Studies of the respiratory center using isolated brainstem-spinal cord preparations

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NEUROSCIENCE RESERRCH Neuroscience Research 21 (1995) 183-190

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Studies of the respiratory center using isolated brainstem-spinal cord preparations Hiroshi Onimaru Department of Physiology, Showa University School of Medicine, 1-5-8 HatanodaL Shinagawa-ku, Tokyo 142, Japan Received 6 October 1994; accepted 9 November 1994

Abstract It has been ten years since a brainstem-spinal cord preparation isolated from a newborn rat was introduced for study of the mammalian respiratory center. Here, I briefly summarize first, these studies, which include the tissue condition of in vitro preparations, respiratory reflexes, pharmacology, rhythm generation, respiratory chemoreception, phrenic motoneurons, regulation from pons, and development of a respiratory center. In the latter half of this paper, I focus on the neural mechanisms of respiratory rhythm generation. A current hypothesis for the central pattern generator of respiration proposed by the author's group is that the respiratory rhythm generator, composed of pre-inspiratory neurons in the rostral ventrolateral medulla, produces the primary rhythm of respiration and triggers an inspiratory pattern generator composed of inspiratory neurons in the rostral and the caudal ventrolateral medulla. Keywords: Respiratory rhythm; Respiratory center; Brainstem; Newborn rat; In vitro

1. Introduction

2. Brief review

A brainstem-spinal cord preparation isolated from a newborn rat was introduced by Suzue (1984) as an in vitro model for study of the mammalian respiratory center. This preparation generates neuronal bursts corresponding to the respiratory rhythm in an experimental chamber under superfusion by a modified Krebs solution. Therefore, we can study the respiratory center after complete removal (or selective retaining) of peripheral inputs without any vascular circulation. Considerable studies related to the respiratory center have been done during the last ten years using in vitro newborn rat preparations. First, I will summarize briefly those resuits, and then discuss, in particular, the neural mechanisms of the respiratory rhythm generation in this preparation.

2.1. Condition o f preparations The preparations were isolated from 0- to 4-day-old rats. The basic methods are described by Suzue et al. (1983) and Suzue (1984). The most noticeable points of the experimental conditions were a higher glucose concentration (30 mM) in the Krebs solution and a lower temperature (25-27°C) of the perfusate. The low respiratory rates in the in vitro preparations compared with those in the in vivo newborn rats could be explained by removal of the vagal afferent inputs and the lower temperature of the preparation (Murakoshi et al., 1985; Smith et al., 1990). The discharge pattern of the motorneurons during the inspiratory phase, i.e. rapidly peaking-slowly decrementing discharge, could also be explained primarily by the de-afferentiation (Smith et al., 1990; H a m a d a et al., 1992). Recently, tissue conditions such as PO2 or p H of the preparation have been measured in detail (Okada et al.,

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1993a; Brockhaus et al., 1993). In the ventrolateral reticular formation at depths of 200-500 #m in which major populations of respiratory neurons are distributed, values of PO2 and pH fell from 190 to 20 mmHg and from 6.98 to 6.74, respectively (Brockhaus et al., 1993). These PO2 values are high enough for aerobic metabolism as compared to in vivo normoxic condition (e.g. Grote et al., 1981). Moreover, Brockhaus et al. (1993) observed transient decreases of PO 2 and concomitant increases of extracellular potassium activity during spontaneous inspiratory and stimulus-evoked neuronal activity. They concluded that the oxygen supply of the ventral respiratory network is sufficient to maintain aerobic neuronal metabolism and network function in the newborn rat preparation. Furthermore, it was suggested that anaerobic metabolism in addition to aerobic metabolism contributes to maintenance of respiratory activity and ionic homeostasis (Ballanyi et al., 1992). On the other hand, Okada et al. (1993a) measured changes in the pH in the extracellular space of the preparation at 100 and 200 #m depth while replacing the superfusing solution from control to acidic; either with increased PCO 2 (respiratory acidosis) or by adding fixed acid (metabolic acidosis). They found that the changes in the extracellular pH were smaller with respiratory acidosis than those with metabolic acidosis, as a result of the buffering of the brain tissue. These findings are important in the study of central chemosensitivity of respiration in the in vitro preparation (see below).

al., 1990, 1991), acetylcholine (Monteau et al., 1990), 5HT (Morin et al, 1990a, 1991a,b), and substance P (Yamamoto et al., 1992) has been investigated in more detail.

2.4. Rhythm generation

To study respiratory reflexes in vitro, Murakoshi and Otsuka (1985) developed a preparation including the lungs and the vagus nerve. In this preparation, C4 (or C5) inspiratory activity was inhibited by lung inflation. They suggested that the inhibitory reflex was mediated by ~,-amino butyric acid (GABA) receptors and/or glycine receptors in the medulla. This kind of preparation is very useful for elucidation of detailed neuronal mechanisms of respiratory reflexes such as the HeringBreuer reflex.

