Respiratory Rhythm Generation Research Articles

Knockout of sodium pump α3 subunit gene (Atp1a3−/−) results in perinatal seizure and defective respiratory rhythm generation

ATP1A3 encodes a neuron-specific human α3 subunit isoform of the sodium pump that plays an important role in neuronal excitability. Point and deletion mutations in ATP1A3 have been recognized in diverse neurological disorders. Three ATP1A3 disorders, alternating hemiplegia of childhood (AHC); apnea; and severe infantile epileptic encephalopathy often appear shortly after birth. To gain insight into the pathophysiology of these disorders and to understand the functional roles of the sodium pump α3 subunit in the brain in vivo during this period of development, we examined the phenotype of Atp1a3 knockout homozygous mouse fetuses (Atp1a3−/−). We focused on fetuses just before birth because at birth, about half of them showed severe seizure, and none could continue effective breathing and died soon after birth, without any gross anatomical anomalies. We examined c-Fos expression in the brains of Atp1a3−/− and found a significantly increased number of c-Fos-expressing cells in various regions of the brains, with unique distribution in the cerebellum, when compared with wild-type littermates (Atp1a3+/+). We also measured contents of monoamine neurotransmitters in the brains and found higher contents, especially of dopamine and noradrenaline, in the brains of Atp1a3−/− compared with those of Atp1a3+/+. In addition, we found various abnormal respiratory rhythms produced in the brainstem of Atp1a3−/−. These results suggest that Atp1a3 plays a critical role in neural function during development and at birth.

0143 CARBACHOL INHIBITS GLUTAMATERGIC INPUT TO MOUSE HYPOGLOSSAL MOTOR NEURONS - A MECHANISM FOR REM SLEEP SUPPRESSION OF GENIOGLOSSUS ACTIVITY

In REM sleep, the genioglossus (GG) muscle undergoes a dramatic suppression of activity. A current hypothesis is that the loss of GG activity during REM sleep is mediated by a combination of 1) monoaminergic disfacilitation and 2) a cholinergic inhibition of hypoglossal motor neurons. Strikingly, blockade of cholinergic receptors in the hypoglossal motor nucleus fully restores REM sleep tonic and inspiratory-modulated components of GG activity (Grace et al., 2013), suggesting that the cholinergic signal is largely responsible for the REM sleep suppression of GG activity. Respiratory rhythm generator neurons of the pre-Bötzinger complex drive the activation of hypoglossal motor neurons through glutamatergic premotor neurons in the parahypoglossal region (PH). In the current study, we investigate how cholinergic signaling affects the PH glutamatergic input to hypoglossal motor neurons. We stereotaxically injected the PH region of vGluT2-cre mice with a cre-dependent AAV-ChR2-mCherry to expressed channelrhodopsin2 (ChR2) in PH glutamatergic premotor neurons. We then performed whole-cell recordings in hypoglossal neurons while photostimulating PH glutamatergic inputs expressing ChR2. Photostimulation of the glutamatergic PH input evoked AMPA-mediated EPSCs in hypoglossal motor neurons. These photoevoked EPSCs were maintained in TTX, indicating direct connectivity between stimulated terminals and recorded hypoglossal motoneurons. Bath application of carbachol strongly inhibited the PH glutamatergic excitation of hypoglossal neurons via muscarinic receptors. The effect of carbachol on photoevoked EPSCs was maintained when 1) we blocked postsynaptic G-mediated effects by adding GDP-β-S in the recording pipette and 2) when we blocked action potential mediated transmission by adding TTX in the extracellular bath solution. These results indicate that carbachol inhibits PH input to hypoglossal motoneurons through a presynaptic mechanism. Our results provide a possible mechanism for cholinergic inhibition of the hypoglossal motor neurons in REM sleep. We propose that the cholinergic presynaptic suppression of the excitatory drive from the PH premotor neurons can be responsible for the reduction in activity of hypoglossal motor neurons in REM sleep. P01HL095491.

