Central Respiratory Rhythm Research Articles

Control of Breathing in Ectothermic Vertebrates.

The ectothermic vertebrates are a diverse group that includes the Fishes (Agnatha, Chondrichthyes, and Osteichthyes), and the stem Tetrapods (Amphibians and Reptiles). From an evolutionary perspective, it is within this group that we see the origin of air-breathing and the transition from the use of water to air as a respiratory medium. This is accompanied by a switch from gills to lungs as the major respiratory organ and from oxygen to carbon dioxide as the primary respiratory stimulant. This transition first required the evolution of bimodal breathing (gas exchange with both water and air), the differential regulation of O2 and CO2 at multiple sites, periodic or intermittent ventilation, and unsteady states with wide oscillations in arterial blood gases. It also required changes in respiratory pump muscles (from buccopharyngeal muscles innervated by cranial nerves to axial muscles innervated by spinal nerves). The question of the extent to which common mechanisms of respiratory control accompany this progression is an intriguing one. While the ventilatory control systems seen in all extant vertebrates have been derived from common ancestors, the trends seen in respiratory control in the living members of each vertebrate class reflect both shared-derived features (ancestral traits) as well as unique specializations. In this overview article, we provide a comprehensive survey of the diversity that is seen in the afferent inputs (chemo and mechanoreceptor), the central respiratory rhythm generators, and the efferent outputs (drive to the respiratory pumps and valves) in this group. © 2022 American Physiological Society. Compr Physiol 12: 1-120, 2022.

Evolution of vertebrate respiratory central rhythm generators

Tracing the evolution of the central rhythm generators associated with ventilation in vertebrates is hindered by a lack of information surrounding key transitions. To begin with, central rhythm generation has been studied in detail in only a few species from four vertebrate groups, lamprey, anuran amphibians, turtles, and mammals (primarily rodents). Secondly, there is a lack of information regarding the transition from water breathing fish to air breathing amniotes (reptiles, birds, and mammals). Specifically, the respiratory rhythm generators of fish appear to be single oscillators capable of generating both phases of the respiratory cycle (expansion and compression) and projecting to motoneurons in cranial nerves innervating bucco-pharyngeal muscles. In the amniotes we find oscillators capable of independently generating separate phases of the respiratory cycle (expiration and inspiration) and projecting to pre-motoneurons in the ventrolateral medulla that in turn project to spinal motoneurons innervating thoracic and abdominal muscles (reptiles, birds, and mammals). Studies of the one group of amphibians that lie at this transition (the anurans), raise intriguing possibilities but, for a variety of reasons that we explore, also raise unanswered questions. In this review we summarize what is known about the rhythm generating circuits associated with breathing that arise from the different rhombomeric segments in each of the different vertebrate classes. Assuming oscillating circuits form in every pair of rhombomeres in every vertebrate during development, we trace what appears to be the evolutionary fate of each and highlight the questions that remain to be answered to properly understand the evolutionary transitions in vertebrate central respiratory rhythm generation.

Brainstem activity, apnea, and death during seizures induced by intrahippocampal kainic acid in anaesthetized rats.

To investigate how prolonged seizure activity affects cardiorespiratory function and activity of pre-Bötzinger complex, leading to sudden death. Urethane-anesthetized female Long-Evans rats were implanted with nasal thermocouple; venous and arterial cannulae; and electrodes for electrocardiography (ECG) and hippocampal, cortical, and brainstem recording. Kainic acid injection into the ventral hippocampus induced status epilepticus. Seizures caused hypertension, tachycardia, and tachypnea punctuated by recurrent transient apneas. Salivation increased considerably: in 11 of 12 rats, liquid with alkaline pH consistent with saliva was expelled from the mouth. Most transient apneas were obstructive: nasal airflow ceased, while, in 83%, efforts to breathe persisted as continued rhythmic activity of respiratory pre-Bötzinger neurons, inspiratory electromyography (EMG), and excursions of the chest wall and abdomen. Blood pressure oscillated in time with respiratory efforts. This pattern also occurred in a minority of cases (16%) of incomplete apnea, but not in rare cases (1%) of transient central apneas. During transient obstructive apneas, the frequency of all inspiratory efforts decreased abruptly by ~30%, suggesting a resetting of the central respiratory rhythm generator. Twenty-two of thirty-one rats died, due either to obstructive apnea (12) or central apnea following progressive slowing of respiration (10). Most rats dying of central apnea had experienced several transient obstructive apneas. Negative DC field potential shifts of the brainstem followed the final breath, consistent with previous reports on spreading depolarization in mouse models. Timing suggests that the DC shift is a consequence rather than cause of respiratory collapse. Cardiac activity continued for tens of seconds. Seizure activity in forebrain induces pronounced autonomic activation and disrupts activity in medullary respiratory centers, resulting in death from either obstructive or central apnea. These results directly inform mechanisms of death in status epilepticus, and indirectly provide clues to mechanisms of sudden unexpected death in epilepsy (SUDEP).

