of St. John and Wang (1977) that central chemoreceptor 'afferent' influences must be ubiquitously distributed to the medullary respiratory neurons. Based on the view that the central chemoreceptors are separate from the medullary respiratory neurons (Leusen, 1950; see reviews by Loeshcke, 1974; Bledsoe and Hornbein, 1981), the nature of the central chemoreceptor influence has therefore been assumed to be synaptic excitation, as opposed to direct excitation, of these neurons. However, as pointed out in recent reviews (Bledsoe and Hornbein, 1981; Cherniack, 1982), experimental studies to date have not provided conclusive evidence for this view, as the specific cellular elements which comprise the central chemosensor have not been identified. Although excitatory influences from both the central and the peripheral chemoreceptors appeared to be equally distributed among the vl-NTS inspiratory neurons when pharmacologic activation of the peripheral chemoreceptors was utilized (St. John, 1981), St. John and Wang (1977) had previously reported that excitatory peripheral chemoreceptor afferent influences (activated via isocapnic hypoxia) were not uniformly distributed within the vl-NTS inspiratory region. However, as the stimulus of isocapnic hypoxia results in a ventilatory alteration which represents the net effect of central chemoreceptor depression and peripheral chemoreceptor excitation of the vI-NTS cells, St. John and Wang (1977) concluded that, in hypoxic situations, the activity of vl-NTS cells will only increase in those neurons receiving adequate excitatory peripheral chemoreceptor input to overcome the direct depression of neuronal activity by brainstem hypoxia. Furthermore, they suggested that, since phrenic motoneurons demonstrate identical changes in activity in hypercapnia and in hypoxia (St. John and Bartlett, 1979), there must be, within the phrenic motoneuron pool, significant integration of BS inspiratory activity since both vI-NTS and r-NRA inspiratory neurons demonstrate such differing responses to hypoxia. Finally, from an evaluation of the mechanisms underlying alterations in the discharge frequencies of these BS inspiratory cells in hypercapnia and hypoxia, St. John and Bianchi

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(1985) concluded that the overall change of membrane potential during the respiratory cycle, as opposed to the absolute level of depolarization, appears to be the primary determinant. This was based on observations of variations of their antidromic latencies during normocapnic hyperoxia as compared to hypercapnia or hypoxia. In the former situation, the latencies for these inspiratory neurons decreased to minima during their active phase with maxima occurring during expiration, while the hypercapnia or hypoxia resulted in unaltered minima with maxima typically increasing for those cells displaying increased discharge frequencies. For only those cells displaying increased discharge frequencies, a strong correlation existed between the increase in discharge frequency and the maximum-minimum latency difference (which also increased); no such correlation was found for neurons whose discharge frequencies decreased in hypoxia and/or hypercapnia. To add to this complexity of inputs, various neuroanatomical and electrophysiological studies have associated a number of other cranial nerves and/or their major branches with the NTS region. These include the facial and trigeminal nerves (Torvik, 1956), the aortic nerve (Biscoe and Sampson, 1970; Ciriello and Calaresu, 1981; Ciriello et al., 1981a), and the cardiovascular and gastrointestinal afferents via the vagus nerve (von Baumgarten et al., 1959; Kalia and Mesulam, 1980a). 2.1.1.5. Projections and synaptic connections

Evidence concerning the medullary and spinal axon projections of the 'late-peak' inspiratory neurons of the vl-NTS have been obtained from both neuroanatomical (Torvik, 1957; Kuypers and Maisky, 1977; Loewy and Burton, 1978; Bystrzycka, 1980; Holstege and Kuypers, 1982; Rikard-Bell et al., 1984, 1985) and neurophysiological (Nakayama and von Baumgarten, 1964; Bianchi, 1971, 1974; Newsom Davis and Plum, 1972; von Euler et al., 1973a, b; Cohen et al., 1974; Merrill, 1975, 1979; Berger, 1.977; Sessle et al., 1978; Lipski et al., 1979, 1983; Graham and Duffin, 1982; Feldman and Speck, 1983a; Fedorko et al., 1983; Ballantyne and Richter, 1984; Hilaire et al., 1984; Lipski and Duflin, 1985) studies. On the basis that numerous studies (Lipski et al., 1979, 1983; Merrill, 1979, 1981; Fedorko et al., 1983; Lipski and Duflin, 1985) have successfully activated from the spinal cord virtually all the inspiratory cells (both R~ and Ra) of the vI-NTS which were under examination (but see Bianchi, 1971, 1974; Hilaire et al., 1984; Ballantyne and Richter, 1984) and that morphological examination of the 'late-peak' vl-NTS neurons (von Euler et al., 1973a; Berger et al., 1983, 1984, 1985) has shown them to be a relatively homogeneous population, it seems that this inspiratory population is solely composed of BS neurons (R~ and Ra types). Using antidromic mapping, Merrill (1975) found that some of these inspiratory neurons (both R~ and Ra types) have extensive collateral aborizations in the ipsilateral inspiratory region of the NRA, with occasional collateral arbors in the contralateral r-NRA as well as the ipsi- and contralateral c-NRA (Fig. 2). Using retrograde HRP labeling (Loewy and Burton, 1978; Bystryzycka, 1980) and anterograde labeling with tritiated amino acids (Loewy and Burton, 1978), the existence of such projections from the vI-NTS region to the rostral and caudal extents of the NRA, to the level of the retrofacial nucleus, was confirmed. However, in contrast to the mainly ipsilateral projection identified by both Merrill (1975) and Loewy and Burton (1978), Bystrzycka (1980) found it to be primarily contralateral. Presumably the inspiratory neurons displaying these medullary projections also had spinal projections, though Merrill (1974a, 1979, 1981) did not indicate whether he performed the two activation tests on the same group of cells. The presence of these vl-NTS inspiratory collaterals in the ipsilateral and, to a lesser extent, contralateral r-NRA inspiratory region, together with the parallel in the time course and pattern of the EPSPs for the 'late-peak' inspiratory neurons of both the vI-NTS and the r-NRA, are suggestive of an excitatory connection from the 'late-peak' inspiratory cells of the vI-NTS to those of the r-NRA. While this conjecture is not supported by the results of a recent cross-correlation study (Feldman and Speck, 1983a), the demonstration of short-term synchronization of discharge for less than 10% (n = 50 pairs) of ipsilateral vI-NTS-r-NRA inspiratory pairs might be due to other reasons. The sample of vI-NTS

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FIG. 2. A sectional view of the medulla and spinal cord illustrating the axonal pathway for a 'late-peak' inspiratory neuron of the vI-NTS. inspiratory neurons under study may not have had the appropriate axon collaterals in the ipsilateral r-NRA. Secondly, even if the vl-NTS cells did arborize in the ipsilateral r-NRA, the random selection of neuronal pairs may, on the basis of probability, account for the small percentage of demonstrated correlations. Finally, as not all inspiratory cells studied by Feldman and Speck (1983a) displayed an augmenting discharge pattern, it is not known if the vI-NTS-r-NRA inspiratory pairs had the appropriate discharge patterns. The possible role of the sparse collateral arborization of vI-NTS inspiratory cells in the c-NRA expiratory region (bilateral) is not known. Due to the dissimilarity between the 'late-peak' discharge pattern of the vI-NTS cells to the declining IPSP pattern of the c-NRA expiratory group (Richter et al., 1979a; Richter and Ballantyne, 1981; Richter, 1982a, b; Ballantyne and Richter, 1984), the vl-NTS inspiratory cells do not appear to be the source of the inspiratory inhibition of the c-NRA expiratory group. Furthermore, the possible existence of synaptic connectivity between vI-NTS inspiratory cells (both R~ and Ra) and c-NRA expiratory neurons is not supported by any correlation of their discharge (Merrill, 1979; note: the cross-correlation histograms were obtained during the period of their overlapping discharge and, for Ra cells, during their activation by lung inflation in the expiratory phase). Some, but not all, of the tested inspiratory cells had the appropriate axon collaterals in the expiratory region. While the vI-NTS inspiratory cells could not be shown to project to the contralateral vI-NTS by electrophysiological means (Merrill, 1979, 1981), neuroanatomical studies indicated otherwise. The results of retrograde transport of H R P (Bystrzycka, 1980) suggested a projection from the contralateral vl-NTS to the ipsilateral vI-NTS, although the uptake of HRP by axons of passage may also account for the observed labelling (i.e. uptake of HRP by axons coursing to the contralateral rostral NRA) (Krishman and Singer, 1973) Little is presently known about the course of the main axons in both the medulla and the spinal cord. As shown by HRP intracellular injection into physiologically identified vl-NTS inspiratory neurons, the main axons course medially from the cell soma, crossing the midline of the medulla rostral to the cell bodies (Berger et al., 1984). The majority of these main axons (both R~ and Ra) eventually descend into the contralateral cervical spinal cord in the ventrolateral funiculus (Torvik, 1957; Kuypers and Maisky, 1977; Bianchi, 1971; Berger, 1977; Holstege and Kuypers, 1982; Loewy and Burton, 1978; Rikard-Bell et al., 1984; Newsom Davis and Plum, 1972; Merrill, 1979; Nakayama and Baumgarten, 1964; Lipski et al., 1979, 1983; Sessle et al., 1978; Fedorko et al., 1983) though some earlier studies (von Euler et al., 1973a, b; Richter et al., 1979b) had been unsuccessful in the spinal antidromic activation of R B types. A small percentage of these axons (R~ and Rp) have collaterals which arborize in the phrenic nucleus, as shown by antidromic mapping

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(Merrill, 1979; Fedorko et al., 1983; Lipski et al., 1983). In addition, the latter technique has shown that the majority of R~ and R~ main axons descend into the thoracic cord (Merrill, 1979; Lipski and Duttin, 1985) where they demonstrate extensive collateral arborizations within the intermediate and ventral grey matter (Lipski and Duffin, 1985). Within the thoracic cord, these axon collaterals branch at multiple spinal levels (Lipski and Duflin, 1985). Recent neuroanatomical studies (Loewy and Burton, 1978; Holstege and Kuypers, 1982; Rikard-Bell et al., 1984, 1985) appear to confirm these cervical and thoracic projections from the vI-NTS region as well as the collateral arborization in the immediate vicinity of the phrenic nucleus and the thoracic respiratory motoneurons (intercostal and abdominal). In particular, the autoradiographic experiment of Loewy and Burton (1978) demonstrated vI-NTS projections which were scattered throughout the medial part of the ventrolateral funiculus at the cervical cord level but, in the thoracic cord, the main distribution of projections was in the lateral funiculus; the concentration of projections appears to decrease towards the lower thoracic and lumbar segments. Furthermore, Loewy and Burton observed silver grains outlining the dendrites of many large neurons (presumably phrenic motoneurons) in the C4-C5 segments; this labeling persisted over this ventral-most region of the ventral horn throughout the thoracic spinal cord. In contrast, the results of Rikard-Bell et al. (1984, 1985), using retrograde transport of HRP, showed projections to the upper and lower thoracic cord to be relatively sparse compared to cervical projections, thereby suggesting that a large number of axons terminate in the cervical cord. As this latter interpretation is not in agreement with either antidromic mapping results (Merrill, 1979; Lipski and Duffin, 1985) or the results of autoradiography (Loewy and Burton, 1978), limitations of the retrograde HRP transport (Rikard-Bell et al., 1984, 1985) over the relatively long distances involved might account for this disparity. Overall, the majority of these anatomically identified spinal projections for vI-NTS appear to be contralateral to the cell body (Loewy and Burton, 1978; Holstege and Kuypers, 1982; Rikard-Bell et al., 1984, 1985). According to Rikard-Bell and colleagues (1984, 1985), this contralateral:ipsilateral spinal projection for vl-NTS cells is approximately 75% : 25%. Whether this bilateral projection is a consequence of bifurcating axons which descend on both sides of the cord or a small percentage of cells which project to the ipsilateral cord only, has not been resolved. The former possibility does, however, receive some support from the recent demonstration that some vI-NTS inspiratory axons bifurcate in the medulla with one branch projecting caudally on the ipsilateral side while the other crossed to the contralateral medulla (Berger et al., 1984; see Section 2.1.1.1 for further details). The interpretation of the results from such neuroanatomical studies, however, is limited by their inability to establish the physiological identity of the labeled cells (i.e. retrograde transport of HRP) and/or labeled projections (i.e. anterograde transport of tritiated amino acids). Unfortunately, the results from Loewy and Burton (1978), as well as from Rikard-Bell and colleagues (1984, 1985), are further complicated by their demonstrations that the size of the labeled vI-NTS cells differed relative to the location of their spinal projections; primarily the medium sized cells of the vI-NTS project to the phrenic nucleus region while large vI-NTS cells project mainly to thoracic motoneuron levels (see Section 2.1.1.1 for further details). Such variability in somal size for the labeled cells is not in agreement with the recent demonstration that 'late-peak' inspiratory neurons of the vI-NTS (both R~ and R Btypes) are an homogeneous population (Berger et al., 1983, 1984, 1985) similar in size to those cells projecting mainly to the thoracic motoneuron region but substantially larger than those projecting to the phrenic nucleus region. However, as Rikard-Bell and colleagues (1984, 1985) observed an anatomical separation of the labeled cells within the vl-NTS relative to their spinal projections (i.e. phrenic vs thoracic motoneuron regions), it may be that the recording locations used by Berger and colleagues (1983, 1984, 1985) resulted in the intracellular staining of primarily large cells which, on a neuroanatomical basis, project primarily to thoracic motoneurons. However, it is equally

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as plausible that the medium sized cells which project to the phrenic nucleus region are not, in fact, part of vI-NTS inspiratory group. Regardless of the physiological identity of the medium sized cells which project to the phrenic nucleus, it is extremely likely that the large cells projecting to the thoracic respiratory motoneuron region are, in fact, inspiratory cells as their cellular morphologies were very similar to those studied by Berger and colleagues (Berger et al., 1983, 1984, 1985; Rikard-Bell et al., 1984, 1985) (see Section 2.1.1.1 for further explanation). In this instance, the observed dorsoventral separation of the cells labeled from upper (T3-T4) and lower (Ts-T9) thoracic HRP injections, respectively, suggests the existence of a somatotopic organization of the vI-NTS inspiratory group (Rikard-Bell et al., 1985). Electrophysiological techniques have been utilized to examine the functional role of these axonal arborizations of the vI-NTS inspiratory group in both the phrenic nucleus and the thoracic intercostal motoneuron region. Cross-correlation of the discharge of vl-NTS inspiratory cells to that of the contralateral phrenic nerve demonstrated the existence of short-term synchronization of their discharges (Cohen et al., 1974; Hilaire and Monteau, 1976; Graham and Duffin, 1982; Feldman and Speck, 1983a; Cohen and Feldman, 1984). Cohen and Feldman (1984) have also demonstrated such correlations for vl-NTS inspiratory cells (R~ and Ra) with the ipsilateral phrenic nerve discharge. The onset of the peak of the neuron to phrenic correlations was delayed by a range of latencies suggestive of monosynaptic excitation of phrenic motoneurons by inspiratory neurons of the vl-NTS; furthermore, such correlations were observed for both R~ and R~ types of vl-NTS cells (Graham and Duffin, 1982; Cohen and Feldman, 1984). These findings were recently supported by the direct demonstration, using spike-triggered averaging (STA) of intracellular potentials, that R~ and R~ inspiratory neurons (approximately 80% of tested neurons, n = 27) make monosynaptic excitatory connections with phrenic motoneurons (Fedorko et al., 1983; Lipski et al., 1983). In addition, the observation that both R~ and R~ neurons had synaptic connections with more than one phrenic motoneuron in the C5 and C6 segments suggests a prominent divergence of synaptic connections (Fedorko et al., 1983; Lipski et al., 1983). For single R~ cells, this divergence of their axonal arborization and their excitatory action within the phrenic nucleus was also confirmed by crosscorrelation of their activity with that recorded from C5 and C6 phrenic rootlets (note: the tested Ra neurons had first been shown to produce individual EPSPs in at least one motoneuron) (Lipski et al., 1983). This occurrence of monosynaptic excitation of phrenic motoneurons by the vI-NTS inspiratory group has also been confirmed by Monteau and colleagues (1985), using spike-triggered averaging. With the demonstrations that short-term synchrony of discharge occurs mainly between phrenic motoneurons with similar recruitment (i.e. both early or both late) (Hilaire et al., 1983) and that the vl-NTS inspiratory neurons (particularly Rt~ types) are characterized by early- and late-inspiratory discharge onsets (Lipski et al., 1983; Averill et al., 1984), further STA analyses (Monteau et al., 1985) were carried out to determine the frequency of occurrence of monosynaptic excitatory connections relative to the recruitments of vl-NTS inspiratory neurons and phrenic motoneurons. As expected, such monosynaptic connections were frequently observed between neurons and motoneurons having similar recruitments (i.e. both early onset or late onset) but were infrequent between pairs having different recruitments (i.e. one early, one late). Hence, it appears that a nonhomogeneous drive from, at least, the vl-NTS inspiratory neurons (early onset and late onset types) may be an important factor in the determination of the recruitment of phrenic motoneurons. However, based on the responses of early and late onset phrenic motoneurons to lung inflation testing, Donnelly and colleagues (1985) have concluded that the inputs to late onset phrenic motoneurons are not predominantly or exclusively from the late onset vI-NTS neurons. In fact, the responses of none of the phrenic motoneurons to lung inflations corresponded to that of the late-onset R~ vI-NTS inspiratory cells. Although Hi!aire and Monteau (1976), utilizing the cross-correlation technique, were unsuccessful in demonstrating a similar connection to the intercostal motoneurons, Lipski

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and Duffin (1985) have recently demonstrated that both R~ and R~ inspiratory vl-NTS neurons monosynaptically evoke EPSPs in the contralateral inspiratory intercostal motoneurons. The evidence for such connectivity was provided both by STA of synaptic noise of intercostal motoneurons (T3-T 4 segments) and by cross-correlation of discharges of inspiratory vl-NTS cells with that of intercostal nerve filaments (Ta-T4 segments). Hence, both cross-correlation and STA analyses have shown the inspiratory neurons (R~ and Ra types) of the vI-NTS to be a source of monosynaptic excitatory drive to the inspiratory motoneurons in the cervical and thoracic spinal cord. Against this view is the recent demonstration of monosynaptic excitatory connections with the inspiratory motoneurons (both phrenic and intercostal) for less than 10% of vl-NTS inspiratory neurons tested (n = 88), utilizing the cross-correlation technique (Sears et al., 1985). Although the severe criteria utilized for identifying monosynaptic connections probably resulted in an underestimation of the synaptic connectivity, Sears and colleagues (1985) concluded that the majority of excitation of inspiratory motoneurons is via interneurons. This was based on their estimations of the number of BS neurons which would be required if motoneuron depolarization was produced by such monosynaptic connections alone, and that such estimates appear to be far in excess of any estimates of actual numbers of neurons which can be made on the basis of recording experience and/or anatomical evidence. Typical conduction velocities for the axons of the vI-NTS inspiratory neurons (n = 16; R~ and Ra not identified) range from 30 to 48 m/sec, as calculated from antidromic latency measurement from a single point in the cervical cord (Bianchi, 1971). Utilizing the same method at the C 6 segment, Lipski and colleagues (1983) reported a mean conduction velocity of 34.6 + 8.2 m/sec (S.D.) for R~ neurons (n = 7); the minimal value for antidromic latency was used to make this calculation. A similar mean conduction velocity [34.4 +__3.01 m/sec (S.E.)] was determined by antidromic latency measurement at the C2--C 3 level (Bianchi and St. John, 1981). In contrast to the medullary and spinal projections of the vI-NTS inspiratory group, little is known about possible outputs from these cells to more rostral regions of the brain. Using HRP and autoradiographic techniques, several studies (Kuypers and Maisky, 1977; Norgren, 1978; Ricardo and Koh, 1978) have demonstrated bilateral projections from the NTS to the parabrachial regions of the pons, though the latter two studies were performed in the rat. Such projections from the NTS to the parabrachial regions have been more specifically localized to the nucleus parabrachialis medialis and the Kolliker-Fuse nucleus, as demonstrated by Kalia (1977), using HRP, and to the nucleus parabrachialis by King (1980), using radioactive amino acids. While these two anatomical studies do not confirm that the vl-NTS inspiratory neurons are the origin of these pontine projections, particularly as the projections originated predominantly from the medial NTS, electrophysiological studies by Cohen (1976) and Bianchi and St. John (1981) have demonstrated that a limited number of respiratory neurons in the region of the vl-NTS project to these pontine nuclei. These bulbopontine respiratory neurons, however, exhibited a respiratory-modulated tonic or phase-spanning activity (Cohen, 1976; Bianchi and St. John, 1981) and could not be activated from the spinal cord (Bianchi and St. John, 1981). Moreover, King and Knox (1982) were unsuccessful in antidromically activating vl-NTS inspiratory neurons from the nucleus parabrachialis. Hence, at present, there is little evidence to suggest that R~ and Ra cells have pontine projections. Finally, in the rat, neurons in the NTS have been shown to send projections directly to limbic forebrain structures (Ricardo and Koh, 1978), however, the neurons were not physiologically identified. 2.1.1.6. Summary The vI-NTS contains a population of 'late-peak' inspiratory neurons which have axonal projections to the contralateral cervical and thoracic spinal cord as well as to the rostral and caudal NRA respiratory regions in the medulla. As the NTS is the terminus for afferent fibres from the IXth and Xth cranial nerves and as these inspiratory cells are a source of monosynaptic excitation of the inspiratory motoneurons, it would appear that

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the vI-NTS cells play a role in the processing and integration of viscerosensory input at the premotor neuron level. Such a role has been directly demonstrated for some of these 'late-peak' cells (Ra types) which receive monosynaptic excitation from pulmonary stretch receptor afferents. Since the Ra 'late-peak' cells have not been shown to differ from the R~ cells on the basis of their morphology, their synaptic connectivity with inspiratory motoneurons, or their synaptic inhibition by BOT expiratory neurons during expiration, it appears that R~ and R~ cells differ only with respect to their connectivity with pulmonary stretch receptors. While the significance of their projections to the rostral and caudal regions of the NRA remains unknown, it has been suggested that the vI-NTS inspiratory neurons provide the patterned excitatory input for the 'late-peak' inspiratory neurons of the r-NRA. Also unresolved is the source of the excitatory drive to these vl-NTS inspiratory neurons during the inspiratory phase, particularly as the short-term synchronization of their discharge patterns has been shown to be due mostly to common afferent input though interneuronal interactions also appear to be responsible. 2.2. NUCLEUS RETROAMBIGUALISRESPIRATORYNEURONS 2.2. I. Rostral nucleus retroambigualis 2.2.1.1. 'Late-peak' inspiratory neurons (a) Location and morphology. As a consequence of electrophysiological (extracellular recording and antidromic stimulation) and histological investigations (Merrill, 1970, 1974a, 1979; Bianchi, 1971), the 'late-peak' inspiratory neurons of the r-NRA have been localized within bilateral, longitudinal columns which extend 4-5 mm rostrally from the level of the obex (Merrill, 1970, 1974a, 1981; Kalia, 1981a). Situated within the ventrolateral medulla between 3-5 mm below the dorsal surface and 3-5 mm lateral to the midline, the columns have a thickness of about 500/zm (Merrill, 1970) (see Fig. 1). Both the most caudal, and the most rostral portions of the r-NRA have been associated with mixed populations of both inspiratory and expiratory neurons (Merrill, 1970, 1974a, 1979). While the caudal expiratory activity is known to belong to the expiratory neurons of the c-NRA, it is likely that the source of the rostral expiratory activity recorded by Merrill in 1970 was the subsequently identified BOT expiratory neurons (Kalia et al., 1979, Bystrzycka, 1980; Lipski and Merrill, 1980). In addition to these rostral and caudal associations, the medial aspect of the r-NRA merges with the NA (Merrill, 1970, 1974a, 1981); however, it appears that the respiratory units of the NA are almost completely inactive in the deeply anaesthetized pentobarbital preparation (Merrill, 1970, 1974a, 1981). A morphological study (Kreuter et al., 1977), using Procion yellow, confirmed the location of the 'late-peak' inspiratory neurons of the r-NRA in the ventrolateral part of the reticular formation, but it failed to show a significant difference between the locations of vagal motoneurons and the r-NRA inspiratory neurons; at the level of the obex they seemed to be intermingled. Utilizing the antidromic activation technique, Kreuter and colleagues (1977) identified two categories of 'late-peak' r-NRA inspiratory neurons: BS and NAA (i.e. not activated from contra- or ipsilateral spinal cord or the ipsilateral vagus nerve). Interestingly, the NAA 'late-peak' inspiratory neurons, displaying either a 'throughout type' or, less commonly, a 'late type' inspiratory discharge, had different morphological features compared to the BS 'late-peak' inspiratory cells. With regard to single cell morphology, the mean surface area of the somata of the NAA neurons were significantly smaller than that of the BS neurons (1814 #m 2 vs 3573 #m2); the length of the dendritic tree was less in the NAA neuron (up to 200 # m) compared with that of the BS neurons (up to 400/~m); and, the NAA neurons appeared to have fewer dendritic trunks (3-5) than their BS counterparts in the NRA (4-8). In addition, examination of neuronal localizations (Kreuter et al., 1977) showed that while the BS inspiratory r-NRA neurons were surrounded by small cells (interneuron type) but clearly separated from all other groups of neurons, including the NAA inspiratory cells, the latter cells were located

