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Control of respiration in the isolated central nervous system of the neonatal opossum, Monodelphis domestica
Brain Research Bulletin, Vol. 53, No. 5, pp. 605– 613, 2000 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
PII S0361-9230(00)00394-4
Control of respiration in the isolated central nervous system of the neonatal opossum, Monodelphis domestica Jaime Eugenı´n1 and John G. Nicholls2* 1
Department of Biological Sciences, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile; and 2Department of Biophysics, International School for Advanced Studies, Trieste, Italy [Received 5 May 2000; Revised 10 July 2000; Accepted 16 August 2000] ulus. For example, an increase in temperature increases the rate of fictive respiration without changing its amplitude, whereas noradrenaline decreases the rate while increasing the amplitude. Thus, changes of timing and amplitude need not go hand in hand. The opossum CNS offers a favorable preparation for the analysis of neural mechanisms that generate and modulate a motor rhythm, as the animal develops from embryonic to adult stages. © 2001 Elsevier Science Inc.
ABSTRACT: Respiration represents an unusual motor activity with respect to its development. As newly born mammals enter the world, their limb movements are not coordinated; time and experience are required for effective performance to be achieved. Yet the rhythm of respiration is of necessity functionally perfected and unfailing at birth. Inspiratory and expiratory motor neurons are already able to fire at appropriate rates, under the command of rhythmically active neurons in the medulla. In this review, we discuss refinements of control present in the newborn opossum, particularly with respect to mechanisms that allow adaptation of respiration to changes in the level of activity or in the outside environment. Our own studies have been aimed at analyzing respiration at the earliest stages, and at establishing the way in which important variables influence inspiration and expiration. To this end, we have used the central nervous system (CNS) of a neonatal opossum, isolated in its entirety and maintained in culture. Although the opossum is unable to walk and highly immature at birth, its respiration is regular and unfailing. The isolated CNS survives, undergoes development, and maintains its neural activity and fine structure in vitro. Moreover, fictive respiration persists for over a day or longer at rates similar to those of the intact pup. The effects of altered pH, of increased temperature, and of drugs known to alter respiratory rhythm in intact animals can be measured directly, by electrical recordings made from medullary neurons or ventral roots. As in a slice, fluids of different composition can be applied focally, through micropipettes to the surface of the ventral medulla, or diffusely to the brainstem, With highly localized application of procaine hydrochloride (2%) to selected areas of the ventral medulla, the respiratory rhythm is reduced or abolished. As in adult mammals, both the rate and the amplitude of respiration simultaneously increase in response to lowered pH (6.5—7.1) or to topical application of 1.0 M carbachol. Conversely, as expected, the rate and amplitude decrease in response to increased pH (pH 7.5–7.7), or 100 M scopolamine. Two characteristic features of the control of respiration in the neonatal opossum are evident from such tests. First, changes in rate are achieved by changes in the duration of the expiratory phase of respiration. This result suggests that the timing of the respiratory cycle in the neonatal opossum is controlled by an expiratory instead of an inspiratory “off-switch”. Second, the rate and the amplitude of the respiratory excursions can be controlled independently, depending on the stim-
KEY WORDS: Respiratory pattern generation, Control of breathing, Marsupials, Central chemoreception, Development.
INTRODUCTION At the time a mammal is born, the neural network that generates the respiratory rhythm is primed and ready for the successful activation of inspiratory and expiratory muscles. From the outset, breathing is rhythmic and synchronized, but, as development proceeds, characteristic patterns of respiration emerge. The respiratory rate and depth are modified to fit the demands of the animal for uptake of oxygen and removal of carbon dioxide. In particular, the interactions of intrinsic respiratory motor programs with sensory inputs become modified, as adult characteristics become defined [51,77]. Changes in Frequency of Respiration Brought About by Altered Duration of Inspiratory Phase The motor programming for breathing can be analyzed in terms of variables for amplitude and for timing. Measurements of the relationship between amplitude and inspiratory and expiratory durations have provided insights into functional mechanisms underlying the respiratory motor pattern. Clark and von Euler [9] showed in anaesthetised cats that an increase in inhaled CO2 increases both the amplitude and the rate of respiration. The shortening of the respiratory cycle consists of a decrease in the inspiratory duration mediated by the vagal reflex that inhibits inspiration (Hering-Breuer reflex). Thus, during chemical stimulation by low pH or raised levels of CO2, it is the duration of the
* Address for correspondence: Dr. John G. Nicholls, International School for Advanced Studies, Via Beirut 2, I-34014 Trieste, Italy. Fax: ⫹39-0403787-528; E-mail: [email protected]
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606 inspiratory phase of respiration that is shortened so as to produce an increase in frequency. At the same time the amplitude of the respiratory excursions increases [4,5,9,11,36,46,97]. These observations have been incorporated into an “inspiratory off-switch hypothesis”. According to this scheme, the generation of the respiratory rhythm depends on two control mechanisms. One compartment, the central inspiratory activator, generates and shapes the inspiratory activity; a second compartment, known as the “inspiratory off-switch”, inhibits the central inspiratory activator. Vagal inputs from slowly adapting pulmonary mechanoreceptors and tonic central activity (probably from pontine nuclei) are assumed to activate the “inspiratory off-switch” [6,12,14,21,96, 97]. Alterations in the Duration of the Expiratory Phase of Respiration In certain preparations, selective changes in the duration of the expiratory, rather than the inspiratory, phase account for changes in rate. These represent mechanisms in which the central pattern generator for respiration is not controlled by vagal inputs. For example, in vagotomized adult rats, anaesthetised and ventilated artificially, the expiratory phase becomes shortened in response to chemical stimulation [38]. Similar phenomena are seen in the in vitro brainstem of adult guinea pigs [69,70]. In conscious humans, breathing at low tidal volumes [13,15,43], vagal afferents are minimally excited and it is the expiratory phase that shortens in response to mild chemical stimuli. A further example is provided by results obtained in pre-term infants. A decrease in respiratory frequency is induced by inhalation of 15% O2, and this results from an increase in the duration of the expiratory phase [51]. Fictive Respiration of Isolated Mammalian Preparations In Vitro Further simplification of preparations for the study of respiration is provided by the use of slices in vitro. Slices used by Feldman, Smith and others [42,44,56,59,81– 83,87,89] offer the advantage of direct access to defined groups of neurons for recording and for application of drugs while respiratory activity is recorded from hypoglossal roots. Inevitably, however, all ascending and descending connections have by definition been cut, and this may explain differences in the patterns of responses to altered pH in slices and intact central nervous system (CNS) preparations [79]. In 1984, Suzue demonstrated that fictive respiration occurs in the isolated brainstem of the neonatal rat in vitro [94]. In this preparation, known as the en bloc preparation, peripheral nerves as well as dorsal and ventral roots are cut, but the regions of the CNS are maintained in their normal relationships. Since this pioneering work, such preparations have been used to study the generation and regulation of the respiratory rhythm in neonatal mammals [1,2,7,16,40,45,49, 50,53,57,58,60,62,64,65,74,75,88,90]. In a series of experiments made on embryonic or neonatal rat CNS in vitro, Hilaire and his colleagues have described the time of onset of respiration and analyzed the effects of serotonin and substance P on respiratory neurons [17–20,52,63,65– 67,80]. In both types of preparations, neonatal rodent CNS in vitro and brainstem slices, the frequency of activity recorded is very low compared to that of the intact animal. The bursts occur at only about 9 per minute [62,79,89]; as a result, problems of interpretation can arise about whether the activity represents respiration or perhaps some other rhythmical function such as gasping [39,41,54,91, 92,99]. Recently, preparations of adult mammalian CNS in vitro have been developed, in which characteristic respiratory re-
EUGENI´N AND NICHOLLS sponses can be obtained by increases in CO2 or lowered pH [68,70,76,78]. Advantages of Neonatal Opossums for Studies of Development of Respiration Marsupials, such as opossums and kangaroos, are born at an extremely immature stage of development. At a time when the cortex is not yet formed and when motor performance is not coordinated, the new-born animal has to produce rhythmical inspiratory and expiratory movements for respiration. This feature has made opossums attractive animals for studying respiratory mechanisms during development. Farber, for example, has described the properties of respiratory control mechanisms and motor neurons in suckling North American opossums (Didelphis virginiana) as they mature, with particular reference to the effects of transmitters, cerebellar stimulation, vagal afferent stimulation and altered load produced by positive pressure breathing [24 –35]. Moreover, the South American opossum, Monodelphis domestica, which corresponds at birth to a 15-day rat or mouse embryo, can be bred in the laboratory. Its respiration can be studied from the first day of life. The CNS at this time already contains neurons with properties and connections resembling those of an adult that enable it to breathe effectively [71,100]. FICTIVE RESPIRATION IN ISOLATED NEONATAL OPOSSUM CNS The central nervous system of the newly born opossum (Monodelphis domestica), isolated in its entirety and maintained in culture, is in many respects similar to the rat preparation described by Suzue. At the same time, the opossum CNS offers certain advantages. The preparation is so small that it survives well for periods of 3 weeks or longer; it maintains its electrical excitability, fine structure, and responses to amino acid transmitters such as ␥-aminobutyric acid, glutamate, and glycine [71,93,95,101] and even shows development in the dish [37,71,85,93]. Of particular interest is that the isolated CNS produces regular, robust and well maintained fictive respiration for periods of a day or more, longerlasting and more consistent than other fictive rhythms in vitro [22,71,100]. As in a slice, it is possible to record from individual neurons in the medulla and to apply stimuli by localised application through a pipette or diffusely in the bathing fluid [22,100]. Two convenient features are the absence of the cerebellum (which does not cover the medulla at birth) and the ability to record inspiratory activity in ventral roots and in the phrenic nerve. Moreover, the rate of respiration of the isolated preparation (in medium gassed with 5% CO2–95% O2, pH 7.4 at 22°C) corresponds roughly to that of the newborn pup, continuing at approximately 45 per min (as compared to about 60 per min in the animal, see [71]). The integrated inspiratory burst recorded from cervical ventral roots exhibits a characteristic ramp shape [22]. In the following sections, we review briefly recent experiments made in the isolated opossum CNS preparation that demonstrate similarities and differences from respiratory mechanisms discovered in other developing and mature mammals. One question concerns the location of the neurons that generate the rhythm and another is the degree of control of respiration at the earliest stages of development. For example, would changes in rate involve changes in the duration of the inspiratory cycle, the expiratory cycle or both? Would separate groups of cells be assigned to control of the rate and the amplitude of respiration?
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FIG. 1. Positions and properties of medullary neurons showing activity correlated with inspiration and expiration in neonatal opossums. (A) and (C) show the positions of respiratory neurons from which the unit recordings in (B) were made. Symbols indicate the phase of respiration during which the unit is active. The timing is established from recordings made in the ventral root of C5 (after [100]).
Techniques for Maintaining Isolated Opossum CNS and Recording Respiratory Activity The methods have been described elsewhere [22,71,100]. In brief, the entire CNS is dissected from opossums Monodelphis domestica (5–12-day-old) anaesthetised with methoxyflurane and maintained in a chamber 0.5 ml in volume superfused with basal medium Eagle’s (BME) oxygenated with 95% O2–5% CO2, pH 7.37–7.40 at room temperature (21–23°C). As shown in Fig. 1,
spontaneous electrical activity from C3–C5 ventral roots, recorded with suction electrodes, consists of bursts of action potentials superimposed on a background of random activity. Rhythmic bursts occurring at about 45 per min correspond to fictive respiration [22,71,100]. The bursts are regular in timing and amplitude [22,71,100]. Simultaneously, records are made from individual neurons in the medulla with extracellular microelectrodes. Chemical stimuli are administered by local application of test solutions
FIG. 2. Fictive respiration recorded from C5 ventral root in the isolated central nervous system (CNS) of a 9-day-old opossum. Scheme for recording from isolated CNS. The partition in the bath makes it possible to provide fluids to the lower brainstem and spinal cord separately. Two inspiratory bursts and their integrated activity are illustrated. Amplitude (A) is measured from integrated activity; TE, expiratory duration; TI, inspiratory duration; Ttot, cycle duration.