Smith and Feldman (1987) and Feldman and Smith (1989) proposed the involvement of an intrinsic pacemaker-driven oscillator in rhythm generation in the in vitro newborn rat preparation because the respiratory rhythm remained after blockade of GABA A or glycine receptor-mediated and GABAB-mediated inhibitory synaptic transmission. Greer et al. (1991) studied the involvement of excitatory amino acids (EAAs) in rhythm generation. They suggested that the rhythm generation in this preparation was dependent on endogenously released EAAs acting at non-NMDA receptors, but NMDA receptors were not directly involved in rhythmogenesis (see also Funk et al., 1993). A preliminary study, however, suggested that NMDA receptors may also contribute to rhythm generation (Onimaru et al., 1991a). The role of NMDA receptors in respiration should be further investigated (see also Kashiwagi et al., 1993b). The firing pattern and distribution of respiratory neurons in the ventrolateral medulla were studied using extracellular recordings (Onimaru and Homma, 1987; Onimaru et al., 1987; Arata et al., 1990; Smith et al., 1990). From earlier experimental results including neuronal responses to electrical stimulation and effects of electrolytic lesion of the ventral medulla, Onimaru et al. (1988) suggested primary respiratory rhythm generation by pre-inspiratory (Pre-I) neurons in the rostral ventrolateral medulla (RVL) (Onimaru and Homma, 1987; Onimaru et al., 1987; see below). Recently, a limited region of the ventrolateral medulla, the pre-B6tzinger complex, was suggested to be important in the rhythm generation (Smith et al., 1991; Feldman et al., 199!; see below). Electrophysiological and morphological properties of respiratory neurons have been analyzed using a whole-cell recording technique (Smith et al., 1991; Onimaru et al., 1992; Smith et al., 1992).

2.3. Pharmacological studies

2.5. Respiratory chemoreception

This in vitro preparation has great advantages for pharmacological study of the respiratory center because it can be maintained in an anaesthetic-free condition and drugs can be applied by superfusion in known concentrations. The effects of antagonists can also be evaluated clearly. Murakoshi et al. (1985) indicated that brainstem applications of dopamine, serotonin (5-HT), histamine, acetylcholine, glutamic acid, substance P, and thyrotropin-releasing hormone (TRH) increased the respiratory rate, whereas noradrenaline, GABA, glycine, and enkephalin decreased the rate. Recently, the role in respiratory control of noradrenaline (Errchidi et

Neuronal mechanisms of central chemosensitivity of respiration are still not fully understood (Bruce and Cherniack, 1987). The in vitro newborn rat preparation may be useful also for study of respiratory chemoreception. Respiratory rhythm in this preparation changes dependent on perfusate pH (Suzue, 1984; Murakoshi et al., 1985). Harada et al. (1985a,b) suggested that external H + and CO2 could affect independently the respiratory rate and magnitude, respectively. In a low Ca/high Mgsynaptic blockade solution, bursting Pre-I neurons in the RVL were sensitive to perfusate pH changes (Onimaru et al., 1989a). As suggested by Fukuda and

2.2. Respiratory reflexes

H. Onimaru/ Neurosci. Res. 21 (1995) 183-190

Loeshcke (1979), cholinergic transmission might be involved in the chemoreceptive responses in the ventral surface layer of adult rats. Monteau et al. (1990) suggested that acetylcholine is implicated in the central respiratory chemosensitivity in the newborn rat preparation. The location of the central chemoreceptors sensitive to changes in PCO 2 was studied in the ventral medulla by Issa and Remmers (1992). They found that chemoreceptive sites were located rostrocaudally in the ventromedial medulla at depths of 100-350/~m below the ventral surface. Okada et al. (1993b) demonstrated that non-respiratory and respiratory neurons which were excited by hypercapnia were found mainly in the rostral chemosensitive area in the ventral medulla. 2.6. Phrenic motoneurons

The role of EAAs in the synaptic transmission of inspiratory drive from bulbospinal neurons to phrenic motoneurons was examined in the spinal cord of in vitro newborn rat preparations. The results suggested that an important component of the neurotransmission of bulbospinal respiratory drive involves endogenous EAAs acting at AP4-sensitive sites and non-NMDA receptors (McCrimmon et al., 1989). This mechanism was more directly confirmed by an intracellular recording study of phrenic motoneurons (Liu et al., 1990). They also suggested that activation of presynaptic AP4sensitive sites blocks the transmission of inspiratory drive (see also Greer et al., 1992a). Analysis of the excitatory postsynaptic currents in neurons in the phrenic motoneuron pool using whole-cell patch-clamp recordings demonstrated the quantal nature of the excitatory synaptic transmission (Liu and Feldman, 1992). Lindsay et al. (1991) showed that newborn rat phrenic motoneurons labeled with HRP have most of the morphological features described for phrenic motoneurons in the adult rat. Morin et al. (1992) demonstrated the existence of differences in central respiratory drive and differences in response to 5-HT between cervical and hypoglossal motoneurons. 5-HT, applied to the spinal cord, increased cell excitability and decreased inspiratory-modulated synaptic current in phrenic motoneurons via different types of receptors (Lindsay and Feldman, 1993). 2. 7. Pons

