Respiratory Rhythm Generation Research Articles

Identification of the hypertension drug niflumic acid as a glycine receptor inhibitor

Glycine is one of the major neurotransmitters in the brainstem and the spinal cord. Glycine binds to and activates glycine receptors (GlyRs), increasing Cl− conductance at postsynaptic sites. This glycinergic synaptic transmission contributes to the generation of respiratory rhythm and motor patterns. Strychnine inhibits GlyR by binding to glycine-binding site, while picrotoxin blocks GlyR by binding to the channel pore. We have previously reported that bath application of strychnine to zebrafish embryos causes bilateral muscle contractions in response to tactile stimulation. To explore the drug-mediated inhibition of GlyRs, we screened a chemical library of ~ 1,000 approved drugs and pharmacologically active molecules by observing touch-evoked response of zebrafish embryos in the presence of drugs. We found that exposure of zebrafish embryos to nifedipine (an inhibitor of voltage-gated calcium channel) or niflumic acid (an inhibitor of cyclooxygenase 2) caused bilateral muscle contractions just like strychnine-treated embryos showed. We then assayed strychnine, picrotoxin, nifedipine, and niflumic acid for concentration-dependent inhibition of glycine-mediated currents of GlyRs in oocytes and calculated IC50s. The results indicate that all of them concentration-dependently inhibit GlyR in the order of strychnine > picrotoxin > nifedipine > niflumic acid.

Retracted: Control of hypoglossal pre-inspiratory discharge.

What is the topic of this review? This review explores the modulatory role of lung vagal afferents and intra-neuraxial and carotid body chemoreceptors upon hypoglossal pre-inspiratory activity. What advances does it highlight? Pre-inspiratory activity manifesting in hypoglossal neural efferent discharge may be potentiated by mechanical interruption of vagal continuity and challenge with administration of a hypoxic and/or hypercapnic gas mixture and attenuated by static and/or dynamic pulmonary stretch. Differential excitability of, or premotoneuronal volleys exhibiting distinct spatiotemporal patterns of discharge arriving at, motoneurons residing within the hypoglossal motor nucleus may emergently generate phase-spanning pre-inspiratory inspiratory activity of hypoglossal neural efferent discharge manifest at the population level. The hypoglossal nerve (XII) innervates muscles mediating excursive movements of the tongue. The population discharge of hypoglossalmotoneuronal axons constituting the hypoglossal nerve precedes and extends through the inspiratory epoch. The epoch subtended between the onsets of hypoglossal and phrenic neural discharge constitutes so-called pre-inspiration. Hypoglossal pre-inspiratory neural discharge serendipitously displaces the tongue along a tensor reducing upper airway resistance anticipative of succeeding inspiratory efforts. Hypoglossal motoneurons exhibiting discharge onset during pre-inspiration experience successive hyperpolarization of membrane voltage and attenuation of unitary spiking frequency, although a subset may, paradoxically and state-dependently, exhibit depolarization of membrane voltage and augmentation of neuronal spiking frequency, by dynamic stretch placed upon the alveolar walls and interstitium. Marked static elevation of positive-end expiratory pressure may induce hypoglossal bursting decoupled from phasic rhythmic phrenic discharge. Augmentation of the amplitude and/or duration of hypoglossal inspiratory discharge during successive pre-inspiratory and inspiratory epochs by inhalation of a hypoxic and/or hypercapnic gas mixture remains restrained in the presence of intact vagal inputs and is potentiated by interruptions of vagal continuity. Unravelling the mechanisms underlying the genesis of pre-inspiratory activity will inform our understanding of respiratory rhythm generation and pattern shaping. In the present work, I seek to explore the mechanisms underlying modulation of hypoglossal pre-inspiratory discharge by hypercapnia, hypoxia and static and dynamic lung stretch placed upon hypoglossal pre-inspiratory activity, the mechanisms underlying the generation of hypoglossal pre-inspiratory activity, and the extent of microanatomical and functional overlap between propriobulbar interneuronal microcircuits generating hypoglossal pre-inspiratory activity and propriobulbar interneuronal microcircuit oscillators generating pre-inspiratory activity inaugurally inducing respiratory rhythmic activity and thus use experimental data from previous work and that developed by other investigators to explore the modulatory role of lung vagal afferents and intra-neuraxial and carotid body chemoreceptors upon hypoglossal pre-inspiratory activity.