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FIG. 3. Effect of pH and procaine on fictive respiration. (A) Effect of focal application of basal medium Eagle’s (BME) pH 6.5 (horizontal bar) to a region of ventral medulla medial to hypoglossal roots in an 8-day-old opossum (site of administration indicated as black spot in the inset); (B) effect of superfusion of the lower brainstem with BME pH 7.1, pH 7.4, and pH 7.6 in a 6-day-old opossum; (C) effects of 30 and 80 s of focal application of 2% procaine hydrochloride to a region medial to hypoglossal roots in a 12-day-old opossum (black spot in central nervous system inset). Note that respiratory effects are opposite to those observed with topical application of pH 6.5 BME [compare with (A)].
to the surface of ventral medulla through a triple barrel micropipette of 100 m outer diameter [22]. Alternatively, by the use of a thin partition at the level of C1–C2 (Fig. 2), the brainstem can be superfused separately from the spinal cord. Changes in the pH of the superfusion medium are achieved by gassing with different CO2:O2 mixtures or by adding bicarbonate to control BME. Figure 2 shows the way in which records are analyzed quantitatively so that the effects of variables can be assessed in terms of altered inspiratory and expiratory phases, as well as amplitude. Location of Medullary Respiratory Neurons in Neonatal Opossum Extracellular recordings were first made by Zou from respiratory-related neurons in the medulla. In his sample of 128 cells, the respiratory neurons were classified as: (1) early inspiratory (n ⫽ 69), (2) inspiratory (n ⫽ 38), (3) post-inspiratory (n ⫽ 17), and (4) expiratory (n ⫽ 4) [100]. These four types showed discharge patterns similar to those recorded in other mammalian preparations [3,11,23,61]. The inspiratory and expiratory cells were intermingled. All were located in the ventral medulla close to the nucleus ambiguus (Fig. 1). Effects of Altered pH on Respiratory Rate and Amplitude As expected, a reduction of pH from 7.4 to 7.1 in the fluid bathing the lower brainstem increases the rate (by approximately 100%) and the amplitude (by approximately 200%). Similarly, localized, topical applications of low pH fluid (pH 6.5) to regions on the surface of ventral medulla (Figs. 3A and 4) increase the rate of respiration by 88% ⫾ 9% and its amplitude by 109 ⫾ 25% (n ⫽
7; SD). All such effects are readily reversible and can be repeated time after time on the same preparation. An increase in the pH of the superfusion medium from 7.4 to 7.6 produces the opposite effect: thus, fluid with a high pH of 7.7 reduces the rate of fictive respiration by approximately 55% and its amplitude by 50% (n ⫽ 5). Superfusion of the brainstem with sufficiently high pH fluid can reversibly abolish the respiratory rhythm. Quantitative analyses of the effects of altered pH on the phases and amplitude of respiration are shown in Figs. 4 –7. That the activity of the neurons identified as respiratory is crucial for the genesis of the rhythm has been shown by highly localized application of procaine hydrochloride (2%) to the surface of ventral medulla at sites known to be H⫹- and acetylcholinesensitive. Procaine abolishes or reduces the rate and amplitude of fictive respiration (by 60% or more). Figure 3C illustrates the reversible depression in rate and amplitude of fictive respiration obtained after unilateral topical application of procaine to a region medial to the hypoglossal roots. Loss of activity following a focal alteration in neuronal function could result from loss of a stimulatory drive to the network as well as from destruction of a key site for rhythmogenesis. Cholinergic Mechanisms for Responses to Altered pH As in other preparations, there is evidence for a cholinergic drive in respiration and responses to altered pH (see Figs. 4, 6 and 7). Thus, carbachol (1 M) applied to sensitive areas of ventral medulla increases the rate of fictive respiration by 66⫾23% and its amplitude by 66⫾15% (n ⫽ 5). After diffuse application of 100
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M scopolamine, the rate decreases by 58⫾4% and the amplitude by 65⫾4%. Hexamethonium, by contrast has no effect. The specificity of these muscarinic effects is further demonstrated by comparison with noradrenaline [22,100], which produces very different effects on rate and amplitude. For example, Figs. 4 –7 show that addition of noradrenaline (30 –50 M) to the medium perfusing the lower brainstem, reproducibly causes the rate to decrease (by 54 ⫾ 15%), while the amplitude of fictive respiration increases (by 103 ⫾ 16.7%). Responses to Changes in Temperature The temperature of the isolated preparation at 22° C is approximately 7°C lower than that of the neonatal pup (N. Saunders, personal communication). When the temperature of the fluid bathing the preparation is increased to 27°C, the rate of fictive respiration increases (by about 47%), with no accompanying change in amplitude (Figs. 4 –7). Changes in Timing and Amplitude FIG. 4. Effects of chemical stimuli and altered temperature on the rate and amplitude of fictive respiration. Changes in rate (filled columns) and amplitude (open columns) correspond to values during stimulation expressed as percentage with respect to basal (100%); bars indicate SEM. Stimuli are represented as follows. ‘low pH (L)’ signifies topical application of basal medium Eagle’s (BME) pH 6.5; ‘low pH (S)’ signifies brainstem superfusion with BME pH 7.1; ‘carbachol’, topical application of 1 M carbachol hydrochloride; ‘temperature’, increase of bath temperature from 19°C to 27°C; ‘high pH’, brainstem superfusion with BME pH 7.6; ‘scopolamine’, brainstem superfusion with 100 M scopolamine hydrochloride; ‘procaine’, topical application of 2% scopolamine hydrochloride; ‘noradrenaline’, brainstem superfusion of 30 –50 M DL-noradrenaline hydrochloride. Significance levels for *p ⬍ 0.05 and for **p ⬍ 0.02 (Wilcoxon signed-rank test). Number of trials indicated in brackets.
As illustrated in Fig. 4, stimuli can be grouped on the basis of their effects on respiratory rate and amplitude: (1) low pH and carbachol increase both respiratory rate and amplitude; (2) high pH, scopolamine, and topical application of procaine reduce the respiratory rate, and at the same time decrease amplitude; (3) noradrenaline decreases the rate while increasing the amplitude; and (4) raised temperature increases respiratory rate without affecting the amplitude. A common feature of these results is apparent from an analysis of inspiratory and expiratory timing. In isolated opossum CNS preparations, changes in rate are brought about by changes in the duration of the expiratory phase (TE). The
FIG. 5. Burst-to-burst time course of respiratory responses. In the upper panels cycle duration (Ttot), inspiratory duration (TI), and expiratory duration (TE) (in seconds) are plotted against the ordinal numbers of respiratory cycles. It is the expiratory duration that changes with these stimuli. In lower panels the amplitude (arbitrary units) is plotted in a similar manner against respiratory cycle number. The duration of stimulation is indicated by horizontal bars. (A) Topical application of basal medium Eagle’s (BME) pH 6.5 to the ventral medulla (5-day-old opossum). (B) Increase of bath temperature from 19 –27°C (9-day-old opossum). (C) Brainstem superfusion with BME containing 50 M noradrenaline hydrochloride (10-day-old opossum).
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FIG. 6. Response in cycle duration (Ttot) as a function of inspiratory and expiratory phases of the respiratory cycle. Ttot vs. TI (open triangles) and Ttot vs. TE (filled squares) relationships are illustrated. The ordinate in each experiment is the duration of the total respiratory cycle; the abscissa is the duration of the inspiratory or the expiratory phase of respiration. (A) Focal application of basal medium Eagle’s (BME) pH 6.5. (B) Lower brainstem superfusion of BME pH 7.1. (C) Effects of topical application of BME pH 6.5 and the brainstem superfusion of BME pH 7.6 in the same preparation. (D) Topical application of 1 M carbachol hydrochloride. (E) Superfusion of 100 M scopolamine hydrochloride. (F) Topical application of 2% procaine to a region medial to hypoglossal roots. (G) Brainstem superfusion of 50 M noradrenaline hydrochloride. (H) Increase of bath temperature from 19°C to 27°C. This is the only variable that can, with prolonged application, alter the duration of the inspiratory phase of respiration to a minor extent. Lines are based on regression equations and correlation coefficients. (Significance levels indicated below). Arrows show the basal values in the absence of stimuli.
inspiratory phase (TI) remains constant in duration. Furthermore, this close association between cycle duration (TTOT) and TE is evident whatever changes occur in amplitude. For example, with application of either low pH or noradrenaline, the amplitude of fictive respiration is increased (Fig. 4). But with
low pH, the total time of the respiratory cycle (TTOT) is reduced through the shortening of TE, while with noradrenaline both TTOT and TE are prolonged (Figs. 5–7). There is one exception to this general picture: although, higher temperatures increase the rate by shortening TE (as in Fig. 5), Fig. 6 shows that
FIG. 7. Relationships between amplitude and phases of the respiratory cycle. Amplitude is plotted against the duration of inspiration (Ti, open triangles) and expiration (TE, filled squares) for the same experiments as those shown in Fig. 6. Amplitude is expressed in arbitrary units and duration in seconds. Arrows again show basal values without stimuli.
