Respiratory network function in the isolated brainstem-spinal cord of newborn rats

Progress in Neurobiology Vol. 59, pp. 583 to 634, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/99/$ - see front matter

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RESPIRATORY NETWORK FUNCTION IN THE ISOLATED BRAINSTEM-SPINAL CORD OF NEWBORN RATS KLAUS BALLANYI*, HIROSHI ONIMARU$} and IKUO HOMMA$ *II Physiologisches Institut, UniversitaÈt GoÈttingen, Humboldtallee 23, D-37073, GoÈttingen, Germany, and $Department of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142, Japan (Received 22 January 1999) AbstractÐThe in vitro brainstem-spinal cord preparation of newborn rats is an established model for the analysis of respiratory network functions. Respiratory activity is generated by interneurons, bilaterally distributed in the ventrolateral medulla. In particular non-NMDA type glutamate receptors constitute excitatory synaptic connectivity between respiratory neurons. Respiratory activity is modulated by a diversity of neuroactive substances such as serotonin, adenosine or norepinephrine. Clÿ-mediated IPSPs provide a characteristic pattern of membrane potential ¯uctuations and elevation of the interstitial concentration of (endogenous) GABA or glycine leads to hyperpolarisation-related suppression of respiratory activity. Respiratory rhythm is not blocked upon inhibition of IPSPs with bicuculline, strychnine and saclofen. This indicates that GABA- and glycine-mediated mutual synaptic inhibition is not crucial for in vitro respiratory activity. The primary oscillatory activity is generated by neurons of a respiratory rhythm generator. In these cells, a set of intrinsic conductances such as P-type Ca2+ channels, persistent Na+ channels and Gi/o protein-coupled K+ conductances mediates conditional bursting. The respiratory rhythm generator shapes the activity of an inspiratory pattern generator that provides the motor output recorded from cranial and spinal nerve rootlets in the preparation. Burst activity appears to be maintained by an excitatory drive due to tonic synaptic activity in concert with chemostimulation by H+. Evoked anoxia leads to a sustained decrease of respiratory frequency, related to K+ channel-mediated hyperpolarisation, whereas opiates or prostaglandins cause longlasting apnea due to a fall of cellular cAMP. The latter observations show that this in vitro model is also suited for analysis of clinically relevant disturbances of respiratory network function. # 1999 Elsevier Science Ltd. All rights reserved

CONTENTS 1. Introduction 2. Respiratory activity 2.1. Nerve recordings 2.2. Relation to eupneic breathing 3. Respiratory neurons 3.1. Classes 3.2. Morphology and projections 3.3. Rostrocaudal distribution 3.4. Connectivity 4. Cellular mechanisms of rhythmic activity 4.1. Neurotransmitters 4.1.1. Excitation 4.1.2. Inhibition 4.2. Second messengers 4.2.1. Ca 4.2.2. cAMP 4.2.3. H 4.3. Intrinsic conductances 4.3.1. K 4.3.2. Ca 4.3.3. Na 4.3.4. Conditional bursting 5. Respiratory pattern generator 5.1. `Noeud vital' 5.2. Rhythm generator

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} Corresponding author. Tel.: +81-3-3784-8113; fax: +81-3-3784-0200. 583

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5.2.1. RVL-Pre-I neuron hypothesis 5.2.2. pre-BoÈtC conditional burster hypothesis 5.3. Inspiratory pattern generator 5.4. Bilateral interactions 5.5. Developmental aspects 6. Modulation of rhythm 6.1. Biogenic amines 6.1.1. (Nor)adrenaline 6.1.2. Serotonin 6.2. Adenosine 6.3. Neuropeptides 6.3.1. Opiates 6.3.2. Substance P 6.3.3. TRH 6.4. Acetylcholine 6.5. Central chemosensitivity 7. Metabolic aspects 7.1. Response to hypoxia 7.2. Role of anaerobic metabolism References

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ABBREVIATIONS ACh AMPA ATP cAMP CVL DRG EAA EPSP Exp GABA 5-HT Hepes HVA Insp IPG IPSP

acetylcholine a-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid adenosine triphosphate cyclic adenosine monophosphate caudal ventrolateral medulla dorsal respiratory group excitatory amino acids excitatory postsynaptic potential expiratory neuron g-amino butyric acid serotonin N-2-hydroxyethlypiperazine-N'-2-ethanesulphonic acid high voltage-activated inspiratory neuron inspiratory pattern generator inhibitory postsynaptic potential

1. INTRODUCTION In mammals, utilisation of aerobic metabolism depends critically on e€ective uptake of O2 and concomitant removal of CO2. Such gas exchange is provided by ventilation of the lungs that is mediated by rhythmic variation of the thorax volume. The two ventilatory phases of inhalation and exhalation are controlled by the three neural phases of inspiration, post-inspiration and active expiration (Richter, 1982, 1996; Feldman, 1986; Bianchi et al., 1995). The distinct neural phases are revealed by monitoring extracellular activity of respiratory muscles like diaphragm or external and internal intercostal muscles, or of their nerves containing axons of rhythmically active motoneurons (Gesell and White, 1938; von Euler, 1986; Monteau and Hilaire, 1991). These motoneurons are activated by (bulbospinal) premotoneurons that receive synaptic input from interneurons of a bilaterally organised respiratory network within the lower brainstem. Respiratory active neurons are found in the nucleus KoÈlliker±Fuse and nucleus parabrachialis (pontine respiratory group, PRG), in the ventrolat-

IVA Kir LVA NMDA P Pre-I pre-BoÈtC PRG PGE PSP RRG RVL TEA TRH TTX VLM VRG

intermediate voltage-activated inwardly rectifying K+ channel low voltage-activated N-methyl-D-aspartate partial pressure pre-inspiratory neuron pre-BoÈtzinger Complex pontine respiratory group prostaglandin E postsynaptic potential respiratory rhythm generator rostral ventrolateral medulla tetraethylammonium thyrotropin-releasing hormone tetrodotoxin ventrolateral medulla ventral respiratory group.

eral aspect of the nucleus of the solitary tract (dorsal respiratory group, DRG), and in the ventral respiratory group (VRG) which is closely associated with the nucleus ambiguus (Cohen, 1979; Richter, 1982; Feldman, 1986; Long and Dun, 1986; Bianchi et al., 1995). Cross-correlation analysis in concert with intracellular recording has revealed that the threephase respiratory rhythm is due to a highly complex pattern of mutual synaptic interaction between di€erent classes of VRG neurons (Cohen, 1979; Richter, 1982, 1996; Long and Dun, 1986; Ezure, 1990; Bianchi et al., 1995). During the inspiratory, post-inspiratory and expiratory phase, each class of these neurons is subject to a characteristic pattern of either pronounced hyperpolarisation due to Clÿdependent inhibitory postsynaptic potentials (IPSPs) or shows spike discharge, elicited by summating excitatory postsynaptic potentials (EPSPs; Richter, 1982, 1996; Bianchi et al., 1995). Despite the potency of the respiratory network to operate autonomously, its activity is controlled by other central nervous structures such as cortex, hypothalamus or pons, and also by peripheral chemosensors for O2, CO2 and H+ of the carotid and

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aortic bodies (Eldridge and Millhorn, 1981; Feldman, 1986; Long and Dun, 1986). Functional adaptation of the respiratory network is furthermore mediated by mechanoreceptors that are, for example, responsible for the lung stretch (`Hering± Breuer') re¯ex (Cohen, 1975; Bonham and McCrimmon, 1990). This ongoing modulation by a multiplicity of excitatory and inhibitory a€erent inputs hampers the analysis of cellular mechanisms, responsible for generation and modulation of the primary respiratory rhythm in the intact animal. Accordingly, there is a demand for reduced in vitro brainstem preparations, in which functions of the center of the respiratory network can be studied (Suzue, 1984; Onimaru and Homma, 1987; Onimaru et al., 1988, 1997; Feldman and Smith, 1989; Feldman et al., 1991; Richter et al., 1992; Onimaru, 1995; Smith et al., 1995). On the basis of in vivo lesion experiments it was hypothesised that the DRG and VRG are not involved in the generation of respiratory rhythm (Speck and Feldman, 1982; Feldman, 1986). This view was proven after establishing an in vitro brainstem-spinal cord preparation from neonatal rats, in which respiratory network functions are retained (Suzue et al., 1983; Suzue, 1984). In this preparation which does not contain the pons and thus the PRG, respiratory activity remains una€ected after removal of the dorsal half of medulla by sectioning (Arata et al., 1990; Smith et al., 1991). Respiratory neurons in the brainstemspinal cord preparation are found in the ventrolateral medulla (VLM) which includes the classical VRG around the nucleus ambiguus/retrofacial nucleus and extends more ventrally near the ventral surface (section 3.3.). Furthermore, respiratory activity disappeared after extensive electrical lesion of the VLM (Onimaru et al., 1987, 1988). These ®ndings consolidate the view that VLM neurons including the VRG (i.e. VLM-VRG neurons) are responsible for generation of respiratory rhythm (Dun et al., 1995). Finally, reduction of this en bloc preparation by transverse sectioning has established that not the entire rostrocaudal extension of the VLM-VRG is involved in generation of the primary rhythm. These experiments showed that respiratory activity is rather produced by VLM-VRG interneurons in a limited rostral region of the ventrolateral medulla (Onimaru et al., 1987; Arata et al., 1990; Smith et al., 1991; Budzinska et al., 1985), con®rming the existence of a discrete respiratory center as a `noeud vital' that was hypothesised more than hundred years ago (Fluorens, 1858). In the present study, we summarise the current understanding of mechanisms involved in generation and maintenance of primary respiratory rhythm as derived from investigations on the isolated brainstem-spinal cord preparation. We concentrate on cellular mechanisms of respiratory network function with an emphasis on the role of neurotransmittergated and intrinsic ion conductances as well as of second messengers, crucial for synaptic interactions and initiation of burst activity of VLM-VRG neurons. It is also shown that a multitude of (endogenous) neuroactive substances like biogenic amines, adenosine, prostaglandins, or neuropeptides such as substance P and somatostatin are involved in modu-

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lation of in vitro respiratory activity. Finally, it is demonstrated that the brainstem-spinal cord preparation has a high potency as an in vitro model for the analysis of cellular mechanisms, responsible for clinically relevant disturbances of respiratory network functions such as hypoxia-induced respiratory depression or opioid and prostaglandin E (PGE)evoked apnea.

2. RESPIRATORY ACTIVITY In this paragraph, experiments are summarised that have established that rhythmic discharge of cranial and spinal ventral nerve rootlets represents inspiratory activity of the respiratory network in the brainstem-spinal cord preparation. It is pointed out that the di€erence of this rhythm with eupnic breathing in intact neonatal rats is secondary to removal of a€erent inputs in the course of the isolation of the medulla and to the decreased in vitro temperature. 2.1. Nerve Recordings A brainstem-spinal cord preparation, isolated from 0- to 4-days-old rats and superfused with arti®cial cerebrospinal with elevated glucose concentration (section 7.2.), was developed by Suzue and coworkers for the in vitro analysis of integrative functions of the mammalian central nervous system (Fig. 1). In an initial study, these authors investigated cranial and spinal re¯exes as well as their pharmacological modulation by opioids, somatostatin and norepinephrine (Suzue et al., 1983). It was only referred to in this report (Suzue et al., 1983), but described in detail later by Suzue (1984) that this preparation, devoid of intact circulation, produces autonomous and highly synchronised rhythmic activity that is recorded from cranial (glossopharyngeal [IX], vagal [X], hypoglossal [XII]) and spinal cervical (C1±8) and thoracic (T1±13) ventral nerve rootlets (Fig. 2). At a reduced in vitro temperature of 25±278C, this rhythm with a frequency of 5±15 bursts minÿ1 is stable for periods of 5±7 h. The pattern of individual bursts consists of a rapidly (about 50 ms) peaking and slowly decrementing discharge envelope with a duration of 0.2±0.8 s (Smith et al., 1990). These authors (1990) have also shown that onset of bursting in the cranial nerves precedes that in cervical rootlets by 50±200 ms, whereas initiation of bursts at the thoracic level is delayed by 20±40 ms (Fig. 2). Such a temporal correlation is similar to that of cranial and spinal respiratory motoneuron discharge in adult mammals in vivo (Feldman, 1986; Cohen et al., 1987). That the in vitro rhythm is indeed adequate to respiratory activity was shown in a preparation, in which the brainstem-spinal cord was isolated together with the dorsal part of the thorax (Fig. 1). Spontaneous upward movement of the thorax was found to occur synchronously with the active phase of the cycle (Suzue et al., 1983). In the latter preparation, it was furthermore demonstrated by simultaneous recording of nerve and muscle activities (Suzue, 1984) that the ®ring of phrenic (C3±C6)

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Fig. 1. Respiratory activity in newborn rats in vitro. Aa, an isolated brainstem-spinal-cord-thorax preparation of a 0-days-old rat shows spontaneous upward movement (note dotted line) of the thorax (right) with regards to the control (left). Ab, upward movement of the thorax (monitored as the tension [in g] from the distal end of the rib) occurs synchronously with neuronal electrical activity (recorded with a suction electrode) of spinal left ventral root (C4) activity. Ac, left C4 activity is synchronous with burst activity of the right phrenic nerve that causes inspiratory contraction of the diaphragm in vivo. Recordings in A are taken from Suzue (1984) by permission. Ba, in the recording chamber for a brainstem-lung preparation the bronchi and lungs are kept separate from the brainstem-spinal cord. The right vagus nerve (covered with vaseline) passes between peripheral respiratory organ and brainstem through a groove on a partition between the two compartments that are superfused separately (black arrows). Intratracheal pressure is increased by applying gas through the tracheal cannula (open arrow). Bb, 1 mM of the GABAA receptor antagonist bicuculline e€ectively blocks the inhibitory (Hering± Breuer) re¯ex, responsible for suppression of inspiration-related C5 activity due to elevation of tracheal pressure (in mmHg). Bc, in a preparation, in which both vagi are connected with the central nervous system, re¯ex inhibition is followed by a pronounced increase in respiratory frequency. Recordings in B are taken from Murakoshi and Otsuka (1985) by permission.

nerve rootlets is synchronous with the contraction of the external intercostal and other inspiratory muscles which move the thorax upward (Figs. 1 and 2; Smith et al., 1990). The similarity with the nerve activities and the thorax movements in intact animals during inspiration suggests that the in vitro rhythm corresponds to inspiratory-like activity. The interval between inspiratory bursts is adequate to the expiratory phase which consists of two distinct subphases (Fig. 2). An early as well as a late expiratory discharge are revealed by (bilateral) recordings

from cranial (glossopharyngeal and vagal) nerves (Smith et al., 1990). The early expiratory (E1) phase discharge consists of a low amplitude decrementing envelope which onsets immediately after cessation of the inspiratory activity. The late (E2) expiratory phase discharge pattern is characterised by a low amplitude augmenting envelope, preceding the inspiratory phase by 0.5±2 s (Fig. 2; Smith et al., 1990). Thus similar to the in vivo situation, the in vitro respiratory rhythm is organised in three neuronal phases. The early expiratory ventral root dis-

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Fig. 2. Organisation of the neuronal respiratory cycle of neonatal rats in vitro. A, traces (taken from di€erent brainstem-spinal cord preparations) show the temporal pattern of spinal (cervical [C4], lower thoracic [T6, T10]) and cranial (glossopharyngeal [IX], vagal [X], and hypoglossal [XII]) motoneuronal activity that coincides with inspiratory-related muscle activity (E6, external intercostal electromyograph recorded from 6th intercostal space). In contrast to the inspiratory (I) phase, the expiratory (E) period consists of an early (E1 i.e. post-inspiratory) and a late (E2) phase. The two traces of IX nerve activity illustrate variations in the pattern of cranial nerve E phase activity. B, cycle-triggered histograms of inspiratory nerve discharge (from di€erent preparations) show that the onset of burst activity is progressively delayed along the rostrocaudal neuraxis. Recordings are taken from Smith et al. (1990) by permission.

charge corresponds to the post-inspiratory period, whereas the late expiratory nerve activity represents active (stage-2) expiration (Richter, 1982, 1996; Feldman, 1986; Smith et al., 1990). It was shown by transection and lesion experiments, and also by using experimental chambers allowing for discrete administration of drugs to the spinal or the cranial aspect of the preparation, that the respiratory activity originates in the medulla (Suzue, 1984; Onimaru and Homma, 1987; Smith and Feldman, 1987a,b). Accordingly, extracellular recording from di€erent classes of neurons within the ventrolaleral medulla has revealed that their rhythmic discharge is in phase with inspiratory nerve activity (Onimaru et al., 1987, 1988; Smith et al., 1990). The pattern of spike discharge of these neurons (section 3.1.), located in medullary regions representing the VLM-VRG in adult mammals (section 3.3.), furthermore shows that the respiratory network in the brainstem-spinal cord preparation of the newborn rat is also organised in three phases (Smith et al., 1990; Feldman et al., 1991; Richter et al., 1992; Bianchi et al., 1995; Onimaru, 1995). Not only interneurons, that are responsible for generation of the rhythm, but also (pre-) motoneurons that feed the respiratory drive into respiratory muscles, remain active in the brainstem-spinal cord preparation. This functional integrity holds true also

for a€erent synaptic inputs from structures, involved in adaptation of respiratory network function in the intact animal. For example, it was demonstrated that electrical stimulation and/or lesion of pontine midline structures, or of the A5 noradrenergic area in the pons, modulates respiratory activity (Hilaire et al., 1989; Morin et al., 1990b). These results, in concert with the observation that respiratory frequency increases after transverse sectioning at the pontomedullary junction (Hilaire et al., 1989; Smith et al., 1990), suggested that the in vitro respiratory network is subject to ongoing depression in brainstem-spinal cord preparations including the pons (section 5.1.; Hilaire et al., 1997). Not only these synaptic connections, but also re¯exes related to respiratory network function are retained in the preparation. As one particular elegant example, it was demonstrated that lung in¯ation leads to inhibition of respiratory rhythm in a brainstem-spinal cord preparation containing the lung (Fig. 1; Murakoshi and Otsuka, 1985). This indicates also that the central nervous components of the lung stretch (`Hering±Breuer') re¯ex as one important mechanism of a€erent control of the respiratory center (Cohen, 1975; Bonham and McCrimmon, 1990) can be analysed in vitro. Although not related to the present study, it should be noted that the brainstem-spinal cord prep-

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aration is also an established model for the analysis of other cranial and spinal motor systems. Administration of neuroactive substances such as Nmethyl-D-aspartate (NMDA) or serotonin (5-HT) evokes alternating rhythmic activity at the lumbar level, corresponding to spinal locomotion (Kudo and Yamada, 1987; Smith and Feldman, 1987a,b; Smith et al., 1988; Cazalets et al., 1995). In contrast, rhythmic activity of hypoglossal nerve rootlets, evoked by administration of NMDA or 5-HT, is attributed to activation of a suckling- or masticatory-like motoactivity (Ballanyi, Pestean and Schwarzacher, in preparation; Nakamura and Katakura, 1995). 2.2. Relation To Eupneic Breathing The above studies have established that the spontaneous rhythm in the brainstem-spinal cord preparation represents respiratory activity of the medullary respiratory network. But the in vitro rhythm di€ers from eupneic breathing of neonatal rats in vivo. Respiratory activity in intact neonatal (and also adult) rats exerts an incrementing rather than a decrementing pattern of inspiratory bursts (Fig. 3) and the frequency (60±120 minÿ1) is about one order of magnitude higher than in vitro. However, bilateral vagotomy in neonate pups in vivo leads to a decrease of respiratory frequency to about 23 minÿ1 (Murakoshi et al., 1985; Fedorko et al., 1988; Smith et al., 1990). This value is only slightly higher than that of respiratory frequency in vitro (Fig. 3). Furthermore, the frequency and also the duration of the inspiratory burst overlap those in vivo after elevation of the in vitro temperature to values in the intact brain (Fig. 3; Smith et al., 1990). As discussed in detail by Smith et al. (1990), the reduction of respiratory frequency by vagotomy is congruent with the established role of pulmonary stretch receptor inputs in adult mammals (Rossignol et al., 1988). The important in¯uence of vagal a€erents is also indicated by the ®nding that an increase in tracheal pressure in the brainstem-lung preparation results in block of respiratory rhythm that is followed by a pronounced increase in respiratory frequency (Fig. 1; Murakoshi and Otsuka, 1985). Also in a variety of other rhythmic motor systems, removal of inputs from mechanosensory a€erents leads to prolongation of the cycle period by yet unknown cellular mechanisms (Feldman and Grillner, 1983; Pearson, 1987). In this context, it was recently described that dea€erentation does not cause a signi®cant reduction in respiratory frequency in the newborn opossum (Eugenõ n and Nicholls, 1997). Possibly as a consequence of the small e€ect of vagal a€erents in this animal, respiratory frequency in the brainstem-spinal cord preparation is similarly high as in vivo. The observation of a low frequency and a decrementing pattern of inspiratory bursts led to the suggestion that the in vitro respiratory activity corresponds to gasping (Lumsden, 1923) rather than eupneic breathing (Suzue, 1984; Murakoshi et al., 1985; St John, 1990, 1998; Wang et al., 1996). However, the results obtained in neonatal rats in vivo not only show that vagal mechanosensory

inputs critically determine the frequency of breathing. It was also found that vagotomy leads to transformation from an incrementing to a decrementing pattern of the inspiratory burst and to an increase in the amplitude of phrenic nerve activity (Fig. 3; Murakoshi et al., 1985; Fedorko et al., 1988; Smith et al., 1990). In contrast to (hypoxic/ischemic) gasping that is independent on vagal inputs (St John, 1990, 1998; St John and Knuth, 1981), the duration of the inspiratory phase is not reduced, but rather increased after vagotomy in vivo as well as after isolation of the brainstem-spinal cord (Fig. 3; Smith et al., 1990). According to these results, the combined e€ects of vagotomy on frequency and pattern of respiratory activity produce a breathing pattern resembling with some respect gasping in response to hypoxia or asphyxia in adult mammals (St John and Knuth, 1981; Mace®eld and Nail, 1987; St John, 1990, 1998) not only in the in vitro medulla, but also in the in vivo neonatal rat (Murakoshi et al., 1985; Fedorko et al., 1988; Smith et al., 1990). Since this pattern in neonatal rats in vivo is observed immediately after vagotomy, it is unlikely that the changes in respiratory rhythm are due to hypoxia (Murakoshi et al., 1985; Mace®eld and Nail, 1987; Smith et al., 1990). However, it is likely that the resulting pronounced overall decrease in ventilation causes secondary hypoxia, leading to death due to respiratory failure in anesthetised vagotomised young animals (Mace®eld and Nail, 1987). Gasping not only occurs in vivo in response to metabolic failures, but also upon transection at the level of the pontomedullary junction (section 5.1.; Lumsden, 1923). This led to the suggestion that medullary mechanisms underlying the neurogenesis of gasping are tonically suppressed by respiratory mechanisms of the pons (St John, 1990). In contrast to this view, neither the pattern nor amplitude of respiratory activity in the brainstem-spinal cord are a€ected after removal of the pons and thus the PRG (Hilaire et al., 1989; Smith et al., 1990). In this in vitro preparation, it was furthermore found that central chemosensitivity is retained and respiratory activity is modulated by neuroactive substances such as opioids, 5-HT or noradrenaline (section 6.) in a manner, that is almost identical to that observed in vivo (Suzue, 1984; Murakoshi et al., 1985; Harada et al., 1985; Onimaru et al., 1997). Since gasping is insensitive to hypercapnea, hypocapnea or other (pharmacological) stimuli (St John and Knuth, 1981; St John, 1990), the activity of the isolated respiratory network basically di€ers from gasping that results from metabolic insults. Gasping is assumed to constitute the terminal response of the respiratory network during severe hypoxia or ischemia (St. John, 1990, 1998). Accordingly, it was reported that the terminal respiratory gasp during anoxia coincides with loss of ion homeostasis, leading to complete impairment of brain function (Hansen, 1977, 1985; Ballanyi et al., 1992). However, in the brainstem-spinal cord preparation baseline levels and activity-related changes of extracellular K+, Ca2+ and pH in the region of the VLM-VRG are stable for several hours (Ballanyi et al., 1992; Brockhaus et al., 1993; VoÈlker et al., 1995) and similar to those observed in vivo

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Fig. 3. Transformation of respiratory pattern in newborn rats during the in vitro isolation. A, in an anesthetised neonatal rat pup section of the left vagus nerve (middle panel) and subsequent bilateral vagotomy (lower panel) result in a progressive decrease in respiratory frequency as recorded by movements of the thorax. Recordings are taken from Murakoshi et al. (1985) by permission. B, in vivo vagotomy also produces an instantaneous change from an incrementing into a decrementing pattern of integrated diaphragm electromyograph (upper two traces) that is very similar to integrated phrenic (C4) inspiratory burst activity (lower two traces). Note that the duration of the inspiratory phase is prolonged upon reduction of the in vitro temperature to 278C, at which respiratory rhythm remains stable for about 7 h. C, a plot of the cycle period reveals that respiratory frequency is almost indiscernible in the vagotomised neonate pup and in the brainstem-spinal cord preparation, kept at an in vitro temperature of 358C corresponding to the in vivo situation. The cycle frequency of the isolated respiratory network is further reduced after lowering the temperature of the superfusate to 278C and in preparations (kept at 278C) that contain the pons. Each boxplot displays summary of the data distribution including median and upper and lower quartiles containing 25% of the data points above and below the median, respectively. Tails represent range of data. Upper and lower adjacent values for distributions shown represent upper and lower bounds of range, respectively. Recordings in B and C are taken from Smith et al. (1990) by permission.