Mechanisms Contributing to the Generation of Mayer Waves.

Mayer waves may synchronize overlapping propriobulbar interneuronal microcircuits constituting the respiratory rhythm and pattern generator, sympathetic oscillators, and cardiac vagal preganglionic neurons. Initially described by Sir Sigmund Mayer in the year 1876 in the arterial pressure waveform of anesthetized rabbits, authors have since extensively observed these oscillations in recordings of hemodynamic variables, including arterial pressure waveform, peripheral resistance, and blood flow. Authors would later reveal the presence of these oscillations in sympathetic neural efferent discharge and brainstem and spinal zones corresponding with sympathetic oscillators. Mayer wave central tendency proves highly consistent within, though the specific frequency band varies extensively across, species. Striking resemblance of the Mayer wave central tendency to the species-specific baroreflex resonant frequency has led the majority of investigators to comfortably presume, and generate computational models premised upon, a baroreflex origin of these oscillations. Empirical interrogation of this conjecture has generated variable results and derivative interpretations. Sinoaortic denervation and effector sympathectomy variably reduces or abolishes spectral power contained within the Mayer wave frequency band. Refractorines of Mayer wave generation to barodeafferentation lends credence to the hypothesis these waves are chiefly generated by brainstem propriobulbar and spinal cord propriospinal interneuronal microcircuit oscillators and likely modulated by the baroreflex. The presence of these waves in unitary discharge of medullary lateral tegmental field and rostral ventrolateral medullary neurons (contemporaneously exhibiting fast sympathetic rhythms [2–6 and 10 Hz bands]) in spectral variability in vagotomized pentobarbital-anesthetized and unanesthetized midcollicular (i.e., intercollicular) decerebrate cats supports genesis of Mayer waves by supraspinal sympathetic microcircuit oscillators. Persistence of these waves following high cervical transection in vagotomized unanesthetized midcollicular decerebrate cats would seem to suggest spinal sympathetic microcircuit oscillators generate these waves. The widespread presence of Mayer waves in brainstem sympathetic-related and non-sympathetic-related cells would seem to betray a general tendency of neurons to oscillate at this frequency. We have thus presented an extensive and, hopefully cohesive, discourse evaluating, and evolving the interpretive consideration of, evidence seeking to illumine our understanding of origins of, and insight into mechanisms contributing to, the genesis of Mayer waves. We have predicated our arguments and conjectures in the substance and matter of empirical data, though we have occasionally waxed philosophical beyond these traditional confines in suggesting interpretations exceeding these limits. We believe our synthesis and interpretation of the relevant literature will fruitfully inspire future studies from the perspective of a more intimate appreciation and conceptualization of network mechanisms generating oscillatory variability in neuronal and neural outputs. Our evaluation of Mayer waves informs a novel set of disciplines we term quantum neurophysics extendable to describing subatomic reality. Beyond informing our appreciation of mechanisms generating sympathetic oscillations, Mayer waves may constitute an intrinsic property of neurons extant throughout the cerebrum, brainstem, and spinal cord or reflect an emergent property of interactions between arteriogenic and neuronal oscillations.

Key role of 5-HT1A receptors in the modulation of the neuronal network underlying the respiratory rhythm generation in lampreys.