RESPIRATION IN NEONATAL OPOSSUM prolonged exposure to 27° can also shorten the duration of the inspiratory phase (TI) to a minor extent. RESPIRATION OF NEONATAL OPOSSUM IN VITRO COMPARED WITH THAT OF OTHER MAMMALIAN PREPARATIONS Respiration of the isolated new-born opossum CNS resembles that of adult mammals in that the rate and the amplitude can be varied independently [4,9,11,12,46,96,97]. Although medullary respiratory neurons are close to the surface and accessible, they are not grouped in discrete inspiratory and expiratory clusters. Similarly, chemosensitive neurons that influence amplitude and timing are inextricably interspersed in such a way that H⫹- and acetylcholine-sensitive regions of the ventral medulla in the isolated CNS of new-born opossums overlap [22]. In spite of the absence of anatomical boundaries, functionally defined compartments that control different aspects of respiration can be postulated. Thus, the respiratory pattern generator appears to consist of two functional compartments, one controlling amplitude and the other controlling timing of respiratory activity [6,12,14,21,96,97]. One unusual control mechanism in the isolated Monodelphis CNS is that the frequency of respiration depends primarily on changes in the duration of expiratory rather than inspiratory events. Accordingly, the functional compartment controlling the timing of the respiratory cycle in the new-born opossum represents an “expiratory off-switch” rather than an “inspiratory off-switch” mechanism. As mentioned in the Introduction, in some mammalian preparations similar selective changes in TE rather than TI can occur in response to chemical stimulation [13,15,38,43,51,69,70]. In adult mammals, the rate component is primarily mediated by vagal feedback, which influences the inspiratory rather than the expiratory phase [9,35,97,98], and which is not present in the isolated opossum preparation. The maturational changes that are responsible for this switch in the respiratory response are still unknown. An additional distinctive feature of the isolated new-born Monodephis CNS is the coupling of amplitude and frequency. Increases in amplitude produced by chemical and cholinergic stimulation are associated with a shortened duration of expiration. This has not been found to be the case in Didelphis virginiana opossums aged 7–20 days. In animals at that age the increased respiration in response to chemical stimulation occurs primarily by increases in depth of ventilation (tidal volume). The association between amplitude and rate becomes evident only at later stages, when the rate component develops [34,35]. In other new-born mammals, the respiratory response to chemical stimulation also consists of increases in the amplitude of inspiration, with little or no change in rate, whether vagal feedback is present or not [8,10,47,73,84,86,98]. Only in adult animals, do the vagal feedback and the rate component become major contributors [35,98]. In isolated brainstem-spinal cord preparations from neonatal rats, separation of rate and amplitude control mechanisms is also apparent: chemical stimulation can change the rate or the amplitude independently [48,49,55,64,72,94]. The clear dissociation between amplitude of respiration and duration of inspiration in the isolated Monodelphis CNS suggests that there is no coupling between the central mechanisms that activate inspiration and that turn it off (the inspiratory off-switch). Rather, the shortening of expiration during chemical stimulation appears to be related to an augmentation of amplitude. The relation is inverse, and resembles the volume threshold curve for inspiration-inhibiting reflexes (Tidal volume vs. TI curve) characteristic of adult mammals with intact vagal feedback [9]. One possible explanation for the findings in opossum could be the presence of an “expiratory off-switch” activated by chemical stimulation of
611 inspiratory generator mechanisms. Modification in amplitude is not per se a main factor determining frequency. (With the exception however, that an increase in the duration of expiration can occur with an increase or no change in amplitude, in response to noradrenaline or lowered temperature.) An attractive scheme for functional organisation of the respiratory pattern generator is that coupled inspiratory and expiratory generators are under the control of inspiratory and expiratory off-switches. Chemical and cholinergic inputs would excite the inspiratory generator and the expiratory off-switch; thermic receptors would activate the expiratory off-switch, with minor effects on the inspiratory on switch; noradrenaline would inhibit the expiratory off-switch, but stimulate the inspiratory activator. SUMMARY In this review, we compare the properties and regulatory mechanisms of fictive respiration in isolated opossum CNS preparations with those found in other in vitro preparations and in intact animals. The principal findings are that at early stages of opossum development, changes of the pH in contact with medullary respiratory neurons give rise to increases and decreases in frequency that depend on the timing of the expiratory phase of respiration. This result suggests that the regulation of respiration in the newborn opossum depends on an “expiratory off-switch”. This is in contrast to other immature mammalian preparations in which modulation of amplitude or changes in the inspiratory phase constitute responses to chemical stimuli. Moreover, changes in the frequency and amplitude of respiration are controlled by separate mechanisms. The principal advantages of the isolated opossum CNS for such studies are its long life, the accessibility of the respiratory command neurons, the normal relations of the different parts of the CNS to each other and a frequency of fictive respiration that is similar to that in intact animals. ACKNOWLEDGEMENTS
We thank Roland Geiser and Urs Berglas for maintaining the colony of Monodelphis and Paul Ba¨ttig for photography. We are particular grateful to Dr. A.R. Martin for his critical reading of the manuscript, and to Dr. W. Adams for his help with the electronics, software, and valuable suggestions throughout the experiments and the preparation of this manuscript. This work was supported by the Swiss Nationalfonds Grant (no. 313626292) and BBW Grant (no. PL 960211) to J. G. N., Fondecyt (no. 1980819) and Dicyt-USACH (no. 029743EL) to J.E.
REFERENCES 1. Arata, A.; Onimaru, H.; Homma, I. Respiration-related neurons in the ventral medulla of newborn rats in vitro. Brain Res. Bull. 24:599 – 604; 1990. 2. Arata, A.; Onimaru, H.; Homma, I. The adrenergic modulation of firings of respiratory rhythm-generating neurons in medulla-spinal cord preparation from newborn rat. Exp. Brain Res. 119:399 – 408; 1998. 3. Bianchi, A. L.; Denavit-Saubie, M.; Champagnat, J. Central control of breathing in mammals: Neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75:1– 45; 1995. 4. Bradley, G. W.; von Euler, C.; Marttila, I.; Roos, B. Steady-state effects of CO2 and temperature on the relationship between lung volume and inspiratory duration (Hering-Breuer threshold curve). Acta Physiol. Scand. 92:351–363; 1974. 5. Bradley, G. W.; von Euler, C.; Marttila, I.; Roos, B. Transient and steady state effects of CO2 on mechanisms determining rate and depth of breathing. Acta Physiol. Scand. 92:341–350; 1974. 6. Bradley, G. W.; von Euler, C.; Marttila, I.; Roos, B. A model of the central and reflex inhibition of inspiration in the cat. Biol. Cybernet. 19:105–116; 1975.
612 7. Brockhaus, J.; Ballanyi, K.; Smith, J. C.; Richter, D. W. Microenvironment of respiratory neurons in the in vitro brainstem-spinal cord of neonatal rats. J. Physiol. 462:421– 445; 1993. 8. Carroll, J. L.; Canet, E.; Bureau, M. A. Dynamic ventilatory responses to CO2 in the awake lamb: Role of the carotid chemoreceptors. J. Appl. Physiol. 71:2198 –2205; 1991. 9. Clark, F. J.; von Euler, C. On the regulation of depth and rate of breathing. J. Physiol. 222:267–295; 1972. 10. Cohen, G.; Xu, C.; Henderson-Smart, D. Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. J. Appl. Physiol. 71:168 –174; 1991. 11. Cohen, M. I. Neurogenesis of respiratory rhythm in the mammal. Physiol. Rev. 59:1105–1173; 1979. 12. Cohen, M. I.; Feldman, J. L. Models of respiratory phase-switching. Fed. Proc. 36:2367–2374; 1977. 13. Cunningham, D. J.; Gardner, W. N. A quantitative description of the pattern of breathing during steady-state CO2 inhalation in man, with special emphasis on expiration. J. Physiol. 272:613– 632; 1977. 14. D’Angelo, E. Control of breathing: Central mechanisms and peripheral inputs. Bull. Eur. Physiopath. Resp. 16:111–112; 1980. 15. Davis, N. J.; Stagg, D. Interrelationships of the volume and time components of individual breaths in resting man. J. Physiol. 245:481– 498; 1975. 16. Di Pasquale, E.; Lindsay, A.; Feldman, J.; Monteau, R.; Hilaire, G. Serotonergic inhibition of phrenic motoneuron activity: An in vitro study in neonatal rat. Neurosci. Lett. 230:29 –32; 1997. 17. Di Pasquale, E.; Monteau, R.; Hilaire, G. In vitro study of central respiratory-like activity of the fetal rat. Exp. Brain Res. 89:459 – 464; 1992. 18. Di Pasquale, E.; Monteau, R.; Hilaire, G. Endogenous serotonin modulates the fetal respiratory rhythm: An in vitro study in the rat. Brain Res. Dev. Brain Res. 80:222–232; 1994. 19. Di Pasquale, E.; Morin, D.; Monteau, R.; Hilaire, G. Serotonergic modulation of the respiratory rhythm generator at birth: An in vitro study in the rat. Neurosci. Lett. 143:91–95; 1992. 20. Errchidi, S.; Monteau, R.; Hilaire, G. Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: An in vitro study. J. Physiol. 443:477– 498; 1991. 21. Eugenin, J. Generation of the respiratory rhythm: Modelling the inspiratory off switch as a neural integrator. J. Theor. Biol. 172:107– 120; 1995. 22. Eugenin, J.; Nicholls, J. G. Chemosensory and cholinergic stimulation of fictive respiration in isolated CNS of neonatal opossum. J. Physiol. 501:425– 437; 1997. 23. Ezure, K. Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog. Neurobiol. 35:429 – 450; 1990. 24. Farber, J. P. Development of pulmonary reflexes and pattern of breathing in Virginia opossum. Respir. Physiol. 14:278 –286; 1972. 25. Farber, J. P. Expiratory motor responses in the suckling opossum. J. Appl. Physiol. 54:919 –925; 1983. 26. Farber, J. P. Motor responses to positive-pressure breathing in the developing opossum. J. Appl. Physiol. 58:1489 –1495; 1985. 27. Farber, J. P. Differential recruitment of expiratory muscles during opossum development. J. Appl. Physiol. 60:841– 845; 1986. 28. Farber, J. P. Medullary inspiratory activity during opossum development. Am. J. Physiol. 254:R578 –R584; 1988. 29. Farber, J. P. Medullary expiratory activity during opossum development. J. Appl. Physiol. 66:1606 –1612; 1989. 30. Farber, J. P. Effects on breathing of rostral pons glutamate injection during opossum development. J. Appl. Physiol. 69:189 –195; 1990. 31. Farber, J. P. GABAergic effects on respiratory neuronal discharge during opossum development. Am. J. Physiol. 264:R331–R336; 1993. 32. Farber, J. P. Maximum discharge rates of respiratory neurons during opossum development. J. Appl. Physiol. 75:2040 –2044; 1993. 33. Farber, J. P. Effects on breathing of medullary bicuculline microinjections in immature opossums. Am. J. Physiol. 269:R1295–R1300; 1995. 34. Farber, J. P.; Bedell, G. N. Responsiveness of breathing control centers to CO2 and neurogenic stimuli. Respir. Physiol. 19:88 –95; 1973.