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close to groups of similarly shaped cells but surrounded by 1-2 cells of larger size. These differences and the fact that vagal inspiratory motoneurons are considerably larger than the BS inspiratory neurons suggest that the NAA 'late-peak' inspiratory neurons are an anatomically and, perhaps, a functionally different type of r-NRA neuron, probably propriobulbar as evidenced by the inability to activate these neurons from the spinal cord or the vagus nerve. [Note: this issue will receive further attention in Section 2.2.1.1 (b).] Both types of the 'late-peak' inspiratory neurons (BS and NAA) showed profuse branching of their primary dendritic trunks. Further staining of these neurons within a single electrode track indicated overlapping of the dendritic trees for different respiratory neurons (Kreuter et al., 1977). In one instance, an inspiratory BS neuron was shown to have an extensive overlap of its dendritic tree with that of an expiratory BS neuron of the c-NRA (the latter cell was located 12 ~m ventral to this inspiratory neuron). Though less extensive, dendritic overlap also occurred for a pair of BS 'late-peak' inspiratory neurons (inter-somal distance of 70 #m) as well as a pair of NAA 'late-peak' inspiratory neurons (inter-somal distance of 30/~m). Furthermore, specific to the BS 'late-peak' inspiratory neurons (and, c-NRA expiratory BS cells), their dendritic trees lie mainly in the transverse plane, with dorsomedial and ventrolateral projections preferentially. As a consequence of this dendritic overlap, and in particular the similarities of the dendritic tree orientations for the BS neurons, the potential exists for modification of their neuronal activity by extracellular field potentials, dendritic chemical synaptic interaction, and/or alteration in the extracellular ionic concentration as a consequence of the activation of a neighbouring neuron (Kreuter et aL, 1977; Richter et al., 1978). However, electrical synapses of the gap junction type do not appear to be present either in the r-NRA inspiratory group or in the region of the N R A at the obex level where inspiratory and expiratory neurons overlap, since dye diffusion from the Procion yellow injected neurons and transport to neighbouring neurons were not observed (Kreuter et al., 1977). Of possible functional importance, there was an absence of axon collaterals up to a stained axonal distance of 800 #m from any one cell body (NAA and BS alike), though this negative finding may be due to an inability of Procion yellow to stain axon collaterals, or to the presence of axon collaterals at a greater distance down the main axon (Kreuter et al., 1977; Berger, 1981). The axons of the BS 'late-peak' inspiratory neurons coursed dorsomedially with myelination not observed until some 30-50 #m away from the cell body (Kreuter et al., 1977). More recently, Rikard-Bell and colleagues (1984, 1985), using retrogradely transported H R P as a marker, identified neurons in the r-NRA region which had spinal axons projecting to the immediate vicinity of the respiratory motoneurons (i.e. motoneurons supplying the diaphragm, intercostals, and abdominals). While the discharge behaviour of these labeled BS neurons in the r-NRA is not known, the results of antidromic mapping studies of the spinal axons of the 'late-peak' r-NRA inspiratory neurons in the cervical and thoracic cord [Merrill, 1970, 1971, 1974a, 1979; Fedorko et al., 1983; Dick and Berger, 1985; Feldman et al., 1985; see Section 2.2.1.1(e) for details] strongly suggest that these retrogradely labeled r-NRA neurons belong to the 'late-peak' BS inspiratory group. This possibility is supported by a comparison of the morphology of the HRP labeled cells with that of the Procion yellow labeled BS 'late-peak' inspiratory neurons (Kreuter et al., 1977) which does demonstrate some similarities: (1) the location of the BS neurons relative to the vagal motoneurons of the NA; (2) the medium size of these neurons relative to the larger vagal motoneurons; (3) the shape of the cells in the transverse plane (compare Fig. 4, Kreuter et al., 1977 and Fig. 5, Rikard-Bell et al., 1984); and (4) the number of primary dendrites and the dendritic tree orientations. While the somal diameters, measured in the transverse plane, of the BS neurons which project to the phrenic nucleus (Rikard-Bell et al., 1984; range 11.2-35.7#m) appear to be smaller than those of the BS 'late-peak' inspiratory neurons (Kreuter et al., 1977; range of minor and major somal diameters 21-53 #m), the range of somal diameters of the NRA neurons projecting to thoracic respiratory motoneurons (Rikard-Bell et al., 1985; 16--47.8 #m) does not appear to be

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significantly different. As the cells which project to the phrenic nucleus were smaller and frequently positioned more ventrolaterally compared to those projecting to the thoracic levels, Rikard-Bell and colleagues (1985) have suggested a separation of the NRA neurons projecting to the phrenic and thoracic motoneurons. However, Merrill's (1971, 1974a) demonstration that all inspiratory axons tested which had phrenic arborizations also had thoracic arborizations supports neither their contention of neuronal organization based on the level of the spinal projection nor the identification of the labeled cells as BS 'late-peak' inspiratory neurons of the r-NRA. Hence, the identity of the retrogradely labeled cells in the region of the N R A remains ambiguous. From the morphological studies of the 'late-peak' inspiratory neurons of the vi-NTS (von Euler et al., 1973a; Berger et al., 1983, 1984, 1985), it is possible to compare the 'late-peak' inspiratory neurons of the vI-NTS and r-NRA on an anatomical basis (see Section 2.1.1.1 for further details re: vl-NTS cells). Comparison of the average minor and major somal diameters and the surface areas of transversely sectioned r-NRA and vl-NTS 'late-peak' inspiratory cells shows the vl-NTS neurons to be intermediate between the two classes of r-NRA inspiratory neurons (i.e. NAA and BS units) (Kreuter et al., 1977; Berger et al., 1983, 1984). Specific to the BS inspiratory neurons, the number of primary dendrites per cell was similar for the two groups, vI-NTS and r-NRA (i.e. 4-10); however, their dendrites were oriented in two different planes (horizontal for vI-NTS, and transverse for r-NRA). Hence, based on the observable differences between these two anatomical localizations of 'late-peak' inspiratory neurons, it appears that the 'late-peak' inspiratory neurons of the r-NRA (BS, in particular) and those of the vI-NTS constitute morphologically distinct classes of inspiratory neurons. (b) Patterns o f activity. ExtraceUular recordings of the 'late-peak' inspiratory neurons of the r-NRA have demonstrated an augmenting discharge pattern, with their peak-discharge frequency occurring near the point of maximum inspiratory effort, late in inspiration (Merrill, 1974a, 1979, 1981; Bianchi, 1974). To differentiate these inspiratory cells from those displaying an early-burst inspiratory pattern, Merrill (1979) used the term 'latepeak'. While the earliest onsets of discharge can occur up to 300 msec before the onset of phrenic nerve activity, some 'late-peak' inspiratory units have onsets up to a second after the onset of the inspiratory phase (Merrill, 1974a; Taylor et al., 1978; Dick and Berger, 1985). With the more recent recognition of a 'post-inspiratory' discharge in the phrenic nerve in some experimental situations, it is now recognized that the termination of discharge in the 'late-peak' inspiratory neurons must be discussed in relation to the presence or absence of a declining phrenic discharge during the post-inspiratory phase. However, in contrast to a slight overlap of discharge between the 'late-peak' inspiratory r-NRA cells and expiratory c-NRA cells at the transition from expiration to inspiration, Merrill (1974a) reported that substantial overlap in their discharges occurs at the transition to the expiratory phase. This latter finding is at odds with the more recent observation that the c-NRA expiratory neurons are not synaptically depolarized until after the postinspiratory phase comes to an end (Ballantyne and Richter, 1985). From closer examination of the discharge patterns of numerous 'late-peak' r-NRA inspiratory neurons, Merrill (1974a) suggested that there may be two types of these cells, one type having an earlier onset and a later termination than the other, but with fairly equal peak discharge rates. As Taylor and colleagues (1978) found these peak discharge rates, as well as the time of occurrence of the peak, to be independent of the onset time, they suggested that the 'late-peak' inspiratory neurons are not simply recruited on a threshold basis. Consequently, from the results of extracellular recordings, it is not clear as to whether there are two functionally distinct groups of 'late-peak' inspiratory r-NRA neurons or these discharge patterns represent two extremes of a unimodal distribution (Merrill, 1974a; Taylor et al., 1978). Subsequent examinations of the postsynaptic potentials of these 'late-peak' inspiratory r-NRA neurons during the respiratory cycle has, indeed, revealed two, if not three, different patterns of post-synaptic activity. Similar to the 'late-peak' inspiratory neurons of the vI-NTS, those of the r-NRA receive an augmenting pattern of EPSPs throughout

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the inspiratory phase followed by an augmenting pattern of IPSPs throughout the expiratory phase (Hildebrandt, 1974; Mitchell and Herbert, 1974b; Richter et al., 1975, 1979a). A declining pattern of EPSPs coincident with the decline of the phrenic nerve discharge during the post-inspiratory phase may also be present (Richter, 1981; Richter and Ballantyne, 1981; Richter, 1982a, b). In addition, there is evidence of two different patterns of inhibitory inputs to these 'late-peak' inspiratory neurons during their active phase: (1) early inspiratory inhibition, characterized by a declining pattern of IPSPs during early inspiration interacting with the 'throughout' augmenting EPSP pattern, and (2) late inspiratory inhibition, characterized by the same 'throughout' augmenting EPSP pattern interacting with an augmenting IPSP pattern late in the inspiratory phase (Richter, 1980, 1981, 1982a, b; Richter and Ballantyne, 1983; Ballantyne and Richter, 1984). It is likely that these two patterns of inspiratory phase inhibition explain the previous extracellular observations of Merrill (1974a) and Taylor and colleagues (1978) concerning the onset of inspiratory neuronal discharge relative to the inspiratory period. Also similar to the 'late-peak' inspiratory neurons of the vI-NTS, Ballantyne and Richter (1984) found that these two different patterns of inspiratory inhibition occur in both BS (n = 8) and NAA (n = 11) 'late-peak' neurons, though rarely do the two inhibitory patterns coexist in the same inspiratory neuron, at least within the tested range of experimental conditions. While it is possible that some of these NAA 'late-peak' neurons actually had spinal axons which were not successfully activated, Ballantyne and Richter (1984) argue that this would only apply to a small percent of the eleven NAA 'late-peak' inspiratory r-NRA neurons. Based on the fact that Kreuter and colleagues (1977) identified NAA 'late-peak' inspiratory neurons within the r-NRA which had either a 'throughout type' or a 'late type' of discharge and were morphologically different from the BS 'late-peak' inspiratory neurons of the r-NRA, it is likely that non-BS, perhaps propriobulbar, 'late-peak' inspiratory neurons coexist with the BS 'late-peak' inspiratory neurons of the r-NRA and that both types of inspiratory neurons receive either early inhibition or late inhibition during the active phase. Closer examination of the three types of inhibitory input to the 'late-peak' inspiratory neurons suggests that the late inspiratory and expiratory IPSPs arise at or close to the cell soma, as shown by the time course of reversal of the IPSPs following either chloride injection or chloride diffusion into the cells and by the increase in input conductance (Richter, 1982a; Ballantyne and Richter, 1984). In contrast, the early inspiratory IPSPs appear to arise at more distal dendrites; this is suggested by the relatively longer period of chloride injection/diffusion before IPSP reversal occurred and by the minimal or absent change in input conductance compared with that for the late inspiratory or expiratory inhibiton (Ballantyne and Richter, 1984). In addition to these complex synaptic inputs to the BS 'late-peak' inspiratory neurons of the r-NRA, it appears that their neuronal excitability is influenced by intrinsic membrane properties. Using the calcium chelator EGTA, Mifflin and colleagues (1985) have recently demonstrated that a calcium activated potassium conductance contributes, at least in part, to an increase in input conductance of these neurons at any given time in the respiratory cycle. During the neuron's active phase, this calcium activated potassium conductance is responsible for the late afterhyperpolarizations (lasting 12-32 msec) which follow both spontaneous and antidromic action potentials; in contrast, the early afterhyperpolarizations (lasting less than 2 msec) do not appear to be sensitive to EGTA. Further, the hyperpolarizations observed after a burst of action potentials (elicited by a depolarizing current pulse) in these neurons (i.e. 'post-tetanic hyperpolarizations') are generated, at least in part, by this calcium activated potassium conductance (Mifflin et al., 1985). Hence, Mifflin and colleagues (1985) speculate that the response of these 'late-peak' inspiratory BS neurons to synaptic input are influenced by modulation of their intrinsic membrane properties (i.e., synaptic alteration of this calcium activated potassium conductance). As mentioned previously, a third pattern of post-synaptic activity appears to exist in some NAA 'late-peak' inspiratory neurons of the r-NRA. (Note: This type of pattern has

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not been reported for the 'late-peak' neurons of the vI-NTS (Richter et al., 1979a, 1985;

Richter, 1982a, b; Richter and Ballantyne, 1983).) On the basis of its 'ramp-like' discharge behaviour during the inspiratory phase, Richter's laboratory (Richter, 1982a, b; Richter et al., 1985) has utilized the term 'ramp' inspiratory neuron. While its postsynaptic pattern during the inspiratory phase appears to be similar to that of the 'late-peak' inspiratory neurons (both r-NRA and vl-NTS) which receive late inspiratory inhibition, closer examination of extra- and intracellular recordings at the onset of inspiration reveals an abrupt depolarization often associated with the discharge of 2-3 spikes before the augmenting inspiratory depolarization begins; this phenomenon resembles rebound excitation (Richter, 1982b; Richter and Ballantyne, 1983; Richter et al., 1985). Furthermore, unlike the 'late-peak' inspiratory neurons (both 'early' and 'late' inhibition types), the 'ramp' neurons display a rapid onset, declining pattern of post-inspiratory inhibition with maximal hyperpolarization occurring at the onset of the phase. Finally, the expiratory phase inhibition of the 'ramp' inspiratory neurons does not display the augmenting IPSP pattern seen in other inspiratory neurons; instead, the 'ramp' neuron's expiratory inhibition has a plateau-like pattern (Richter et al., 1979a, 1985; Richter, 1982a, b; Richter and Ballantyne, 1983). Interestingly, Richter (1982b) points out that the time-intensity profile of this expiratory inhibition in the 'ramp' inspiratory neuron is very similar to that of the discharge of expiratory laryngeal motoneurons (see Barillot and Bianchi, 1971; Barillot and Dussardier, 1976; Cohen, 1977). As a result of this observation and the fact that vagal inspiratory motoneuron activity was intracellularly recorded in at least one of these experiments from Richter's laboratory (i.e. see Richter et al., 1979a), the possibility that this 'ramp' neuron is actually a NAA inspiratory vagal motoneuron cannot be ruled out. On the other hand, if this 'ramp' neuron is actually a nonvagal, r-NRA inspiratory neuron, there is no evidence to date that qualifies the 'ramp' neuron as a class of neurons which are morphologically distinct from the other NAA 'late-peak' inspiratory r-NRA neurons. In this instance, we suggest that the variation in postsynaptic activity might simply be a result of variations in synaptic input and/or experimental conditions. This suggestion is reinforced by the following two experimental observations. First, the 'rebound excitation' phenomenon associated specifically with the 'ramp' inspiratory neurons (Richter, 1982b; Richter and Ballantyne, 1985; Richter et al., 1985), likely a consequence of the delayed activation of a voltage dependent potassium current (the A current), can be experimentally induced in other respiratory neurons when steep depolarization is preceded either by a period of long-lasting hyperpolarization or by release from strong synaptic inhibition. Second, in a physiological situation such as an increased inspiratory drive, this 'rebound excitation' phenomenon can even be seen in the phrenic nerve discharge (Richter et al., 1985). From these observations it would appear that the 'rebound excitation' phenomenon may occur in any respiratory neuron under some circumstances. Consequently, we argue that the distinct pattern of postsynaptic potentials recorded from 'ramp' inspiratory neurons is not adequate, by itself, to classify them as a morphologically and functionally distinct class of r-NRA inspiratory neurons. In summary, if one combines the results of the aforementioned intracellular studies with those of the antidromic mapping and morphology studies of the 'late-peak' inspiratory neurons of the r-NRA, it appears that the existence of the BS 'late-peak' cells has been adequately verified. On the other hand, while both the preliminary morphological identification of 'late-peak' cells of smaller somal diameter than the 'late-peak' BS ceils and the inability to antidromically activate some 'late-peak' neurons from the spinal cord support the existence of propriobulbar 'late-peak' inspiratory neurons in the r-NRA, further experimentation is required to confirm their existence as a group of cells which are anatomically and functionally (i.e. nonbulbospinal) distinct from their BS 'late-peak' counterpart in the r-NRA. This point is particularly relevant as Merrill (1974a, 1979) found that essentially all 'late-peak' inspiratory neurons of the r-NRA have spinal axons (n = 150; Merrill, 1974a). Finally, the NAA 'late-peak' inspiratory neurons, which have

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been classified as 'ramp inspiratory' neurons solely on the basis of their distinctive patterns of post-synaptic activity, may not exist as a separate neuronal population. Unfortunately, much of the remaining information concerning the 'late-peak' inspiratory neurons of the r-NRA is confounded by this 'neuronal identification' dilemma. In many experiments, particularly the earlier ones, the 'late-peak' inspiratory r-NRA neurons were only identified on the basis of their extracellular discharge pattern and not by their 'bulbospinal' character. Consequently, as the identity of the recorded 'late-peak' inspiratory neurons is often ambiguous, the remaining discussion of the 'late-peak' r-NRA inspiratory neurons is not always specific to neurons which have been electrophysiologically confirmed to have spinal projections. (c) Functional interrelations. Few (10%, n = 36 pairs) short-time scale correlations of discharge pattern have been shown for 'late-peak' inspiratory neurons of the r-NRA (spinal axons not identified) which were separated by approximately 1-4 mm (Vachon and Duffin, 1978). In contrast, for a small sample size (n = 6 pairs), Feldman and colleagues (1980) found that more than 65% of near-neighbouring neurons had a positive crosscorrelation but these results were based on single electrode techniques where false positive correlations are easily obtained. A second study from this laboratory (Feldman and Speck, 1983a), using interelectrode spacings ranging from 0 to 2000 #m, reported that approximately 15% (n = 216 pairs) of inspiratory pairs of the r-NRA displayed short-time scale correlations and suggested that near neighbours were no more likely to interact than distant cells (up to 2 mm apart), a finding in contrast to the results of others (Vachon and Duffin, 1978; Long and Duffin, 1984). Further, their indirect identification of these inspiratory neurons as BS based on the correlation of unit discharge with the contralateral phrenic nerve activity led them to suggest that BS-BS neuron pairs were more likely to display short-term synchronization of discharge compared to BS-nonbulbospinal neuron pairs. While these 'nonbulbospinal' inspiratory neurons might belong to the group of NAA, morphologically distinct 'late-peak' inspiratory neurons observed by Kreuter and colleagues (1977), it is equally possible that they are inspiratory vagal motoneurons as is suggested by the presence of a decrementing discharge pattern for some inspiratory neurons in a preparation in which nucleus ambiguus activity was not suppressed (i.e. chloralose urethane; Feldman and Speck, 1983a). Nonetheless, the results of both Vachon and Duffin (1978) and Feldman and Speck (1983a) suggest that short-term synchronization of discharge is not a frequent occurrence for this inspiratory neuron population. While Feldman and Speck (1983a) suggest that the source of short-time scale correlation is common input, unequivocal differentiation between common input and neuronal interaction cannot be made from a cross-correlation based on spontaneous neuronal discharges alone (see Long and Duffin, 1984; Graham and Duffin, 1985). More recently, a cross-correlation study (Long and Duffin, 1984) has shown the existence of short-term synchronization of firing for 40% of neighbouring neuronal pairs, though the sample size was small (n = 20 pairs). Typically, the primary feature of these positive cross-correlation histograms was dissimilar peaks on either side within one millisecond of time zero, one low and broad, the other high and narrow. In an attempt to determine the mechanism of such short-term synchronization, further cross-correlation histograms were computed for one neuron of a given pair during spinal antidromic activation of the other (for details of the technique, see Long and Duffin, 1984). Due to a limitation of this latter technique and the fact that an interneuronal interaction was demonstrated for only one of two tested pairs, these results are only suggestive of reciprocal interactions in the 'late-peak' inspiratory neuron population which are likely due to dendrodendritic or electronic synapses (i.e. very short latencies, less than 0.6 msec). This suggestion, however, is supported by the previous demonstration of profuse branching and intermingling of dendritic trees among these neurons (Kreuter et al., 1977). Unfortunately, it is ambiguous as to whether the pairs of neurons displaying such short-term synchronization of discharge were both BS or whether one of the neurons was a nonbulbospinal, perhaps propriobulbar inspiratory neuron for the following reasons. Kreuter and colleagues (1977) have identified morphologically distinct 'late-peak' inspira-

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tory neurons in the r-NRA which could not be antidromically activated from the spinal cord. Secondly, for the correlated pairs, the present study (Long and Duffin, 1984) either only antidromically activated (from C2 or C3) one neuron of the pair or, in one instance, activated one neuron recorded on each of the electrode pair, but each electrode was recording from more than one inspiratory neuron. Hence, firm conclusions concerning the functional interrelations among 'late-peak' inspiratory neurons of the r-NRA await the examination of discharge patterns of neurons which are precisely identified according to their axonal projections and/or morphological characteristics. (d) Afferents (i) Brain stem and spinal cord. Both neuroanatomical and electrophysiological examinations of the medullary afferent projections to the r-NRA indicate that these 'late-peak' inspiratory neurons may be synaptically influenced by ipsi- and contralateral 'late-peak' inspiratory cells of the r-NRA as well as by other medullary respiratory neurons. The former connection is supported by the electrophysiological demonstration (Merrill, 1971, 1974a, 1979) that the medullary collaterals of these 'late-peak' BS cells arborize in the ipsiand contralateral r-NRA regions. However, short-time scale correlations for the discharge patterns of randomly selected ipsilateral-contralateral r-NRA 'late-peak' inspiratory neuron pairs (Feldman et al., 1980) have not been found. While cross-correlation results for pairs of such cells located within the ipsilateral r-NRA are suggestive of synaptic interaction (Long and Duffin, 1984), the proximity of the tested neuronal pairs (i.e. 350#m) together with the short-latency of the correlations are supportive of dendrodendritic or electrotonic synapses rather than connection via axon collaterals. Consequently, the possibility that the 'late-peak' inspiratory neurons of both the ipsiand contralateral r-NRA are synaptically interconnected by axon collaterals remains unresolved. Alternatively, the 'late-peak' inspiratory neurons of the vI-NTS may synapse with the r-NRA 'late-peak' cells. This possibility is suggested by the presence of vl-NTS inspiratory axon arbors in the ipsilateral and, to a lesser extent, the contralateral r-NRA regions (Merrill, 1974a, 1979; Bystrzycka, 1980). In particular, the proposal that the Re neurons are a source of the IPSPs occurring in those 'late-peak' inspiratory cells receiving lateinspiratory phase inhibition (Baker and Remmers, 1980; Ballantyne and Richter, 1984), based on the association of Ra discharge with graded inhibition of inspiration (Younes et al., 1978), suggests that this pathway from the vI-NTS to the r-NRA, at least for the late onset R e neurons, might be inhibitory. While cross-correlation studies (Feldman et al., 1980) do not support either an inhibitory or an excitatory connection between vI-NTS and r-NRA 'late-peak' inspiratory neurons, the failure to detect such neuronal interaction might be due to the small sample size (n = 17) and/or the fact that the vI-NTS cells were cross-correlated with the contralateral r-NRA cells, to which they have been shown to have few axonal projections (Merrill, 1979), So, despite the similarities of the time course and patterns of PSPs in the 'late-peak' inspiratory neurons of the vI-NTS and r-NRA (i.e. augmenting EPSPs interacting with either early, decrementing IPSPs or late, augmenting IPSPs), the possibility of synaptic interaction between vl-NTS and r-NRA 'late-peak" inspiratory cells remains unresolved. For the 'late-peak' inspiratory neurons receiving early inspiratory inhibition (Ballantyne and Richter, 1984), the similarities in the time course and pattern of IPSPs in these 'late-peak' cells and the firing pattern of 'early-burst' inspiratory neurons suggest that the 'early-burst' neurons are inhibitory to the 'late-peak' inspiratory neurons (Ballantyne and Richter, 1984, 1985), similar to the vl-NTS 'late-peak' inspiratory neurons. However, in contrast to such a hypothesis for the vI-NTS cells, this hypothesis for the r-NRA cells is further supported by the presence of both the 'early-burst' cells within the r-NRA and the 'early-burst' axon collaterals in the ipsi- and contralateral r-NRA (Merrill, 1974a, 1979, 1981). By the same argument, the similarity of the time-intensity profile of the postinspiratory phase IPSPs of all 'late-peak' inspiratory r-NRA neurons with that of the discharge of 'post-inspiratory' cells, together with the localization for these ~post-