In mammals, 5-HTexcitatory respiratory effects imply 5-HT1A receptor-mediated disinhibition of pre-Bötzinger complex neurons. In the lamprey, 5-HT1A receptors are involved in the neural control of locomotion, but their role in the respiratory regulation, particularly at the level of the putative respiratory rhythm generator, the paratrigeminal respiratory group (pTRG), is not known. We here investigate the respiratory function of inhibitory 5-HT1A receptors within the pTRG of the isolated brainstem of the adult lamprey. The 5-HT1A receptor agonists either bath applied or microinjected into the pTRG did not cause significant effects. However, the selective 5-HT1A receptor antagonist (S)-WAY 100135 bath applied or microinjected into the pTRG induced depressing respiratory effects or even apnoea, thus revealing that 5-HT exerts a 5-HT1A receptor-mediated potent tonic influence on respiration and contributes to maintain baseline levels of respiratory activity. Microinjections of strychnine or bicuculline, either alone or in combination, into the pTRG prevented (S)-WAY 100135-induced apnoea. In addition, immunohistochemical studies corroborate the present findings suggesting that 5-HT1A receptors are widely expressed in close apposition to the soma of glycine-immunoreactive cells located within the pTRG region. The results show that in the lamprey respiratory network, 5-HT exerts a tonic influence on respiration by a potent inhibitory control on both GABAergic and glycinergic mechanisms. The observed disinhibitory effects resemble the excitatory respiratory modulation exerted by 5-HT1A receptor-mediated inhibition of glycinergic and/or GABAergic neurons present in mammals, supporting the notion that some features of the neuronal network subserving respiratory rhythm generation are highly conserved throughout phylogeny.

Interleukin 1-beta but not the interleukin-1 receptor antagonist modulates inspiratory rhythm generation in vitro

Interleukin 1-beta (IL-1β) is a cytokine that modulates breathing when applied systemically or directly into the brain. IL-1β is expressed, along with its receptors, in IL-1β-sensitive respiratory-related circuits, which likely include the inspiratory rhythm generator (the preBötzinger complex, preBötC). Thus, considering that IL-1β might directly modulate preBötC function, we tested whether IL-1β and its endogenous antagonist IL1Ra modulate inspiratory rhythm generation in the brainstem slice preparation containing the preBötC. We found that IL-1β reduces, in a concentration-dependent manner, the amplitude of the fictive inspiratory rhythm generated by the preBötC, which is prevented by IL1Ra. Only a negligible effect on the rhythm frequency was observed at one of the concentrations tested (10 ng/mL). In sum, these findings indicate that IL-1β modulates respiratory rhythm generation. In contrast, IL1Ra did not produce a major effect but slightly increased burst amplitude regularity of the fictive respiratory rhythm. Our findings show that IL-1β modulates breathing by directly modulating the inspiratory rhythm generation. This modulation could contribute to the respiratory response to inflammation in health and disease.

Opioid sensitivity of projection‐specific Kölliker‐Fuse neurons

Opioid‐induced respiratory depression is the primary cause of death in individuals who overdose on opioids. Respiration is controlled by a highly interconnected neural network located in the pons and medulla. The Kölliker‐Fuse nucleus (KF), located in the dorsolateral pons, is an essential part of this network and projects to core respiratory nuclei in the ventrolateral medulla. Opioids injections into the KF disturb respiratory rate, volume, and pattern. Deletion of opioid receptors from KF neurons attenuates morphine‐induced respiratory depression. These studies suggest that the KF is a critical site in opioid‐induced respiratory depression. Mu opioid receptor agonists hyperpolarize a population of neurons in the KF due to activation of G protein‐mediated inwardly rectifying conductance. However, we do not know the projection target of the opioid‐sensitive KF neurons. Given the extensive projections from the KF to medullary respiratory rhythm generators, we hypothesize that opioid‐sensitive KF neurons project to respiratory rhythm generators. Fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into the Bötzinger complex (BötC), pre‐Bötzinger complex (preBötC), and rostral ventral respiratory group (rVRG) of mice. Whole‐cell voltage‐clamp recordings were made from retrograde‐labeled KF neurons contained in brain slices. Mu opioid receptor‐mediated outward currents were identified in KF neurons that project to the preBötC, and rVRG, but not in KF neurons that project to the BötC. Opioid‐sensitive connections between the KF and respiratory rhythm generators may be a possible mechanism by which opioids reduce respiratory rate.Support or Funding InformationR00, DA038069

Postictal Modulation of Breathing by the Central Amygdala (CeA) Can Induce Sudden Unexpected Death in Epilepsy (SUDEP).