(Richter et al., 1978; Richter and Acker, 1989; Trippenbach et al., 1990; Richter and Ballanyi, 1996). The ®nding that ion homeostasis in the VLM-VRG is not pathologically disturbed due to presumed hypoxia (Suzue, 1984; St John, 1990) was con®rmed by microelectrode measurements of tissue oxygen. These recordings revealed a pO2 in the VLM-VRG that is sucient to provide full utilisation of aerobic metabolism (Ballanyi et al., 1992; Brockhaus et al., 1993; VoÈlker et al., 1995). That the respiratory neurons are indeed under aerobic conditions is also indicated by studies in which aerobic metabolism in the entire en bloc preparation was blocked by superfusion of hypoxic solution (Ballanyi et al., 1994a) or addition of cyanide

(Ballanyi, 1999b). In the majority of VLM-VRG neurons, these procedures result in a persistent, K+ channel-mediated hyperpolarisation as well as in block of rhythmic membrane potential ¯uctuations (section 7.1.). These hyperpolarisations were found to be associated with a biphasic change (initially accelerating, late slowing) of respiratory rhythm (Ballanyi et al., 1992; VoÈlker et al., 1995; Ballanyi, 1999b) that is also a characteristic feature of the hypoxia response of intact newborn mammals including infants (Rigatto, 1984; Richter and Ballanyi, 1996; Martin et al., 1998). Evoked anoxia was also found to result in block of Clÿ-mediated inhibition (Ballanyi et al., 1994a; Richter and Ballanyi, 1996; Richter et al., 1999) that is a characteristic feature

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of the vast majority of VLM-VRG neurons (section 3.1.) in the brainstem-spinal cord preparation superfused with oxygenated saline (Onimaru and Homma, 1992; Smith et al., 1992; Onimaru et al., 1996a; Brockhaus and Ballanyi, 1998). The above ®ndings demonstrate that the reduction of respiratory frequency and the transformation of discharge pattern in the brainstem-spinal cord compared to the intact animal is primarily due to removal of peripheral a€erent inputs in the course of the in vitro isolation. The pattern of both the in vitro activity and the in vivo respiratory rhythm in vagotomised neonatal rats share some features with gasping in adult mammals. However, gasping constitutes a pathological activity of the respiratory network in the terminal period of metabolic disturbance that is insensitive to chemostimuli or neuromodulators. In contrast, the respiratory rhythm in dea€erented neonatal rats as well as the in vitro respiratory activity appear to represent a particular type of enforced breathing (Cherniack et al., 1981). Although this enforced pattern of respiration is not identical with eupneic breathing, it retains sensitivity to neuromodulation of the neuronal network that produces the primary respiratory rhythm. Therefore the isolated medulla preparation is well suited for the analysis of the cellular mechanism of generation and modulation of the primary respiratory rhythm.

3. RESPIRATORY NEURONS Respiratory neurons are classi®ed according to the occurrence of rhythmic spike discharge with respect to the three-phase activity of respiratory nerves (Richter, 1982, 1996; Feldman, 1986; Bianchi et al., 1995). So far, basically three classes of respiratory neurons have been characterised in the VLMVRG of the brainstem-spinal cord preparation. Each of these classes of neonatal respiratory neurons, namely pre-inspiratory (Pre-I), inspiratory (Insp) and expiratory (Exp) cells consists of subtypes, according to the pattern of rhythmic membrane potential oscillations, spike discharge characteristics and hyperpolarisation after termination of the active phase (Onimaru and Homma, 1987, 1992; Smith et al., 1990, 1992; Homma et al., 1993; Onimaru et al., 1996a, 1997; Brockhaus and Ballanyi, 1998). 3.1. Classes Pre-I neurons show membrane depolarisation leading to initiation of spike discharge by up to several hundred milliseconds prior to onset of inspiration-related nerve discharge (Onimaru and Homma, 1987, 1992). The vast majority of these VLM-VRG neurons is hyperpolarised by up to 30 mV due to GABAA and/or glycine receptormediated IPSPs during the inspiratory phase (section 4.1.2.; Onimaru and Homma, 1992; Onimaru et al., 1990, 1996a; Brockhaus and Ballanyi, 1998). After termination of inspiratory nerve discharge, corresponding to the post-inspiratory phase, these cells ®re again for up to several seconds (Fig. 4).

Due to lack of inspiration-related Clÿ-mediated inhibition, about 20% of Pre-I neurons do not show a biphasic bursting pattern. The ongoing activity of such so-called `throughout' Pre-I neurons (Onimaru and Homma, 1987, 1992) starts prior to onset of the inspiratory phase and persists for up to several seconds after its termination (Fig. 4). The duration of pre- or post-inspiratory activity of Pre-I neurons varies considerably between di€erent cells, and even from cycle to cycle in individual neurons (Fig. 4; Onimaru et al., 1997). It should be noted that these neonatal respiratory neurons have also been classi®ed as `biphasic expiratory' cells (Smith et al., 1990; Ballanyi et al., 1994a; Brockhaus and Ballanyi, 1998). A di€erent class of neonatal Pre-I neurons (so called `pre-I4I' neurons) was ®rst described by Smith et al. (1990). In these cells, spike discharge starts before onset of inspiratory nerve activity, but spiking declines and ceases with the inspiratory phase (Smith et al., 1990). The latter type of Pre-I neurons could also represent a subclass of Insp neurons (Onimaru et al., 1997). There are few reports for typical biphasic Pre-I neurons in adult mammals (Schwarzacher et al., 1995), while monophasic Pre-I neurons have been described more frequently (Cohen 1969; Homma et al., 1990; Schwarzacher et al., 1995; Sun and Reis, 1996; Paton, 1996; see also Fukuda, 1992, for cranial nerve discharge pattern). Hence, it is possible that some of the biphasic Pre-I neurons transform into monophasic Pre-I neurons, whereas others di€erentiate into post-inspiratory neurons during postnatal development. Insp neurons (Fig. 4) are characterised by action potential discharge during the inspiratory phase. This class of neonatal VLM-VRG neurons consists of three subtypes as determined by the pattern of postsynaptic potentials (PSPs) during the pre- and post-inspiratory phase (Onimaru and Homma, 1992). Type I Insp (Insp-I) neurons exert a high probability of occurrence of subthreshold EPSPs in the pre- and post-inspiratory phase. The probability of EPSPs increases during the pre-inspiratory phase, thus producing a ramp-like depolarisation that triggers a burst of spikes at the onset of the inspiratory nerve discharge (Fig. 4). Since the pattern of the subthreshold EPSPs parallels the bursting activity of Pre-I neurons, Insp-I neurons probably receive excitatory synaptic inputs from these cells (section 3.4.). A small number of Insp-I neurons, that starts to ®re during the pre-inspiratory phase due to large EPSPs (Fig. 4; Onimaru et al., 1997), might be equivalent to `pre-I4I' neurons recorded extracellularly by Smith et al. (1990). Type II Insp (Insp-II) neurons (Fig. 4) show neither a high probability of EPSPs nor IPSPs during the pre- or post-inspiratory period. This suggests lack of synaptic connectivity with Pre-I neurons. In some of these neurons, EPSPs or IPSPs are revealed during the expiratory phase, indicating synaptic inputs from expiratory neurons (Arata et al., 1998b). Type III Insp (InspIII) neurons (Fig. 4) are hyperpolarised by IPSPs within both the pre- and post-inspiratory phase. Thus they appear to receive inhibitory synaptic inputs from Pre-I neurons (Onimaru and Homma, 1992). These IPSPs are reversed by Clÿinjection and

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Fig. 4. Classes of respiratory neurons in the brainstem-spinal cord preparation. A, pre-inspiratory (PreI) neurons are characterised by synaptic drive potentials and spike discharge which onset prior to and terminate after cessation of inspiratory spinal (C4) nerve discharge. Clÿ-mediated IPSPs during the inspiratory phase are a feature of most Pre-I neurons Aa, whereas inhibition is not observed in a subclass of these cells (Ab). Ac and Ad exemplify that the duration of the pre- or post-inspiratory activity phase varies between individual cells. B, type-I inspiratory (Insp-I) neurons are characterised by spike discharge during C4 burst activity and by subthreshold (Ba) or spike-evoking EPSPs (Bb) within the peri-inspiratory period. Type-III Insp neurons are hyperpolarised by Clÿ-mediated IPSPs during the peri-inspiratory phase (Bd), whereas peri-inspiratory PSPs are not observed in type-II Insp cells (Bc). C, expiratory (Exp) neurons are hyperpolarised by Clÿ-mediated IPSPs either during the inspiratory (Expi, Ca) or the peri-inspiratory (Exp-p-i, Cb) phase. Recordings are taken from Onimaru et al. (1997) by permission.

blocked by suppression of Clÿ-mediated IPSPs with bicuculline and/or strychnine (section 4.1.2.; Onimaru et al., 1996a; Brockhaus and Ballanyi, 1998; see Fig. 8). Intermediate subtypes of Insp-I and Insp-III neurons, which show both EPSPs and IPSPs during both, pre- and post-inspiratory phase, have also been described (Onimaru et al., 1997). In addition to the above classi®cation according to the pattern of respiration-related PSPs, Insp neurons can be classi®ed according to membrane poten-

tial trajectories related to `intrinsic' membrane conductances (section 4.3.). After termination of the inspiratory burst, a subpopulation of all types of Insp neurons shows a transient hyperpolarisation with a slow decay (Smith et al., 1992; Rekling et al., 1996a,b). It is yet not clear, whether this afterhyperpolarisation is due to a Ca2+-activated K+ current (Ballanyi, Onimaru and Homma, in preparation), a slowly inactivating G protein-coupled inwardly rectifying K+ current (Johnson et al., 1996) or other

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intrinsic ion conductances, such as the h-type cation current (Onimaru et al., 1997; section 4.3.). In line with the typical decrementing pattern of inspiratory nerve activity (section 2.1.), the inspiration-related synaptic depolarisation shows a rapid onset, followed by a gradual repolarisation in many Insp neurons. In consequence, ®ring frequency decreases during inspiratory bursts in these VLM-VRG neurons, whereas it remains almost constant in other cells, in particular Insp-III neurons (Fig. 4). According to the extracellular spike pattern, two types of expiratory (Exp) neurons were originally described in the brainstem-spinal cord preparation, namely tonic and late Exp neurons (Smith et al., 1990). Intracellular (perforated-patch) recordings showed that these VLM-VRG cells are hyperpolarised and thus inhibited by Clÿ-mediated IPSPs during the inspiratory phase (Smith et al., 1992; Arata et al., 1998b; Brockhaus and Ballanyi, 1998). One of the latter studies provided evidence that a major portion of Exp neurons receives inhibitory synaptic inputs from Pre-I neurons as well as from Insp neurons (Arata et al., 1998b). As derived from the pattern of these IPSPs, Exp neurons are classi®ed into two subtypes; Exp-i neurons (Fig. 4) in which spike discharge is inhibited only during the inspiratory phase, and Exp-p-i neurons (Fig. 4) in which inhibition of ®ring starts with the pre-inspiratory phase and terminates after the post-inspiratory phase. Exp-p-i neurons might be equivalent to late Exp (or E2) neurons in adult mammals, in which ®ring is inhibited for up to several hundred milliseconds before onset of inspiratory discharge (Klages et al., 1993; Paton, 1996). Some Exp-p-i neurons appear to form inhibitory synaptic connections with Insp-II neurons (Arata et al., 1998b). Consequently, it was proposed that pre-inspiratory phase is distinguishable from other three respiratory phases in the central respiratory activity in the newborn rat preparation in vitro (Onimaru et al., 1997; Arata et al., 1998b). It may turn out in future studies that the neuronal respiratory cycle consists of four instead of three phases, namely the pre-inspiratory, the inspiratory, the post-inspiratory and the active expiratory phase. Recently developed medullary block preparations from (newborn) mice allow for analysis of the e€ects of gene manipulation on respiratory network function (Jaquin et al., 1996; Rekling et al., 1996a,b). In these reports, three distinct types of Insp neurons were characterised, whereas the authors did not describe Pre-I and Insp-III neurons. In contrast, Arata et al. (1997) found all types of respiratory neurons (namely Pre-I, Insp-I±III, Exp-i, Exp-p-i) in the VLM-VRG of the brainstem-spinal cord preparation from newborn mice under experimental conditions such as used for the preparation from newborn rats. In the mouse preparation, the latter authors furthermore described (1997) a novel subtype of Pre-I neurons, in which burst activity is restricted to the pre-inspiratory phase. 3.2. Morphology And Projections Since the region of VLM-VRG contains interneurons as well as (pre-) motoneurons of diverse

function and structure, analysis of morphology (Fig. 5) and location (Fig. 6) of recorded respiratory active neurons is of ultimate relevance. In the neonatal rat, cell bodies of respiratory VLM-VRG neurons are located 50±600 mm (main distribution 200±500 mm) below the ventral surface of the medulla (Smith et al., 1990; Arata et al., 1990; Onimaru and Homma, 1992; Kawai et al., 1996). The somata of the deepest neurons recorded are closely associated with the nucleus ambiguus or retrofacial nucleus (Fig. 6). The shape of the respiratory neurons is either pyramidal, multipolar or fusiform. Insp-III neurons are characterised by small and mainly fusiform cell bodies (20 mm long diameter and 10±15 mm short diameter), whereas Insp-I neurons are comparatively large (15±30 mm). Some of these cells exert a high density of dendrites near the soma (Fig. 5; Onimaru and Homma, 1992; Arata et al., 1993a). Axons primarily project towards the dorsomedial medulla, in particular to the ipsilateral and/or contralateral area of the nucleus of the solitary tract, or medially towards the contralateral ventral medulla through the raphe. In some Insp neurons, axons run into glossopharyngeal or vagal nerve rootlets indicating that these cells are cranial motoneurons. Dendrites project in ventromedialdorsolateral and/or dorsoventral direction. In some Pre-I neurons, length of dendrites can approach 1 mm (Arata et al., 1993a; Onimaru et al., 1995). Many respiratory neurons posses dendrites that terminate close to the ventral surface of the medulla (Onimaru and Homma, 1992; Kawai et al., 1996). This led to the assumption that these super®cial dendritic regions might be important for central chemosensitivity (section 6.5.; Kawai et al., 1996). A similar projection of dendrites was described for respiratory neurons in the RVL of adult rats in vivo (Pilowsky et al., 1990). It was found that the input resistance of neonatal respiratory neurons depends on the size of cell soma and the diameter of the primary dendrites. The size of the dendritic ®eld and the numbers of dendritic varicosities appears to be related to the degree of postsynaptic activity (Onimaru and Homma, 1992; Arata et al., 1993a). 3.3. Rostrocaudal Distribution Pre-I neurons are distributed rostrocaudally in the reticular formation from the most rostral level of the retrofacial nucleus (or the caudal part of the facial nucleus) to the level of the rostral end of the lateral reticular nucleus (Fig. 6). The ventral extension of distribution was found to range from surface regions to the ventral aspect of the nucleus ambiguus or retrofacial nucleus (at depth of 50±500 mm; Onimaru et al., 1987; Arata et al., 1990). This area is called the rostral ventrolateral medulla (RVL), which corresponds to the nucleus reticularis rostroventrolateralis (nRVL; Ross et al., 1984). In the RVL, the distribution of Insp neurons basically overlaps that of Pre-I neurons, but extends into more caudal regions of the ventrolateral medulla (CVL). These results are principally consistent with those of Smith et al. (1990), whereas former results (Onimaru et al., 1987; Arata et al., 1990) showed a more rostral and ventral extension of the distri-

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Fig. 5. Morphology of respiratory neurons in the brainstem-spinal cord preparation. After electrophysiological identi®cation (a) of a particular type of neonatal respiratory neuron (A±D), cells that were ®lled via the patch electrode with lucifer yellow, were histologically analysed and displayed as camera lucida drawings (b). Graphs in c indicate location of the cell in the medulla. Arrow heads denote axons. Data are taken from Onimaru, Arata and Homma, unpublished.

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Fig. 6. Distribution of respiratory neurons in the brainstem-spinal cord preparation. A, ventral view of the brainstem-spinal cord preparation, indicating the distribution of Pre-I and Insp neurons (according to Onimaru et al. (1987) and Arata et al. (1990)) in the ventrolateral medulla (VLM). RVL, rostral ventrolateral medulla; CVL, caudal ventrolateral medulla. Dashed line indicates level of transection that results in block of C4 burst activity (Onimaru and Homma, 1987). nVII, facial nucleus; IX±XII, cranial nerves, C1±C4, cervical ventral nerves. B, left side, optical image corresponding to the inspiratory burst activity (taken from Onimaru et al., (1996b)). The active region was found to be ventral to the nucleus ambiguus or retrofacial nucleus (AMB/RFN). VS, ventral surface. Right side, distribution of Pre-I (solid circles) and Insp (open circles) neurons, similar to level of section on left side (plotted from data of Arata et al. (1990) and Kashiwagi et al., (1993)). C, D, perturbations of respiratory motor pattern with serial microsections of neonatal rat medulla showing pre-BoÈtzinger Complex and neighboring regions (SO, superior olive; 7, facial nucleus; LRN, lateral reticular nucleus; RFN, retrofacial nucleus; rVRG, rostral ventral respiratory group; cNA, caudal (semicompact division) of nucleus ambiguus. Consecutive 75 mm sectioning from the rostral to caudal direction does not a€ect the cycle frequency, recorded from C4 rootlet (D) until the hatched area in C is reached. A further cut (section 8) profoundly decreases the cycle frequency, whereas rhythm is irreversibly abolished after a further section (9). Circle represent data from 15 cycles of the preparation. C and D are taken from Smith et al. (1991) by permission.

bution of Pre-I (`biphasic expiratory') and Insp neurons than the latter. Cell bodies of Insp-III neurons are located more closely to the ventral surface than those of Insp-I neurons in the RVL, nevertheless with considerable overlap (H. Onimaru, A. Arata and I. Homma, unpublished observation). Exp neurons are found in similar VLM regions in which Pre-I or Insp neurons are located (Brockhaus et al., 1993; Smith et al., 1990). Smith et al. (1990) reported that Insp, biphasic expiratory (classi®ed as Pre-I in the present study) and Exp neurons represented 65, 10 and 25%, respectively, of all neurons mapped in the ventrolateral reticular formation at

medullary levels, extending from the pyramidal decussation to the caudal pole of facial nucleus. The RVL does not fully correspond to the rostral part of the VRG and BoÈtzinger complex as described for the adult cat (Lipski and Merrill, 1980; Merrill et al., 1983; see also von Euler, 1986; Feldman, 1986 for review). Respiratory neurons in the isolated brainstem of neonatal rats are also found more ventrally than the VRG-BoÈtzinger complex, overlapping partially the area of C1 adrenergic neurons in the RVL (Onimaru et al., 1987; Arata et al., 1990). Their distribution resembles that of respiratory neurons in the adult rat (Ezure et al., 1988;

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Pilowsky et al., 1990). A study by Kanjhan et al. (1995) suggests that adjacent pre-sympathetic neurons and BoÈtzinger complex Exp neurons in adult rats form two anatomically and functionally distinct neuronal subpopulations in the RVL. Recently, optical methods using a voltage-sensitive dye revealed inspiratory burst activity in the VLM of medullary block preparations (Onimaru et al., 1996b). The active area in the latter study (Fig. 6) coincides well with regions of a high density of respiratory neurons in the brainstem-spinal cord preparation. One particular region in the VLM, that is located just caudal to the BoÈtzinger complex (BoÈtC; i.e. most rostral part of the VRG), is the so-called preBoÈtzinger complex (pre-BoÈtC; Fig. 6; Smith et al., 1991). The pre-BoÈtC, that has a high density of interneurons (Smith et al., 1991), seems to correspond to the rostral part of the CVL, in which the rostral end of the lateral reticular nucleus appears (compare Fig. 1 in Smith et al., 1991 and Fig. 14 in Smith et al., 1990 with Fig. 2b±d in Arata et al., 1990 and Fig. 1 in Onimaru et al., 1993; Koshiya and Guyenet, 1996). In the adult cat or rat, subtypes of respiratory neurons in the VLM-VRG have a basically similar characteristic rostrocaudal distribution. In particular, Exp neurons are located in the BoÈtC, Insp neurons in the rostral VRG and bulbospinal Exp neurons in the caudal VRG (Feldman, 1986). Di€erent types of respiratory neurons also are found in other VRG regions (Bianchi et al., 1995). 3.4. Connectivity The patterns of burst activity and PSPs of respiratory neurons described above indicate highly complex mutual synaptic connections between di€erent classes of neonatal VLM-VRG neurons. These are in particular excitatory as well as inhibitory synaptic connections from Pre-I to Insp neurons, inhibitory synaptic connections from Insp to Pre-I and Exp neurons, inhibitory synaptic connections from Pre-I to Exp neurons, and inhibitory synaptic inputs from Exp to Insp neurons. The latter ®ndings show on the one hand that (Clÿ-mediated) inhibition is functional in the respiratory network already at birth, which is in contrast to the excitatory action of GABAA and glycine receptors in other neonatal brain regions (section 4.1.2.). On the other hand they show that inhibitory connections are a predominant feature of the synaptic interaction within the respiratory network not only in adult mammals, but also in neonates (Fig. 7). Pulse-cross correlation analysis revealed a high incidence of mono- or oligo-synaptic excitatory connections between paired Pre-I or paired Insp neurons or shared inputs to them (Kashiwagi et al., 1993a). Such synaptic connections, that are most likely mediated by excitatory amino acid (EAA) receptors, may be important for synchronisation of respiratory neurons (Onimaru et al., 1991). Cross-correlation histograms, indicating reciprocal excitatory connections between recorded neurons, were found in bilateral pairs of Insp neurons in the RVL (Kashiwagi et al., 1993a) and in the CVL (Onimaru et al., 1993). This reciprocal excit-

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atory connectivity is probably responsible for maintenance of inspiratory burst activity as suggested for adult cats (Ezure and Manabe, 1989). Spike-triggered averaging also revealed excitatory synaptic connections from Pre-I neurons to Insp neurons in the VLM (Onimaru et al., 1992). Some Pre-I and Insp (types I and II) neurons in the VLM were found to project to the ipsilateral or the contralateral spinal cord (Onimaru and Homma, 1992; Kashiwagi et al., 1993a; Onimaru et al., 1993), whereas Insp-III neurons are possibly propriobulbar.