Chemoreflexes : A Major Component of Adaptation to High Altitude/Hypoxic Environment

The decrease in O2 pressure in hypoxia, either at high altitude or in disease, induces physiological responses mediated through oxygen sensing systems. Among those responses, chemoreflexes play a major role, especially during the initial phase of exposure to hypoxia. Peripheral chemoreceptors (carotid bodies) and central receptors (retrotrapezoid nucleus) are sensitive to hypoxia and hypercapnia and modulate the activity of the Respiratory rhythm generator. Our aim was to determine the role of erythropoietin (Epo) on those mechanisms in two animal models: transgenic anemic mice under‐expressing Epo (EpoTagh), and the Pika (Ochotona curzoniae) a lagomorph genetically adapted to high altitude on the Tibetan plateau. We showed that Epo are necessary for ventilatory acclimatization in EpoTagh mice and that nNOS and NMDA receptors are overexpressed in the medulla of Pikas. In humans, the sensitivity to hypoxia is a determinant factor for acclimatization to high altitude. A prospective cohort study on 1017 subjects has shown that the ventilatory response to hypoxia at exercise is predictive of the occurrence of severe high altitude illnesses (SHAI). From clinical evaluation and physiological response to hypoxia measured during a hypoxia exercise test, a predictive score of SHAI was calculated, which allowed the detection of high‐risk subjects. Instability of the control of ventilation has been evidenced during exercise in hypoxia, with oscillations of ventilation with a period of 11–12 seconds. The mechanisms of this instability is linked to the gain of the O2 and CO2 response, as well as other factors such as dead space and cardiac output.Support or Funding InformationLaboratory of Excellence GrEx

Intermittent Hypoxia during Gestation Disrupts Respiratory Rhythm and Neuroplasticity in Newborn Rat Brainstem‐spinal cord Preparations In Vitro

Pregnant women with sleep‐disordered breathing experience intermittent hypoxia, especially during late pregnancy. To test whether intermittent hypoxia exposures during pregnancy (G‐IH; gestational intermittent hypoxia) alter breathing in newborn rats, pregnant rats were exposed to intermittent hypoxia (2 min 10.5% oxygen / 2 min normoxia for 8 h) from gestational day 10 to 21 (exposures completed prior to birth). Newborn rats (P0–P3) were anesthetized and their brainstem‐spinal cords isolated under in vitro conditions. Spontaneous respiratory motor bursts were recorded on cervical spinal roots (C4–C5). G‐IH preparations from P0–P1 rats had a lower baseline burst frequency (8.2 +/− 0.6 bursts/min; n=10) compared to normoxic controls (11.0 +/− 0.3 bursts/min; n=21). After 90 min, burst frequency decreased with time nearly 2× times greater in G‐IH preparations compared to controls. In most G‐IH preparations, there were frequent “pauses” in the rhythm, which resembled spontaneous apneas. With respect to respiratory neuroplasticity, dihydroxyflavone (DHF) application (5 micromolar, 9 min; TrkB receptor agonist) induced long‐lasting 19% increase in respiratory motor burst amplitude in control preparations (n=5) after 90 min. However, only 2/4 G‐IH preparations expressed a DHF‐dependent increase in motor burst amplitude. Also, episodic substance‐P application (100 nanomolar, 3 min on / 7 min wash ×3) induced a long‐lasting (60‐min) increase in respiratory burst frequency in control preparations. However, there was no substance‐P induced frequency plasticity in any G‐IH preparations (n=4). These data suggest that gestational intermittent hypoxia disrupts respiratory rhythm generation and reduces the capacity for expressing respiratory neuroplasticity in newborn rats.Support or Funding InformationNIH Grant: NS085226

Respiratory rhythm generation: triple oscillator hypothesis.

Breathing is vital for survival but also interesting from the perspective of rhythm generation. This rhythmic behavior is generated within the brainstem and is thought to emerge through the interaction between independent oscillatory neuronal networks. In mammals, breathing is composed of three phases – inspiration, post-inspiration, and active expiration – and this article discusses the concept that each phase is generated by anatomically distinct rhythm-generating networks: the preBötzinger complex (preBötC), the post-inspiratory complex (PiCo), and the lateral parafacial nucleus (pF L), respectively. The preBötC was first discovered 25 years ago and was shown to be both necessary and sufficient for the generation of inspiration. More recently, networks have been described that are responsible for post-inspiration and active expiration. Here, we attempt to collate the current knowledge and hypotheses regarding how respiratory rhythms are generated, the role that inhibition plays, and the interactions between the medullary networks. Our considerations may have implications for rhythm generation in general.

