Richard J. A. Wilson

Evidence that ventilatory rhythmogenesis in the frog involves two distinct neuronal oscillators

In Rana catesbeiana the upper airways are used for two distinct yet highly coordinated ventilatory behaviours: buccal ventilation and lung inflation cycles. How these behaviours are generated and coordinated is unknown. The purpose of this study was to identify putative rhythmogenic brainstem loci involved in these ventilatory behaviours. We surveyed the isolated postmetamorphic brainstem to determine sites where local depolarization, produced by microinjecting the non‐NMDA glutamate receptor agonist, AMPA, augmented the ventilatory motor patterns. Two sites were identified: a caudal site, at the level of cranial nerve (CN) X, where AMPA injections caused increased buccal burst frequency but abolished lung bursts, and a rostral site, between the levels of CN VIII and IX, where injections increased the frequency of both types of ventilatory bursts. These two sites were further examined using GABA microinjections to locally inhibit cells. GABA injected into the caudal site suppressed the buccal rhythm but the lung rhythm continued, albeit at a different frequency. When GABA was injected into the rostral site the lung bursts were abolished but the buccal rhythm continued. When the two sites were physically separated by transection, both rostral and caudal brainstem sections were capable of rhythmogenesis. The results suggest the respiratory network within the amphibian brainstem is composed of at least two distinct but interacting oscillators, the buccal and lung oscillators. These putative oscillators may provide a promising experimental model for studying coupled oscillators in vertebrates.

Sudden neonatal death in PACAP‐deficient mice is associated with reduced respiratory chemoresponse and susceptibility to apnoea

Pituitary adenylate cyclase‐activating polypeptide (PACAP)‐deficient mice are more prone to sudden death during postnatal weeks 1–3 than wild‐type littermates. Given that PACAP is localized in brainstem regions associated with respiratory chemosensitivity, we examined whether PACAP‐null neonates have reduced respiratory responses to hypoxia and hypercapnia. Using unrestrained, whole‐body, flow‐through plethysmography we found that, by postnatal day 4, the PACAP‐null neonates had significantly reduced ventilation during baseline breathing, and blunted responses to both hypoxia (10% O2–90% N2) and hypercapnia (8% CO2–92% air). To determine whether the respiratory phenotype of the PACAP‐null mice may contribute to their greater neonatal mortality, we used ECG to examine respiration and cardiovascular function of littermates. We demonstrate that, under conditions that exacerbate mortality of knockout but not wild‐type animals, PACAP‐deficient mice experience prolonged apnoeas that precede atrio‐ventricular block. Both apnoeas and atrio‐ventricular block were absent in wild‐type littermates. These data suggest that PACAP‐deficiency results in higher neonatal mortality primarily as a result of respiratory control defects and raise the possibility that mutations in genes encoding components of the PACAP signalling pathways may contribute to neonatal breathing disorders in humans.

Respiratory activity in neonatal rats

In neonatal animals in vitro preparations have been employed widely to study the central control of respiration. These preparations have limitations in that reflex afferent inputs and kinesiological studies cannot be performed. Here, we describe an alternative in situ experimental model for studying both peripheral and central control of the respiratory system in neonatal rats. Using technology based on adult mammals, we introduce an intra-arterially perfused working heart-brainstem preparation (WHBP) that permits studies on eupnoeic respiration in neonatal rats from within a few hours of birth. Using this preparation we demonstrate a three-phase respiratory rhythm as revealed by the activity in phrenic and recurrent laryngeal motor nerves, the respiratory modulation of laryngeal resistance and the firing patterns of respiratory neurones recorded from the ventrolateral medulla. We conclude that the neonatal rat WHBP is an in situ preparation because it produces a respiratory rhythm similar to that of adult in vivo mammal preparations but distinct from in vitro preparations.

A negative interaction between brainstem and peripheral respiratory chemoreceptors modulates peripheral chemoreflex magnitude

Interaction between central (brainstem) and peripheral (carotid body) respiratory chemosensitivity is vital to protect blood gases against potentially deleterious fluctuations, especially during sleep. Previously, using an in situ arterially perfused, vagotomized, decerebrate preparation in which brainstem and peripheral chemoreceptors are perfused separately (i.e. dual perfused preparation; DPP), we observed that the phrenic response to specific carotid body hypoxia was larger when the brainstem was held at 25 Torr P  CO 2 compared to 50 Torr P  CO 2. This suggests a negative (i.e. hypo‐additive) interaction between chemoreceptors. The current study was designed to (a) determine whether this observation could be generalized to all carotid body stimuli, and (b) exclude the possibility that the hypo‐additive response was the simple consequence of ventilatory saturation at high brainstem P  CO 2. Specifically, we tested how steady‐state brainstem P  CO 2 modulates peripheral chemoreflex magnitude in response to carotid body P  CO 2 and P  O 2 perturbations, both above and below eupnoeic levels. We found that the peripheral chemoreflex was more responsive the lower the brainstem P  CO 2 regardless of whether the peripheral chemoreceptors received stimuli which increased or decreased activation. These findings demonstrate a negative interaction between brainstem and peripheral chemosensitivity in the rat in the absence of ventilatory saturation. We suggest that a negative interaction in humans may contribute to increased controller gain associated with sleep‐related breathing disorders and propose that the assumption of simple addition between chemoreceptor inputs used in current models of the respiratory control system be reconsidered.