EUGENI´N AND NICHOLLS 35. Farber, J. P.; Hultgren, H. N.; Tenney, S. M. Development of the chemical control of breathing in the Virginia opossum. Respir. Physiol. 14:267–277; 1972. 36. Feldman, J. L. Neurophysiology of breathing in mammals. In: Bloom, F. E., ed. American Physiological Society: Handbook of physiology. Bethesda, MD: Williams & Wilkins; 1986:463–524. 37. Fernandez, J.; Nicholls, J. G. Fine structure and development of dorsal root ganglion neurons and Schwann cells in the newborn opossum Monodelphis domestica. J. Comp. Neurol. 396:338 –350; 1998. 38. Fukuda, Y.; Honda, Y. Effects of hypocapnia on respiratory timing and inspiratory activities of the superior laryngeal, hypoglossal, and phrenic nerves in the vagotomized rat. Jpn. J. Physiol. 33:733–742; 1983. 39. Fung, M. L.; Wang, W.; Darnall, R. A.; St. John, W. M. Characterization of ventilatory responses to hypoxia in neonatal rats. Respir. Physiol. 103:57– 66; 1996. 40. Fung, M. L.; Wang, W.; St. John, W. M. Medullary loci critical for expression of gasping in adult rats. J. Physiol. 480:597– 611; 1994. 41. Fung, M. L.; Wang, W.; St. John, W. M. Medullary loci critical for expression of gasping in adult rats. J. Physiol. 480:597– 611; 1994. 42. Funk, G. D.; Smith, J. C.; Feldman, J. L. Generation and transmission of respiratory oscillations in medullary slices: Role of excitatory amino acids. J. Neurophysiol. 70:1497–1515; 1993. 43. Gardner, W. N. The relationship between tidal volume and inspiratory and expiratory times during steady-state carbon dioxide inhalation in man. J. Physiol. 272:591– 611; 1977. 44. Gray, P. A.; Rekling, J. C.; Bocchiaro, C. M.; Feldman, J. L. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex. Science 286:1566 –1568; 1999. 45. Greer, J. J.; Smith, J. C.; Feldman, J. L. Respiratory and locomotor patterns generated in the fetal rat brain stem-spinal cord in vitro. J. Neurophysiol. 67:996 –999; 1992. 46. Grunstein, M. M.; Younes, M.; Milic-Emili, J. Control of tidal volume and respiratory frequency in anesthetized cats. J. Appl. Physiol. 35:463– 476; 1973. 47. Guthrie, R. D.; Standaert, T. A.; Hodson, W. A.; Woodrum, D. E. Sleep and maturation of eucapnic ventilation and CO2 sensitivity in the premature primate. J. Appl. Physiol. 48:347–354; 1980. 48. Harada, Y.; Kuno, M.; Wang, Y. Z. Differential effects of carbon dioxide and pH on central chemoreceptors in the rat in vitro. J. Physiol. 368:679 – 693; 1985. 49. Harada, Y.; Wang, Y. Z.; Kuno, M. Central chemosensitivity to H⫹ and CO2 in the rat respiratory center in vitro. Brain Res. 333:336 – 339; 1985. 50. Hilaire, G.; Bou, C.; Monteau, R. Rostral ventrolateral medulla and respiratory rhythmogenesis in mice. Neurosci. Lett. 224:13–16; 1997. 51. Hilaire, G.; Duron, B. Maturation of the mammalian respiratory system. Physiol. Rev. 79:325–360; 1999. 52. Hilaire, G.; Monteau, R.; Errchidi, S. 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; 1989. 53. Hilaire, G.; Monteau, R.; Gauthier, P.; Rega, P.; Morin, D. Functional significance of the dorsal respiratory group in adult and newborn rats: In vivo and in vitro studies. Neurosci. Lett. 111:133–138; 1990. 54. Huang, Q.; Zhou, D.; St. John, W. M. Lesions of regions for in vitro ventilatory genesis eliminate gasping but not eupnea. Respir. Physiol. 107:111–123; 1997. 55. Issa, F. G.; Remmers, J. E. Identification of a subsurface area in the ventral medulla sensitive to local changes in PCO2. J. Appl. Physiol. 72:439 – 446; 1992. 56. Johnson, S. M.; Smith, J. C.; Funk, G. D.; Feldman, J. L. Pacemaker behavior of respiratory neurons in medullary slices from neonatal rat. J. Neurophysiol. 72:2598 –2608; 1994. 57. Kawai, A.; Ballantyne, D.; Muckenhoff, K.; Scheid, P. Chemosensitive medullary neurones in the brainstem—Spinal cord preparation of the neonatal rat. J. Physiol. 492:277–292; 1996. 58. Kawai, A.; Okada, Y.; Muckenhoff, K.; Scheid, P. Theophylline and hypoxic ventilatory response in the rat isolated brainstem-spinal cord. Respir. Physiol. 100:25–32; 1995. 59. Koshiya, N.; Smith, J. C. Neuronal pacemaker for breathing visualized in vitro. Nature 400:360 –363; 1999.
RESPIRATION IN NEONATAL OPOSSUM 60. Liu, G.; Feldman, J. L. Quantal synaptic transmission in phrenic motor nucleus. J. Neurophysiol. 68:1468 –1471; 1992. 61. Long, S.; Duffin, J. The neuronal determinants of respiratory rhythm. Prog. Neurobiol. 27:101–182; 1986. 62. McLean, H. A.; Remmers, J. E. Respiratory motor output of the sectioned medulla of the neonatal rat. Respir. Physiol. 96:49 – 60; 1994. 63. Monteau, R.; Morin, D.; Hennequin, S.; Hilaire, G. Differential effects of serotonin on respiratory activity of hypoglossal and cervical motoneurons: An in vitro study on the newborn rat. Neurosci. Lett. 111:127–132; 1990. 64. Monteau, R.; Morin, D.; Hilaire, G. Acetylcholine and central chemosensitivity: In vitro study in the newborn rat. Respir. Physiol. 81:241–254; 1990. 65. Monteau, R.; Ptak, K.; Broquere, N.; Hilaire, G. Tachykinins and central respiratory activity: An in vitro study on the newborn rat. Eur. J. Pharmacol. 314:41–50; 1996. 66. Morin, D.; Hennequin, S.; Monteau, R.; Hilaire, G. Serotonergic influences on central respiratory activity: An in vitro study in the newborn rat. Brain Res. 535:281–287; 1990. 67. Morin, D.; Monteau, R.; Hilaire, G. 5-Hydroytryptamine modulates central respiratory activity in the newborn rat: An in vitro study. Eur. J. Pharmacol. 192:89 –95; 1991. 68. Morin-Surun, M. P.; Boudinot, E.; Sarraseca, H.; Fortin, G.; DenavitSaubie, M. Respiratory network remains functional in a mature guinea pig brainstem isolated in vitro. Exp. Brain Res. 90:375–383; 1992. 69. Morin-Surun, M. P.; Boudinot, E.; Schafer, T.; Denavit-Saubie, M. Brain stem chemosensitivity: Its implication in central respiratory regulation. Biol. Neonate 65:166 –170; 1994. 70. Morin-Surun, M. P.; Boudinot, E.; Schafer, T.; Denavit-Saubie, M. Localization of chemosensitive structures in the isolated brainstem of adult guinea-pig. J. Physiol. 485:203–212; 1995. 71. Nicholls, J. G.; Stewart, R. R.; Erulkar, S. D.; Saunders, N. R. Reflexes, fictive respiration and cell division in the brain and spinal cord of the newborn opossum, Monodelphis domestica, isolated and maintained in vitro. J. Exp. Biol. 152:1–15; 1990. 72. Okada, Y.; Mu¨ckenhoff, K.; Scheid, P. Hypercapnia and medullary neurons in the isolated brain stem-spinal cord of the rat. Respir. Physiol. 93:327–336; 1993. 73. Olsen, G. D.; Weil, J. A. Noninvasive breathing analysis: Repetitive chloral hydrate in neonatal guinea pigs. Pediatr. Pulmonol. 13:227– 234; 1992. 74. Onimaru, H. Studies of the respiratory center using isolated brainstem-spinal cord preparations. Neurosci. Res. 21:183–190; 1995. 75. Onimaru, H.; Arata, A.; Homma, I. Neuronal mechanisms of respiratory rhythm generation: An approach using in vitro preparation. Jpn. J. Physiol. 47:385– 403; 1997. 76. Paton, J. F.; Ramirez, J. M.; Richter, D. W. Functionally intact in vitro preparation generating respiratory activity in neonatal and mature mammals. Pflugers Arch. 428:250 –260; 1994. 77. Paton, J. F.; Richter, D. W. Maturational changes in the respiratory rhythm generator of the mouse. Pflugers Arch. 430:115–124; 1995. 78. Paton, J. F. R. The ventral medullary respiratory network of the mature mouse studied in a working heart brainstem preparation. J. Physiol. 493:819 – 831; 1996. 79. Peever, J. H.; Tian, G. F.; Duffin, J. Temperature and pH affect respiratory rhythm of in-vitro preparations from neonatal rats. Respir. Physiol. 117:97–107; 1999. 80. Ptak, K.; Hilaire, G. Central respiratory effects of substance P in neonatal mice: An in vitro study. Neurosci. Lett. 266:189 –192; 1999. 81. Ramirez, J. M.; Quellmalz, U. J.; Richter, D. W. Postnatal changes in the mammalian respiratory network as revealed by the transverse brainstem slice of mice. J. Physiol. 491:799 – 812; 1996. 82. Rekling, J. C.; Feldman, J. L. PreBotzinger complex and pacemaker
613
83. 84. 85.
86. 87.
88. 89.
90.
91. 92. 93.
94. 95.
96. 97.
98.
99. 100. 101.
neurons: Hypothesized site and kernel for respiratory rhythm generation. Annu. Rev. Physiol. 60:385– 405; 1998. Richter, D. W.; Pierrefiche, O.; Lalley, P. M.; Polder, H. R. Voltageclamp analysis of neurons within deep layers of the brain. J. Neurosci. Methods 67:121–123; 1996. Saetta, M.; Mortola, J. P. Breathing pattern and CO2 response in newborn rats before and during anesthesia. J. Appl. Physiol. 58: 1988 –1996; 1985. Saunders, N. R.; Balkwill, P.; Knott, G.; Habgood, M. D.; Mollgard, K.; Treherne, J. M.; Nicholls, J. G. Growth of axons through a lesion in the intact CNS of fetal rat maintained in long-term culture. Proc. R. Soc. Lond. B Biol. Sci. 250:171–180; 1992. Segal, B. S.; Inman, J. D.; Moss, I. R. Respiratory responses of piglets to hypercapnia during postnatal development: Effects of opiods. Pediatr. Pulmonol. 11:113–119; 1991. Shao, X. M.; Feldman, J. L. Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: Differential roles of glycinergic and GABAergic neural transmission. J. Neurophysiol. 77:1853–1860; 1997. Smith, J. C.; Ballanyi, K.; Richter, D. W. Whole-cell patch-clamp recordings from respiratory neurons in neonatal rat brainstem in vitro. Neurosci. Lett. 134:153–156; 1992. Smith, J. C.; Ellenberger, H. H.; Ballanyi, K.; Richter, D. W.; Feldman, J. L. Pre-Bo¨tzinger complex: A brainstem region that may generate respiratory rhythm in mammals. Science 254:726 –729; 1991 Smith, J. C.; Greer, J. J.; Liu, G.; Feldman, J. L. 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; 1990. St. John, W. M. Neurogenesis, control, and functional significance of gasping. J. Appl. Physiol. 68:1305–1315; 1990. St. John, W. M. Medullary regions for neurogenesis of gasping: Noeud vital or noeuds vitals? J. Appl. Physiol. 81:1865–1877; 1996. Stewart, R. R.; Zou, D. J.; Treherne, J. M.; Mollgard, K.; Saunders, N. R.; Nicholls, J. G. The intact central nervous system of the newborn opossum in long-term culture: Fine structure and GABAmediated inhibition of electrical activity. J. Exp. Biol. 161:25– 41; 1991. Suzue, T. Respiratory rhythm generation in the in vitro brain stemspinal cord preparation of the neonatal rat. J. Physiol. 354:173–183; 1984. Treherne, J. M.; Woodward, S. K.; Varga, Z. M.; Ritchie, J. M.; Nicholls, J. G. Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture. Proc. Natl. Acad. Sci. USA 89:431– 434; 1992. von Euler, C. The functional organization of the respiratory phaseswitching mechanisms. Fed. Proc. 36:2375–2380; 1977. von Euler, C. Brain stem mechanisms for generation and control of breathing pattern. In: Cherniack, N. S.; Widdicombe, J. G., eds. American Physiological Society: Handbook of physiology. Baltimore, MD: Williams & Wilkins; 1986:1– 68. Zhou, D.; Huang, Q.; Fung, M. L.; Li, A.; Darnall, R. A.; Nattie, E. E.; St. John, W. M. Phrenic response to hypercapnia in the unanesthetized, decerbrate, newborn rat. Respir. Physiol. 104:11–22; 1996. Zhou, D.; Wasicko, M. J.; Hu, J. M.; St. John, W. M. Differing activities of medullary respiratory neurons in eupnea and gasping. J. Appl. Physiol. 70:1265–1270; 1991. Zou, D. J. Respiratory rhythm in the isolated central nervous system of newborn opossum. J. Exp. Biol. 197:201–213; 1994. Zou, D. J.; Treherne, J. M.; Stewart, R. R.; Saunders, N. R.; Nicholls, J. G. Regulation of GABA B receptors by histamine and neuronal activity in the isolated spinal cord of neonatal opossum in culture. Proc. R. Soc. Lond. B Biol. Sci. 246:77– 82; 1991.
