- Email: [email protected]
A commentary on eupnoea and gasping
Respiratory Physiology & Neurobiology 139 (2003) 105 /111 www.elsevier.com/locate/resphysiol
A commentary on eupnoea and gasping James Duffin * Departments of Physiology and Anaesthesia, University of Toronto, Toronto, Ont., Canada M5S 1A8 Accepted 20 May 2003
Abstract This commentary discusses the differences between patterns of bursting activity recorded from the phrenic nerves of different species, in several experimental preparations and under differing conditions. The spectrum of bursting activity patterns varies from that termed eupnoic to that termed gasping. Taking the pattern of activity recorded in the least reduced preparation as a standard for normality, i.e. eupnoea, consideration is given to the possible factors affecting the pattern of bursting activity in progressively reduced preparations. An examination of the conditions of these preparations leads to the conclusion that tissue gas exchange is a major determinant of bursting pattern, and consideration is given to the possible differences in respiratory rhythm generation that can be inferred from these different patterns. # 2003 Elsevier B.V. All rights reserved. Keywords: Control of breathing, central pattern; Disease, dyspnea; Pattern of breathing, central, eupnea, bursting, gasping
1. Introduction To ventilate the lungs the diaphragm requires a pattern of phrenic nerve activity that is physiologically suitable. Inspiration requires a ramp of increasing phrenic activation of the diaphragm to overcome the increasing elastic recoil of the lungs. Expiration in its early part requires a declining ramp of phrenic activation of the diaphragm to act as a brake on expiratory flow, so as to maintain lung volume and effect an expiratory pause. This expiratory pause assists the distribution of air within the lungs, facilitating the matching of ventilation and perfusion in the various regions. * Tel.: /1-416-978-6379; fax: /1-416-978-4940. E-mail address: [email protected] (J. Duffin).
Expiration in its later part then requires a period of phrenic and diaphragm inactivity to allow the completion of expiratory outflow. These physiological requirements, therefore, dictate a phrenic activity that takes the form of an augmenting ramp during inspiration, a decreasing ramp during the first part of expiration and a silent period during the later part of expiration. Just such a pattern of activity is observed, and it has been termed ‘‘eupnoea’’, from the Greek ‘‘eu’’ (normal) and ‘‘pnein’’ (to breathe). There is, therefore, an obvious link between the observed phrenic pattern of activity and its physiological function; one implies the other. This link is similar to that between anatomical description and physiological function; indeed one is often used to imply the other, and this interplay can be a
1569-9048/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1569-9048(03)00194-0
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J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111
valuable tool in deciphering how systems operate. For example the anatomical description of the carotid body by de Castro and Rubio (1968) produced a theory of carotid body function as a peripheral chemoreceptor that is still relevant (Torrance, 1996). However, applying such a linkage is not without peril, and like all assumptions of implication it sometimes fails. For example, despite the neuroanatomical demonstration of extensive neuronal projections from upper cervical inspiratory neurons to respiratory motoneurons, (Lipski and Duffin, 1986; Lipski et al., 1993) which imply an important role, our experiments to determine the function of these projections have had only limited success, so that their physiological role remains mysterious (Tian and Duffin, 1996). Just as it is necessary to view the physiological implications of anatomy with care, so it is also necessary to treat the inferential link between observed patterns of phrenic nerve activity and the physiological operation of the respiratory rhythm generator with caution. With this caution in mind; from the description of the three phases of phrenic activity observed during eupnoea it can be inferred that the respiratory rhythm generator also functions as a threephase oscillator in eupnoeic breathing (Richter, 1982), and a number of models of such an oscillator, based on mutual inhibition between populations of neurons, have been simulated (e.g. Duffin et al., 1995; Ryback et al., 1997). However, because it is difficult to determine functional interconnections between respiratory neurons and their cellular characteristics in largely intact animals, these models remain theoretical, and investigators have sought reduced preparations that will allow more intensive examination of the respiratory rhythm generator. However, if reduced preparations are to be used to examine the eupnoeic operation of the respiratory rhythm generator it becomes important to apply the following comparison paradigm.