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inspiratory' cells, intermingled amongst other"r-NRA inspiratory cells, has led to the suggestion that the latter cells inhibit these 'late-peak' cells during the post-inspiratory phase (Richter, 1982b). The failure to identify axon collaterals within the r-NRA from c-NRA expiratory neurons (Merrill, 1971, 1974a, 1979; Kalia et al., 1979) suggests that they are not the source of the inhibition of 'late-peak' inspiratory neurons during the expiratory phase, despite the similarities of the time course and pattern of their discharge and PSPs, respectively. The more recently identified BOT expiratory neurons, in contrast, appear to be likely candidates for the expiratory inhibition of the 'late-peak' inspiratory cells as they have not only the appropriate time-intensity profile of their discharge but also the required axonal arbors within the r-NRA regions (Bystrzycka, 1980; Bianchi and Barillot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a). Though not documented in the literature, Fedorko and Merrill (1984b) have reported, at the symposium entitled Neurogenesis of Central Respiratory Rhythm, that BOT expiratory neurons are monosynaptically inhibitory to the contralateral r-NRA 'late-peak' inspiratory neurons during the expiratory phase. In addition to these afferent inputs from medullary respiratory neurons, other brain stem sites have been shown to send axonal projections to the r-NRA region. The retrograde transport of H R P (Bystrzycka, 1980) has demonstrated projections arising from the nucleus paragigantocellularis lateralis bilaterally; the contralateral ventral part of the nucleus reticularis pontis oralis; the nucleus locus coeruleus complex bilaterally; the ipsilateral parabrachial nucleus; and the ipsilateral Kolliker-Fuse nucleus. The latter projection to the r-NRA appears to be quite strong. Furthermore, electrical stimulation in the rostral pons has been shown to orthodromically activate a small percentage of inspiratory r-NRA neurons (Baker and Remmers, 1982). While Schmid and colleagues (1985) also found that rostral pontine stimulation (nucleus parabrachialis medialis and locus coeruleus) orthodromically influenced the discharge of r-NRA inspiratory cells in the rabbit; inhibitory afferent connections were found to be more prevalent than excitatory ones. However, only a few of the projections from the region of the nucleus parabrachialis medialis and Kolliker-Fuse nucleus have been shown to originate from respiratory neurons, as determined electrophysiologically (Bianchi and St. John, 1982). Furthermore, the largest portion of these pontobulbar respiratory neurons had phase-spanning patterns of activity, as was similarly shown for the pontobulbar projections to the vl-NTS region. More recently, cross-correlation analysis of medullary-pontine pairs of inspiratory neurons (Lindsey et al., 1985) failed to demonstrate synaptic interaction (i.e. less than 1%); such results are in agreement with the earlier demonstration that few respiratory and, in particular, inspiratory neurons of the pons project to the r-NRA. Electrophysiological examination of the brainstem projections of respiratory muscle afferents has yielded results suggesting that, in addition to the inspiratory neurons of the vl-NTS, these afferents also influence those of the r-NRA. As a consequence of electrical stimulation of the central end of the phrenic nerve, two laboratories (Macron et al., 1985; Speck et al., 1985) recorded short latency evoked potentials (3 msec) in the region of the r-NRA, and Speck and colleagues (1985) found that this afferent stimulation also evoked activity in the ipsilateral r-NRA inspiratory neurons (discharge pattern not identified) at latencies of 5-15 msec. The other laboratory (Macron et al., 1985), however, found the respiratory neuron discharge in the N R A to be unaffected by phrenic nerve afferent activation, except in one instance. Hence, it remains unclear as to whether phrenic nerve afferents do influence the discharge of r-NRA inspiratory neurons. In contrast to the reported augmentation of r-NRA inspiratory neuron activity in response to phrenic nerve stimulation (Speck et al., 1985), it appears that similar activation of intercostal (internal and external) and abdominal muscle proprioceptors and thoracic wall cutaneous receptors results in a transient reduction of the discharge rate, or a premature termination of activity for the inspiratory r-NRA neurons (Shannon and Freeman, 1981), as was previously described for the vl-NTS inspiratory neurons. Subsequently, the inspiratory neuronal responses to selective excitation of Group Ia (via muscle vibration) and Group Ib (via ventral root elicited intercostal muscle contraction) J.P.N. 27/2<

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afferents of the internal and external intercostal muscles have demonstrated that increased Group Ib (golgi tendon organ) activity is responsible for an inhibition of the r-NRA inspiratory neurons while activation of muscle spindle afferents (Group Ia) appears to have no modulating effect on their neuronal discharge (Bolser et al., 1983, 1984; Shannon et al., 1985b). Also similar to the response characteristics of the vI-NTS region, neuronal activity in close proximity to the r-NRA inspiratory cells was evoked by such respiratory muscle proprioceptor stimulation, and was associated with changes in the inspiratory neuron activity produced by such stimulation (Shannon and Lindsey, 1983). This led to the proposal that these nonspontaneously active cells are interneurons which act to transmit respiratory muscle afferent input to the r-NRA inspiratory cells. While these results from Shannon's laboratory suggest that proprioceptors of the intercostal and abdominal muscles act on the medullary inspiratory neurons (vI-NTS and r-NRA) to reduce or terminate inspiratory drive to the inspiratory motoneurons, such an interpretation is based on nonphysiological stimuli (i.e. electrical stimulation, mechanical vibration). Utilizing inspiratory mechanical loading in vagotomized cats as a method for increasing the activity of inspiratory muscle proprioceptors, the r-NRA inspiratory neurons were found to have a prolongation of their discharge to the first loaded breath, though not all neurons were affected (Shannon et al., 1985a). Unexplained is the fact that a previous study from the same laboratory (Shannon et al., 1972) reported a small reduction in discharge rate and/or delay to the onset of activity for some r-NRA neurons in response to similar inspiratory mechanical loading; this was not seen in the recent study. While these conflicting results do confound the interpretation of r-NRA inspiratory neuron responses to inspiratory loading, the demonstration that sectioning of both the cervical and thoracic dorsal roots eliminated any neuronal response to the loading strongly suggests that the diaphragm and the inspiratory intercostal muscles afferents are responsible for'the 'loading' reflex effects on medullary inspiratory neurons. (ii) Cranial nerves and central chemoreceptors. As shown by numerous neuroanatomical studies, the major central projections of the afferents of the IXth and Xth cranial nerves, and their various branches, are to the NTS whereas similar projections to the NRA region have not been found (Cottle, 1964; Berger, 1979a; Kalia and Mesulam, 1980a, b; Kalia and Welles, 1980; Ciriello and Calaresu, 1981; Ciriello et al., 1981a, b; Davies and Kalia, 1981). Hence, it appears that the NTS is the site of this viscero-sensory afferent input and that any alteration of the discharge patterns of r-NRA inspiratory neurons, in response to both physiological and nonphysiological stimulation of these cranial nerves, or their branches, is probably not a consequence of direct synaptic connection. Consequently, much of the evidence concerning the influence of these cranial nerve afferents on the 'late-peak' r-NRA inspiratory neurons is indirect, having been obtained from the observations of neuronal discharge during physiological (i.e. manipulation of the lung volume or carbon dioxide level) or during nonphysiological stimulation of these afferents. Lung volume manipulation studies have shown that maintained lung inflation inhibits the inspiratory BS r-NRA neurons (Bianchi and Barillot, 1975) while withheld inflations (i.e. no-inflation test) have no effect on their discharge pattern (Cohen, 1979; Dick and Berger, 1985). Similarly, based on the results of lung inflations and tracheal occlusions at the onset of inspiration, Seaman and colleagues (1983) found the discharge of r-NRA cells to be unaffected or inhibited. However, it has been reported that some BS r-NRA inspiratory neurons are excited by lung inflation (i.e. R e type response), as demonstrated by lung inflation testing (Bianchi and Barillot, 1975) and, the 'no-inflation' test (Dick and Berger, 1985). This latter response is unlikely to be mediated by monosynaptic excitation from pulmonary stretch receptors as pulmonary vagal afferents have not been shown to project to the NRA region (Kalia and Mesulam, 1980b). These results suggest that the response to pulmonary stretch receptor afferent activation for the majority of the 'late-peak' inspiratory neurons of the r-NRA corresponds to that for the R, inspiratory neurons of the vl-NTS. Also similar to the late-peak inspiratory vl-NTS neurons, the r-NRA inspiratory cells were excited by hyperinflation of the lungs during expiration (i.e.

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Head's paradoxical reflex) (Bianchi and Barillot, 1975; Bowden, 1978), presumably by the activation of the rapidly adapting pulmonary receptors. More recently, Seaman and colleagues (1983) have utilized mechanical loading to examine the influence of vagally mediated volume information on the discharge profile of r-NRA inspiratory cells. For three types of loading--resistive, elastic and tracheal occlusion, application during inspiration significantly increased the inspiratory duration with no significant change in the rate of increase of neuronal discharge. Similar expiratory resistive loading produced a prolongation of the expiratory duration but had no effect on the discharge pattern of the r-NRA cells during the subsequent, unloaded inspiration. Larger expiratory loads, however, did prolong the subsequent, unloaded inspiration. Bilateral cervical vagotomy eliminated these responses. Although only a proportion of carotid sinus nerve afferent fibres have been found to project ventrolaterally as far as the NRA (Davies and Edwards, 1973, 1975; Davies and Kalia, 1981), stimulation of the carotid chemoreceptor results in excitation of r-NRA inspiratory neurons (Koepchen et al., 1973; Mitchell and Herbert, 1974a; St. John and Wang, 1977; St. John, 1981; Lipski et al., 1984). Further, such peripheral chemoreceptor activation produces an increased amplitude of the depolarizing wave during inspiration, and this results in an increased firing frequency. Also of interest, Lipski and colleagues (1984) noted that, on occasion, the latency of the responses of single inspiratory cells to carotid chemoreceptor stimulation (via close arterial injections of CO2-equilibrated saline) was not simultaneous with, but instead was longer than that of the phrenic nerve. This may indicate a nonuniformity of peripheral chemoreceptor input to r-NRA inspiratory neurons. Although it is likely that interneurons, as yet unidentified, are involved in this excitatory chemoreceptor input, the projection of a small proportion of the chemoreceptor afferent fibres ventrolaterally as far as the NRA region suggests that some responses might be due to direct monosynaptic excitation. Comparison of the effects of carotid chemoreceptor (pharmacologic activation) and central chemoreceptor (hyperoxic hypercapnia) stimulation on the discharge of the r-NRA inspiratory cells during equivalent elevations of ventilatory activity (i.e. peak integrated phrenic nerve activity) has shown that the augmentation in neuronal discharge was only significant for the central chemoreceptor activation (St. John, 1981). These results also suggest that, unlike the vI-NTS inspiratory neurons, the r-NRA inspiratory neurons do not uniformly receive excitatory peripheral chemoreceptor input but, similar to the vI-NTS group, they receive an equally distributed input from the central chemoreceptors. This suggestion of a nonuniform delivery of carotid chemoreceptor input to these cells is further supported by the recent findings of Lipski and colleagues (1984) (as discussed above). On the other hand, such a homogeneous influence of the central chemoreceptors may occur via synaptic, or direct, excitation (see Section 2.1.1.4 for references). An earlier study (St. John and Wang, 1977) which also compared the responses of r-NRA inspiratory neurons to stimulation of the central chemoreceptors (via hyperoxic hypercapnia) and the peripheral chemoreceptors (via isocapnic hypoxia) reported similar results though the proportion of inspiratory neurons in which activity either did not increase or was depressed during isocapnic hypoxic activation of carotid chemoreceptors was higher than during pharmacologic activation (St. John, 1981). Neurons which did not show an increase (i.e. unchanged or depressed activity) were more prevalent in the r-NRA than in the vl-NTS region. Overall, this nonuniform effect of isocapnic hypoxia on the r-NRA inspiratory cells, taken with the dual hypoxic effect of central chemoreceptor depression and peripheral chemoreceptor excitation, suggests that an increase in neuronal discharge occurs only in those cells receiving adequate excitatory peripheral chemoreceptor input to overcome the generalized medullary hypoxic depression (St. John and Wang, 1977; St. John and Bianchi, 1985). Alternatively, the inhibitory response of hypoxia may be mediated by some inhibitory interneurons located in the NTS, which have shown a response pattern to chemoreceptor stimulation similar to that previously described by Lipski et al. (1976) (Lipski et al., 1984). Such projections from the NTS to the r-NRA region have been demonstrated on an anatomical basis (Loewy and Burton, 1978).

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Intracellular examination of these cells during specific carotid chemoreceptor activation (i.e. NaCN injection), as well as during generalized chemoreceptor stimulation (normoxic hypercapnia), has shown that the augmentation of inspiratory discharge was produced by an increased amplitude of the depolarizing wave (Mitchell and Herbert, 1974a). While hypercapnia also resulted in greater hyperpolarization during expiration, carotid chemoreceptor stimulation (by NaCN) had no significant effect on the resting membrane potential during expiration. Hypocapnia produces effects in the opposite direction, that is less depolarization in inspiration and less hyperpolarization during expiration. This is consistent with the results of antidromic latency measurements for the inspiratory cells during eucapnic breathing and hyperventilation apnea (Merrill, 1974a). As the excitatory effect of chemoreceptor stimulation (peripheral as well as central) was found to be effective only during inspiration, Mitchell and Herbert (1974a) concluded that the excitatory chemoreceptor effect on these cells is 'gated'. Perhaps the BOT expiratory neurons which have been reported to monosynaptically inhibit contralateral r-NRA inspiratory neurons (Fedorko and Merrill, 1984b, as reported at the symposium entitled Neurogenesis of Central Respiratory Rhythm) are responsible for such expiratory gating of chemoreceptor excitation during the expiratory phase. As previously discussed, Lipski and colleagues (1984) have concluded that these BOT expiratory neurons may be responsible for the expiratory inhibition of the vl-NTS inspiratory cells produced by carotid chemoreceptor stimulation. More recently, from observations of the variations of antidromic latencies of the r-NRA inspiratory cells during normocapnic hyperoxia as compared to hypercapnia or hypoxia, St. John and Bianchi (1985) have concluded that the overall change of membrane potential (and hence the rate of change) during the respiratory cycle, as opposed to the absolute level of depolarization, primarily defines the discharge frequencies of these cells (for further details, see Section 2.1.1.4). This conclusion, however, appears to be at odds with the findings of Mitchell and Herbert (1974a) which associated the increased firing frequency with the increased magnitude of depolarization during inspiration, at least, in the instance of carotid chemoreceptor excitation. (e) Projections and synaptic connections. Numerous neuroanatomical (Kuypers and Maisky, 1977; Holstege and Kuypers, 1982; Feldman and Speck, 1983b; Rikard-Bell et al., 1984, 1985; Feldman et al., 1985) and neurophysiological (Bianchi, 1971; Merrill, 1971, 1974a, 1979; Newsom Davis and Plum, 1972; Hukuhara, 1973; Fedorko et al., 1983; Ballantyne and Richter, 1984; Hilaire et al., 1984; Feldman and Speck, 1983a; Long and Dutiin, 1984; Dick and Berger, 1985) have shown the 'late-peak' inspiratory neurons of the r-NRA to have varied efferent projections. However, examination of these axonal projections is complicated by the identification of two types of 'late-peak' inspiratory neurons which have different axonal pathways (bulbospinal vs nonbulbospinal) and which appear to be morphologically distinct (i.e. Kreuter et al., 1977; Ballantyne and Richter, 1984; Hilaire et al., 1984). All 'late-peak' inspiratory neurons (Merrill, 1971, 1974a, 1979, 1981) appear to have spinal axons (i.e., only 6 of approximately 150 tested cells could not be antidromically activated from the cervical cord). Furthermore, Merrill reported that some of these BS cells had medullary collaterals which arborized in the ipsi- and contralateral r-NRA inspiratory region and, less commonly, in the contralateral c-NRA expiratory region (Merrill, 1971, 1974a, 1979) (Fig. 3). The role of such collaterals is at present unknown. No collaterals in either the ipsilateral c-NRA expiratory region or the vI-NTS inspiratory region, bilaterally, were found (Merrill, 1974a, 1975, 1979, 1981). Though Kalia and colleagues (1979) demonstrated such projections from the rostral portion of the NRA, in the vicinity of the retrofacial nucleus, Lipski and Merrill (1980) have subsequently confirmed these projections as belonging to the expiratory population of the Botzinger complex. In contrast, the axonal projections of the morphologically distinct, nonbulbospinal 'late-peak' inspiratory neurons can only be inferred from the inability to antidromically activate these cells from either the spinal cord or the ipsilateral vagus nerve (Kreuter et al., 1977). This negative finding as well as their smaller cell size (i.e. compared with their

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FIG. 3. A sectional view of the medulla and spinal cord illustrating the axonal pathway for a

'late-peak' inspiratory neuron of the r-NRA. BS counterpart) suggest these neurons have restricted intramedullary projections (i.e. propriobulbar) or ascending projections (i.e. bulbopontine) (Kreuter et al., 1977; Richter, 1982b; Richter and Ballantyne, 1983; Ballantyne and Richter, 1984). With regard to the more infrequent axonal arborization of 'late-peak' r-NRA cells in the contralateral c-NRA, the possible existence of synaptic connectivity with the expiratory neurons has not been supported by any correlation of their discharges (Merrill, 1979; note: the cross-correlation histograms were obtained from the period of their overlapping discharge at the transition from inspiration to expiration). The presence of the appropriate axon collaterals in the c-NRA region was demonstrated for some but not all of the tested 'late-peak' inspiratory cells. Furthermore, the dissimilarity between the 'late-peak' discharge of the r-NRA inspiratory cells and the declining IPSP pattern of the c-NRA expiratory group (Merrill, 1974a; Mitchell and Herbert, 1974b; Richter et al., 1975, 1979a) does not suggest the 'late-peak' inspiratory cells are the source of inspiratory inhibition of the c-NRA expiratory group. Detailed antidromic mapping studies (Merrill, 1971, 1974a, 1979) of the BS 'late peak' inspiratory neurons have shown variety in their spinal anatomy. Most spinal axons have a contralateral projection, although approximately 5-10% project to the ipsilateral cord. For the contralateral projections, some axons cross the midline dorsal to the central canal while others cross under the fourth ventricle at various depths; the crossing occurs between 4 mm caudal and more than 5 mm rostral to the obex. While the inspiratory axons are largely concentrated in the ventrolateral white matter, some can be found in the most medial part of the ventral funiculus as well as in the most dorsal part of the lateral funiculus. The approximate positions of these axons at C 3 are in the lateral and ventral parts, respectively, of the ventral and lateral funiculi (Fig. 3). A recent antidromic mapping study of these inspiratory neurons (n = 25) (Dick and Berger, 1985) also found their axons in both the lateral and ventral columns at C3, but the majority were aggregated directly below the ventral horn. Individual axons (n = 10) maintained their positions in either of the two descending columns from rostral C3 to mid C4 segments. The axons from five of six neighbouring neuron pairs (300/~m dorsoventral separation) travelled within close proximity of each other (within 350/~m) in the cervical cord. While the one exception involved a neuronal pair with very different firing patterns (i.e. early-onset vs late-onset discharge relative to the phrenic activity), each of the other pairs had similar discharge patterns (i.e. both early-onset or both late-onset). These results led Dick and Berger (1985) to suggest the existence of a microorganization of the descending inspiratory tracts, with the axons of adjacent neurons travelling in close proximity to one another; the similarity of discharge (early-onset vs late-onset) of adjacent neurons may be a determinant of such microorganization. However, there appeared to be

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no relationship between the type of discharge pattern and the preference for a particular descending inspiratory tract (ventral vs lateral column) of individual neurons in the r-NRA. In addition, antidromic mapping studies have shown that approximately 25% of the spinal axons have collaterals which arborize in the phrenic nucleus (C4-C6) (Merrill, 1971, 1974a, 1979; Fedorko et al., 1983) while virtually all inspiratory main axons continue their spinal descent to arborize in the thoracic cord, usually over several segments; none continue into the lumbar cord (Merrill, 1971, 1974a, 1979). Although the inspiratory arborizations are primarily restricted to the intermediate and ventral horns of one side, some arbors do cross the midline, but only within the cervical cord segments (i.e. phrenic nucleus) (Merrill, 1971, 1974a; Merrill and Lipski, personal communication). Finally, the thoracic projections of these inspiratory cells do not appear to be somatotopic as shown by the ability to antidromically activate approximately equal numbers of neurons at all r-NRA levels from the T 8 level (Merrill and Lipski, personal communication). However, Merrill and Lipski (personal communication) caution that the latter observation does not preclude the existence of a slight preference of axons from one region of the r-NRA to project to particular spinal segmental levels or, perhaps, of restricted synaptic interactions at only some of the segmental levels in which the axons arborize. The existence of such spinal pathways from the r-NRA region within the lateral and ventral funiculi of the cervical cord have been confirmed by neuroanatomical studies (Kalia, 1977; Kuypers and Maisky, 1977; Holstege and Kuypers, 1982; Feldman and Speck, 1983b; Rikard-Bell et al., 1984, 1985; Feldman et al., 1985). However, some of these latter studies (i.e. Holstege and Kuypers, 1982; Rikard-Bell et al., 1984, 1985; Feldman et aL, 1985) have shown a far greater ipsilate~ral contribution than was electrophysiologically demonstrated (i.e. less than 10%) by Merrill (1971, 1974a, 1979, 1981). Rikard-Bell and colleagues (1984, 1985), using retrograde transport of HRP from the phrenic nucleus and the thoracic respiratory motoneuron regions, found a relative axonal contribution from the ipsi- and contralateral NRA (relative to the cell soma) of approximately 35% and 65%, respectively. Although some of the pronounced ipsilateral labeling might be the result of inspiratory axon collaterals which cross the midline of the spinal cord (Merrill, 1971, 1974a), it is perhaps more likely that since such neuroanatomical studies do not establish the physiological identity of the labeled cells, some of the ipsilateral (and contralateral as well) projections may have belonged to nonrespiratory cells. In an attempt to minimize this 'neuroanatomical' limitation, Feldman and colleagues (Feldman and Speck, 1983b; Feldman et al., 1985) studied the anterograde transport of tritiated amino acids which had been injected into a physiologically identified inspiratory region in the r-NRA (n = 5 cats), with subsequent histological confirmation of the injection sites. Though the possibility of active uptake of the amino acids by quiescent neurons within the inspiratory region and/or neurons in regions adjacent to the inspiratory region could produce false positive results, the resulting axonal projections in the cervical and thoracic cord are remarkably similar to the electrophysiological mapping results (Merrill, 1971, 1974a, 1979, 1981; Dick and Berger, 1985) with one major exception. Within the cervical cord, Feldman and colleagues (1985) found a bilateral projection which became restricted to the contralateral cord at approximately C6. Yet, Merrill had reported that all spinal axons, even the few ipsilateral ones, descended into the thoracic cord. Nonetheless, this autoradiographic study did find the projection to the phrenic nucleus to be primarily contralateral, with that to the thoracic levels being solely contralateral. Interestingly, although other neuroanatomical studies (i.e. Rikard-Bell et al., 1984, 1985) had found bilateral projections to both the cervical and thoracic cord from the NRA region (i.e. both rostral and caudal parts), Feldman and colleagues (1985) did not confirm this ipsilateral thoracic projection for either the inspiratory or expiratory NRA regions. The locations of the axons in the white matter of the cervical cord (particularly at C3-C4 segments), as well as the occurrence of axonal arbors and terminal synaptic fields in both the phrenic nucleus (C4.-C6) and the ventral horn of thoracic but not upper lumbar segments (Feldman et al., 1985), are in close agreement with the results of antidromic

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mapping studies (Merrill, 1971, 1974a, 1979, 1981; Dick and Berger, 1985). As Feldman et ai. (1985) found that the projections in the lateral funiculus diminished significantly in the C4--C6 region, they suggested that the r-NRA axons in the lateral funiculus project primarily to the phrenic nucleus, while those in the ventral funiculus project to the thoracic grey matter (i.e. intercostal motoneuron regions) and may project to the phrenic nucleus. Against this view is the electrophysiological demonstration that thoracic arborizations occurred for all inspiratory axons which had phrenic arborizations (Merrill, 1971, 1974a). Regardless of the possible existence of such anatomical organization of the axonal tracts, the contralateral ventral tract of axons gradually dissipated as it travelled through the thoracic cord and is no longer apparent by T~2 (Feldman et al., 1985). Perhaps both the discontinuation of the lateral tract projections (C4--C6) and this dissipation of axons in the thoracic cord are associated with the less extensive occurrence of arborizations in thoracic white matter as compared to that in the phrenic nucleus (Feldman et al., 1985). Finally, in agreement with Merrill's identification (1974a) of collaterals which crossed the midline in the lower cervical cord, ipsilateral arbors and terminal fields were neuroanatomically identified (Feldman et al., 1985) in the C5-C6 segments, although these could have originated from the axons labeled in the ipsilateral cervical cord. Overall, such electrophysiological and neuroanatomical studies have established the contralateral projections to and extensive arborizations within both the phrenic nucleus and the thoracic ventral horn for the 'late-peak' BS inspiratory r-NRA neurons. The functional importance of such projections has been investigated by both cross-correlation and STA methods. While Hilaire and Monteau (1976) suggested that approximately 61% of tested cells (n = 18) were monosynaptically connected to phrenic motoneurons, as revealed by neuron to phrenic nerve cross-correlation, more recent examination of possible interaction with phrenic motoneurons has revealed a much lower incidence of such correlation. Feldman and Speck (1983a) demonstrated phrenic nerve correlations for approximately 35% of all r-NRA inspiratory cells (n > 350), with the latencies of the correlation peaks being similar to antidromic latencies (at the C5--C6 levels) previously reported for r-NRA inspiratory cells (i.e. 1.2-6.0msec for the correlation peak; 1.2-5.7 msec for the antidromic activation, as determined by Fedorko et al., 1983). As Feldman and Speck (1983a) examined such a large sample, it appears that the occurrence of such monosynaptic excitatory connections between r-NRA inspiratory cells and phrenic motoneurons is much less frequent than was originally suggested by the data from the small sample size examined by Hilaire and Monteau (1976). Although an incidence of neuron to phrenic nerve correlation (27%, n = 26 cells), similar to that reported by Feldman and Speck (1983a), has recently been demonstrated by Fedorko and colleagues (1983), the latencies of these peaks were considerably longer than the antidromic latencies for r-NRA from the C5~C6 segments (i.e. 3.0-8.5 msec range for peak latencies vs 1.2-5.7 msec range for antidromic activation). These results, taken with the direct demonstration of monosynaptic excitatory connections with phrenic motoneurons for only 2 to 7% (n = 58) of 'late-peak' inspiratory cells, using the STA technique, suggest that r-NRA inspiratory neurons make only rare monosynaptic connections with phrenic motoneurons. Providing further substantiation of this interpretation, Fedorko and colleagues (1983) noted that the absence of PSPs in the remaining 93% (n = 54) of the STAs occurred even when summated phrenic nerve activity showed positive correlations. These investigators concluded that the majority of interactions indicated by such extracellular cross-correlation analysis must be due to scarce monosynaptic connections and/or to di- and oligosynaptic inputs. This conclusion is, of course, surprising since approximately 25 % of r-NRA inspiratory axons arborize in the phrenic nucleus (Merrill, 1971, 1974a, 1979). However, as Fedorko et al. (1983) did not verify the presence of arborizations near the phrenic nucleus for the entire sample of neurons utilized in the STA and cross-correlation analyses (i.e. only 18 of 57 neurons were tested; such arborizations were found for only 5 (28%) neurons), the failure to demonstrate significant monosynaptic connections might be due to the absence of the appropriate axon arbors for such synaptic contact. Yet, even if the cells which