Some pontine structures are thought to be involved in the maintenance of a normal breathing pattern in the adult rat (Wang et al., 1993; Jodkowski et al., 1994) as well as other mammals (reviews by von Euler, 1986; Feldman, 1986). The in vitro newborn rat preparation is also useful for the study of cellular and network mechanisms of descending regulations of the respiratory center in the medulla from the pons or upper brainstem structure. In the newborn rat preparation, it was reported that pontine neuronal activity depresses con-

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tinuously the respiratory rhythm generator in the medulla (Hilaire et al., 1989; Smith et al., 1990; Hamada et al., 1994). The noradrenergic A5 area is proposed as one of the pontine structures producing such inhibitory drives (Hilaire et al., 1989). Electrical and chemical stimulation of the midline pontine raphe nuclei increased the respiratory rate and depressed the amplitude of the hypoglossal nerve discharge. These responses were suggested to be induced via a release of endogenous 5-HT (Morin et al., 1990b). 2.8. Development

A fetal rat brainstem-spinal cord preparation was used for analysis of fetal central respiratory activity (Di Pasquale et al., 1992a; Greer et al., 1992b). It was suggested that the RVL is a crucial site in respiratory rhythm generation in the last stage (D20-21) of fetal rats as well as in newborn rats (Di Pasquale et al., 1994). Developmental change of respiratory networks in a postnatal period after 4 days old could be studied in the in vitro condition using perfused brainstem preparations (Morin-Surun et al., 1989; Hayashi et al., 1991; Kuwana et al., 1992; Ballanyi et al., 1992) or tilted-sagittal slices including major respiratory networks (Paton et al., 1994). Rhythm generation in developed respiratory networks is suggested to require inhibitory synaptic connections (Hayashi and Lipski, 1992; Paton et al., 1994; see Richter et al., 1992 for review).

3. Rhythm generation 3.1. Sites

Electrolytic lesion of the dorsal area of the medulla did not suppress the respiratory output in in vitro newborn rat preparations (Hilaire et al., 1990). Furthermore, respiratory rhythm remained after complete removal of the dorsal half of the medulla (Arata et al., 1990). It was, therefore, concluded that the basic rhythm of respiration could be generated in the ventral medulla. Several types of respiratory neurons are found in the ventrolateral medulla (VLM) (Onimaru and Homma, 1987; Smith et al., 1990; Fig. 1). Inspiratory neurons fire during C4/C5 inspiratory bursts (Fig. IA). Pre-inspiratory (Pre-I) neurons fire preceding inspiratory bursts (Fig. 1A). The Pre-I neurons are usually inhibited during the inspiratory phase, mediated by GABA A and/or glycine receptors (Onimaru et al., 1990), and fire again for a while during the post-inspiratory phase. The activity of some Pre-I neurons, however, was not inhibited during the inspiratory phase (Fig. 1B). Tonic expiratory neurons fire tonically and are inhibited during the inspiratory phase (Fig. IF). Inspiratory and Pre-I neurons have subtypes showing tonic activity during the burst to burst periods (Fig. IC-E). Pre-I neurons were distributed rostrocaudally in a reticular formation from the most rostral level of the

H. Onimaru/Neurosci. Res. 21 (1995) 183-190

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Fig. 1. The firing patterns of several respiration-related neurons in the ventrolateral medulla of the newborn rat preparation together with C4 inspiratory activity. A: Typical Pre-I and inspiratory (Insp) neurons. B: Pre-I neuron of which firing is not inhibited during the Insp phase. C: Tonic Pre-I neuron that fires virtually continuously, but whose activity increases just before and after the Insp phase. D: Tonic Pre-I neuron of which firing is only weakly depressed during the Insp phase. E: Tonic Insp neuron that fires almost continuously, but whose activity increases during the lnsp phase. F: Tonic expiratory (Exp) neuron that fires continuously, but whose activity is inhibited during the Insp phase. (Modified from Homma et al., 1993).

1990). These distributions resemble those of respiratory neurons in the adult rat (Ezure et al., 1988; Pilowsky et al., 1990). Onimaru and H o m m a (1987) indicated that C4 bursts ceased after transection o f the medulla at the level o f the most rostral roots of the X I I nerves (see also Mclean and Remmers, 1994) and spontaneously bursting neurons could be recorded in the ventrolateral part of the block of the rostral medulla. Therefore, more rostral medulla than this cutting level is necessary for respiratory rhythm generation (Fig. 2). Electrical stimulation in the R V L reset the respiratory rhythm and electrolytic lesions in the R V L slowed or stopped the respiratory outputs (Onimaru et al., 1987). Results from some pharmacological experiments reconfirmed the crucial role of the R V L on rhythm generation: Microinjections o f noradrenaline into the R V L depressed the respiratory rhythm (Errchidi et al., 1991). Furthermore, local applications o f 5-HT to the R V L increased the respiratory rate (Di Pasquale et al., 1992b). In addition to the RVL, the C V L is important in the inspiratory pattern generation because electrolytic lesions of the C V L caused a disappearance o f the inspiratory burst without disturbing the burst activity of rhythm generating neurons in the R V L (Onimaru et al., 1988). Recently, Smith et al. (1991) suggested that the preB6tzinger complex is an important part o f the medulla