2. Comparing phrenic bursting patterns It is one of the classic paradigms of research to compare measurements before and after an inter-
vention. Such a comparison applies to the investigation of many physiological systems including respiration. Thus, the term eupnoea describes the pattern of breathing activity in a normal human or animal, and any intervention, be it surgical or pharmacological, can then be judged to affect respiration if the pattern changes from eupnoea. Progressively greater changes in the pattern by more and more invasive interventions can be used to infer what alterations have been made to the system by the intervention and illustrate how particular system aspects determine overall function: still keeping in mind the caution about inferential links. Early examples of this approach to examine respiratory rhythm generation were brainstem ablation and sectioning experiments (Lumsden, 1923; Pitts, 1946; Wang et al., 1957). The experimenters noted changes from the eupnoeic breathing pattern and coined new terms such as apneustic and gasping to describe the resulting pattern. Similarly, exposure of the whole adult animal to anoxia produces a change from the eupnoeic pattern to a gasping pattern (St. John, 1998, 1999). Use of the comparison paradigm is dependent on definitions of eupnoea and gasping that are simple, and as a result, easily recognized. To be widely understood their definition requires a consensus process, and while most of us have a definition in mind, to my knowledge it has not been the subject of explicit review such as this one. So, what are the distinguishing characteristics of each? Perhaps the conditions that produce these patterns are an obvious starting point and could be described as follows. The eupnoeic condition is that of the whole adult animal at rest and the gasping condition is that of the whole adult animal during anoxia. However, nerve recordings cannot be made without interventions, which already depart from these initial conditions; the animal must be anaesthetized or decerebrate and surgical exposure of nerves made to record them, and these may include recording from the proximal ends of cut motor nerves, in which case any afferent information ceases to be transmitted. Nevertheless, these are the minimal interventions that are necessary to record from phrenic nerves, and so
J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111
these conditions can be used to define the patterns for eupnoea and gasping. Fig. 1 shows familiar patterns of activity observed in the phrenic nerves of a pentobarbitone anaesthetized adult cat, a decerebrate vagotomized adult cat, and a decerebrate vagotomized adult rat. The similarities are more obvious than the differences in this figure, and it therefore, appears that neither decerebration nor vagotomy substantially alters the pattern from eupnoea, and that the rat and cat are not substantially different except in cycle period and burst duration. These latter descriptors, although obvious choices for defining eupnoea, are therefore, not appropriate for a simple and obvious comparison definition if it is to be applicable across species, unless a scaling for
Fig. 1. Patterns of phrenic nerve activity recorded in a pentobarbitone anaesthetized adult cat (top trace) a decerebrate vagotomized cat (centre trace) and a decerebrate vagotomized rat (bottom trace). Modified from Duffin and van Alphen (1995), Iscoe and Duffin (1996) and Duffin et al. (2000), respectively.
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animal size is used; but they can be used within a species. The three phase nature of the phrenic discharge, an augmenting ramp during inspiration followed by a declining ramp during early expiration and then silence during late expiration is mostly apparent (e.g. see Fig. 1 decerebrate rat) but sometimes not (e.g. see Fig. 1 anaesthetized cat); so for a definition of eupnoea that is widely applicable a description of the eupnoeic pattern as three-phase does not meet our requirements that it be obvious and simple. Likewise, the same argument applies to the often but not always observed abrupt cessation of activity at the junction between inspiration and expiration (e.g. see Fig. 1 decerebrate cat). The most prominent common feature of all the patterns is an augmenting ramp during inspiration. Combined with the species-specific duration of the ramp and the cycle time, these are the key features and they define the eupnoeic pattern for phrenic activity. This definition can then be used to examine the effects of interventions using the comparison paradigm.
Fig. 2. Patterns of phrenic nerve activity recorded in a decerebrate rat (top trace) and an in vitro brainstem /spinal cord preparation from a neonatal rat (bottom trace). Modified from Duffin et al. (2000) and Peever et al. (1999), respectively.
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Fig. 2 shows phrenic recordings from a decerebrate vagotomized adult rat and a neonatal rat brainstem /spinal cord in vitro preparation. The patterns of activity are markedly different; with respect to the features of eupnoea defined above, the in vitro phrenic activity is no longer an augmenting ramp but a declining one, the duration of the activity is much shorter, and the cycle time much longer. This pattern is similar to the gasping pattern of phrenic activity observed in adult rats during anoxia (St. John, 1998, 1999). To use the comparison paradigm we first need to ask what differences between the two preparations are responsible for the change in pattern. While both preparations are from decerebrate and vagotomized rats, the in vitro preparation is from a neonatal aged rat, at a reduced temperature and without vascular perfusion to facilitate tissue gas exchange. Further, while decerebration and vagotomy in the adult rat do not alter the pattern from the eupnoeic, they may do so in the neonatal rat.