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arborize in the phrenic nucleus do make monosynaptic connections, on the basis of probability, one would have expected more significant results than were observed in the study by Fedorko and colleagues (1983). Similar examinations of the functional significance of r-NRA inspiratory arborizations within the vicinity of thoracic respiratory motoneurons have been made. For a small sample size (n = 18), cross-correlation analysis (Hilaire and Monteau, 1976) provided indirect evidence of monosynaptic excitatory connections to contralateral external intercostal motoneurons for 83% of tested r-NRA neurons; some of these cells also made monosynaptic connections to contralateral phrenic motoneurons. However, Merrill and Lipski (personal communication) failed to find many significant correlations (approximately 10%) between inspiratory units and four different contralateral external intercostal nerves (segments Ts-Ts). Moreover, no significant correlations were observed for specific cells which were demonstrated to have extensive axon arborization in the segments of origin of the tested nerves. Spike-triggered averages computed from pairs of r-NRA inspiratory cells and contralateral external intercostal motoneurons (n = 51) (Lipski and Merrill, 1983; Merrill and Lipski, personal communication) failed to demonstrate any monosynaptic excitatory connections, though two of the STAs displayed 'PSP' patterns which probably represent disynaptic excitatory connections. A similar study of pairs of r-NRA inspiratory cells (n = 17) and contralateral internal intercostal motoneurons (Ts-T6, T8-T9) revealed no evidence of IPSPs, thereby indicating that r-NRA inspiratory neurons do not act to synaptically inhibit expiratory intercostal motoneurons during the inspiratory phase, at least not via mono- or disynaptic pathways (Lipski and Merrill, 1983). These results concerning the paucity of monosynaptic connections of these inspiratory cells with the inspiratory motoneurons (phrenic and intercostal), however, are not supported by the recent cross-correlation results from Sears and colleagues (1985). For 28% of the tested r-NRA cells (n = 127), the correlation peaks were interpreted as indicating monosynaptic excitatory connections with inspiratory motoneurons (phrenic and external intercostal). Furthermore, due to the severe criteria used for identifying monosynaptic connections, Sears and colleagues (1985) suggest that they have probably underestimated the connectivity for some of the tested pairs. Nonetheless, in line with the interpretation made from STA experiments (Fedorko et al., 1983; Merrill and Lipski, personal communication), Sears et al. (1985) conclude that the majority of excitation is disynaptic, i.e. via interneurons. [Note: Sears et al. (1985) make this same conclusion for vI-NTS inspiratory cells which have been shown to monosynaptically excite inspiratory motoneurons (Fedorko et al., 1983; Lipski et al., 1983; Lipski and Duffin, 1985); for an explanation of the rationale for this 'interneuron' suggestion, see Section 2.1.1.5.] In a di- or oligosynaptic pathway from the r-NRA inspiratory neurons to the inspiratory motoneurons, the interposed interneurons could be segmental, located in the vicinity of the motoneuron pools, or propriospinal, some segments away from the motoneurons. As these r-NRA inspiratory cells have axonal arborizations in the region of both the phrenic and intercostal motoneurons, segmental interneurons would, theoretically, be the most likely candidates to be involved in the synaptic pathway of r-NRA cells. The anatomical localization of interneurons in the phrenic nucleus (Lamina IX) (Keswani et al., 1954), together with the recording of inspiratory discharges dorsal to the phrenic nucleus (Baumgarten et al., 1963), suggests that such interneurons may exist at the phrenic nucleus level. Similarly, recordings of inspiratory activity were observed dorsal and slightly medial to the intercostal motor nuclei (Merrill and Lipski, personal communication). Based on their location, as well as on the failure to antidromically activate these neurons with intercostal nerve stimulation, Merrill and Lipski suggest their recordings were made from interneurons. Interestingly, these neurons appear to receive segmental inputs in addition to inputs from descending respiratory drives. In addition to the possible existence of the appropriate segmental inspiratory interneurons, propriospinal inspiratory neurons have been located in the upper cervical cord (C~-C2 segments, in particular) (Aoki, 1982; Aoki et al., 1980, 1983a, b, 1984; Lipski and

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Duffin, 1986). As these upper cervical inspiratory neurons appear to be involved in the control of phrenic and intercostal motoneurons, probably through a disynaptic pathway involving segmental interneurons (Lipski and Duffin, 1986), they, too, might be interposed interneurons in the descending di- or oligosynaptic pathway from the r-NRA inspiratory neurons to the respiratory motoneurons. Certainly the location of these upper cervical inspiratory neurons, near the lateral border of the intermediate grey matter and the adjacent lateral funiculus (Aoki, 1982; Aoki et al., 1980, 1983a; Lipski and Duffin, 1986) provides the anatomical substrate for synaptic interaction with r-NRA inspiratory axons as they descend in the lateral and, to a lesser extent, ventral funiculi. Furthermore, it has been reported that these upper cervical neurons can be orthodromically activated from the inspiratory region of the NRA (Aoki et al., 1983a, b). For further details concerning the upper cervical group, see Section 2.4. Several methods have been utilized to determine the average conduction velocity of the r-NRA inspiratory BS axons. For a sample of these r-NRA inspiratory cells (n = 25; plus one inspiratory vI-NTS cell), Dick and Berger (1985) have recently compared the following four methods: (1) single point determinations based on antidromic activation (AA) of BS neurons, 'single-point AA'; (2) single point determinations based on STA of extracellular field potentials, 'single-point STA'; (3) determinations based on antidromic activation at two spinal levels, 'two-point AA'; (4) determinations based on STA at two spinal levels, 'two-point STA'. These latter two determinations are calculated from the difference in conduction time and the difference in conduction distance from two selected points in the spinal cord. Dick and Berger (1985) found that the 'single-point AA' method produced estimates of mean axonal conduction velocity which were as much as 42% less than the 'single-point STA' method at both C3 and C4 levels; these differences were statistically significant. In turn, both types of single-point estimates were less than determinations by either of the 'two-point' determination methods. These latter techniques (two points--rostral C3 and mid-C4) estimated average conduction velocities as 55.4 ___13.1 m/sec (S.D.) using AA and 53.3_ 13.1 m/sec (S.D.) using STA; these two estimates are not significantly different. These results led Dick and Berger (1985) to conclude that two-point determinations of axonal conduction velocity are most accurate while the 'single-point AA' method, in combination with poor estimates of conduction distance, provides the least accurate estimates of conduction velocity (for more specific details see Dick and Berger, 1985). As a consequence of this study, it appears that previous conduction velocity estimates of inspiratory r-NRA BS axons made from 'single-point AA' (Richter et al., 1975; Kreuter et al., 1977; Bianchi and St. John, 1981) may have underestimated the true value for two reasons. The antidromic conduction times derived from a single point include an axon utilization time thereby overestimating this value, while the axonal conduction distance is underestimated as these BS axons usually project rostrally from the cell soma before crossing the medulla and descending into the spinal cord (Merrill, 1971, 1974a). For these reasons, it is also difficult to compare the mean conduction velocity of the r-NRA inspiratory BS axons (see above values, as calculated from the 'two-point' method) to that of the vI-NTS inspiratory BS axons since the measurements for the latter were based on 'single-point AA' (Bianchi, 1971; Bianchi and St. John, 1981; Lipski et al., 1983). As expected, the estimated values suggest that the mean conduction velocity is considerably slower for vl-NTS axons (i.e. approximately 35 m/sec) compared to that of r-NRA inspiratory axons (i.e. approximately 55 m/sec). The possibility of efferent projections of the r-NRA 'late-peak' inspiratory group to more rostral sites has only recently received attention. As for the NTS, the neurohistochemical study of Kalia (1977) has demonstrated the existence of efferent pathways from the N R A - N A region to both the ipsilateral and contralateral nucleus parabrachialis medialis and Kolliker-Fuse nucleus in the pons, with a greater density of projection to the ipsilateral side; this projection, however, is much less as compared to that from the vI-NTS. More recently, King (1980), using both H R P and autoradiographic techniques, was only able to demonstrate projections to the nucleus parabrachialis from cells near (i.e. lateral

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tegmental field) but not in the NRA or NA. Subsequent electrophysiological work by Bianchi and St. John (1981) has shown that, although a limited number of respiratory neurons in the r-NRA do project ipsilaterally to these nuclei in the pons, the greater majority of these bulbopontine neurons exhibited a respiratory-modulated tonic or phase-spanning activity. While these cells were intermingled among other respiratory cells (i.e. bulbospinal), none of the bulbopontine neurons could be antidromically activated from either the spinal cord or the vagus nerve. Similarly, King and Knox (1982) were unable to antidromically activate any inspiratory cells in the r-NRA from the nucleus parabrachialis, though such activation of quiescent cells in the NRA region was possible. As these latter cells could not be induced to discharge by increasing respiratory afferent inputs (i.e. hypercapnia, activation of Hering-Breuer reflexes, vagal stimulation), it appears that these inactive bulbopontine cells were not respiratory neurons which were below discharge threshold. More recently, in the rabbit, none of the tested r-NRA inspiratory BS neurons could be antidromically activated from the rostral pons (nucleus parabrachialis medialis and locus coeruleus (Schmid et al., 1985). Finally, cross-correlation analysis between inspiratory neurons (discharge pattern not identified) of the r-NRA and those of the rostral pons (nucleus parabrachialis medialis and Kolliker-Fuse nucleus (Lindsey et al., 1985) demonstrated an absence of synaptic connections (i.e. less than 1%, n = 67). Hence, there is no evidence to suggest that 'late-peak' inspiratory r-NRA neurons project to the 'respiratory-related' regions of the pons. (f) Summary. While a large proportion of the 'late-peak' inspiratory neurons of the r-NRA are bulbospinal, it appears that other 'late-peak' r-NRA inspiratory cells may exist which have only intramedullary projections (i.e. nonbulbospinal, nonvagal) and, in addition, which are morphologically distinct from their BS counterparts. Those neurons which project to phrenic and external intercostal motoneurons appear to be a source of excitatory drive during the inspiratory phase, but the pathway for such input seems to be mainly dior oligosynaptic rather than monosynaptic. In contrast, the role of the intramedullary projections (ipsi- and contralateral NRA) of both the BS and nonbulbospinal (i.e. propriobulbar) 'late-peak' cells is not presently known. While the r-NRA region is not the termination site for afferent fibres from the IXth and Xth cranial nerves, the 'late-peak' r-NRA neurons do appear to be influenced by these viscerosensory inputs, likely via interneurons. Similar to the 'late-peak' inspiratory neurons of the vl-NTS, however, the source of excitation that results in the periodic discharge of these 'late-peak' cells during the inspiratory phase continues to remain in question, although neuronal interaction has been suggested to account for short-term synchronization of discharge in a few neuronal pairs studied to date. While the 'early-burst' inspiratory neurons of the r-NRA are purported to be the source of early inspiratory inhibition for some of these 'late-peak' cells, the source of late inspiratory inhibition for other 'late-peak' cells has not been identified. During the post-inspiratory phase, the decrementing discharge pattern of these neurons has been attributed to the 'postinspiratory' neurons but this has not been electrophysiologically demonstrated. Finally, in depth examination of the discharge pattern and projections of the BOT expiratory neurons and the results of recent STA experiments have shown that these cells are the source of monosynaptic inhibition of the 'late-peak' inspiratory neurons of the r-NRA during the expiratory phase. 2.2.1.2. 'Early-burst' inspiratory neurons (a) Location and morphology. The 'early-burst' inspiratory propriobulbar neurons are found in clusters in the r-NRA among the 'late-peak' inspiratory neurons (Bianchi, 1971, 1974; Merrill, 1972b, 1974a, 1979, 1981). Based on extracellular recordings, Merrill (1974a) proposed that the 'early-burst' inspiratory neurons constitute only a small fraction of the r-NRA inspiratory population, perhaps only 10-20%. Recently, neurons exhibiting an early burst, decreasing inspiratory discharge pattern have been associated with the retrofacial nucleus (Bianchi and Barillot, 1982; Bianchi, 1985; Remmers et al., 1985a).

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However, because these neurons were intermingled with 'late-peak' inspiratory neurons in a location which corresponds to the most rostral part of the NA-NRA (Kalia et al., 1979; Bystrzycka, 1980; Merrill et al., 1983), we suggest that these retrofacial early-burst inspiratory neurons may be anatomically and functionally identical to those 'early-burst' inspiratory neurons of the r-NRA. Because of the short duration of extracellular recording times and the small spike amplitudes encountered for 'early-burst' inspiratory cells, Merrill (1974a) has suggested that they are likely much smaller or have more limited dendritic fields compared to other inspiratory neurons in the r-NRA. Although the morphology of the respiratory neurons of the N R A has been studied using Procion yellow (Kreuter et al., 1977), these investigators were unable to locate and stain any inspiratory neurons having an 'early type' of discharge. Consequently, the shape and absolute size of the 'early-burst' cells, as well as the orientation and extent of their dendritic fields, remain unknown. (b) Patterns o f activity. Characteristically, this type of neuron begins its discharge coincident with the termination of activity in the expiratory neurons of the c-NRA and the onset of activity in 'late-peak' inspiratory r-NRA neurons; its peak discharge rate (usually above 50/sec) occurring early in the inspiratory phase with a subsequent decline in discharge until termination in mid to late inspiration (Bianchi, 1974; Merrill, 1974a, Taylor et al., 1978). These observations, taken with the electrophysiological demonstration of excitatory connections to 'early-burst' neurons from contralateral 'early-burst' cells [Merrill, 1974a; see Section 2.2.1.2 (c) for details], led Merrill (1974a) to conclude: (1) that the timing of the 'early-burst' activity is extrinsically determined by the same source as for the 'late-peak' inspiratory r-NRA neurons (as suggested by their coincident onsets of firing after the termination of expiration), and (2) that the details of the discharge pattern are determined by both the excitatory interconnections with other 'early-burst' cells and the accomodative properties of the individual cells. Recently, however, Ballantyne and Richter (1985) have observed that the discharge behaviour of the 'early-burst' neurons is not fixed but variable; this variation occurs over a fairly wide range and can be related to the prevailing pattern of phrenic nerve activity. From intracellular recordings, the 'early-burst' inspiratory neurons have been shown to exhibit a rapid onset, and a decrementing pattern of inspiratory membrane depolarization with a much greater rate of decline during the later part of the inspiratory phase; this is associated with a reduction in both the intensity of the synaptic noise and neuron input resistance (R. A. Mitchell and D. A. Herbert, unpublished observations as cited in Mitchell and Berger, 1975; Richter, 1982a; Ballantyne and Richter, 1985; Richter et al., 1985). There is no evidence, as yet, for the existence of rebound excitation at the onset of inspiration (Richter, 1982b). The transition to the post-inspiratory phase is marked by a low synaptic noise, rapid onset of membrane hyperpolarization to a maximum potential, followed by membrane depolarization at a slow, fairly consistent rate throughout the expiratory phase (Richter et al., 1985). While this behaviour would suggest that the 'early-burst' neuron receives a declining pattern of EPSPs and IPSPs during the inspiratory and postinspiratory phases, respectively, with less prominent inhibition during the expiratory phase, Richter and colleagues (1985) contend that this behaviour is, in part, the result of interaction between postsynaptic activities and intrinsic membrane properties (i.e. membrane conductance). In particular, the decline of membrane depolarization during the inspiratory phase, as well as the post-inspiratory hyperpolarization, could result from a potassium current which is activated by a calcium influx during the early part of the inspiratory phase (Richter et al., 1985). This contention is supported by the following experimental observations: (1) calcium activity in the extracellular region of inspiratory and expiratory neurons decreases during their respective discharge periods (Acker and Richter, 1985); (2) the decrease in extracellular calcium is, at least partly, a result of a calcium influx into respiratory neurons, as revealed by intraceUular analyses (Richter et al., 1985); (3) following EGTA blockage of a voltage-dependent calcium influx, inspiratory neurons with an augmenting discharge pattern demonstrate both a steepening of membrane depolarization in response to EPSPs,

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and a disappearance of the transient hyperpolarization which follows the inspiratory depolarization in the control period (Richter et al., 1985); and (4) the occurrence of temporal changes in the afterhyperpolarization amplitudes of the 'early-burst' inspiratory action potentials, as suggested by the gradual increase in their antidromic latencies during the burst (Merrill, 1974a). From these data, Richter and colleagues (1985) have proposed that such a calcium current, activated during early inspiration, may result in a slow repolarization of the membrane potential during the second half of inspiration and, therefore, the 'accomodation' of discharge described by Merrill (1974a), even if excitatory synaptic input (EPSPs) to the early-burst cells persists. (c) Afferents. On an anatomical basis, the 'early-burst' inspiratory neurons of the r-NRA may be synaptically influenced by collaterals of: the 'late-peak' inspiratory neurons of the vl-NTS (mainly ipsilateral), the 'late-peak' inspiratory neurons of the r-NRA (bilateral), the expiratory neurons of the BOT (bilateral), as well as the 'early-burst' inspiratory interneurons of the r-NRA (predominantly contralateral) (Merrill, 1971, 1974a, 1975, 1979, 1981; Bystrzycka, 1980; Bianchi and Barillot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a; Bianchi, 1985). Of these potential inputs, only the excitatory afferent connections from the contralateral 'early-burst' cells have been demonstrated; for a small sample (n = 3), Merrill (1974a) was successful in activating 'early burst' cells both antiand orthodromically from a contralateral r-NRA site characterized by 'early-burst' inspiratory activity. The possible significance of this synaptic connection to the discharge of the 'early-burst' inspiratory neuron has been discussed in the preceding section. Whether synaptic inputs from the 'late-peak' inspiratory neurons of both the vI-NTS and the r-NRA, as well as from the expiratory neurons of the BOT, actually occur is presently not known. However, since a preliminary report (Fedorko and Merrill, 1984b) has shown the BOT expiratory neurons to be a source of monosynaptic inhibition of inspiratory neurons of the contralateral r-NRA, it is plausible that they are also the source of the expiratory inhibition of these 'early-burst' inspiratory interneurons (Richter, 1982a; Richter et al., 1985). Experimental demonstration of afferent inputs to r-NRA from the upper and lower airway receptors as well as the peripheral chemoreceptors has been hampered by the failure of neuroanatomical studies to identify projections of the vagal and carotid sinus afferents to this region of the medulla (Cottle, 1964; Kalia and Mesulam, 1980a, b; Ciriello and Calaresu, 1981; Ciriello et al., 1981a, b; Davies and Kalia, 1981). Functionally, however, there is indirect evidence that the 'early-burst' inspiratory neuron discharge is influenced by pulmonary stretch receptors, superior laryngeal afferents, and chemoreceptors (peripheral and/or central). Cohen's laboratory (Cohen and Feldman, 1977; Cohen, 1979) has provided indirect evidence that the 'early-burst' inspiratory neurons are inhibited by pulmonary stretch receptors, as shown by the fact that a no-inflation inspiration caused an increase of discharge frequency as compared with a control inspiration. In contrast to this effect, stimulation of the chemoreceptors (via carbon dioxide) appears to have an excitatory effect on these neurons (Merrill, 1974a; Cohen, 1979) while hypocapnia, experimentally induced by passive hyperventilation, resulted in the silencing of (apparently) all inspiratory cells of the r-NRA (Merrill, 1974a). However, whether this excitatory effect on the 'early-burst' cells is restricted to only the inspiratory phase is not known. More recently, Ballantyne and Richter (1985) have demonstrated that electrical stimulation of the superior laryngeal nerve excited 'earlyburst' neurons during the inspiratory phase; the 'typical' rapid onset and decrementing discharge pattern was converted to a rapid onset, plateau-like or incrementing pattern with a late peak. Whether any of these effects are the result of afferent connections to the 'early-burst' neurons or via other respiratory neurons such as 'late-peak' inspiratory neurons is not known. (d) Projections and synaptic connections. In contrast to the scant indirect evidence for afferent pathways to the 'early-burst' inspiratory cells, the projections of these cells have been directly demonstrated using the antidromic mapping technique (Merrill, 1972b, 1974a, 1979, 1981). The 'early-burst' inspiratory neurons have rich medullary arboriz-

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FIG. 4. A sectional view of the medulla illustrating the axonal pathway for an 'early-burst'

inspiratory neuron.

ations, predominantly to the contralateral NRA inspiratory and expiratory regions, with the ipsilateral expiratory region of the NRA receiving a very sparse projection (Fig. 4). These cells do not appear to have projections to the inspiratory group in the vI-NTS, though Merrill (1979) qualifies that this issue has not yet been carefully examined using the antidromic mapping technique. Because antidromic activation from the spinal cord has so far been unsuccessful, it is assumed that the 'early-burst' axonal projections are confined to the medulla. Overall, the knowledge of their apparent small somal size, their lack of spinal projections, their extensive arborizations within the expiratory region of the NRA, and their distinctive discharge pattern led Merrill (1972a, b, 1974a, 1979) to conclude that the 'early-burst' inspiratory neurons of the r-NRA are interneurons which supply a potent inhibitory input to the expiratory neurons of the c-NRA. Since then, the results from intracellular studies of postsynaptic potentials in various respiratory neurons have suggested that the 'early-burst' inspiratory neurons may constitute a source of early° inspiratory synaptic inhibition not only to the expiratory c-NRA neurons, but also to the 'post-inspiratory' r-NRA interneurons and to some of the 'late-peak' inspiratory neurons (r-NRA and vl-NTS) (i.e. not all inspiratory cells with a late-peak discharge pattern receive an early inspiratory inhibition; see Section 2.1.1.2) (Richter and Ballantyne, 1981, 1983; Ballantyne and Richter, 1982, 1984, 1985; Richter, 1982a, b; Remmers et al., 1985a). This hypothesis of the 'early-burst' neuron's inhibitory role is based on the similarities between the time-intensity profiles of the early inspiratory inhibition observed in the expiratory c-NRA neurons, the post-inspiratory r-NRA neurons, and some of the 'late-peak' inspiratory neurons (r-NRA and vl-NTS) and, the time course and declining discharge of the 'early-burst' inspiratory interneurons (Ballantyne and Richter, 1984, 1985). Ballantyne and Richter (1985), however, point out that, while the similarity of the time-intensity profiles for both expiratory and 'post-inspiratory' neurons exists over a wide range of conditions (i.e. superior laryngeal nerve stimulation), the coincident similarity of the time-intensity profile for the 'late-peak' inspiratory neurons (r-NRA and vI-NTS) has only been confirmed for a limited range of conditions. In any event, the possibility of such common inhibitory input to the r-NRA (both the 'post-inspiratory' neurons and the 'late-peak' inspiratory neurons) and the c-NRA (expiratory neurons) is supported by the previous electrophysiological demonstration of the appropriate arborizations of the 'early-burst' cells; unfortunately, the possibility of such input to the 'late-peak' inspiratory neurons of the vI-NTS has not, to date, received any anatomical support for the presence of the appropriate arborizations, although this has not been carefully examined (Merrill, 1974a, 1979). To date, only one study has attempted to document the postulated inhibitory action

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of the 'early-burst' inspiratory neurons on other respiratory neurons. Using STA of post-synaptic noise, Remmers and colleagues (1985a) have shown a correlation between the 'early-burst' inspiratory neuron discharge and the membrane potential of 'postinspiratory' neurons. However, they suggest that, due to the large contribution of high frequency oscillations to the waveforms, the interpretation of monosynaptic inhibition from 'early-burst' inspiratory neurons to 'post-inspiratory' neurons cannot be made. Nonetheless, this study does verify the precise temporal relationship of the 'early-burst' inspiratory neuron discharge to the 'post-inspiratory' neuron membrane potential. [Note: although these investigators identified these 'early-burst' inspiratory neurons as belonging to the retrofacial nucleus, we have classified them within the r-NRA; see Section 2.2.1.2 (a) for details]. In support of the connection from 'early-burst' inspiratory neurons to the expiratory c-NRA neurons, Ballantyne and Richter (1985) have recently shown (using low intensity stimulation of the superior laryngeal nerve) that similar changes occur in the time-intensity profiles of the 'early-burst' inspiratory discharge pattern and the expiratory c-NRA neuron membrane potential pattern during the inspiratory phase; both convert from the typical rapid onset, decrementing pattern to a rapid onset, plateau-like or incrementing pattern. This coincident and parallel change in the expiratory c-NRA neuron inhibition is the type of behaviour which lends support to the contention that expiratory c-NRA neurons are inhibited by the 'early-burst' inspiratory neurons. In addition to these postulated inhibitory synaptic effects of the 'early-burst' inspiratory neurons on other medullary respiratory neurons, Bainton and colleagues 0985) have suggested that they are also responsible for the depression of sympathetic activity (recorded from cardiac and renal nerves) during early inspiration. This postulation is reinforced by the observation in one cat, that the early inspiratory depression of sympathetic activity was superimposed on an otherwise tonic pattern. The function of such early inspiratory modulation of sympathetic activity is not known at present. (e) Summary. The 'early-burst' inspiratory neurons, located in clusters amongst the 'late-peak' inspiratory neurons of the r-NRA, display a decrementing discharge pattern during the inspiratory phase. While the timing of the discharge onset of the 'early-burst' cell appears to be determined by an unknown extrinsic source, the discharge pattern is thought to be determined by excitatory interconnections with other 'early-burst' neurons and intrinsic membrane properties (accommodation). Their rich intrameduUary arborizations, as well as the similarity of the time-intensity profiles of their discharge pattern to that of the IPSPs in various respiratory neurons, suggest that the 'early-burst' inspiratory neurons function as propriobulbar inhibitory neurons with divergent outputs to the expiratory c-NRA neurons, the 'post-inspiratory' r-NRA neurons, and, at least, some of the 'late-peak' inspiratory neurons in the r-NRA and vl-NTS during the inspiratory phase (Merrill, 1974a; Ballantyne and Richter, 1984, 1985). Indeed, Ballantyne and Richter (1985) surmised that such shared distribution of the 'early-burst' inspiratory synaptic inhibition to the three respiratory neuron types "might reasonably be expected to exert some synchronizing, as well as a delaying, action on their discharge". Certainly the role for such inspiratory inhibition of the expiratory and 'post-inspiratory' neurons is to prevent their discharge and allow inspiration to proceed. The role for early inspiratory inhibition of some 'late-peak' inspiratory neurons by the 'early-burst' units is less clear; perhaps it is involved in the shaping of the depolarizing trajectory of the inspiratory output neurons from the medulla. 2.2.1.3. ' Post-inspiratory' neurons (a) Location and morphology. This population of bulbar respiratory neurons, first described by Richter and Ballantyne (1981), constitutes a moderately large group of cells, intermingled with inspiratory neurons predominantly in the region of the r-NRA (Ballantyne and Richter, 1982; Richter and Ballantyne, 1983; Remmers et al., 1985a, b). Assuming that the early-expiratory ('post-ramp') neurons recorded by Merrill and colleagues (1983) belonged to this 'post-inspiratory' population, it seems that these neurons, together with