RationaleSUDEP is the most common cause of death in refractory epilepsy patients. The MORTality in Epilepsy Monitoring Unit Study (MORTEMUS) reported monitored SUDEP cases, and fatal apnea preceded terminal asystole in all patients. The mechanisms involved in seizure‐induced apnea are not known, but evidence suggests the amygdala may be a critical node in the pathway by which seizures spread from the forebrain to the brainstem respiratory network. Depth electrode recordings from the amygdala of epilepsy patients showed that prolonged apneas occur when seizures spread into the CeA. Apneas also occur when the CeA is electrically stimulated in awake patients. Here we examined the role of the amygdala in peri‐ictal apnea and its involvement in SUDEP in two epilepsy models: Scn1aR1407X/+ (Dravet Syndrome; DS) mice and DBA/1 mice.MethodsBaseline breathing, hypercapnic ventilatory response (HCVR) and hypoxic ventilatory response (HVR) were assessed using whole‐body plethysmography in DS (n=7) and DBA/1 (n=8) mice before and after lesioning the CeA. Under surgical anesthesia (1–2% isoflurane), electrolytic lesions were induced in the CeA, after which mice were instrumented with EEG, ECG and EMG electrodes. After 5 days of recovery, mice were tethered to a preamplifier and placed in a plethysmography chamber. Seizures were induced in DS mice (n=22) by increasing body temperature 0.5°C per minute using a heat lamp and in DBA/1 mice (n=13) with loud white noise.In a separate cohort of DS mice (n=4), the CeA was stimulated under light anesthesia (0.5–1% isoflurane) by injecting current with stereotactically guided monopolar electrodes while measuring breathing using head‐out plethysmography. Electrode placement was verified post hoc using histology.In a third cohort of DS mice (n=12), CeA lesions or sham lesions were made and mice were video‐monitored for 100 days. A subset of these mice had spontaneous seizures and SUDEP.ResultsCeA lesions did not affect baseline breathing, HCVR or HVR, and did not prevent seizures in DS or DBA/1 mice. Apnea and death occurred in control (sham‐lesioned) DS mice following heat‐induced seizures in 54.4% of cases (n=11). In contrast, only 9.1% of DS mice with CeA lesions showed respiratory arrest and death after heat‐induced seizures (n=11; p=0.016).Stimulation of the rostral CeA produced apnea that was dependent on the frequency and amplitude of stimulation, without causing seizures. At 50 Hz and 500 μA, apnea was induced for 1–4 minutes without causing death (n=4) or reversal of anesthetic immobility, suggesting lack of an arousal response.Kaplan‐Meier survival curves showed that mice with CeA lesions had higher survival (64.5%) than sham lesions (28.3%) (p=0.015).ConclusionOur data suggest that the CeA can induce apnea but, is not directly involved in baseline respiratory rhythm generation or the ventilatory response to hypercapnia or hypoxia. CeA lesions reduce the risk of apnea during seizures. Therefore, the CeA is a key component of the neural pathway through which seizures spread from the forebrain to respiratory centers in the brainstem to elicit ventilatory arrest after seizures.Support or Funding InformationNIH/NINDS U01‐NS0904143 SUDEP Research Alliance

Activation of PACAP expression in mouse RTN neurons at birth protects postnatal breathing

