Respiratory-modulated Neurons Research Articles

Phase synchronization as a flexible definition of the respiratory pattern: Application to pontine‐dependent control of the respiratory pattern

A distinct pattern of inspiratory, post‐inspiratory and expiratory activities that are expressed in respiratory‐modulated neurons and motor outputs during stationary breathing are the foundation of most contemporary models for respiratory rhythm generation and pattern formation. Models and experiments show that impaired synaptic inhibition in the respiratory circuitry of the brainstem is associated with neurogenic breathing disorders in Rett syndrome (inspiratory efforts against a closed glottis) or with paradoxical vocal cord movement (glottal closure during inspiration). Previously, we reported that the pontine Kölliker‐Fuse nuclei (KFn) couple the spinal and cranial motor patterns and control respiratory pattern variability. Here, we test the hypothesis that the KFn is critical for expression of the phase relationships among respiratory motor activities. In arterially‐perfused brainstem preparations (n=7), we recorded respiratory activity from the hypoglossal, cervical vagal, phrenic and iliohypogastric nerves before and after bilateral bicuculline (a GABA(A)R antagonist, 50nl, 10mM) microinjections in the KFn. Disinhibition of KFn increased the variability of the respiratory rhythm (coefficient of variation (CV) of cycle duration (TTOT before, 0.10 ± 0.01 versus after bicuculline 0.50 ± 0.26, p<0.01). The increase in variability was associated with unpredictable timing of phase durations and transitions. Standard metrics such as TTOT failed to quantitatively describe the complexity in the changes of the respiratory motor pattern. Here, we introduce a quantitative definition of the respiratory motor pattern based on the phase synchronization of respiratory motor outputs or its underlying neuronal activities. Power spectral analysis revealed that the respiratory motor activities had a periodicity indicating that a coherent respiratory‐pattern persisted. Quantifying phase synchronization of respiratory motor outputs revealed a significant reduction in coupling strengths, without a change in their relative phase relationships (mean relative phase at baseline, 1.16 ± 0.07 rad versus after disinhibition, 1.17 ± 0.05 rad; and phase coherence at baseline, 0.87 ± 0.01 versus after disinhibition, 0.31 ± 0.03, p=3.68×10−4). Our data further support the concept that respiratory pattern formation requires reciprocal interaction between pontine and medullary respiratory groups. Focal pontine disinhibition resulted in reduced coupling strength between ponto‐medullary synaptic interactions that consequently disrupted the motor pattern, while the undisturbed medullary aspects are able to uphold the periodicity of respiration. Taken together, our results illustrate that medullary respiratory rhythm generating circuit require the pons to convert its periodicity into a viable and vital respiratory motor pattern.Support or Funding InformationSupported by a grant from the Australian Research Council.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The physiological motor patterns produced by neurons in the nucleus retroambiguus in the rat and their modulation by vagal, peripheral chemosensory, and nociceptive stimulation.

The nucleus retroambiguus (NRA) is a neuronal cell group in the medullary ventrolateral tegmentum, rostrocaudally between the obex and the first cervical spinal segment. NRA neurons are premotor interneurons with direct projections to the motoneurons of soft palate, pharynx, and larynx in the nucleus ambiguus in the lateral medulla as well as to the motoneurons in the spinal cord innervating diaphragm, abdominal, and pelvic floor muscles and the lumbosacral motoneurons generating sexual posture. These NRA premotor interneurons receive very strong projections from the periaqueductal gray (PAG) in the context of basic survival mechanisms as fight, flight, freezing, sound production, and sexual behavior. In the present study in rat we investigated the physiological motor patterns generated by NRA neurons, as the result of vagal, peripheral chemosensory, and nociceptive stimulation. The results show that the NRA contains phasic respiratory modulated neurons, as well as nonphasic tonically modulated neurons. Stimulation in the various rostrocaudal levels of the NRA generates site-specific laryngeal, respiratory, abdominal, and pelvic floor motor activities. Vagal and peripheral chemosensory stimulation induces both excitatory and inhibitory modulation of phasic NRA-neurons, while peripheral chemosensory and nociceptive stimulation causes excitation and inhibition of nonphasic NRA-neurons. These results are in agreement with the concept that the NRA represents a multifunctional group of neurons involved in the output of the emotional motor system, such as vomiting, vocalization, mating, and changes in respiration.