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the 'early-burst' inspiratory neurons, are slightly ventral to the inspiratory neurons of the NRA. The cellular morphology of the 'post-inspiratory' group has not yet been reported in the literature. (b) Patterns of activity. The discharge of the 'post-inspiratory' neurons occurs only during the post-inspiratory period of the neural respiratory cycle (i.e. the period of declining discharge in the phrenic nerve). The onset of their discharge is marked by an early peak firing rate with a subsequent decline in activity (Richter and Ballantyne, 1981, 1983; Ballantyne and Richter, 1982; Richter, 1982a, b) and this time-intensity discharge profile closely parallels that of the phrenic motoneurons during the post-inspiratory period (Richter and Ballantyne, 1983; Remmers et aL, 1985b). Their declining discharge pattern has similarities to that of the 'early expiratory' neurons previously described by Feldman and Cohen (1978), but in contrast, the 'post-inspiratory' neurons do not continue to fire during the expiratory phase (i.e. during the augmenting discharge of expiratory neurons). Similarly, it is unlikely that the 'early expiratory' neurons identified in the vicinity of the retrofacial nucleus (Bianchi and Barillot, 1982) belong to this 'post-inspiratory' group. Unfortunately, no comparison of the time course of discharge of the 'early expiratory' neurons with that of the phrenic nerve could be made from the published data of Bianchi and Barillot (1982). Intracellular recordings have demonstrated that the 'post-inspiratory' neurons receive a prominent, decrementing pattern of IPSPs throughout the inspiratory phase; their membrane potential is often maximally hyperpolarized early in the phase (Richter and Ballantyne, 1981, 1983; Ballantyne and Richter, 1982, 1985; Richter, 1982a, b; Remmers et al., 1985a). The transition from the inspiratory to the post-inspiratory phase is marked by disinhibition (i.e. cessation of the inspiratory phase inhibition) together with a rapid depolarization from a level of membrane hyperpolarization, with an overshoot of discharge often occurring during the initial period of depolarization (Richter et al., 1985). Similar to the explanation for the overshoot seen in the 'ramp' inspiratory neurons, Richter and colleagues (1985) suggest that, with the rapid onset of EPSPs following the inspiratory hyperpolarization period, the A conductance (a voltage-dependent potassium conductance) is activated with a delay, thereby allowing the transient overshoot to occur before reducing the neuron's excitability [see Section 2.2.1.1 (b) for further details]. Furthermore, the behaviour of the membrane depolarization (i.e. decaying pattern) of the 'post-inspiratory' neuron during its active phase does not appear to be simply the result of excitatory synaptic activity, but also of a potassium current which is activated by a calcium influx during the early part of the post-inspiratory phase. This calcium conductance also appears to play a role in determining the discharge pattern of the 'early-burst' inspiratory neurons of the r-NRA. For the reasons previously discussed [see Section 2.2.1.2 (b) for details], such a current could result in a slow repolarization of the membrane potential and a declining pattern of discharge (Richter et al., 1985). The onset of the expiratory phase is marked by the cessation of the declining pattern of discharge coincident with the arrival of an augmenting pattern of IPSPs, similar to that received by the 'late-peak' inspiratory BS neurons and the 'early-burst' inspiratory neurons of the r-NRA (Richter et al., 1979a; BaUantyne and Richter, 1982, 1985; Richter, 1982a, b; Richter and Ballantyne, 1983). Overall, these intracellular results prompted Richter (1982b) to suggest that the 'post-inspiratory' neurons would be tonically active if rhythmic inspiratory and expiratory activities were to fail. (c) Afferents. Much of the information concerning afferent connections to the 'postinspiratory' neurons of the r-NRA has been obtained from measurements of their membrane potential in relation either to the activity of other medullary respiratory neurons or to nonphysiological (and, in one instance, physiological) stimulation of various nerve or brain stem regions associated with respiration. Two factors--the similarity of the timeintensity profile of the 'early-burst' inspiratory neurons' discharge to that of the IPSPs in 'post-inspiratory' neurons during the inspiratory phase and the impressive arborization of the 'early-burst' inspiratory axon teminals in the contralateral r-NRA (the location of the 'post-inspiratory' neurons) (Merrill, 1974a, 1979, 1981)--suggest that the 'early-burst'

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inspiratory neurons monosynaptically inhibit 'post-inspiratory' neurons during the inspiratory phase. While a recent study using the STA technique showed a high incidence of correlation between 'early-burst' inspiratory neuron spikes and the membrane potential of'post-inspiratory' neurons (Remmers et al., 1985a), these investigators submit that, due to the large contribution of high frequency oscillation to the waveform, the correlation cannot be interpreted as evidence of a monosynaptic inhibitory connection. Nonetheless, this study does document the precise temporal relationship of the 'early-burst' inspiratory action potentials to the 'post-inspiratory' membrane potential. [Note: Remmers et al. (1985a), identified the 'early-burst' inspiratory neurons as belonging to the retrofacial nucleus; due to the proximity of this nucleus to the r-NRA, we assume that these 'early-burst' inspiratory neurons are anatomically and functionally identical to those described by previous investigators (Merrill, 1974a, 1979, 1981: Bianchi, 1971, 1974; see Section 2.2.1.2 (a) for further details).] The remainder of information concerning synaptic inputs from other medullary respiratory neurons is quite indirect. The fact that the time course and augmenting pattern of the 'post-inspiratory' neuron's membrane potential matches that of the 'late-peak' expiratory neuron discharge in both the BOT and the c-NRA is suggestive of an inhibitory connection from medullary expiratory neurons to the 'post-inspiratory' neurons. Further support for the existence of this inhibitory synaptic connection between the 'postinspiratory' and expiratory neurons is the similarity of their responses to spontaneous or induced changes in the duration of the expiratory duration; for example, prolongation of the expiratory phase is associated with a similar prolongation of both the expiratory neuron discharge and the 'post-inspiratory' neuron inhibition (Ballantyne and Richter, 1982). Although no medullary collaterals have been demonstrated for the expiratory neurons of the c-NRA (Merrill, 1974a, 1979; Kalia, 1977; Kalia et al., 1981), the axonal projections of the BOT expiratory neurons to the r-NRA (Bianchi and Barillot, 1982; Fedorko and Merrill, 1984a) might conceivably be the source of the expiratory inhibition of the 'post-inspiratory' neurons (Ballantyne and Richter, 1985). In addition to the possibility of respiratory neuronal input to the 'post-inspiratory' neurons, the results of electrical stimulation experiments suggest that these neurons receive excitatory synaptic input from rostral pontine areas which are involved in inspiratory termination and expiratory prolongation, as well as from peripheral chemoreceptors (carotid sinus nerve), and laryngeal receptors (superior laryngeal nerve) (Richter, 1982a; Remmers et al., 1985b). While single shock stimulation of each of these sites evoked short latency compound EPSPs in 'post-inspiratory' neurons, tetanic stimulation at the onset of the post-inspiratory phase augmented 'post-inspiratory' neuronal discharge and lengthened the phase duration; the durations of the inspiratory and expiratory phases were not significantly changed from that of the control. Physiological stimuli such as subglottic pressure changes, insufflation of smoke into the upper airway, or application of water to the larynx produced similar effects (Remmers et al., 1985b). Hence, afferent inputs from the larynx as well as stimulation of the carotid sinus nerve and the rostral pons result in excitation of the 'post-inspiratory' cells. The possible influence of pulmonary vagal afferents on the 'post-inspiratory' neurons has not been as well examined. While lung stretch receptors do not appear to have a strong influence on these cells (Richter, 1982a), the unpublished data of D. Ballantyne, S. Mifflin, S. Backman and D. W. Richter (as cited in Ballantyne and Richter, 1985) led Ballantyne and Richter (1985) to suggest that stimulation of presumed 'irritant' laryngeal receptors results in excitation of the 'post-inspiratory' cells; however, this is based on two assumptions: (1) 'post-inspiratory' neurons are inhibitory to the expiratory neurons of the c-NRA; (2) changes in the time-intensity profile of IPSPs in the expiratory neurons of the c-NRA in response to 'irritant' laryngeal receptor stimulation are the result of change in the activity pattern of the 'post-inspiratory' neurons. Finally, from an anatomical viewpoint only, these 'post-inspiratory' neurons may be synaptically influenced by collaterals from the 'late-peak' inspiratory neurons of both the vI-NTS (mainly ipsilateral) and the r-NRA (bilateral) (Merrill, 1971, 1974a, 1975, 1979,

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1981; Loewy and Burton, 1978). However, the dissimilarity of the time-intensity profile of the discharge of the 'late-peak' inspiratory neurons (both vl-NTS and r-NRA) to that of the decrementing IPSPs in 'post-inspiratory' neurons during the inspiratory phase is not supportive of such a synaptic connection. (d) Projections and synaptic connections. The inability to antidromically activate 'postinspiratory' neurons from the contra- and ipsilateral vagus nerves (Remmers et al., 1985b) or from the spinal cord (Richter and BaUantyne, 1983; Remmers et al., 1985b) strongly suggests an absence of axonal projections to these peripheral sites. While these results lend support to the original contention (Ballantyne and Richter, 1982; Richter, 1982a, b) that the 'post-inspiratory' neurons are 'higher order' respiratory interneurons, the issue of their medullary axonal projections remains unresolved. Consequently, the following examination of possible 'post-inspiratory' axonal projections to and synaptic connections with various medullary respiratory neurons is indirect, based primarily on a comparison of the characteristics of the discharge patterns of the former with the postsynaptic membrane potentials of the latter. First, for the 'late-peak' inspiratory neurons (vl-NTS and r-NRA) which are active during the post-inspiratory phase, the similarity of both the time course and the declining pattern of EPSPs of these neurons during the post-inspiratory phase to that of the 'post-inspiratory' neurons (Richter and Ballantyne, 1981) suggests that the 'late-peak' inspiratory cells are synaptically excited by the 'post-inspiratory' neurons. Furthermore, the fact that the reflex responsiveness of the discharge pattern of the phrenic nerve closely parallels that of the 'post-inspiratory' neuron (Ballantyne and Richter, 1982) lends support to the contention that the 'post-inspiratory' neurons excite 'late-peak' inspiratory BS neurons (see Sections 2.1.1.4. and 2.2.1.1.(d) for further details). Likewise, the existence of a declining pattern of IPSPs in the 'early-burst' inspiratory neurons during the post-inspiratory phase is likely to be a consequence of the 'postinspiratory' neurons (Richter et al., 1979a; Richter, 1982b; Richter and Ballantyne, 1983; Ballantyne and Richter, 1985). Hence, the overall similarity of the time-intensity profiles of the 'known' medullary inspiratory neurons to that of the 'post-inspiratory' neurons suggests that the latter neurons play a role in determining the presence or absence of activity in the various medullary inspiratory neuron populations during the postinspiratory phase. Similarly, the 'post-inspiratory' neurons may play a role in the inhibition of the BS expiratory neurons of the c-NRA during the post-inspiratory phase. This is based on the close parallel between the time-intensity profile of the post-inspiratory IPSPs of the expiratory neurons and the abrupt onset, decrementing, pattern of discharge shown by the 'post-inspiratory' neurons (Ballantyne and Richter, 1985). Moreover, the synaptic actions of the 'post-inspiratory' neurons do not appear to be limited to these classically defined respiratory neurons. Rhythmic inhibition during the post-inspiratory phase has been demonstrated in otherwise tonically active neurons in the medullary reticular formation; the time-intensity profile of this inhibition closely paralleled that of the post-inspiratory activity (Richter, 1982a, b; Richter and Ballantyne, 1983). As the post-inspiratory activity in the phrenic nerve has been associated with a phasic inhibition of sympathetic activity (renal nerve) (Ballantyne and Richter, 1983; Bainton et al., 1985), Ballantyne and Richter (1983) have suggested that this post-inspiratory inhibition of reticular neurons may serve a more general function of eliminating excitatory inputs to respiratory neurons and possibly also to cardiovascular neurons. (e) Summary. The 'post-inspiratory' neurons appear to be a population of bulbar respiratory neurons which are intermingled amongst the inspiratory neurons of the r-NRA. Actively inhibited during both the inspiratory and expiratory phases, these neurons display a decrementing discharge pattern which parallels the time-intensity profile of phrenic nerve activity during the post-inspiratory phase. Examination of the timeintensity profiles of the IPSPs received during the other two respiratory phases suggests that the 'post-inspiratory' neurons are inhibited by the 'early-burst' inspiratory neurons of the r-NRA and the BOT expiratory neurons during the inspiratory and expiratory JP.N. 27/2--D

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phases, respectively. Likewise, during the post-inspiratory phase, examination of the time-intensity profiles of the postsynaptic patterns in various respiratory neurons suggests that the 'post-inspiratory' neurons are excitatory to, at least, some 'late-peak' inspiratory BS neurons of the vI-NTS and r-NRA, yet inhibitory to both the BS expiratory neurons of the c-NRA and the 'early-burst' inspiratory neurons of the r-NRA. While it is suggested that the 'post-inspiratory' neuron is the indirect source of the post-inspiratory activity in the phrenic nerve (i.e. via excitation of the 'late-peak' inspiratory BS neurons), the sources of excitation which result in the periodic firing of the 'post-inspiratory' neuron during its active phase are not known.

2.2.2. Caudal nucleus retroambigual& 2.2.2.1. 'Late-peak' expiratory neurons (a) Location and morphology. The 'late-peak' expiratory neurons of the c-NRA are concentrated caudal to the inspiratory neurons of the r-NRA, with some overlap of the two populations occurring at the level of the obex (Merrill, 1970, 1974a, 1979, 1981; Bianchi, 1971, 1974). Electrophysiological and histological studies (Merrill, 1970, 1974a, 1981) have further shown that the expiratory cells, located in the region of grey matter between the nucleus of the spinal tract of the trigeminal nerve and the lateral reticular nucleus, extend from approximately the level of the obex to the first cervical rootlets. Situated in a 500#m thick column in the ventrolateral medulla, the stereotaxic coordinates (lateral and depth) for localization of these expiratory cells are similar to that of the 'late-peak' inspiratory neurons of the r-NRA (Merrill, 1970, 1981) [see Section 2.2.1.1 (a)] (Fig. 5). Following Nissl staining, Merrill (1974a) described the expiratory neurons of the c-NRA as multipolar or fusiform in shape with somal diameters ranging from 15-35 #m (i.e. small to medium-sized). A subsequent morphological study of c-NRA expiratory cells (Kreuter et al., 1977), using Procion yellow, reported a range of minor and major somal diameters of 20 to 66 # m for expiratory BS units (n = 5) and 16 to 36/~m for expiratory NAA units (n = 2); the somata of the BS units were spindle-shaped. As Merrill (1974a) found that virtually all expiratory neurons of the c-NRA have spinal axons, there appears to be a discrepancy between the somal diameters reported by Merrill (1974a) and those of the BS units examined by Kreuter and colleagues (1977). Ironically, Merrill's expiratory BS cells had somal diameters similar to those of the two NAA expiratory units of the c-NRA (i.e. nonvagal; nonbulbospinal) studied by Kreuter and colleagues (1977). The latter investigators reported that the mean surface areas of the expiratory neurons of the c-NRA were 4029/tm 2 and 1933 #m 2 for the BS and NAA somata, respectively. The intercellular distances between stained BS neurons (including inspiratory and expiratory neurons of the NRA) and neighbouring cells of a similar size ranged between 30-200 # m (Kreuter et al., 1977). In one instance, an expiratory BS neuron of the c-NRA was shown to have a rich overlap of its dendritic tree with that of an inspiratory BS neuron of the r-NRA (12 #m dorsal to the expiratory neuron). Such dendritic overlap also existed between two expiratory neurons of the c-NRA (one BS cell and one NAA cell) with an inter-soma distance of 80 #m. Having 4--8 primary trunks per cell, the dendritic trees of the BS expiratory neurons spread mainly in the transverse plane, predominantly in ventrolateral and dorsomedial directions; they extend over a length of up to 400/~m and up to 200 #m for BS and NAA neurons, respectively. Because of the dendritic overlap of the respiratory neurons of the NRA and the similarities of their dendritic tree orientations (both expiratory and inspiratory neurons), the possibility exists that the activity of respiratory cells can be modified by extracellular field potentials, dendritic chemical synaptic interaction, and/or alteration in the extracellular ionic concentration as a consequence of the activation of a neighbouring neuron (Kreuter et al., 1977). Electrical synapses of the gap junction type do not appear to be present either in the c-NRA expiratory group or in the overlap region of the inspiratory and expiratory neurons (obex

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level) as suggested by the failure of Procion yellow to stain cells neighbouring the Procion yellow injected neurons (Kreuter et al., 1977). The main axons of the BS expiratory neurons of the c-NRA coursed dorsomedially (Fig. 5) and acquired a myelin sheath approximately 30-50#m away from the cell soma (Kreuter et al., 1977). The fact that no axon collaterals were observed for up to a stained axonal distance of 800 gm from NRA respiratory neurons (including the expiratory BS neurons) might suggest either that the Procion yellow technique is incapable of staining collaterals or that collaterals arise at considerable distances away from the cell body (Kreuter et al., 1977). However, Merrill's (1974a, 1979) inability to antidromicaUy identify axon collaterals for the BS expiratory neurons of the c-NRA strongly suggests the absence of any medullary collaterals (i.e. in 1979 Merrill stated that he was able to demonstrate collaterals for the other medullary respiratory cells with relative ease). Following iontophoretic injection of HRP into the phrenic nucleus (Rikard-Bell et al., 1984) and in the vicinity of both inspiratory and expiratory motoneuron activity in the upper (T3-T4) and lower (Ts-T9) thoracic segments (Rikard-Bell et al., 1985), labeled medullary neurons were concentrated in the region of the NRA (rostral and caudal to the obex); presumably some of these labeled cells belong to the BS expiratory population of the c-NRA as their axons course through both the cervical and thoracic spinal cord (Merrill, 1971). In particular, the presence of terminal arborizations of the expiratory axons within the intermediate and ventral horns of the thoracic cord (Merrill, 1971) suggests that the neurons of the c-NRA which were labeled following injection into the vicinity of thoracic respiratory motoneurons are, in fact, the BS expiratory cells. The somal diameters reported by Rikard-Bell and colleagues (1985) for the labeled neurons of the NRA (mean 29.5 #m, range 16-41.7 #m for lower thoracic injection; mean 28.9 #m, range 18.4-47.8 #m for upper thoracic injection) are within the range of those BS expiratory neurons studied by Merrill (1974a) but only within the lower range of those examined by Kreuter and colleagues (1977). Although Rikard-Bell and co-workers (1985) found that the labeled 'unidentified' cells of the NRA had only up to four primary dendrites, these dendrites did assume the dorsomedial and ventrolateral orientation previously described by Kreuter et al. (1977). Finally, these retrogradely labeled NRA cells were oval or round in the instance of lower thoracic injection, but were frequently triangular in shape following the upper thoracic injection. Based on the assumption that the labeled NRA cells belong to the respiratory BS group, it appears that the upper and lower thoracic respiratory motoneurons do not receive arborizations from the same individual neurons. Yet, Merrill (1974a) has shown that axons of individual NRA respiratory neurons arborize within as many as seven thoracic segments. Overall, while there are several similarities in the morphology of the expiratory BS neurons of the c-NRA (Merrill, 1974a; Kreuter et al., 1977) and that of the retrogradely labeled neurons of the NRA studied by Rikard-Bell and colleagues (1985), the data from the latter study is confounded by two issues. The labeled neurons, though likely to be respiratory neurons, were not physiologically identified by their discharge patterns. Secondly, even if these neurons were BS respiratory neurons of the NRA, the localization of the labeled cells within the vicinity of the obex (1 mm rostral and 1.5 mm caudal to the obex) would not ensure that any of the labeled cells were necessarily expiratory as opposed to inspiratory although the higher density of labeling in the caudal portion of the NRA suggests that they are more likely to be expiratory cells. (b) Patterns o f activity. The BS expiratory neurons of the c-NRA have an incrementing discharge frequency characterized by a gradual increase to a plateau or peak late in the expiratory phase (Merrill, 1974a, Bianchi, 1974); discharge never occurs at the very onset of the post-inspiratory phase (Richter, 1982b). The termination of discharge occurs synchronously throughout the expiratory population approximately 150 msec before the beginning of the inspiratory phase (as monitored by chest wall movement); overlap of firing between the expiratory neurons and the 'late-peak' inspiratory neurons at the transition from the expiratory to inspiratory phase is slight (Merrill, 1974a). Merrill (1974a) suggested that the expiratory population is functionally uniform, with the

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variation in discharge rates between cells accounted for by a wide distribution of 'threshold'. A more recent study of their discharge pattern (Taylor et al., 1978), however, reported that, while these expiratory cells exhibit peak-firing frequencies dependent on their recruitment time, the overlap of their onset and offset times does not support the concept of recruitment on purely a threshold basis. The characteristic burst of action potentials arises from a pattern of augmenting EPSPs occurring throughout the expiratory phase; in contrast to the 'late-peak' inspiratory neurons of the vl-NTS and r-NRA, no IPSP interaction is evident during the active phase of the expiratory cells (Hildebrandt, 1974; Mitchell and Herbert, 1974b; Richter et al., 1979a; Ballantyne and Richter, 1982, 1985; Richter, 1982b). During the inspiratory phase, these neurons are strongly inhibited (Mitchell and Herbert, 1971, 1974b; Merrill, 1974b; Richter et al., 1975, 1979a; Ballantyne and Richter, 1985), however, the shape of the inspiratory wave of IPSPs is strongly dependent on the trajectory of the phrenic nerve inspiratory activity (Ballantyne and Richter, 1985). The pattern of the synaptic inputs to the c-NRA expiratory neurons during the inspiratory phase is linked to the pattern of synaptic events present in the expiratory neurons during the subsequent post-inspiratory phase (Ballantyne and Richter, 1985). For example, in the instance of a smoothly rising pattern of phrenic discharge, the expiratory neurons receive a rapid onset, decrementing pattern of IPSPs with maximum hyperpolarization occurring early in the inspiratory phase and ceasing (i.e. disinhibition) coincident with the end of the inspiratory phase, and during the subsequent postinspiratory phase, the expiratory neurons are subject to a weak, decrementing pattern of IPSPs. Only when the post-inspiratory phase comes to an end are the expiratory neurons synaptically depolarized by the smoothly rising pattern of EPSPs during the expiratory phase (Ballantyne and Richter, 1985). However, if the smoothly rising pattern of phrenic nerve activity is converted to a steeply rising one, then the decrementing inspiratory inhibition of the expiratory cells changes to a rapid onset, plateau-like or augmenting pattern with a late peak intensity. During the subsequent post-inspiratory phase, the large increase in the intensity and, usually, the time course of the phrenic nerve activity is paralleled by a similar change in the magnitude and duration of the IPSPs of the BS expiratory neurons of the c-NRA. The onset of the expiratory neuron's synaptic depolarization, however, still remains coincident with the cessation of both the postinspiratory phase and its associated post-inspiratory inhibition. Previous to these observations that the BS expiratory neurons are subject to synaptic inhibition during the post-inspiratory phase (Ballantyne and Richter, 1985), Richter's laboratory (Ballantyne and Richter, 1982; Richter, 1982b; Richter and Ballantyne, 1983) had suggested that, particularly in the presence of prolonged post-inspiratory activity in the phrenic nerve, the expiratory neurons of the c-NRA were 'disfacilitated' by the 'post-inspiratory' neurons (i.e. antecedent gating of the excitatory input) thereby delaying the activation of the BS expiratory neurons and, consequently, the onset of the expiratory phase for a given cycle. However, it now appears that the BS expiratory neurons of the c-NRA are inhibited throughout both the inspiratory and the post-inspiratory phases. In addition to these patterns of postsynaptic potentials, it appears likely that these expiratory neurons are influenced by a calcium activated potassium conductance, similar to that described for the BS 'late-peak' inspiratory neurons of the r-NRA [see Section 2.2.1.1 (b) for details]. During all phases of the respiratory cycle, this conductance is responsible for a lowering of the input resistance of these neurons (Mifflin et al., 1985). Furthermore, the late afterhyperpolarizations and the 'post-tetanic hyperpolarizations' observed following a spontaneous action potential and a burst of action potentials (elicited by a depolarizing current pulse), respectively, are, at least in part, a consequence of a potassium conductance activated by an increase in intracellular calcium. Whether modulation of this conductance is critical to the regulation of the discharge behaviour of the expiratory neurons of the c-NRA is not yet known. (c) Functional interrelations. Cross-correlation analysis of the 'late-peak' expiratory neurons of the c-NRA for near neighbouring pairs (approximately 250-350 #m apart,