Thoracic sympathetic chain stimulation modulates and entrains the respiratory pattern.

Stimulation of thoracic sympathetic chain (TSC) afferents has been shown to slow the respiratory rhythm in dogs, monkeys and humans. However, sparse information exists about the physiological role of TSC afferents in modulating respiration or the central pathways of these afferents. Here, we sought to investigate whether the perfused preparation of juvenile rats is a suitable experimental model to study the role of TSC-afferents in the modulation of respiration. We show that tonic (30s) TSC stimulation initially triggered either prolonged post-inspiratory vagal nerve discharge, or when the stimulus onset occurred in the second half of expiration, TSC stimulation also modulated late-expiratory abdominal nerve activity. Independent of the timing of the TSC-stimulation the net effect was lengthening of the expiratory interval and subtle shortening of inspiration. TSC evoked respiratory modulation showed progressive habituation during the stimulus period. Importantly, high thoracic spinal cord transections abolished the TSC-evoked respiratory modulation, indicating that TSC afferents are likely to be relayed within the thoracic spinal cord. Next, we repeatedly applied 400 ms trains of stimuli at an inter-burst interval near that of the intrinsic respiratory rate and show that rhythmic TSC stimulation has a strong potential to entrain the central respiratory rhythm. Importantly, under the imposed rhythm, TSC stimuli became aligned with the late expiratory phase. The entrainment pattern supports the hypothesis that the TSC pathway may convey extra-pulmonary visceral mechano-sensory feedback that might be sensitive to visceral mass movements during locomotion. The latter was previously discussed to significantly contribute to the locomotor-respiratory coupling in various mammalian species.

Chapter 22 - Clinical neurophysiology of apnea

Understanding the clinical neurophysiology of apnea generation encompasses discussion of the neuroanatomic aspects of central respiratory rhythm and pattern generation, including the central respiratory control networks, central and peripheral chemoreceptors, mechanisms of respiratory muscles, and sleep state dependent differences. Anatomical and functional links to apnea also involve central respiratory motor output recruited from the hypoglossal nerve, which has led to novel treatments for obstructive sleep apnea. Autonomic fluctuations occur in relation to sleep-wake and sleep states (i.e., REM vs NREM sleep), with both parasympathetic and sympathetic contributions. Finally, our understanding of the pathophysiology of obstructive sleep apnea now includes concepts of critical closing pressure of the upper airway, increased loop gain as reflected by high responsiveness to external perturbations, inadequate responsiveness of upper airway muscle recruitment, and reductions in arousal threshold leading to ventilatory instability. In turn, these concepts have led to the development of novel therapies such as hypoglossal nerve stimulation and targeting key culprit physiologic mechanisms specific to the individual.

The rostral medulla of bullfrog tadpoles contains critical lung rhythmogenic and chemosensitive regions across metamorphosis