x,'~xl.~ retrofacial nucleus (or the caudal part of the facial nucleus) to the level of the rostral end o f the lateral reticular nucleus, and ventrally from the ventral surface area to the ventral area o f the nucleus ambiguus or retrofacial nucleus (at depths of 50-500 #m) (Arata et al., 1990). This area is called the rostral ventrolateral medulla (RVL), which seems to correspond almost identically to the nucleus reticularis rostroventrolateralis (nRVL) by Ross et al. (1984). The distribution o f inspiratory neurons overlapped approximately the distribution of Pre-I neurons in the RVL, and the inspiratory neurons were also distributed near the nucleus ambiguus in the more caudal ventrolateral medulla (CVL) (Arata et al., 1990; Fig. 2). The R V L does not completely correspond to the rostral part of the ventral respiratory group (VRG) and B6tzinger complex described in the adult cat (Lipski and Merill, 1980; Merill et al., 1983; see also von Euler, 1986; Feldman, 1986), because respiratory neurons in in vitro newborn rat preparations are distributed more ventrally, overlapping the area of C1 adrenaline neurons in the R V L (Onimaru et al., 1987; A r a t a et al.,

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Fig. 2. Ventral view of brainstem-spinal cord preparation, indicating distribution of Pre-I and inspiratory (Insp) neurons in the ventrolateral medulla (VLM), and a model of the generation of rhythmic respiratory activity. The distribution was determined based on Onimaru et al. (1987) and Arata et al. (1990). RVL, rostral ventrolateral medulla; CVL, caudal ventrolateral medulla. The dashed line shows the level of transection which stopped the C4 burst (Onimaru and Homma, 1987). The respiratory rhythm generator (RRG), composed of Pre-I neurons (including intrinsic bursters) in the RVL, produces primary rhythm of respiration and triggers the inspiratory pattern generator (IPG). IPG, composed of inspiratory neurons in the RVL and CVL, generates patterned inspiratory activity which drives phrenic motoneurons (Phr.) and inhibits RRG. nVlI, facial nucleus; IX-XII, cranial nerves; C1-C4, cervical ventral nerves.

H. Onimaru/Neurosci. Res. 21 (1995) 183-190

for the rhythm generation. The pre-B6tzinger complex was described to be located caudally to B6tzinger complex and rostrally to the rostral VRG (Smith et al., 1990, 1991; Feldman et al., 1991; see also Connelly et al., 1992 for adult cats). As mentioned above, however, respiratory neurons which might be essential to rhythm generation are found also more ventrally and rostrally in the RVL. A recent study using medullary slice preparations which generate rhythmic hypoglossal nerve activity suggested that more ventral area is important in the determination of frequency of the respiratory rhythm (Funk et al., 1993). We believe that analysis of neuron networks and cellular mechanisms of rhythm generation in the RVL is indispensable for understanding primary respiratory rhythm generation in the medulla. 3.2. Neuronal mechanisms On the basis of results of reset, lesion and stimulation experiments, Onimaru et al. (1988) proposed a hypothesis that the central pattern generator of respiration consists of a rhythm generator (RRG) and an inspiratory pattern generator (IPG): The RRG, composed of Pre-I neurons in the RVL, generates a primary respiratory rhythm and triggers periodically IPG. IPG is composed of inspiratory neurons in the RVL and CVL, and produces inspiratory burst activity (see also Onimaru and Homma, 1987; Fig. 2). Experimental grounds for this hypothesis include the following points: (1) The phase of the respiratory rhythm was reset dependent on induction of a premature Pre-I burst when the RVL was stimulated electrically; (2) Change in the rhythm of Pre-I neurons induced change in the rhythm of the inspiratory burst; (3) The rhythmic Pre-I burst was retained whether inspiratory activity was present or not. About half of the Pre-I neurons retained their rhythmic bursts after chemical synaptic transmission was blocked in a low Ca/high Mg solution (Onimaru et al., 1989a). Pre-I, inspiratory, and C4 bursting activity were also retained under blockade of GABA A (and/or glycine) receptors or during reduction of the CI- concentration of the perfusate (Onimaru et al., 1990). These results suggested that respiratory rhythm is primarily generated in an excitatory synaptic network including Pre-I intrinsic bursting cells (i.e., pacemaker neurons) in the RVL. Voltage-dependent, intrinsic burst generating properties of Pre-I neurons were more directly demonstrated using nystatin-perforated whole-cell recordings (Onimaru et al., 1992a). Recently, it was suggested that cAMP is involved in the regulation of the intrinsic burst generating properties of Pre-I neurons (Arata et al., 1993). Smith et al. (1991) identified neurons with voltage-dependent oscillatory properties in the preB6tzinger complex in slice preparations (Smith et al., 1991). The relation between these intrinsic bursting neurons and Pre-I neurons is unknown.