4. CELLULAR MECHANISMS OF RHYTHMIC ACTIVITY In a variety of neuronal tissues, sets of conductances have been identi®ed that cooperate to mediate rhythmic bursting. Since about ten years ago, results from nerve recordings have presented indirect evidence that rhythmic activity of the respiratory network in the brainstem-spinal cord preparation is a€ected by a variety of drugs that are known to either act on receptors for neurotransmitters, modulate cellular second messenger systems, or directly a€ect ion channels. Only since quite recently, intracellular recordings on VLM-VRG neurons in the latter preparation (and in transverse slices containing subdivisions of the VLM-VRG) have revealed that neonatal respiratory neurons possess a complex set of transmitter-regulated or voltage-dependent ion conductances that are involved in the generation and modulation of respiratory rhythm. 4.1. Neurotransmitters 4.1.1. Excitation As in most other brain regions, synaptic transmission via glutamate and related EAA receptors is essential for generation and transmission of rhythmic activity of respiratory neurons. Accordingly, it was shown that complete block of EAA receptors causes respiratory arrest in the adult cat. In contrast, suppression of either the NMDA or nonNMDA, i.e. kainate and/or g-amino-3-hydroxy-5methyl-4-isoxalone propionic acid (AMPA) subtype of EAA receptors appears to be rather well tolerated (Pierre®che et al., 1994b). However, in vagotomised cats systemic administration of NMDA receptor antagonists was found to produce prolonged inspiration (apneusis). This suggests NMDA receptormediated interaction of pulmonary vagal a€erent inputs with a central respiratory mechanism (Foutz et al., 1988a,b). As blockers of non-NMDA receptors do not prolong the inspiratory phase (Pierre®che et al., 1994b), it was concluded that NMDA receptors are in particular important for the inspiratory o€-switch (section 5.1.; Foutz et al., 1988a; Feldman et al., 1992). A recent report indicates that several classes of respiratory neurons are disfacilitated during their active phase (Haji et al., 1996c). Thus, a decreased inhibitory action (normally mediated by these cells) probably plays a major role in the apneustic prolongation of inspi-

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Fig. 7. Generation of respiratory rhythm. A, neuronal organisation of the central pattern generator of respiration according to data obtained in the brainstem-spinal cord preparation. RRG, respiratory rhythm generator, composed of Pre-I neurons in the rostral ventrolateral medulla (RVL). IPG, inspiratory pattern generator, composed of Insp in the RVL and in the caudal ventrolateral medulla (CVL). Type-I Insp neurons receive excitatory synaptic inputs (open circles), type-III Insp (and also Exp-p-i) cells are subjected to inhibition (closed circles) from Pre-I neurons. Insp neurons inhibit both Pre-I and Exp cells. Modi®ed after Onimaru et al. (1997) by permission. B, this model of the connectivity pattern of respiratory neurons in adult cats in vivo is based on the analysis of discharge pro®les and postsynaptic activities. Only early-inspiratory and post-inspiratory neurons seem to constitute the primary rhythm generator. Under physiological conditions these two neuron types are modulated by synaptic feedback from expiratory neurons. I, inspiratory neurons; E-2, expiratory neurons. Taken from Richter et al. (1992) by permission.

ration (Feldman et al., 1992; Pierre®che et al., 1994b; Haji et al., 1996c). In contrast to these in vivo reports, NMDA receptor-dependent apneusis was not observed in the brainstem-spinal cord preparation of newborn rats (Greer et al., 1991; Otsuka et al., 1994). This is unlikely to be related to a possible immaturity of the respiratory NMDA mechanism, since NMDA receptor antagonists produce apneusis not only in adult vagotomised cats (Foutz et al., 1988b; Pierre®che et al., 1994b), but also in newborn kitten (Schweitzer

et al., 1990). This discrepancy on the involvement of NMDA receptors in respiratory pattern generation is also not due to species di€erences as NMDA receptor blockade results in apneusis also in adult rats (Connelly et al., 1992) and guinea pigs (MorinSurun et al., 1995). In the latter study, NMDA receptor-dependent apneusis was, in contrast to the in vivo experiments, not observed in an arterially perfused medulla preparation of guinea pigs. This indicates that supramedullary structures are involved in the NMDA receptor-dependent apneu-

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Fig. 8. Clÿ-mediated inhibition in respiratory neurons of the brainstem-spinal cord preparation. A, 9 min after giga-seal formation the nystatin (Nys) perforated-patch recording of membrane potential (Vm) of an expiratory (Exp-p-i) neuron reveals hyperpolarising IPSPs during the (peri-) inspiratory phase (indicated by spinal [C4] nerve activity). After 22 min, the polarity of IPSPs has reversed due to di€usion of Clÿfrom the CsCl-containing patch electrode into the cell. Recordings are taken from Arata et al. (1998) by permission. B, in an inspiratory (Insp-III) neuron, spike discharge is suppressed by peri-inspiratory IPSPs immediately after establishing the whole-cell con®guration with a KCl-containing patch electrode. After further 15 min, the discharge pattern of the cell has basically changed due to dialysis of the cell with Clÿ. C, in an Insp-III neuron, recorded with a low (4 mM) Clÿ patch electrode solution, bath-application of the GABAB receptor agonist baclofen (7 mM) does neither produce a major e€ect on Vm nor on input resistance (measured by hyperpolarising current pulses) despite block of respiratory rhythm. After washout of baclofen, glycine (4 mM) and also the GABAA receptor agonist muscimol (10 mM) e€ectively hyperpolarise the cell and decrease its input resistance, accompanied by suppression of respiratory rhythm. Recordings in B and C are taken from Brockhaus and Ballanyi (1998) by permission.

sis. This is also suggested by the ®nding that pontine lesions produce apneusis in rats in vivo (section 5.1.; Morrison et al., 1994; Fung and St. John, 1995). Furthermore, preliminary results in the brainstemspinal cord preparation show that electrical stimulation of the nucleus parabrachialis complex induces premature termination of inspiratory burst discharge which depends on NMDA and also on GABA receptors (Arata et al., 1996). In particular Smith, Feldman and colleagues (Greer et al., 1991; Funk et al., 1993; Smith et al., 1995) found that generation of respiratory rhythm in vitro depends on endogenously released EAAs, that act primarily on non-NMDA receptors. It was, for example, demonstrated that the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3dione (CNQX) evokes a site-speci®c block of rhyth-

mogenesis in vitro (Smith et al., 1991; Funk et al., 1993), whereas block of NMDA receptors has only minor e€ects (Greer et al., 1991; Otsuka et al., 1994). However, the observation by Otsuka et al. (1994) should be noted that rhythmic ®ring of medullary inspiratory neurons persists after block of non-NMDA receptors. These authors (1994) concluded that non-NMDA receptors are not crucial for generation of respiratory rhythm. Despite apparent lack of a major role of NMDA receptors in generation of the primary respiratory rhythm, it was reported on the one hand that inhibition of NMDA receptors produces slowing of respiratory rhythm in the perfused medulla of guinea pigs (Morin-Surun et al., 1995). On the other hand, activation of this subtype of glutamate receptor increases respiratory frequency (Greer et al., 1991) and also induces or

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potentiates bursting of VLM-VRG neurons in the brainstem-spinal cord preparation (Kashiwagi et al., 1993b). In summary, the speci®c role of pre- and postsynaptic EAA receptors in generation of respiratory rhythm remains speculative at present. In contrast, rather direct evidence exists for the mechanism of synaptic transmission from medullary respiratory pre-motoneurons to spinal (phrenic, intercostal; McCrimmon et al., 1989; Liu et al., 1990; Greer et al., 1991) or cranial (hypoglossal; Funk et al., 1993) motoneurons. The results from the latter in vitro studies suggest that non-NMDA EAA receptors mediate inspiratory drive transmission to all respiratory motoneurons with only a minor contribution of NMDA receptors. Furthermore, it was revealed that presynaptic `AP4-sensitive' (presumably metabotropic) EAA receptors strongly attenuate inspiratory drive transmission to spinal, but not to cranial respiratory motoneurons (Greer et al., 1992; Funk et al., 1993). The latter ®nding indicates that AP4 receptor activation is not essential for rhythm generation. Together with the above described lack of block of NMDA receptors on respiratory rhythm in vitro, this suggests that excitatory synaptic transmission in the center of the respiratory network is mediated by ionotropic non-NMDA type EAA receptors (Smith et al., 1995). 4.1.2. Inhibition The importance of Clÿ-dependent inhibition for generation of the primary respiratory rhythm is yet not clear. On the one hand, in vivo reports on adult cats led to the hypothesis that mutual synaptic inhibition by GABAergic and glycinergic IPSPs is important for generation of respiratory rhythm (Fig. 7; Ballantyne and Richter, 1986; Bianchi et al., 1995; Richter, 1996; Schmid et al., 1996; Ramirez et al., 1997). On the other hand, studies originally done on the isolated brainstem-spinal cord preparation of newborn rats have shown that respiratory rhythm persists after block of both GABAergic and glycinergic inhibition (Feldman and Smith, 1989; Onimaru et al., 1990). The inecacy of these blockers to a€ect the in vitro respiratory rhythm is not due to lack of functional expression of GABA or glycine receptors. Extracellular (Onimaru et al., 1990) and intracellular recordings (Onimaru and Homma, 1992; Smith et al., 1992; Ballanyi et al., 1994a; Paton and Richter, 1995b; Onimaru et al., 1996a; Ramirez et al., 1996; Shao and Feldman, 1997; Arata et al., 1998b; Brockhaus and Ballanyi, 1998) have shown that Clÿ-mediated IPSPs determine the characteristic pattern of membrane potential ¯uctuations of di€erent types of neonatal VLMVRG neurons (Figs. 4, 8, 9, 10) similar to the situation in adult mammals (Ballantyne and Richter, 1986; Haji et al., 1992; Bianchi et al., 1995; Richter, 1996). An important functional role of Clÿ-mediated inhibition in vitro is suggested by the ®nding that lung in¯ation, leading to activation of mechanosensory a€erents, results in bicuculline-sensitive suppression of respiratory rhythm in the brainstemspinal cord preparation (Fig. 1; section 2.1.; Murakoshi and Otsuka, 1985).

There is increasing evidence that GABA and glycine responses are depolarising and thus excitatory in diverse regions of the neonatal brain (Kaila, 1994; Ben-Ari et al., 1997). This is assumed to be due to the action of an inwardly-directed Clÿpump, often in cooperation with bicarbonate e‚ux through the receptor-coupled Clÿchannels (Kaila, 1994). It was, however, demonstrated with perforated patch as well as with high Clÿwhole-cell recordings that respiration-related IPSPs are hyperpolarising in VLM-VRG neurons of neonatal rats (Fig. 8; Onimaru et al., 1996a; Arata et al., 1998b; Brockhaus and Ballanyi, 1998). This agrees well with previous ®ndings of hyperpolarising respiratory IPSPs in kitten in vivo (Schwarzacher et al., 1993). The mean reversal potential (ÿ60 to ÿ75 mV) of spontaneous IPSPs (Smith et al., 1992; Shao and Feldman, 1997) and also of hyperpolarisations due to bath-application of the GABAA receptor agonist muscimol and of glycine (ÿ65 mV, Fig. 8; Shao and Feldman, 1997; Brockhaus and Ballanyi, 1998) is more positive than the calculated equilibrium potential for Clÿ. This discrepancy is possibly due to the depolarising action of HCOÿ3 e‚ux, since the shape of the inspiration-related hyperpolarisation is modi®ed after changing to nominally CO2/HCOÿ3-free, N-2-hydroxyethlypiperazine-N'-2-ethanesulphonic acid (Hepes) pH-bu€ered saline in the brainstemspinal cord preparation (Brockhaus and Ballanyi, 1998). Accordingly, it was demonstrated in neurons of the region of the VRG in thin slices from neonatal rats that bicarbonate e‚ux in response to GABA and glycine evokes a prominent intracellular acidosis (LuÈckermann et al., 1997). The latter results con®rmed earlier assumptions that periodic decreases of intracellular pH in VRG neurons of the in vivo cat are caused by e‚ux of HCOÿ3 through GABAA and/or glycine receptors (Ballanyi et al., 1994a). It was recently described that inspiration-related IPSPs in tonic expiratory neurons of rhythmic brainstem slices from neonatal rats are abolished after block of glycine receptors with strychnine (Shao and Feldman, 1997). However, combined application of strychnine and of the GABAA receptor blocker bicuculline was found necessary to block inspiration-related IPSPs in post-inspiratory neurons of rhythmic brainstem slices from juvenile mice (Ramirez et al., 1996, 1997). The latter results agree well with recordings from a subpopulation of VLMVRG neurons in the brainstem-spinal cord preparation (Brockhaus and Ballanyi, 1998). In other neonatal respiratory neurons of the latter study, administration of either of the blockers was sucient to abolish inspiration-related IPSPs (Fig. 9; Onimaru et al., 1990). This discrepancy might be related to the fact that Shao and Feldman (1997) recorded from expiratory neurons of the pre-BoÈtC, which possibly exclusively receive glycinergic inputs in contrast to more caudal or rostral expiratory cells (section 3.3). Interestingly, peripherally-induced bicuculline-blockable IPSPs in bulbar respiratory neurons of decerebrate cats were found to be insensitive to strychnine (Haji et al., 1996b). In both rhythmic slices of mice (Ramirez et al., 1997) and the brainstem-spinal cord preparation of newborn

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Fig. 9. Persistence of respiratory rhythm in the brainstem-spinal cord preparation after block of IPSPs. A, in a (tonic) pre-inspiratory (Pre-I) neuron, bicuculline induces reversible suppression of inspirationrelated IPSPs without a major e€ect on inspiratory motor output (C1 nerve activity) of the in vitro respiratory network. B, respiratory rhythm also persists after suppression of Clÿ-mediated inhibition by combined administration of bicuculline and strychnine, although the drugs profoundly changes the activity pattern of a Pre-I cell. Recordings in A and B are taken from Brockhaus and Ballanyi (1998) by permission. C, in this preparation block of GABAA, GABAB and glycine receptors with 50 mM bicuculline, 200 mM 2-OH-saclofen and 10 mM strychnine, respectively, induces seizure-like spinal nerve activity. Suppression of such activity upon bath-application of 500 mM adenosine reveals that respiratory rhythm persists after block of IPSPs.

rats, pharmacological (Brockhaus and Ballanyi, 1998) or anoxia-induced (Ballanyi, 1999b) block of Clÿ-mediated IPSPs unmasks inspiration-related excitation. In this situation, post-inspiratory (mice) or Exp (rats) neurons undergo an apparent functional transformation resulting in discharge characteristics typical for (Pre-) Insp neurons (Fig. 9). It might be that at least some of the presumed Exp neurons rather represent Pre-I neurons that show tonic spike discharge during the expiratory phase, possibly due to an increase in tonic drive to the network. This shows that not only inhibitory, but also (glutamatergic) excitatory synaptic connections are established between antagonistically active VLM-VRG neurons. The in vitro respiratory rhythm in the brainstemspinal cord preparation is not profoundly perturbed after block of glycine receptors, whereas bicuculline induces massive burst discharge masking respiratory activity in ventral nerve rootlets (Feldman and Smith, 1989; Onimaru et al., 1990; Brockhaus and

Ballanyi, 1998, 1999; Paton and Richter, 1995b; Ramirez et al., 1996; Shao and Feldman, 1997). This activity is most probably due to disinhibition of spinal motor networks (Smith et al., 1988; Paton and Richter, 1995a,b; Bracci et al., 1996; Brockhaus and Ballanyi, 1998, 1999). The perturbed rhythm resembles epileptiform activity in cortical brain regions seen after block of GABAergic inhibition (Schwartzkroin and Prince, 1980). In the cortex, adenosine was found to e€ectively suppress the (bicuculline-induced) epileptiform discharge (Thompson et al., 1992). Similar results were obtained in the brainstem-spinal cord preparation (section 6.2.; Brockhaus and Ballanyi, 1999). In the presence of adenosine, it is evident that the in vitro respiratory rhythm is not a€ected by block of glycine and GABAA/GABAB receptors with a mixture of strychnine, bicuculline and 2-OH-saclofen (Fig. 9; Brockhaus and Ballanyi, 1999). These in vitro ®ndings are not in line with previous observations in the

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Fig. 10. Metabolic disturbance of the respiratory network in the brainstem-spinal cord preparation. A, anoxia (evoked by superfusion of hypoxic solution) leads to reversible slowing of inspiration-related cervical (C4) nerve activity, accompanied by a decrease in input resistance (measured by regular hyperpolarising current pulses) and a concomitant hyperpolarisation in a pre-inspiratory (Pre-I) neuron (Aa). The presentation in Ab of the recording in Aa at extended time and membrane potential (Vm) scales shows that the anoxic hyperpolarisation is accompanied by suppression of respiration-related Vm ¯uctuations. B, the glycolytic blocker iodoacetate (5 mM) irreversibly abolishes respiratory rhythm and elicits a prominent depolarisation (preceded by a hyperpolarisation and resistance decrease) in an inspiratory neuron. C, in a neuron in the region of the ventral respiratory group (VRG) in a thin brainstem slice 1 mM CNÿ evokes a persistent outward current and resistance decrease. The response is e€ectively suppressed by 200 mM tolbutamide, a blocker of ATP-sensitive K+ channels. D, in this preparation CNÿ induces a slowing of respiratory rhythm that is stable for more than 30 min. This e€ect is accompanied by a stable hyperpolarisation of an inspiratory neuron (see left panel), a fall of input resistance and attenuation of inspiratory EPSPs. The metabolic hyperpolarisation and resistance decrease as well as the slowing of respiratory rhythm are antagonised by 1 mM Ba2+, whereas 400 mM of thyrotropin-releasing hormone (TRH) is only e€ective to reverse the CNÿ e€ects on respiratory activity, but not on the membrane properties of the recorded cell. Recordings are taken from Ballanyi (1999b).

brainstem of mature rodents where respiratory rhythm is abolished or at least profoundly perturbed after suppression of Clÿ-mediated inhibition (Hayashi and Lipski, 1992; Paton and Richter, 1995a,b). Similarly, bicuculline and strychnine were shown to block respiration upon injection into the pre-BoÈtC of adult cats (Pierre®che et al., 1998). To explain this discrepancy, it was hypothesised that the increased dependence on synaptic inhibition is due to a larger number of inhibitory neurons in vivo (Ramirez et al., 1997). On the other hand, it was assumed that the importance of synaptic inhibitory mechanisms for rhythm generation increases during perinatal development (section 5.5.; Smith et al.,

1991; Feldman et al., 1991; Richter et al., 1992; Funk and Feldman, 1995; Paton and Richter, 1995a,b). The observation that respiratory rhythm persists upon complete block of IPSPs does not allow the conclusion that the isolated respiratory network of neonatal rats is not modulated by inhibitory amino acids. Blockers of GABA or glycine uptake mechanisms, that lead to endogenous elevation of interstitial levels of these inhibitory amino acids, suppress respiratory rhythm in the brainstem-spinal cord preparation (Brockhaus and Ballanyi, 1998). In the same study, it was found that bath-application of the GABAA receptor agonist muscimol or of glycine

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leads to frequency depression of respiratory rhythm that results in respiratory arrest at higher concentrations (Feldman and Smith, 1989). This e€ect is due to a prominent hyperpolarisation and conductance increase of the majority of VLM-VRG neurons that is e€ectively antagonised by bicuculline or strychnine, respectively (Brockhaus and Ballanyi, 1998; Shao and Feldman, 1997). Very similar responses were also seen in respiratory neurons of adult perfused rats (Hayashi and Lipski, 1992) or in vivo cats (Haji et al., 1992). However, in particular in Insp-I and Insp-II neurons, GABA and glycine were rather ine€ective to evoke a major hyperpolarisation or conductance increase (Brockhaus and Ballanyi, 1998). In these cells, the pattern of rhythmic membrane potential oscillations did also not change upon dialysis with high Clÿsolution, which led to reversal of the polarity of respiration-related hyperpolarising IPSPs in the other types of VLMVRG neurons (section 3.1.; Fig. 8; Brockhaus and Ballanyi, 1998; Arata et al., 1998b). This suggests that mutual Clÿ-dependent synaptic inhibition is not established between all classes of neonatal VLMVRG neurons. In contrast to the direct inhibitory e€ects of GABAA or glycine receptor activation on most neonatal respiratory neurons, GABAB receptormediated depression of the respiratory network, as elicited by the agonist baclofen (Feldman and Smith, 1989), appears to be caused by a rather indirect e€ect. Upon administration of the GABAB receptor agonist baclofen, membrane potential or conductance of most respiratory neurons in the brainstem-spinal cord preparation remains almost una€ected (Fig. 8). In contrast, the drug evokes a prominent hyperpolarisation with a reversal potential close to ÿ90 mV in the majority of (tonic) nonrespiratory neurons in the region of the VLM-VRG (Brockhaus and Ballanyi, 1998). These hyperpolarisations are most likely mediated by Gi/o proteincoupled, Ba2+-sensitive K+ channels (Smith et al., 1995; Misgeld et al., 1995; Johnson et al., 1996). However, it was shown in pre-motoneurons of neonatal rats that baclofen can also activate a Ba2+insensitive K+ conductance (Wagner and Dekin, 1993). The baclofen-induced inhibition of in vitro inspiratory nerve activity is antagonised by 2-OHsaclofen or phaclofen (Feldman and Smith, 1989; Arata et al., 1998a; Brockhaus and Ballanyi, 1998). In contrast, 2-OH-saclofen (or phaclofen) alone do neither exert major e€ects on respiratory rhythm (Feldman and Smith, 1989; Arata et al., 1998a), nor on respiration-related IPSPs of VLM-VRG neurons (Brockhaus and Ballanyi, 1998). This is consistent with the ®nding that ionophoretically applied baclofen or phaclofen does not profoundly change membrane potential or conductance of VRG neurons in the in vivo cat (Haji and Takeda, 1993; but see Lalley, 1986). On the basis of these results, it was hypothesised that the GABAB receptor-related respiratory depression is caused by inhibition of tonically active medullary neurons that provide ongoing excitatory drive to the respiratory network (sections 4.3.4.; 5.2.; Smith et al., 1995; Ballanyi and Brockhaus, 1998). However, it might well be the case that direct depression of (a limited number of)

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VLM-VRG cells, involved in rhythm generation, is sucient to block the entire respiratory network. Furthermore, inhibition of other conductances like voltage-gated Ca2+ channels might also contribute to the GABAB receptor-induced block of respiratory rhythm (Zhang et al., 1997a). In conclusion, it is evident that hyperpolarising GABAergic and glycinergic IPSPs critically determine the pattern of membrane potential ¯uctuations of respiratory neurons not only in intact mammals, but also in reduced in vitro preparations from newborn or even rather mature rodents. It is obvious that increased interstitial levels of GABA or glycine block the in vitro respiratory rhythm due to activation of Clÿ conductances of VLM-VRG neurons and of a Gi/o protein-coupled K+ conductance that is presumably characteristic for neurons providing excitatory drive to the network. It is also evident that block of GABAA, GABAB and glycine receptors does not a€ect the primary respiratory rhythm despite considerable changes in discharge characteristics of several classes of VLM-VRG neurons in the reduced preparations. It remains to be analysed, whether the sensitivity of the in vivo respiratory network of (adult) mammals to blockers of synaptic inhibition is related to a higher number of inhibitory neurons or rather caused by an in¯uence of peripheral or supramedullary structures that are absent or functionally inactive in the in vitro preparations. 4.2. Second Messengers The above ®ndings on functional expression of metabotropic glutamate (section 4.1.1.) and GABAB (section 4.1.2.) receptors indicate that second messengers are involved in respiratory network functions. However, knowledge on the mechanisms of the interaction between particular second-messengers and membrane conductances of respiratory neurons is rather limited yet. Besides the well-established stimulatory action of CO2 and H+ on respiratory network function, corresponding to central chemosensitivity (section 6.5.), it appears that the excitability of respiratory neurons is critically determined by the cellular concentration of cyclic adenosine monophosphate (cAMP). It is also discussed below that periodic rises of intracellular Ca2+ during rhythmic activity of respiratory neurons might have second messenger function. 4.2.1. Ca2+ It was shown in respiratory neurons of adult cats that injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) increases the steepness and amplitude of excitatory drive potentials (Richter et al., 1993a) and prolongs action potential discharge due to a decrease in membrane conductance and, thus, adaptation (Pierre®che et al., 1994a). It was hypothesised that a prominent excitation-related rise of intracellular Ca2+ activates (di€erent types of) Ca2+-activated K+ channels (Sah, 1996), involved in termination of inspiration (Pierre®che et al., 1994a). The assumption of occurrence of major rises in intracellular Ca2+ was veri®ed by the recent obser-

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vation that rhythmic depolarisations of inspiratory VLM-VRG neurons in transverse slice preparations from juvenile rodents are accompanied by Ca2+ transients with a magnitude of up to several hundred nM (Frermann et al., 1999; Koshiya and Smith, 1997). Since these Ca2+ transients are strongly attenuated in voltage-clamp, a major component of these responses appears to be secondary to Ca2+ in¯ux via voltage-gated Ca2+ channels (section 4.3.2.; Frermann et al., 1999). However, only in a minority of Pre-I or Insp neurons in VLM-VRG of the neonatal rat termination of the excitatory phase is followed by a signi®cant (non-synaptically mediated) hyperpolarisation (Fig. 4). Furthermore, the characteristic oscillatory membrane trajectories are apparently not changed when VLM-VRG neurons in the brainstem-spinal cord preparation are whole-cell recorded with electrodes containing either 1 mM (Brockhaus and Ballanyi, 1998) or 11 mM (Smith et al., 1992; Onimaru et al., 1996a) BAPTA or 10±11 mM ethylene glycol-bis(b-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA; Onimaru and Homma, 1992; Onimaru et al., 1995, 1998) as Ca2+ chelators. These results indicate that this particular second messenger function of Ca2+, which is important for adaptation of excitability in a variety of neuronal systems (Sah, 1996), is not crucial for generation of primary rhythm in the in vitro respiratory network, at least not in neonatal rats. Although further putative second messenger roles of Ca2+ in respiratory network function deserve to be analysed, it should be noted that ca€eine has been found to excite neonatal VLM-VRG neurons in particular after respiratory depression, evoked by PGE or opiates (section 6.3.; Meyer et al., 1998, 1999). It is not clear yet, whether this action of ca€eine and other methylxanthines such as theophylline and isobutylmethylxanthine (IBMX; Ballanyi et al., 1997; Meyer et al., 1998, 1999) is related to Ca2+ release from intracellular stores or elevation of cAMP. 4.2.2. cAMP 2+

