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POINT-COUNTERPOINT COMMENTS
Medical College of Wisconsin
The following letters are in response to the Point:Counterpoint series "The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal" that appears in this issue.
To the Editor: Unique data from three of our past studies on awake goats are relevant to the point:counterpoint cited below (3). First, when we cooled the rostral ventrolateral medullary (RVLM) surface of awake goats (n = 6) with chronically implanted thermodes, diaphragm phasic activity was sustained but abdominal expiratory muscle activity was eliminated during eucapnia, hypercapnia, and hypoxia (2). Thus a mechanism near the RVLM surface is critical for activation of expiratory muscles, but inspiratory muscle activity is not dependent on this mechanism. Second, when we injected a glutamate receptor agonist in awake goats (n = 2) through microtubules chronically implanted near the RVLM surface at the caudal border of the facial nucleus, there was marked activation of airway constrictor muscles during expiration and a highly fractionated diaphragm activity and inspiratory airflow (1). Thus it appears an expiratory muscle mechanism was activated that interfered with inspiratory muscle activity. Third, when we bilaterally substantially destroyed the preBotzC with injections of a neurotoxin in the awake state (n = 7), there was no phasic diaphragm activity but there was phasic abdominal muscle activity that generated a low level of passive inspiratory airflow (4). Thus expiratory muscle phasic activity can occur independent of inspiratory muscle phasic activity. These three studies support the concept that during physiological conditions in adult mammals, respiratory rhythm and pattern are generated by dual medullary oscillators, but the preBotzC is the dominant determinant of overall respiratory muscle activity.
REFERENCES
University of Wisconsin School of Medicine and Public Health
To the Editor: There is substantial evidence that both regions play important roles in the regulation of rhythm (1). However there are at present more persuasive findings that, for the sake of this debate, point to the preBötC as the primary pacemaker. In vitro experiments have shown that over 80% of the NK-1 receptor-bearing preBötC neurons are rhythmically active in inspiration, 25% of which are autorhythmic (5). Ataxic breathing and diaphragmatic EMG disturbances persist in conscious adult rats after NK-1 receptor-bearing preBötC neurons are eliminated, as noted by Feldman and Janczewski. By contrast, elimination of NK-1 receptor neurons in the pFRG depresses tidal volume and CO2 responsiveness without disrupting rhythm (3). So, how do the autorhythmic neurons in the pFRG region function? Feldman and Janczewski propose that the pFRG is an expiratory rhythm generator. Apparently, >40% NK-1R cell death might be necessary to disrupt this function (3, 4). They also propose that the pFRG maintains breathing at birth, when an opiate surge depresses the preBötC. Does rhythmogenesis emanate exclusively from the preBötC and pFRG? It doesn't seem so. Monnier et al.'s (2) precise, functional mapping of the ventrolateral respiratory column with DLH microinjections indicates that rhythm generation extends beyond the preBötC. Furthermore, µ-opiate application in the dorsolateral Pons produces rhythm slowing like that seen after iv administration in several species (1). As functional imaging and other technologies improve, the issue of where the critical sites for respiratory rhythm generation are in all mammals, including humans, will be hopefully resolved.
REFERENCES
University of Alberta
To the Editor: The structural formation of the preBötC in the rat fetus coincides with the inception of inspiratory rhythmogenesis in vitro (brain stem- spinal cord and medullary slice preparations) and fetal breathing movements in utero (1). Anatomical, electrophysiological, and calcium- imaging data demonstrate the same correlation in fetal mouse in vitro preparations (5). In contrast, rhythmic respiratory activity within the pFRG commences later in gestation (2). This would suggest that, according to the schema proposed by Onimaru/Homma (3), the system undergoes a fundamental reorganization prior to birth with the pFRG essentially overriding the preBötC. Alternatively, the preBötC continues its primary inspiratory rhythmogenic role and the emergence of the pFRG coincides with the onset of phasic expiratory activity, as suggested by the counterpoint perspective (3). Examining perinatal brain stem-spinal cord preparations for the relative ontogeny of inspiratory/expiratory motor discharge should provide data toward testing the latter hypothesis.