Developmental disinhibition: Turning off inhibition turns on breathing in vertebrates

Development requires age-dependent changes in essential behaviors. While the mechanisms determining the developmental expression of such behavior in vertebrates remain largely unknown, a few studies have identified permissive mechanisms in which the appearance of promoting signals activates pre-established networks. Here we report a different developmental process. Specifically, we show that the neuronal substrate that produces putative lung breathing in tadpoles is formed early in development, but remains more or less inactive until metamorphosis because of suppression mediated by a GABA(B) receptor-dependent mechanism. Blocking this suppression using 2-hydroxy-saclofen, a GABA(B) receptor antagonist, results in the precocious production of the putative lung breathing motor pattern. This blocker failed to augment putative lung breaths after metamorphosis. Thus, our results suggest that loss of an inhibitory signal during development (i.e., developmental disinhibition) is responsible for the developmental expression of air breathing.

Brainstem PCO2 modulates phrenic responses to specific carotid body hypoxia in an in situ dual perfused rat preparation

Inputs from central (brainstem) and peripheral (carotid body) respiratory chemoreceptors are coordinated to protect blood gases against potentially deleterious fluctuations. However, the mathematics of the steady‐state interaction between chemoreceptors has been difficult to ascertain. Further, how this interaction affects time‐dependent phenomena (in which chemoresponses depend upon previous experience) is largely unknown. To determine how central PCO2 modulates the response to peripheral chemostimulation in the rat, we utilized an in situ arterially perfused, vagotomized, decerebrate preparation, in which central and peripheral chemoreceptors were perfused separately (i.e. dual perfused preparation (DPP)). We carried out two sets of experiments: in Experiment 1, we alternated steady‐state brainstem PCO2 between 25 and 50 Torr in each preparation, and applied specific carotid body hypoxia (60 Torr PO2 and 40 Torr PCO2) under both conditions; in Experiment 2, we applied four 5 min bouts (separated by 5 min) of specific carotid body hypoxia (60 Torr PO2 and 40 Torr PCO2) while holding the brainstem at either 30 Torr or 50 Torr PCO2. We demonstrate that the level of brainstem PCO2 modulates (a) the magnitude of the phrenic responses to a single step of specific carotid body hypoxia and (b) the magnitude of time‐dependent phenomena. We report that the interaction between chemoreceptors is negative (i.e. hypo‐additive), whereby a lower brainstem PCO2 augments phrenic responses resulting from specific carotid body hypoxia. A negative interaction may underlie the pathophysiology of central sleep apnoea in populations that are chronically hypocapnic.

Phylogeny of vertebrate respiratory rhythm generators: the Oscillator Homology Hypothesis.

A revolution is underway in our understanding of respiratory rhythm generation in mammals. Until recently, a major focus of research within the field has centered around the question of locating and elucidating the mechanism of rhythmogenesis of a single respiratory neuronal oscillator which is reiterated bilaterally within the brainstem. Now it appears that each hemisection may contain at least two oscillators that interact to generate the respiratory rhythm in mammals. Comparative studies have hinted at the existence of multiple respiratory oscillators in non-mammalian vertebrates for some time, raising the possibility of homologous oscillators. Here, we consider available tools to identify neuronal oscillators and critically review the evidence for the importance and existence of multiple respiratory oscillators in vertebrates. First focusing on a comparison between frogs and mammals, we then evaluate the hypothesis that ventilatory oscillators in extant tetrapods evolved from ancestral oscillators present in fish (the Oscillator Homology Hypothesis). While supporting data are incomplete, the Oscillator Homology Hypothesis will likely serve as a useful framework to motivate further studies of respiratory rhythm generation in lower vertebrates.