The brainstem systems that regulate breathing are established prenatally but they remain immature and vulnerable to failure at the time of birth; this reflects, in part, underdevelopment of the respiratory chemosensory systems that typically monitor blood gases (O2, CO2) to regulate breathing. In the mouse retrotrapezoid nucleus (RTN), Phox2b‐expressing respiratory chemoreceptor neurons are a nexus for integration of multiple respiratory‐related inputs, homeostatically regulating blood gases by providing CO2‐modulated drive to brainstem respiratory rhythm generator and pre‐motor circuits. In this work, we demonstrate that postnatal expression of a neuropeptide in chemosensitive RTN neurons is precisely tuned to influence breathing at the time of birth. First, by using single cell qRT‐PCR and multiplex in situ hybridization, we define a striking postnatal burst of pituitary adenylate cyclase‐activating peptide (PACAP) expression in RTN neurons that is activated in neonates within minutes of exposure to the external environment. Further, by using whole body plethysmography, we show that mouse pups (P2‐P12) lacking PACAP in Phox2b neurons (Phox2b::Cre;PACAPfl/fl) have severely blunted CO2‐stimulated ventilation (reduced by >50%) and a striking ~3‐fold greater incidence of apneic episodes. PACAP expression in RTN neurons persists into adulthood, albeit at apparently lower levels. PACAP deletion/depletion in adult RTN neurons (in Phox2b::Cre;PACAPfl/fl mice or by shRNA‐mediated knockdown) also depresses CO2‐regulated breathing (by ~30–40%) and increases apneas (~3‐fold), while viral‐mediated re‐expression of PACAP in RTN neurons corrects these deficits. After PACAP knockdown in RTN, CO2‐dependent Fos expression was retained in RTN neurons but decreased in cells of the preBötzinger complex (preBötC), which contains respiratory rhythm‐generating neurons targeted by the RTN. Consistent with the preBötC as a site of action for RTN‐derived PACAP, we show that: 1) the PACAP receptor, PAC1, is expressed in preBötC; 2) PACAP injection directly into the preBötC stimulated central respiratory output, both in vitro and in vivo; and 3) shRNA‐mediated depletion of PAC1 in the preBötC phenocopied breathing deficits observed with RTN deletion of PACAP. Notably, PACAP variants are associated with sudden infant death syndrome (SIDS), and with a SIDS‐like phenotype in PACAP‐null mice. Collectively, these data demonstrate that well‐timed PACAP expression by RTN neurons protects the developmentally immature respiratory control system from hypoventilation and potentially dangerous apneas at birth, and identifies key molecular components of a circuit that supports breathing at this particularly vulnerable period in life.Support or Funding InformationR01 HL108609; R01 HL074011

Modulation of respiratory network activity by forelimb and hindlimb locomotor generators.

Early at the onset of exercise, breathing rate accelerates in order to anticipate the increasing metabolic demand resulting from the extra effort produced. Accordingly, the respiratory neural networks are the target of various input signals originating either centrally or peripherally. For example, during locomotion, the activation of muscle sensory afferents is able to entrain and thereby increase the frequency of spontaneous respiratory rhythmogenesis. Moreover, the lumbar spinal networks engaged in generating hindlimb locomotor rhythms are also capable of activating the medullary respiratory generators through an ascending excitatory command. However, in the context of quadrupedal locomotion, the influence of other spinal cord regions, such as cervical and thoracic segments, remains unknown. Using isolated brainstem-spinal cord preparations from neonatal rats and mice, we show that cervicothoracic circuitry may also contribute to locomotion-induced acceleration of respiratory cycle frequency. As previously observed for the hindlimb CPGs, the pharmacological activation of forelimb locomotor networks produces episodes of fictive locomotion that in turn increase the ongoing respiratory rhythm. Thoracic neuronal circuitry may also participate indirectly in this modulation via the activation of both cervical and lumbar CPG neurons. Furthermore, using light stimulation of CHR2-expressing glutamatergic neurons, we found that the modulation of the respiratory rate during locomotion involves lumbar glutamatergic circuitry. Our results demonstrate that during locomotion, the respiratory rhythm-generating networks receive excitatory ascending inputs from the spinal circuits responsible for generating and coordinating fore- and hindlimb movements. This constitutes a distributed central mechanism that contributes to matching breathing rate to the speed of locomotion.

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).

Effect of magnetic flow and external forcing current on mixed bursting in the pre-Bötzinger complex**Project supported by the National Natural Science Foundation of China (Grant Nos. 11772069 and 11872003).

The pre-Bötzinger complex (pre-BötC) in mammalian brainstem is essential for the generation of respiratory rhythms. Most dynamic studies on the pre-BötC neuron have been focused on its firing activities modulated by the ion conductances rather than that by the electromagnetic radiation or the external forcing current. In this paper, by adding the electromagnetic radiation and external forcing current to Park and Rubin’s model, we mainly investigate the influences of those two factors on the mixed bursting (MB) of single pre-BötC neuron. First, we explore how the variation of external forcing current affects the MB patterns of the system with non-vanishing magnetic flux. We classify the MB patterns and show their dynamic mechanism through fast-slow decomposition and bifurcation analysis. Then, by modifying the feedback coefficient, we further analyze the sole effect of electromagnetic radiation on the firing activities of the system. Our results may be instructive in understanding the dynamical behavior of pre-BötC neuron.