Developmental studies of mutant mouse models with respiratory dysfunction are also relevant to the debate. For example, a necdin-deficient mouse model of Prader-Willi Syndrome has a grossly abnormal breathing pattern and dies shortly after birth (4). The irregular inspiratory rhythm, featuring prolonged apneas, is present in the brain stem-spinal cord preparation. Importantly, direct recordings from medullary slice preparations (i.e., without the pFRG) clearly demonstrate that the rhythmogenic dysfunction occurs within the preBötC per se and thus provides a mechanistic basis for the pathological breathing observed in vivo that is consistent with the Feldman/Janczewski proposal. The future development of mutant models with specific pFRG deletions should provide further insights into the controversy.
REFERENCES
Dartmouth Medical School
To the Editor 1) The RTN/pFRG region is not functionally homogeneous. It has many types of neurons, e.g., pre-I, E, and chemosensitive (1–3, 5); many of the pre-I neurons appear to be located more laterally (4), a site that is difficult to inhibit or lesion in vivo in conscious rats. 2) Lesion or nonopioid inhibition of the RTN in anesthetized animals causes apnea and a severely reduced CO2 response (1, 2). The RTN drives breathing in anesthesia; the PBC does not "take over" after RTN lesion/inhibition. 3) In the conscious rat, lesions/inhibition of the RTN reduces CO2 sensitivity and ventilation without any change in respiratory rhythm (1, 2, 5). Lesions of the PBC also reduce CO2 sensitivity and ventilation and produce a fragmented, ataxic respiration (1). The PBC provides a strong influence on rhythm; the RTN provides "drive." The RTN (and other sites) cannot restore normal rhythm when the PBC is lesioned but the PBC (and other sites) cannot restore normal ventilation and chemosensitivity when the RTN is lesioned. 4) We believe that the RTN is a primary site for chemoreception that also modulates inputs from the carotid body and in sum provides an important "drive" to breathe (2, 5). Part of this "drive" is mediated via the PBC; part directly influences the pattern generator "outside" of the PBC. Other sites participate in this chemosensitive "drive" (1, 2).
REFERENCES
The College of William and Mary
To the Editor: Onimaru and Homma provide a thorough summary of the rhythm-generating capabilities of Pre-I neurons and the RTN/pFRG region (5). But there are three issues that must be addressed, which pertain directly to their hypothesis that Pre-I neurons comprise the fundamental RRG, which periodically triggers inspiratory activity in the preBötC.
First, rhythmic discharge of neurons in the RTN/pFRG disappears in the absence of active expiration, such as during lung deflation (4) and in anesthetized in vivo preparations (3), where the degree of excitability is lowered. These observations suggest that the RTN/pFRG triggers rhythmic expiration and are inconsistent with a role for the RTN/pFRG in periodically triggering inspiratory activity, which persists in both cases.
Second, lesions that ablate preBötC neurons disrupt normal breathing in rats (2). According to Onimaru and Homma's hypothesis, ataxic breathing must result from indirect and/or secondary effects that impact rostral respiratory circuits, because the RTN/pFRG is unaffected by the saporin lesioning method. Although this is not inconceivable, a more parsimonious interpretation is that the inspiratory breathing pattern actually originates in the preBötC, which is directly targeted by the lesion.
Third, the authors assert that activity in the preBötC is "masked" in vitro and in vivo, where "masking" refers to silencing the rhythmogenic role of the preBötC, but there are no data to demonstrate whether or how this occurs in either condition.
Conversely, masking would apply to Hoxa1–/– and Krox-20–/– mice (1), which lack RTN/pFRG circuits and succumb to fatal apneas when preBötC activity is "masked" by endogenous opiates that silence preBötC neurons.
REFERENCES
Department of Anatomy and Neurobiology, Washington University School of Medicine
To the Editor: The preBötzinger Complex (preBötC) is necessary for normal breathing in intact, adult animals, but may play a secondary role in respiratory timing under specific in vivo and in vitro conditions (4). The ability to actually test whether para-facial respiratory group (pFRG) neurons are also necessary for normal breathing has been hampered by inconsistent and inaccurate anatomical analyses of the location and identity of these neurons (4).