Tissue PO2 and the effects of hypoxia on the generation of locomotor-like activity in the in vitro spinal cord of the neonatal mouse

The neonatal mouse en bloc spinal cord-brainstem preparation used in combination with advances in mouse genomics provides a novel strategy for studying the spinal control of locomotion. How well the mouse en bloc preparation is oxygenated however, is unknown. This is an important consideration given that (a) other superfused mammalian en bloc preparations have anoxic cores and (b) hypoxia can have profound effects on neuronal activity. Here we measure the level of tissue oxygenation in the mouse preparation and determine how neuronal activity within the spinal cord is influenced by poor superfusion and/or low oxygen. To measure tissue oxygenation, oxygen depth profiles were obtained (P0-1 and P2-3; Swiss Webster mice). At P0-1, spinal cords were oxygenated throughout under resting conditions. When fictive locomotor activity was evoked (5-HT 10 microM, dopamine 50 microM, NMA 5 microM), there was a substantial reduction in tissue PO(2) starting within 5 min of drug application. Following washout, the PO(2) slowly returned to control levels over a period of 30 min. The experiments described above were repeated using P2-3 preparations. In this older age group, the spinal cord preparations had a hypoxic/anoxic core that was exacerbated during metabolically demanding tasks such as drug-evoked rhythmic activity. To examine how an anoxic core affects neuronal activity within the spinal cord we either altered the flow-rate or manipulated superfusate PO(2). When the flow-rate was reduced a transient disruption in the rhythmicity of drug-induced locomotion occurred during the first 15 min (P0-1 preparations). However, the motor output adapted and stabilized. During prolonged superfusion with hypoxic artificial cerebrospinal fluid on the other hand, both the motor bursts in spinal nerves and the activity of most neurons near the center of the tissue were abolished.Overall, this study suggests that while oxygenation of P0-P1 preparations is adequate for studies of locomotor function, oxygenation of older preparations is more problematic. Our data also show that neonatal spinal neurons require oxygen to maintain activity; and the spinal locomotor rhythm generator continues to function providing the peripheral tissue of the cord is oxygenated. Together, these results are consistent with the results of a previous study which suggest that the locomotor pattern generator is located close to the surface of the spinal cord.

Rapid-Onset Obesity With Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation: Analysis of Hypothalamic and Autonomic Candidate Genes

Rapid-onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD) is a rare and complex pediatric disorder. Despite increased identification and advancing knowledge of the disease course, the variable onset and timing of phenotypic features in ROHHAD often result in delayed or missed diagnosis, potentially leading to fatal central hypoventilation, cardiorespiratory arrest, and impaired neurocognitive development. The 5-hydroxytryptamine receptor 1A (HTR1A), orthopedia (OTP), and pituitary adenylate cyclase activating polypeptide (PACAP) genes were targeted in the etiology of ROHHAD based on their roles in the embryologic development of the hypothalamus and autonomic nervous system. We hypothesized that variations of HTR1A, OTP, and/or PACAP would be associated with ROHHAD. All coding regions and intron-exon boundaries of the HTR1A, OTP, and PACAP genes, in addition to the promoter region of the HTR1A gene, were analyzed by standard sequencing in 25 ROHHAD cases and 25 matched controls. Thirteen variations, including six protein-changing mutations, were identified. None of these variations were significantly correlated with ROHHAD. This report provides evidence that variation of the HTR1A, OTP, and PACAP genes are not responsible for ROHHAD. These results represent a further step in the investigation of the genetic determinants of ROHHAD.

Respiratory Control in Neonatal Rats Exposed to Prenatal Cigarette Smoke

RATIONALE Prenatal cigarette smoke (CS) exposure, increased environmental temperature, and hypoxic episodes have been postulated as major risk factors for sudden infant death syndrome. OBJECTIVES To test the hypothesis that maternal CS exposure disrupts eupneic breathing and depresses breathing responses of neonatal rats to thermal and hypoxic challenges. METHODS Experiments were performed on 1-week-old rat pups exposed prenatally to CS (n = 39) or room air (sham; n = 30). Breathing patterns were recorded by whole-body plethysmography during thermoneutral or hyperthermic states under normoxic and hypoxic conditions. MEASUREMENTS AND MAIN RESULTS Mean pup weight, breaths per minute, and gasping respiratory patterns were measured for both smoke- and sham-exposed groups during thermoneutral and hyperthermic states under normoxic and hypoxic conditions. Under thermoneutral conditions, hypoxia caused gasping in CS-exposed animals but not in sham-exposed animals. Furthermore, under hyperthermic conditions, whereas hypoxia induced gasping in both groups, only CS-exposed animals exhibited a pronounced and longer lasting respiratory depression after the termination of hypoxia. CONCLUSIONS We show that prenatal CS exposure increases the likelihood of gasplike respiration and provide the first experimental evidence that the combined effects of prenatal CS exposure and hyperthermia dramatically prolong the time required for neonates to return to eupneic breathing after hypoxia. These observations provide important evidence of how prenatal CS exposure, hypoxic episodes, and hyperthermia might place infants at higher risk for sudden infant death syndrome.