The development of amphibian breathing provides insight into vertebrate respiratory control mechanisms. Neural oscillators in the rostral and caudal medulla drive ventilation in amphibians, and previous reports describe ventilatory oscillators and CO2 sensitive regions arise during different stages of amphibian metamorphosis. However, inconsistent findings have been enigmatic, and make comparisons to potential mammalian counterparts challenging. In the current study we assessed amphibian central CO2 responsiveness and respiratory rhythm generation during two different developmental stages. Whole-nerve recordings of respiratory burst activity in cranial and spinal nerves were made from intact or transected brainstems isolated from tadpoles during early or late stages of metamorphosis. Brainstems were transected at the level of the trigeminal nerve, removing rostral structures including the nucleus isthmi, midbrain, and locus coeruleus, or transected at the level of the glossopharyngeal nerve, removing the putative buccal oscillator and caudal medulla. Removal of caudal structures stimulated the frequency of lung ventilatory bursts and revealed a hypercapnic response in normally unresponsive preparations derived from early stage tadpoles. In preparations derived from late stage tadpoles, removal of rostral or caudal structures reduced lung burst frequency, while CO2 responsiveness was retained. Our results illustrate that structures within the rostral medulla are capable of sensing CO2 throughout metamorphic development. Similarly, the region controlling lung ventilation appears to be contained in the rostral medulla throughout metamorphosis. This work offers insight into the consistency of rhythmic respiratory and chemosensitive capacities during metamorphosis.

Respiratory Variability during Sleep in Methadone Maintenance Treatment Patients.

Methadone maintenance treatment (MMT) patients have a high prevalence of central sleep apnea and ataxic breathing related to damage to central respiratory rhythm control. However, the quantification of sleep apnea indices requires laborious manual scoring, and ataxic breathing pattern is subjectively judged by visual pattern recognition. This study proposes a semi-automated technique to characterize respiratory variability in MMT patients. Polysomnography, blood, and functional outcomes of sleep questionnaire (FOSQ) from 50 MMT patients and 20 healthy subjects with matched age, sex, and body mass index, were analyzed. Inter-breath intervals (IBI) were extracted from the nasal cannula pressure signal. Variability of IBI over 100 breaths was quantified by standard deviation (SD), coefficient of variation (CV), and scaling exponent (α) from detrended fluctuation analysis. The relationships between these variability measures and blood methadone concentration, central sleep apnea index (CAI), apnea-hypopnea index (AHI), and clinical outcome (FOSQ), were then examined. MMT patients had significantly higher SD and CV during all sleep stages. During NREM sleep, SD and CV were correlated with blood methadone concentration (Spearman R = 0.52 and 0.56, respectively; p < 0.01). SD and CV were also correlated with CAI (R = 0.63 and 0.71, p < 0.001, respectively), and AHI (R = 0.45 and 0.58, p < 0.01, respectively). Only α showed significant correlation with FOSQ (R = -0.33, p < 0.05). MMT patients have a higher respiratory variability during sleep than healthy controls. Semi-automated variability measures are related to apnea indices obtained by manual scoring and may provide a new approach to quantify opioid-related sleep-disordered breathing.

Disturbances in central respiratory rhythm generation may contribute to breathing disturbances in prematurely born mice

Nearly one million premature infants die annually, due to respiratory distress. While immaturity of the lungs is a major contributor to morbidity and mortality, much less is known about the potential contribution of disturbances in central neuronal networks. Here we tested the hypothesis that central neuronal mechanisms contribute to neonatal apneas and breathing disturbances occurring within the first few hours of life. We used an animal model of prematurity, in which we administered lipopolysaccharide (LPS) into pregnant dams eliciting a maternal inflammatory response that resulted in mouse pups being born prematurely. This experimental model parallels the human condition because a large fraction of human premature births are associated with Chorioamnionitis and other forms of maternal infections. Administration of 5 micrograms of LPS to C57BL/6 mice at G17.5 resulted in delivery of pups at G18.5, about half of which were viable for at least 2 h post delivery. In a flow, whole‐body plethysmograph, premature mice from LPS‐treated dams showed abnormally large breathing patterns occurring at low frequency, which in some pups transitioned to more mature eupneic respiratory activity. Transverse, rhythmically active slices containing the pre‐Bötzinger complex, the putative respiratory rhythm generator, obtained within 30 minutes after birth from LPS pups, showed large amplitude bursts occurring at a slow and irregular frequency. Conversely term and C‐sectioned pups exhibited respiratory rhythmic activity with a smaller and more regular frequency and amplitude. Bath applied caffeine effectively stimulated and regularized burst activity. The in vitro preparations will enable us to compare the cellular mechanisms that underlie the abnormally large breathing patterns seen primarily in premature mouse pups, with the respiratory patterns generated in term and c‐sectioned pups. Understanding the central mechanisms that underlie disordered breathing in prematurity may lead to novel treatment strategies for neonatal apneas and breathing failures in human infants.Support or Funding InformationInvestigators Inter‐center seed funds