The major characteristic of pFRG neurons is their peri-inspiratory activity (pre-I firing.) Summary images of the location of these cells in papers has progressively moved their location from the rostral end of the pbc to recent proposals they lie primarily at the level of the facial motor nucleus (1, 4). These summary images, however, have been in contrast to the mapped location of individual pre-I neurons that can be found clearly extending into the preBötC (1, 3). Thus either a pre-I firing pattern is not an exclusive marker for pFRG neurons, or the anatomical boundaries of this region are much broader than has been proposed. Conversely, however, the published recordings and imaging of pre-I neurons are clearly distinguishable from any published data for the location of the retrotrapazoid nucleus (RTN) or regions of Krox20 expression (2, 5). Thus suggestions the pFRG, RTN, and rhombomere 5 may be equivalent populations are without anatomical support.
It was 10 years between the identification of the preBötC and the efforts by a number of labs addressing the anatomical organization of the preBötC that enabled testing its necessity in vivo. Similar application of strict anatomical analyses may provide us with the ability to test the necessary role of pFRG neurons in the near future.
REFERENCES
Department of Neuro and Sensory Physiology
Georg August University of Goettingen
To the Editor: Respiratory rhythm is generated by a complex neuronal network whose behavior and operating rhythmogenic mechanism are state dependent (2, 3, 5). The state of the respiratory network (and hence the rhythmogenic mechanism expressed) depends on many factors, including metabolic conditions (e.g., hypoxia may suppress inhibition) and influences of other regions (e.g., pons whose absence in reduced preparations implies its postulated unimportance for rhythm generation). Therefore, the comparison of rhythmogenic mechanisms suggested from different preparations requires a cautious analysis of conditions and the caveat that pharmacological manipulations or removal of "unimportant" regions may change the state of the network and release rhythmogenic mechanisms not operating in an undisturbed, intact network in vivo. The metabolic conditions (e.g., oxygenation) in the isolated brain stem-spinal cord preparation used by Onimaru and Homma are very different from those in vivo. Hence the role of rhythmic activity in parafacial/retro-trapezoidal nucleus (pF/RTN) may be specific for this preparation and its metabolic conditions. In turn, pharmacological manipulations (e.g., fentanyl) or pons removal may suppress the postinspiratory activity, providing phasic inhibition within the respiratory network and release a specific rhythmic activity in pF/RTN leading to a two-generator oscillations in the network (1) that are normally suppressed. Thus the real challenge is not identifying which of the mechanisms suggested from different preparations is more realistic, but identifying the role of different network compartments in different states and under different conditions, i.e., the state dependency of the respiratory rhythm generation.
REFERENCES
University of Wisconsin
To the Editor: The Point:Counterpoint article debating the site(s) of respiratory rhythmogenesis in mammals (4) raises an important question: what are the evolutionary origins of respiratory rhythm-generating neuronal structures? The coupled oscillator hypothesis postulates that the pFRG generates expiratory rhythm whereas the pre-BötC generates inspiratory rhythm (4). A key evolutionary link for this hypothesis is thought to arise from the conservation of dual gill/buccal and lung oscillators in frogs that respond similarly to opiates as the pFRG and preBötC. Although intriguing, similar responses do not necessarily imply structural or functional homology. The evolution of neural circuits that subserve a specific behavior (e.g., breathing) is difficult to determine, even in relatively simple nervous systems (2). Therefore, an accurate understanding of non-mammalian breathing is critical and may provide new insights into pFRG and preBötC function. With this in mind, certain features of breathing in amphibians and reptiles need to be clarified. For example, expiration and inspiration are active motor processes in some amphibians (2) and reptiles (1); inspiration is not passive as postulated (4). Also, some neurotransmitters do not alter amphibian gill/buccal and lung oscillator function in the same way as they affect pFRG and preBötC oscillator function (2), which is inconsistent with proposed structural and functional homologies (4). Although our understanding of respiratory neurobiology in ectothermic vertebrates lags far behind compared with mammals, carefully targeted studies incorporating a phylogenetic analysis can provide valuable information that leads us closer to a proper evolutionary perspective on the neural control of breathing (5).