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n = 22) (Graham and Duffin, 1981), as well as for neuronal pairs (n = 31) recorded by electrodes with tip separations from 0.1 to 1.0 mm (Merrill, 1978), showed no evidence for short-term synchronization of their discharge patterns. Although Feldman and colleagues (1980) reported considerable short-time scale correlation of near neighbouring expiratory cells (62%, n = 45 pairs), they utilized a single electrode recording technique which can give false positive cross-correlations. Hence, as the firing of these expiratory neurons of the c-NRA does not seem to exhibit short-term synchrony, then neither common synaptic afferent input nor interactions between neurons are likely to be responsible for the generation of their periodic discharge. This absence of neuronal interactions is further supported by the fact that antidromic activation of near neighbours does not appear to alter the probability of antidromic invasion of a tested respiratory neuron (Merrill, 1974b). Consequently, the source of the EPSPs that result in the periodic firing of the expiratory population of the c-NRA continues to remain in question. (d) Afferents. (i) Brain stem and spinal cord. Since Merrill (1970) first located these expiratory neurons within the c-NRA, progress in the identification of afferent connections to these neurons has been slow. Electrophysiological and neuroanatomical methods have identified extensive afferent pathways from other medullary respiratory neurons. Initial antidromic mapping studies (Merrill, 1972b, 1974a) established that the 'early-burst' inspiratory neurons of the r-NRA have extensive medullary axonal arborizations within the contralateral c-NRA and a very sparse projection to the ipsilateral c-NRA (Merrill, 1979a). Selective neuroanatomical examination of projections to the c-NRA (Kalia, Sommer, and Cohen, as cited by Kalia, 1981a) has subsequently confirmed the presence of such pathways from the ipsi- and contralateral r-NRA (ipsilateral being more prominent), presumably from the 'early-burst' cells. This projection together with the complementary nature of the time-intensity profiles of the postsynaptic potentials during the inspiratory phase strongly suggests that the 'early-burst' cells are the source of the IPSPs in these expiratory cells (Merrill, 1972a, b, 1974a, 1979; Richter, 1982b; Ballantyne and Richter, 1985). Furthermore, the results of microstimulation, sagittal medullary lesions, and antidromic latency measurements (Merrill, 1972b, Bainton et al., 1978) support this inhibitory connection from 'early-burst' cells to expiratory neurons. Similarly, there appears to be an inhibitory connection from 'post-inspiratory' neurons in the r-NRA to the expiratory c-NRA group. This inference is based on the parallel between the time course and the declining pattern of discharge of the 'post-inspiratory' neurons and that of the IPSPs in the expiratory neurons (Ballantyne and Richter, 1985). The r-NRA location of this 'post-inspiratory' group (Ballantyne and Richter, 1982; Richter and Ballantyne, 1983; Remmers et al., 1985a, b) suggests that the neuroanatomically identified projections from the contra- and ipsilateral r-NRA (ipsilateral being more prominent) (Kalia, Sommer, and Cohen, as cited in Kalia, 198 la; Kalia et al., 1981) may represent the axonal projections of the 'post-inspiratory' neurons in addition to those of the 'early-burst' group. However, this has yet to be confirmed by an antidromic mapping study of the 'post-inspiratory' axonal pathways. Furthermore, the contralateral r-NRA projection to c-NRA may also be due to the presence of 'late-peak' inspiratory collaterals which have been electrophysiologically located within the c-NRA (Merrill, 1974a, 1979, 1981). In addition to a projection from the 'late-peak' inspiratory neurons of the r-NRA to the c-NRA expiratory region (mainly contralateral), the 'late-peak' inspiratory neurons of the vl-NTS has collaterals which project to this region bilaterally (ipsilateral being more prominent). However, the collateral arborizations within the expiratory region are not extensive for either the r-NRA or vl-NTS inspiratory cells (Merrill, 1974a, 1979, 1981). The existence of such projections from the 'late-peak' inspiratory units of the r-NRA and the vI-NTS has received neuroanatomical support (Bystrzycka, 1980; Kalia, Sommer and Cohen, as cited in Kalia, 1981a; Kalia et al., 1981) though the ipsilateral r-NRA and the contralateral vl-NTS projections were found to be more prominent than was shown by the

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antidromic mapping studies (Merrill, 1974a, 1979, 1981). While the function of these projections to the c-NRA expiratory regions is not known, the possibility of synaptic interconnection between the 'late-peak' inspiratory and expiratory cells is not supported by the results of cross-correlation analysis (Merrill, 1979); such correlations were made using the discharge overlap which occurs at the transition from inspiration to expiration and, for Ra cells, during their activation by lung inflation during the expiratory phase. Neuroanatomical and electrophysiological studies (Bystrzycka, 1980; Kalia et al., 1981; Bianchi and Barillot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a; Bianchi, 1985) have shown that the BOT complex 'late-peak' expiratory neurons project to both the ipsiand contralateral c-NRA, with axonal arborizations intermingled within the c-NRA expiratory neurons (Fedorko and Merrill, 1984a). Without substantiation, it has been suggested that the BOT expiratory neurons synaptically drive the expiratory neurons of the c-NRA (L. Fedorko, J. Lipski and E. G. Merrill, unpublished observations, as cited in Merrill, 1981). While the presence of appropriate collateral arbors together with the similarity of the time-intensity profile of their discharge patterns provide indirect support for the hypothesis that BOT expiratory neurons provide a source of common input to the c-NRA group, the failure of cross-correlation studies of neighbouring expiratory neurons to demonstrate short-term synchrony of discharge (Merrill, 1978; Graham and Duffin, 1981) does not support this hypothesis, at least for the near neighbouring expiratory neurons. Furthermore, a recent cross-correlation study (Hilaire et al., t984) failed to demonstrate any evidence for such synaptic connections from the BOT expiratory neurons to the c-NRA expiratory neurons though this absence of correlation may be due to other reasons (see Section 2.3.1.4 for details). Other more rostral regions, the ipsilateral and contralateral parabrachial nucleus of the pons (ipsilateral projection being more intense) (Bystrzycka, 1980; Kalia, Sommer and Cohen, as cited in Kalia, 1981a) and the ipsilateral Kolliker-Fuse nucleus (Bystrzycka, 1980) also project to the c-NRA expiratory region. Furthermore, electrical stimulation in the rostral pons (nucleus parabrachialis medialis and locus coeruleus) in the rabbit (Schmid et al., 1985) resulted in orthodromic activation and, in a few instances, orthodromic inhibition of the c-NRA expiratory neurons. However, such neuroanatomical and electrophysiological results have not identified whether these afferent projections actually originate from respiratory pontine neurons. As for the vl-NTS and the r-NRA regions, Bianchi and St. John (1982) demonstrated that a few respiratory neurons (mostly phase-spanning discharge type) in the areas of the nucleus parabrachialis medialis and Kolliker-Fuse nucleus project to the c-NRA. However, it is not known if such pontine afferents have synaptic connections with medullary respiratory neurons. In particular, a recent analysis of pontine-medullary respiratory neuron interactions, using the crosscorrelation technique, suggests there are few synaptic interactions, particularly for phasic expiratory and inspiratory neurons of the pons and medulla (i.e. most of the positive correlations occurred between tonically active respiratory neurons) (Lindsey et al., 1985). Other brainstem afferent projections to the c-NRA region arise from the contralateral nucleus paragigantocellularis dorsalis (weak) (Bystrzycka, 1980), the ipsi- and contralateral nucleus paragigantocellularis lateralis (Bystrzycka, 1980), the contralateral ventral part of the nucleus reticularis pontis oralis (Bystrzycka, 1980), and the reticular formation between the vl-NTS and the r-NRA (Kalia et al., 1981), as demonstrated by neuroanatomical methods; the identity of the neurons which make these projections is not known. In contrast to the responses in the r-NRA inspiratory neurons evoked by phrenic nerve (central cut end) stimulation, the discharge of expiratory c-NRA neurons was not modified by such perturbations, although the phrenic nerve afferents do appear to project to the c-NRA region, at least as suggested by the occurrence of evoked responses to such phrenic afferent activation (Macron et al., 1985; Speck et al., 1985). Stimulation of intercostal and abdominal muscle afferents, however, does appear to inhibit the activity of some expiratory c-NRA neurons though it is not apparent whether this is a direct action of the proprioceptor afferents or secondary to the inhibitory action of these afferents on the inspiratory neurons of the r-NRA and vI-NTS (Shannon and Freeman, 1981).

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(ii) Cranial nerves and central chemoreceptors. Similar to the r-NRA region, experimental demonstration of afferent inputs to the c-NRA from the upper and lower airway receptors as well as the peripheral chemoreceptors has been hampered by the failure of neuroanatomical studies to identify projections of either the vagal (Cottle, 1964; Kalia and Mesulam, 1980a, b; Ciriello et al., 1981b; Kalia et al., 1981; Donoghue et al., 1982) or the peripheral chemoreceptor (Cottle, 1964; Berger, 1979a; Kalia and Welles, 1980; Ciriello and Calaresu, 1981; CirieUo et al., 1981a, b; Davies and Kalia, 1981) afferents to this region of the medulla, although a small proportion of chemoreceptor fibres have been reported to project ventrolaterally as far as the NRA region (Davies and Edwards, 1975; Davies and Kalia, 1981). Functionally, however, there is indirect evidence that the c-NRA expiratory neuron discharge is influenced by pulmonary vagal receptors and chemoreceptors (peripheral and/or central). Observations of the discharge of these expiratory neurons during the application of phasic or maintained (Bianchi and Barillot, 1975; Bowden, 1978) lung inflation in the expiratory phase suggests they are excited by slowly adapting pulmonary stretch receptor input but inhibited by rapidly adapting pulmonary receptor input; identification of the type of receptor activated was based on the presence or absence of a hyperinflation gasp response. While other investigators (Batsel, 1965; Cohen et al., 1982) have reported that phasic lung inflations delivered during the expiratory phase usually caused an inhibition of neuron discharge (i.e. transitory reduction in frequency), this inhibitory effect cannot be related to the activation of a specific type of pulmonary receptor as the size of the inflation and the presence/absence of a gasp response were not reported. During a maintained expiratory inflation whereby exhalation is prevented at the end of the preceding inspiration, small increases above the experimental functional residual capacity produced either no change (Cohen et al., 1982) or an increase (Bianchi and Barillot, 1975; Bowden, 1978; Cohen et al., 1982) in expiratory neuron discharge frequency while larger increases resulted in a reversal of this effect (i.e. change from excitation to inhibition). Although interpretation of such modulation of expiratory neuron activity in relation to the activation of a specific type of pulmonary receptor is complicated by the marked overlap between the thresholds of individual slowly adapting stretch and rapidly adapting pulmonary receptors (Knowlton and Larrabee, 1946), from the present results it seems likely that expiratory neurons are excited by slowly adapting pulmonary stretch receptors (low maintained volumes) and inhibited by rapidly adapting pulmonary receptors (large maintained volume), as previously suggested by Bowden (1978). That this maintained expiratory inflation resulted in a lengthening of the discharge duration with a monotonic relation between the size of the inflation and the duration of the discharge burst suggests that this prolongation of expiratory neuron discharge is secondary to a delayed inspiratory onset (see Lipski et al., 1984) and not a result of pulmonary vagal afferent influence. How these effects on both the duration and frequency of expiratory neuron discharge are mediated is presently not known, though the apparent absence of pulmonary vagal projections to the c-NRA region does not support a direct connection. With the demonstrations of monosynaptic excitatory connections between slowly adapting pulmonary stretch receptors and the Ra inspiratory neurons of the vI-NTS (Averill et al., 1984; Backman et al., 1984), as well as projections from Ra neurons to the c-NRA region (Merrill, 1979a, 1981), it appears that any lung volume induced alterations of the c-NRA expiratory neuron excitability during the inspiratory phase might be mediated via the vI-NTS inspiratory neurons. This view is supported by the demonstration that withholding of lung inflation during the inspiratory phase resulted in an increased delay in the onset of discharge, though the ensuing burst of the neuron was similar to that in the control phase, reaching about the same maximum frequency at the end of the expiratory phase (note: withholding inflation resulted in a prolongation of the expiratory phase) (Cohen et al., 1982). However, to date, direct examination of the excitability changes of these expiratory neurons during the controlled manipulation of lung volume during their silent phase (i.e. inspiration) has not been made.

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Excitation of these expiratory neurons by activation of the peripheral chemoreceptors has been reported (Trzebski and Peterson, 1964; Koepchen et al., 1973; Davies and Edwards, 1975; St. John and Wang, 1977; Bainton and Kirkwood, 1979; St. John, 1981; Lipski et aL, 1984). However, Lipski and colleagues (1984) found that identical chemoreceptor stimuli (close arterial injection of CO2 equilibrated saline) produced three different effects--excitation (primarily), inhibition, and no response--in different expiratory neurons. While the cells displaying no alteration in their discharge frequency were observed to have a prolongation of the discharge burst, the latter cannot be interpreted as an excitation but, rather, as a secondary effect due to delayed expiratory termination (Lipski et aL, 1984). Overall, these results led Lipski and colleagues (1984) to conclude that the population of c-NRA expiratory neurons is nonhomogeneous with respect to their responsiveness to carotid chemoreceptor input. A similar examination of the response of these neurons to peripheral, carotid chemoreceptor stimulation (pharmacologic activation) (St. John, 1981) also demonstrated the three effects described by Lipski and colleagues (1984), with an excitatory effect predominating. St. John's laboratory (St. John and Wang, 1977; St. John, 1981; St. John and Bianchi, 1985) concluded that the varying effects are due to a limited and unequal distribution of excitatory peripheral chemoreceptor afferent inputs among the expiratory c-NRA neuron population. Further to this, the fact that discharge activity did not increase for a greater proportion of expiratory neurons during normocapnic hypoxic peripheral chemoreceptor stimulation (St. John and Wang, 1977) as compared to pharmacologic stimulation (St. John, 1981) led them to suggest that, in hypoxia, discharge activity will only increase in those neurons receiving sufficient excitatory peripheral chemoreceptor input to overcome the direct depression of neuronal activity by brainstem hypoxia. While it is likely that interneurons, as yet unidentified, are involved in the mediation of the excitatory response, a direct monosynaptic excitatory response cannot be excluded for at least some of these neurons, since a small proportion of the chemoreceptor afferent fibres have been found to project ventrolaterally as far as the NRA (Davies and Edwards, 1973, 1975; Davies and Kalia, 1981). Comparison of the effects of stimuli acting primarily on the peripheral chemoreceptors (pharmacologic activation) and central chemoreceptors (hyperoxic hypercapnia) on the discharge of the c-NRA expiratory cells during equivalent elevations of ventilatory activity (i.e. peak integrated phrenic nerve activity) has shown that the increase in discharge was of greater magnitude during the central chemoreceptor stimulation (St. John, 1981). Furthermore, in contrast to the three different responses of individual cells to peripheral chemoreceptor stimulation, all cells were excited by activation of the central chemoreceptors. These results suggest that, similar to the 'late-peak' inspiratory neurons of the vl-NTS and r-NRA, the c-NRA expiratory cells receive an equally distributed input from the central chemoreceptors. Further examination of the effect of hyperoxic hypercapnia (i.e. central chemoreceptor stimulation) has shown that these cells are subject to a tonic carbon dioxide dependent excitation (Bainton and Kirkwood, 1979). This graded effect of carbon dioxide on their tonic discharge activity progressively increases as carbon dioxide (i.e. chemoreceptor drive) is increased over its physiological range. Within that range, the tonic activity gradually transforms to rhythmic respiratory activity via the onset of periodic synaptic inhibition. These observations are supported by the results of intracellular recordings of these cells during hypocapnia as well as over the physiological range of carbon dioxide levels (Mitchell, 1977). Moreover, the latter study showed, that as the carbon dioxide level increased, the hyperpolarization during inspiration increased, and the discharge frequency during expiration increased even though the peak depolarization at end-expiration was not significantly altered. Whether such excitatory input from the central chemoreceptors results from monosynaptic interaction with the central chemoreceptors (as yet unidentified), or by direct, nonsynaptic chemical action (i.e. carbon dioxide or hydrogen ion), has not been resolved, though the absence of short-term synchronization of the discharge of neighbouring expiratory cells (Merrill, 1978; Graham and Duffin, 1981) does

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not support the possibility of a distributed synaptic input. While the source of inhibition of these cells during the inspiratory phase also remains unresolved, the level of inhibition does appear to be directly related to the chemical drive. According to Mitchell (1977), the mechanism underlying the increase in the discharge frequency of the expiratory cells during increased chemical drive (i.e., hypercapnia) is the rate of depolarization during expiration, and not the absolute magnitude of depolarization. In turn, he suggested that the magnitude of hyperpolarization during inspiration determines the rate of depolarization. More recently, from observations of the variations of antidromic latencies of the c-NRA expiratory cells during normocapnic hyperoxia as compared to hypercapnia or hypoxia, St. John and Bianchi (1985) have drawn a similar conclusion; that is, that the discharge frequencies of these cells are primarily determined by the overall change of membrane potential during the respiratory cycle (for further details, see Section 2.1.1.4). The latter study also suggested that this was the primary determinant for the discharge frequencies of the 'late-peak' inspiratory neurons of the vl-NTS and r-NRA. Finally, some investigators (Folgering and Smolders, 1979; Baker et al., 1979) have found the c-NRA expiratory cells to be less responsive to increases in chemical drive than are the medullary inspiratory neurons. While the results from St. John's laboratory (St. John and Wang, 1977; St. John, 1981) for the respiratory neuron response to hyperoxic hypercapnia and carotid chemoreceptor stimulation are generally in agreement with those of the study by Folgering and Smolders (1979), the results from St. John's laboratory did not confirm Folgering and Smolders' finding that expiratory neurons respond less to changes in chemical drive. St. John (1981) suggests that this difference "most probably reflects the different data treatment of the various studies". However, still unexplained is the finding that transient hypoxia and hypercapnia sufficient to increase ventilation do not increase the discharge rate of c-NRA expiratory neurons (Baker et al., 1979). Hence, whether chemic~' drive has differential effects on the augmentation of discharge of medullary inspiratory and expiratory neuron populations remains unresolved. (e) Projections and synaptic connections. Evidence concerning the axonal projections of the c-NRA expiratory neurons has been obtained primarily as a result of neurophysiological studies (i.e. Nakayama and Baumgarten, 1964; Hukuhara et al., 1968; Merrill, 1970, 1971, 1972c, 1974a, 1979; Bianchi, 1971, 1974; Newsom Davis and Plum, 1972; Hukuhara, 1973; Richter et al., 1975; Kreuter et al., 1977; Merrill and Fedorko, 1984). Despite intensive investigation using antidromic activation, no medullary axon collaterals have been demonstrated for these neurons (Merrill, 1971, 1974a, 1979, 1981; Merrill et al., 1983; Lipski et al., 1984). Two further tests, using STA of extracellular expiratory activity in the vI-NTS and of postsynaptic potentials in vI-NTS inspiratory neurons, were also unsuccessful in demonstrating any projections of the expiratory group to the ipsi- and contralateral vl-NTS region (Merrill et al., 1983). The failure to demonstrate short-term synchronization of discharge for pairs of contralateral c-NRA expiratory neurons (0%, n = 97 pairs) (Feldman et al., 1980) is in agreement with the antidromic mapping results. Recent neuroanatomical studies (Kalia, 1977; Kreuter et al., 1977; Kalia, Sommer and Cohen, as cited in Kalia, 1981a; Kalia et al., 1981) seem to confirm this absence of medullary collaterals. While these negative results do not preclude the existence of collaterals shorter than 0.5 mm (Merrill, 1974a; Mulloney and Selverston, 1972), neither cross-correlation (Merrill, 1978; Graham and Duffin, 1981) nor antidromic testing of neighbouring expiratory neurons (Merrill, 1971) have yielded results in support of 'short axon collateral' synaptic interconnections between cells in the c-NRA. Virtually all of the c-NRA expiratory neurons have projections to the contralateral spinal cord (Merrill, 1970, 1971, 1972c, 1974a, 1979, 1981; but see Bianchi, 1971, 1974). In fact,~for a large sample size (n = 240), Merrill (1974a) successfully activated more than 95% of these cells from the spinal cord. Intensive antidromic mapping (Fig. 5) indicates that the decussation of their axons takes a dorso-medial course, crossing the midline near the central canal, slightly rostral to their cell bodies; the crossings take place between 1 mm caudal to the obex and the C2 segment (Merrill, 1971, 1974a). As they course laterally, the

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// FIG. 5. A sectional view of the medullaand spinal cord illustrating the axonal pathway for a 'late-peak' expiratory neuron of the c-NRA.

axons abruptly turn ventral, descending into the spinal cord in the ventral column slightly ventral to the tip of the ventral horn. At C3, the expiratory axons form a discrete tract at approximately two thirds the distance from the tip of the ventral horn to the ventral surface. In the lower cervical cord, however, this tract gradually becomes diffuse, so that by the C8 segment, expiratory axons can be found throughout the ventrolateral white matter, intermixed with the inspiratory BS axons (Merrill, 1974a). No axon collaterals of these neurons at the level of the phrenic nucleus have been demonstrated by antidromic mapping (Merrill, 1971, 1974a). In the thoracic (T3-TI2) and upper lumbar (L1-L3) levels, these axons arborize extensively in the intermediate and ventral gray matter, contralateral to the cell body (Merrill, 1971, 1974a; Merrill and Lipski, personal communication). Arborizations from individual axons occupied several adjacent segments and, in fact, the occurrence of such axonal arborization over the length of the exposed cord (i.e. 6-8 segments) suggests widespread arborizations for many individual expiratory axons (Merrill, 1971, 1974a, 1979). Recently, Merrill and Lipski (personal communication) found some expiratory axons to have arbors at both T 5 and L2-L 3 levels. The existence of such terminal arbors along much of the length of the thoracic and upper lumbar cord does not suggest a somatotopic projection for these expiratory cells. Furthermore, the ability to antidromically activate expiratory cells at all levels of the c-NRA from L~, just lateral to the ventral horn tip, suggests that no particular region in the c-NRA appears to be concerned with lower thoracic/upper lumbar cord projection (Merrill and Lipski, personal communication). However, Merrill and Lipski (personal communciation) caution that these observations do not preclude the existence of a slight preference of axons from one region of the c-NRA to project to particular spinal segmental levels or, perhaps, restricted synaptic interactions at only some of the segmental levels in which the axons arborize. While the data from neuroanatomical studies (Kuypers and Maisky, 1977; Kalia, 1977; Kalia et al., 1981; Holstege and Kuypers, 1982; Feldman and Speck, 1983b; Rikard-Bell et al., 1984, 1985; Feldman et al., 1985) support the existence of spinal axonal projections from the c-NRA expiratory region, such axons do not appear to be solely contralateral. For example, the data from retrograde transport of HRP from the phrenic nucleus (Rikard-Bell et al., 1984) and the region of the thoracic respiratory motoneurons (Rikard-Bell et al., 1985) suggest that the axonal projections from the NRA respiratory region were approximately 35% ipsilateral and 65% contralateral (relative to cell soma). As this estimate includes both the inspiratory and expiratory NRA regions, the ipsilateral labeling could be due to both the inspiratory axon collaterals which cross the midline of the spinal cord (Merrill, 1971, 1974a) and the inspiratory cells which project to the ipsilateral cord (only 5-10% according to Merrill, 1971, 1974a). Furthermore, such

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neuroanatomical studies do not establish that the labeled cells belong, in fact, to the NRA respiratory group. It is interesting to note, however, that the thoracic ventral horn projection from the c-NRA region appears stronger than that from the r-NRA and the vl-NTS regions (Rikard-Bell et al., 1985). More specific neuroanatomical examination of the expiratory c-NRA neurons has been provided by an autoradiographic study (Feldman and Speck, 1983b; Feldman et al., 1985) whereby tritiated amino acids were injected into the middle of the caudal expiratory group as identified by both electrophysiological mapping and subsequent anatomical identification. Although the projections for only two cats were studied, the results suggest that the axons descend in both the lateral and ventral funiculi of the upper cervical cord, bilaterally. In agreement with Merrill's work, at the mid to lower cervical levels (C3-C6), the axons form a discrete tract below the ventral horn but eventually disperse throughout the ventrolateral white matter in the thoracic and upper lumbar cord. The axonal projections beyond C6 are, however, restricted to primarily the contralateral side (Feldman and Speck, 1983b; Feldman et al., 1985). The occurrence of bilateral labeling in the ventral horns of the C4~C6 segments in the vicinity of the phrenic nucleus, as well as in the ventral horns of the thoracic and lumbar segments (Feldman et al., 1985), demonstrates the existence of axonal arbors and possible terminal synaptic fields for the c-NRA cells. Electrophysiological studies (Merrill, 1971, 1974a), however, have failed to demonstrate such axonal arbors in the phrenic nucleus. As ipsilateral axonal projections were not demonstrated beyond the C6 segment, the ipsilateral ventral horn label in the thoracic and lumbar segments is presumed to be the result of collaterals which cross the midline of the spinal cord though Merrill (1971, 1974a) failed to demonstrate such arborizations on both sides of the cord. The extent of arborization within the ventral horn of the contralateral cervical segments appeared to be greater than that of the ispilateral counterpart but similar to that of the contralateral thoracic segments (Feldman et al., 1985). Though this recent neuroanatomical study (Feldman et al., 1985) could produce false positive results by the labeling of nonexpiratory neurons in regions adjacent to the physiologically identified c-NRA expiratory cells, the numerous similarities of these axonal projections and arborizations with those previously identified electrophysiologically by Merrill (1971, 1974a) strongly suggest that the labeled projections belong to the c-NRA expiratory group. However, the neuroanatomical identification (Feldman et al., 1985) of ipsilateral axonal projections (at least, within the cervical cord); axonal arborizations within the phrenic nucleus vicinity; and ipsilateral arborizations in the ventral horn of thoracic segments for c-NRA neurons awaits physiological verification that these identified projections/arborizations belong to expiratory cells of the c-NRA. In general, the results of both electrophysiological and neuroanatomical studies appear to confirm the contralateral projections to and extensive arborizations within the intermediate and ventral horns of the thoracic and upper lumbar cord for the expiratory c-NRA neurons. The neuroanatomically identified axonal arbors within the phrenic nucleus bilaterally (Feldman et al., 1985) have not been electrophysiologically identified as belonging to expiratory neurons of the c-NRA (Merrill, 1971, 1974a). Moreover, the failure of the STA technique to reveal either mono- or disynaptic connection of these expiratory cells with phrenic motoneurons (Merrill, 1981; Merrill and Fedorko, 1984) is consistent with this apparent absence of terminal arborizations within the phrenic nucleus. While such negative STA results do not necessarily rule out c-NRA expiratory neuronal synaptic inhibition of phrenic motoneurons (i.e. at least 30% are actively inhibited during expiration; Berger, 1979b) via polysynaptic pathways (Watt et al., 1976), the necessary expiratory populations of interneurons having similar discharge patterns and projecting to or located in the cervical cord have not been identified (Merrill and Fedorko, 1984). It appears, therefore, that c-NRA neurons do not synaptically act on the phrenic motoneurons. The earliest examination of the functional significance of the c-NRA expiratory arborizations in the thoracic and upper lumbar grey matter involved two methods: (1)