3. Factors affecting phrenic bursting pattern First, consider the developmental age of the rat as a factor; there are numerous developmental changes that can impact the generation of respiratory rhythm (Richter and Spyer, 2001). However, recordings from anaesthetized neonatal rats (Smith et al., 1990; Wang et al., 1996) show an augmenting burst of activity with a longer duration and a shorter cycle length than that of the in vitro preparation. The pattern in the anaesthetized neonatal rat is, therefore, essentially eupnoeic, and so developmental age alone cannot account for the different patterns of Fig. 2. Second, consider the effects of temperature in the neonatal rat brainstem /spinal cord in vitro preparation. Fig. 3 shows that the reduced temperature accounts for an increase in cycle time, and increasing the temperature modifies the declining ramp towards an augmenting ramp, although it does not fully restore the pattern to a eupnoeic one. Thus, although temperature may be a contributing factor, it too does not account for the different patterns in Fig. 2.
Fig. 3. The effect of increasing the temperature of the artificial cerebrospinal fluid, bathing an in vitro brainstem /spinal cord preparation from a neonatal rat, on phrenic nerve discharge. (A) Changes in phrenic bursting frequency with temperature. (B) The pattern of phrenic nerve discharge, raw (top traces) and integrated (bottom traces) at temperatures of 25 and 35 8C. Modified from Peever et al. (1999).
The third consideration is the effect of vagotomy, which although not a factor altering the phrenic pattern of activity from the eupnoeic in the adult, has been argued to do so in the neonate (Ballanyi et al., 1999). Patterns of phrenic nerve activity recorded from an anaesthetized neonatal rat before and after vagotomy by Smith et al. (1990) demonstrate a change from an augmenting to a declining ramp on vagotomy, although Wang et al. (1996) did not find such a change. In these preparations breathing often fails to provide adequate gas exchange after vagotomy (Fedorko et al., 1988), and so it is possible that oxygenation is inadequate in the vagotomized neonatal rat. In that case the gasping pattern of activity observed in some preparations could be due to anoxia. The problem of providing adequate oxygenation is
J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111
avoided in vascularly perfused preparations, and the phrenic activity recorded in perfused neonatal rats displays a eupnoeic pattern (Dutschmann et al., 2000). Although the vagi were not sectioned in these experiments, phasic pulmonary stretch receptor feedback was absent because the lungs were not rhythmically inflated. Further, albeit in perfused juvenile not neonatal rats, bilateral vagotomy did not produce a change in the pattern of phrenic activity from the eupnoeic (St. John and Paton, 2000). These observations, therefore, suggest that vagotomy is unlikely to account for the observed differences in the pattern of phrenic activity between neonatal in vitro preparations and adults shown in Fig. 2. The last major difference between the two preparations compared in Fig. 2 to be considered is the means of supplying oxygen to tissues; the in vitro preparation is not vascularly perfused, and oxygen is supplied via the bathing medium by diffusion. Several investigators have measured the oxygen levels in the in vitro preparation and concluded that it is sufficient for normal aerobic metabolism (Ballanyi et al., 1992; Brockhaus et al., 1993; Volker et al., 1995), although others did not (Okada et al., 1993). Of relevance to the question is the observation that in the artificially perfused preparation of neonatal rats the phrenic activity has an augmenting ramp pattern, instead of the declining ramp pattern of the neonatal in vitro preparation (Dutschmann et al., 2000). Further, albeit in perfused juvenile not neonatal rats, tissue oxygen levels have been shown to be well maintained (Wilson et al., 2001), and the phrenic nerve discharge may be converted from an augmenting ramp to a declining ramp by manipulating the oxygen level of the vascular perfusion solution just as in adult rats (St. John and Paton, 2000).
4. Conclusions Thus, the difference between the adult rat and the neonatal rat brainstem /spinal cord in vitro preparations that accounts for the differences in the patterns of phrenic activity shown in Fig. 2 appears to be that of tissue gas exchange. This difference in the conditions of the preparations is
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important, because the next stage in the application of the comparison paradigm is to infer differences in the physiological systems producing the observed patterns; in this case the system generating respiratory rhythm. As discussed earlier, the adult intact rat, with a eupnoeic pattern of phrenic activity, implies a three-phase network model of rhythm generation, but supporting evidence is difficult to obtain. By contrast, it is less difficult to determine how the rhythm is generated in neonatal in vitro preparations, and considerable evidence has accumulated to support the concept of an oscillator dependent on the intrinsic bursting or ‘‘pacemaker’’ properties of specialized neurons (Gray et al., 1999; Smith et al., 1991); but the oscillator produces a gasping pattern of activity. Differences between preparations may be crucial to the investigation of the respiratory rhythm generator. For example, if the difference in the pattern of activity was due simply to vagotomy then it could be assumed that the in vitro preparation behaves like a vagotomized intact neonate and the respiratory rhythm generator remains essentially the same in the two preparations. However, if the difference is due to the condition of tissue oxygenation as the previous assessment concluded, then the in vitro preparation produces a pattern of phrenic activity that is not eupnoeic and resembles the gasping pattern observed during anoxia in adult preparations. In that case, the respiratory rhythm generator has assumed an alternative mode of operation, gasping, either by a reconfiguration of the eupnoeic oscillator (Lieske et al., 2000), or by a silencing of the eupnoeic oscillator and replacement by a gasping oscillator (St. John, 1996; St. John and Rybak, 2002). In either case the respiratory rhythm generator in the in vitro preparation is not a replica of that in an oxygenated animal. Finally, although the previous discussion has been restricted to phrenic nerve activity similar arguments may be made about the hypoglossal nerve, as Fig. 4 illustrates, and are especially relevant to the use of the neonatal rat in vitro brainstem slice preparation as a model of the respiratory rhythm generator.