PII S0361-9230(00)00394-4
Control of respiration in the isolated central nervous system of the neonatal opossum, Monodelphis domestica Jaime Eugenı´n1 and John G. Nicholls2* 1
Department of Biological Sciences, Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile; and 2Department of Biophysics, International School for Advanced Studies, Trieste, Italy [Received 5 May 2000; Revised 10 July 2000; Accepted 16 August 2000] ulus. For example, an increase in temperature increases the rate of fictive respiration without changing its amplitude, whereas noradrenaline decreases the rate while increasing the amplitude. Thus, changes of timing and amplitude need not go hand in hand. The opossum CNS offers a favorable preparation for the analysis of neural mechanisms that generate and modulate a motor rhythm, as the animal develops from embryonic to adult stages. © 2001 Elsevier Science Inc.
ABSTRACT: Respiration represents an unusual motor activity with respect to its development. As newly born mammals enter the world, their limb movements are not coordinated; time and experience are required for effective performance to be achieved. Yet the rhythm of respiration is of necessity functionally perfected and unfailing at birth. Inspiratory and expiratory motor neurons are already able to fire at appropriate rates, under the command of rhythmically active neurons in the medulla. In this review, we discuss refinements of control present in the newborn opossum, particularly with respect to mechanisms that allow adaptation of respiration to changes in the level of activity or in the outside environment. Our own studies have been aimed at analyzing respiration at the earliest stages, and at establishing the way in which important variables influence inspiration and expiration. To this end, we have used the central nervous system (CNS) of a neonatal opossum, isolated in its entirety and maintained in culture. Although the opossum is unable to walk and highly immature at birth, its respiration is regular and unfailing. The isolated CNS survives, undergoes development, and maintains its neural activity and fine structure in vitro. Moreover, fictive respiration persists for over a day or longer at rates similar to those of the intact pup. The effects of altered pH, of increased temperature, and of drugs known to alter respiratory rhythm in intact animals can be measured directly, by electrical recordings made from medullary neurons or ventral roots. As in a slice, fluids of different composition can be applied focally, through micropipettes to the surface of the ventral medulla, or diffusely to the brainstem, With highly localized application of procaine hydrochloride (2%) to selected areas of the ventral medulla, the respiratory rhythm is reduced or abolished. As in adult mammals, both the rate and the amplitude of respiration simultaneously increase in response to lowered pH (6.5—7.1) or to topical application of 1.0 M carbachol. Conversely, as expected, the rate and amplitude decrease in response to increased pH (pH 7.5–7.7), or 100 M scopolamine. Two characteristic features of the control of respiration in the neonatal opossum are evident from such tests. First, changes in rate are achieved by changes in the duration of the expiratory phase of respiration. This result suggests that the timing of the respiratory cycle in the neonatal opossum is controlled by an expiratory instead of an inspiratory “off-switch”. Second, the rate and the amplitude of the respiratory excursions can be controlled independently, depending on the stim-
KEY WORDS: Respiratory pattern generation, Control of breathing, Marsupials, Central chemoreception, Development.
INTRODUCTION At the time a mammal is born, the neural network that generates the respiratory rhythm is primed and ready for the successful activation of inspiratory and expiratory muscles. From the outset, breathing is rhythmic and synchronized, but, as development proceeds, characteristic patterns of respiration emerge. The respiratory rate and depth are modified to fit the demands of the animal for uptake of oxygen and removal of carbon dioxide. In particular, the interactions of intrinsic respiratory motor programs with sensory inputs become modified, as adult characteristics become defined [51,77]. Changes in Frequency of Respiration Brought About by Altered Duration of Inspiratory Phase The motor programming for breathing can be analyzed in terms of variables for amplitude and for timing. Measurements of the relationship between amplitude and inspiratory and expiratory durations have provided insights into functional mechanisms underlying the respiratory motor pattern. Clark and von Euler [9] showed in anaesthetised cats that an increase in inhaled CO2 increases both the amplitude and the rate of respiration. The shortening of the respiratory cycle consists of a decrease in the inspiratory duration mediated by the vagal reflex that inhibits inspiration (Hering-Breuer reflex). Thus, during chemical stimulation by low pH or raised levels of CO2, it is the duration of the
* Address for correspondence: Dr. John G. Nicholls, International School for Advanced Studies, Via Beirut 2, I-34014 Trieste, Italy. Fax: ⫹39-0403787-528; E-mail: [email protected]
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606 inspiratory phase of respiration that is shortened so as to produce an increase in frequency. At the same time the amplitude of the respiratory excursions increases [4,5,9,11,36,46,97]. These observations have been incorporated into an “inspiratory off-switch hypothesis”. According to this scheme, the generation of the respiratory rhythm depends on two control mechanisms. One compartment, the central inspiratory activator, generates and shapes the inspiratory activity; a second compartment, known as the “inspiratory off-switch”, inhibits the central inspiratory activator. Vagal inputs from slowly adapting pulmonary mechanoreceptors and tonic central activity (probably from pontine nuclei) are assumed to activate the “inspiratory off-switch” [6,12,14,21,96, 97]. Alterations in the Duration of the Expiratory Phase of Respiration In certain preparations, selective changes in the duration of the expiratory, rather than the inspiratory, phase account for changes in rate. These represent mechanisms in which the central pattern generator for respiration is not controlled by vagal inputs. For example, in vagotomized adult rats, anaesthetised and ventilated artificially, the expiratory phase becomes shortened in response to chemical stimulation [38]. Similar phenomena are seen in the in vitro brainstem of adult guinea pigs [69,70]. In conscious humans, breathing at low tidal volumes [13,15,43], vagal afferents are minimally excited and it is the expiratory phase that shortens in response to mild chemical stimuli. A further example is provided by results obtained in pre-term infants. A decrease in respiratory frequency is induced by inhalation of 15% O2, and this results from an increase in the duration of the expiratory phase [51]. Fictive Respiration of Isolated Mammalian Preparations In Vitro Further simplification of preparations for the study of respiration is provided by the use of slices in vitro. Slices used by Feldman, Smith and others [42,44,56,59,81– 83,87,89] offer the advantage of direct access to defined groups of neurons for recording and for application of drugs while respiratory activity is recorded from hypoglossal roots. Inevitably, however, all ascending and descending connections have by definition been cut, and this may explain differences in the patterns of responses to altered pH in slices and intact central nervous system (CNS) preparations [79]. In 1984, Suzue demonstrated that fictive respiration occurs in the isolated brainstem of the neonatal rat in vitro [94]. In this preparation, known as the en bloc preparation, peripheral nerves as well as dorsal and ventral roots are cut, but the regions of the CNS are maintained in their normal relationships. Since this pioneering work, such preparations have been used to study the generation and regulation of the respiratory rhythm in neonatal mammals [1,2,7,16,40,45,49, 50,53,57,58,60,62,64,65,74,75,88,90]. In a series of experiments made on embryonic or neonatal rat CNS in vitro, Hilaire and his colleagues have described the time of onset of respiration and analyzed the effects of serotonin and substance P on respiratory neurons [17–20,52,63,65– 67,80]. In both types of preparations, neonatal rodent CNS in vitro and brainstem slices, the frequency of activity recorded is very low compared to that of the intact animal. The bursts occur at only about 9 per minute [62,79,89]; as a result, problems of interpretation can arise about whether the activity represents respiration or perhaps some other rhythmical function such as gasping [39,41,54,91, 92,99]. Recently, preparations of adult mammalian CNS in vitro have been developed, in which characteristic respiratory re-
EUGENI´N AND NICHOLLS sponses can be obtained by increases in CO2 or lowered pH [68,70,76,78]. Advantages of Neonatal Opossums for Studies of Development of Respiration Marsupials, such as opossums and kangaroos, are born at an extremely immature stage of development. At a time when the cortex is not yet formed and when motor performance is not coordinated, the new-born animal has to produce rhythmical inspiratory and expiratory movements for respiration. This feature has made opossums attractive animals for studying respiratory mechanisms during development. Farber, for example, has described the properties of respiratory control mechanisms and motor neurons in suckling North American opossums (Didelphis virginiana) as they mature, with particular reference to the effects of transmitters, cerebellar stimulation, vagal afferent stimulation and altered load produced by positive pressure breathing [24 –35]. Moreover, the South American opossum, Monodelphis domestica, which corresponds at birth to a 15-day rat or mouse embryo, can be bred in the laboratory. Its respiration can be studied from the first day of life. The CNS at this time already contains neurons with properties and connections resembling those of an adult that enable it to breathe effectively [71,100]. FICTIVE RESPIRATION IN ISOLATED NEONATAL OPOSSUM CNS The central nervous system of the newly born opossum (Monodelphis domestica), isolated in its entirety and maintained in culture, is in many respects similar to the rat preparation described by Suzue. At the same time, the opossum CNS offers certain advantages. The preparation is so small that it survives well for periods of 3 weeks or longer; it maintains its electrical excitability, fine structure, and responses to amino acid transmitters such as ␥-aminobutyric acid, glutamate, and glycine [71,93,95,101] and even shows development in the dish [37,71,85,93]. Of particular interest is that the isolated CNS produces regular, robust and well maintained fictive respiration for periods of a day or more, longerlasting and more consistent than other fictive rhythms in vitro [22,71,100]. As in a slice, it is possible to record from individual neurons in the medulla and to apply stimuli by localised application through a pipette or diffusely in the bathing fluid [22,100]. Two convenient features are the absence of the cerebellum (which does not cover the medulla at birth) and the ability to record inspiratory activity in ventral roots and in the phrenic nerve. Moreover, the rate of respiration of the isolated preparation (in medium gassed with 5% CO2–95% O2, pH 7.4 at 22°C) corresponds roughly to that of the newborn pup, continuing at approximately 45 per min (as compared to about 60 per min in the animal, see [71]). The integrated inspiratory burst recorded from cervical ventral roots exhibits a characteristic ramp shape [22]. In the following sections, we review briefly recent experiments made in the isolated opossum CNS preparation that demonstrate similarities and differences from respiratory mechanisms discovered in other developing and mature mammals. One question concerns the location of the neurons that generate the rhythm and another is the degree of control of respiration at the earliest stages of development. For example, would changes in rate involve changes in the duration of the inspiratory cycle, the expiratory cycle or both? Would separate groups of cells be assigned to control of the rate and the amplitude of respiration?
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FIG. 1. Positions and properties of medullary neurons showing activity correlated with inspiration and expiration in neonatal opossums. (A) and (C) show the positions of respiratory neurons from which the unit recordings in (B) were made. Symbols indicate the phase of respiration during which the unit is active. The timing is established from recordings made in the ventral root of C5 (after [100]).
Techniques for Maintaining Isolated Opossum CNS and Recording Respiratory Activity The methods have been described elsewhere [22,71,100]. In brief, the entire CNS is dissected from opossums Monodelphis domestica (5–12-day-old) anaesthetised with methoxyflurane and maintained in a chamber 0.5 ml in volume superfused with basal medium Eagle’s (BME) oxygenated with 95% O2–5% CO2, pH 7.37–7.40 at room temperature (21–23°C). As shown in Fig. 1,
spontaneous electrical activity from C3–C5 ventral roots, recorded with suction electrodes, consists of bursts of action potentials superimposed on a background of random activity. Rhythmic bursts occurring at about 45 per min correspond to fictive respiration [22,71,100]. The bursts are regular in timing and amplitude [22,71,100]. Simultaneously, records are made from individual neurons in the medulla with extracellular microelectrodes. Chemical stimuli are administered by local application of test solutions
FIG. 2. Fictive respiration recorded from C5 ventral root in the isolated central nervous system (CNS) of a 9-day-old opossum. Scheme for recording from isolated CNS. The partition in the bath makes it possible to provide fluids to the lower brainstem and spinal cord separately. Two inspiratory bursts and their integrated activity are illustrated. Amplitude (A) is measured from integrated activity; TE, expiratory duration; TI, inspiratory duration; Ttot, cycle duration.
