Facts and challenges in respiratory neurobiology.

Respiratory neurobiology has been a lead discipline in the field of neuroscience for almost a century. Despite this, research studies on the fundamental synaptic and cellular processes underlying the generation and modulation of breathing movements suffered a significant decline during the last decade. We still believe that respiratory neurobiology is one of the most exciting and imperative fields of neuroscience. With the first white paper concerned with the central control of breathing, we want to celebrate the global importance of breathing research.

Blockade of dorsolateral pontine 5HT1A receptors destabilizes the respiratory rhythm in C57BL6/J wild-type mice.

The neurotransmitter serotonin (5HT) acting via 5HT1a receptors (5HT1aR) is a potent determinant of respiratory rhythm variability. Here, we address the 5HT1aR-dependent control of respiratory rhythm variability in C57BL6/J mice. Using the in situ perfused preparation, we compared the effects of systemic versus focal blockade of 5HT1aRs. Blocking 5HT1aRs in the Kölliker-Fuse nucleus (KFn) increased the occurrence of spontaneous apneas and accounted for the systemic effects of 5HT1aR antagonists. Further, 5HT1aRs of the KFn stabilized the respiratory rhythm's response to arterial chemoreflex perturbations; reducing the recovering time, e.g., the latency to return to the baseline pattern. Together, these results suggest that the KFn regulates both intrinsic and sensory determinants of respiratory rhythm variability.

The effect of red blood cell transfusion on intermittent hypoxemia in ELBW infants.

OBJECTIVE: To test the hypothesis that the effect of red blood cell (RBC) transfusion on intermittent hypoxemia (IH) in extremely low birth weight (ELBW) infants is dependent on postnatal age. STUDY DESIGN: Oxygen saturation of 130 ELBW infants, who required transfusion, was monitored continuously for the first 8 weeks of life. We compared the characteristics of IH (SpO2⩽80% for ⩾4 s and ⩽3 min), 24 h before and both 24 h and 24 to 48 h after each RBC transfusion at three distinct time periods: Epoch 1, 1 to 7 days; Epoch 2, 8 to 28 days; and Epoch 3, >28 days. RESULT: In Epoch 1, the frequency and severity of IH events were not significantly different before and after transfusion. In both Epochs 2 and 3 there was a decrease in IH frequency and severity 24 h after RBC transfusion that persisted for 48 h. In addition, there was a decrease in the overall time spent with SpO2 ⩽80% which persisted for 24 h after transfusion in Epochs 1 and 3, and for 48 h in Epoch 3. CONCLUSION: The benefit of RBC transfusion on IH is age dependent as improvement in the frequency and severity of IH after transfusion only occurs beyond the first week of life. These observations will aid clinician's decision making by clarifying the benefit of RBC transfusions on patterns of oxygenation in preterm infants.

Vagal-dependent nonlinear variability in the respiratory pattern of anesthetized, spontaneously breathing rats.

Physiological rhythms, including respiration, exhibit endogenous variability associated with health, and deviations from this are associated with disease. Specific changes in the linear and nonlinear sources of breathing variability have not been investigated. In this study, we used information theory-based techniques, combined with surrogate data testing, to quantify and characterize the vagal-dependent nonlinear pattern variability in urethane-anesthetized, spontaneously breathing adult rats. Surrogate data sets preserved the amplitude distribution and linear correlations of the original data set, but nonlinear correlation structure in the data was removed. Differences in mutual information and sample entropy between original and surrogate data sets indicated the presence of deterministic nonlinear or stochastic non-Gaussian variability. With vagi intact (n = 11), the respiratory cycle exhibited significant nonlinear behavior in templates of points separated by time delays ranging from one sample to one cycle length. After vagotomy (n = 6), even though nonlinear variability was reduced significantly, nonlinear properties were still evident at various time delays. Nonlinear deterministic variability did not change further after subsequent bilateral microinjection of MK-801, an N-methyl-D-aspartate receptor antagonist, in the Kölliker-Fuse nuclei. Reversing the sequence (n = 5), blocking N-methyl-D-aspartate receptors bilaterally in the dorsolateral pons significantly decreased nonlinear variability in the respiratory pattern, even with the vagi intact, and subsequent vagotomy did not change nonlinear variability. Thus both vagal and dorsolateral pontine influences contribute to nonlinear respiratory pattern variability. Furthermore, breathing dynamics of the intact system are mutually dependent on vagal and pontine sources of nonlinear complexity. Understanding the structure and modulation of variability provides insight into disease effects on respiratory patterning.

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.

Reconfiguration of the pontomedullary respiratory network: a computational modeling study with coordinated in vivo experiments.