Besides Ca , cAMP is established to play a crucial second messenger role in modulation of neuronal functions (Hille, 1992). For example, a nonspeci®c cAMP-dependent cation (CAN) conductance has been hypothesised to be involved in bursting of respiratory neurons in vivo (Richter et al., 1992) as demonstrated in snail neurons (Partridge et al., 1990). Smith et al. (1995) detected no e€ects of cAMP elevating drugs like forskolin, db-cAMP and 8-Br-cAMP on in vitro rhythmic activity in the brainstem-spinal cord preparation, whereas an acceleration of respiratory frequency due to such drugs was reported by Arata et al. (1993b). In the latter study, the stimulatory e€ect of cAMP elevating drugs was in particular evident in preparations with a rather low burst rate, whereas no e€ect or even a slight inhibitory action was revealed in preparations with a high frequency. It might well be that under the experimental conditions of the study by Smith et al. (1995), cellular cAMP levels were already high enough to produce maximal stimulation of the respiratory network. Arata et al. (1993b) also showed that forskolin reversed the frequency depression of

the in vitro respiratory rhythm elicited by clonidine that is known to lower cAMP via activation of a2 adrenoceptors (section 6.1.; UhleÂn and Wikberg, 1988). The assumption of a stimulatory action of cAMP on the respiratory network gains further support by the ®nding that cAMP elevating drugs also antagonise respiratory depression (and even in vitro apnea), evoked by opiates or PGE-induced activation of IP3 prostanoid receptors (sections 5.5.; 6.3.; Ballanyi et al., 1997; Meyer et al., 1998, 1999). It was, however, also demonstrated that cAMP elevating agents have a direct excitatory e€ect on the intrinsic burst rate of Pre-I neurons (Arata et al., 1993b). Recently, it was shown that elevation of cAMP increases the excitability of expiratory neurons in vivo via stimulation of protein kinase A (Lalley et al., 1997b; Haji et al., 1996a). The authors discussed that protein kinase A could mediate the stimulating e€ect by phosphorylation of a multitude of possible targets. This results in inhibition of di€erent types of K+ channels, of GABAergic and glycinergic conductances in enhancement of glutamate-mediated excitability. Whatever mechanism will turn out to be primarily responsible for the overall increase in excitability of respiratory neurons after elevation of cAMP (and also for the block of respiratory rhythm when cAMP levels fall below a critical level), it is clear that cAMP is of ultimate importance for respiratory rhythm generation (Arata et al., 1993b; Ballanyi et al., 1997; Meyer et al., 1998, 1999). This is of prospective therapeutic importance for the treatment of pathological disturbances of breathing. 4.2.3. H+ In expiratory neurons of adult cats it was demonstrated that intracellular pH falls by about 0.2 pH units during the IPSP-mediated hyperpolarisation within the inspiratory phase (Ballanyi et al., 1994b). Also application of GABA or glycine in brainstem slices from neonatal rats revealed an intracellular acidosis by a maximal of 0.5 pH units in neurons in the region of the VLM-VRG (LuÈckermann et al., 1997). As described above (section 4.1.2.), these decreases of intracellular pH are likely to be caused by bicarbonate e‚ux through the receptor-coupled Clÿchannels. In their study, LuÈckermann et al. (1997) furthermore showed that a Ca2+/H+ pump mediates a depolarisation-induced sustained fall of intracellular pH in the VLM-VRG neurons. This plasmalemmal ion pump is activated by the rise of intracellular Ca2+ secondary to activation of voltage-gated Ca2+ channels. Thus, both inhibitory and excitatory processes produce a substantial acidosis during neuronal activity (Ballanyi and Kaila, 1998). Since these types of activity-related intracellular pH changes are caused by a transmembrane net in¯ux of acid equivalents, an extracellular alkalosis is expected to occur in the vicinity of the respiratory neurons as was demonstrated for other neuronal tissues (Kaila and Ransom, 1998). In reverse, such changes in intracellular and/or extracellular pH are likely to a€ect the excitability of respiratory neurons as shown for other central neurons (Kaila and Ransom, 1998). This is mainly due to the

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pH dependence of the activity of a diversity of ion channels, in particular several types of voltage-gated conductances like Na+, Ca2+ or K+ channels, but also neurotransmitter receptors. In most cases, pH a€ects speci®c target sites. For example, glutamate or GABA receptors are insensitive to changes in intracellular pH, whereas their function is highly sensitive to changes in extracellular pH. On the other hand, certain types of voltage-gated Ca2+ channels are blocked by intracellular acidosis (Kaila and Ransom, 1998). According to these considerations, it is most probable that H+ ions have a second messenger function in the respiratory network during neuronal activity. However, as their activity produces periodic changes of extra- and intracellular pH per se, it is questionable whether (dendritic) structures of respiratory neurons are well suited as chemosensor structures for hypercapnia- or hypocapnia-induced changes of pH in the brainstem. The receptors mediating such central chemosensitivity

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might rather be located on tonically active neurons close to ventral surface of the medulla (section 6.5.).

4.3. Intrinsic Conductances The following paragraph deals with ion conductances that are not directly regulated by neurotransmitters and that are thought to be involved in generation, modulation or transmission of rhythmic bursting in VLM-VRG neurons of the brainstemspinal cord preparation. It turns out that neonatal respiratory neurons possess most of the (voltagegated) conductances characteristic for other central neurons that are capable of generating rhythmic activity. It will be pointed out that the interaction of speci®c inwardly rectifying K+ channels (Kir) with voltage-gated channels mediating excitation might play a central role in generation of respiratory-related burst activity.

Fig. 11. E€ects of apamin on a type-III Insp neuron. A, the speci®c blocker of SK-type Ca2+-dependent K+ channels apamin (1 mM) results in transient perturbation of inspiratory motor output as recorded from cervical (C4) nerve rootlets, but has no major e€ect on the regularity of rhythmic membrane potential (Vm) ¯uctuations of the medullary respiratory neuron. However, as evident from individual inspiratory bursts, shown at higher time resolution in B, the drug leads to potentiation of inspiratory drive potential. C, apamin (0.5 mM) also e€ectively blocks after-hyperpolarisation following an electrically-evoked action potential. Recordings taken from Ballanyi, Onimaru and Homma (in preparation).

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4.3.1. K+ Channels Neuronal resting potential is typically dominated by K+ conductances. Since respiratory neurons in the intact animal are subject to massive ongoing phasic and/or tonic excitatory and inhibitory synaptic inputs, determination of their resting potential is dicult. Under in vitro conditions, gross synaptic activity can be abolished by suppression of Na+ spikes with tetrodotoxin (TTX). Under these conditions, resting potential of VLM-VRG neurons stabilises at around ÿ50 mV (Figs. 12 and 14; Onimaru et al., 1995, 1996a; Rekling et al., 1996a; Brockhaus and Ballanyi, 1998). These rather low resting potentials are certainly not due to imperfect recording conditions as input resistances are similar (300±700 MO) to those of other central neurons and the cells show Na+ spikes that overshoot to more than +30 mV in the standard TTX-free solution (Smith et al., 1992; Onimaru and Homma, 1992; Onimaru et al., 1996a). Furthermore, during inspiration-related inhibition, Pre-I and Exp neurons can hyperpolarise by more than 20 mV (Figs. 4 and 9) and, muscimol or glycine evoke hyperpolarisations to a maximal of ÿ75 mV (Fig. 8; Brockhaus and Ballanyi, 1998). These observations indicate that resting K+ conductance of neonatal VLM-VRG neurons is rather low. Accordingly, opening of K+ channels during block of aerobic metabolism evokes a stable hyperpolarisation with a maximum of 25 mV in these cells (Fig. 10; Ballanyi et al., 1994a; Ballanyi, 1999b). The origin of this metabolism-related increase in resting K+ conductance is not clear yet. ATP-sensitive K+ channels (section 7.1.) might be involved, since the sulphonylurea blocker of such channels tolbutamide reverses the frequency depression of respiratory activity upon block of aerobic metabolism (section 7.1.; Ballanyi, 1999b). Furthermore, tolbutamide blocks CNÿ-induced outward K+ currents in neurons of the region of the VLM-VRG from thin brainstem slices (Fig. 10; see also Mironov et al., 1998). The anoxic slowing of respiratory rhythm is also reversed by submicromolar concentrations of thyrotropin-releasing hormone (TRH) or by 0.5-l mM Ba2+ (Fig. 10; Ballanyi, 1999b). Interestingly, such Ba2+ concentrations block a novel type of Kir (Kir2.4), that is predominantly expressed in cranial (including hypoglossal) motoneurons (ToÈpert et al., 1998). Kir2.4 also appears to be present in the region of the VLM-VRG, although at low densities (C. Karschin and A. Karschin, personal communication). Kirs can be associated with a receptor-Gi/o protein complex (Smith et al., 1995; Johnson et al., 1996; ToÈpert et al., 1998). Pertussistoxin (and Ba2+) -sensitive Kirs have recently been considered crucial for generation and/or modulation of respiratory rhythm in vitro as Ba2+ was found to e€ectively attenuate GABAB or m-opioid receptor-mediated frequency depression (Smith et al., 1995; Johnson et al., 1996). The Ba2+-induced reversal of anoxiaevoked respiratory frequency depression is, indeed, accompanied by a depolarisation of VLM-VRG neurons (Fig. 10; Ballanyi, 1999b). In contrast, the stimulatory e€ect of TRH on respiratory rhythm after anoxic slowing occurs without a major e€ect

on membrane potential or conductance of the vast majority of VLM-VRG neurons (Fig. 10; Ballanyi, 1999b). However, it was demonstrated that TRH depolarises in particular so-called `type-I' Insp neurons (Rekling et al., 1996b) as well as hypoglossal motoneurons (Bayliss et al., 1992) in medullary slices of neonatal rodents. TRH and Ba2+ also reverse the opioid- or PGE-induced in vitro apnea (Meyer et al., 1998, 1999). A similar stimulation of the neonatal respiratory network after depression by opioids or PGEs is seen upon application of muscarine (Lalley et al., 1998; Meyer et al., 1998, 1999). Thus, it appears that several types of receptor-G proteincoupled K+ conductances, including M-type K+ channels, are important for generation and modulation of in vitro respiratory activity (section 6.; Smith et al., 1995). In contrast to these K+ channels that are thought to determine resting potential (ToÈpert et al., 1998), less is known about the expression or properties of Ca2+- or voltage-dependent K+ currents in neonatal respiratory neurons. The hyperpolarisationactivated A-type K+ current is a characteristic feature of non-respiratory medullary neurons (Champagnat et al., 1986; Trapp and Ballanyi, 1995). So far, there are only two studies hinting at the existence of this current in neurons (in the region) of the VLM-VRG (Johnson and Getting, 1991; Rekling et al., 1996a). Also delayed recti®er K+ channels, which are certainly present in the neonatal VLM-VRG neurons, are yet not analysed. It was, however, shown that the excitability of neonatal VLM-VRG neurons is greatly increased by the K+ channel blockers Ba+, Cs2+ or tetraethylammonium (TEA; Onimaru et al., 1996a; Jaquin et al., 1997), thus revealing regenerative Ca2+ spikes (section 4.3.2.). Ca2+-activated K+ channels appear to be functional in neonatal VLM-VRG neurons as Ba2+ was found to suppress a transient hyperpolarisation after termination of depolarising current pulses (Onimaru et al., 1996a; Rekling et al., 1996a). Furthermore, in some VLM-VRG cells apamin leads to potentiation of the excitatory drive potential and also e€ectively reduces the hyperpolarisation after termination of individual spikes (Fig. 11; Ballanyi, Onimaru and Homma, in preparation). Accordingly, Ca2+-activated K+ channels of the `SK-type' (Sah, 1996) might be important for attenuation of the synaptic drive potential. This drive potential tends to become excessive due to mutual excitatory synaptic connections between Insp neurons (Onimaru et al., 1993; Kashiwagi et al., 1993a) and ampli®cation of synaptic responses by voltage-gated Ca2+ channels (section 4.3.2.; De Schutter and Bower, 1994; see also Fig. 15). However, Ca2+-activated K+ channels related to burst afterhyperpolarisation do not appear to be a typical feature of neonatal VLM-VRG neurons. Despite considerable rises of intracellular Ca2+ during the excitatory phase of the respiratory cycle (Frermann et al., 1999; Koshiya and Smith, 1997), a major hyperpolarisation after termination of the excitatory phase is only seen in some VLM-VRG cells (Figs. 4 and 5). Furthermore, membrane potential ¯uctuations are almost identical when VLM-VRG neurons in the brainstem-spinal cord preparation

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Fig. 12. Ca2+ responses of neonatal respiratory neurons. A, 20 min after addition of 0.2 mM tetrodotoxin (TTX), depolarising current pulses (lower traces) do not elicit action potentials in an Insp-III neuron (recorded with a K+ gluconate-®lled patch electrode). Subsequent exposure to 2 mM Ba2+ and 20 mM tetraethylammonium (TEA) evokes all-or-none Ca2+ spikes and post-stimulus after-depolarisations (arrowheads). B, hyperpolarising current pulses of increasing amplitude reveal a graded low voltage activated (LVA) rebound depolarisation in an Insp-I, cell. C, the superimposed traces show that 0.4 mM o-agatoxin-IVA leads to irreversible suppression of the intermediate voltage activated (IVA) after-depolarisation, whereas it does not substantially reduce pulse-evoked high voltage activated (HVA) Ca2+ spikes. Subsequent addition of 10 mM nifedipine suppresses the Ca2+ spikes, but does not further reduces the after-depolarisation. D, in this cell o-agatoxin-IVA not only suppresses the IVAmediated after-depolarisation (arrows), but also reduces the slope of the initial IVA depolarisation (arrowheads), causing a delay in triggering a HVA Ca2+ spike. E, in the presence of 2 mM extracellular Ba2+, a depolarising voltage ramp (upper trace, holding potential ÿ60 mV) produces an N-shaped current, indicating activation of the IVA Ca2+ response at a threshold of about ÿ47 mV (arrow) in a PreI neuron. Recordings in B±E are done with Cs+/TEA+/Clÿ-®lled patch electrodes in TTX-containing superfusate. Horizontal arrows indicate zero current levels. Recordings are taken from Onimaru et al. (1996a) by permission.

are whole-cell recorded with electrodes containing di€erent types of Ca2+ chelators which a€ect intracellular Ca2+ and, thus, Ca2+-activated K+ channels in vivo (section 4.2.1.). Finally, apamin has only a minor disturbing e€ect on respiratory activity and does not block hyperpolarisations after termination

of respiration-related bursts in neonatal VLM-VRG neurons, whereas it e€ectively blocks spike afterhyperpolarization of these cells (Fig. 11; K. Ballanyi, H. Onimaru and I. Homma, in preparation). Respiratory rhythm is also not perturbed upon block of the two types (SK and BK) of Ca2+-acti-

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vated K+ channels upon combined administration of apamin and charybdotoxin (Smith et al., 1995). 4.3.2. Ca2+ Channels The functional expression of voltage-gated Ca2+ channels in di€erent classes of VLM-VRG neurons in the brainstem-spinal cord preparation of neonatal rats was studied in detail after block of Na+ channels with TTX and suppression of K+ channels with Ba2+, Cs+ and TEA (Onimaru et al., 1996a). Under these conditions, all-or-none repetitive or plateautype action potentials with a mean activation threshold of ÿ33 mV were seen in all cells. These responses are due to high voltage-activated (HVA) Ca2+ channels, presumably of the `L-type' and `Ntype' (Llinas, 1988; Swandulla et al., 1991; Olivera et al., 1994), since both nifedipine and o-conotoxinGVIA lead to reduction of these responses, whereas o-agatoxin-IVA has no e€ect (Fig. 12; Onimaru et al., 1996a; see also Mironov and Richter, 1998). Also low voltage-activated (LVA) Ca2+ spikes (Huguenard, 1996; Llinas, 1988) with a threshold potential of between ÿ60 and ÿ70 mV were revealed after termination of hyperpolarising pulses (Fig. 12; see also Rekling et al., 1996a). However, these graded rebound responses, which are potentiated by Ba2+ and blocked by Co2+, were only detected in less than 20% of neonatal VLM-VRG neurons with a slight preference to Exp cells (Onimaru et al., 1996a). The latter ®nding is in contrast to observations in adult cats, in which LVA Ca2+ responses were found in many inspiratory and almost all expiratory neurons (Richter et al., 1993a). It was speculated that the functional relevance of LVA (`Ttype') Ca2+ currents increases during ontogenetic changes in the mechanisms of respiratory rhythm generation within the perinatal period (section 5.5.). T-type Ca2+ currents are considered as important for rhythm generation in adult mammals, in which prominent membrane potential ¯uctuations are characteristic for all types of respiratory neurons (Richter, 1982, 1996; Richter et al., 1992, 1997). In contrast, VLM-VRG cells of neonatal rats have a resting potential which is rather stable at around ÿ50 mV (Figs. 4 and 5; section 4.3.1.). At such potentials, LVA Ca2+ channels (if at all present in a particular neonatal respiratory neuron) should be inactivated. With the exception of Insp-III neurons, neonatal VLM-VRG neurons depolarise during (Insp cells) or prior to (Pre-I cells) inspiratory burst onset without any preceding hyperpolarisation that could deinactivate LVA Ca2+ channels. Intermediate voltage-activated (lVA) Ca2+ responses are a characteristic feature of Pre-I and Insp-III cells. These IVA Ca2+ currents are activated in the range of the normal membrane potential ¯uctuations of the neonatal VLM-VRG neurons (Fig. 12; Onimaru et al., 1996a). The mean activation threshold of these o-agatoxin-IVA-sensitive `P-type' (Olivera et al., 1994) IVA Ca2+ channels is ÿ42 mV. Therefore, a regenerative slow depolarisation can be initiated in response to minor depolarisations from resting potential. Accordingly, P-type Ca2+ currents might play a potentiating role in maintenance and shaping (and possibly also in in-

itiation) of burst discharge of Pre-I and Insp-III neurons (section 4.3.4.). In accordance with this assumption, it was previously suggested that voltage-gated Ca2+ channels contribute to potentiation of synaptic drive potentials in post-inspiratory neurons of cats (Takeda and Haji, 1993; see also De Schutter and Brower, 1994). Thus it is likely that IVA Ca2+ channels signi®cantly contribute to the intracellular Ca2+ rise during the respiration-related drive potential (Frermann et al., 1999; Koshiya and Smith, 1997) in addition to that caused by activation of LVA and, in particular, HVA Ca2+ channels (Richter et al., 1993a). Besides these `classical' types of voltage-gated Ca2+ channels, yet not identi®ed Ca2+ channels might, at least partly, be responsible for the low resting potentials (section 4.3.1.) of neonatal VLM-VRG neurons. This assumption is based upon the observation that the non-selective blocker of voltage-gated Ca2+ channels Co2+ produces a steady hyperpolarisation (Fig. 13; Onimaru et al., 1996a; K. Ballanyi, H. Onimaru and I. Homma, in preparation). In a recent study, selective Ca2+ channel blockers (Swandulla et al., 1991; Olivera et al., 1994) were used to evaluate the functional role of HVA and IVA Ca2+ channels in burst generation of neonatal respiratory neurons (Ballanyi, Onimaru and Homma, in preparation). The P-type (IVA) Ca2+ channel blocker o-agatoxin-IVA was found to attenuate the burst amplitude of Pre-I and Insp-III neurons (Fig. 13), whereas the L-type (HVA) Ca2+ channel blocker nifedipine shortened burst duration. These results con®rm the above assumption that IVA and HVA Ca2+ channels contribute to potentiation of burst activity in neonatal respiratory neurons. In contrast, the N-type (HVA) Ca2+ channel blocker, o-conotoxin-GVIA increased the burst amplitude, and caused partial inactivation of spike discharge in some type-III Insp neurons (Fig. 13). Since the latter toxin reduced the hyperpolarisation after termination of single evoked spikes similar to apamin (Fig. 11), the increase in burst amplitude is likely to be related to lack of activation of Ca2+activated K+ channels due to reduced Ca2+ in¯ux. In addition to these e€ects on spontaneous bursting and (evoked) action potentials, it was found that oconotoxin-GVIA reduces stimulus-induced PSPs by about 20% and also attenuates phrenic nerve re¯ex responses by more than 30% (Fig. 13). In contrast, nifedipine did not induce any signi®cant change of these responses, but decreased burst duration (K. Ballanyi, H. Onimaru and I. Homma, in preparation). Despite an initial frequency perturbation, respiratory rhythm is not rapidly abolished after concomitant administration of nifedipine, o-conotoxin-GVIA and o-agatoxin-IVA (Fig. 13). This inecacy to suppress respiratory rhythm might be related to restricted tissue di€usion of these high molecular weight voltage-gated Ca2+ channel blockers. As an alternative explanation, R-type Ca2+ currents, which represent those voltage-gated Ca2+ channels that are not a€ected by selective blockers of L-, N- or P-type Ca2+ channels (Wu et al., 1998), might be sucient to maintain synaptic transmission for extended periods. In contrast to nifedipine and

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Fig. 13. E€ects of Ca2+ channel blockers. A, Co2+ leads to rapid suppression of both inspiratory spinal (C4) nerve activity and rhythmic ¯uctuations of membrane potential (Em) of a Pre-I neuron. Note that this unselective Ca2+ channel blocker evokes a reversible hyperpolarisation. B, time course of changes in burst amplitude (upper trace) and burst interval (middle trace) of an Insp-III neuron, and of spinal (C4) nerve burst amplitude (lower trace) upon administration of the L-, N, and P-type Ca2+ channel blockers nifedipine (Nif), o-conotoxin-GVIA (o-CgTX) and o-agatoxin-IVA (o-Aga), respectively. With the exception of reduction of burst duration (B'b), nifedipine did not exert a major e€ect, whereas the conotoxin irreversibly increased neuronal burst amplitude, and decreased C4 burst amplitude to about 50% (see original recordings in B'). Subsequent administration of the agatoxin depressed (the conotoxin-induced potentiation of) neuronal burst amplitude and attenuated C4 activity. C, conotoxin-induced decrease of the amplitudes of C4 burst and EPSP, evoked in an Insp-III neuron by electrical stimulation of the contralateral ventrolateral medulla. Note that respiratory frequency was not a€ected by administration of either of the drugs. Data taken from Ballanyi, Onimaru and Homma, in preparation.