A Randomized Clinical Trial Comparing the Effects of Antitussive Agents on Respiratory Center Output in Patients With Chronic Cough

Cough is produced by the same neuronal pool implicated in respiratory rhythm generation, and antitussive drugs acting at the central level, such as opioids, may depress ventilation. Levodropropizine is classified as a nonopioid peripherally acting antitussive drug that acts at the level of airway sensory nerves. However, the lack of a central action by levodropropizine remains to be fully established. We set out to compare the effects of levodropropizine and the opioid antitussive agent dihydrocodeine on the respiratory responses to a conventional CO2 rebreathing test in patients with chronic cough of any origin. Twenty-four outpatients (aged 39-70 years) with chronic cough were studied. On separate runs, each patient was randomly administered 60mg levodropropizine, 15mg dihydrocodeine, or a matching placebo. Subsequently, patients breathed a mixture of 93%oxygen and 7%CO2 for 5 min. Fractional end-tidal CO2 (Fetco2) and inspiratory minute ventilation (V˙i) were continuously monitored. Changes in breathing pattern variables were also assessed. At variance with dihydrocodeine, levodropropizine and placebo did not affect respiratory responses to hypercapnia (P< .01). The ventilatory increases by hypercapnia were mainly accounted for by a rise in the volume components of the breathing pattern. The results are consistent with a peripheral action by levodropropizine; the assessment of ventilatory responses to CO2 may represent a useful tool to investigate the central respiratory effects of antitussive agents. European Union Clinical Trials Register (EudraCT No.: 2013-004735-68); URL: https://www.clinicaltrialsregister.eu/.

Respiratory Rhythm Generation: The Whole Is Greater Than the Sum of theParts.

Breathing is a continuous behavior essential for life in mammals and one of the few behaviors that can be studied in vivo in intact animals awake, anesthetized or decerebrated and in highly reduced in vitro and in situ preparations. The preBötzinger complex (preBötC) is a small nucleus in the brainstem that plays an essential role in normal breathing and is widely accepted as the site necessary and sufficient for generation of the inspiratory phase of the respiratory rhythm. Substantial advances in understanding the anatomical and cellular basis of respiratory rhythmogenesis have arisen from in vitro and in vivo studies in the past 25years; however, the underlying cellular mechanisms remain unknown.

Neural Network Reconfigurations: Changes of the Respiratory Network by Hypoxia as an Example.

Neural networks, including the respiratory network, can undergo a reconfiguration process by just changing the number, the connectivity or the activity of their elements. Those elements can be either brain regions or neurons, which constitute the building blocks of macrocircuits and microcircuits, respectively. The reconfiguration processes can also involve changes in the number of connections and/or the strength between the elements of the network. These changes allow neural networks to acquire different topologies to perform a variety of functions or change their responses as a consequence of physiological or pathological conditions. Thus, neural networks are not hardwired entities, but they constitute flexible circuits that can be constantly reconfigured in response to a variety of stimuli. Here, we are going to review several examples of these processes with special emphasis on the reconfiguration of the respiratory rhythm generator in response to different patterns of hypoxia, which can lead to changes in respiratory patterns or lasting changes in frequency and/or amplitude.

Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO2-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90min after the application of 50/100μM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50μM, 70-90min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.

Deficiency of CPEB2-Confined Choline Acetyltransferase Expression in the Dorsal Motor Nucleus of Vagus Causes Hyperactivated Parasympathetic Signaling-Associated Bronchoconstriction.