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Fig. 4. Patterns of activity recorded from hypoglossal (XII) and phrenic nerves in a decerebrate rat (top traces), the hypoglossal nerve at 25 and 35 8C in a brainstem /spinal cord in vitro preparation from a neonatal rat (middle two traces), and the hypoglossal nerve in a transverse medullary slice in vitro preparation from a neonatal rat. Modified from Peever et al. (2001, 1999), respectively.
Acknowledgements This research was suppoted by the Canadian Institutes of Health Research.
References Ballanyi, K., Kuwana, S., Vo¨lker, A., Morawietz, G., Richter, D.W., Volker, A., 1992. Developmental changes in the hypoxia tolerance of the in vitro respiratory network of rats. Neurosci. Lett. 148, 141 /144. Ballanyi, K., Onimaru, H., Homma, I., 1999. Respiratory network function in the isolated brainstem /spinal cord of newborn rats. Prog. Neurobiol. 59, 583 /634. Brockhaus, J., Ballanyi, K., Smith, J.C., Richter, D.W., 1993. Microenvironment of respiratory neurons in the in vitro
brainstem /spinal cord of neonatal rats. J. Physiol., London 462, 421 /445. de Castro, F., Rubio, M., 1968. The anatomy and innervation of the blood vessels of the carotid body and the role of chemoreceptive reactions in the autoregulation of the blood flow. In: Torrance, R.W. (Ed.), Arterial Chemoreceptors. Blackwell, Oxford, pp. 267 /277. Duffin, J., van Alphen, J., 1995. Cross-correlation of augmenting expiratory neurons of the Bo¨tzinger complex in the cat. Exp. Brain Res. 103, 251 /255. Duffin, J., Ezure, K., Lipski, J., 1995. Breathing rhythm generation: focus on the rostral ventrolateral medulla. NIPS 10, 133 /140. Duffin, J., Tian, G., Peever, J.H., 2000. Functional synaptic connections among respiratory neurons. Respir. Physiol. 122, 237 /246. Dutschmann, M., Wilson, R.J., Paton, J.F., 2000. Respiratory activity in neonatal rats. Auton. Neurosci. 84, 19 /29. Fedorko, L., Kelly, E.N., England, S.J., 1988. Importance of vagal afferents in determining ventilation in newborn rats. J. Appl. Physiol. 65, 1033 /1039. Gray, P.A., Rekling, J.C., Bocchiaro, C.M., Feldman, J.L., 1999. Modulation of respiratory frequency by peptidergic input to rhythmogenic neurons in the preBo¨tzinger complex. Science 286, 1566 /1568. Iscoe, S., Duffin, J., 1996. Effects of stimulation of phrenic afferents on cervical respiratory interneurones and phrenic motoneurones in cats. J. Physiol., London 497, 803 /812. Lieske, S.P., Thoby-Brisson, M., Telgkamp, P., Ramirez, J.M., 2000. Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps. Nat. Neurosci. 3, 600 /607. Lipski, J., Duffin, J., 1986. An electrophysiological investigation of propriospinal inspiratory neurons in the upper cervical cord of the cat. Exp. Brain Res. 61, 625 /637. Lipski, J., Duffin, J., Kruszewska, B., Zhang, X., 1993. Upper cervical inspiratory neurons in the rat: an electrophysiological and morphological study. Exp. Brain Res. 95, 477 / 487. Lumsden, T., 1923. Observations on the respiratory centres. J. Physiol., London 57, 354 /367. Okada, Y., Mu¨ckenhoff, K., Holtermann, G., Acker, H., Scheid, P., 1993. Depth profiles of pH and PO2 in the isolated brainstem /spinal cord of the neonatal rat. Respir. Physiol. 93, 315 /326. Peever, J.H., Tian, G.F., Duffin, J., 1999. Temperature and pH affect respiratory rhythm of in vitro preparations from neonatal rats. Respir. Physiol. 117, 97 /107. Peever, J.H., Mateika, J.H., Duffin, J., 2001. Respiratory control of hypoglossal motoneurones in the rat. Pflu¨gers Arch. 442, 78 /86. Pitts, R.F., 1946. Organisation of the respiratory centre. Physiol. Rev. 26, 609 /630. Richter, D., 1982. Generation and maintenance of the respiratory rhythm. J. Exp. Biol. 100, 93 /107.