Optogenetic analysis of respiratory neuronal networks in the ventral medulla of neonatal rats producing channelrhodopsin in Phox2b-positive cells.

Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.

Effects of persistent sodium current blockade in respiratory circuits depend on the pharmacological mechanism of action and network dynamics.

The mechanism(s) of action of most commonly used pharmacological blockers of voltage-gated ion channels are well understood; however, this knowledge is rarely considered when interpreting experimental data. Effects of blockade are often assumed to be equivalent, regardless of the mechanism of the blocker involved. Using computer simulations, we demonstrate that this assumption may not always be correct. We simulate the blockade of a persistent sodium current (INaP), proposed to underlie rhythm generation in pre-Bötzinger complex (pre-BötC) respiratory neurons, via two distinct pharmacological mechanisms: (1) pore obstruction mediated by tetrodotoxin and (2) altered inactivation dynamics mediated by riluzole. The reported effects of experimental application of tetrodotoxin and riluzole in respiratory circuits are diverse and seemingly contradictory and have led to considerable debate within the field as to the specific role of INaP in respiratory circuits. The results of our simulations match a wide array of experimental data spanning from the level of isolated pre-BötC neurons to the level of the intact respiratory network and also generate a series of experimentally testable predictions. Specifically, in this study we: (1) provide a mechanistic explanation for seemingly contradictory experimental results from in vitro studies of INaP block, (2) show that the effects of INaP block in in vitro preparations are not necessarily equivalent to those in more intact preparations, (3) demonstrate and explain why riluzole application may fail to effectively block INaP in the intact respiratory network, and (4) derive the prediction that effective block of INaP by low concentration tetrodotoxin will stop respiratory rhythm generation in the intact respiratory network. These simulations support a critical role for INaP in respiratory rhythmogenesis in vivo and illustrate the importance of considering mechanism when interpreting and simulating data relating to pharmacological blockade.

Robustness of respiratory rhythm generation across dynamic regimes.

A central issue in the study of the neural generation of respiratory rhythms is the role of the intrinsic pacemaking capabilities that some respiratory neurons exhibit. The debate on this issue has occurred in parallel to investigations of interactions among respiratory network neurons and how these contribute to respiratory behavior. In this computational study, we demonstrate how these two issues are inextricably linked. We use simulations and dynamical systems analysis to show that once a conditional respiratory pacemaker, which can be tuned across oscillatory and non-oscillatory dynamic regimes in isolation, is embedded into a respiratory network, its dynamics become masked: the network exhibits similar dynamical properties regardless of the conditional pacemaker node’s tuning, and that node’s outputs are dominated by network influences. Furthermore, the outputs of the respiratory central pattern generator as a whole are invariant to these changes of dynamical properties, which ensures flexible and robust performance over a wide dynamic range.

V2a Neurons Constrain Extradiaphragmatic Respiratory Muscle Activity at Rest.

Breathing requires precise control of respiratory muscles to ensure adequate ventilation. Neurons within discrete regions of the brainstem produce oscillatory activity to control the frequency of breathing. Less is understood about how spinal and pontomedullary networks modulate the activity of respiratory motor neurons to produce different patterns of activity during different behaviors (i.e., during exercise, coughing, swallowing, vocalizing, or at rest) or following disease or injury. Here, we use a chemogenetic approach to inhibit the activity of glutamatergic V2a neurons in the brainstem and spinal cord of neonatal and adult mice to assess their potential roles in respiratory rhythm generation and patterning respiratory muscle activity. Using whole-body plethysmography (WBP), we show that V2a neuron function is required in neonatal mice to maintain the frequency and regularity of respiratory rhythm. However, silencing V2a neurons in adult mice increases respiratory frequency and ventilation, without affecting regularity. Thus, the excitatory drive provided by V2a neurons is less critical for respiratory rhythm generation in adult compared to neonatal mice. In addition, we used simultaneous EMG recordings of the diaphragm and extradiaphragmatic respiratory muscles in conscious adult mice to examine the role of V2a neurons in patterning respiratory muscle activity. We find that silencing V2a neurons activates extradiaphragmatic respiratory muscles at rest, when they are normally inactive, with little impact on diaphragm activity. Thus, our results indicate that V2a neurons participate in a circuit that serves to constrain the activity of extradiaphragmatic respiratory muscles so that they are active only when needed.