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Fig. 14. Intrinsic bursting of a Pre-I neuron. Aa, after block of respiratory rhythm with a solution of 0.2 mM Ca2+, 5 mM Mg2+ and 0.1 mM Cd2+ membrane potential (Vm) of a Pre-I neuron shows spontaneous oscillations (see also faster sweep of the response in Ab) that are suppressed by currentinduced hyperpolarisation by about 8 mV (lower trace). Ac, after current-induced hyperpolarisation, a depolarising pulse reveals a slow active depolarisation (see arrowheads). B, in the same cell addition of 1 mM Ba2+ to the Cd2+-containing low Ca2+/high Mg2+ solution results (in the absence of rhythmic spinal [C4] nerve activity) in pronounced bursting activity whose rate is decreased upon consecutive hyperpolarisation (Bb Bc). Such spontaneous bursting is abolished with 0.5 mM tetrodotoxin (TTX, Ba). Data taken from Ballanyi, Onimaru and Homma, in preparation).

the toxins, Co2+ or Cd2+ lead to rapid (and reversible) suppression of respiratory rhythm (Fig. 13). 4.3.3. Na+ Channels Action potential-mediated signaling between the di€erent classes of VLM-VRG neurons involves the `classical' TTX-sensitive `Hodgkin/Huxley-type' voltage-gated Na+ channels (Richter et al., 1992; Smith et al., 1995). However, one particular feature of these channels (at least in neonatal rats) is their capability to display a full Na+ spike which overshoots to more than +30 mV, even if resting potential is less negative than ÿ50 mV. Similar large amplitude Na+ spikes are, for example, characteristic for medullary dorsal vagal neurons (Trapp and Ballanyi, 1995; Ballanyi et al., 1996a). It remains to be determined whether the steady-state (in-)activation current-voltage relation of these Na+ channels di€ers from those of other central neurons (Hille, 1992). TTX was found to abolish membrane potential oscillations of neonatal VLM-VRG neurons (Figs. 12 and 14; Onimaru et al., 1995, 1996a). In contrast, voltage-dependent regenerative activity (of Pre-I neurons) is revealed in TTX-free saline after block of synaptic transmission with a low

Ca2+/high Mg2+ solution with Cd2+ added (Onimaru and Homma, 1997). Further application of Ba2+ greatly potentiates these responses. The resulting rhythmic discharge with a single burst duration of up to several seconds is abolished by TTX (Fig. 14). These results indicate that TTX-sensitive persistent Na+ channels (Crill, 1996; Llinas, 1988) are functional in respiratory neurons as was assumed earlier (Richter et al., 1992). These ®ndings agree with recent observations that the persistent Na+ conductance is functional in inspiratory neurons of rhythmic brainstem slices from neonatal mice after postnatal day 2 (Zhang et al., 1997b). The combined operation of such persistent Na+ channels with P-type Ca2+ channels might play a central role in burst generation of VLM-VRG neurons in conjunction with neuromodulator-sensitive, G protein-coupled Kirs (Fig. 15; section 4.3.4.). 4.3.4. Conditional Bursting As described above (section 4.1.2.), inspiratory nerve activity persists in the brainstem-spinal cord preparation after block of inhibitory synaptic transmission (Feldman and Smith, 1989; Onimaru et al., 1990). Furthermore, it was found that rhythmic ®r-

Neonatal Respiratory Network

ing continues in a major proportion of Pre-I neurons after block of synaptic transmission with low Ca2+/high Mg2+ solution (Onimaru et al., 1989). These observations led to the assumption that generation of the primary rhythm is mediated by particular pacemaker neurons with intrinsic bursting properties (Fig. 15; section 5.2.; Onimaru et al., 1989; Feldman and Smith, 1989). More recent (intracellular) studies have con®rmed that membrane conductances, that allow for generation of endogenous rhythmic activity, are a feature of several types of VLM-VRG neurons, in particular Pre-I and Insp cells (Smith et al., 1991; Johnson et al., 1994; Di Pasquale et al., 1994b; Onimaru et al., 1995; Rekling et al., 1996a). Since not all neurons of a given class remain active after block of synaptic transmission, bursting neurons represent subclasses of the active populations of VLM-VRG neurons in vitro (Di Pasquale et al., 1994b; Johnson et al., 1994; Onimaru et al., 1995; Rekling et al., 1996a; Rekling and Feldman, 1998). Furthermore, also non-respiratory neurons in the region of the preBoÈtC have been demonstrated to be capable of endogenous bursting (Smith et al., 1991). In the latter studies, it was demonstrated by intracellular current injection that rhythmic ®ring is typically evoked at membrane potentials less negative than ÿ60 mV (Fig. 14). Further depolarisation leads to an increase in burst frequency until bursting is masked by or transformed into tonic activity at potentials of around ÿ40 mV (Smith et al., 1991; Onimaru et al., 1995; Rekling et al., 1996a). Due to

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the voltage-dependence of their bursting behaviour, the cells are classi®ed as conditional burster neurons. It is assumed that the VLM-VRG burster neurons deserve ongoing excitatory drive (Figs. 7 and 15). This drive might stem from stimulatory e€ects of H+ (Onimaru et al., 1989; sections 4.2.3.; 6.5.), cAMP (Arata et al., 1993b; Ballanyi et al., 1997; section 4.2.2.) or (glutamatergic) inputs from tonic non-respiratory cells (Smith et al., 1995; Funk et al., 1993; Richter et al., 1992). In addition, agents such as H+, adrenaline or cAMP were found to directly stimulate intrinsic bursting of Pre-I neurons, as revealed in the absence of synaptic transmission in low Ca2+/high Mg2+ solution (Arata et al., 1998a; Yamamoto et al., 1992). As pointed out by Johnson et al. (1994), the above described procedure to suppress synaptic transmission with low Ca2+/high Mg2+ solution (containing Cd2+) might fail to identify those bursting neurons that could contribute to rhythmogenesis. These solutions may perturb the intrinsic oscillatory and spiking capabilities in some pacemaker neurons by altering Ca2+, mixed cation or Ca2+ activated K+ channels. Furthermore, block of synaptic transmission could lead to displacement of membrane potential into a range, in which rhythmic bursting is not maintained. It is also known that synaptically released neuromodulators and/or transmitters are crucial for activation of some burst-mediating conductances (Harris-Warick and Flamm, 1987). This agrees with the ®nding that (Pre-I) VLM-VRG neuronal activity is modulated by cAMP-elevating drugs in the

Fig. 15. Putative role of ion channels in bursting activity of neonatal rhythm generating respiratory neurons. During the activity phase, high frequency spiking mediated by Hodgkin±Huxley-type Na+ channels (NaHH) provides Ca2+ in¯ux through L- and N-type of voltage-gated Ca2+ channels (CaL,N). Thus, termination of the activity phase might be supported by Ca2+-activated K+ channels (KCa). Inwardly-rectifying K+ (Kir) and possibly also h- (i.e. Q-type) non-selective cation (H) channels provide a slow depolarisation. P-type (and presumably also T-type) Ca2+ channels (CaP,t) as well as persistent Na+ channels (Nap) are likely to be involved in burst initiation. The steepness of the slow depolarisation and, thus the frequency of bursting, is determined by the level of excitatory drive from tonically active cells of the reticular formation (that may be identical with central chemosensitive neurons). Resting potential of these cells, but also of the burst-generating respiratory neurons themselves, is determined by the concert of a variety of neuromodulators with exclusive excitatory (++), inhibitory (ÿÿ), or biphasic action (+ÿ), mediated via G proteins on the Kirs. Abbreviations: ACh, acetylcholine; cAMP, cyclic adenosine monophosphate; 5-HT, serotonin; (N)E, (nor-) epinephrine; PGE, prostaglandin; SST, somatostatin; TRH, thyrotropin releasing hormone).

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absence of synaptic transmission (Arata et al., 1993b) and, accordingly, most probably also by a variety of neuromodulators a€ecting cAMP (sections 4.2.2.; 6.). So far, there is no experimental evidence for the cellular mechanism that mediates intrinsic bursting in respiratory neurons. As described in detail by Smith et al. (1995), this mechanism appears to be di€erent from those providing endogenous bursting in other oscillatory neurons. It was shown that LVA Ca2+ currents in concert with either Ca2+-activated K+ channels or the `queer' h-type cation current mediate neuronal oscillations in a variety of neurons (Llinas, 1988; Pape, 1996; Huguenard, 1996). However, at least in the neonatal rat LVA Ca2+ currents are only expressed in a minor population of VLM-VRG neurons. Furthermore, in these cells LVA Ca2+ channels are subject to ongoing inactivation due to the low resting potential (section 4.3.2.). As discussed above (section 4.3.1.), Ca2+activated K+ channels are also unlikely to be involved in rhythmogenesis. In the brainstem, the h (or Q)-type current (Pape, 1996) is a feature of mature hypoglossal motoneurons (Berger et al., 1996) and proposed interneurons in the region of the VRG (Johnson and Getting, 1991). However, this hyperpolarisation-activated current has only been found so far in one type of neonatal VLMVRG cells, the so-called `type-2' Insp neurons in slices from mice (Rekling et al., 1996a). Due to the delayed onset of inspiratory activity in these cells, the authors (1996a) hypothesised that this class of VRG cells is not involved in initiation of respiratory rhythm. The non-speci®c CAN current (section 4.2.2.) was shown to be important for rhythmic bursting of snail neurons (Partridge et al., 1990). It was speculated that this current also contributes to the generation of theta-rhythm in the EEG (Partridge et al., 1994). It was furthermore hypothesised that the CAN current participates in potentiation of bursting in respiratory neurons (Richter et al., 1992). However, this current is suppressed by intracellular cAMP (Partridge et al., 1994), whereas the excitability of respiratory neurons in vitro (Arata et al., 1993b; Ballanyi et al., 1997) and in vivo (Lalley et al., 1997a,b) is increased by this secondmessenger (section 4.2.2.). The involvement of IVA or HVA Ca2+ currents in initiation of burst activity is also questionable as bursting of VLM-VRG neurons persists in low Ca2+/high Mg2+ solutions. However, it cannot be excluded that the Ca2+ gradient might still be sucient to provide an excitatory action responsible for burst generation under these conditions (Onimaru, 1995; Onimaru et al., 1995, 1997). The recent observation that TTX suppresses bursting of Pre-I neurons, induced by Ba2+ after block of synaptic transmission with combined administration of Cd2+ and low Ca2+/high Mg2+ solution (Fig. 14) suggests that persistent Na+ channels play an essential role in burst initiation (Fig. 15; Onimaru and Homma, 1997). It is not clear, which conductances produce an initial depolarisation (or decrease of resting conductance) of sucient magnitude to activate the voltage-dependent persistent Na+ channels. Possibly, Kirs are

activated by release of neuromodulators during the inspiratory excitatory phase, thereby causing hyperpolarisation. Termination of the action of the neuromodulators by di€usion or uptake might lead to progressive inactivation of these K+ channels. This would cause a slow and steady depolarisation until the activation threshold of the persistent Na+ channels is reached and the next cycle starts. In this phase, IVA and HVA Ca2+ currents might play a crucial role for ampli®cation of bursting and for increasing intracellular Ca2+ to exert second-messenger functions (section 4.2.1.). In support of this hypothesis, `Rekling et al. type-1 Insp' neurons show a slowly decrementing hyperpolarisation after termination of inspiration (Rekling et al., 1996a; see also Fig. 14). At least in neonatal mice, only the latter type of VLM-VRG neurons is directly depolarised by the neuromodulator TRH (Rekling et al., 1996b). Interestingly, TRH is one of the most e€ective stimulators of in vitro respiratory rhythm, in particular within the prenatal period (Greer et al., 1996b; Meyer et al., 1998, 1999). Furthermore, TRH was found to e€ectively antagonise the opioidor PGE-induced apnea in the brainstem-spinal cord preparation of neonatal rats (Meyer et al., 1998, 1999). In this context, it was shown that TRH and also Ba2+ cause depolarisation and increase in excitability of hypoglossal motoneurons, presumably by blocking yet not identi®ed K+ channels (section 4.3.1.; Bayliss et al., 1992). It was assumed that tonically active neurons in the region of the VLM-VRG provide the excitatory drive to the respiratory rhythm generator (section 5.2.). In this case, the adaptive e€ects of a variety of neuromodulators (section 6.) on respiratory rhythm could, at least partly, be due to modulation of the `slope' of the inactivation of the TRH-sensitive K+ conductance (which might be a particular Kir) of the proposed rhythm generating neurons by inputs from the nonrespiratory cells (Smith et al., 1995; Brockhaus and Ballanyi 1998).

5. RESPIRATORY PATTERN GENERATOR 5.1. `Noeud Vital' Due to the pronounced e€ects of particular lesions on breathing, it was hypothesised by Fluorens (1858) that a `noeud vital' constitutes the respiratory center in the lower brainstem. This view was supported by the studies of Lumsden (1923), who demonstrated that transverse sections of the brainstem lead to consecutive transformation of breathing from eupnea to apneusis, gasping and, ®nally, apnea. This led to the view, that is established in textbooks, of a hierarchical rostrocaudal organisation of the medullary respiratory network. This model proposes two regions in the pons, that critically determine the pattern of breathing. These are a rostral `pneumotaxic center' (in the parabrachialis medialis and KoÈlliker±Fuse nuclei), that is responsible for eupneic breathing, and a more caudal `apneustic center' (Lumsden, 1923). However, a variety of other investigators were unable to reproduce these results of Lumsden. As reviewed previously

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(Feldman, 1986; Bianchi et al., 1995), such profound e€ects of pontine lesions on respiration are only observed in vagotomised animals. In this context, it was demonstrated that vagotomised cats with chronic pneumotaxic area lesions are capable of normal respiratory rhythm in the awake state, but exhibit apneusis under anaesthesia (St John et al., 1972). Nevertheless, PRG neurons have a powerful in¯uence on the pattern of breathing in the intact animal. This is easy to understand as the rostral pons receives from the nucleus of the solitary tract, the area postrema, and the ventral surface of the medulla numerous inputs, related to the regulation of visceral, cardiovascular or neuroendocrine functions (Bianchi et al., 1995). Surprisingly, little respiratory-related neuronal activity appears to be present in the pons of decerebrate cats with intact vagi. In contrast, tonic pontine activity is assumed to produce respiratory modulation when volume-related sensory feedback is suppressed (Bianchi et al., 1995). Accordingly, it was suggested that PRG neurons promote inspiratory termination (`o€-switch') by increasing their phasic activity in the absence of pulmonary feedback (for references, see Bianchi et al., 1995). In particular the nucleus parabrachialis complex of the PRG appears to be involved in mediation of inspiratory o€-switch in the intact animal, as local application of NMDA antagonists or lesion of this area induces apneusis in vagotomised newborn or adult animals in vivo (section 4.1.1.). Due to technical diculties of in vivo recording from PRG neurons, there is only very restricted information on cellular mechanisms of pontine modulation of the medullary RRG (Dick et al., 1994). In contrast, intracellular recordings can routinely be obtained from pontine respiration-related neurons in the brainstem-spinal cord preparation including the pons (Oyamada et al., 1998). That the ponsbrainstem-spinal cord preparation is, indeed, well suited for the analysis of pontine in¯uences on the medullary RRG is suggested by a variety of previous reports. For example, it was shown that local application of NMDA antagonists to the parabrachialis complex or lesion of this area does not produce apneusis (Greer et al., 1991), similar to observations in the in vitro arterially perfused brainstem of adult guinea pigs (Morin-Surun et al., 1995). The latter authors concluded that the duration of the inspiratory phase does no longer depend on NMDA receptor activation in reduced in vitro preparations. On the other hand, electrical stimulation in the nucleus parabrachialis complex in the pons-brainstem-spinal cord preparations induces premature termination of inspiratory burst discharge that depends on both NMDA and GABA receptors (Arata et al., 1996). These results suggest the presence of an NMDA-dependent pathway for inspiratory burst termination in this pontine area in the brainstem-spinal cord preparation. They also indicate that VLM-VRG neurons, involved in the generation of primary rhythm or inspiratory pattern (section 5.2.) must be subject to both, inhibitory and excitatory modulation from pontine neuronal structures (Arata et al., 1996).

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Transection of the in vitro preparation at the pontomedullary junction results in acceleration of respiratory frequency (Fig. 16; Hilaire et al., 1989; Monteau et al., 1989; Smith et al., 1990; Hamada et al., 1992; Grebenstein et al., 1993; Okada et al., 1998). This observation in concert with ®ndings from lesion experiments revealed that the medullary RRG is tonically inhibited by a structure located in the caudal ventrolateral pons, in an area containing the A5 noradrenergic nucleus (Hilaire et al., 1989, 1997; Smith et al., 1990; Hamada et al., 1992). That (nor-) adrenaline is involved in such tonic depression of the RRG is suggested by the ®nding that electrical stimulation of the caudal ventrolateral pons, that inhibits respiratory rhythm in control solution, becomes ine€ective after preincubation with a2 antagonists (section 6.1.; Hilaire et al., 1989). Besides, it was suggested that non-noradrenergic neurons of ventral reticular aspects of the caudal pons function to promote respiratory rhythm in mice (Jaquin et al., 1996; Borday et al., 1997). Furthermore, serotonergic neurons in the midline pontine raphe nuclei might modulate in vitro rhythmic activity of the RRG and also of the hypoglossal motonucleus via diverse subtypes of 5-HT receptors (6.1.; Morin et al., 1990a,b). The lack of occurrence of a major transformation of the pattern of rhythmic nerve bursting after removal of the pons consolidates the conclusion from in vivo work (Feldman, 1986; Bianchi et al., 1995) that pontine structures are not pivotal for generation or maintenance of the primary respiratory rhythm. Thus, it must be assumed that the noeud vital is located within the bilateral column of the medullary VLM-VRG. Accordingly, reduction of the brainstem-spinal cord preparation in rostrocaudal direction by transverse sectioning showed that the in vitro respiratory rhythm is perturbed and, ®nally, irreversibly blocked after removal of de®ned aspects of the VLM-VRG. At present, there is disagreement on the precise location of the site, that is responsible for generation of the in vitro respiratory rhythm. Whereas one study (Onimaru and Homma, 1987) demonstrated that spinal inspiratory activity is blocked after cutting just caudal to the RVL, a further report showed that respiratory rhythm is suppressed only after cutting immediately caudal to the BoÈtzinger complex (Smith et al., 1991). Based upon the present experimental ®ndings, it cannot be argued, whether the RVL/CVL or rather the pre-BoÈtC (which might partially or fully overlap the CVL) constitutes the noeud vital. Besides this uncertainty on the precise anatomical site of the respiratory center, it is generally accepted (Onimaru and Homma, 1987; Feldman and Smith, 1989; Feldman et al., 1991; Smith et al., 1995; Bianchi et al., 1995; Onimaru et al., 1997) that the respiratory network is functionally organised by a central pattern generator that consists of a respiratory rhythm generator (RRG) and an inspiratory pattern generator (IPG). 5.2. Rhythm Generator As pointed out in the review of Bianchi and colleagues (1995), the primary processes of rhythm gen-

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eration even in `simple' neuronal networks, such as that mediating swimming in Tritonia, are poorly understood. Therefore, it can only be speculated at present, which (inter-) cellular mechanisms are involved in generation of rhythmic neuronal activity in the mammalian respiratory network. There exists a variety of diverging hypotheses on how respiratory rhythm is generated in vivo. As example, one model proposes that the primary oscillator is constituted by two particular types of medullary interneurons

(Fig. 7), which feed their activity into the remaining classes of VLM-VRG neurons (Richter, 1982, 1996; Richter et al., 1992). As stated by Bianchi et al. (1995), this model might be incomplete, as it has to take into account independent control of inspiration and expiration (e.g. apneusis). Nevertheless, from Fig. 7B, which is based upon analysis of the pattern of membrane potential oscillations in adult VLMVRG neurons, it is obvious that the vast amount of synaptic interaction between individual populations

Fig. 16. (Pontine in¯uence on) serotonin-induced modulation of the isolated respiratory network. A, in a brainstem-spinal cord preparation with the pons attached, regular inspiratory activity is recorded from spinal (C4) nerves (Ba). Respiratory frequency considerably increases after cutting-induced removal of the pons (dashed line in Ba). Bb, bath-application of 10 mM serotonin (5-HT) leads to acceleration of respiratory rhythm in a preparation with intact pons, whereas the drug rather decreases the frequency in a di€erent preparation with removed pons. Bc, in both types of preparations 5-HT induces, in addition to its e€ects on respiratory frequency, pronounced tonic activity. Recordings in A, B taken from Grebenstein, Ballanyi and Schwarzacher, unpublished. C, in a brainstem-spinal cord preparation without pons attached 5-HT evokes an initial frequency increase of respiratory rhythm, re¯ected by acceleration of rhythmic discharge of an intracellularly recorded Insp-I cell (upper trace) and an extracellularly recorded Pre-I neuron of the contralateral RVL (middle trace). In the later phase of 5-HT administration, in which tonic activity partly obscures respiratory nerve discharge, it is evident from the rhythmic discharge of the slightly depolarised Insp-I cell that the drug decreases the frequency of respiratory rhythm. Note that the Pre-I neuron shows discharge out of phase with respiratory rhythm. Recordings in C taken from Onimaru et al. (1998) by permission.

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of respiratory neurons is due to GABAA-ergic and glycinergic IPSPs. Accordingly, it was proposed that the RRG is constituted by a neuronal circuitry, whose activity critically depends on mutual synaptic inhibition (Richter, 1982, 1996; Richter et al., 1992), as is also assumed for other rhythmically active motor networks (Feldman and Grillner, 1983; Pearson, 1987; Rossignol et al., 1988; Bracci et al., 1996; Grillner et al., 1998). However, inspiratory activity was found to persist upon block of synaptic inhibition in in vitro preparations of the respiratory network, including the brainstem-spinal cord from newborn rats (Fig. 9; section 4.1.2.). This led to the hypothesis that the RRG consists of a hybrid with both, pacemaker and network properties. In the reduced preparations, intrinsic (or `conditional') burster neurons are assumed to generate and transmit rhythmic activity to follower neurons that do not depend on mutual inhibition (Feldman and Smith, 1989; Onimaru et al., 1989, 1990, 1995; Smith et al., 1991, 1995). As explanation for the discrepancy with the in vivo results, an increased number of inhibitory neurons might be responsible for the dependence of rhythm generation in the intact animal on Clÿ-mediated IPSPs (Ramirez et al., 1996; Ramirez and Richter, 1996; Pierre®che et al., 1998). At present there are two major hypotheses on the location of the RRG and the cellular mechanisms of rhythm generation, as derived from ®ndings in the in vitro isolated respiratory network of newborn rats. As outlined above (section 5.1.), there is cur-

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rent disagreement on the exact site of the noeud vital: The group of Onimaru, Homma and coworkers assumes that the RRG is constituted by an excitatory network of Pre-I neurons in the RVL that periodically triggers the IPG composed of Insp neurons in the RVL and CVL. In contrast Smith, Feldman and colleagues propose that neurons of the pre-BoÈtC are responsible for generation of respiratory rhythm. In the following, these models are brie¯y summarised. 5.2.1. RVL-Pre-I Neuron Hypothesis Onimaru, Homma and colleagues observed in the brainstem-spinal cord preparation that spinal inspiratory nerve activity is always preceded by ®ring of Pre-I neurons, although Pre-I neuron activity is occasionally not followed by inspiratory nerve activity (Onimaru and Homma, 1987). Single-pulse stimulation in the RVL was found to induce premature Pre-I neuron bursting and to reset the phase of the respiratory rhythm, whether phrenic activity is induced or not (Fig. 17; Onimaru et al., 1987, 1988). Electrolytic lesions within the RVL lead to reduction of the rate of phrenic activity that can terminate in respiratory arrest (Onimaru et al., 1987). The latter result is consistent with recent ®ndings that microinjection of antagonists of excitatory amino acids (EAA) into the RVL produces apnea in adult rats in vivo (Sun and Reis, 1996). It was furthermore observed that vagal stimulation inhibits inspiratory nerve activity, but does not usually hamper the activity of Pre-I neurons (Onimaru and

Fig. 17. Stimulus-induced resetting of in vitro respiratory rhythm. A, ventral aspect of the brainstemspinal cord preparation including sites of recording and stimulation; dashed line shows level of decerebration. V±XII, cranial nerves; C1 ±C4 cervical ventral nerves. Unit activity of neurons in rostral ventrolateral medulla (RVL) was recorded extracellularly from shaded area with a microelectrode. Inspiratory motor output was monitored with a glass suction electrode. B, right RVL was stimulated (2 mA, 100 ms) via a tungsten electrode. Activity of an individual Pre-I neuron was recorded from left medulla (RVL traces) and C4 activity from right ventral root. Arrows indicate time of stimulation. Delay of stimulation increased from upper to lower pairs of recordings. Note that the stimulation elicited Pre-I neuron activity and reset respiratory phase whether C4 activity was evoked (B3) or not (B1). Taken from Onimaru et al. (1987) by permission.

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Homma, 1987). Bilateral lesions or cooling of the rostral part of the CVL, which may correspond to pre-BoÈtC (section 3.3.) abolish phrenic nerve activity, whereas rhythmic activity of Pre-I neurons in the RVL is preserved (Onimaru et al., 1988, 1997). The latter observations, that indicate an essential role of the CVL in inspiratory burst generation (section 5.3.), are consistent with conclusions from recent results in adult rats in vivo (Koshiya and Guyenet, 1996). Pre-I neuron-like rhythmic activity was found to persist in the RVL, while phrenic activity was abolished after complete transection between the rostral medulla (including the RVL) and the caudal medulla (Onimaru and Homma, 1987). In this context, McLean and Remmers (1994) showed that transection of the medulla at the intermediate level of the RVL in the brainstem-spinal cord preparation consistently decreases phrenic burst rate. Furthermore, Pre-I neurons were demonstrated to possess intrinsic membrane conductances that provide conditional bursting as a precondition of rhythm generation (section 4.3.4.). In about 50% of Pre-I neurons, rhythmic bursting is retained after block of synaptic transmission in low Ca2+/high Mg2+ solution (Onimaru et al., 1989). The view that the in vitro RRG is constituted by Pre-I neurons is further supported by the observation that their activity is preserved upon pharmacological inhibition of GABAA and/or glycine receptors, during reduction of the Clÿ concentration of the superfusate, as well as after block of synaptic inhibition (Onimaru et al., 1990, 1995; Brockhaus and Ballanyi, 1998). Finally, intrinsic bursting of Pre-I neurons was found to be modulated by a variety of

substances such as (nor)adrenaline (Arata et al., 1998a), 5-HT (Onimaru et al., 1998), NMDA (Kashiwagi et al., 1993b), substance P (Yamamoto et al., 1992) or H+ (A. Kawai, H. Onimaru and I. Homma, unpublished observations). Fig. 18 illustrates a model for primary rhythm generation by the excitatory synaptic network consisting of Pre-I neurons with intrinsic bursting properties. The mechanism for generation of intrinsic bursting of Pre-I neurons might involve persistent Na+ and P-type IVA Ca2+ channels (Fig. 15; sections 4.3.2, 4.3.3). 5.2.2. pre-BoÈtC Conditional Burster Hypothesis Smith, Feldman and colleagues (1991) reported that consecutive reduction of the brainstem-spinal cord preparation in rostrocaudal direction by transverse sectioning leads to perturbation of in vitro respiratory rhythm. In this study, the rhythm was irreversibly blocked after removal of aspects of the VLM-VRG, immediately caudal to the BoÈtzinger complex. This led to the assumption that the preBoÈtC (section 3.3.) contains all the neuronal elements that are essential for generation of respiratory rhythm (Smith et al., 1991). Accordingly, transverse brainstem slices containing the pre-BoÈtC have been developed that produce spontaneous inspiration-related hypoglossal nerve activity (Smith et al., 1991; Funk et al., 1993; Johnson et al., 1996) which is indiscernible from that in the brainstemspinal cord preparation (Grebenstein et al., 1993). These in vitro ®ndings led to the assumption that the pre-BoÈtC constitutes the `kernel' of the respiratory network (Smith et al., 1991; Feldman et al., 1991; Richter et al., 1992; Rekling and Feldman, 1998).