J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111 Richter, D.W., Spyer, K.M., 2001. Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models. Trends Neurosci. 24, 464 /472. Ryback, I.A., Paton, J.F.R., Schwaber, J.S., 1997. Modeling neural mechanisms for genesis of respiratory rhythm and pattern. II. Network models of the central respiratory pattern generator. J. Neurophysiol. 77, 2007 /2026. Smith, J.C., Greer, J.J., Liu, G.S., Feldman, J.L., 1990. Neural mechanisms generating respiratory pattern in mammalian brain stem /spinal cord in vitro. I. Spatiotemporal patterns of motor and medullary neuron activity. J. Neurophysiol. 64, 1149 /1169. Smith, J.C., Ellenberger, H.H., Ballanyi, K., Richter, D.W., Feldman, J.L., 1991. Pre-Bo¨tzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254, 726 /729. St. John, W.M., 1996. Medullary regions for neurogenesis of gasping: noeud vital or noeuds vitals. J. Appl. Physiol. 81, 1865 /1877. St. John, W.M., 1998. Neurogenesis of patterns of automatic ventilatory activity. Prog. Neurobiol. 56, 97 /117. St. John, W.M., 1999. Rostral medullary respiratory neuronal activities of decerebrate cats in eupnea, apneusis and gasping. Respir. Physiol. 116, 47 /65.
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St. John, W.M., Paton, J.F., 2000. Characterizations of eupnea, apneusis and gasping in a perfused rat preparation. Respir. Physiol. 123, 201 /213. St. John, W.M., Rybak, I.A., 2002. Influence of levels of carbon dioxide and oxygen upon gasping in perfused rat preparation. Respir. Physiol. 129, 279 /287. Tian, G.F., Duffin, J., 1996. Connections from upper cervical inspiratory neurons to phrenic and intercostal motoneurons studied with cross-correlation in the decerebrate rat. Exp. Brain Res. 110, 196 /204. Torrance, R.W., 1996. Prolegomena. Chemoreception upstream of transmitters. Adv. Exp. Med. Biol. 410, 13 /38. Volker, A., Ballanyi, K., Richter, D.W., 1995. Anoxic disturbance of the isolated respiratory network of neonatal rats. Exp. Brain Res. 103, 9 /19. Wang, S.C., Ngai, S.H., Frumin, M.J., 1957. Organisation of central respiratory mechanisms in the brain stem of the cat. Am. J. Physiol. 190, 333 /342. Wang, W., Fung, M.L., Darnall, R.A., St. John, W.M., 1996. Characterizations and comparisons of eupnoea and gasping in neonatal rats. J. Physiol., London 490, 277 /292. Wilson, R.J., Remmers, J.E., Paton, J.F., 2001. Brain stem PO2 and pH of the working heart /brain stem preparation during vascular perfusion with aqueous medium. Am. J. Physiol. 281, R528 /R538.
A commentary on eupnoea and gasping James Duffin * Departments of Physiology and Anaesthesia, University of Toronto, Toronto, Ont., Canada M5S 1A8 Accepted 20 May 2003
Abstract This commentary discusses the differences between patterns of bursting activity recorded from the phrenic nerves of different species, in several experimental preparations and under differing conditions. The spectrum of bursting activity patterns varies from that termed eupnoic to that termed gasping. Taking the pattern of activity recorded in the least reduced preparation as a standard for normality, i.e. eupnoea, consideration is given to the possible factors affecting the pattern of bursting activity in progressively reduced preparations. An examination of the conditions of these preparations leads to the conclusion that tissue gas exchange is a major determinant of bursting pattern, and consideration is given to the possible differences in respiratory rhythm generation that can be inferred from these different patterns. # 2003 Elsevier B.V. All rights reserved. Keywords: Control of breathing, central pattern; Disease, dyspnea; Pattern of breathing, central, eupnea, bursting, gasping
1. Introduction To ventilate the lungs the diaphragm requires a pattern of phrenic nerve activity that is physiologically suitable. Inspiration requires a ramp of increasing phrenic activation of the diaphragm to overcome the increasing elastic recoil of the lungs. Expiration in its early part requires a declining ramp of phrenic activation of the diaphragm to act as a brake on expiratory flow, so as to maintain lung volume and effect an expiratory pause. This expiratory pause assists the distribution of air within the lungs, facilitating the matching of ventilation and perfusion in the various regions. * Tel.: /1-416-978-6379; fax: /1-416-978-4940. E-mail address: [email protected] (J. Duffin).