I11 Respiratory Function Decreases With Progression Of Huntington's Disease

<h3>Background</h3> There is some evidence of altered central respiratory rhythm and upper airway obstruction in people with Huntington’s disease (HD) (Antczak <i>et al</i>. 2013) yet little known of the consequences of HD pathology on respiratory function. <h3>Aims</h3> In order to inform our knowledge of respiratory problems in people with HD, this study assessed respiratory function across the progression of the disease and compared it to healthy control participants. <h3>Methods</h3> Data from 67 people with HD (20 pre-manifest) and 39 healthy control participants were evaluated for differences. Respiratory function was assessed as lung volume, peak cough flow, and respiratory muscle strength. Data were analysed using Kruskall-Wallis and Mann-Whitney-U for post hoc comparisons; correlations were used to explore respiratory function and disease progression. Flow volume curves were analysed descriptively. <h3>Results</h3> Respiratory function was significantly decreased in people with manifest HD compared to healthy control participants (inspiratory muscle strength mean difference 57.4 cm H₂O, CI 45.97, 68.84) and people with pre-manifest HD (inspiratory muscle strength mean difference 61.4 cm H₂O, CI 48.23, 74.58), with no differences between healthy control participants and people with pre-manifest HD. Respiratory function declined linearly with disease progression measured by total functional capacity scale (p &lt; 0.001), in particular, peak cough flow was abnormal at the middle stage of the disease. Flow volume curves indicated upper airway resistance to flow. <h3>Conclusion</h3> This study confirms that respiratory function is different in HD compared with healthy controls and that respiratory function decreases linearly with disease progression. These findings have implications for monitoring and managing respiratory problems throughout HD disease progression with decreased lung volume and peak cough flow increasing the likelihood of pneumonia. Reduced respiratory muscle strength may result in decreased exercise capacity whilst increased upper airways resistance may influence sleep apnoea. Further research is needed to explore the underlying cause of respiratory dysfunction in HD. Antczak J, <i>et al</i>. Huntington’s Disease and Sleep Related Breathing Disorders. <i>Hygeia</i>2013;48(4):pp. 449-455

Pathophysiology of human ventilatory control.

We review the substantial recent progress made in understanding the underlying mechanisms controlling breathing and the applicability of these findings to selected human diseases. Emphasis is placed on the sites of central respiratory rhythm and pattern generation as well as newly described functions of the carotid chemoreceptors, the integrative nature of the central chemoreceptors, and the interaction between peripheral and central chemoreception. Recent findings that support critical contributions from cortical central command and muscle afferent feedback to exercise hyperpnoea are also reviewed. These basic principles, and the evidence supporting chemoreceptor and ventilatory control system plasticity during and following constant and intermittent hypoxaemia and stagnant hypoxia, are applied to: 1) the pathogenesis, consequences and treatment of obstructive sleep apnoea; and 2) exercise hyperpnoea and its control and limitations with ageing, chronic obstructive pulmonary disease and congestive heart failure.

Cardiorespiratory coupling of sympathetic outflow in humans: a comparison of respiratory and cardiac modulation of sympathetic nerve activity to skin and muscle