Fig. 18. Model of rhythm generation by Pre-I neuron network. In the absence of synaptic connectivity, individual Pre-I neurons of the rostral ventrolateral medulla with intrinsic burst generating properties exhibit independent rhythmic activity with comparatively weak bursting and considerable variation of burst rate. Enhanced burst activity by activation of excitatory synaptic interconnections produces synchronised activity of the entire rhythm generating network. Enhanced Pre-I neuron activity triggers the inspiratory pattern generator of the rostral and caudal ventrolateral medulla which, in turn, inhibits the Pre-I neuron network activity. Thus, many Pre-I neurons show biphasic (i.e. pre- and post-inspiratory) activity.

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That this area is, indeed, critical for respiratory network function is indicated by recent ®ndings that block of Clÿ-mediated inhibition within the preBoÈtC causes respiratory arrest in adult cats in vivo (Pierre®che et al., 1998; Ramirez et al., 1998). Smith, Feldman and colleagues hypothesise that excitatory synaptic coupling between the pre-BoÈtC pacemaker neurons synchronises their depolarisation and bursting within the bilaterally distributed cell population. According to the results from modelling studies, Smith et al. (1995) presumed a spatial and temporal dispersion of onset times of spiking within the population of pacemaker neurons, with a fraction of neurons bursting early (`pre-I4I' neurons; section 3.1.) at the initiation site within the population. The latter sequence of events as hypothesised by Smith, Feldman and colleagues seems to be basically similar to that assumed by Onimaru, Homma et al. (1987, 1989, 1990, 1995) for initiation of inspiratory bursting by the RVL Pre-I neurons. In the models of both groups, the activity of neurons in the preinspiratory phase is thought to be crucial for initiation of in vitro respiratory rhythm. Membrane potential recordings from `pre-I4I' neurons (Fig. 4) imply that this neuron type receives strong excitatory synaptic inputs from Pre-I cells (Onimaru et al., 1997), whereas electrophysiological properties and functional relevance of `pre-I4I' neurons remain to be analysed. As further common assumption in the (otherwise diverging) models by these groups, and also in models by others (e.g. Bianchi et al., 1995; Richter et al., 1992), it is believed that the RRG deserves ongoing extrinsic input from tonically active (beating) neurons (see also Figs. 7 and 15). 5.3. Inspiratory Pattern Generator Independent on whether RVL Pre-I neurons or pre-BoÈtC conditional burster cells generate the primary rhythm in the in vitro preparations of newborn rats, there is agreement on the assumption that the synchronised rhythmic activity is synaptically fed into the network of the follower neurons of the IPG (Fig. 7). The IPG, that is thought to be composed of Insp neurons, is responsible for inspiratory motor output in the isolated neonatal respiratory network (section 2.1.). Inspiratory activity starts when excitatory synaptic inputs from the RRG to the IPG reach the activation threshold by temporal and/or spatial summation (Onimaru et al., 1992; Onimaru, 1995). Burst activity is maintained by excitatory coupling among Insp neurons (Kashiwagi et al., 1993a; Onimaru et al., 1993) and related activation of `intrinsic' conductances, despite a marked reduction of inputs from the RRG by feedback inhibition from the IPG. Insp-II and Insp-III neurons may participate in maintenance of burst activity and also function to distinguish the inspiratory phase from pre- and post-inspiratory phases. The IPG shows a transition from the active to the silent phase as soon as network activity decreases below a certain level, owing to gradual increase in the ®ring threshold of Insp neurons during burst activity. Thus termination of the inspiratory burst is prob-

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ably independent on inhibitory synaptic inputs to the IPG (Onimaru et al., 1990; section 4.1.2.). Immediately after inspiratory o€-switch, the IPG is subject to a strong refractory period, and the threshold gradually decreases to permit a new inspiratory burst after several seconds (Onimaru et al., 1997). These intrinsic properties of the IPG could result in a higher trigger threshold and a smaller output amplitude of in vitro phrenic activity at shorter cycle time, i.e. at higher burst rate. The cellular mechanisms responsible for termination of inspiratory burst and refractoriness during the postinspiratory phase are yet unknown. These properties of the IPG might not only be due to changes in membrane conductances of VLM-VRG neurons localised in the region of the soma and/or axon, which are closely related to initiation and maintenance of spike discharge (section 4.3.). They might also depend on pre- and/or post-synaptic changes in the ecacy of synaptic transmission. Insp neurons which show after-hyperpolarisations after termination of bursting (section 3.3.1.; Onimaru et al., 1997; Rekling et al., 1996a) might be responsible for generation of refractory period of the IPG. Although the RRG and IPG interact via mutual synaptic connections, it is possible that they constitute networks that can operate independent of each other (Onimaru et al., 1988). The rate of inspiratory burst activity could be a€ected by modulation of properties of both, the (Pre-I-mediated) RRG and (Insp-mediated) IPG. At least three mechanisms could be responsible for pharmacologically-induced frequency reduction of inspiratory output activity of the respiratory network in the brainstem-spinal cord preparation: (i) a decrease in the rate of Pre-I neuron bursts observed upon administration of adrenaline (Arata et al., 1998a) or GABA (Onimaru et al., 1990); (ii) a decrease in the intraburst ®ring frequency of Pre-I neurons, resulting in failure to trigger the IPG during 5-HT (Onimaru et al., 1998); and (iii) a direct (postsynaptic) or indirect (presynaptic) inhibition of the IPG (vagal stimulation, Onimaru and Homma, 1987; opiate- or PGEinduced respiratory depression, Ballanyi et al., 1997). Depending on the level of rostral transection of the medulla, rhythmic brainstem slices contain more rostral parts of the VLM and thus a larger population of RVL Pre-I neurons. It is presumable that in such slices the Pre-I neuron network serves as a main source to trigger inspiratory burst generation (Onimaru et al., 1988; 1997). In slices that lack a major proportion of aspects rostral to the rostral end of the CVL, conditional bursting of pre-BoÈtC cells within the network of Insp IPG neuron may substitute for generation of rhythmic activity (Smith et al., 1991; Funk et al., 1993; Johnson et al., 1996). 5.4. Bilateral Interactions A bilateral distribution of VLM-VRG neurons (including the noeud vital) is in agreement with a basically bilateral organisation of respiratory (e.g. intercostal) muscles and thus spinal motoneurons. In the reduced in vitro preparations it is possible to analyse whether the bilateral connectivity of both columns of the VLM-VRG is essential for gener-

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ation of respiratory rhythm. It was found that midsagittal sections in the brainstem-spinal cord preparation, extending the entire length of the medulla and thus separating the preparation into two halves, abolishes rhythmic activity in both, spinal and hypoglossal nerve roots (McLean and Remmers, 1994). Furthermore, midline incision from the ventral surface at the CVL level (1 mm deep, 0.5 mm long) decreases the amplitude of spinal inspiratory activity without a€ecting its frequency or burst duration (H. Onimaru, A. Arata and I. Homma, unpublished observations). Finally, unilateral lesion in the CVL causes disappearance of spinal inspiratory activity, although it gradually recovers about 10 min after the lesion (Onimaru et al., 1988). These results are consistent with the idea that mutual excitatory connections between bilateral groups of Insp neurons in the VLM are important for generation and maintenance of inspiratory burst activity (Loeschcke, 1982; Feldman, 1986; Ezure and Manabe, 1989; Kashiwagi et al., 1993a; Onimaru et al., 1993; see also Funk et al., 1993). Similarly, bilateral synaptic interactions contribute to synchronisation of burst activity of Pre-I neurons and therefore to trigger the IPG (Onimaru et al., 1987, 1988). In contrast, intrinsic bursting of individual Pre-I neurons is independent on bilateral synaptic connections (Onimaru et al., 1989, 1991). 5.5. Developmental Aspects In many brain areas such as the visual cortex or the auditory system, major changes in the structure and synaptic organisation occur in the early postnatal period in the context of learning and plasticity. In contrast, the respiratory network deserves to produce a stable rhythm immediately after birth to provide sucient oxygen supply of the body. Therefore, it can be assumed that the basic connectivity and functional organisation of the network is established at birth. As example, Clÿ-mediated IPSPs are hyperpolarising in the neonatal respiratory network, whereas IPSPs are depolarising in most other brain regions at birth (section 4.1.2.). Nevertheless, due to postnatal changes, for example in retractory forces of the lung or in functional adaptation of breathing (see response to hypercapnia; section 6.5.) some changes are expected to occur also in the organisation of the respiratory network. Indeed, it was shown that the motor pattern of hypoglossal nerve, and also the strength of coupling between inspiratory burst activity of hypoglossal nerve and preBoÈtC neurons changes considerably within the ®rst three postnatal weeks in rodents (Paton et al., 1994; Paton and Richter, 1995a; Ramirez et al., 1996). Furthermore, it was assumed that the relevance of synaptic inhibition for generation of respiratory rhythm increases profoundly after birth. This assumption is based upon the ®nding that respiratory rhythm in the brainstem-spinal cord preparation from neonatal rats persists after block of inhibition, whereas rhythmic activity is abolished under these conditions in adult mammals in vivo (section 4.1.2.). Indeed, one particular comparative in vivo/in vitro developmental study presented evidence for a profound increase in the relevance of glycinergic inhi-

bition for rhythm generation (Paton and Richter, 1995b). The same report proposed that the functional role of GABAA receptors in the respiratory network of rodents changes over the ®rst two weeks. Other studies on medullary slices have, however, presented evidence that Clÿ-mediated inhibition is not essential for maintenance of respiratory activity in rather mature mice (Ramirez et al., 1996; Greer et al., 1996a). As pointed out by Ramirez and Richter (1996), there is strong evidence that in vitro preparations of the respiratory network generate rhythmic activity in the absence of synaptic inhibition, irrespective of age. Accordingly, it was hypothesised that the higher relevance of synaptic inhibition in the intact animal is due to an increased number of inhibitory neurons (Ramirez and Richter, 1996; Ramirez et al., 1997). In contrast to the uncertainty on a developmental change in the relevance of Clÿ-mediated inhibition for rhythm generation, it is established that the tolerance of the respiratory network of rodents to metabolic disturbances decreases dramatically within about ten days after birth. As will be pointed out in detail below (section 7.2.), these di€erences are primarily due to a decrease in the ability of respiratory neurons to utilise anaerobic metabolism (Ballanyi et al., 1992; 1996a,b). There is also increasing evidence that the sensitivity of the respiratory network of rodents to neuromodulators changes considerably within the perinatal period. As one example, both TRH and 5-HT exert a strong stimulatory e€ect on respiratory frequency in isolated medulla preparations from embryonic rats, whereas only a modest acceleration (or even a slowing) of rhythmic activity is seen in newborn pups (Di Pasquale et al., 1994a; Greer et al., 1996b; Meyer et al., 1999; Onimaru et al., 1998). Furthermore, it was recently revealed that PGE1 has a potent excitatory action on respiratory rhythm in brainstem-spinal cord preparations from embryonic (E) age E18±E19 rats, whereas the drug, at the same nanomolar concentrations, evokes longlasting in vitro apnea in E20±P1 rats (Ballanyi et al., 1997; Ballanyi, 1999a; Lalley et al., 1998; Meyer et al., 1998, 1999). In accordance with the view that PGE might serve to suppress the respiratory network in utero shortly before birth, it was found that the blocking e€ect of the drug profoundly decreases in brainstem-spinal cords from pups older than 1 day (Meyer et al., 1998, 1999). These observations agree well with the clinically relevant observation of a depressing action of prostaglandins on preterm, but not on older infants (Ballanyi et al., 1997). The developmental state of the brain of neonatal rats is comparable with that of the fetal human brain (Alling, 1985). This led to the hypothesis that the brainstem-spinal cord preparation from neonatal rats is an excellent model for the analysis of the human respiratory network in particular within the perinatal period (Meyer et al., 1998, 1999). These examples show that the developmental state of animals used for in vitro models of the respiratory needs to be considered in addition to species di€erences (Ballanyi, 1999a). Neonatal rats di€er considerably from mice, and even distinct strains of mice (e.g. inbred vs outbred strains) are genetically

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not closely related with each other. For example, respiratory activity is not revealed in brainstemspinal cord preparations from precocial mice that are born at a much more mature state compared to other neonatal rodents (Greer et al., 1996a). This might be due to the low tolerance of this en bloc preparation to anoxia, since rhythmic slices from the same precocial mice, most likely experiencing improved oxygen and substrate supply (Brockhaus et al., 1993), produce a stable respiratory rhythm (Greer et al., 1996a). A further example for species and developmental di€erences of in vitro respiratory activity is the opossum. In isolated en bloc preparations from animals with an age of up to two weeks, regular respiratory rhythm with a frequency of around 40 bursts minÿ1 is recorded (Eugenõ n and Nicholls, 1997). With regard to the primary process of rhythmogenesis, it cannot be resolved at present in the di€erent established in vivo and recent in vitro preparations, whether respiratory rhythm in intact or reduced neonatal or adult mammals is better described by a pacemaker or a multiphase network model or a pacemaker-network hybrid (Feldman and Smith, 1989; Onimaru et al., 1989; Smith et al., 1991; Richter et al., 1992). It appears, however, that the primary mechanisms of rhythm generation may not substantially change during life (Funk and Feldman, 1995).

6. MODULATION OF RHYTHM 6.1. Biogenic Amines Respiratory network functions are subject to ongoing (state-dependent) modulation by biogenic amines such as 5-HT, adrenaline, noradrenaline or dopamine (Eldridge and Millhorn, 1981; Bianchi et al., 1995). Several in vivo reports presented evidence that these substances predominantly exert their actions on the respiratory network via modulation of G protein-induced e€ects on second messengers (Haji et al., 1996a; Lalley et al., 1994a,b, 1995; Richter et al., 1997; section 4.2.). Very similar complex responses of the biogenic amines as seen in vivo are revealed in the isolated respiratory network in the brainstem-spinal cord preparation. Due to involvement of a variety of receptor subtypes, acting on di€erent pathways, the overall response of the in vitro respiratory network to these neuromodulators is critically determined by their concentration. For example, administration of 10 mM dopamine results in an increase of respiratory rate, whereas a biphasic response (initial frequency stimulation, late frequency depression) is observed after addition of 30 mM of the drug (Murakoshi et al., 1985). As discussed for the sensitivity of the brainstem-spinal cord preparation to chemostimuli (section 6.5.), the response to biogenic amines (and other neuromodulators) seems to depend on the metabolic state of and/or isolation procedure for the preparation, and also on whether the pons is retained (section 5.1.). Accordingly, it was found by transection experiments that a solely stimulating e€ect of 5-HT on in vitro respiratory frequency in a subpopulation of brainstem-spinal cords is transformed into a de-

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pressing action after removal of the pons (Fig. 16; Di Pasquale et al., 1992; Grebenstein et al., 1993). In contrast to 5-HT and (nor)adrenaline (see below), the e€ects of dopamine on the isolated respiratory network have not been analysed in much detail until now. In addition to the above mentioned stimulatory e€ect of the drug on the burst rate of the in vitro respiratory rhythm, it was recently demonstrated that the selective D1 receptor agonist 6-chloro-APB e€ectively antagonises long-lasting in vitro apnea in response to opioids or PGE1 (Ballanyi et al., 1997). In this study, it was also shown that the drug evokes a slow non-respiratory rhythm in addition to reactivating respiratory activity. Recent analysis has shown that the slow 6chloro-APB-evoked rhythm corresponds to ®ctive locomotion, that is generated in the spinal aspect of the preparation (S. HeibuÈlt, E. Schomburg and K. Ballanyi, in preparation). 6.1.1. (Nor)adrenaline A5 neurons in the pons are assumed to constitute a major source of noradrenaline (norepinephrine). It was shown by Hilaire et al. (1989) that the a2-adrenergic receptor antagonists yohimbine and idazoxan accelerate the respiratory rhythm in brainstemspinal cord including the pons, whereas the drugs have no e€ect in preparations lacking the pons. In the latter study, it was furthermore found that a depressing e€ect of electrical stimulation of the caudal ventral pons on respiratory rhythm is blocked by the a2 receptor antagonists. These observations indicate that the mechanisms underling the biosynthesis of noradrenaline in the A5 region remain functional in vitro, and that the medullary RRG is subject to a tonic noradrenergic inhibitory drive that originates in this area of the pons (see also Di Pasquale et al., 1992; section 5.1.). This assumption was substantiated by the observation that ionophoretic injection of noradrenaline into the A5 region (Hilaire et al., 1989), or bath-application of the drug to the pontine compartment in double-bath experiments (Errchidi et al., 1991), leads to a rise of respiratory frequency, that is most likely related to inhibition of A5 noradrenergic neurons. In contrast to the excitatory e€ect of pontine administration of noradrenaline on the RRG, bath-application of the drug in brainstem-spinal cord preparations lacking the pons has a primarily depressing action on respiratory activity (Hilaire et al., 1989; Errchidi et al., 1991). Based upon results from electrical stimulation, lesions and local application of noradrenaline, it was suggested that a2 receptors are involved in such depression of respiratory rate, and that the main site of the action of noradrenaline is located in the RVL (Errchidi et al., 1991). In this context, a depressing action on respiratory frequency was also found upon bath-application of noradrenaline or during pressure injection into the pre-BoÈtC of rhythmic medullary slices from neonatal rats (Al-Zubaidy et al., 1996). Errchidi et al. (1991) demonstrated that (nor)adrenaline induces a1 receptor-mediated tonic phrenic nerve activity in vitro and hypothesised that this response is of medullary origin. In contrast with that view, a

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recent study presented evidence that (nor)adrenaline-induced tonic spinal nerve activity could also well be mediated by neuronal structures in the spinal cord (Arata et al., 1998a). In the latter report, it was furthermore demonstrated that the RRG is more sensitive to depression by adrenaline than noradrenaline and that the inhibitory e€ect is mediated by a2 receptors. In addition, the authors revealed an indirect inhibitory e€ect of these amines that involves a1-adrenergic activation of inhibitory GABAergic neurons. It was furthermore demonstrated that a1 receptor activation directly excites Pre-I VLM-VRG neurons, whereas their rhythmic activity is inhibited by activation of a2 adrenergic receptors (Arata et al., 1998a). In this report, adrenaline was found to induce rhythmic bursting in some Pre-I neurons after suppression of synaptic transmission with low Ca2+/high Mg2+ solution. Therefore, (nor)adrenaline acts as a strong burst-inducing factor as shown for sympathetic preganglionic neurons in the upper thoracic cord of the cat (Yoshimura et al., 1987). The long-lasting depression of respiratory rhythm, produced by the a2 agonist clonidine was reversed by forskolin, an activator of adenylyl cyclase (Arata et al., 1993b). Thus, adrenaline might regulate the Pre-I network by a€ecting intracellular cAMP levels (section 4.2.2.). In line with the latter ®ndings of involvement of both stimulatory a1 receptors and inhibitory a2 receptors, it was revealed that subpopulations of Pre-I cells were either excited or depressed upon bath-application of adrenaline (Arata et al., 1998a). Which type of response is elicited upon the latter unselective activation of several subtypes of adrenaline receptors might be due to di€erences in the regulatory state of the G protein-regulated (K+) conductances in a given cell (North et al., 1987; ToÈpert et al., 1998). These ®ndings of diverging responses of the (nor)adrenergic modulation even in VLM-VRG neurons of one particular type in the isolated respiratory network help to explain previous, apparently contradicting, in vivo observations. These studies showed that catecholamines on the one hand stimulate ventilation, whereas they typically depress rhythmic activity of medullary respiratory neurons (Champagnat et al., 1979). 6.1.2. Serotonin Similar to (nor)adrenaline, 5-HT appears to control respiratory network functions by highly complex mechanisms. 5-HT containing axons from neurons of the raphe complex, that show ongoing respiratory-modulated activity, terminate in close proximity to VLM-VRG neurons (Lalley et al., 1994a,b, 1995, 1997). Accordingly, electrical or chemical stimulation of the raphe complex was found to produce either excitation or inhibition of respiratory neurons, the e€ect being dependent on the site of stimulation. When 5-HT analogues are applied systemically, intraventricularly or extracellularly, the discharge of bulbar respiratory neurons is mostly depressed, whereas synaptically coupled motoneurons are generally activated. Finally, ionophoresis of 5-HT near to respiratory neurons has similar diverse e€ects on VLM-VRG neurons (Arita

and Ochiishi, 1991; Lalley et al., 1994a,b, 1995, 1997). As suggested for (nor)adrenaline (see above), the mechanisms for biosynthesis of 5-HT seem to remain functional in the brainstem-spinal cord preparation from newborn rats. Ongoing release of endogenous 5-HT appears to exert a steady excitatory action on the medullary RRG (Morin et al., 1990a,b). Electrical or chemical stimulation of the midline raphe nuclei in the pons, which should lead to release of endogenous 5-HT, was found to increase respiratory rate (Morin et al., 1990a). In contrast with the consistent excitatory e€ect of raphe stimulation on in vitro respiratory activity (Al-Zubaidy et al., 1996), bath-application of 5-HT produces a rather non-uniform e€ect. Several groups found that the drug consistently induces a biphasic (initial accelerating, secondary slowing) modulation of inspiratory frequency (Fig. 16; Murakoshi et al., 1985; Onimaru et al., 1998). Although such a biphasic e€ect was also revealed in a subpopulation of brainstem-spinal cords in the study of Grebenstein et al. (1993), these authors showed that bath-applied 5-HT can also elicit either a solely stimulating or inhibitory e€ect in other preparations (Fig. 16). Finally, Hilaire, Monteau and colleagues described an exclusively accelerating e€ect on the RRG (Monteau et al., 1990b; Morin et al., 1990a). These complex and diverging responses might be secondary due to a di€erent activity state of the in vitro respiratory network under resting conditions. In accordance with that view, it was found that the stimulating e€ect of the biphasic response to 5-HT was more prominent, when control burst rate was low (Onimaru et al., 1998). In contrast, the depressing e€ect was more obvious when baseline respiratory frequency was high (Onimaru et al., 1998). Due to the presumed pivotal role of cellular cAMP levels for the RRG (section 4.2.2.), it might well be that the depressing e€ect of 5-HT is seen in preparations, in which cellular levels of cAMP are high enough to provide maximal stimulation of the in vitro respiratory network, whereas in `slow' preparations a rise of cAMP, causing frequency stimulation, is induced by particular 5-HT receptor subtypes (Onimaru et al., 1998). One study reported that the (monophasic) stimulatory e€ect of bath-applied 5-HT on respiratory frequency is abolished after removal of the pons (Di Pasquale et al., 1992; see also Fig. 16). However, other groups rather reported a biphasic 5-HT e€ect in brainstem-spinal cord preparations with or without pontine structures attached (Murakoshi et al., 1985; Grebenstein et al., 1993; Onimaru et al., 1998). To further disprove the assumption by Di Pasqale et al. (1992) that the divergence in the response to 5-HT depends on the rostrocaudal extension of the preparation, the biphasic e€ect was also detected in transverse slices containing the pre-BoÈtC (Ballanyi et al., 1998b). In the latter study, a subpopulation of rhythmic slices responded with frequency depression to bath-applied 5-HT. Slowing of respiratory activity (if rhythm was at all a€ected) has been described as the predominant modulatory action of 5-HT in the slice study of Johnson et al. (1996). In contrast, respiratory rate was found to