Expiration in its later part then requires a period of phrenic and diaphragm inactivity to allow the completion of expiratory outflow. These physiological requirements, therefore, dictate a phrenic activity that takes the form of an augmenting ramp during inspiration, a decreasing ramp during the first part of expiration and a silent period during the later part of expiration. Just such a pattern of activity is observed, and it has been termed ‘‘eupnoea’’, from the Greek ‘‘eu’’ (normal) and ‘‘pnein’’ (to breathe). There is, therefore, an obvious link between the observed phrenic pattern of activity and its physiological function; one implies the other. This link is similar to that between anatomical description and physiological function; indeed one is often used to imply the other, and this interplay can be a
1569-9048/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1569-9048(03)00194-0
106
J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111
valuable tool in deciphering how systems operate. For example the anatomical description of the carotid body by de Castro and Rubio (1968) produced a theory of carotid body function as a peripheral chemoreceptor that is still relevant (Torrance, 1996). However, applying such a linkage is not without peril, and like all assumptions of implication it sometimes fails. For example, despite the neuroanatomical demonstration of extensive neuronal projections from upper cervical inspiratory neurons to respiratory motoneurons, (Lipski and Duffin, 1986; Lipski et al., 1993) which imply an important role, our experiments to determine the function of these projections have had only limited success, so that their physiological role remains mysterious (Tian and Duffin, 1996). Just as it is necessary to view the physiological implications of anatomy with care, so it is also necessary to treat the inferential link between observed patterns of phrenic nerve activity and the physiological operation of the respiratory rhythm generator with caution. With this caution in mind; from the description of the three phases of phrenic activity observed during eupnoea it can be inferred that the respiratory rhythm generator also functions as a threephase oscillator in eupnoeic breathing (Richter, 1982), and a number of models of such an oscillator, based on mutual inhibition between populations of neurons, have been simulated (e.g. Duffin et al., 1995; Ryback et al., 1997). However, because it is difficult to determine functional interconnections between respiratory neurons and their cellular characteristics in largely intact animals, these models remain theoretical, and investigators have sought reduced preparations that will allow more intensive examination of the respiratory rhythm generator. However, if reduced preparations are to be used to examine the eupnoeic operation of the respiratory rhythm generator it becomes important to apply the following comparison paradigm.
2. Comparing phrenic bursting patterns It is one of the classic paradigms of research to compare measurements before and after an inter-
vention. Such a comparison applies to the investigation of many physiological systems including respiration. Thus, the term eupnoea describes the pattern of breathing activity in a normal human or animal, and any intervention, be it surgical or pharmacological, can then be judged to affect respiration if the pattern changes from eupnoea. Progressively greater changes in the pattern by more and more invasive interventions can be used to infer what alterations have been made to the system by the intervention and illustrate how particular system aspects determine overall function: still keeping in mind the caution about inferential links. Early examples of this approach to examine respiratory rhythm generation were brainstem ablation and sectioning experiments (Lumsden, 1923; Pitts, 1946; Wang et al., 1957). The experimenters noted changes from the eupnoeic breathing pattern and coined new terms such as apneustic and gasping to describe the resulting pattern. Similarly, exposure of the whole adult animal to anoxia produces a change from the eupnoeic pattern to a gasping pattern (St. John, 1998, 1999). Use of the comparison paradigm is dependent on definitions of eupnoea and gasping that are simple, and as a result, easily recognized. To be widely understood their definition requires a consensus process, and while most of us have a definition in mind, to my knowledge it has not been the subject of explicit review such as this one. So, what are the distinguishing characteristics of each? Perhaps the conditions that produce these patterns are an obvious starting point and could be described as follows. The eupnoeic condition is that of the whole adult animal at rest and the gasping condition is that of the whole adult animal during anoxia. However, nerve recordings cannot be made without interventions, which already depart from these initial conditions; the animal must be anaesthetized or decerebrate and surgical exposure of nerves made to record them, and these may include recording from the proximal ends of cut motor nerves, in which case any afferent information ceases to be transmitted. Nevertheless, these are the minimal interventions that are necessary to record from phrenic nerves, and so
J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111
these conditions can be used to define the patterns for eupnoea and gasping. Fig. 1 shows familiar patterns of activity observed in the phrenic nerves of a pentobarbitone anaesthetized adult cat, a decerebrate vagotomized adult cat, and a decerebrate vagotomized adult rat. The similarities are more obvious than the differences in this figure, and it therefore, appears that neither decerebration nor vagotomy substantially alters the pattern from eupnoea, and that the rat and cat are not substantially different except in cycle period and burst duration. These latter descriptors, although obvious choices for defining eupnoea, are therefore, not appropriate for a simple and obvious comparison definition if it is to be applicable across species, unless a scaling for
Fig. 1. Patterns of phrenic nerve activity recorded in a pentobarbitone anaesthetized adult cat (top trace) a decerebrate vagotomized cat (centre trace) and a decerebrate vagotomized rat (bottom trace). Modified from Duffin and van Alphen (1995), Iscoe and Duffin (1996) and Duffin et al. (2000), respectively.