A large body of data suggests that the pontine respiratory group (PRG) is involved in respiratory phase-switching and the reconfiguration of the brain stem respiratory network. However, connectivity between the PRG and ventral respiratory column (VRC) in computational models has been largely ad hoc. We developed a network model with PRG-VRC connectivity inferred from coordinated in vivo experiments. Neurons were modeled in the "integrate-and-fire" style; some neurons had pacemaker properties derived from the model of Breen et al. We recapitulated earlier modeling results, including reproduction of activity profiles of different respiratory neurons and motor outputs, and their changes under different conditions (vagotomy, pontine lesions, etc.). The model also reproduced characteristic changes in neuronal and motor patterns observed in vivo during fictive cough and during hypoxia in non-rapid eye movement sleep. Our simulations suggested possible mechanisms for respiratory pattern reorganization during these behaviors. The model predicted that network- and pacemaker-generated rhythms could be co-expressed during the transition from gasping to eupnea, producing a combined "burst-ramp" pattern of phrenic discharges. To test this prediction, phrenic activity and multiple single neuron spike trains were monitored in vagotomized, decerebrate, immobilized, thoracotomized, and artificially ventilated cats during hypoxia and recovery. In most experiments, phrenic discharge patterns during recovery from hypoxia were similar to those predicted by the model. We conclude that under certain conditions, e.g., during recovery from severe brain hypoxia, components of a distributed network activity present during eupnea can be co-expressed with gasp patterns generated by a distinct, functionally "simplified" mechanism.

Modeling the ponto-medullary respiratory network.

The generation and shaping of the respiratory motor pattern are performed in the lower brainstem and involve neuronal interactions within the medulla and between the medulla and pons. A computational model of the ponto-medullary respiratory network has been developed by incorporating existing experimental data on the medullary neural circuits and possible interactions between the medulla and pons. The model reproduces a number of experimental findings concerning alterations of the respiratory pattern following various perturbations/stimulations applied to the pons and pulmonary afferents. The results of modeling support the concept that eupneic respiratory rhythm generation requires contribution of the pons whereas a gasping-like rhythm (and the rhythm observed in vitro) may be generated within the medulla and involve pacemaker-driven mechanisms localized within the medullary pre-Botzinger Complex. The model and experimental data described support the concept that during eupnea the respiration-related pontine structures control the medullary network mechanisms for respiratory phase transitions, suppress the intrinsic pacemaker-driven oscillations in the pre-BotC and provide inspiration-inhibitory and expiration-facilitatory reflexes which are independent of the pulmonary Hering-Breuer reflex but operate through the same medullary phase switching circuits.

Invited review: Neural network plasticity in respiratory control.

Respiratory network plasticity is a modification in respiratory control that persists longer than the stimuli that evoke it or that changes the behavior produced by the network. Different durations and patterns of hypoxia can induce different types of respiratory memories. Lateral pontine neurons are required for decreases in respiratory frequency that follow brief hypoxia. Changes in synchrony and firing rates of ventrolateral and midline medullary neurons may contribute to the long-term facilitation of breathing after brief intermittent hypoxia. Long-term changes in central respiratory motor control may occur after spinal cord injury, and the brain stem network implicated in the production of the respiratory rhythm could be reconfigured to produce the cough motor pattern. Preliminary analysis suggests that elements of brain stem respiratory neural networks respond differently to hypoxia and hypercapnia and interact with areas involved in cardiovascular control. Plasticity or alterations in these networks may contribute to the chronic upregulation of sympathetic nerve activity and hypertension in sleep apnea syndrome and may also be involved in sudden infant death syndrome.

Ventilatory behavior after hypoxia in C57BL/6J and A/J mice.

Given the environmental forcing by extremes in hypoxia-reoxygenation, there might be no genetic effect on posthypoxic short-term potentiation of ventilation. Minute ventilation (VE), respiratory frequency (f), tidal volume (VT), and the airway resistance during chemical loading were assessed in unanesthetized unrestrained C57BL/6J (B6) and A/J mice using whole body plethysmography. Static pressure-volume curves were also performed. In 12 males for each strain, after 5 min of 8% O2 exposure, B6 mice had a prominent decrease in VE on reoxygenation with either air (-11%) or 100% O2 (-20%), due to the decline of f. In contrast, A/J animals had no ventilatory undershoot or f decline. After 5 min of 3% CO2-10% O2 exposure, B6 exhibited significant decrease in VE (-28.4 vs. -38.7%, air vs. 100% O2) and f (-13.8 vs. -22.3%, air vs. 100% O2) during reoxygenation with both air and 100% O2; however, A/J mice showed significant increase in VE (+116%) and f (+62.2%) during air reoxygenation and significant increase in VE (+68.2%) during 100% O2 reoxygenation. There were no strain differences in dynamic airway resistance during gas challenges or in steady-state total respiratory compliance measured postmortem. Strain differences in ventilatory responses to reoxygenation indicate that genetic mechanisms strongly influence posthypoxic ventilatory behavior.