Neonatal Respiratory Network

increase in a di€erent report on such transverse medullary slices (Al-Zubaidy et al., 1996). In the latter study, modulatory e€ects on in vitro respiratory frequency, identical with those upon bath-applied 5HT, were observed upon selective application via pressure injection into the pre-BoÈtC. In agreement with the suggestion by these and other authors (Di Pasquale et al., 1992; Johnson et al., 1996) that the serotonergic modulation of the respiratory network is due to a localised action on particular VLM-VRG neurons, it was found that 5-HT has direct e€ects on the excitability of Pre-I neurons that are proposed to constitute the RRG (Onimaru et al., 1998). In these RVL cells, 5-HT induced a time-dependent modulation of burst properties, that consisted of an increase in burst rate and a delayed decrease in intraburst ®ring frequency and amplitude (Fig. 16; Onimaru et al., 1998). Under the assumption that Pre-I neurons trigger the IPG (section 5.3.; Onimaru et al., 1992; 1988), a biphasic modulation of respiratory rhythm by 5-HT could be explained by an initial increase in the burst rate of Pre-I neurons. This increase causes a rise in the burst rate of Insp neurons, while the intraburst ®ring frequency of Pre-I cells is still high enough to trigger burst generation of Insp neurons. Subsequent reduction of the intraburst ®ring frequency of Pre-I neurons and disturbance of synchronised activity of Pre-I neuron bursts may lead to a decrease in the temporal and spatial summation of excitatory synaptic inputs to type-I Insp neurons from Pre-I neurons. This might result in failure to trigger Insp burst generation, thereby causing a decrease in the Insp neuron burst rate and, thus, respiratory rate. It was suggested that the initial excitatory e€ect is mediated by 5HT2A receptors, whereas the 5-HT2C receptor subtype is responsible for the inhibitory e€ects of 5-HT (Onimaru et al., 1998). In the latter study, it was found that 5-HT leads to a membrane depolarisation of both, RVL Pre-I (Fig. 16) and Insp neurons. This depolarisation is proposedly caused by an increase in a mixed Ca2+/ Na+ conductance in addition to a decrease in a K+ conductance (Onimaru et al., 1998). Also one minor subpopulation of pre-BoÈtC neurons of rhythmic slices was found to be subjected to a moderate depolarisation of less than 5 mV amplitude in response to 5-HT (Ballanyi, Pestean and Schwarzacher, in preparation). It remains to be analysed, whether these depolarising responses of neonatal VLM-VRG neurons are mediated by 5-HT2 receptors, as shown for expiratory and post-inspiratory neurons in vivo (Lalley et al., 1994b). In contrast to depolarisation of RVL Pre-I neurons and of some pre-BoÈtC neurons, most pre-BoÈtC neurons in rhythmic slices did not respond to bath-applied 5-HT, or to injection of the drug into the pre-BoÈtC, or were hyperpolarised by less than 5 mV. These responses to 5-HT were not accompanied by a noticeable change in membrane conductance (K. Ballanyi, A. Pestean and S. Schwarzacher, in preparation). Such lack of occurrence of a major 5-HT-induced hyperpolarisation in neonatal respiratory neurons is in contrast with observations in VLM-VRG neurons in vivo (Lalley et al., 1994a,b). This is certainly not due to inactivation or washout of second messengers in the neo-

619

natal neurons by dialysis via the patch electrode. Non-respiratory neurons, recorded under identical in vitro conditions, were found to respond with a prominent hyperpolarisation and conductance increase to activation of G protein coupled GABAB receptors (Brockhaus and Ballanyi, 1998). Although detailed information on the intracellular signal transduction cascades of 5-HT e€ects on respiratory neurons is lacking at present, it is important to note that 5-HT1A receptors can either be negatively or positively coupled to adenyl cyclase (reviewed in Hoyer et al., 1994). The resulting modulation of cAMP levels was previously suggested to be important in regulation of in vitro respiratory rhythm (section 4.2.2.; Arata et al., 1993b; Ballanyi et al., 1997; Meyer et al., 1998, 1999). Accordingly, it was shown that activation of 5-HT1A receptors leads to G protein-dependent activation of an inwardly rectifying K+ conductance (Umemiya and Berger, 1995). In contrast, 5-HT2 receptor activation was demonstrated to stimulate phosphatidyl inositol metabolism, thus causing an increase in IP3 production (Martin and Humphrey, 1994). Excitatory e€ects by 5-HT2 receptor activation are suggested to be a result of decrease in resting K+ conductance and/or enhancement of Ih current (Aghajanian and Andrade, 1997). In contrast to the moderate e€ects of 5-HT on VLM-VRG neurons, the drug produces a depolarisation by up to 20 mV of both spinal (phrenic) and cranial (hypoglossal) motoneurons. The accompanying tonic motoneuronal discharge can obscure respiratory nerve activity in vitro (Fig. 16; Morin et al., 1990a; Monteau et al., 1990b; Onimaru et al., 1998). Since non-phasic action potential activity is typically not observed in the VLM-VRG cells during 5-HT (Grebenstein et al., 1993), the tonic XII nerve activity is most likely due to a direct postsynaptic e€ect on the motoneurons and not caused by an increase in excitatory drive from the respiratory network. The ®nding that ketanserine and methysergide blocked the 5-HT-induced depolarisation of individual hypoglossal cells as well entire tonic hypoglossal nerve activity (Al-Zubaidy et al., 1996; Ballanyi, Pestean and Schwarzacher, in preparation) indicates involvement of 5-HT1C/2 receptor-mediated block of K+ channels (Lindsay and Feldman, 1993). In the presence of the latter drugs, it becomes evident that 5-HT does not diminish inspiration-related XII nerve burst activity or rhythmic discharge of hypoglossal motoneurons, as was proposed by others (Monteau et al., 1990b; Morin et al., 1992). That inspiratory drive potentials of these cells are not profoundly a€ected by 5-HT is also obvious when tonic spike discharge of the recorded cell is suppressed by current-induced hyperpolarisation (Ballanyi, Pestean and Schwarzacher, in preparation). In contrast, it was found that 5-HT rather potentiates subthreshold inspiratory drive potentials, thus leading to rhythmic bursting in some hypoglossal cells. The latter observation of an excitatory e€ect of 5-HT on the drive potentials indicates that presumed presynaptic inhibition of hypoglossal motoneurons, mediated by 5-HT1B receptors (Singer et al., 1996) plays only a minor role under the experimental condition of a global increase in 5-HT con-

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centration in the functioning isolated respiratory network. These ®ndings are in line with those of recent in vivo studies, which demonstrated that administration of 5-HT to the hypoglossal motonucleus leads to strong activation of hypoglossalinnervated muscles and that (endogenous) 5-HT provides a prominent excitatory drive to the XII motoneurons (Khater-Boidin et al., 1996; Kubin et al., 1992; Rose et al., 1996). A similar excitatory e€ect of serotonin was reported for inspiratory active cervical motoneurons in the brainstem-spinal cord preparation of neonatal rats. (Morin et al., 1990a, 1991, 1992). 6.2. Adenosine Adenosine, which plays a key role in energy metabolism (section 7.1.), has been demonstrated to reduce neuronal excitability in nervous structures, associated with higher brain functions (Greene and Haas, 1991). Since interstitial levels of adenosine increase considerably during neuronal activity, it is assumed that this inhibitory neuromodulator serves as a mediator of negative feedback interaction between metabolism and electroresponsiveness (Greene and Haas, 1991). In a variety of neurons, the inhibitory e€ect of adenosine is mediated by preand/or postsynaptic A1 receptors, resulting in activation of a G protein-coupled K+ channels (Greene and Haas, 1991; Thompson et al., 1992; Umemiya and Berger, 1994). Accordingly, it was found in adult cats in vivo that adenosine produces an 8cyclopentyl-1,3-dipropylxanthine (DPCPX)-sensitive and, thus, Al receptor-mediated postsynaptic hyperpolarisation as well as suppression of spontaneous and evoked PSPs of VLM-VRG neurons (Schmidt et al., 1995). Also in the brainstem-spinal cord preparation from newborn rats, adenosine exerts an inhibitory e€ect on PSPs. The drug produces DPCPX-sensitive depression of subthreshold EPSPs of VLM-VRG neurons occurring within the expiratory phase. In contrast, inspiration-related EPSPs and, thus, drive potentials are only slightly reduced and IPSPs are not a€ected (Brockhaus and Ballanyi, 1999). This (presynaptic) e€ect was found by the latter authors (1999) to stabilise the in vitro respiratory rhythm. This stabilising action is in particular evident, when rhythmic activity is perturbed by elevation of the K+ concentration of the superfusate. As further example for this action of adenosine, the drug suppresses Dl dopamine (section 6.3.1.) receptorinduced non-respiratory tonic activity as well as a slow rhythm that represents ®ctive locomotion (Brockhaus and Ballanyi, 1999). In the latter study, it was also shown that adenosine antagonises bicuculline-induced spinal epileptiform discharge without disturbing respiratory rhythm (Fig. 9). In contrast, block of GABAA-ergic inhibition with bicuculline did not evoke seizure-like discharge in hypoglossal nerve rootlets after removal of the spinal aspect from the brainstem-spinal cord preparation (Brockhaus and Ballanyi, 1999). The authors hypothesised (1999) that elevation of interstitial adenosine acts as an endogenous anticonvulsant in this isolated respiratory network as described

for other (isolated) brain tissues (Greene and Haas, 1991). In contrast with these ®ndings, Herlenius et al. (1997) described that A1 receptor agonists as well as block of adenosine uptake decrease extracellularly recorded spike activity and also phrenic burst rate. It was furthermore shown that administration of A1 agonists to the spinal aspect of the brainstem-spinal cord preparation does not a€ect respiratory burst rate, whereas phrenic burst amplitude as well as inspiratory drive currents of phrenic motoneurons are attenuated (Dong and Feldman, 1995). The latter authors (1995) also found that A1 receptor activation does not increase a postsynaptic K+ conductance, as was also reported for hypoglossal motoneurons in non-rhythmic slices (Bellingham and Berger, 1994). Similar to lack of a direct postsynaptic action of adenosine on spinal and cranial motoneurons in vitro, the drug does not have a major direct e€ect on resting potential and conductance in all types of neonatal VLM-VRG neurons of the brainstem-spinal cord preparation (Brockhaus and Ballanyi, 1999). It should, however, be noted that an apparent hyperpolarisation of Pre-I neurons by adenosine might be secondary to reduction of non-respiratory related EPSPs (Brockhaus and Ballanyi, 1999). Findings from several in vivo studies suggested that adenosine is involved in respiratory depression during anoxia (Richter and Ballanyi, 1996). However, as discussed in detail in the chapter on metabolic disturbances (section 7.2.1.), inhibitory actions of adenosine do not appear to have a major contribution to either the anoxic slowing of respiratory rhythm, or to the accompanying hyperpolarisation of neonatal VRG-VLM neurons. 6.3. Neuropeptides Neuropeptides exert a multitude of modulatory actions on respiration, as was elaborated in an extensive amount of studies (Murakoshi et al., 1985; Yamamoto et al., 1988, 1992; Greer et al., 1995, 1996b; Johnson et al., 1996). In particular opioid peptides (Florez et al., 1980; Morin-Surun et al., 1984, 1992; Shook et al., 1990) and somatostatin (Harfstrand et al., 1985; Yamamoto et al., 1988; Chen et al., 1991) have been shown to act as potent respiratory depressants, whereas breathing was found to be stimulated by substance P (Chen et al., 1991, 1996) and TRH (Hedner et al., 1981). The `classical' studies by Suzue and colleagues have established that these drugs a€ect the in vitro respiratory activity in the brainstem-spinal cord preparation in the same manner as the respiratory network in the intact animal (Suzue, 1984; Murakoshi et al., 1985). Subsequent to the observation by Suzue (1984) that somatostatin produces apnea, that cannot be reversed by the opioid antagonist naloxone, there is no study that analysed the cellular mechanism of the action of this potent inhibitor on in vitro respiration (Harfstrand et al., 1985; Chen et al., 1991). One intracellular study on neurons of the solitary tract complex has, however, indicated that the drug depresses neuronal excitability through hyperpolarisation and augmentation of an M-type K+ current (Jaquin et al., 1988). As

Neonatal Respiratory Network

suggested by the ®ndings of North and colleagues, the somatostatin receptor might be coupled to the same Kir channel that is also activated by (nor)adrenaline, baclofen or opioids (North et al., 1987; Tatsumi et al., 1990). As outlined below the inhibitory e€ects of opioid receptor agonists as well as the excitatory e€ects of substance P and TRH have been analysed in more detail. 6.3.1. Opiates Some cases of periodic breathing and apnea in newborn humans might be due to activation of opiate receptors, since endogenous opioid levels are assumed to be higher in respiratory regions of the brain in neonates than in adult animals (Shook et al., 1990; Greer et al., 1995; Ballanyi et al., 1997). in vivo experiments on adult mammals have established that both, m- and d-types of opiate receptors have a depressant e€ect on respiration (Morin-Surun et al., 1984). In the brainstem of rats, quantitative receptor autoradiography has elucidated that m receptors are expressed at high density in cardiorespiratory-related nuclei at birth, whereas d receptors are very scarce (Xia and Haddad, 1991). Accordingly, the study of Greer et al. (1995) presented evidence that the depressing e€ect of opiates on respiration in the isolated brainstem-spinal cord preparation, and also in intact neonatal rats, is exclusively mediated by m receptors. As determined in the same study in intact animals, d receptors contribute to such depression after about the ®rst postnatal week. That d receptor activation is ine€fective in newborn rats was also suggested by the ®ndings of Takita et al. (1998) in the brainstem-spinal cord preparation. In contrast, it was reported that the d agonist Ala-Leu-Enk produces long-lasting depression of respiratory frequency in this preparation and that this depression is reversed by the selective d receptor antagonist naltrindole (Ballanyi et al., 1997). The latter study furthermore showed that frequency depression or in vitro apnea, induced by the opiates, was not accompanied by a major e€ect on membrane potential or conductance in the vast majority of neonatal VLM-VRG neurons. It was concluded by these authors that the inhibitory action of these drugs might be due to presynaptic e€ects on synaptic transmission, possibly by reduction of voltage-gated Ca2+ currents. Since it was found that tonic nonrespiratory neurons in the region of the VRG are directly hyperpolarised by the opiates (Fig. 19; Lalley et al., 1998), it might also be the case that the inhibitory action is related to reduction of the excitatory drive to the RRG. It was furthermore found by the group of Ballanyi and colleagues that the opioid-induced respiratory depression in the brainstem-spinal cord preparation is not only antagonised by selective opiate receptor antagonists, but also by cAMP-elevating drugs, such as rolipram, forskolin or ca€eine, and also by muscarine, Ba2+ or by lowering of the pH of the superfusate (Fig. 19; Ballanyi et al., 1997; Lalley et al., 1998; Meyer et al., 1999). 6.3.2. Substance P Application of capsaicin, which is known to induce release of substance P from primary a€erent

621

neurons, was described to produce a long-lasting stimulatory e€ect on respiratory frequency in the brainstem-spinal cord preparation, similar to that induced by the peptide itself (Murakoshi et al., 1985; Johnson et al., 1996). In the report by Murakoshi et al. (1985), and also in a more recent study (Yamamoto et al., 1992), it was revealed that substance P produces an excitatory e€ect mainly in preparations with a low baseline frequency, whereas it can even reduce respiratory burst rate in preparations with a high frequency. Observations made with selective agonists and antagonists of tachykinin receptors indicate that NK1 and NK3 receptors are involved in the stimulatory e€ect on the isolated respiratory network (Monteau et al., 1996). It was also demonstrated that substance P is e€ective to reverse depression of respiratory frequency, induced by the a2 (nor)adrenergic agonist clonidine (Yamamoto et al., 1992). In the latter study, it was furthermore seen that the drug induces tonic nerve activity at higher (>0.1 mM) concentrations, as was also shown by Murakoshi et al. (1985). Finally, substance P was shown to restore or potentiate extracellularly recorded burst activity of Pre-I neurons after suppression of synaptic transmission (Yamamoto et al., 1992). Although it was speculated that a cAMPelevating second-messenger mechanism (section 4.2.2.) is involved (Yamamoto et al., 1992), the underlying cellular mechanism of the excitatory e€ect of substance P on neonatal respiratory neurons remains to be analysed. In contrast, it was demonstrated for non-respiratory neurons of the RVLM (Li and Guyenet, 1997) and pons (Shen and North, 1992) that the excitatory e€ect of substance P is caused by opening of cation channels in combination with closure of K+ channels.

6.3.3. TRH Intracerebroventricular injection of TRH was demonstrated to have a potent stimulatory e€ect on breathing in adult rodents (Hedner et al., 1981; Homma et al., 1984). Similar excitatory e€ects of the tripeptide, that were mainly due to a potentiating action on respiratory frequency, were seen in fetal, neonatal and adult animals of a diversity of species (Greer et al., 1996b). However, pressure injection of TRH in the region of the nucleus ambiguus in the VLM did not a€ect respiratory activity in vivo, although ambigual neurons were demonstrated to depolarise upon application of the drug (Johnson and Getting, 1992). The latter contradicting results might be explained by the ®nding that TRH immunoreactive boutons form close appositions with respiratory neurons only in particular subregions of the VLM-VRG (Sun et al., 1996; see also McCown et al., 1986). Accordingly, it was demonstrated in rhythmic slices from newborn rats that the accelerating e€ect of TRH on in vitro frequency is seen after selective application to the preBoÈtC (Greer et al., 1996b). In contrast, similar administration of the drug to the pre-BoÈtC of rhythmic slices from neonatal mice did not lead to a rise in frequency of the respiratory cycle (Funk et al., 1994). In the latter study, it was found that TRH

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potentiates the amplitude of hypoglossal nerve discharge with increasing postnatal age. In the brainstem-spinal cord preparation from newborn rats, it was ®rst described by Murakoshi et al. (1985) that TRH accelerates respiratory frequency. In the report of Greer et al. (1996b), it was further elucidated that TRH has a strong excitatory e€ect in fetal preparations, in which baseline frequency of respiratory rhythm is much slower than in those from newborn rats. The assumption by these authors that the rate of rhythm before birth depends on the tonic excitatory drive to the VLMVRG neurons, generating respiratory bursts (Greer et al., 1996b), is consolidated by the recent ®ndings that not only TRH, but also other depolarising drugs, such as Ba2+ or muscarine accelerate respiratory activity in fetal rats (Meyer et al., 1999). The latter authors (1999) suggested that the action of yet not identi®ed neuromodulators might produce ongoing depression of the RRG, that is e€ectively antagonised by cAMP-elevating and/or depolarising drugs such as rolipram, muscarine, Ba2+, and also TRH. In accordance with the assumption of a rather indirect e€ect of TRH on the RRG, it was found that reversal of a cyanide-induced slowing of respiratory rhythm in the brainstem-spinal cord preparation is not accompanied by a direct depolarising e€ect on VLM-VRG neurons (Ballanyi, 1999b). However, the excitatory action of TRH might be restricted to a limited number of RRG neurons, as it was demonstrated that only subpopulations of VLM-VRG neurons are depolarised by the drug (Rekling et al., 1996b). The cellular mechanism of the TRH-induced depolarisation of VLM-VRG neurons is not analysed so far, but there is indication that the substance blocks a particular type of Kir channels (section 4.3.1.). 6.4. Acetylcholine Acetylcholine (ACh) is responsible for transferring the motor output of the respiratory network to the respiratory muscles. However, this neurotransmitter is not involved in generation of the medullary primary rhythm, at least in vitro, as respiratory nerve activity persists after combined administration of blockers of the nicotinic and muscarinic subtypes of ACh receptors (Murakoshi et al., 1985; Monteau et al., 1990a). Nevertheless, in vivo experiments of the groups of Loeschcke and Mitchell (Loeschcke, 1982) have, for example, demonstrated that administration of nicotine to those aspects of the ventral medulla, that are thought to contain H+-sensitive receptors, has a strong stimulatory e€ect on respiration. Interestingly, this area of central chemosensitivity has a dense packing of cholinergic cells and all cells in which H+ increases (tonic) ®ring are also stimulated by local application of ACh (Loeschcke, 1982). Such experiments have established the view that cholinergic cells and/or mechanisms constitute a key link between central chemosensitivity and respiratory network function (section 6.5.). In agreement with the latter in vivo results, it was found that bath-application of ACh produces an increase in the frequency of respiratory activity in the brainstem-spinal cord preparation (Murakoshi

et al., 1985). In extension of these studies, Monteau et al. (1990a) observed that selective administration of ACh to the pons has no e€ect, whereas application in the vicinity of the ventral surface of the medulla produces the typical frequency acceleration. This stimulating e€ect of ACh was markedly diminished, but not completely abolished by atropine, whereas the nicotinic antagonist dihydro-b-erythroidine was ine€ective. Therefore, the stimulating e€ect of ACh was thought to be predominantly mediated by muscarinic receptors (Murakoshi et al., 1985; Monteau et al., 1990a). The latter studies furthermore suggested that endogenous cholinergic mechanisms are functioning in the isolated brainstem, since both, edrophonium and physostigmine as blockers of Ach esterase exert an accelerating e€ect on the respiratory rhythm similar to that of exogenous ACh. This assumption of involvement of endogenous cholinergic mechanisms in modulation of the RRG in the isolated brainstem was consolidated by the ®ndings that inhibition of ACh synthesis depresses, while facilitation of ACh synthesis stimulates inspiratory phrenic output (Burton et al., 1995). Furthermore, the latter authors (1995) showed that microinjection of an inhibitor of ACh synthesis into the rostral region of the RVL decreases bursting frequency, while microinjection in the CVL or caudal region of the RVL decreases bursting amplitude. They suggested that separate neuronal pools may be responsible for e€ects on respiratory rate and depth, which is in agreement with the idea on the organisation of the central pattern generator of respiration proposed by Onimaru et al. (1988). Similar to the other neuromodulators, described in this section, neither Ach nor muscarine have a major e€ect on the in vitro respiratory rhythm in brainstem-spinal cord preparations with a high resting burst rate (Meyer et al., 1999). In the course of the latter reports, it was furthermore found that the stimulating e€ect of muscarine on respiratory frequency increases with decreasing age within the perinatal period. The e€ect is much more evident at embryonic (E) age 18±21 rats than at postnatal (P) age P2±4. Furthermore, in the fetal rats, submicromolar concentrations of muscarine produce an atropin-sensitive tonic activity (probably due to excitation of spinal motoneuronal networks) whereas the tonic e€ect is not observed in the postnatal animals (Meyer et al., 1999). It was also described that bath-application of either muscarine, nicotine or neostigmine is e€ective to reverse in vitro apnea, evoked by either PGE1 or the m opioid receptor fentanyl (Lalley et al., 1998; Meyer et al., 1998). This stimulating e€ect of muscarine was found to be related to an atropin-sensitive depolarisation of a subpopulation of VLM-VRG neurons and/or tonically active cells in the region of the VLM-VRG (Fig. 19). Although not analysed so far, it is likely that inactivation of the M-type K+ current (Champagnat et al., 1986; Bianchi et al., 1995) is underlying the excitatory action of muscarine, whereas the cellular mechanism of the nicotinic stimulation remains obscure at present. Finally, it was revealed in the context of disturbance of in vitro respiratory network function that muscarine fully

Neonatal Respiratory Network

antagonises both the hyperpolarisation and depression of respiratory frequency upon block of aerobic metabolism (section 7.1.; Ballanyi, 1999b). 6.5. Central Chemosensitivity It is established in the textbooks that peripheral chemoreceptors, sensitive to O2, CO2, or H+ are crucial for maintenance of homeostasis of oxygen supply and acid-base balance by nervous control of the activity of the cardio-respiratory network (Peers and Buckler, 1995). However, the observation that increases in [H+] or partial pressure of CO2 (PCO2) evoke forced breathing even after complete chemodenervation hints at the existence of central nervous chemoreceptors. In particular the work of Mitchell, Loeschcke and colleagues (see Loeschcke, 1982; Harada et al., 1985; Millhorn and Eldridge, 1986; Bruce and Cherniack, 1987), showed that these central chemoreceptors are located in the vicinity of the ventral surface of the medulla in a rostral (Mitchell's) and a caudal (Loeschcke's) area, that partially overlap the bilateral columns of VLMVRG neurons (compare Fig. 8 in Loeschcke, 1982 with Fig. 6 of the present study). In vivo perfusion with acidic solution of constant PCO2 was found to stimulate respiration, whereas perfusion with high PCO2 at constant pH failed to increase respiratory activity. This established the assumption that the primary stimulus of the central chemoreceptors is H+ rather than CO2, as was also hypothesised for peripheral chemoreceptors (Peers and Buckler, 1995). It is still under debate, whether the chemoinduced changes in respiratory activity correlate with the kinetics of the pH change in the interstitial space of the VLM-VRG or of the cerebrospinal ¯uid (Loeschcke, 1982; Harada et al., 1985; Issa and Remmers, 1992; Voipio and Ballanyi, 1997). As also discussed below, it is not clear yet whether the chemoreceptive structures are located on subcellular structures in the vicinity of the medullary surface or in the somatic regions within the VLM-VRG (Loeschcke, 1982; Fukuda, 1983; Kawai et al., 1996; VoÈlker et al., 1995; Voipio and Ballanyi, 1997). In addition to the responsiveness to `classical' neuromodulators such as biogenic amines or peptides as described above, the isolated respiratory network in the brainstem-spinal cord preparation of newborn rats retains the sensitivity to chemostimuli. After the initial observation by Suzue (1984) that inspiratory nerve activity is enhanced by superfusion of low pH solution and depressed by high pH solution, Harada et al. (1985) presented experimental evidence that the mammalian central chemoreceptor for respiratory control is responsive independently to H+ and CO2 (and also HCOÿ3). In the same study, H+ and CO2 were assumed to exert di€erential e€ects on the respiratory center in terms of frequency and magnitude of nerve discharge. A basically similar modulation of both, strength and frequency of in vitro phrenic activity was also observed in related reports (Monteau et al., 1990a; Okada et al., 1993b). In most studies on the brainstem-spinal cord preparation, hypercapnia as evoked by increasing the gassing of the superfusate with CO2 led to a rise in respiratory frequency rather