What is the central question of this study?Muscle sympathetic nerve activity (MSNA) is well known to be modulated by the arterial baroreceptors and respiration, but what are the magnitudes of cardiac and respiratory modulation of skin sympathetic nerve activity (SSNA), which primarily subserves thermoregulation?What is the main finding and what is its importance?Using direct microelectrode recordings of MSNA and SSNA in awake humans, we show that the magnitude of respiratory modulation of SSNA is identical to that of MSNA, the primary difference between the two sources of sympathetic outflow being the greater cardiac modulation of MSNA. This emphasises the role of the baroreceptors in entraining sympathetic outflow to muscle. It is well known that microelectrode recordings of skin sympathetic nerve activity (SSNA) in awake human subjects reveal spontaneous bursts of activity with no overt modulation by changes in blood pressure or respiration, in contrast to the clear cardiac and respiratory modulation of muscle sympathetic nerve activity (MSNA). However, cross-correlation analysis has revealed that, like individual muscle vasoconstrictor neurones, the firing of individual cutaneous vasoconstrictor neurones is temporally coupled to both the cardiac and respiratory rhythms during cold-induced cutaneous vasoconstriction, and the same is true of single sudomotor neurones during heat-induced sweating. Here, we used cross-correlation analysis to determine whether SSNA exhibits cardiac and respiratory modulation in thermoneutral conditions and to compare respiratory and cardiac modulation of SSNA with that of MSNA. Oligounitary recordings of spontaneous SSNA (n = 20) and MSNA (n = 18) were obtained during quiet, unrestrained breathing. Respiration was recorded by a strain-gauge transducer around the chest and ECG recorded by surface electrodes. Respiratory and cardiac modulation of SSNA and MSNA were quantified by fitting polynomial equations to the cross-correlation histograms constructed between the sympathetic spikes and respiration or ECG. The amplitude of the respiratory modulation (52.5 ± 3.4%) of SSNA was not significantly different from the amplitude of the cardiac modulation (46.6 ± 3.2%). Both were comparable to the respiratory modulation of MSNA (47.7 ± 4.2%), while cardiac modulation of MSNA was significantly higher (89.8 ± 1.5%). We conclude that SSNA and MSNA share similar levels of respiratory modulation, the primary difference between the two sources of sympathetic outflow being the marked cardiac modulation of MSNA provided by the baroreceptors.

Irregular Breathing in Mice following Genetic Ablation of V2a Neurons

Neural networks called central pattern generators (CPGs) generate repetitive motor behaviors such as locomotion and breathing. Glutamatergic neurons are required for the generation and inhibitory neurons for the patterning of the motor activity associated with repetitive motor behaviors. In the mouse, glutamatergic V2a neurons coordinate the activity of left and right leg CPGs in the spinal cord enabling mice to generate an alternating gait. Here, we investigate the role of V2a neurons in the neural control of breathing, an essential repetitive motor behavior. We find that, following the ablation of V2a neurons, newborn mice breathe at a lower frequency. Recordings of respiratory activity in brainstem-spinal cord and respiratory slice preparations demonstrate that mice lacking V2a neurons are deficient in central respiratory rhythm generation. The absence of V2a neurons in the respiratory slice preparation can be compensated for by bath application of neurochemicals known to accelerate the breathing rhythm. In this slice preparation, V2a neurons exhibit a tonic firing pattern. The existence of direct connections between V2a neurons in the medial reticular formation and neurons of the pre-Bötzinger complex indicates that V2a neurons play a direct role in the function of the respiratory CPG in newborn mice. Thus, neurons of the embryonic V2a lineage appear to have been recruited to neural networks that control breathing and locomotion, two prominent CPG-driven, repetitive motor behaviors.

Metabotropic glutamate receptors (mGluR5) activate transient receptor potential canonical channels to improve the regularity of the respiratory rhythm generated by the pre‐Bötzinger complex in mice

Metabotropic glutamate receptors (mGluRs) are hypothesized to play a key role in generating the central respiratory rhythm and other rhythmic activities driven by central pattern generators (e.g. locomotion). However, the functional role of mGluRs in rhythmic respiratory activity and many motor patterns is very poorly understood. Here, we used mouse respiratory brain-slice preparations containing the pre-Bötzinger complex (pre-BötC) to identify the role of group I mGluRs (mGluR1 and mGluR5) in respiratory rhythm generation. We found that activation of mGluR1/5 is not required for the pre-BötC to generate a respiratory rhythm. However, our data suggest that mGluR1 and mGluR5 differentially modulate the respiratory rhythm. Blocking endogenous mGluR5 activity with 2-Methyl-6-(phenylethynyl)pyridine (MPEP) decreases the inspiratory burst duration, burst area and frequency, whereas it increases the irregularity of the fictive eupneic inspiratory rhythm generated by the pre-BötC. In contrast, blocking mGluR1 reduces the frequency. Moreover, the mGluR1/5 agonist 3,5-dihydroxyphenylglycine increases the frequency and decreases the irregularity of the respiratory rhythm. Based on previous studies, we hypothesized that mGluR signaling decreases the irregularity of the respiratory rhythm by activating transient receptor potential canonical (TRPC) channels, which carry a non-specific cation current (ICAN). Indeed, 3,5-dihydroxyphenylglycine (DHPG) application reduces cycle-by-cycle variability and subsequent application of the TRPC channel blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365) hydrochloride reverses this effect. Our data suggest that mGluR5 activation of ICAN-carrying TRPC channels plays an important role in governing the cycle-by-cycle variability of the respiratory rhythm. These data suggest that modulation of TRPC channels may correct irregular respiratory rhythms in some central neuronal diseases.