This study first generated and characterized cpeb2 gene-deficient mice. CPEB2-knock-out (KO) mice are born alive but most die within 3 d after birth showing no overt defects in anatomy. We found that the KO neonates showed severe apnea and altered respiratory pattern. Such respiratory defects could be recapitulated in mice with pan-neuron-specific or cholinergic neuron-specific ablation of the cpeb2 gene. Further investigation revealed that cholinergic transmission in the KO dorsal motor nucleus of vagus was overactivated because KO mice lack CPEB2-suppressed translation of the rate-limiting enzyme in the production of acetylcholine (i.e., choline acetyltransferase). Consequently, increased parasympathetic signaling leads to hyperactivated bronchoconstriction and abnormal respiration in the KO neonates.

Dynamics of in-phase and anti-phase bursting in the coupled pre-Bötzinger complex cells.

Activity of neurons in the pre-Bötzinger complex within the mammalian brain stem has an important role in the generation of respiratory rhythms. Previous experimental results have shown that the dynamics of sodium and calcium within each cell may be responsible for various bursting mechanisms. In this paper, we study the bursting dynamics of the two-coupled pre-Bötzinger complex neurons. Using a combination of fast-slow decomposition and two-parameter bifurcation analysis, we explore the possible forms of dynamics that the model network can produce as well the transitions of in-phase and anti-phase bursting respectively.

Carotid body chemoreflex: a driver of autonomic abnormalities in sleep apnoea.

What is the topic of this review? This article presents emerging evidence for heightened carotid body chemoreflex activity as a major driver of sympathetic activation and hypertension in sleep apnoea patients. What advances does it heighlight? This article discusses the recent advances on cellular, molecular and epigenetic mechanisms underlying the exaggerated chemoreflex in experimental models of sleep apnoea. The carotid bodies are the principal peripheral chemoreceptors for detecting changes in arterial blood oxygen concentration, and the resulting chemoreflex is a potent regulator of the sympathetic tone, blood pressure and breathing. Sleep apnoea is a disease of the respiratory system that affects several million adult humans. Apnoeas occur during sleep, often as a result of obstruction of the upper airway (obstructive sleep apnoea) or because of defective respiratory rhythm generation by the CNS (central sleep apnoea). Patients with sleep apnoea exhibit several co-morbidities, with the most notable among them being heightened sympathetic nerve activity and hypertension. Emerging evidence suggests that intermittent hypoxia resulting from periodic apnoea stimulates the carotid body, and the ensuing chemoreflex mediates the increased sympathetic tone and hypertension in sleep apnoea patients. Rodent models of intermittent hypoxia that simulate the O2 saturation profiles encountered during sleep apnoea have provided important insights into the cellular and molecular mechanisms underlying the heightened carotid body chemoreflex. This article describes how intermittent hypoxia affects the carotid body function and discusses the cellular, molecular and epigenetic mechanisms underlying the exaggerated chemoreflex.

El complejo pre-Bötzinger: generación y modulación del ritmo respiratorio

IntroductionIn mammals, the preBötzinger complex (preBötC) is a bilateral and symmetrical neural network located in the brainstem which is essential for the generation and modulation of respiratory rhythm. There are few human studies about the preBötC and, its relationship with neurological diseases has not been described. However, the importance of the preBötC in neural control of breathing and its potential participation in neurological diseases in humans, has been suggested based on pharmacological manipulation and lesion of the preBötC in animal models, both in vivo and in vitro. MethodIn this review, we describe the effects of some drugs on the inspiratory activity in vitro in a transverse slice that contains the preBötC, as well as some in vivo experiments. Drugs were classified according to their effects on the main neurotransmitter systems and their importance as stimulators or inhibitors of preBötC activity and therefore for the generation of the respiratory rhythm. ConclusionClinical neurologists will find this information relevant to understanding how the central nervous system generates the respiratory rhythm and may also relate this information to the findings made in daily practice.

Emergent Network Topology within the Respiratory Rhythm-Generating Kernel Evolved In Silico.