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Changes of IPG-network properties dependent on the respiratory cycle is important in the determination of the inspiratory burst pattern. The IPG could be discussed in the following 4 stages (Onimaru et al., 1991b): (1) Burst initiation. Abrupt transition from the silent phase to the burst phase occurs when excitatory inputs from RRG reach the threshold by temporal and/or spatial summation (Onimaru et al., 1992b); (2) Burst maintenance. Burst activity is maintained by excitatory coupling among inspiratory neurons (see below), despite a marked reduction of inputs from the R R G by feedback inhibition from the IPG; (3) Burst termination. IPG moves from the burst to the silent phase when neuronal activity in the network decreases below a certain threshold level; (4) Refractory period. After a burst, the IPG enters a refractory period, and the threshold gradually decreases to permit a new burst (Onimaru et al., 1989b). Details of the neuronal mechanisms of termination of the inspiratory burst and refractoriness during the post-inspiratory phase are unknown, although changes in efficiency of synaptic transmission during burst activity might be important. 3.3. Network organization Synaptic connections between Pre-I neurons and between inspiratory neurons were studied by pulse crosscorrelation (PCC) analysis (Kashiwagi et al., 1993a). The results indicated a high incidence of mono- or oligosynaptic excitatory connections between paired neurons or shared inputs. Cross-correlation histograms consistent with reciprocal excitatory connections between recorded neurons (Ezure and Manabe, 1989) were found in the pairs of bilateral inspiratory neurons in the RVL (Kashiwagi et al., 1993a) and in the CVL (Onimaru et al., 1993). These results suggested short-term synchronization among medullary respiratory neurons via excitatory coupling. Synaptic connections mediated by EAA receptors may be important in the synchronization between respiratory neurons (Onimaru et al., 1991a). Analysis of neuronal connections using spike-triggered averaging revealed excitatory synaptic connections from Pre-I neurons to inspiratory neurons in the VLM (Onimaru et al., 1992b). Some Pre-I and inspiratory neurons in the VLM project to the ipsilateral or the contralateral spinal cord (Onimaru and Homma, 1992; Kashiwagi et al., 1993a; Onimaru et al., 1993) Studies by whole-cell recordings demonstrated that inspiratory neurons were classified into three subtypes from the patterns of synaptic potentials (Onimaru and Homma, 1992): Type I neurons had a high probability of excitatory postsynaptic potentials in the pre-and postinspiratory phases, so that they probably receive excitatory synaptic inputs from Pre-I neurons. Type II neurons showed no sign of postsynaptic potentials suggesting innervation from Pre-I neurons. Type III neurons were hyperpolarized by inhibitory postsynaptic

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functions from pons or upper brainstem structures are also interesting subjects.

Acknowledgments The author thanks Prof. Ikuo Homma for his helpful advice and discussion, and Dr. Akiko Arata for commenting on the manuscript. The author is grateful to Ms. Suzanne Knowlton for improving the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

References

Fig. 3. Neuronal organization of the central pattern generator of respiration in the medulla. RRG, respiratory rhythm generator, composed of Pre-I neurons in the RVL. IPG, inspiratory pattern generator, composed of inspiratory neurons in the RVL and CVL (see Fig. 2). Type I inspiratory neurons receive excitatory synaptic inputs (open triangle) and type II1 neurons receive inhibitory synaptic inputs (solid triangle) from Pre-I neurons. Pre-I neurons receive inhibitory synaptic inputs from inspiratory neurons.

potentials in the pre- and post-inspiratory phases, thus they are thought to receive inhibitory synaptic inputs from Pre-I neurons. Type I inspiratory neurons are believed to be important in initiating inspiratory bursts. The roles of type II and III neurons is unknown. These neurons may be involved in maintaining inspiratory bursts. On the other hand, these results suggested the presence of two different types of Pre-I neurons, excitatory and inhibitory (Fig. 3).

4. Concluding remarks One of the advantages of the in vitro newborn rat preparation in the study of brainstem functions is that we can analyze and discuss a series of characteristics of neurons, including network or synaptic properties, receptors, membrane excitability (or ionic channels), and intracellular mechanisms such as a second messenger system, after identifying the neurons in the whole brainstem network. Application of patch clamp recordings gives a full advantage of this preparation on these studies. Previous research has revealed a basic neuronal organization for respiratory rhythm generation in this preparation. In the future, important areas concerning rhythm generation to be researched are analyses of ionic channels involved in the burst generation, cellular mechanisms contributing to network properties, and developmental changes in cellular and network properties. New findings would be expected for neuronal mechanisms concerning respiratory reflexes and central chemoreceptions. Descending regulations of medullary