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FIG. 3. Effect of pH and procaine on fictive respiration. (A) Effect of focal application of basal medium Eagle’s (BME) pH 6.5 (horizontal bar) to a region of ventral medulla medial to hypoglossal roots in an 8-day-old opossum (site of administration indicated as black spot in the inset); (B) effect of superfusion of the lower brainstem with BME pH 7.1, pH 7.4, and pH 7.6 in a 6-day-old opossum; (C) effects of 30 and 80 s of focal application of 2% procaine hydrochloride to a region medial to hypoglossal roots in a 12-day-old opossum (black spot in central nervous system inset). Note that respiratory effects are opposite to those observed with topical application of pH 6.5 BME [compare with (A)].
to the surface of ventral medulla through a triple barrel micropipette of 100 m outer diameter [22]. Alternatively, by the use of a thin partition at the level of C1–C2 (Fig. 2), the brainstem can be superfused separately from the spinal cord. Changes in the pH of the superfusion medium are achieved by gassing with different CO2:O2 mixtures or by adding bicarbonate to control BME. Figure 2 shows the way in which records are analyzed quantitatively so that the effects of variables can be assessed in terms of altered inspiratory and expiratory phases, as well as amplitude. Location of Medullary Respiratory Neurons in Neonatal Opossum Extracellular recordings were first made by Zou from respiratory-related neurons in the medulla. In his sample of 128 cells, the respiratory neurons were classified as: (1) early inspiratory (n ⫽ 69), (2) inspiratory (n ⫽ 38), (3) post-inspiratory (n ⫽ 17), and (4) expiratory (n ⫽ 4) [100]. These four types showed discharge patterns similar to those recorded in other mammalian preparations [3,11,23,61]. The inspiratory and expiratory cells were intermingled. All were located in the ventral medulla close to the nucleus ambiguus (Fig. 1). Effects of Altered pH on Respiratory Rate and Amplitude As expected, a reduction of pH from 7.4 to 7.1 in the fluid bathing the lower brainstem increases the rate (by approximately 100%) and the amplitude (by approximately 200%). Similarly, localized, topical applications of low pH fluid (pH 6.5) to regions on the surface of ventral medulla (Figs. 3A and 4) increase the rate of respiration by 88% ⫾ 9% and its amplitude by 109 ⫾ 25% (n ⫽
7; SD). All such effects are readily reversible and can be repeated time after time on the same preparation. An increase in the pH of the superfusion medium from 7.4 to 7.6 produces the opposite effect: thus, fluid with a high pH of 7.7 reduces the rate of fictive respiration by approximately 55% and its amplitude by 50% (n ⫽ 5). Superfusion of the brainstem with sufficiently high pH fluid can reversibly abolish the respiratory rhythm. Quantitative analyses of the effects of altered pH on the phases and amplitude of respiration are shown in Figs. 4 –7. That the activity of the neurons identified as respiratory is crucial for the genesis of the rhythm has been shown by highly localized application of procaine hydrochloride (2%) to the surface of ventral medulla at sites known to be H⫹- and acetylcholinesensitive. Procaine abolishes or reduces the rate and amplitude of fictive respiration (by 60% or more). Figure 3C illustrates the reversible depression in rate and amplitude of fictive respiration obtained after unilateral topical application of procaine to a region medial to the hypoglossal roots. Loss of activity following a focal alteration in neuronal function could result from loss of a stimulatory drive to the network as well as from destruction of a key site for rhythmogenesis. Cholinergic Mechanisms for Responses to Altered pH As in other preparations, there is evidence for a cholinergic drive in respiration and responses to altered pH (see Figs. 4, 6 and 7). Thus, carbachol (1 M) applied to sensitive areas of ventral medulla increases the rate of fictive respiration by 66⫾23% and its amplitude by 66⫾15% (n ⫽ 5). After diffuse application of 100
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M scopolamine, the rate decreases by 58⫾4% and the amplitude by 65⫾4%. Hexamethonium, by contrast has no effect. The specificity of these muscarinic effects is further demonstrated by comparison with noradrenaline [22,100], which produces very different effects on rate and amplitude. For example, Figs. 4 –7 show that addition of noradrenaline (30 –50 M) to the medium perfusing the lower brainstem, reproducibly causes the rate to decrease (by 54 ⫾ 15%), while the amplitude of fictive respiration increases (by 103 ⫾ 16.7%). Responses to Changes in Temperature The temperature of the isolated preparation at 22° C is approximately 7°C lower than that of the neonatal pup (N. Saunders, personal communication). When the temperature of the fluid bathing the preparation is increased to 27°C, the rate of fictive respiration increases (by about 47%), with no accompanying change in amplitude (Figs. 4 –7). Changes in Timing and Amplitude FIG. 4. Effects of chemical stimuli and altered temperature on the rate and amplitude of fictive respiration. Changes in rate (filled columns) and amplitude (open columns) correspond to values during stimulation expressed as percentage with respect to basal (100%); bars indicate SEM. Stimuli are represented as follows. ‘low pH (L)’ signifies topical application of basal medium Eagle’s (BME) pH 6.5; ‘low pH (S)’ signifies brainstem superfusion with BME pH 7.1; ‘carbachol’, topical application of 1 M carbachol hydrochloride; ‘temperature’, increase of bath temperature from 19°C to 27°C; ‘high pH’, brainstem superfusion with BME pH 7.6; ‘scopolamine’, brainstem superfusion with 100 M scopolamine hydrochloride; ‘procaine’, topical application of 2% scopolamine hydrochloride; ‘noradrenaline’, brainstem superfusion of 30 –50 M DL-noradrenaline hydrochloride. Significance levels for *p ⬍ 0.05 and for **p ⬍ 0.02 (Wilcoxon signed-rank test). Number of trials indicated in brackets.
As illustrated in Fig. 4, stimuli can be grouped on the basis of their effects on respiratory rate and amplitude: (1) low pH and carbachol increase both respiratory rate and amplitude; (2) high pH, scopolamine, and topical application of procaine reduce the respiratory rate, and at the same time decrease amplitude; (3) noradrenaline decreases the rate while increasing the amplitude; and (4) raised temperature increases respiratory rate without affecting the amplitude. A common feature of these results is apparent from an analysis of inspiratory and expiratory timing. In isolated opossum CNS preparations, changes in rate are brought about by changes in the duration of the expiratory phase (TE). The
FIG. 5. Burst-to-burst time course of respiratory responses. In the upper panels cycle duration (Ttot), inspiratory duration (TI), and expiratory duration (TE) (in seconds) are plotted against the ordinal numbers of respiratory cycles. It is the expiratory duration that changes with these stimuli. In lower panels the amplitude (arbitrary units) is plotted in a similar manner against respiratory cycle number. The duration of stimulation is indicated by horizontal bars. (A) Topical application of basal medium Eagle’s (BME) pH 6.5 to the ventral medulla (5-day-old opossum). (B) Increase of bath temperature from 19 –27°C (9-day-old opossum). (C) Brainstem superfusion with BME containing 50 M noradrenaline hydrochloride (10-day-old opossum).
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FIG. 6. Response in cycle duration (Ttot) as a function of inspiratory and expiratory phases of the respiratory cycle. Ttot vs. TI (open triangles) and Ttot vs. TE (filled squares) relationships are illustrated. The ordinate in each experiment is the duration of the total respiratory cycle; the abscissa is the duration of the inspiratory or the expiratory phase of respiration. (A) Focal application of basal medium Eagle’s (BME) pH 6.5. (B) Lower brainstem superfusion of BME pH 7.1. (C) Effects of topical application of BME pH 6.5 and the brainstem superfusion of BME pH 7.6 in the same preparation. (D) Topical application of 1 M carbachol hydrochloride. (E) Superfusion of 100 M scopolamine hydrochloride. (F) Topical application of 2% procaine to a region medial to hypoglossal roots. (G) Brainstem superfusion of 50 M noradrenaline hydrochloride. (H) Increase of bath temperature from 19°C to 27°C. This is the only variable that can, with prolonged application, alter the duration of the inspiratory phase of respiration to a minor extent. Lines are based on regression equations and correlation coefficients. (Significance levels indicated below). Arrows show the basal values in the absence of stimuli.
inspiratory phase (TI) remains constant in duration. Furthermore, this close association between cycle duration (TTOT) and TE is evident whatever changes occur in amplitude. For example, with application of either low pH or noradrenaline, the amplitude of fictive respiration is increased (Fig. 4). But with
low pH, the total time of the respiratory cycle (TTOT) is reduced through the shortening of TE, while with noradrenaline both TTOT and TE are prolonged (Figs. 5–7). There is one exception to this general picture: although, higher temperatures increase the rate by shortening TE (as in Fig. 5), Fig. 6 shows that
FIG. 7. Relationships between amplitude and phases of the respiratory cycle. Amplitude is plotted against the duration of inspiration (Ti, open triangles) and expiration (TE, filled squares) for the same experiments as those shown in Fig. 6. Amplitude is expressed in arbitrary units and duration in seconds. Arrows again show basal values without stimuli.