We hypothesize that the network topology within the pre-Bötzinger Complex (preBötC), the mammalian respiratory rhythm generating kernel, is not random, but is optimized in the course of ontogeny/phylogeny so that the network produces respiratory rhythm efficiently and robustly. In the present study, we attempted to identify topology of synaptic connections among constituent neurons of the preBötC based on this hypothesis. To do this, we first developed an effective evolutionary algorithm for optimizing network topology of a neuronal network to exhibit a ‘desired characteristic’. Using this evolutionary algorithm, we iteratively evolved an in silico preBötC ‘model’ network with initial random connectivity to a network exhibiting optimized synchronous population bursts. The evolved ‘idealized’ network was then analyzed to gain insight into: (1) optimal network connectivity among different kinds of neurons—excitatory as well as inhibitory pacemakers, non-pacemakers and tonic neurons—within the preBötC, and (2) possible functional roles of inhibitory neurons within the preBötC in rhythm generation. Obtained results indicate that (1) synaptic distribution within excitatory subnetwork of the evolved model network illustrates skewed/heavy-tailed degree distribution, and (2) inhibitory subnetwork influences excitatory subnetwork primarily through non-tonic pacemaker inhibitory neurons. Further, since small-world (SW) network is generally associated with network synchronization phenomena and is suggested as a possible network structure within the preBötC, we compared the performance of SW network with that of the evolved model network. Results show that evolved network is better than SW network at exhibiting synchronous bursts.

Voltage-Dependent Rhythmogenic Property of Respiratory Pre-Bötzinger Complex Glutamatergic, Dbx1-Derived, and Somatostatin-Expressing Neuron Populations Revealed by Graded Optogenetic Inhibition.

The rhythm of breathing in mammals, originating within the brainstem pre-Bötzinger complex (pre-BötC), is presumed to be generated by glutamatergic neurons, but this has not been directly demonstrated. Additionally, developmental expression of the transcription factor Dbx1 or expression of the neuropeptide somatostatin (Sst), has been proposed as a marker for the rhythmogenic pre-BötC glutamatergic neurons, but it is unknown whether these other two phenotypically defined neuronal populations are functionally equivalent to glutamatergic neurons with regard to rhythm generation. To address these problems, we comparatively investigated, by optogenetic approaches, the roles of pre-BötC glutamatergic, Dbx1-derived, and Sst-expressing neurons in respiratory rhythm generation in neonatal transgenic mouse medullary slices in vitro and also more intact adult perfused brainstem-spinal cord preparations in situ. We established three different triple-transgenic mouse lines with Cre-driven Archaerhodopsin-3 (Arch) expression selectively in glutamatergic, Dbx1-derived, or Sst-expressing neurons for targeted photoinhibition. In each line, we identified subpopulations of rhythmically active, Arch-expressing pre-BötC inspiratory neurons by whole-cell recordings in medullary slice preparations in vitro, and established that Arch-mediated hyperpolarization of these inspiratory neurons was laser power dependent with equal efficacy. By site- and population-specific graded photoinhibition, we then demonstrated that inspiratory frequency was reduced by each population with the same neuronal voltage-dependent frequency control mechanism in each state of the respiratory network examined. We infer that enough of the rhythmogenic pre-BötC glutamatergic neurons also have the Dbx1 and Sst expression phenotypes, and thus all three phenotypes share the same voltage-dependent frequency control property.

Inhibitory control of ascending glutamatergic projections to the lamprey respiratory rhythm generator

Neurons within the vagal motoneuron region of the lamprey have been shown to modulate respiratory activity via ascending excitatory projections to the paratrigeminal respiratory group (pTRG), the proposed respiratory rhythm generator. The present study was performed on in vitro brainstem preparations of the lamprey to provide a characterization of ascending projections within the whole respiratory motoneuron column with regard to the distribution of neurons projecting to the pTRG and related neurochemical markers. Injections of Neurobiotin were performed into the pTRG and the presence of glutamate, GABA and glycine immunoreactivity was investigated by double-labeling experiments. Interestingly, retrogradely labeled neurons were found not only in the vagal region, but also in the facial and glossopharyngeal motoneuron regions. They were also present within the sensory octavolateral area (OLA). The results show for the first time that neurons projecting to the pTRG are immunoreactive for glutamate, surrounded by GABA-immunoreactive structures and associated with the presence of glycinergic cells. Consistently, GABAA or glycine receptor blockade within the investigated regions increased the respiratory frequency. Furthermore, microinjections of agonists and antagonists of ionotropic glutamate receptors and of the GABAA receptor agonist muscimol showed that OLA neurons do not contribute to respiratory rhythm generation. The results provide evidence that glutamatergic ascending pathways to the pTRG are subject to a potent inhibitory control and suggest that disinhibition is one important mechanism subserving their function. The general characteristics of inhibitory control involved in rhythmic activities, such as respiration, appear to be highly conserved throughout vertebrate evolution.