The logic behind neural control of breathing pattern

The respiratory rhythm generator is spectacular in its ability to support a wide range of activities and adapt to changing environmental conditions, yet its operating mechanisms remain elusive. We show how selective control of inspiration and expiration times can be achieved in a new representation of the neural system (called a Boolean network). The new framework enables us to predict the behavior of neural networks based on properties of neurons, not their values. Hence, it reveals the logic behind the neural mechanisms that control the breathing pattern. Our network mimics many features seen in the respiratory network such as the transition from a 3-phase to 2-phase to 1-phase rhythm, providing novel insights and new testable predictions.

Reduced computational modelling of Kölliker-Fuse contributions to breathing patterns in Rett syndrome.

Reduced computational models are used to test effects of loss of inhibition to the Kölliker-Fuse nucleus (KFn). Three reduced computational models that simulate eupnoeic and vagotomized respiratory rhythms are considered. All models exhibit the emergence of respiratory perturbations associated with Rett syndrome as inhibition to the KFn is diminished. Simulations suggest that application of 5-HT1A agonists can mitigate the respiratory pathology. The three models can be distinguished and tested based on their predictions about connections and dynamics within the respiratory circuit and about effects of perturbations on certain respiratory neuron populations. Rett syndrome (RTT) is a developmental disorder that can lead to respiratory disturbances featuring prolonged apnoeas of variable durations. Determining the mechanisms underlying these effects at the level of respiratory neural circuits would have significant implications for treatment efforts and would also enhance our understanding of respiratory rhythm generation and control. While experimental studies have suggested possible factors contributing to the respiratory patterns of RTT, we take a novel computational approach to the investigation of RTT, which allows for direct manipulation of selected system parameters and testing of specific hypotheses. Specifically, we present three reduced computational models, developed using an established framework, all of which successfully simulate respiratory outputs across eupnoeic and vagotomized conditions. All three models show that loss of inhibition to the Kölliker-Fuse nucleus reproduces the key respiratory alterations associated with RTT and, as suggested experimentally, that effects of 5-HT1A agonists on the respiratory neural circuit suffice to alleviate this respiratory pathology. Each of the models makes distinct predictions regarding the neuronal populations and interactions underlying these effects, suggesting natural directions for future experimental testing.

Determining the opioid sensitivity of projections from the Kölliker‐Fuse to respiratory rhythm generators

Opioid‐induced respiratory suppression is the primary cause of death in individuals who overdose on opioids, but the mechanism remains elusive. Mu opioid receptor agonists have been shown to hyperpolarize a population of neurons in the Kölliker‐Fuse nucleus (KF) due to activation of G protein‐mediated inwardly rectifying (GIRK) conductance (Levitt et al., J Physiol, 2015). The KF, required for normal eupneic breathing, adjusts respiratory rhythm and rate by modulating respiratory rhythm generators. Given the interconnectivity of the KF and respiratory rhythm generators, we hypothesize that KF neurons projecting to respiratory rhythm generators are opioid‐sensitive and thus a possible mechanism by which opioids reduce respiratory rate. The goal of these experiments was to determine if opioid‐sensitive KF neurons project to respiratory rhythm generators. Fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into respiratory rhythm generators, including pre‐Bötzinger complex (preBötC) and post‐inspiratory complex (PiCo), of mice. Whole‐cell voltage‐clamp recordings were made from retrograde‐labeled neurons in the KF. Mu opioid receptor‐mediated outward currents were identified in KF neurons that project to the preBötC. Therefore, opioid‐sensitive connections between the KF and respiratory rhythm generators may be involved in opioid‐induced respiratory suppression.Support or Funding InformationSupported by NIH grant DA038069.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neurophysiological Mechanisms of Respiratory Activity in Cyclostomes and Fish during Aquatic Breathing