Arata, A., Onimaru, H. and Homma, I. (1990) Respiration-related neurons in the ventral medulla of newborn rats in vitro. Brain Res. Bull., 24: 599-604. Arata, A., Onimaru, H. and Homma, I. (1993) Effects of cAMP on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rat. Brain Res., 605: 193-199. Ballanyi, K., Kuwana, S., V61ker, A., Morawietz, G. and Richter, D.W. (1992) Developmental changes in the hypoxia tolerance of the in vitro respiratory network of rats. Neurosci. Lett., 148: 141-144. Brockhaus, J., Ballanyi, K., Smith, J.C. and Richter, D.W. (1993) Microenvironment of respiratory neurons in the in vitro brainstem-spinal cord of neonatal rats. J. Physiol., 462: 421-445. Bruce, E.N. and Cherniack, N.S. (1987) Central chemoreceptors. J. Appl. Physiol., 62: 389-402. Connelly, C.A., Dobbins, E.G. and Feldman, J.L. (1992) PreB6tzinger complex in cats: respiratory neuronal discharge patterns. Brain Res., 590: 337-340. Di Pasquale, E., Monteau, R. and Hilaire G. (1992a) In vitro study of central respiratory-like activity of the fetal rat. Exp. Brain Res., 89: 459-464. Di Pasquale, E., Morin, D., Monteau, R. and Hilaire, G. (1992b) Serotonergic modulation of the respiratory rhythm generator at birth: an in vitro study in the rat. Neurosci. Lett., 143: 91-95. Di Pasquale, E., Monteau, R. and Hilaire, G. (1994) Involvement of the rostral ventro-lateral medulla in respiratory rhythm genesis during the peri-natal period: an in vitro study in newborn and fetal rats. Dev. Brain Res., 78: 243-252. Euler, C. yon (1986) Brainstem mechanisms for generation and control of breathing pattern. In: A.P. Fishman (Ed.), The Respiratory System, Handbook of Physiology, Section 3, Waverly, MD, pp. 1-67. Errchidi, S., Hilaire, G. and Monteau, R. (1990) Permanent release of noradrenaline modulates respiratory frequency in the newborn rat: an in vitro study. J. Physiol., 429: 497-510. Errchidi, S., Monteau, R. and Hilaire, G. (1991) Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: an in vitro study. J. Physiol., 443: 477-498. Ezure, K., Manabe, M. and Yamada, H. (1988) Distribution of medullary respiratory neurons in the rat. Brain Res., 455: 262-270. Ezure, K. and Manabe, M. (1989) Monosynaptic excitation of medullary inspiratory neurons by bulbospinal inspiratory neurons of the ventral respiratory group in the cat. Exp. Brain Res., 74:501-511. Feldman, J.L. (1986) Neurophysiology of breathing in mammals. In: F.E. Bloom (Ed.), The Nervous System, Volume IV, Intrinsic Regulatory Systems of the Brain, Handbook of Physiology, Section 1, American Physiological Society, Bethesda, MD, pp. 463-524. Feldman, J.L. and Smith, J.C. (1989) Cellular mechanisms underlying modulation of breathing pattern in mammals. In: M. Davis, B.L.

H. Onimaru/ Neurosci. Res. 21 (1995) 183-190

Jacobs and R.I. Schpenfeld, (Eds.), Modulation of Defined Vertebrate Neural Circuits. Ann. NY Acad. Sci., 563:114-130. Feldman, J.L., Smith, J.C. and Liu, G. (1991) Respiratory pattern generation in mammals: in vitro en bloc analyses. Curr. Opin. Neurobiol., 1: 590-594. Fukuda, Y. and Loeshcke, H.H. (1979) A cholinergic mechanism involved in the neuronal excitation by H + in the respiratory chemosensitive structures of the ventral medulla oblongata of rats in vitro. Pflfigers Arch., 379: 125-135. Funk, G.D., Smith, J.C. and Feldman, J.L. (1993) Generation and transmission of respiratory oscillations in medullary slices: role of excitatory amino acids. J. Neurophysiol., 70: 1497-1515. Greet, J.J., Smith, J.C. and Feldman, J.L. (1991) Role of excitatory amino acids in the generation and transmission of respiratory drive in neonatal rat. J. Physiol., 437: 727-749. Greer, J.J., Smith, J.C. and Feldman, J.L. (1992a) Glutamate release and presynaptic action of AP4 during inspiratory drive to phrenic motoneurons. Brain Res., 576: 355-357. Grecr, J.J., Smith, J.C. and Feldman, J.L. (1992b) Respiratory and locomotor patterns generated in the fetal rat brain stem-spinal cord in vitro. J. Neurophysiol., 67: 996-999. Grote, J., Zimmer, K. and Schubert, R. (1981) Effects of severe arterial hypocapnia on regional blood flow regulation, tissue PO 2 and metabolism in the brain cortex of cats. Pflfigers Arch., 391: 195-199. Hamada, O., Garcia-Rill, E. and Skinner, R.D. (1992) Respiration in vitro: I. Spontaneous activity. Somatosen. Motor Res., 9:313-326. Hamada, O., Garcia-Rill, E. and Skinner, R.D. (1994) Electrical activation and inhibition of respiration in vitro. Neurosci. Res., 19: 131-142. Harada, Y., Wang, Y.Z. and Kuno, M. (1985a) Central chemosensitivity to H + and CO 2 in the rat respiratory center in vitro. Brain Res., 333: 336-339. Harada, Y., Kuno, M. and Wang, Y.Z. (1985b) Differential effects of carbon dioxide and pH on central chemoreceptors in the rat in vitro. J. Physiol., 368: 679-693. Hayashi, F., Jiang, C. and Lipski, J. (1991) lntracellular recording from respiratory neurones in the perfused in situ rat brain. J. Neurosci. Meth., 36: 63-70. Hayashi, F. and Lipski, J. (1992) The role of inhibitory amino acids in control of respiratory motor output in an arterially perfused rat. Resp. Physiol., 89: 47-63. Hilaire, G., Monteau, R. and Errchidi, S. (1989) Possible modulation of the medullary respiratory rhythm generator by the noradrenergic A5 area: an in vitro study in the newborn rat. Brain Res., 485: 325-332. Hilaire, G., Monteau, R., Gauthier, P., Rega, P. and Morin, D. (1990) Functional significance of the dorsal respiratory group in adult and newborn rats: in vivo and in vitro studies. Neurosci. Lett., 111: 133-138. Homma, 1., Arata, A. and Onimaru, H. (1993) Respiratory rhythm generation in the ventral medulla. In: Speck, D.F., Dekin, M.S., Revelette, W.R. and Frazier, D.T. (Eds.), Respiratory Control: Central and Peripheral Mechanisms, The University Press of Kentucky, Lexington, KY, pp. 39-42. Issa, F.G. and Remmers, J.E. (1992) Identification of a subsurface area in the ventral medulla sensitive to local changes in PCO 2. J. Appl. Physiol., 72: 439-446. Jodkowski, J.S., Coles, S.K. and Dick, T.E. (1994) A 'pneumotaxic centre' in rats. Neurosci. Lett., 172: 67-72. Kashiwagi, M., Onimaru, H. and Homma, I. (1993a) Correlation analysis of respiratory neuron activity in ventrolateral medulla of brainstem-spinal cord preparation isolated from newborn rat. Exp. Brain Res., 95: 277-290. Kashiwagi, M., Onimaru, H. and Homma, I. (1993b) Effects of NMDA on respiratory neurons in newborn rat medulla in vitro. Brain Res. Bull., 32: 65-69.