Prenatal nicotine exposure alters the response of the mouse in vitro respiratory rhythm to hypoxia

Prenatal nicotine exposure is associated with deficiencies in the ability to respond to life threatening stressors such as hypoxia. Although many of these deficiencies appear to originate from defects in the brainstem respiratory network, the specific effects of prenatal nicotine exposure on the brainstem respiratory network are not well understood. We have tested the effects of prenatal nicotine exposure on the respiratory rhythm using an in vitro mouse brainstem slice preparation containing the pre-Bötzinger Complex, a region of the ventral respiratory group that is the postulated site of inspiratory rhythm generation. We found that nicotine exposure during pre- and early postnatal development led to a lower frequency of baseline fictive respiratory discharges from rhythmic slices and a reduction in the ability of the slice to maintain a respiratory rhythm during exposure to severe hypoxia compared to controls. These impairments of the central respiratory rhythm could potentially affect the ability to survive a period of exposure to severe hypoxia in vivo.

Identification of Minimal Neuronal Networks Involved in Flexor-Extensor Alternation in the Mammalian Spinal Cord

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated invitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.

Teashirt 3 Regulates Development of Neurons Involved in Both Respiratory Rhythm and Airflow Control

Neonatal breathing in mammals involves multiple neuronal circuits, but its genetic basis remains unclear. Mice deficient for the zinc finger protein Teashirt 3 (TSHZ3) fail to breathe and die at birth. Tshz3 is expressed in multiple areas of the brainstem involved in respiration, including the pre-Bötzinger complex (preBötC), the embryonic parafacial respiratory group (e-pF), and cranial motoneurons that control the upper airways. Tshz3 inactivation led to pronounced cell death of motoneurons in the nucleus ambiguus and induced strong alterations of rhythmogenesis in the e-pF oscillator. In contrast, the preBötC oscillator appeared to be unaffected. These deficits result in impaired upper airway function, abnormal central respiratory rhythm generation, and altered responses to pH changes. Thus, a single gene, Tshz3, controls the development of diverse components of the circuitry required for breathing.

Respiratory and Mayer wave-related discharge patterns of raphé and pontine neurons change with vagotomy

Previous models have attributed changes in respiratory modulation of pontine neurons after vagotomy to a loss of pulmonary stretch receptor "gating" of an efference copy of inspiratory drive. Recently, our group confirmed that pontine neurons change firing patterns and become more respiratory modulated after vagotomy, although average peak and mean firing rates of the sample did not increase (Dick et al., J Physiol 586: 4265-4282, 2008). Because raphé neurons are also elements of the brain stem respiratory network, we tested the hypotheses that after vagotomy raphé neurons have increased respiratory modulation and that alterations in their firing patterns are similar to those seen for pontine neurons during withheld lung inflation. Raphé and pontine neurons were recorded simultaneously before and after vagotomy in decerebrated cats. Before vagotomy, 14% of 95 raphé neurons had increased activity during single respiratory cycles prolonged by withholding lung inflation; 13% exhibited decreased activity. After vagotomy, the average index of respiratory modulation (eta(2)) increased (0.05 +/- 0.10 to 0.12 +/- 0.18 SD; Student's paired t-test, P < 0.01). Time series and frequency domain analyses identified pontine and raphé neuron firing rate modulations with a 0.1-Hz rhythm coherent with blood pressure Mayer waves. These "Mayer wave-related oscillations" (MWROs) were coupled with central respiratory drive and became synchronized with the central respiratory rhythm after vagotomy (7 of 10 animals). Cross-correlation analysis identified functional connectivity in 52 of 360 pairs of neurons with MWROs. Collectively, the results suggest that a distributed network participates in the generation of MWROs and in the coordination of respiratory and vasomotor rhythms.