The review addresses the features of the establishment of respiratory activity in the ancient taxa, cyclostomes and fish, which allowed them to adapt to aquatic habitats with a low oxygen level. The brainstem of cyclostomes and fish contains a basic set of nuclei that provides the formation of adequate respiratory activity. The latter is mediated by the universal excitatory and inhibitory neurotransmitters (glutamate, GABA, glycine), suggesting the existence of pivotal, evolutionarily conserved mechanisms to reproduce respiratory oscillations. The qualities of water as a habitat with a reduced oxygen tension determine a high significance of branchial and extrabranchial oxygen-sensitive chemoreceptors which share similar features with highly specialized mammalian oxygen receptors. Neurophysiological details of the respiratory rhythm generator machinery as well as peculiarities of respiratory adaptation to fluctuations in water oxygen tension (PwO2) in agnathans (cyclostomes) and gnathostome fish support the concept of a close relationship between the evolutionarily “verified” adaptive mechanisms regardless of the level of organization of individual vertebrate classes.

Respiratory rhythm generation and pattern formation: oscillators and network mechanisms.

The respiratory rhythm is generated by the interaction of oscillators disparately distributed throughout the pons, medulla, and spinal cord. According to the classic model, the interaction amongst preBötzinger complex (preBötzC) spontaneously bursting preinspiratory units and Bötzinger complex (BötzC) expiratory cells generates the principal respiratory rhythm, thence relayed caudally to the pattern generating elements and premotoneurons of the rostral and caudal divisions of the ventral respiratory group and bulbospinal units of the dorsal respiratory group. Rhythm and pattern generating elements in the ventrolateral medulla receive powerful phasic and tonic modulatory inputs from diencephalic structures, midbrain, Kölliker-Fuse, and parabrachial nuclei, retrotrapezoid nucleus, parafacial respiratory group, ventrolateral metencephalon, nucleus tractus solitarius, and brainstem reticular formation, collectively shaping the normal eupneic discharge. Empirical and computational studies have generated models of respiratory rhythmogenesis and pattern formation variously predicated upon pacemaker, network, or hybrid pacemaker network mechanisms to explain oscillatory behavior and regularity. Network mechanisms critically require the integrity and functionality of inhibitory synaptic neurotransmission. The operation and contribution of inhibitory elements in respiratory rhythm generation and pattern formation are well demonstrated empirically and incorporated in computational network and hybrid models of breathing. Fast inhibitory synaptic neurotransmission utilizes GABAAergic and glycinergic mediated activation of receptor linked chloride conductances, generating an inwardly directed flux of chloride ions mediating membrane voltage hyperpolarization and is required to generate eupneic respiratory patterns in vivo and situ. Persistence of rhythmicity in the presence of synaptic antagonism of GABAA and glycine receptor mediated fast inhibitory neurotransmission indicates pacemaker generating mechanisms sufficiently capable of independently generating this behavior in vivo and transected intact preparations maintaining the preBötzC as the most rostrally preserved structure. The role of GABAB receptor mediated neuromodulation in respiratory rhythm generation and pattern formation is comparatively significantly less investigated. GABABergic activation of postsynaptic and presynaptic membrane receptors variably upregulates potassium conductances and downregulates calcium conductances. Respiratory rhythm and pattern are powerfully modulated in vivo, in situ, and in vitro by superfusion or localized microinjections of GABABergic agonists and antagonists, though are typically not abolished by these experimental interventions. Directionality and magnitude of these effects exhibit maturational changes. The relative depolarization of chloride reversal potentials during the early neonatal period, with gradual shifts towards normal hyperpolarizing values during development, suggests GABABergic signaling may mediate the inhibitory neurotransmission necessary to generate triphasic eupnea. We review and discuss the role of spontaneously bursting oscillators and network mechanisms predicating upon fast inhibitory synaptic neurotransmission in contributing to respiratory rhythmogenesis and pattern formation.