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sagittal medullary lesion (C~ to 1 mm caudal to the obex) to selectively interrupt descending expiratory axons and (2) microstimulation among c-NRA expiratory neurons (Merrill, 1972c). The former perturbation resulted in immediate cessation of all activity in both internal intercostal alpha motoneurons and activity in the internal intercostal muscles (plus most of the respiratory component in rhythmically active internal intercostal gamma motoneurons), while the latter method evoked activity in the contralateral thoracic expiratory motoneurons (stimulus applied during expiration) but suppressed activity in inspiratory intercostal motoneurons (stimulus applied during expiration). These results suggest that c-NRA expiratory neurons are a source of excitatory drive to expiratory intercostal motoneurons and inhibitory drive to inspiratory intercostal motoneurons, during the expiratory phase. Direct evidence for monosynaptic excitatory connections to internal intercostal motoneurons (Ts) from expiratory c-NRA neurons has been provided by Kirkwood and Sears (1973), using the STA technique. However, they neither identified their sample size nor gave estimates of the frequency of occurrence of this connection. Utilizing the same technique, Lipski and Merrill (1983) found that such monosynaptic connections between expiratory cells and contralateral expiratory intercostal motoneurons (Ts-T 6 and T8-T 9 segments) are rare (less than 1%, n --- 57 c-NRA cells) despite the presence of collateral arbors from the trigger neurons as well as good intercostal nerve spontaneous activity for the appropriate motoneuron segment (Merrill and Lipski, personal communication). In the instance of the two monosynaptic connections between expiratory cells and internal intercostal motoneurons, further STA of either near neighbouring c-NRA cells with the same internal intercostal motoneuron or the same c-NRA cell with a neighbouring internal intercostal motoneuron produced negative results, thereby supporting the absence of a gross somatotopic projection from c-NRA expiratory cells to the thoracic cord (Merrill and Lipski, personal communication). The expiratory axons of the c-NRA cells also do not appear to make monosynaptic connections with the contralateral external intercostal motoneurons (Ts-T6, T8-Tg), as demonstrated by STA (Lipski and Merrill, 1983). This negative result suggests that these expiratory neurons do not act to synaptically inhibit inspiratory motoneurons during the expiratory phase, at least not via mono- or disynaptic pathways. These STA results are supported by recent cross-correlation analyses (Sears et al., 1985) of the discharges of expiratory cells with those of the contralateral intercostal (T2-T 9 segments) filaments and of the phrenic nerve; these results were suggestive of few monosynaptic connections (i.e. less than 1% n = 80 cells). Despite the earlier suggestion by these same investigators (Kirkwood et al., 1982) that intercostal motoneuron synchronization over 2 to 3 adjacent segments results from synaptic connections with axon collaterals from descending medullary respiratory drives, they concluded that the majority of excitation of expiratory intercostal motoneurons is via interneurons which transmit the respiratory drive over several adjacent segments. Yet, while the STA data from Lipski and Merrill (1983) do not support the existence of a disynaptic pathway, an oligosynaptic pathway cannot be ruled out. As previously discussed [see for further details Section 2.2.1. l.(e)], interneurons which might be involved in such a di- or oligosynaptic pathway have been located dorsal and slightly medial to the intercostal motor nuclei (Merrill and Lipski, personal communication). In addition to segmental interneurons, propriospinal interneurons might also act as interposed interneurons in the pathway to the intercostal motoneurons, however, no other population of expiratory interneurons has, as yet, been identified in the spinal cord. Conduction velocities for the c-NRA expiratory BS axons have been estimated either from the onset of EPSPs in internal intercostal motoneurons via STA or by the 'singlepoint' antidromic activation method. Using the former method, Kirkwood and Sears (1973) reported conduction velocities ranging between 35 and 93 m/sec (sample size unidentified). For a mixed population of NRA BS neurons (n = 40; 50% were expiratory cells), 'singlepoint' antidromic activation from the C2-C4 spinal level resulted in an average conduction velocity estimate of 55.7 + 12.5 m/sec (S.D.) (Richter et al., 1975). In contrast, a sample of

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30 N R A respiratory BS neurons of which only four were expiratory cells demonstrated a considerably lower average conduction velocity [34.4 _ 3.01 m/sec (S.E.)] when calculated from 'single-point' antidromic activation at the C2-C3 spinal level (Bianchi and St. John, 1981). Recently, Merrill and Lipski (personal communication) have made 'single-point' antidromic activation determinations of conduction velocity at varying levels throughout the cervical, thoracic and lumbar cord. Allowing for a mean soma invasion time of 0.3msec, they reported the following mean conduction velocities: 36m/sec (range 10.8-66.6 m/sec, C4); 42 m/sec (range 5.6--40 m/sec, T2-T3); 29 m/sec (range 4.1-30.8 m/sec, Ts); and 29m/sec (range 2.0-16m/sec, L2-L3). However, utilization of their data to determine mean conduction velocity via 'two-point' antidromic activation results in considerably larger values. For example, expiratory axons (n = 8) examined at two cervical levels demonstrated mean conduction velocities from 31 to 105 m/sec (mean, 63.6 m/sec), as calculated from the differenceg in the two antidromic latencies (Merrill and Lipski, personal communication). These values should reflect the conduction velocities of their main axons as expiratory axons appear to arborize very little, rostral to T3 (Merrill, 1971, 1974a; Merrill and Lipski, personal communication; but see Feldman et al., 1985). In contrast, the decrease in the average conduction velocities (from single point determinations) from 42 m/sec at T2-T 3 to 29 m/sec at both T5 and L2-L3 is probably a result of slowing of conduction due to extensive axonal arborization (Merrill and Lipski, personal communication). Based on the recent demonstration (Dick and Berger, 1985) that two-point determinations of axonal conduction velocity are most accurate, it appears that Merrill and Lipski's 'two-point' antidromic activation estimates (personal communication) which ranged from 31 to 105 m/sec (mean, 63.6 m/sec) are the most reliable values available for the c-NRA expiratory BS axons, at least within the cervical cord. Efferent projections from the c-NRA region to more rostral regions have only recently been demonstrated. Following injection of HRP into the nucleus parabrachialis medialis and Kolliker-Fuse nucleus, Kalia (1977) observed labeling in both the ipsi- and contralateral c-NRA (ipsilateral projection was more prominent). In contrast, neuroanatomical examination of the medullary projections to the nucleus parabrachialis (King, 1980) demonstrated that cells near but not in the c-NRA (i.e. lateral tegmental field cells) provide such afferent pathways. A subsequent electrophysiological study (Bianchi and St. John, 1981) demonstrated few bulbopontile expiratory neurons in the c-NRA (i.e. 9%, n = 45); none of these cells could be antidromically activated from the spinal cord. Within the c-NRA region, however, some phase-spanning neurons could be antidromically activated from the rostral pons. Recently, Schmid and colleagues (1985) found similar results during electrical stimulation of the nucleus parabrachialis and the locus coeruleus in rabbits; however, none of the tested BS expiratory neurons could be antidromically activated from these nuclei. Overall, the failure to antidromically activate BS expiratory neurons (n = 4) from the rostral pons, taken with Merrill's antidromic mapping results (1971, 1974a), strongly suggests these c-NRA cells have neither medullary nor pontile axon collaterals. Finally, this apparent absence of pontine projections is further supported by the demonstration that short-time scale correlations for pairs of c-NRA-pontile respiratory neurons occurred primarily for tonically active respiratory neurons (Lindsey et al., 1985). (f) Summary. The c-NRA contains a population of 'late-peak' expiratory neurons which have axonal projections to the contralateral cervical, thoracic and upper lumbar spinal cord; no brainstem collaterals have been identified. While the discharge of these neurons is influenced by lung volume, chemical and respiratory muscle proprioceptor inputs, the mechanisms responsible for the mediation of such expiratory neuronal responses have not been identified. These expiratory cells are chemically driven by central CO2 throughout the respiratory cycle, and their silent periods during the inspiratory and post-inspiratory phases have been attributed to synaptic inhibition from the 'early-burst' inspiratory neurons and the 'post-inspiratory' neurons, respectively, though this has not been electrophysiologically demonstrated.

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2.3. BOTZINGER COMPLEX RESPIRATORY NEURONS 2.3.1. ' L a t e - p e a k ' expiratory neurons 2.3.1.1. Location and morphology Recently, the existence of a bilateral population of predominantly expiratory neurons at the most rostral extreme of the N A - N R A has been shown by both neuroanatomical (Kalia et al., 1979; Bystrzycka, 1980) and electrophysiological (Lipski and Merrill, 1980) studies. Identified as the Botzinger complex (Lipski and Merrill, 1980; Kalia, 1981b; Merrill, 1981), it is situated approximately 4-5 mm rostral to the obex, 3-3.5 mm lateral to the midline, and at a depth of 4~.5 mm below the dorsal surface (Fig. 6) (Lipski and Merrill, 1980; Bianchi and Barillot, 1982; Merrill et al., 1983; Fedorko and Merrill, 1984a; Lipski et al., 1984; Hilaire et al., 1984). Histological techniques have located this complex of expiratory neurons in proximity to the medial border of the retrofacial nucleus, close to the ponto-medullary junction (Kalia et al., 1979; Bystrzycka, 1980; Lipski and Merrill, 1980; Bianchi and Barillot, 1982; Merrill et al., 1983; Fedorko and Merrill, 1984a). This anatomical association with the retrofacial nucleus has prompted some investigators (Bianchi and Barillot, 1982; Hilaire et al., 1984; Bianchi, 1985; Remmers et al., 1985a) to refer to this complex as the retrofacial respiratory group. Although the morphology of these expiratory neurons, to date, has not received direct attention, the neuroanatomical study of Kalia and co-workers (1979) has identified a dense cluster of large neurons (soma diameter greater than 50/~m), multipolar in shape, in the vicinity of the retrofacial nucleus. As these neurons were retrogradely labeled following deposit of HRP in the inspiratory region of the contralateral vI-NTS, it is not known if the labeled neurons were, in fact, expiratory neurons. However, the subsequent electrophysiological demonstration that BOT expiratory neurons make monosynaptic inhibitory connections with the inspiratory BS neurons of the vl-NTS (Merrill et al., 1983) strongly suggests that the above morphological description does apply to BOT expiratory neurons. 2.3.1.2. Patterns o f activity Extracellular recordings of the BOT expiratory neurons (Lipski and Merrill, 1980; Bianchi and Barillot, 1982; Richter, 1982b; Merrill et al., 1983; Fedorko and Merrill, 1984a; Lipski et al., 1984; Merrill and Fedorko, 1984; Bianchi 1985; Remmers et al., 1985a) have demonstrated an augmenting discharge pattern with the occurrence of fairly high peak firing rates (up to 127 Hz) late in the expiratory phase. Discharge begins at or soon after the termination of phrenic nerve activity and ceases abruptly, coincident with the initiation of activity in the earliest medullary inspiratory neurons (Merrill et al., 1983; Lipski et al., 1984). This discharge pattern is similar to that of the 'late-peak' expiratory BS neurons of the c-NRA (Merrill, 1970, 1974a; Lipski and Merrill, 1981; Richter, 1982b; Merrill and Fedorko, 1984). However, in the absence of any intracellular studies of the BOT expiratory neurons, it is not known whether their postsynaptic potentials are also similar to those of the expiratory neurons in the c-NRA. In addition to these 'late-peak' expiratory neurons, other types of respiratory neurons have been recorded in the vicinity of the BOT. In barbiturate-anaesthetized cats, Merrill's group (Lipski and Merrill, 1980; Merrill et al., 1983) recorded inspiratory, early burst inspiratory, early expiratory (post-ramp), and phase-spanning neurons at locations somewhat dorsal to the locus of BOT expiratory activity. Similarly, other investigators, using decerbrate preparations, have recorded inspiratory neurons with an augmenting discharge pattern (Bianchi and Barillot, 1982; Hilaire et al., 1984; Bianchi, 1985; Remmers et al., 1985a); early burst inspiratory neurons with a decrementing discharge pattern (Bianchi and Barillot, 1982; Bianchi, 1985; Remmers et al., 1985a); early expiratory neurons with a decrementing discharge pattern (Bianchi and Barillot, 1982); and phasespanning neurons (Bianchi and Barillot, 1982; Hilaire et al., 1984; Bianchi, 1985) in the vicinity of the retrofacial nucleus. Bianchi's group (Bianchi and Barillot, 1982; Bianchi, 1985) have therefore suggested

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that, in addition to the BOT expiratory neurons, and with the exception of the early burst inspiratory neurons, these other types of respiratory neurons should be classified as retrofacial neurons. However, since the BOT complex is a rostral extension of the N A - N R A (Merrill, 1981; Merrill et al., 1983; Bystrzcka, 1980; Kalia et al., 1979), many of the additional types of respiratory neurons could have been recorded from the rostral pole of the NA-NRA. The discharge patterns are very similar to those of the N A - N R A (Merrill, 1970, 1974a, 1979; Bianchi, 1974; Taylor et al., 1978) and many of these neurons, could be antidromically activated from either the vagus nerve or the contralateral NRA and/or the contralateral spinal cord (Bianchi and Barillot, 1982; Bianchi, 1985). Furthermore, Merrill and colleagues (Merrill et al., 1983; Fedorko and Merrill, 1984a) report very few neurons with inspiratory or phase spanning patterns intermingled amongst the BOT expiratory neurons in the barbiturate-anaesthetized spontaneously breathing cat, and while Bianchi (1985) suggests that this latter finding can be explained by differences in the experimental techniques (Bianchi's group utilized decerebrate cats), the absence of such neurons in the spontaneously breathing preparation suggests that they are not critical to the genesis of respiratory rhythm. In consequence of these considerations and for the purposes of this review, the BOT complex will be assumed to consist solely of expiratory neurons. 2.3.1.3. Afferents To date, evidence for afferent connections to the BOT neurons is both limited and indirect. While axonal projections to the region of the retrofacial nucleus from other medullary respiratory nuclei have not been identified, this negative finding may be due to the fact that past antidromic mapping studies of the medullary collaterals of the respiratory neurons of the vI-NTS and N R A (Merrill, 1971, 1974a, 1975, 1979) did not test for the possibility of such collaterals because the expiratory activity in the BOT had not yet been discovered. However, as Merrill (1974a, 1979) was unable to identify any rostral medullary collaterals of the expiratory neurons of the c-NRA, a projection from these neurons to the BOT appears to be unlikely. A projection from various subnuclei in the tractus solitarius, including the ventrolateral subnucleus, to the retrofacial region has been demonstrated by neuroanatomical techniques (Loewy and Burton, 1978) but is is not known if these projections are from inspiratory neurons or nonrespiratory neurons in the vI-NTS. Evidence concerning the influence of pulmonary vagal afferents on these neurons is also indirect, having been obtained from observations of neuronal discharge during external manipulation of lung volume. During a maintained expiratory inflation whereby exhalation is prevented at the end of the inspiratory phase (Cohen et al., 1982), small increases in lung volume produced either no change or an increase in expiratory neuron discharge frequency while larger increases in volume produced a reversal of this effect (i.e. change from excitation to inhibition). That this maintained expiratory inflation resulted in a lengthening of the duration of the discharge burst with a monotonic relation between the size of the inflation and the duration of the discharge burst suggests that this prolongation of expiration is secondary to a delayed expiratory termination (see Lipski et al., 1984). In addition, it appears that brief lung inflations delivered during the expiratory phase have similar effects on neuron discharge frequency (Kubin and Lipski, 1980; Cohen et al., 1982) and duration (Cohen et al., 1982). Unfortunately, it is not possible to interpret such modulation of BOT expiratory neuron activity in relation to the activation of a specific type of pulmonary receptor (slowly adapting stretch vs rapidly adapting) since there is a marked overlap between the threshold of individual slowly adapting stretch and rapidly adapting receptors (Knowlton and Larrabee, 1946). Furthermore, while lung hyperinflations eliciting the gasp response (Head's paradoxical reflex) are associated with the activation of rapidly adapting pulmonary receptors (Knowlton and Larrabee, 1946; Larrabee and Knowlton, 1946; Sellick and Widdicombe, 1970), it is not known whether the large expiratory inflations applied by Cohen and associates (1982) elicited gasp responses. Finally, as the major

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central projections of the pulmonary vagal afferents and, in particular, lung slowly adapting stretch and rapidly adapting receptors terminate in the NTS (Kalia and Mesulam, 1980a, b; Ciriello et al., 1981b; Donoghue et al., 1982a; Kubin and Davies, 1984, 1985; Spyer, 1985), the mechanism which produces the BOT expiratory new ~nal responses to lung volume manipulation remains unknown. With the demonstrations of monosynaptic excitatory connections between slowly adapting pulmonary stretch receptors and the Ra inspiratory neurons of the vI-NTS (Averill et al., 1984; Backman et al., 1984) and of projections from neurons in the vI-NTS to the retrofacial nucleus region (Loewy and Burton, 1978), it appears that any lung volume induced alterations of the BOT expiratory neuron excitability during inspiration may be mediated via the NTS. The discharge of BOT expiratory neurons is also influenced by peripheral chemoreceptor stimulation (Kubin and Lipski, 1980; Lipski et al., 1984). Carotid chemoreceptor excitation (CO2) during the expiratory phase (close arterial injection of CO2 equilibrated saline) was associated with excitation (increased peak firing frequency) of approximately 45% (4/9) of the BOT expiratory neurons which were tested (Lipski et al., 1984). In addition, 3 of these 4 units projected to the contralateral vl-NTS. These results together with the previous demonstration of a monosynaptic inhibitory connection from BOT expiratory neurons to the R, and R~ inspiratory units of the vI-NTS (Merrill et al., 1983) provide an explanation for the reduced excitability of the R~ and R/~ inspiratory neurons during the application of brief chemoreceptor sitmuli in the expiratory phase (Lipski et al., 1977). As all of the tested BOT expiratory neurons were not excited by identical chemoreceptor stimuli applied during expiration, the BOT expiratory neurons seem to be heterogenous with respect to their afferent inputs (Lipski et al., 1984; for an analysis of their heterogeneity with respect to efferent projections see the next section). Finally, the fact that the chemoreceptor stimulation during expiration resulted in a prolongation of the discharge burst of all the tested neurons (n = 9) led Lipski and colleagues (1984) to interpret this, not as an excitation, but as a secondary effect due to a delayed expiratory termination. 2.3.1.4. Projections and synaptic connections Evidence for axonal projections from the 'late-peak' expiratory neurons of the BOT is considerable. Originally, neuroanatomical studies identified the existence of axonal projections from cells in the vicinity of the retrofacial nucleus to the contralateral (Kalia et al., 1979; Bystrzycka, 1980) and ipsilateral (Bystrzycka, 1980) inspiratory regions of the vI-NTS and to the contra- and ipsilateral r-NRA (Bystrzycka, 1980; Kalia et al., 1981). Following Lipski and Merrill's electrophysiological demonstration (1980) that expiratory neurons were the origins of these projections, the axonal projections and synaptic connections of the BOT expiratory neurons have been the subject of extensive investigation. Further antidromic mapping experiments have demonstrated that the BOT expiratory neurons project bilaterally to the vl-NTS (Bianchi and Barillot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a; Bianchi, 1985). While the studies from Bianchi's laboratory (Bianchi and Barillot, 1982; Bianchi, 1985) suggest that the contralateral projection is far more common, Fedorko and Merrill (1984a) report a similar percentage of projections to the contralateral and ipsilateral sides. However, the number of neurons tested in the latter study was small (n = 4) so that examination of a larger sample size will be necessary to resolve this issue. Data from Lipski and Merrill (1980) suggests that there is a point-topoint projection from particular parts of the BOT complex to a particular rostrocaudal level in the contralateral vl-NTS. Furthermore, the BOT expiratory neurons appear to have terminal arborizations within the inspiratory region of the NTS, as indicated by the antidromic mapping technique (Lipski and Merrill, 1980; Merrill et al., 1983; Fedorko and Merrill, 1984a). Using STA of intracellular potentials, Merrill and colleagues (1983) were successful in

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demonstrating monosynaptic inhibitory connections from BOT expiratory neurons to the R~ and Ra inspiratory neurons of the contralateral vI-NTS; it is likely that the projections to the ipsilateral vl-NTS are also inhibitory although this has not been demonstrated. Similarly, the bilateral projections from the retrofacial nucleus region to the N R A (Bystrzycka, 1980) have been shown to originate from the BOT expiratory neurons (Bianchi and Barillot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a; Bianchi, 1985); projections to the contra- and ipsilateral N R A seem to occur with similar frequency (Fedorko and Merrill, 1984a; Bianchi, 1985). More detailed antidromic mapping within the N R A has revealed the existence of axonal arborizations of the BOT expiratory neurons in both the inspiratory and expiratory regions (Bianchi and Barillot, 1982; Fedorko and Merrill, 1984a). While the significance of these axonal arborizations in the N R A has not yet been reported in the literature, Fedorko and Merrill (1984b), at the symposium entitled Neurogenesis of Central Respiratory Rhythm, reported that the BOT expiratory projections to the contralateral r-NRA are the source of monosynaptic inhibition to the inspiratory (presumably 'late-peak', BS) neurons of this nucleus during the expiratory phase. Furthermore, the preliminary observations of L. Fedorko, J. Lipski and E. G. Merrill (unpublished observations, as cited in Merrill, 1981) suggest that BOT expiratory neurons provide the augmenting excitatory input to the expiratory cells of the c-NRA; the presence of the appropriate collateral arbors within the c-NRA region together with the similarity of their discharge patterns provide indirect support for this hypothesis. However, a recent cross-correlation study (Hilaire et al., 1984) failed to demonstrate any evidence for such synaptic connections from the BOT expiratory neurons to the expiratory BS neurons of the c-NRA. Moreover, this absence of short term synchrony in their discharges suggests that a common synaptic input to the BOT and c-NRA expiratory neurons is not likely to be responsible for the similarity of their discharge patterns. While this absence of correlation between expiratory neurons of the BOT complex and c-NRA is supported in part by the failure of previous cross-correlation studies to demonstrate common synaptic inputs to the expiratory neurons of the c-NRA (Merrill, 1978; Feldman et al., 1980; Graham and Duffin, 1981), Hilaire et al.'s failure to demonstrate a correlation between BOT expiratory and c-NRA expiratory neurons may be due to other reasons. Firstly, the small sample size (n = 8 neuronal pairs) as well as the random selection of neuronal pairs may, on the basis of probability, account for the flat cross-correlograms. Secondly, the sample of BOT expiratory neurons under study may not have had the appropriate axon collaterals in the c-NRA. Thirdly, Hilaire and colleagues (1984) did not identify whether the eight BOT expiratory neurons had the appropriate, augmenting discharge pattern, since neurons with an early decreasing expiratory pattern have been recorded in the retrofacial region in a similar type of experimental preparation (Bianchi and Barillot, 1982). Hence, at present, the role for the BOT expiratory axonal projections bilaterally to the c-NRA remains unresolved. In addition to these medullary projections, the BOT expiratory neurons send axons to the cervical spinal cord, as identified by antidromic mapping studies (Bianchi and BariUot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a; Bianchi, 1985). A recent neuroanatomical study (Rikard-Bell et al., 1984), utilizing retrograde transport of H R P from the phrenic nucleus, appears to confirm this cervical projection. From a sample of 72 BOT expiratory neurons, Fedorko and Merrill (1984a) reported spinal projections (C~C 6 segments) in more than 72% of the tested neurons; projections contra- and ipsilateral to their cell somas appear to occur with equal frequency. As some neurons (n = 2) had bilateral projections to the cervical cord, Fedorko and Merrill (1984a) suggested that close to 100% of all BOT axons project to the cervical cord (for a mathematical analysis, see Fedorko and Merrill, 1984a, p. 491) and that their small sample size underestimates the number of bilateral projections. While the results from Bianchi's laboratory (Bianchi and Barillot, 1982; Bianchi, 1985) do confirm the presence of contralateral, ipsilateral and bilateral projections of BOT expiratory neurons to the cervical cord, the proportions of projections noted in their study are not in agreement with those of Fedorko and Merrill &P.N. 27/2--E

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(1984a). Bianchi's laboratory reports that only 40% of BOT expiratory neurons have spinal axons (Bianchi and Barillot, 1982) and that the contralateral projection is far more common than the ipsilateral one (Bianchi and Barillot, 1982; Bianchi, 1985). Both laboratories offer explanations for the differences in the results of these antidromic mapping studies (for details, see Fedorko and Merrill, 1984a; Bianchi, 1985). Utilizing the antidromic mapping technique, Fedorko and Merrill (1984a) localized the axons of the BOT expiratory neurons in the contra- and ipsilateral dorsal and medial parts of the lateral funiculus in the C4-C5 segments; the axons are scattered and do not form a discrete bundle comparable to the axons of the c-NRA expiratory neurons at the C3 segment and are generally found much more dorsal. Recently, Merrill's laboratory (Merrill, 1982b; Merrill and Fedorko, 1984), using the STA technique, have demonstrated a monosynaptic inhibitory connection from BOT expiratory neurons to contralateral phrenic motoneurons during the expiratory phase; synaptic connections from BOT expiratory neurons to ipsilateral phrenic motoneurons were not investigated. Furthermore, the findings that not all phrenic motoneurons are strongly inhibited during expiration (Berger, 1979b; Merrill and Fedorko, 1984) and that more than 30% of randomly chosen BOT expiratory neuron-phrenic motoneuron pairs (n = 37) demonstrated inhibitory connections suggest that the BOT expiratory neurons are responsible for much of the expiratory phase inhibition in phrenic motoneurons (Merrill and Fedorko, 1984). It is likely that the BOT expiratory axons make synaptic contact with the dendrites of phrenic motoneurons which extend beyond the phrenic nucleus (Cameron et al., 1983) as only a small percentage (25%) of the BOT spinal axons have arbors in the phrenic nucleus, at least at the C5 segment (Fedorko and Merrill, 1984a). Spinal axonal projections from the BOT to the upper thoracic cord (T~-T2 segments) appear to be rare (i.e. 2/19 neurons); in both cases, the axon was ipsilateral to the cell soma (Fedorko and Merrill, 1984a). This observation led Fedorko and Merrill (1984a) to suggest that most spinal axons of the BOT expiratory neurons end in the cervical cord, probably at the level of the phrenic nucleus. Overall, the extensive antidromic mapping studies (Bianchi and Barillot, 1982; Fedorko, 1982; Fedorko and Merrill, 1984a; Bianchi, 1985) have demonstrated that the majority of the BOT expiratory neurons have axonal branches in more than one medullary respiratory nucleus, with terminal arborizations in the vl-NTS and in both the inspiratory and expiratory regions of the NRA, as well as in the cervical spinal cord; such medullary and spinal branchings occur both contra- and ipsilateral to the cell soma (Fig. 6) (i.e. Fedorko and Merrill, 1984a, identified one cell which could be activated from four different places in the medulla and spinal cord). In addition, Bianchi's laboratory (Bianchi and Barillot, 1982; Bianchi, 1985) has identified a projection from a retrofacial expiratory neuron (n = l) to the ipsilateral rostral pons; however, the discharge pattern of this expiratory neuron was described as early decreasing. As Bianchi and Barillot (1982) found no projections to either the ipsilateral pons or the ipsilateral vagus nerve from retrofacial expiratory neurons (n--30) having an augmenting discharge, it seems that the BOT expiratory axonal projections are limited to the medulla and spinal cord, although a neuroanatomical study (Kalia, 1977, 1981a) has demonstrated bilateral efferent projections from the BOT region which terminate in the region of the pontine respiratory nuclei (i.e. nucleus parabrachialis medialis and Kolliker-Fuse nucleus). Typical conduction velocities for the axons of the BOT expiratory neurons range from 18.5 to 44.4 m/sec (mean 26.5 m/sec), as calculated from single-point antidromic activation from the cervical cord (Bianchi and Barillot, 1982). A similar range of axonal conduction velocities (8.7 to 48 m/sec) has been reported by Fedorko and Merrill (1984a), utilizing the same method at the C5 segment; these investigators noted that no corrections for stimulus utilization time or soma invasion times were made in the calculations. Although Bianchi and Barillot (1982) reported typical axonal conduction velocities for the medullary axons of the BOT expiratory neurons (2.4 to 19.4 m/sec; mean 8.32 m/sec), accurate conduction velocity estimates cannot be made for medullary branches for two reasons. First, stimulation in the medulla can result in antidromic activation in an arbor rather than in