189

Kuwana, S., Morawietz, G. and Richter, D.W. (1992) The respiratory network in isolated perfused brainstem of rat. Pflfigers Arch., 420: R125. Lindsay, A.D., Greer, J.J. and Feldman, J.L. (1991) Phrenic motoneuron morphology in the neonatal rat. J. Comp. Neurol., 308: 169-179. Lindsay, A.D. and Feldman, J.L. (1993) Modulation of respiratory activity of neonatal rat phrenic motoneurones by serotonin. J. Physiol., 461: 213-233. Lipski, J. and Merrill, E.G. (1980) Electrophysiological demonstration of the projection from expiratory neurons in rostral medulla to contralateral dorsal respiratory group. Brain Res., 197: 521-524. Liu, G., Feldman, J . L and Smith, J.C. (1990) Excitatory amino acidmediated transmission of inspiratory drive to phrenic motoneurons. J. Neurophysiol., 64: 423-436. Liu, G. and Feldman, J.L. (1992) Quantal synaptic transmission in phrenic motor nucleus. J. Neurophysiol., 68: 1468-1471. McLean, H.A. and Remmers, J.E. (1994) Respiratory motor output of the sectioned medulla of the neonatal rat. Resp. Physiol., 96: 49-60. McCrimmon, D.R., Smith, J.C. and Feldman, J.L (1989) Involvement of excitatory amino acids in neurotransmission of inspiratory drive to spinal respiratory motoneurons. J. Neurosci., 9: 1910-1921. Merrill, E.G., Lipski, J., Kubin, L. and Fedorko, L. (1983) Origin of the expiratory inhibition of nucleus tractus solitarius inspiratory neurons. Brain Res., 263: 43-50. Monteau, R., Morin, D. and Hilaire, G. (1990) Acetylcholine and central chemosensitivity: in vitro study in the newborn rat. Resp. Physiol., 81: 241-254. Morin, D., Hennequin, S., Monteau, R. and Hilaire, G. (1990a) Serotonergic influences on central respiratory activity: an in vitro study in the newborn rat. Brain Res., 535: 281-287. Morin, D., Hennequin, S., Monteau, R. and Hilaire, G. (1990b) Depressant effect of raphe stimulation on inspiratory activity of the hypoglossal nerve: in vitro study in the newborn rat. Neurosci. Lett., 116: 299-303. Morin, D., Monteau, R. and Hilaire, R. (1991a) 5-Hydroxy-tryptamine modulates central respiratory activity in the newborn rat: an in vitro study. Eur. J. Pharmacol., 192: 89-95. Morin, D., Monteau, R. and Hilaire, G. (1991b) Serotonin and cervical respiratory motoneurones: intracellular study in the newborn rat brainstem-spinal cord preparation. Exp. Brain Res., 84: 229-232. Morin, D., Monteau, R. and Hilaire, G. (1992) Compared effects of serotonin on cervical and hypoglossal inspiratory activities: an in vitro study in the newborn rat. J. Physiol., 451: 605-629. Morin-Surun, M.P. and Denavit-Saubi6, M. (1989) Rhythmic discharges in the perfused isolated brainstem preparation of adult guinea pig. Neurosci. Lett., 101: 57-61. Murakoshi, T. and Otsuka, M. (1985) Respiratory reflexes in an isolated brainstem-lung preparation of the newborn rat: possible involvement of ~,-aminobutyric acid and glycine. Neurosci. Lett., 62: 63-68. Murakoshi, T., Suzue, T. and Tamai, S. (1985) A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat. Br. J. Pharmac., 86: 95-104. Okada, Y., Miickenhoff, K., Holtermann, G., Acker, H. and Scheid, P. (1993a)Depth profiles of pH and PO 2 in the isolated brain stem-spinal cord of the neonatal rat. Respir. Physiol., 93:315-326. Okada, Y., Miickenhoff, K. and Scheid, P. (1993b) Hypercapnia and medullary neurons in the isolated brain stem-spinal cord of the rat. Respir. Physiol., 93: 327-336. Onimaru, H. and Homma, I. (1987) Respiratory rhythm generator neurons in medulla of brainstem-spinal cord preparation from newborn rat. Brain Res., 403: 380-384. Onimaru, H., Arata, A., and Homma, I. (1987) Localization of respiratory rhythm-generating neurons in the medulla of brainstem-