Loss of the Persistent Sodium Current Elicits Homeostatic Plasticity in Respiratory Rhythm Generation

The neural networks underlying the generation of breathing rhythms must remain stable and dynamic throughout the lifetime of an organism. In many neural networks, it is hypothesized that mechanisms of homeostatic plasticity regulate synaptic and/or intrinsic properties of neurons to maintain stability. The neural network giving rise to the inspiratory phase of breathing, termed the preBötzinger Complex (preBötC), utilizes a combination of intrinsic membrane conductances, such as the persistent sodium current (INaP) and the nonspecific cation current (ICAN), as well as synaptic properties to generate and shape the inspiratory rhythm. When isolated in brainstem slices, the preBötC continues to produce remarkably stable rhythmic population activity in phase with inspiratory motor output. We hypothesized that homeostatic mechanisms within the preBötC maintain stability of the inspiratory rhythm. Slices containing the preBötC were incubated overnight (~16hrs) in 10μM DNQX or 20μM riluzole to block excitatory synaptic transmission or the persistent sodium current, respectively. Upon washout, we observed that DNQX did not significantly alter the inspiratory rhythm. However, in slices incubated with riluzole, inspiratory burst amplitude was increased and frequency was decreased compared to controls (ACSF only). To explore this finding further, rhythmic activity was recorded for the duration of the riluzole exposure. Following ~30–40min, riluzole perturbed the rhythm such that inspiratory frequency dramatically slowed or stopped. Surprisingly, after ~7hrs the rhythm came back spontaneously, indicating plasticity had occurred to overcome the loss of INaP. The rhythm and burst shape were altered such that amplitude and burst rise slope were increased (~300% and 220% baseline, respectively) and frequency was slowed (~30% baseline). Following thorough washout of riluzole (1–3hrs), the new large and slow inspiratory pattern remained, suggesting long lasting changes in network properties had occurred. In a subset of preBotC slices, riluzole was reapplied following washout. In contrast to naïve slices, there was no effect on inspiratory frequency following &gt;1hrs in riluzole, indicating that the new rhythm was insensitive to loss of INaP. However, burst amplitude was reduced suggesting INaP remained present in preBötC neurons. These studies demonstrate that the preBötC can compensate for long‐lasting changes in network properties with mechanisms of homeostatic plasticity and begin to provide a basis for understanding how the neural networks underlying breathing rhythms may adapt to maintain stability in health and disease.Support or Funding InformationNIH RO1 HL 126523

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

Effects of a quaternary lidocaine derivative, QX-314, on the respiratory activity in brainstem-spinal cord preparation from newborn rats

In the clinical setting, the use of QX-314 (a quaternary derivative of lidocaine) has been proposed to achieve the selective inhibition of nociceptors that express transient receptor potential vanilloid 1 (TRPV1) channels with fewer motor deficits. However, it has been also reported that QX-314 may produce systemic CNS toxicities with relative potencies that are approximately twice as high as those of lidocaine. There are no reports concerning the effects of extracellular QX-314 on the rhythm-generating neurons in the respiratory center. In the present study, we examined the effects of QX-314 on respiratory rhythm generation in brainstem-spinal cord preparations from newborn rats. The extracellular application of QX-314 (200μM) decreased the C4 burst rate, amplitude and slope during the initial rising phase, and the effects slowly developed with a half-decay time of approximately 20min. The combined application of capsaicin (10 or 100μM) with QX-314 (100μM) showed no additional effect. The intracellular application of QX-314 (100μM) to respiratory neurons depressed the action potentials with a half-decay time of around 5min. Our findings could explain one of the mechanisms underlying the central toxicities that occur after the systemic application of QX-314.

The peptidergic control circuit for sighing.

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.