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FIG. 6. A sectional view of the medulla and spinal cord illustratingthe axonal pathway for an expiratory neuron of the BOT complex.

the main axon (Bianchi and Barillot, 1982). Second, the antidromic latency of a medullary neuron is dominated by the i.s.-s.d, invasion time (approximately 250/~s) when activation occurs within the medulla (Fedorko and Merrill, 1984a). Consequently, the estimates from the spinal cord antidromic latencies are likely to be more accurate and suggest that the BOT expiratory neurons have large myelinated axons (Fedorko and Merrill, 1984a). 2.3.1.5. Summary The BOT, located medial to the retrofacial nucleus, contains a population of predominantly 'late-peak' expiratory neurons which have extensive axonal projections bilaterally to the vI-NTS, NRA, and the cervical cord. This neuronal complex has been shown to be a source of monosynaptic inhibition of the R~ and R e inspiratory neurons of the vI-NTS, the inspiratory neurons of the r-NRA, and the phrenic motoneurons, at least on the contralateral side, during the expiratory phase. Presuming that the BOT expiratory projections have similar inhibitory connections with the ipsilateral inspiratory medullary and cervical spinal nuclei, it appears that the BOT expiratory complex provides monosynaptic inhibition of all known inspiratory neuronal networks in the medulla and the lower cervical cord during the expiratory phase; the possibility of such a connection to the upper cervical inspiratory neurons has not yet been examined. This dual expiratory action on both the inspiratory pre-motor neurons and the phrenic motoneurons likely acts to improve the inhibition of the phrenic motor output to the diaphragm during the expiratory phase (Fedorko and Merrill, 1984a; Merrill and Fedorko, 1984). While the significance of the bilateral projections to the expiratory region of the c-NRA remains unknown, it has been suggested that the BOT expiratory neurons provide the patterned excitatory input for the caudal expiratory neurons. This hypothesis, together with the evidence for bilateral connections to the inspiratory neuron populations of the medulla, suggests that the bilateral projections of the BOT neurons within the medulla may play a role in synchronizing respiratory rhythm on both sides of the medulla (Fedorko and Merrill, 1984a). However, the origin of the periodic discharge patterns of the BOT expiratory neurons themselves is not known. 2.4. UPPER CERVICALRESPIRATORYNEURONS 2.4.1. Upper cervical inspiratory neurons 2.4.1.1. Location and morphology Subsequent to the initial demonstrations that rhythmic respiratory movements occur in spinalized animals (Coglianese et al., 1977; Aoki et al., 1978, 1980), Aoki and co-workers

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FIG.7. A sectionalviewof the medullaand spinalcordillustratingthe axonalpathwayfor an upper cervical inspiratoryneuron.

(Aoki et al., 1980, 1983a; Aoki, 1982) recorded inspiratory activity, both extra- and intracellularly, from neurons in the upper cervical cord in lightly anaesthetized, nonspinalized cats. Histologically, Aoki and colleagues (Aoki et al., 1980, 1983a; Aoki, 1982) located these neurons within the intermediate zone of the spinal grey matter of the Cj-C2 segments. More recently, Lipski and Duff]n (1986), using electrolytic lesioning and histology, confirmed their presence within the intermediate grey matter (corresponding to Rexed's lamina VII) (Rexed, 1954), with specific localization near its lateral border. In addition, some neurons were located (particularly in C2) in the adjacent lateral funiculus (Lipski and Duff]n, 1986) (see Fig. 7). That these extracellular recordings within the white matter were made from neurons and not passing axons is supported by several electrophysiological observations (i.e. stability of the recordings, spike amplitudes; see Lipski and Duff]n, 1986 for details). From a large sample size (n = 224) of inspiratory neuronal recordings, it appears that these upper cervical inspiratory neurons form bilateral columns extending from the caudal end of the NRA at the C1 segment to at least the rostral third of the C3 segment (Lipski and Duff]n, 1986). Using the dorsal root entry zones as surface markers, the columns are located at depths ranging from 1.5 to 2.3 mm below the dorsal surface. As a comparison, Aoki and colleagues (1980) encountered the inspiratory neurons in the Cj-C2 segments of the cord, 3-4 mm lateral to the midline, and 2.5-3.4 mm below the dorsal surface. The difference in the depth recordings between the two laboratories could possibly be explained by the weight ranges of the preparations; the cats utilized by the latter investigators were considerably larger (2.3-4.5 kg, Aoki et al., 1980; 1.6-2.4 kg, Lipski and Duff]n, 1986). Lipski and Duff]n suggest that this localization of the upper cervical inspiratory neurons may correspond with that of the nucleus ambiguus inferior, described by Winkler and Potter (1914). Morphologically, the only study of these neurons, to date, involved the retrograde transport of HRP following injection into the phrenic motor nucleus (segments C5-C6) (Aoki et al., 1984). The HRP-labeled neurons, found within the lateral border of the ipsilateral intermediate grey matter in the C I - C 2 segments, were multipolar and demonstrated a wide variation in shape; the largest diameter was 20-30/~m. However, while it remains unknown as to whether these retrogradely labeled neurons actually belong to the CI-C2 inspiratory neuron population, preliminary electrophysiological studies (Aoki, 1982; Aoki et al., 1983a, b, 1984; Lipski and Duff]n, 1986) have identified the appropriate axonal projections from the Ct-C2 inspiratory neurons to the lower cervical segments.

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2.4.1.2. Patterns o f activity Extracellular recordings of the upper cervical inspiratory neurons in anaesthetized, nonspinalized cats have demonstrated an augmenting firing pattern with the firing frequency accelerating rapidly at first and then more gradually during the inspiratory phase (Lipski and Duffin, 1986), with similar discharge patterns to those of the medullary (vI-NTS and r-NRA) 'late-peak' inspiratory neurons during simultaneous extracellular recordings (Aoki et al., 1983a, b). While their mean discharge frequency ranged from 17.8 to 35.5 (mean 26.8) impulses per second (Aoki et al., 1980; Aoki, 1982), their peak discharge frequency (27.6-66.7 impulses per second; mean 46.2) did not occur until the latter part of the burst (Aoki et al., 1980). An autocorrelation of their discharge (Lipski and Duflin, 1986) demonstrated that the regularity of discharge within the burst varies from very regular to nearly random, similar to that of the BS 'late-peak' inspiratory neurons (cf. Graham and Duffin, 1982; Long and Duffin, 1984). The number of spikes within a burst ranged between 21 and 62 (mean 39.8) (Aoki et al., 1980). While Lipski and Duffin (1986) reported that these neurons begin to discharge shortly before inspiratory activity begins in the T3 external intercostal nerve filament, Aoki and colleagues (Aoki, 1982; Aoki et al., 1980) found that some of these inspiratory neurons did not initiate their discharge until 10--70 msec after the onset of the inspiratory phase, monitored by an abdominal pneumograph. This variation in the initiation of discharge in the upper cervical inspiratory neurons relative to the onset of the inspiratory phase is most likely due to the monitoring techniques utilized in the two experiments. Termination of their discharge is abrupt and coincident with the cessation of external intercostal nerve activity (Lipski and Duffin, 1986). Intracellular recordings of these inspiratory neurons have been reported by Aoki et al. (1983a), but only in abstract form, so no information concerning the patterns of postsynaptic potentials occurring during the respiratory cycle is available at present.

2.4.1.3. Afferents To date, evidence for afferent connections to the upper cervical inspiratory neurons is limited and indirect. Aoki et al. (1983a, b) were able to orthodromically activate these neurons from the inspiratory regions of both the vl-NTS and the r-NRA; activations from contralateral nuclei were far more common than that from ipsilateral ones (i.e. vI-NTS: 72% contralateral vs 45% ipsilateral; r-NRA: 93% contralateral vs 54% ipsilateral). The orthodromic latencies (vl-NTS contralateral 4.0--15.0 msec, ipsilateral 5.0-7.0 msec; r-NRA contralateral 2.0-8.0 msec, ipsilateral 2.0-7.0 msec) reported by Aoki and coworkers (1983a, b) are considerably longer than the latencies for these medullary inspiratory neurons when antidromically activated from the C5-C6 level. For example, Fedorko and associates (1983) have reported antidromic latencies from C 5 of 1.5-1.9 msec and 1.2-5.7msec for the inspiratory neurons of the contralateral vl-NTS and r-NRA, respectively. Utilizing the same technique, Lipski and Duffin (1986) also observed synaptic excitation of CI-C 2 inspiratory neurons following stimulation in the contralateral vI-NTS; they did not attempt activation from any other medullary inspiratory nuclei. The latency of this synaptic excitation (1.3-2.0 msec) differs considerably from that reported by Aoki et ai. (1983a, b). In further support of this connection from the contralateral vl-NTS inspiratory neurons to the CI-C 2 inspiratory group, preliminary cross-correlations (n = 2; Duflin and Hoskin, unpublished observations) demonstrate peaks after an appropriate time delay for conduction and synaptic transmission of excitation. The response of the upper cervical inspiratory neurons to lung inflations from the ventilator in the paralyzed cat (Lipski and Dutfin, 1986), closely resembles that of some BS inspiratory neurons (vI-NTS R~ and r-NRA) as well as the phrenic and intercostal inspiratory discharges. Finally, the discharge of these cervical inspiratory neurons appears to be influenced by the carbon dioxide level (Lipski and Dutfin, 1986).

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2.4.1.4. Projections and synaptic connections Originally, Aoki and co-workers (Aoki, 1982; Aoki et al., 1983a, b, 1984) reported that most (92%) of the upper cervical inspiratory neurons could be antidromically activated from the ipsilateral C5~6 ventral funiculus, close to the ventral horn. Following a retrograde HRP study Aoki et al. (1984) suggested that these neurons project to the ipsilateral phrenic motor nucleus (C5~C6 segments). In contrast, Lipski and Duffin (1986), utilizing the antidromic mapping technique, localized the axons of the C~--C2 inspiratory neurons (87% of tested units) within the ipsilateral lateral funiculus at the C6-C 7 segments and reported only minimal collateral branching within the region of the ventral grey matter (11%) at C 5 (Fig. 7). Using the antidromic mapping technique, Lipski and Duffin (1986) also demonstrated that the upper cervical inspiratory neurons (85% of tested units) have axons in the ipsilateral funiculus of the upper thoracic segments (T3-Ts) (Fig. 7). Approximately two-thirds of thoracic level axons have collaterals within the grey matter of at least one thoracic segment and often within two or three adjacent segments as well; some collaterals cross the midline. The majority of the collaterals were found dorsal and medial to the intercostal motor nuclei. In addition, some C~-C2 inspiratory neurons project to, at least, the Tl0 segment (Lipski and Duffin, 1986). Conduction velocities for the axons of these upper cervical inspiratory neurons range from 25-45 m/sec, using the single stimulus site estimate of conduction velocity at C5-C 6 (Aoki et al., 1983a, b, 1984). Utilizing the same technique, Lipski and Duffin (1986) reported conduction velocities ranging from 5-70 m/sec; their distribution was unimodal, with a mean of 32 m/sec. The possibility of synaptic connections of the descending upper cervical inspiratory axons with the ipsilateral phrenic and intercostal motoneurons was investigated with the cross-correlation and intracellular STA techniques (Lipski and Duffin, 1986). No synaptic connections with ipsilateral phrenic motoneurons were demonstrated by either crosscorrelation of the inspiratory neuron discharges with the activity of the C5 phrenic root, or STA of the post-synaptic noise recorded intracellularly from phrenic motoneurons. Such a result indicates that synaptic connections (mono- and disynaptic) between C~-C2 inspiratory neurons and phrenic motoneurons are not frequent. Although collateral, branching of C~-C2 inspiratory axons within the phrenic nucleus may not be frequent, the extensive dendritic field of the phrenic motoneurons (Cameron et al., 1983; Lipski et al., 1985) allows the possibility of monosynaptic connections with phrenic motoneurons. Moreover, the identification of inspiratory interneurons dorsal to the phrenic nucleus (Baumgarten et al., 1963) gives support to possible disynaptic connections with phrenic motoneurons. As only a small sample of upper cervical inspiratory neurons (n = 9) were tested for such connections, the issue of synaptic connectivity with phrenic motoneurons requires further investigation. In contrast, the results of STA experiments with intercostal motoneurons together with those of the cross-correlation of Cj-C 2 inspiratory neuron activity with the discharge of intercostal nerve filaments are suggestive of an excitatory connection, probably disynaptic and mediated through interneurons. Such inspiratory interneurons have been observed (Merrill and Lipski, personal communication) in the thoracic ventral horn (Ts-T 6 or TT-T8), dorsal and slightly medial to the intercostal motor nuclei. With the localization of C~-C2 inspiratory axon collaterals in the same vicinity of the intercostal motor nuclei (Lipski and Duffin, 1986), the appropriate anatomical organization is available to allow for disynaptic connectivity between the upper cervical inspiratory neurons and the inspiratory intercostal motoneurons. 2.4.1.5. Summary The upper cervical inspiratory neurons have been located near the lateral border of the intermediate grey matter and the adjacent lateral funiculus. These propriospinal neurons have axonal projections in the ipsilateral lateral funiculus in both the cervical and thoracic

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cord (to at least Tt0); collateral branchings within the vicinity of the ipsilateral phrenic nucleus appear to be sparse but are extensive in the region of the intercostal nuclei. Electrophysiological studies suggest that the upper cervical inspiratory neurons are involved in the excitation of intercostal motoneurons, probably through disynaptic pathways involving segmental interneurons. Although no synaptic connections with phrenic motoneurons have been demonstrated, the possibility of di- or oligosynaptic connections requires further examination. It would appear that the upper cervical inspiratory neurons comprise a propriospinal system involved in the control of intercostal and, possibly, some phrenic motoneurons. In particular, these inspiratory neurons may serve as a relay of the inspiratory BS synaptic output (vl-NTS and r-NRA) perhaps together with other, as yet unidentified, supraspinal pathways and upper cervical afferent inputs. 3. Conclusion

The present understanding of the neuronal determinants of respiratory rhythm, based upon the experimental evidence cited in this review, can be summarized within the framework of the three defined phases of respiration (inspiratory, post-inspiratory, and expiratory). The inspiratory phase appears to be determined by at least two categories of neurons: the 'early-burst' inspiratory neurons of the r-NRA and the 'late-peak' inspiratory BS neurons of both the vl-NTS and the r-NRA. Present experimental results suggest that the 'early-burst' inspiratory neurons are inhibitory to at least some of the 'late-peak' inspiratory BS neurons, In addition, it has been proposed that two other categories of propriobulbar inspiratory neurons, the 'late-peak' and the 'ramp', exist in the r-NRA. The 'late-peak' inspiratory BS neurons (vI-NTS and r-NRA) have been identified as the pre-motor neurons, which provide the excitatory drive to the inspiratory alpha motoneurons. It appears that these pre-motor cells may also be a source of drive to the upper cervical inspiratory neurons. While the role of the latter cells has not yet been resolved, it has been suggested that they comprise a propriospinal system involved in the control of intercostal and, possibly, some phrenic motoneurons. Throughout the inspiratory phase of the cycle, all other identified respiratory neurons in the medulla, that is the 'post-inspiratory' neurons and the 'late-peak' expiratory BS neurons of both the c-NRA and the BOT, are synaptically inhibited. The 'early-burst' inspiratory neurons of the r-NRA are believed to be the source of inhibition for these 'post-inspiratory' and expiratory neurons. During the subsequent post-inspiratory phase, the 'late-peak' inspiratory BS neurons (vI-NTS and r-NRA) as well as the inspiratory alpha motoneurons display a declining pattern of discharge, and the 'post-inspiratory' neurons of the r-NRA are believed to be the source of this activity. Simultaneously, the 'late-peak' expiratory BS neurons (c-NRA and BOT) experience a delay in the onset of their augmenting discharge pattern. This delay is the result of synaptic inhibition for the c-NRA expiratory BS neurons, and the 'post-inspiratory' neurons are presumed to be the source of this inhibition. This aspect has yet to be studied for the BOT expiratory BS neurons. Finally, the absence of discharge of the 'early-burst' inspiratory neurons of the r-NRA is believed to be the result of interaction between intrinsic membrane properties and synaptic inhibition, and again the 'post-inspiratory' neurons of the r-NRA are believed to be responsible for the latter. The third and final phase of the cycle, the expiratory phase, is characterized by an augmenting discharge of the 'late-peak' expiratory BS neurons in both the c-NRA and the BOT. Throughout this phase, the 'late-peak' inspiratory BS neurons (vI-NTS and r-NRA), the 'early-burst' inspiratory neurons, and the 'post-inspiratory' neurons are actively inhibited. While the BOT expiratory neurons have been shown to be the source of the monosynaptic inhibition of the 'late-peak' inspiratory BS neurons (vl-NTS and r-NRA), the origin of the IPSPs in the other two categories of neurons remains unknown. At the spinal level, the expiratory alpha motoneurons receive excitatory input from the c-NRA expiratory neurons while the phrenic motoneurons are monosynaptically inhibited by the

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xl ~ ,,3k~ ,

]

: ....:~ :....... "-,.D .,............. ~ _1->'

F16. 8. A sectional view of the medulla and spinal cord combining all of the previous figures (2 7).

B O T e x p i r a t o r y neurons. A t the end o f this phase, the discharge o f the ' l a t e - p e a k ' e x p i r a t o r y n e u r o n s ( c - N R A a n d BOT) a b r u p t l y ceases c o n c o m i t a n t with the a b r u p t onset o f discharge o f the ' e a r l y - b u r s t ' i n s p i r a t o r y n e u r o n s a n d the r e s p i r a t o r y cycle repeats itself. A very limited i d e a o f the c o m p l e x i t y o f these n e u r o n a l n e t w o r k s involved in r e s p i r a t o r y r h y t h m generation, as discussed above, is given by Fig. 8 which is a c o m b i n a t i o n o f all the previous sectional views (Figs 1-7). Yet, despite the c o n s i d e r a b l e k n o w l e d g e gained in recent years, as cited in this review, an a c c e p t a b l e m o d e l o f r e s p i r a t o r y r h y t h m g e n e r a t i o n has n o t been f o r t h c o m i n g . T a b l e 1 shows why. It lists the m a i n types o f r e s p i r a t o r y n e u r o n s a n d the k n o w n , o r strongly inferred d e t e r m i n a n t s o f their activities with respect to r e s p i r a t o r y r h y t h m generation. As the table shows, t o o m a n y o f the possible interc o n n e c t i o n s r e m a i n u n k n o w n o r u n p r o v e n , for the p r o p o s a l o f o t h e r t h a n speculative models. The n e u r o n s with p e r h a p s the best u n d e r s t o o d firing p a t t e r n s are the c - N R A ' l a t e - p e a k ' e x p i r a t o r y neurons, whose r e s p i r a t o r y r h y t h m a p p a r e n t l y results from the ' e a r l y - b u r s t ' i n s p i r a t o r y i n h i b i t i o n o f a c o n s t a n t central CO2 drive. This simplistic model, o f a c o n s t a n t drive p e r i o d i c a l l y i n t e r r u p t e d , m a y well characterize the r e s p i r a t o r y r h y t h m g e n e r a t o r as a whole, since a central CO2 drive has been s h o w n to be essential for r h y t h m to occur, a n d m a n y r e s p i r a t o r y n e u r o n s are excited by elevated CO2. T h e a p p l i c a t i o n o f this k i n d o f m o d e l to o t h e r r e s p i r a t o r y n e u r o n s serves to p o i n t o u t the gaps in c u r r e n t knowledge. F o r example, the ' l a t e - p e a k ' i n s p i r a t o r y BS n e u r o n s o f the v l - N T S a n d r - N R A are driven by CO2, a n d inhibited d u r i n g the e x p i r a t o r y p e r i o d by the B O T ' l a t e - p e a k ' e x p i r a t o r y

TABLE 1.

From To r-NRA vI-NTS c-NRA BOT e-I p-I CO 2 r-NRA +? +? 0 -? -'~ + vI-NTS 0 +? 0 _ _? _o + c-NRA 0? 0? 0 +? - ? - ? + BOT 0? 0? 0 + e-I 0 +? +? -? +? p-I 0 _9 _? +? UCIN +? +? r-NRA = late-peak, BS, inspiratory neurons of the r-NRA; vI-NTS = late-peak, BS, inspiratory neurons of the vl-NTS; c-NRA = latepeak, BS, expiratory neurons of the c-NRA; BOT = expiratory, BS, neurons of the BOT; e-I = early-burst, propriobulbar neurons of the r-NRA; p-I = post-inspiratory, propriobulbar neurons of the r-NRA; UCIN = upper cervical inspiratory neurons; CO2 = central and peripheral chemical drive; + = excitatory connection; - = inhibitory connection; 0 = no connection; ? = unproven.

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neurons. However, these inspiratory neurons, critical parts of the respiratory oscillator, lack a convincing mechanism for the termination of their firing at the end of the inspiratory phase. Still less is understood about the control of the BOT expiratory neurons' firing patterns which provide such a widespread and powerful inhibition of inspiratory neurons during expiration; and little is known about the determinants of the firing patterns of the 'early-burst' inspiratory neurons in the r-NRA, except that their decrementing frequency during their burst may be due to intrinsic characteristics. While the post-inspiratory neurons appear to be driven by CO2 and inhibited during both inspiratory and expiratory phases, by 'early-burst' inspiratory neurons and BOT expiratory neurons, respectively, no confirmation for these connections has been found to date. The identification of the other propriobulbar neurons, the 'late-peak', and 'ramp' inspiratory types of the r-NRA, is too recent to allow definition of their roles in respiratory rhythm generation. Clearly, further experimentation may provide the details of connections among these respiratory neurons, and such knowledge should allow the construction of a model of respiratory rhythm generation which is based upon the properties of the network. Such a model will, however, still face the problem of constructing a slow oscillator from fast elements, and this suggests that information about the intrinsic properties of the respiratory neurons could be of crucial importance. The dynamics of cellular chemistry are more suited to the slow rhythm of respiration than the rapid events of electrophysiology. The slow changes in the extracellular ionic environment surrounding respiratory neurons, limited in rate of change by diffusion and uptake, may prove to be more than a secondary effect of activity and a partial determinant of that activity. Similarly, the intrinsic characteristics of respiratory neurons, such as accommodation in the 'early-burst' type may be crucial in determining the neuron's activity. Therefore, a combination of network connections and specific neuron characteristics is likely the key to explaining how the oscillator works. Another problem that a model must address is the coordination of populations of neurons. For example, the c-NRA expiratory neurons have similar patterns of firing in that they all fire during expiration with firing frequencies which increase over the burst and then halt at the start of inspiration. Such overall coordination might be due to interconnections among the population or to a homogeneous distribution of an excitatory drive such as that from the central chemoreceptors. In either case, short-term synchronization of firing within the population should result. Such synchronization cannot be detected. Similarly the synchronization among other populations of respiratory neurons, although detectable, is not as yet convincingly strong enough to explain the coordination of the population. Consequently, within the given framework of the known respiratory neurons, it appears that a workable model of respiratory rhythm generation must await further investigation of these basic unanswered questions. Moreover, it must be kept in mind that the neuronal determinants of respiratory rhythm may include other as yet unidentified neurons. Certainly the foresight of Merrill's suggestion (1981) that "perhaps... we have not yet identified all of the important elements of the medullary generator" has been evidenced by the more recent identifications of both the BOT 'late-peak' expiratory neurons and the 'post-inspiratory' neurons. Furthermore, the existence of nonspiking neurons with periodic membrane potential changes which are involved in various invertebrate rhythmic systems could have implications for the generation of respiratory rhythm. This latter possibility poses an investigative problem as neurons of this type would go undetected in extracellular recordings, and might be difficult to find using intracellular recording techniques. These aspects of respiratory rhythm generation are not new problems for the modeller; they have been considered for a long time, and continue to inspire investigative effort. It is our hope that this review has served to summarize the current state of knowledge and thereby aid in that effort.

Acknowledgements We are indebted to Seward Hung, medical artist for the illustrations, and to Jean Bilyk for the preparation of the manuscript.

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