107
animal size is used; but they can be used within a species. The three phase nature of the phrenic discharge, an augmenting ramp during inspiration followed by a declining ramp during early expiration and then silence during late expiration is mostly apparent (e.g. see Fig. 1 decerebrate rat) but sometimes not (e.g. see Fig. 1 anaesthetized cat); so for a definition of eupnoea that is widely applicable a description of the eupnoeic pattern as three-phase does not meet our requirements that it be obvious and simple. Likewise, the same argument applies to the often but not always observed abrupt cessation of activity at the junction between inspiration and expiration (e.g. see Fig. 1 decerebrate cat). The most prominent common feature of all the patterns is an augmenting ramp during inspiration. Combined with the species-specific duration of the ramp and the cycle time, these are the key features and they define the eupnoeic pattern for phrenic activity. This definition can then be used to examine the effects of interventions using the comparison paradigm.
Fig. 2. Patterns of phrenic nerve activity recorded in a decerebrate rat (top trace) and an in vitro brainstem /spinal cord preparation from a neonatal rat (bottom trace). Modified from Duffin et al. (2000) and Peever et al. (1999), respectively.
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J. Duffin / Respiratory Physiology & Neurobiology 139 (2003) 105 /111
Fig. 2 shows phrenic recordings from a decerebrate vagotomized adult rat and a neonatal rat brainstem /spinal cord in vitro preparation. The patterns of activity are markedly different; with respect to the features of eupnoea defined above, the in vitro phrenic activity is no longer an augmenting ramp but a declining one, the duration of the activity is much shorter, and the cycle time much longer. This pattern is similar to the gasping pattern of phrenic activity observed in adult rats during anoxia (St. John, 1998, 1999). To use the comparison paradigm we first need to ask what differences between the two preparations are responsible for the change in pattern. While both preparations are from decerebrate and vagotomized rats, the in vitro preparation is from a neonatal aged rat, at a reduced temperature and without vascular perfusion to facilitate tissue gas exchange. Further, while decerebration and vagotomy in the adult rat do not alter the pattern from the eupnoeic, they may do so in the neonatal rat.
3. Factors affecting phrenic bursting pattern First, consider the developmental age of the rat as a factor; there are numerous developmental changes that can impact the generation of respiratory rhythm (Richter and Spyer, 2001). However, recordings from anaesthetized neonatal rats (Smith et al., 1990; Wang et al., 1996) show an augmenting burst of activity with a longer duration and a shorter cycle length than that of the in vitro preparation. The pattern in the anaesthetized neonatal rat is, therefore, essentially eupnoeic, and so developmental age alone cannot account for the different patterns of Fig. 2. Second, consider the effects of temperature in the neonatal rat brainstem /spinal cord in vitro preparation. Fig. 3 shows that the reduced temperature accounts for an increase in cycle time, and increasing the temperature modifies the declining ramp towards an augmenting ramp, although it does not fully restore the pattern to a eupnoeic one. Thus, although temperature may be a contributing factor, it too does not account for the different patterns in Fig. 2.
Fig. 3. The effect of increasing the temperature of the artificial cerebrospinal fluid, bathing an in vitro brainstem /spinal cord preparation from a neonatal rat, on phrenic nerve discharge. (A) Changes in phrenic bursting frequency with temperature. (B) The pattern of phrenic nerve discharge, raw (top traces) and integrated (bottom traces) at temperatures of 25 and 35 8C. Modified from Peever et al. (1999).