RESPIRATION IN NEONATAL OPOSSUM prolonged exposure to 27° can also shorten the duration of the inspiratory phase (TI) to a minor extent. RESPIRATION OF NEONATAL OPOSSUM IN VITRO COMPARED WITH THAT OF OTHER MAMMALIAN PREPARATIONS Respiration of the isolated new-born opossum CNS resembles that of adult mammals in that the rate and the amplitude can be varied independently [4,9,11,12,46,96,97]. Although medullary respiratory neurons are close to the surface and accessible, they are not grouped in discrete inspiratory and expiratory clusters. Similarly, chemosensitive neurons that influence amplitude and timing are inextricably interspersed in such a way that H⫹- and acetylcholine-sensitive regions of the ventral medulla in the isolated CNS of new-born opossums overlap [22]. In spite of the absence of anatomical boundaries, functionally defined compartments that control different aspects of respiration can be postulated. Thus, the respiratory pattern generator appears to consist of two functional compartments, one controlling amplitude and the other controlling timing of respiratory activity [6,12,14,21,96,97]. One unusual control mechanism in the isolated Monodelphis CNS is that the frequency of respiration depends primarily on changes in the duration of expiratory rather than inspiratory events. Accordingly, the functional compartment controlling the timing of the respiratory cycle in the new-born opossum represents an “expiratory off-switch” rather than an “inspiratory off-switch” mechanism. As mentioned in the Introduction, in some mammalian preparations similar selective changes in TE rather than TI can occur in response to chemical stimulation [13,15,38,43,51,69,70]. In adult mammals, the rate component is primarily mediated by vagal feedback, which influences the inspiratory rather than the expiratory phase [9,35,97,98], and which is not present in the isolated opossum preparation. The maturational changes that are responsible for this switch in the respiratory response are still unknown. An additional distinctive feature of the isolated new-born Monodephis CNS is the coupling of amplitude and frequency. Increases in amplitude produced by chemical and cholinergic stimulation are associated with a shortened duration of expiration. This has not been found to be the case in Didelphis virginiana opossums aged 7–20 days. In animals at that age the increased respiration in response to chemical stimulation occurs primarily by increases in depth of ventilation (tidal volume). The association between amplitude and rate becomes evident only at later stages, when the rate component develops [34,35]. In other new-born mammals, the respiratory response to chemical stimulation also consists of increases in the amplitude of inspiration, with little or no change in rate, whether vagal feedback is present or not [8,10,47,73,84,86,98]. Only in adult animals, do the vagal feedback and the rate component become major contributors [35,98]. In isolated brainstem-spinal cord preparations from neonatal rats, separation of rate and amplitude control mechanisms is also apparent: chemical stimulation can change the rate or the amplitude independently [48,49,55,64,72,94]. The clear dissociation between amplitude of respiration and duration of inspiration in the isolated Monodelphis CNS suggests that there is no coupling between the central mechanisms that activate inspiration and that turn it off (the inspiratory off-switch). Rather, the shortening of expiration during chemical stimulation appears to be related to an augmentation of amplitude. The relation is inverse, and resembles the volume threshold curve for inspiration-inhibiting reflexes (Tidal volume vs. TI curve) characteristic of adult mammals with intact vagal feedback [9]. One possible explanation for the findings in opossum could be the presence of an “expiratory off-switch” activated by chemical stimulation of
611 inspiratory generator mechanisms. Modification in amplitude is not per se a main factor determining frequency. (With the exception however, that an increase in the duration of expiration can occur with an increase or no change in amplitude, in response to noradrenaline or lowered temperature.) An attractive scheme for functional organisation of the respiratory pattern generator is that coupled inspiratory and expiratory generators are under the control of inspiratory and expiratory off-switches. Chemical and cholinergic inputs would excite the inspiratory generator and the expiratory off-switch; thermic receptors would activate the expiratory off-switch, with minor effects on the inspiratory on switch; noradrenaline would inhibit the expiratory off-switch, but stimulate the inspiratory activator. SUMMARY In this review, we compare the properties and regulatory mechanisms of fictive respiration in isolated opossum CNS preparations with those found in other in vitro preparations and in intact animals. The principal findings are that at early stages of opossum development, changes of the pH in contact with medullary respiratory neurons give rise to increases and decreases in frequency that depend on the timing of the expiratory phase of respiration. This result suggests that the regulation of respiration in the newborn opossum depends on an “expiratory off-switch”. This is in contrast to other immature mammalian preparations in which modulation of amplitude or changes in the inspiratory phase constitute responses to chemical stimuli. Moreover, changes in the frequency and amplitude of respiration are controlled by separate mechanisms. The principal advantages of the isolated opossum CNS for such studies are its long life, the accessibility of the respiratory command neurons, the normal relations of the different parts of the CNS to each other and a frequency of fictive respiration that is similar to that in intact animals. ACKNOWLEDGEMENTS
We thank Roland Geiser and Urs Berglas for maintaining the colony of Monodelphis and Paul Ba¨ttig for photography. We are particular grateful to Dr. A.R. Martin for his critical reading of the manuscript, and to Dr. W. Adams for his help with the electronics, software, and valuable suggestions throughout the experiments and the preparation of this manuscript. This work was supported by the Swiss Nationalfonds Grant (no. 313626292) and BBW Grant (no. PL 960211) to J. G. N., Fondecyt (no. 1980819) and Dicyt-USACH (no. 029743EL) to J.E.
REFERENCES 1. Arata, A.; Onimaru, H.; Homma, I. Respiration-related neurons in the ventral medulla of newborn rats in vitro. Brain Res. Bull. 24:599 – 604; 1990. 2. Arata, A.; Onimaru, H.; Homma, I. The adrenergic modulation of firings of respiratory rhythm-generating neurons in medulla-spinal cord preparation from newborn rat. Exp. Brain Res. 119:399 – 408; 1998. 3. Bianchi, A. L.; Denavit-Saubie, M.; Champagnat, J. Central control of breathing in mammals: Neuronal circuitry, membrane properties, and neurotransmitters. Physiol. Rev. 75:1– 45; 1995. 4. Bradley, G. W.; von Euler, C.; Marttila, I.; Roos, B. Steady-state effects of CO2 and temperature on the relationship between lung volume and inspiratory duration (Hering-Breuer threshold curve). Acta Physiol. Scand. 92:351–363; 1974. 5. Bradley, G. W.; von Euler, C.; Marttila, I.; Roos, B. Transient and steady state effects of CO2 on mechanisms determining rate and depth of breathing. Acta Physiol. Scand. 92:341–350; 1974. 6. Bradley, G. W.; von Euler, C.; Marttila, I.; Roos, B. A model of the central and reflex inhibition of inspiration in the cat. Biol. Cybernet. 19:105–116; 1975.
612 7. Brockhaus, J.; Ballanyi, K.; Smith, J. C.; Richter, D. W. Microenvironment of respiratory neurons in the in vitro brainstem-spinal cord of neonatal rats. J. Physiol. 462:421– 445; 1993. 8. Carroll, J. L.; Canet, E.; Bureau, M. A. Dynamic ventilatory responses to CO2 in the awake lamb: Role of the carotid chemoreceptors. J. Appl. Physiol. 71:2198 –2205; 1991. 9. Clark, F. J.; von Euler, C. On the regulation of depth and rate of breathing. J. Physiol. 222:267–295; 1972. 10. Cohen, G.; Xu, C.; Henderson-Smart, D. Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. J. Appl. Physiol. 71:168 –174; 1991. 11. Cohen, M. I. Neurogenesis of respiratory rhythm in the mammal. Physiol. Rev. 59:1105–1173; 1979. 12. Cohen, M. I.; Feldman, J. L. Models of respiratory phase-switching. Fed. Proc. 36:2367–2374; 1977. 13. Cunningham, D. J.; Gardner, W. N. A quantitative description of the pattern of breathing during steady-state CO2 inhalation in man, with special emphasis on expiration. J. Physiol. 272:613– 632; 1977. 14. D’Angelo, E. Control of breathing: Central mechanisms and peripheral inputs. Bull. Eur. Physiopath. Resp. 16:111–112; 1980. 15. Davis, N. J.; Stagg, D. Interrelationships of the volume and time components of individual breaths in resting man. J. Physiol. 245:481– 498; 1975. 16. Di Pasquale, E.; Lindsay, A.; Feldman, J.; Monteau, R.; Hilaire, G. Serotonergic inhibition of phrenic motoneuron activity: An in vitro study in neonatal rat. Neurosci. Lett. 230:29 –32; 1997. 17. Di Pasquale, E.; Monteau, R.; Hilaire, G. In vitro study of central respiratory-like activity of the fetal rat. Exp. Brain Res. 89:459 – 464; 1992. 18. Di Pasquale, E.; Monteau, R.; Hilaire, G. Endogenous serotonin modulates the fetal respiratory rhythm: An in vitro study in the rat. Brain Res. Dev. Brain Res. 80:222–232; 1994. 19. Di Pasquale, E.; Morin, D.; Monteau, R.; Hilaire, G. Serotonergic modulation of the respiratory rhythm generator at birth: An in vitro study in the rat. Neurosci. Lett. 143:91–95; 1992. 20. Errchidi, S.; Monteau, R.; Hilaire, G. Noradrenergic modulation of the medullary respiratory rhythm generator in the newborn rat: An in vitro study. J. Physiol. 443:477– 498; 1991. 21. Eugenin, J. Generation of the respiratory rhythm: Modelling the inspiratory off switch as a neural integrator. J. Theor. Biol. 172:107– 120; 1995. 22. Eugenin, J.; Nicholls, J. G. Chemosensory and cholinergic stimulation of fictive respiration in isolated CNS of neonatal opossum. J. Physiol. 501:425– 437; 1997. 23. Ezure, K. Synaptic connections between medullary respiratory neurons and considerations on the genesis of respiratory rhythm. Prog. Neurobiol. 35:429 – 450; 1990. 24. Farber, J. P. Development of pulmonary reflexes and pattern of breathing in Virginia opossum. Respir. Physiol. 14:278 –286; 1972. 25. Farber, J. P. Expiratory motor responses in the suckling opossum. J. Appl. Physiol. 54:919 –925; 1983. 26. Farber, J. P. Motor responses to positive-pressure breathing in the developing opossum. J. Appl. Physiol. 58:1489 –1495; 1985. 27. Farber, J. P. Differential recruitment of expiratory muscles during opossum development. J. Appl. Physiol. 60:841– 845; 1986. 28. Farber, J. P. Medullary inspiratory activity during opossum development. Am. J. Physiol. 254:R578 –R584; 1988. 29. Farber, J. P. Medullary expiratory activity during opossum development. J. Appl. Physiol. 66:1606 –1612; 1989. 30. Farber, J. P. Effects on breathing of rostral pons glutamate injection during opossum development. J. Appl. Physiol. 69:189 –195; 1990. 31. Farber, J. P. GABAergic effects on respiratory neuronal discharge during opossum development. Am. J. Physiol. 264:R331–R336; 1993. 32. Farber, J. P. Maximum discharge rates of respiratory neurons during opossum development. J. Appl. Physiol. 75:2040 –2044; 1993. 33. Farber, J. P. Effects on breathing of medullary bicuculline microinjections in immature opossums. Am. J. Physiol. 269:R1295–R1300; 1995. 34. Farber, J. P.; Bedell, G. N. Responsiveness of breathing control centers to CO2 and neurogenic stimuli. Respir. Physiol. 19:88 –95; 1973.