623

than to an increase in the amplitude of nerve bursting (Fig. 19; Suzue, 1984; Monteau et al., 1990a; Okada et al., 1993b; Kawai et al., 1996; Voipio and Ballanyi, 1997; see also Issa and Remmers, 1992). This is in contrast with in vivo observations that hypercapnia primarily evokes a rise in tidal volume with less prominent increase of respiratory frequency (Loeschcke, 1982; Millhorn and Eldridge, 1986; Bruce and Cherniack, 1987). The attenuated in vitro e€ects on the amplitude of phrenic nerve bursting might be related to the fact that the in vitro rhythm most likely represents a state of enforced breathing (Cherniack et al., 1981), associated with removal of a€erent sensory input. As discussed above (section 2.2.), phrenic amplitude and, thus, tidal volume increases considerably in neonatal rats immediately after bilateral vagotomy in vivo (Fig. 3; Murakoshi et al., 1985; Fedorko et al., 1988; Smith et al., 1990). Despite such a putative dea€erentation-related limitation in the dynamics of the chemoresponse with respect to phrenic amplitude, the output magnitude of the in vitro respiratory network is not maximal in the isolated preparation. The amplitude and thus strength of both, cranial and spinal inspiratory nerve discharge can increase by up to 100% after slowing of the in vitro rhythm, induced by either anoxia or administration of opioids or PGE1. Increases in the amplitude of nerve discharge are also in particular evident upon reversal from the latter types of respiratory depression, induced by a variety of drugs such as rolipram or naloxone (section 7.; Meyer et al., 1998). For interpretation of the e€ects of variation of the CO2 content or pH on neuronal properties, it needs to be considered that ongoing CO2 production by aerobic metabolism results in a considerable extracellular acidosis (Voipio and Kaila, 1993). Accordingly, extracellular pH values in intact animals can be up to 0.4 pH units lower than those of arterial blood (Chesler, 1990; Morawietz et al., 1995; Voipio and Ballanyi, 1997; Kaila and Ransom, 1998). It can be derived from these low pH values, that tissue PCO2 levels are considerably higher in the vicinity of the neurons than the value of 35 mmHg of arterial blood. This suggests that venous transport of CO2 is equally insucient to provide `ideal' PCO2 and pH levels as is arterial transport to maintain a high level of PO2 (section 7.). Due to these considerations, it could be assumed that tissue pH in the brainstem-spinal cord preparation substantially di€ers from that of the superfusate. Accordingly, measurements with CO2/H+sensitive microelectrodes revealed a tissue gradient for both, pH and PCO2 (Voipio and Ballanyi, 1997). With increasing recording depths within the boundaries of the main distribution of VLM-VRG neurons (200±500 mm below ventral surface; section 3.2.), PCO2 was found to increase from about 70 to 90 mmHg, whereas extracellular pH falls from 7.1 to 6.9 at steady state in standard saline equilibrated with 5% CO2 and 95% O2 (Fig. 19; Voipio and Ballanyi, 1997; see also Brockhaus et al., 1993; Okada et al., 1993a). Since the precise location of the chemoreceptive structures is not identi®ed at present, it cannot be decided whether the kinetics of the chemoresponse of the respiratory network in the

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brainstem-spinal cord is determined by the large experimentally-induced variations of surface pH and/ or PCO2 or rather follows the time course of the smaller changes (due to higher baseline levels) deeper in the tissue. It was proposed by group of Scheid and colleagues that the chemo-induced changes in respiratory activity in this preparation follow the change in surface pH/PCO2 (Okada et al., 1993b; Kawai et al., 1996). This is in contrast to results from Issa and Remmers (1992) who showed that respiratory frequency only increases when CO2 is injected locally at depths between 100 and 350 mm below the ventral surface (independent on the rostrocaudal position of the electrode within the VLMVRG). These results agree with those of the group of Ballanyi which reported that the respiratory changes follow the kinetics of pH changes in the center of the VLM-VRG at a depth of about 300 mm (VoÈlker et al., 1995; Voipio and Ballanyi, 1997; Ballanyi, 1999a). The latter group has recently demonstrated that a major portion of the elevated PCO2 under steadystate conditions is secondary to titration of bicarbonate by protons. Accordingly, it was shown by Voipio and Ballanyi (1997) that PCO2 in the VLMVRG decreases to less than 20 mmHg in CO2/ HCOÿ3-free, Hepes pH-bu€ered solutions. The authors furthermore demonstrated in the latter study that PCO2 falls to less than 1 mmHg during evoked anoxia in the absence of CO2/HCOÿ3. These results show that a signi®cant component of the elevated levels of tissue PCO2 (and [H+]) is due to CO2 that is produced by aerobic metabolism. Using oxygenated superfusates, a fall of tissue pH by 0.15 pH units, resulting from the low pH bu€ering power of Hepes, was found to accompany the fall of PCO2 in response to removal of CO2/HCOÿ3 from the superfusate (Voipio and Ballanyi, 1997). This rather small extracellular acidosis is most likely responsible for the modest increase in respiratory frequency during the prominent fall of tissue PCO2 upon removal of CO2/HCOÿ3 (Voipio and Ballanyi, 1997). This shows that CO2 has per se no major in¯uence on the discharge frequency of the isolated respiratory network within a broad range. This consolidates previous assumptions that rather a rise in [H+] than in PCO2 is the primary stimulus responsible for central chemosensitivity (Loeschcke, 1982; Millhorn and Eldridge, 1986; Bruce and Cherniack, 1987). Such a view is further supported by the ®nding that a comparable fall of extracellular pH in the VLM-VRG, induced by either increased gassing of the superfusate with CO2 or by lowering the bicarbonate content of the solution, evokes an almost identical rise of respiratory frequency, despite an opposite e€ect on tissue PCO2 (Fig. 19; Voipio and Ballanyi, 1997). However, it was revealed that addition of CO2/ HCOÿ3 (in the absence of oxygen) leads to reappearance of respiratory activity after in vitro apnea, caused by exposure to anoxic, CO2/HCOÿ3-free superfusate. During sustained anoxia, such addition of CO2/HCOÿ3 partly reverses the major (>0.5 pH units) extracellular acidosis that is possibly due to enhanced anaerobic metabolism and, thus, lactate formation (Voipio and Ballanyi, 1997). Since extracellular alkalosis (evoked in oxygenated saline) typi-

cally results in respiratory depression (Harada et al., 1985), this stimulatory e€ect of CO2-containing solution under anoxia is possibly due to a direct excitatory action of CO2 (Voipio and Ballanyi, 1997). As shown above for most of the other neuromodulators, the stimulating e€ect of CO2/H+evoked extracellular acidosis is in particular evident in brainstem-spinal cord preparations that exhibit a slow rhythm under control conditions. Accordingly, the frequency of inspiratory nerve activity does not increase upon administration of acidic solution to preparations that discharge at a frequency of more than about 12 bursts minÿ1 (Lalley et al., 1998). This suggests that the cellular mechanisms of chemostimulation might involve a rise in cAMP, as hypothesised for the stimulating action of 5-HT or (nor)adrenaline (section 6.1.). That this might be, indeed, the case is indicated by the ®nding that slowing of respiratory rhythm in the brainstem-spinal cord preparation, evoked by opioids or PGE1 is not only antagonised by drugs leading to a rise in cAMP, such as ca€eine, forskoline, IBMX or rolipram (Ballanyi et al., 1997; Meyer et al., 1998), but also by acidic solutions (Fig. 19; Lalley et al., 1998). However, also a mechanism that might not depend on cAMP and that involves di€erent types of stimulation of G protein-coupled K+ channels might contribute to central chemosensitivity in vitro. It was described recently that the opioid- or PGE-induced respiratory depression is e€ectively reversed by muscarine and that this e€ects is atropine-sensitive (Fig. 19; Lalley et al., 1998; Meyer et al., 1998). This suggests that muscarinic ACh receptors are not only involved in chemostimulation of the isolated respiratory network in the control solution (Monteau et al., 1990a; for details see section 6.4.), but also during respiratory disturbances (section 7.). With regards to the cellular mechanism underlying chemostimulation in the brainstem-spinal cord preparation, it was shown that reversal of the opioid- or PGE1-evoked depression of inspiratory nerve activity with muscarine is associated with a depolarisation and decrease of conductance of respiratory as well as of tonic neurons in the region of the VLM-VRG (Fig. 19; Lalley et al., 1998; Meyer et al., 1998). This supports the view that the stimulatory e€ect is mediated by K+ channels, coupled to muscarinic ACh receptors. In untreated preparations, it was found that a major population of VLM-VRG cells is depolarised during hypercapnia (Fig. 19; Kawai et al., 1996; A. Kawai, H. Onimaru and I. Homma, in preparation; Onimaru et al., 1989). However, a variety of in vitro studies, mainly done on non-rhythmic medullary slices, has presented evidence that also non-respiratory cells in the ventrolateral medulla increase the frequency of their tonic ®ring pattern during chemostimulation (Fukuda, 1983; Jarolimek et al., 1990; Richerson, 1995). This led to the assumption that central chemosensitivity might primarily be due to an increased drive to the rhythm generating neurons by chemosensitive non-respiratory neurons in the ventrolateral medulla (Richerson, 1998). However, a subpopulation of non-respiratory and also VLMVRG neurons was found to hyperpolarise during hypercapnia (Kawai et al., 1996). The latter study

Neonatal Respiratory Network

Fig. 19. Central chemosensitivity. A, extracellular acidi®cation in the region of the ventral respiratory group (VRG) gives rise to an increase in the frequency of inspiratory-related spinal (C2) nerve bursts despite opposite changes in PCO2 in the VRG upon superfusion of two solutions of pH 7.0 (control pH 7.4) having low [HCOÿ3] (constant PCO2 of 41 mmHg) or high PCO2 (constant [HCOÿ3] of 25 mM). Recording taken from Voipio and Ballanyi (1997) by permission. B, in vitro apnea as induced by the mopioid receptor agonist fentanyl is e€ectively reversed by acidic solution (pH 7.0), see upper trace of mini-electrode recording of pH in the recording chamber) of either elevated (from 5%) CO2 content or reduced (from 25 mM) [HCOÿ3]. Respiratory rhythm is also re-activated upon bath-application of the K+ channel blocker muscarine that also reverses the fentanyl-induced hyperpolarisation of a tonically active cell in the region of the VRG. In contrast, the acidic solutions had no major e€ect on membrane potential (Vm). Recording taken from Meyer, Hoch, Lalley, Richter and Ballanyi, in preparation. C, alkaline solution (due to decrease of CO2 content from 5 to 2%) decreases respiratory frequency and also attenuates the activity of a Pre-I neuron. In the same cell, subsequent exposure to acidic saline of elevated CO2 content accelerates inspiratory-related nerve discharge and potentiates synaptic activity and bursting. Recording taken from Kawai, Onimaru, Arata and Homma, in preparation).

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showed that most cells, that were sensitive to hypercapnia, project their dendrites to the close vicinity of the ventral surface of the medulla, where the chemosensitive areas are located. Nevertheless, based on the results of the latter study it cannot be decided whether the e€ects of chemostimuli are mediated by these distal processes or rather by structures close to the soma that was in most cases located in the VLM-VRG. This study of the group of Scheid and colleagues furthermore demonstrated that the hypercapnia-induced membrane potential changes are intrinsic to the recorded neuron as they persist after block of synaptic transmission with TTX. The cellular mechanisms that underlie the hyperpolarising or depolarising responses remain to be elucidated. In conclusion, a variety of in vitro reports on the isolated brainstem-spinal cord preparation has established that the mechanisms of central chemosensitivity are functional in mammals at birth. Due to the potentiating e€ect of dea€erentation in the course of isolation of the preparation on phrenic nerve amplitude, the chemoresponse is primarily evident as a change in respiratory frequency. The cellular mechanisms seem to include both cAMPdependent pathways and more direct stimulation of (muscarinic) ACh receptor-coupled K+ channels. It is not clear, whether the stimulating e€ect is caused by direct excitation of rhythm generating VLMVRG neurons or secondary to an increase in tonic drive to the network by stimulation of tonically active chemosensitive neurons with receptive structures close to the ventral surface of the medulla.

7. METABOLIC ASPECTS 7.1. Response To Hypoxia Metabolic disturbances such as hypoxia, disglycemia or ischemia induce neuronal disfunctions that can result in irreversible brain damage (Kristian and SiesjoÈ, 1996; Haddad and Jiang, 1993). In contrast to the vulnerability of most nervous structures in mature animals, the brain of neonates exerts a high tolerance to oxygen depletion (Hansen, 1985; Haddad and Jiang, 1993). Not only brain regions, associated with higher functions, but also the respiratory network of newborns is in particular resistant to hypoxia (Richter and Ballanyi, 1996). Respiratory movements were found to persist in intact neonatal rats for more than 1 h during anoxia as evoked by exposure to nitrogen, whereas apnea occurred within several minutes of anoxia in rats older than 1 week (Fazekas et al., 1941; Adolph, 1969; Du€y et al., 1975). In line with these in vivo observations, anoxia was reported to result in reversible suppression of inspiratory nerve activity in an arterially-perfused brainstem preparation from adult rats (Morawietz et al., 1995; SchaÈfer et al., 1993) as is also typical for the in vivo cat (Richter et al., 1991, 1993b). In the study of Morawietz et al. (1995), it was furthermore shown that the anoxic apnea is accompanied by a major perturbation of extracellular K+ and Ca2+ and by a progressive interstitial acidosis in the region of the VLM-VRG as is also observed in vivo (Richter and Acker,

1989). Although a similar acidosis was revealed in the region of the VLM-VRG in the brainstem-spinal cord preparation of neonatal rats, extracellular K+ and Ca2+ were found to remain almost una€ected even during sustained periods of anoxia (Ballanyi et al., 1992; Brockhaus et al., 1993; VoÈlker et al., 1995; Voipio and Ballanyi, 1997). Similarly, only a minor disturbance of ion homeostasis was detected in the VLM-VRG of newborn rabbits (Trippenbach et al., 1990). In addition to the minor e€ects of anoxia on extracellular ion activities, in vitro respiratory rhythm in the brainstem-spinal cord preparation persists, although at reduced frequency, for periods of more than 1 h during anoxia or upon block of aerobic metabolism with cyanide (Ballanyi et al., 1992; VoÈlker et al., 1995; Ballanyi, 1999b; see also Greer and Carter, 1995). In the majority (>60%) of neonatal respiratory neurons of all classes (section 3.1.) anoxia, evoked by superfusion of hypoxic, N2-gassed superfusate, led to a persistent hyperpolarisation. The anoxic hyperpolarisation was accompanied by block of respiration-related membrane potential ¯uctuations in about 50% of cells (Fig. 10; Ballanyi et al., 1994a; Ballanyi, 1999b). This `functional inactivation' does certainly not re¯ect pathological impairment of synaptic transmission or membrane excitability. Action potentials could still be evoked in these cells, and in particular drive potentials of a subpopulation of inspiratory VLM-VRG neurons remained almost unaltered under anoxia (Ballanyi et al., 1994a; Ballanyi, 1999b). This anoxia resistance of excitatory synaptic transmission coinrons and incides with the ability to generate spiking in VLM-VRG interneurons as well as in (bulbospinal) motoneurons. The cellular mechanisms remain to be determined that maintain excitatory transmission in those neurons that provide rhythm generation and motor output of the respiratory network during anoxia, whereas rhythmic EPSPs of the functionally inactivated cells are e€ectively suppressed. Similar to the anoxia sensitivity of rhythmic EPSPs in the latter population of neonatal VLM-VRG neurons, respiration-related Clÿ-mediated IPSPs of almost all respiratory neurons were suppressed within the initial phase of anoxic exposure of the brainstemspinal cord preparation (Ballanyi et al., 1994a; Ballanyi, 1999b). A similar observation was made in VRG neurons of adult cats in vivo where anoxia, besides evoking a stable moderate depolarisation, induced suppression of IPSPs (Richter et al., 1991, 1993b). That inspiratory activity in the brainstemspinal cord preparation of newborn rats persists despite such systemic anoxia-related suppression of IPSPs supports the above (section 4.1.2.) assumption that (Clÿ-mediated) inhibition is not pivotal for rhythm generation in vitro. In expiratory neurons, anoxic block of inspiration-related IPSPs unmasked EPSPs as was also shown for VLM-VRG neurons of rhythmic brainstem slices from mice (Ramirez et al., 1997; see also Richter et al., 1991, 1999; Richter and Ballanyi, 1996). The early onset of suppression of IPSPs might contribute to the initial frequency increase that precedes anoxic depression of respiratory frequency in a considerable percentage of brainstem-spinal cords (Ballanyi et al., 1992; Brockhaus

Neonatal Respiratory Network

et al., 1993; VoÈlker et al., 1995; Ballanyi, 1999b). The ®nding that this early stimulatory response to oxygen depletion is not only a characteristic feature of intact neonatal mammals including infants (Rigatto, 1984; Neubauer et al., 1990; Martin et al., 1998), but also of the dea€erented medulla preparation shows that this phenomenon is not exclusively due to excitation of peripheral chemoreceptors, but implies a central nervous component (VoÈlker et al., 1995; Richter and Ballanyi, 1996). Although yet not proven, it is likely that hyperpolarisation of those burster-type VLM-VRG neurons, involved in rhythm generation, and/or of tonically active cells, that provide the drive to the network (sections 4.3.4.; 5.2.), is responsible for frequency depression in the later phase of anoxia. This assumption gains support from the observation in the brainstem-spinal cord preparation that anoxiainduced respiratory depression is e€ectively reversed by drugs such as muscarine, Ba2+ and TRH that are known to block (G protein-coupled) K+ channels (Fig. 10; Ballanyi, 1999b). Since anoxic slowing of the in vitro respiratory rhythm is also antagonised by tolbutamide (Ballanyi, 1999b), it is possible that ATP-sensitive K+ channels contribute to such frequency depression (Mironov and Richter, 1998). In contrast, there is no evidence that an anoxiamediated rise of interstitial adenosine, that is formed during degradation of ATP (Greene and Haas, 1991), contributes to the anoxia response of the isolated respiratory network of neonatal rats. Neither antagonise blockers of (A1-type) adenosine receptors such as DPCPX or theophylline the anoxic hyperpolarisation or frequency depression, nor does adenosine mimic the latter responses (Ballanyi, 1999b; see also section 6.2.). In contrast to the latter observations, it was reported that adenosine antagonists attenuate hypoxic depression in the brainstem-spinal cord including the pons and that this e€ect is mediated by adenosine (Kawai et al., 1995). However, it is questionable whether a rise of interstitial adenosine during anoxia as measured in the region of the VLM-VRG of adult cats (Richter et al., 1993b) is likely to occur during anoxia in the neonatal brainstem-spinal cord. A major portion of the extracellular increase of adenosine during oxygen depletion seems to be due to degradation of ATP (Greene and Haas, 1991). However, it is assumed that cellular levels of ATP remain almost unaltered in the brainstem-spinal cord preparation of newborn rats during anoxia due to enhanced glycolysis (section 7.2.). Furthermore, in vivo studies suggested that release of endorphins contributes to respiratory depression due to oxygen depletion (De Boeck et al., 1984; Richter and Ballanyi, 1996; Richter et al., 1999). However, this does not seem to be the case in the brainstem-spinal cord preparation since the anoxic depression of respiratory frequency reverses within several minutes upon re-oxygenation, whereas the opiate-induced slowing of the rhythm persists for up to several hours after washout of the drug (compare Ballanyi et al., 1997; Ballanyi 1999b). The same considerations as for the opioids are valid to exclude a major contribution of prostaglandins that are formed in the course of oxygen de-

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pletion in anoxia-vulnerable tissues (Ballanyi, 1999a). PGE or agonists of EP3 receptors cause a long-lasting depression of respiratory activity in the brainstem-spinal cord preparation of newborn rats that persists after washout of the drugs (Ballanyi et al., 1997; Lalley et al., 1998; Meyer et al., 1998). Interestingly, these depressing e€ects of PGE are maximal within 2 days prior to and after birth, whereas a prominent stimulatory action on the isolated respiratory network is revealed at fetal days E18±19 (Meyer et al., 1998, 1999). These results suggest that a yet undetermined mediator that acts on (G protein regulated) K+ channels, is released under anoxia. As an alternative, cellular processes such as change in phosphorylation or redox state might be responsible for activation of K+ channels and, thus, for slowing of rhythm (Ballanyi, 1999a). In contrast to the extreme tolerance of the preparation to block of aerobic metabolism, respiratory rhythm is irreversibly suppressed and ion homeostasis is severely perturbed after complete metabolic arrest as induced by the glycolytic inhibitor iodoacetate (Ballanyi et al., 1996b). These e€ects are accompanied by a progressive depolarisation of the neonatal VLM-VRG neurons that is in most cases preceded by a signi®cant hyperpolarisation (Fig. 10; Ballanyi, 1999b). These responses of the neonatal respiratory network to iodoacetate, that mimics in some respect ischemia in vitro, were very similar to those of anoxia on (cortical) neurons (Hansen, 1985; Kristian and SiesjoÈ, 1996; Haddad and Jiang, 1993). In summary, the response of the isolated neonatal respiratory network to anoxia is an adaptational mechanism that serves to reduce energy consumption (section 7.1.2.) rather than represents a pathological consequence of block of aerobic metabolism. 7.2. Role Of Anaerobic Metabolism The ®nding that both, rhythmic activity of respiratory neurons and ion homeostasis in the region of the VLM-VRG in the brainstem-spinal cord tolerate sustained periods of block of aerobic metabolism suggests that anoxia-related stimulation of anaerobic glycolysis (`Pasteur e€ect') is sucient for longterm maintenance of function of the isolated neonatal respiratory network (Bomont et al., 1992). In agreement with the eciency of the Pasteur e€ect, it is assumed that 50% of ATP production under normoxic conditions is due to anaerobic metabolism in neonates, in contrast to less than 25% in adults (Hansen, 1985). Accordingly, a high lactate dehydrogenase activity indicates a predominant role of anaerobic glycolysis during the perinatal period (Booth et al., 1980). In contrast, the activity of cytochrome C oxidase, an indicator of oxidative glucose utilisation, is found to increase only after 12±17 days after birth (Wong-Riley, 1989). Due to the low eciency of anaerobic ATP production, survival of neurons during extended (>1 h) periods of anoxia is only possible, since the metabolic rate of neonatal brain tissue is very low, i.e. in 1-day-old rats below 5% of that in adults (Du€y et al., 1975; Hansen, 1985). Thus, in the perinatal period, cerebral glucose consumption appears to be less than 10% of that in adult rats (Vannucci

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and Vannucci, 1978). Despite this quantitative di€erence to adult animals, basic metabolic rate appears to be considerable in the brainstem-spinal cord preparation as respiratory rhythm is blocked and extracellular K+ progressively increases within 30 min after glucose removal from the superfusate (Ballanyi et al., 1996b) or after pharmacological block of glycolysis (Fig. 10; Ballanyi, 1999b). That on-going glucose utilisation is, indeed, a characteristic of the preparation is also suggested by the ®nding that 10 mM glucose, instead of 30 mM, is not sucient to provide long-term maintenance of respiratory activity, at least not in brainstem-spinal cord preparations from rats older than 1 day (Ballanyi et al., 1996b; Suzue et al., 1983). However, due to an expected tissue gradient for glucose, similar to that for oxygen, CO2, pH and K+ (see above), it is likely that extracellular glucose concentration in the area of the VLM-VRG is close to 6 mM, which corresponds to the value measured in the blood of normoglycemic rats in vivo (Hansen, 1985; Bomont et al., 1992). (Ballanyi et al., 1994, 1996)

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