Modulation of diaphragm action potentials by K(+) channel blockers.

K(+) channels regulate diaphragm contractility. The present study examined the electrophysiological mechanisms accounting for diversity among K(+) channel blockers in their inotropic actions on the diaphragm. Rat diaphragmatic muscle fibers were recorded intracellularly in vitro at 37 degrees C. Apamin and charybdotoxin (Ca2+)-activated K(+) channel blockers) did not alter resting membrane potential or action potentials. Glibenclamide (ATP-sensitive K(+) channel blocker) slowed action potential repolarization by 12% (P<0.05) and increased action potential area by 25% (P<0.005). Tetraethylammonium (which blocks several types of K(+) channels) increased action potential overshoot by 20% (P<0.01) and prolonged action potential rise time by 17% (P<0.02). 4-Aminopyridine and 3,4-diaminopyridine (which also block several types of K(+) channels) slowed action potential repolarization by 163% (P<0.0001) and 253% (P<0.0001), and increased action potential area by 183% (P<0.0001) and 298% (P<0.0001), respectively. Slowing of repolarization for the aminopyridines was especially marked at voltages approaching resting membrane potential, thereby changing action potential repolarization from a first to a second order decay. Previously reported variability in inotropic effects among K(+) channel blockers correlated significantly with the extent to which they slowed action potential repolarization and increased action potential area, but not with changes in other action potential properties.

Heterogeneity within geniohyoid motor unit subpopulations in firing patterns during breathing.

Respiratory motor units (MU) segregate into subpopulations, which differ in firing patterns during resting and stimulated breathing. For phrenic/diaphragm MUs, diversity also exists within subpopulations, and is greater for late than early-onset MUs. The present study characterized the extent of diversity within upper airway respiratory MU subpopulations by recording geniohyoid MUs in anesthetized cats. Inspiratory MUs (I-MU, n=21) had a wide range of firing durations (coefficient of variation (CV)=42%). In contrast, inspiratory-expiratory MUs (I/E-MU, n=19) had a narrow range of firing durations during inspiration (CV=13%), but a wide range of firing durations during expiration (CV=36%). Mean firing frequency had similar degrees of diversity among units for I-MU and I/E-MU (CV=31-40%). For I-MU firing duration correlated with mean firing frequency, whereas no such relationship was apparent for I/E-MU. Single-breath end-expiratory airway occlusion decreased heterogeneity in firing duration during inspiration and increased it during expiration, whereas end-inspiratory airway occlusion decreased heterogeneity during expiration. In conclusion, (a) there is considerable diversity within geniohyoid MU subpopulations receiving respiratory drive; (b) the degree of diversity within subpopulations differs for I-MU and I/E-MU; and (c) diversity within subpopulations in timing of activity is modulated by single-breath airway occlusion.

Incorporating inheritance into models for understanding ventilatory behavior.

Ventilation and its components (frequency and tidal volume) appear to be determined to a significant extent by inheritance. Gene manipulation, gene identification, and functional genomics now offer powerful tools to identify the strength and mode of inheritance for ventilatory behavior under steady-state and non-steady-state conditions, in health and in disease. Conscious integration of genetic principles into existing explanatory models may increase the likelihood of detecting traits that correlate with protein systems responsible for the structures and the functional components of respiration.

Ventrolateral pons mediates short-term depression of respiratory frequency after brief hypoxia.

The respiratory response to hypoxia is dynamic in the adult anesthetized Sprague-Dawley rat. Hypoxia elicits acute increases in both tidal volume (VT) and respiratory frequency (fR) followed by short-term increases in VT and short-term decreases in fR. After brief hypoxia (<1 min), recovery of the breathing pattern is again dynamic, where both VT and fR decrease immediately, but where VT remains above, and fR drops below, baseline. These acute changes are followed by a short-term progressive decrease in VT and increase in fR to baseline. We have identified a potential neural mechanism that depends on the integrity of the ventrolateral (vl) pons. Our studies show that: (a) blockade of activity in the vl pons prevents the short-term decrease in fR after hypoxia (b) stimulation of the vl pons decreases fR, and (c) vl pontine expiratory neurons are activated after hypoxia. These neurons may not be acting through alpha(2) -adrenergic receptors, but their effect does depend on NMDA-type receptor function. We conclude that the vl pons is a critical element in the pontomedullary network that generates and modulates the fR response to acute hypoxia.

A role for NMDA receptors in posthypoxic frequency decline in the rat.