REFERENCES
Departments of Physiology and Pediatrics
University of Alberta
To the Editor: Analysis of the respiratory-related behavior of in vitro rodent preparations has been and remains central in the evolution of current hypotheses that respiratory rhythm derives from the interaction of at least two distinct, coupled oscillators. The proposal by Onimaru/Homma (4) that the pFRG is a pre-I generator that drives inspiration is best supported by the demonstration using voltage-sensitive dyes in vitro that RTN/pFRG activity precedes PBC discharge. Although an impressive technical accomplishment that focused attention on the RTN/pFRG as a second oscillator, neither these data, the presence of pre-I pacemaker neurons in the RTN/pFRG, nor the demonstration that RTN/pFRG slices are rhythmogenic (5) establish the RTN/pFRG as the primary oscillator. In vitro data are equally consistent with the schema of Feldman/Janczewski (4) that the PBC is the primary inspiratory oscillator (and RTN/pFRG is an expiratory oscillator). Unequivocal definition of phase/function requires approaches where unambiguous indexes of phase are available. In this context, two observations in vivo support the PBC as the primary oscillator; lesioning of PBC neurons irreversibly disrupts inspiration, not expiration (2, 4); transection between the RTN/pFRG and PBC abolishes expiration, not inspiration (3, 4). What remains is to localize the site responsible for the transection effects; does complete, selective lesioning of the RTN/pFRG abolish expiration? It may also be instructive to explore across multiple states in vivo (or in situ) the relationship between RTN/pFRG activity and active expiration; a prediction of the Feldman/Janczewski schema is that the RTN/pFRG, the generator of active expiration, may be silent during quiet breathing (1).
REFERENCES
University of Calgary
To the Editor: The controversy over whether the preBötC or the pFRG is the primary site or respiratory generation in part stems from the delineation of what determines eupnea—specifically, is inspiration a sufficient marker for respiration? Although it appears that the preBötC is necessary and sufficient to generate an inspiratory rhythm (2), it may be that both sites are necessary to generate eupnea.
In the frog, gill/buccal and lung oscillators, propositionally homologous to the pFRG and preBötC (3), both contribute to the eupneic rhythm. The lung oscillator alone, like the preBötC, appears to be necessary and sufficient to generate inspiratory bursts (4). However, the gill/buccal oscillator appears to generate critical neural activity necessary for the inflation of the lung (3). In the frog, the eupneic respiratory burst is comprised of outputs from both oscillators—although inspiration can be identified from the output of the single inspiratory oscillator, a normal breath requires the output of both oscillators.
Janczewski and Feldman proposed distinct oscillators for inspiration and expiration in the rat, with their expiratory oscillator corresponding to Onimaru and Homma's pFRG (1). However, given that these two respiratory phases are intimately related and required for normal respiration and that each phase follows the next, it is somewhat inappropriate to describe either phase as the lead phase, or either oscillator as the primary generator of respiratory rhythm. Rather, an approach synthesizing the combined output of both oscillators may be most appropriate in describing the respiratory rhythm generator.
REFERENCES
University of Calgary, HBI
To the Editor: Two different models of the respiratory rhythm generator are presented (3). Onimaru et al. propose a single rhythm generator: a parafacial oscillator (pFRG) that transmits rhythm to inspiratory (preBötzinger complex; PBC) and expiratory pattern generating elements elsewhere in the network. Similar to our earlier demonstration of two spatially distinct but coupled oscillators in juvenile frogs (5), Feldman and Janczewski proposed the mammalian rhythm generator comprises two coupled oscillators, one inspiratory (PBC), the other expiratory (pFRG).
Both mammalian models assume a parafacial rhythm generator, but only the latter dictates an essential rhythmogenic role for the PBC in eupnea. Whether the PBC has an essential rhythmogenic role in eupnea, and/or serves as a bottleneck, relaying inspiratory activity generated elsewhere, remains controversial (3, 4).