EUGENI´N AND NICHOLLS 35. Farber, J. P.; Hultgren, H. N.; Tenney, S. M. Development of the chemical control of breathing in the Virginia opossum. Respir. Physiol. 14:267–277; 1972. 36. Feldman, J. L. Neurophysiology of breathing in mammals. In: Bloom, F. E., ed. American Physiological Society: Handbook of physiology. Bethesda, MD: Williams & Wilkins; 1986:463–524. 37. Fernandez, J.; Nicholls, J. G. Fine structure and development of dorsal root ganglion neurons and Schwann cells in the newborn opossum Monodelphis domestica. J. Comp. Neurol. 396:338 –350; 1998. 38. Fukuda, Y.; Honda, Y. Effects of hypocapnia on respiratory timing and inspiratory activities of the superior laryngeal, hypoglossal, and phrenic nerves in the vagotomized rat. Jpn. J. Physiol. 33:733–742; 1983. 39. Fung, M. L.; Wang, W.; Darnall, R. A.; St. John, W. M. Characterization of ventilatory responses to hypoxia in neonatal rats. Respir. Physiol. 103:57– 66; 1996. 40. Fung, M. L.; Wang, W.; St. John, W. M. Medullary loci critical for expression of gasping in adult rats. J. Physiol. 480:597– 611; 1994. 41. Fung, M. L.; Wang, W.; St. John, W. M. Medullary loci critical for expression of gasping in adult rats. J. Physiol. 480:597– 611; 1994. 42. Funk, G. D.; Smith, J. C.; Feldman, J. L. Generation and transmission of respiratory oscillations in medullary slices: Role of excitatory amino acids. J. Neurophysiol. 70:1497–1515; 1993. 43. Gardner, W. N. The relationship between tidal volume and inspiratory and expiratory times during steady-state carbon dioxide inhalation in man. J. Physiol. 272:591– 611; 1977. 44. Gray, P. A.; Rekling, J. C.; Bocchiaro, C. M.; Feldman, J. L. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBotzinger complex. Science 286:1566 –1568; 1999. 45. Greer, J. J.; Smith, J. C.; Feldman, J. L. Respiratory and locomotor patterns generated in the fetal rat brain stem-spinal cord in vitro. J. Neurophysiol. 67:996 –999; 1992. 46. Grunstein, M. M.; Younes, M.; Milic-Emili, J. Control of tidal volume and respiratory frequency in anesthetized cats. J. Appl. Physiol. 35:463– 476; 1973. 47. Guthrie, R. D.; Standaert, T. A.; Hodson, W. A.; Woodrum, D. E. Sleep and maturation of eucapnic ventilation and CO2 sensitivity in the premature primate. J. Appl. Physiol. 48:347–354; 1980. 48. Harada, Y.; Kuno, M.; Wang, Y. Z. Differential effects of carbon dioxide and pH on central chemoreceptors in the rat in vitro. J. Physiol. 368:679 – 693; 1985. 49. Harada, Y.; Wang, Y. Z.; Kuno, M. Central chemosensitivity to H⫹ and CO2 in the rat respiratory center in vitro. Brain Res. 333:336 – 339; 1985. 50. Hilaire, G.; Bou, C.; Monteau, R. Rostral ventrolateral medulla and respiratory rhythmogenesis in mice. Neurosci. Lett. 224:13–16; 1997. 51. Hilaire, G.; Duron, B. Maturation of the mammalian respiratory system. Physiol. Rev. 79:325–360; 1999. 52. Hilaire, G.; Monteau, R.; Errchidi, S. 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; 1989. 53. Hilaire, G.; Monteau, R.; Gauthier, P.; Rega, P.; Morin, D. Functional significance of the dorsal respiratory group in adult and newborn rats: In vivo and in vitro studies. Neurosci. Lett. 111:133–138; 1990. 54. Huang, Q.; Zhou, D.; St. John, W. M. Lesions of regions for in vitro ventilatory genesis eliminate gasping but not eupnea. Respir. Physiol. 107:111–123; 1997. 55. Issa, F. G.; Remmers, J. E. Identification of a subsurface area in the ventral medulla sensitive to local changes in PCO2. J. Appl. Physiol. 72:439 – 446; 1992. 56. Johnson, S. M.; Smith, J. C.; Funk, G. D.; Feldman, J. L. Pacemaker behavior of respiratory neurons in medullary slices from neonatal rat. J. Neurophysiol. 72:2598 –2608; 1994. 57. Kawai, A.; Ballantyne, D.; Muckenhoff, K.; Scheid, P. Chemosensitive medullary neurones in the brainstem—Spinal cord preparation of the neonatal rat. J. Physiol. 492:277–292; 1996. 58. Kawai, A.; Okada, Y.; Muckenhoff, K.; Scheid, P. Theophylline and hypoxic ventilatory response in the rat isolated brainstem-spinal cord. Respir. Physiol. 100:25–32; 1995. 59. Koshiya, N.; Smith, J. C. Neuronal pacemaker for breathing visualized in vitro. Nature 400:360 –363; 1999.
RESPIRATION IN NEONATAL OPOSSUM 60. Liu, G.; Feldman, J. L. Quantal synaptic transmission in phrenic motor nucleus. J. Neurophysiol. 68:1468 –1471; 1992. 61. Long, S.; Duffin, J. The neuronal determinants of respiratory rhythm. Prog. Neurobiol. 27:101–182; 1986. 62. McLean, H. A.; Remmers, J. E. Respiratory motor output of the sectioned medulla of the neonatal rat. Respir. Physiol. 96:49 – 60; 1994. 63. Monteau, R.; Morin, D.; Hennequin, S.; Hilaire, G. Differential effects of serotonin on respiratory activity of hypoglossal and cervical motoneurons: An in vitro study on the newborn rat. Neurosci. Lett. 111:127–132; 1990. 64. Monteau, R.; Morin, D.; Hilaire, G. Acetylcholine and central chemosensitivity: In vitro study in the newborn rat. Respir. Physiol. 81:241–254; 1990. 65. Monteau, R.; Ptak, K.; Broquere, N.; Hilaire, G. Tachykinins and central respiratory activity: An in vitro study on the newborn rat. Eur. J. Pharmacol. 314:41–50; 1996. 66. Morin, D.; Hennequin, S.; Monteau, R.; Hilaire, G. Serotonergic influences on central respiratory activity: An in vitro study in the newborn rat. Brain Res. 535:281–287; 1990. 67. Morin, D.; Monteau, R.; Hilaire, G. 5-Hydroytryptamine modulates central respiratory activity in the newborn rat: An in vitro study. Eur. J. Pharmacol. 192:89 –95; 1991. 68. Morin-Surun, M. P.; Boudinot, E.; Sarraseca, H.; Fortin, G.; DenavitSaubie, M. Respiratory network remains functional in a mature guinea pig brainstem isolated in vitro. Exp. Brain Res. 90:375–383; 1992. 69. Morin-Surun, M. P.; Boudinot, E.; Schafer, T.; Denavit-Saubie, M. Brain stem chemosensitivity: Its implication in central respiratory regulation. Biol. Neonate 65:166 –170; 1994. 70. Morin-Surun, M. P.; Boudinot, E.; Schafer, T.; Denavit-Saubie, M. Localization of chemosensitive structures in the isolated brainstem of adult guinea-pig. J. Physiol. 485:203–212; 1995. 71. Nicholls, J. G.; Stewart, R. R.; Erulkar, S. D.; Saunders, N. R. Reflexes, fictive respiration and cell division in the brain and spinal cord of the newborn opossum, Monodelphis domestica, isolated and maintained in vitro. J. Exp. Biol. 152:1–15; 1990. 72. Okada, Y.; Mu¨ckenhoff, K.; Scheid, P. Hypercapnia and medullary neurons in the isolated brain stem-spinal cord of the rat. Respir. Physiol. 93:327–336; 1993. 73. Olsen, G. D.; Weil, J. A. Noninvasive breathing analysis: Repetitive chloral hydrate in neonatal guinea pigs. Pediatr. Pulmonol. 13:227– 234; 1992. 74. Onimaru, H. Studies of the respiratory center using isolated brainstem-spinal cord preparations. Neurosci. Res. 21:183–190; 1995. 75. Onimaru, H.; Arata, A.; Homma, I. Neuronal mechanisms of respiratory rhythm generation: An approach using in vitro preparation. Jpn. J. Physiol. 47:385– 403; 1997. 76. Paton, J. F.; Ramirez, J. M.; Richter, D. W. Functionally intact in vitro preparation generating respiratory activity in neonatal and mature mammals. Pflugers Arch. 428:250 –260; 1994. 77. Paton, J. F.; Richter, D. W. Maturational changes in the respiratory rhythm generator of the mouse. Pflugers Arch. 430:115–124; 1995. 78. Paton, J. F. R. The ventral medullary respiratory network of the mature mouse studied in a working heart brainstem preparation. J. Physiol. 493:819 – 831; 1996. 79. Peever, J. H.; Tian, G. F.; Duffin, J. Temperature and pH affect respiratory rhythm of in-vitro preparations from neonatal rats. Respir. Physiol. 117:97–107; 1999. 80. Ptak, K.; Hilaire, G. Central respiratory effects of substance P in neonatal mice: An in vitro study. Neurosci. Lett. 266:189 –192; 1999. 81. Ramirez, J. M.; Quellmalz, U. J.; Richter, D. W. Postnatal changes in the mammalian respiratory network as revealed by the transverse brainstem slice of mice. J. Physiol. 491:799 – 812; 1996. 82. Rekling, J. C.; Feldman, J. L. PreBotzinger complex and pacemaker
613
83. 84. 85.
86. 87.
88. 89.
90.
91. 92. 93.
94. 95.
96. 97.
98.
99. 100. 101.
neurons: Hypothesized site and kernel for respiratory rhythm generation. Annu. Rev. Physiol. 60:385– 405; 1998. Richter, D. W.; Pierrefiche, O.; Lalley, P. M.; Polder, H. R. Voltageclamp analysis of neurons within deep layers of the brain. J. Neurosci. Methods 67:121–123; 1996. Saetta, M.; Mortola, J. P. Breathing pattern and CO2 response in newborn rats before and during anesthesia. J. Appl. Physiol. 58: 1988 –1996; 1985. Saunders, N. R.; Balkwill, P.; Knott, G.; Habgood, M. D.; Mollgard, K.; Treherne, J. M.; Nicholls, J. G. Growth of axons through a lesion in the intact CNS of fetal rat maintained in long-term culture. Proc. R. Soc. Lond. B Biol. Sci. 250:171–180; 1992. Segal, B. S.; Inman, J. D.; Moss, I. R. Respiratory responses of piglets to hypercapnia during postnatal development: Effects of opiods. Pediatr. Pulmonol. 11:113–119; 1991. Shao, X. M.; Feldman, J. L. Respiratory rhythm generation and synaptic inhibition of expiratory neurons in pre-Botzinger complex: Differential roles of glycinergic and GABAergic neural transmission. J. Neurophysiol. 77:1853–1860; 1997. Smith, J. C.; Ballanyi, K.; Richter, D. W. Whole-cell patch-clamp recordings from respiratory neurons in neonatal rat brainstem in vitro. Neurosci. Lett. 134:153–156; 1992. Smith, J. C.; Ellenberger, H. H.; Ballanyi, K.; Richter, D. W.; Feldman, J. L. Pre-Bo¨tzinger complex: A brainstem region that may generate respiratory rhythm in mammals. Science 254:726 –729; 1991 Smith, J. C.; Greer, J. J.; Liu, G.; Feldman, J. L. 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; 1990. St. John, W. M. Neurogenesis, control, and functional significance of gasping. J. Appl. Physiol. 68:1305–1315; 1990. St. John, W. M. Medullary regions for neurogenesis of gasping: Noeud vital or noeuds vitals? J. Appl. Physiol. 81:1865–1877; 1996. Stewart, R. R.; Zou, D. J.; Treherne, J. M.; Mollgard, K.; Saunders, N. R.; Nicholls, J. G. The intact central nervous system of the newborn opossum in long-term culture: Fine structure and GABAmediated inhibition of electrical activity. J. Exp. Biol. 161:25– 41; 1991. Suzue, T. Respiratory rhythm generation in the in vitro brain stemspinal cord preparation of the neonatal rat. J. Physiol. 354:173–183; 1984. Treherne, J. M.; Woodward, S. K.; Varga, Z. M.; Ritchie, J. M.; Nicholls, J. G. Restoration of conduction and growth of axons through injured spinal cord of neonatal opossum in culture. Proc. Natl. Acad. Sci. USA 89:431– 434; 1992. von Euler, C. The functional organization of the respiratory phaseswitching mechanisms. Fed. Proc. 36:2375–2380; 1977. von Euler, C. Brain stem mechanisms for generation and control of breathing pattern. In: Cherniack, N. S.; Widdicombe, J. G., eds. American Physiological Society: Handbook of physiology. Baltimore, MD: Williams & Wilkins; 1986:1– 68. Zhou, D.; Huang, Q.; Fung, M. L.; Li, A.; Darnall, R. A.; Nattie, E. E.; St. John, W. M. Phrenic response to hypercapnia in the unanesthetized, decerbrate, newborn rat. Respir. Physiol. 104:11–22; 1996. Zhou, D.; Wasicko, M. J.; Hu, J. M.; St. John, W. M. Differing activities of medullary respiratory neurons in eupnea and gasping. J. Appl. Physiol. 70:1265–1270; 1991. Zou, D. J. Respiratory rhythm in the isolated central nervous system of newborn opossum. J. Exp. Biol. 197:201–213; 1994. Zou, D. J.; Treherne, J. M.; Stewart, R. R.; Saunders, N. R.; Nicholls, J. G. Regulation of GABA B receptors by histamine and neuronal activity in the isolated spinal cord of neonatal opossum in culture. Proc. R. Soc. Lond. B Biol. Sci. 246:77– 82; 1991.