The third consideration is the effect of vagotomy, which although not a factor altering the phrenic pattern of activity from the eupnoeic in the adult, has been argued to do so in the neonate (Ballanyi et al., 1999). Patterns of phrenic nerve activity recorded from an anaesthetized neonatal rat before and after vagotomy by Smith et al. (1990) demonstrate a change from an augmenting to a declining ramp on vagotomy, although Wang et al. (1996) did not find such a change. In these preparations breathing often fails to provide adequate gas exchange after vagotomy (Fedorko et al., 1988), and so it is possible that oxygenation is inadequate in the vagotomized neonatal rat. In that case the gasping pattern of activity observed in some preparations could be due to anoxia. The problem of providing adequate oxygenation is
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avoided in vascularly perfused preparations, and the phrenic activity recorded in perfused neonatal rats displays a eupnoeic pattern (Dutschmann et al., 2000). Although the vagi were not sectioned in these experiments, phasic pulmonary stretch receptor feedback was absent because the lungs were not rhythmically inflated. Further, albeit in perfused juvenile not neonatal rats, bilateral vagotomy did not produce a change in the pattern of phrenic activity from the eupnoeic (St. John and Paton, 2000). These observations, therefore, suggest that vagotomy is unlikely to account for the observed differences in the pattern of phrenic activity between neonatal in vitro preparations and adults shown in Fig. 2. The last major difference between the two preparations compared in Fig. 2 to be considered is the means of supplying oxygen to tissues; the in vitro preparation is not vascularly perfused, and oxygen is supplied via the bathing medium by diffusion. Several investigators have measured the oxygen levels in the in vitro preparation and concluded that it is sufficient for normal aerobic metabolism (Ballanyi et al., 1992; Brockhaus et al., 1993; Volker et al., 1995), although others did not (Okada et al., 1993). Of relevance to the question is the observation that in the artificially perfused preparation of neonatal rats the phrenic activity has an augmenting ramp pattern, instead of the declining ramp pattern of the neonatal in vitro preparation (Dutschmann et al., 2000). Further, albeit in perfused juvenile not neonatal rats, tissue oxygen levels have been shown to be well maintained (Wilson et al., 2001), and the phrenic nerve discharge may be converted from an augmenting ramp to a declining ramp by manipulating the oxygen level of the vascular perfusion solution just as in adult rats (St. John and Paton, 2000).
4. Conclusions Thus, the difference between the adult rat and the neonatal rat brainstem /spinal cord in vitro preparations that accounts for the differences in the patterns of phrenic activity shown in Fig. 2 appears to be that of tissue gas exchange. This difference in the conditions of the preparations is
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important, because the next stage in the application of the comparison paradigm is to infer differences in the physiological systems producing the observed patterns; in this case the system generating respiratory rhythm. As discussed earlier, the adult intact rat, with a eupnoeic pattern of phrenic activity, implies a three-phase network model of rhythm generation, but supporting evidence is difficult to obtain. By contrast, it is less difficult to determine how the rhythm is generated in neonatal in vitro preparations, and considerable evidence has accumulated to support the concept of an oscillator dependent on the intrinsic bursting or ‘‘pacemaker’’ properties of specialized neurons (Gray et al., 1999; Smith et al., 1991); but the oscillator produces a gasping pattern of activity. Differences between preparations may be crucial to the investigation of the respiratory rhythm generator. For example, if the difference in the pattern of activity was due simply to vagotomy then it could be assumed that the in vitro preparation behaves like a vagotomized intact neonate and the respiratory rhythm generator remains essentially the same in the two preparations. However, if the difference is due to the condition of tissue oxygenation as the previous assessment concluded, then the in vitro preparation produces a pattern of phrenic activity that is not eupnoeic and resembles the gasping pattern observed during anoxia in adult preparations. In that case, the respiratory rhythm generator has assumed an alternative mode of operation, gasping, either by a reconfiguration of the eupnoeic oscillator (Lieske et al., 2000), or by a silencing of the eupnoeic oscillator and replacement by a gasping oscillator (St. John, 1996; St. John and Rybak, 2002). In either case the respiratory rhythm generator in the in vitro preparation is not a replica of that in an oxygenated animal. Finally, although the previous discussion has been restricted to phrenic nerve activity similar arguments may be made about the hypoglossal nerve, as Fig. 4 illustrates, and are especially relevant to the use of the neonatal rat in vitro brainstem slice preparation as a model of the respiratory rhythm generator.
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Fig. 4. Patterns of activity recorded from hypoglossal (XII) and phrenic nerves in a decerebrate rat (top traces), the hypoglossal nerve at 25 and 35 8C in a brainstem /spinal cord in vitro preparation from a neonatal rat (middle two traces), and the hypoglossal nerve in a transverse medullary slice in vitro preparation from a neonatal rat. Modified from Peever et al. (2001, 1999), respectively.
Acknowledgements This research was suppoted by the Canadian Institutes of Health Research.
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