Even the most elegant ablation experiments cannot distinguish between these possibilities. Indeed, reliance on "necessity tests" to identify>vital circuit components in vertebrates may lead to misguided notions of network architecture. For example, necessity tests are unlikely to work well in distributed networks endowed with redundancy (1, 2); in a distributed system, ablations may have no functional effect. The only effects will be seen at bottlenecks.
We should instead seek areas 1) whose activity is correlated with breathing and 2) that demonstrate an ability to increase breathing frequency when stimulated (sufficient test). Such areas are likely part of a distributed rhythm-generating network and will include most of the VRG, NTS, and pons.
If the PBC rules, it likely rules within a parliament of many.
REFERENCES
University of Alberta
To the Editor: The preBötC and pFRG were originally identified using brain stem-spinal cord preparations from newborn rats, a model in which mutual synaptic interaction between these respiratory centres provides a multiphase rhythm (1).
Both groups contributing to a Point:Counterpoint article on the site of respiratory rhythm generation propose that the pFRG may drive respiratory rhythm in this en bloc preparation (2). However, pFRG removal by sectioning does not affect the rhythm (5). Furthermore, preBötC-containing slices generate inspiratory rhythm with no pFRG-like activity in superfusates with physiological (3 mM) (5) or modestly elevated (5–7 mM) [K+] (4). In contrast, Onimaru et al. (3) showed that transection between the pFRG and preBötC reveals facial rhythm in a pFRG-containing slice whereas cervical rhythm in the remaining preBötC-containing brain stem-spinal cord is depressed under their standard conditions, e.g., 6.2 mM [K+]. Although sectioning levels were determined in the latter study, there is yet no histological information on rostrocaudal boundaries of preBötC slices critical for rhythm generation. Such analysis is pivotal for studies devoted to analyze functions of the isolated preBötC because the pFRG may extend caudally close to, or even to some extent into, the preBötC (1, 2).
Differences in anesthesia or tissue isolation techniques may also contribute to the conflicting in vitro results. Also, findings can only be compared directly between studies using rather similar composition and administration procedures of superfusate. In this regard, efforts should be made to mimic in vivo conditions as closely as possible, at least by using an appropriate composition of the bulk solution.
REFERENCES
Kosair Children's Hospital Research Institute
University of Louisville
To the Editor: The existence of multiple rhythmogenic sites suggests that "the quest to locate the mammalian RRG" may have been a search for an unbiological abstraction. As a motor pattern, breathing is simple: there are no joints, no crossed inhibition, and, to a first approximation, one muscle. By contrast, as a homeostatic behavior, breathing is extraordinarily complex: it must adapt itself to other ongoing behaviors (olfaction, thermoregulation, locomotion, ...) and must continuously adjust amplitude and frequency to maintain blood gas homeostasis (3, 5). Adaptive homeostatic regulation eludes simple solution and predicts the emergence of a regulatory superstructure within which "minimal circuits" may be embedded, but are of limited behavioral relevance (1). Thus in apparently asymptomatic rats lesioned with SP-saporin, mild hyperoxia, hypoxia, or anesthesia are lethal, even while eupneic breathing persists (2). In this context, the emerging picture that respiratory rhythm arises out of the interaction of multiple, structurally distinct elements, each capable of producing qualitatively the same behavior, may be telling us something important about the system that is lost if we try to force these elements into a hierarchy. In the absence of hierarchy, the behavior is spared even if one mechanism fails. More generally, distinct mechanisms for rhythmogenesis imply different dynamics in response to perturbations, thereby simplifying the problem of meeting the conflicting demands of adaptiveness and robustness (4).
REFERENCES
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  R. L. Horner and T. D. Bradley Update in Sleep and Control of Ventilation 2006 Am. J. Respir. Crit. Care Med., March 1, 2007; 175(5): 426 - 431. [Full Text] [PDF]
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 J. L. Feldman and W. A. Janczewski The Last Word: Point:Counterpoint authors respond to commentaries on "The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal" J Appl Physiol, August 1, 2006; 101(2): 689 - 689. [Full Text] [PDF]
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