Conditional expression of central respiratory rhythm in developing fat‐tailed dunnarts (Marsupialia: Sminthopsis crassicaudata)

Marsupials offer unparalleled opportunities for developmental studies as the young are born small and immature, with most development occurring in the pouch where the young are readily accessible. The fat‐tailed dunnart gives birth to one of the smallest newborns of any mammal (~4mm long, weighing 13‐15mg). Newborn dunnarts do not typically breathe until 3‐4 days postnatal (P), achieving gas exchange largely across the skin (Mortola et al., Nature, 1999). Currently, little is known about the development of respiratory rhythmogenesis in this species. We examined central respiratory rhythm in neonatal dunnarts from the day of birth up to 3 weeks of age (206‐261mg) using the in vitro brainstem‐spinal cord preparation. Under normocapnic conditions (5% CO2/95% O2, pH 7.4), fictive breathing was present at P0 and frequency increased with development until about P12. In P0 preparations, under normocapnia, fictive respiratory bursts occurred in episodes but became regular with increased drive (8% CO2, pH 7.2). In P5 and older preparations, under normocapnia, breathing was regular and fictive respiratory frequency correlated with the level of drive at all ages. These data indicate that dunnarts on the day of birth are capable of generating central respiratory rhythm even though expression of the rhythm does not occur for several days. (Supported by NSERC of Canada).

Riluzole disrupts autoresuscitation from hypothermic respiratory arrest in neonatal hamsters but not rats

We examined the effect of riluzole on expression of the central respiratory rhythm and the ability of neonates to autoresuscitate from hypothermic respiratory arrest using in vitro brainstem-spinal cord preparations of rats and hamsters. At a constant temperature of 27 degrees C, riluzole (5-200 microM) decreased the burst amplitude of respiratory-related motor discharge, but had little effect on the fictive respiratory frequency in rat preparations. In contrast, in hamster preparations, riluzole reduced fictive respiratory frequency, but had little effect on burst amplitude. Hamster preparations were more cold-tolerant than rat preparations, with respiratory arrest and autoresuscitation occurring at lower temperatures during cooling of the preparation. This difference was removed by incubation with riluzole (5 microM); riluzole significantly increased the temperature at which fictive respiration arrested and restarted in hamster preparations, but had no effect in rat preparations. The species differences observed in this study may reflect fundamental differences in the relative role of riluzole-sensitive mechanisms in the expression of the respiratory rhythm in early development of an altricial vs. a more precocial species.

Ponto‐medullary transection attenuates sympathorespiratory coupling and eliminates cardiac sinus arrhythmia in the in situ rat

Sympathetic nerve activity obtained from experimental animals demonstrates a pronounced modulation with the central respiratory rhythm. This modulation is mirrored in heart rate (respiratory sinus arrhythmia) and arterial pressure (Hering‐Traube waves) that are common to all mammals. The neural substrate responsible for sympatho‐respiratory coupling has not yet been defined. Based on recent evidence, we hypothesized that pontine neuron populations may be key components for this synchrony. Using the arterially perfused in situ preparation of rat, we recorded neural activities from the left phrenic nerve, central end of the vagus nerve and thoracic sympathetic chain (T8‐T10), as well as heart rate. Following a control period, the brainstem was transected transversely at the rostral edge of the facial nucleus, separating the pons and medulla. Respiration remained rhythmic but apneustic, with an increase of inspiratory duration. Both post‐inspiratory activity in the vagus nerve and the cardiac sinus arrhythmia were abolished while the synchronization of sympathetic outflow with the central respiratory signal was reduced substantially both in amplitude and duration. We surmise that descending drives from the pons are critical for the normal coupling of respiratory related modulations of sympathetic outflows destined for the heart and vasculature. Funded by the Leverhulme Trust (University of Bristol).