190

H. Onimaru/Neurosci. Res. 21 (1995) 183-190

spinal cord preparations from newborn rats. Neurosci. Lett., 78: 151-155. Onimaru, H., Arata, A. and Homma, I. 0988) Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat. Brain Res., 445: 314-324. Onimaru, H., Arata, A. and Homma, I. (1989a) Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro. Exp. Brain Res., 76: 530-536. Onimaru, H., Arata, A. and Homma, I. (1989b) Electrophysiological properties and localization of inspiratory pattern generator in medulla isolated from newborn rats. Jpn. J. Physiol., 39: s249. Onimaru, H., Arata, A. and Homma, I. (1990) Inhibitory synaptic inputs to the respiratory rhythm generator in the medulla isolated from newborn rats. Pflfigers Arch., 417: 425-432. Onimarn, H., Arata, A. and Homma, I. (1991a) The role of a putative excitatory amino acid transmitter in the generation of respiratory rhythm in medulla isolated from newborn rat. Adv. Biosci., 79: 57-59. Onimaru, H., Arata, A., Kashiwagi, M. and Homma, I. (1991b) Network properties of inspiratory pattern generator in medulla isolated from newborn rats. Comp. Physiol. Biochem., 8: 160. Onimarn, H. and Homma, I. (1992) Whole cell recordings from respiratory rfeurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Pflfigers Arch., 420: 399-406. Onimaru, H., Arata, A. and Homma, I. (1992a) Analysis of pacemaker properties of respiratory neurons using 'perforated' whole-cell recordings. Neurosci. Res., 17: s212. Onimaru, H., Homma, I. and Iwatsuki, K. (1992b) Excitation of inspiratory neurons by preinspiratory neurons in rat medulla in vitro. Brain Res. Bull., 29: 879-882. Onimaru, H., Kashiwagi, M., Arata, A. and Homma, I. (1993) Possible mutual excitatory couplings between inspiratory neurons in caudal ventrolateral medulla of brainstem-spinal cord preparation isolated from newborn rat. Neurosci. Lett., 150: 203-206. Paton, J.F.R., Ramirez, J.M. and Richter, D.W. (1994) Mechanisms of respiratory rhythm generation change profoundly during early life in mice and rats. Neurosci. Lett., 170: 167-170. Pilowsky, P.M., Jiang, C. and Lipski, J. (1990) An intracellular study

of respiratory neurons in the rostral ventrolateral medulla of rat and their relationship to catecholamine-containing neurons. J. Comp. Neurol., 301: 604-617. Richter, D.W., Ballanyi, K. and Schwarzacher, S. (1992) Mechanisms of respiratory rhythm generation. Curr. Opin. Neurobiol., 2: 788-793. Ross, C.A., Ruggiero, D.A., Joh, T.H., Park, D.H. and Reis, D.J. (1984) Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing Cl adrenaline neurons. J. Comp. Neurol., 228: 168-185. Smith, J.C. and Feldman, J.L. (1987) Central respiratory pattern generation studied in an in vitro mammalian brainstem-spinal cord preparation. In: G.C. Sieck, S.G. Gandevia and W.E. Cameron (Eds.), Respiratory Muscles and their Neuromotor Control, Liss, New York, pp. 27-36. Smith, J.C., Greet, J.J., Liu, G. and Feldman, J.L. (1990) Neural mechanisms generating respiratory pattern in mammalian brain stem-spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol., 64: 1149-1169. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W. and Feldman, J.L. (1991) Pre-B6tzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science, 254: 726-729. Smith, J.C., Bailanyi, K. and Richter, D.W. (1992) Whole-cell patchclamp recordings from respiratory neurons in neonatal rat brainstem in vitro. Neurosci. Lett., 134: 153-156. Suzue, T., Murakoshi, T. and Tamai, S. (1983) Electrophysiology of reflexes in an isolated brainstem-spinal cord preparation of the newborn rat. Biomed. Res., 4:611-614. Suzue, T. (1984) Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J. Physiol., 354: 173-183. Yamamoto, Y., Onimaru, H. and Homma, I. (1992) Effect of substance P on respiratory rhythm and pre-inspiratory neurons in the ventrolateral structure of rostral medulla oblongata: an in vitro study. Brain Res., 599: 272-274. Wang, W., Fung, M.-L. and St. John, W.M. (1993) Pontile regulation of ventilatory activity in the adult rat. J. Appl. Physiol., 74: 2801-281 I.