Posthypoxic frequency decline (PHFD) refers to the undershoot in respiratory frequency that follows brief hypoxic exposures. Lateral pontine neurons are required for PHFD. The neurotransmitters involved in the circuit that activate and/or are released by these pontine neurons regulating PHFD are unknown. We hypothesized that N-methyl-D-aspartate (NMDA) receptors are required for PHFD, because of the similarity in respiratory pattern after blocking lateral pontine activity or NMDA receptors. Furthermore, we hypothesized that the location of these NMDA receptors could be visualized by optimizing binding affinity with spermidine. In vagotomized, anesthetized rats (n = 16), cardiorespiratory responses to hypoxia (8% O2, 30-90 s) were recorded before and after dizocilpine (10 microg-1 mg/kg iv), and NMDA receptors were mapped with [3H]dizocilpine (n = 6). Dizocilpine elicited a dose-related effect on PHFD, blocking PHFD at high doses. Resting arterial blood pressure and breathing frequency decreased with high doses of dizocilpine, but the respiratory response to hypoxia remained intact. Our novel anatomical data indicate that NMDA receptors were widespread but distributed differentially in the brain stem. We conclude that NMDA receptors are located in pontine and medullary respiratory-related regions and that PHFD requires NMDA-receptor activation.

Persistence of the biphasic ventilatory response to hypoxia in preterm infants.

OBJECTIVE: To characterize postnatal maturation of the biphasic ventilatory response to hypoxia in order to determine whether it persists beyond the first weeks of life in preterm infants, and the contributions of respiratory frequency and tidal volume to this response. METHODS: Stable preterm infants were studied at two postnatal ages, 2 to 3 weeks (n = 12) and 4 to 8 weeks (n = 12), before hospital discharge at 35 weeks (range, 33 to 38 weeks) of postconceptional age. Infants were exposed to 5 minutes of 15% (or 13%) inspired oxygen; ventilation, oxygen saturation, end-tidal partial pressure of carbon dioxide, and heart rate were simultaneously recorded. RESULTS: Minute ventilation exhibited a characteristic biphasic response to hypoxia at both postnatal ages, regardless of the development of periodic breathing. At both ages there was a transient increase in tidal volume, which peaked at 1 minute, accompanied by a sustained decrease in respiratory frequency as a result of significant prolongation of expiratory time. CONCLUSION: The characteristic biphasic ventilatory response to hypoxia persists into the second month of postnatal life in preterm infants. We speculate that this finding is consistent with the prolonged vulnerability of such infants to neonatal apnea.

Post-hypoxic frequency decline does not depend on alpha2-adrenergic receptors in the adult rat.

The aim of this study was to determine whether post-hypoxic frequency decline (PHFD) requires central activation of alpha2-adrenergic receptors. PHFD is defined as the undershoot in respiratory frequency that occurs immediately following brief hypoxic periods. Adult anesthetized, vagotomized rats were exposed to hypoxia (8% O2, mean=45 s) before and after intracerebroventricular (i.c.v.) infusion of vehicle or alpha2-antagonist. The efficacy of the i.c.v. antagonist was assessed by recording the response to intravenous injection of alpha2-agonist before and after the infusion. We compared breathing frequencies before, during, and after hypoxia, both before and after treatments. The decline in breathing frequency after hypoxia was not prevented by the alpha2-antagonists, RX 821002 or SK&F-86466. Guanabenz, an alpha2-agonist, prolonged baseline expiration and potentiated PHFD. Prior treatment with SK&F-86466 blocked the agonist-evoked response which was also reversed by subsequent administration of SK&F-86466. We conclude that PHFD does not require the activation of alpha2-adrenergic receptors, but that alpha2-adrenergic receptors can modulate resting and post-hypoxic respiratory frequency.

Prolongation in expiration evoked from ventrolateral pons of adult rats.

Activation of neurons in the ventrolateral (vl) pons was hypothesized to alter the breathing pattern because previous studies demonstrated apneusis after inhibiting neuronal activity with bilateral muscimol (10 mM) microinjections into the vl pons (17). The excitatory amino acid L-glutamate (10 mM) was microinjected (10-100 nl) into the vl pons in anesthetized, vagotomized, paralyzed, and ventilated adult rats (n = 8). In four of these animals, the target site was approached from the ventral surface of the pons to avoid penetrating the dorsolateral (dl) pons. The expiratory phase was prolonged transiently and concurrently with the microinjection. The location of the injection sites included the A5 area, was independent of the approach, and was distinct from the dl pons. These results complement our previous data and indicate that neurons located in the vl pons influence respiration specifically by prolonging expiration when activated and by delaying the inspiratory-to-expiratory phase transition when inhibited.