Commentaries on Viewpoint: Central chemoreception is a complex system function that involves multiple brain stem sites

to the editor: Amniote vertebrates adjust the acid-base status of the blood through ventilatory alterations of PaCO2 and, to some degree, by renal modulation of bicarbonate concentration. Receptor systems for the ventilatory acid-base regulation has been studied mainly in mammals, in which the

Multielectrode recording of respiratory neuronal activities in pontine microcircuits

Recent studies showed that the pontine pneumotaxic region plays an important role in integrating inputs from vagally-mediated pulmonary mechanoreceptors that modulate the respiratory rhythm. To characterize the neuronal microcircuits underlying this process, we recorded simultaneously the extracellular activities of multiple pontine pneumotaxic neurons using a tungsten microelectrodes array with a multichannel data acquisition system from urethane-anesthetized, bi-vagotamized, paralyzed and servo-ventilated adult rats. The neuronal responses to vagal electrical stimulation (80 Hz, 20-40 μA, 1 min) were analyzed. Of a total of 452 dorsolateral pontine neurons recorded in 22 rats, 61 units showed respiratory-modulated rhythmic activities with either inspiratory, expiratory, or phase-spanning discharge patterns, sometimes with superimposed tonic discharge. Pairwise cross-correlation analysis identified 16 (out of total 256) neuronal pairs showing short-latency offset peaks suggesting functional correlations among them. Vagal stimulation attenuated or suppressed the rhythmic activities in most respiratory-modulated neurons (n=53) and promoted tonic discharges in others (n=8). Gravitational transformation of ensemble spike-train activities before, during and after vagal stimulation revealed functional convergence or divergence of respiratory neuronal responses. Results showed that pontine respiratory neurons displayed a multiplicity of rhythmic patterns with differing responsiveness to vagal stimulation. Supported by NIH grants HL67966 and HL072849.

Nasal trigeminal inputs release the A5 inhibition received by the respiratory rhythm generator of the mouse neonate.

Experiments were performed on neonatal mice to analyze why, in vitro, the respiratory rhythm generator (RRG) was silent and how it could be activated. We demonstrated that in vitro the RRG in intact brain stems is silenced by a powerful inhibition arising from the pontine A5 neurons through medullary alpha(2) adrenoceptors and that in vivo nasal trigeminal inputs facilitate the RRG as nasal continuous positive airway pressure increases the breathing frequency, whereas nasal occlusion and nasal afferent anesthesia depress it. Because nasal trigeminal afferents project to the A5 nuclei, we applied single trains of negative electric shocks to the trigeminal nerve in inactive ponto-medullary preparations. They induced rhythmic phrenic bursts during the stimulation and for 2-3 min afterward, whereas repetitive trains produced on-going rhythmic activity up to the end of the experiments. Electrolytic lesion or pharmacological inactivation of the ipsilateral A5 neurons altered both the phrenic burst frequency and occurrence after the stimulation. Extracellular unitary recordings and trans-neuronal tracing experiments with the rabies virus show that the medullary lateral reticular area contains respiratory-modulated neurons, not necessary for respiratory rhythmogenesis, but that may provide an excitatory pathway from the trigeminal inputs to the RRG as their electrolytic lesion suppresses any phrenic activity induced by the trigeminal nerve stimulation. The results lead to the hypothesis that the trigeminal afferents in the mouse neonate involve at least two pathways to activate the RRG, one that may act through the medullary lateral reticular area and one that releases the A5 inhibition received by the RRG.

Role of the cerebellar deep nuclei in respiratory modulation.

The cerebellum contains three deep nuclei, i.e., the fastigial, interposed and lateral nucleus. Recent studies demonstrate that these nuclei play different roles in respiratory modulation. Activation of fastigial nuclear neurons predominantly increases ventilation via elevation of respiratory frequency and/or tidal volume. Ablation of the fastigial nucleus did not significantly alter eupneic breathing, but did markedly attenuate the respiratory response to medium and severe hypercapnia as well as hypoxia. The fastigial nucleus contains respiratory-modulated neurons and about 25% of these neurons do not show their respiratory-related phasic activity until exposed to hypercapnia. The fastigial nucleus also contains CO2/H+ chemosensitive sites that contributed to the respiratory response to hypercapnia. Therefore, it is concluded that fastigial nuclear facilitatory influence on chemoreflexes emerges during hypercapnia via recruiting intrinsic chemoreception and respiratory-modulated neurons. Full expression of the fastigial nucleus-mediated respiratory responses depends on the integrity of the medullary gigantocellular nucleus at least partially via monosynaptic projections. Additionally, the fastigial nucleus receives inhibitory inputs primarily from Purkinje cells located in the medial vermis and recent observations indicate that simulation of these Purkinje cells inhibits respiration. As compared to chemoreflexes, fastigial nuclear role in the respiratory mechanoreflexes is not significant. The studies related to the role of the interposed and lateral nucleus in eupneic breathing are limited and the results appear controversial. However, there is evidence to show that the interposed nucleus contains respiratory-modulated neurons and is involved in coughing motor control.

Integral-differential calculus computations by short-term potentiation and depression in NTS-pontine pathways

Neural integrators are a class of neural networks that afford extended temporal summation of neural activity or, equivalently, low-pass frequency filtering of neurotransmission [1]. In the respiratory system, a biphasic neural integrator in the carotid chemoreflex response is demonstrated by the classic short-term potentiation (STP) and afterdischarge of phrenic activity [2]. Recently, a new class of neural networks called neural differentiators, which afford high-pass frequency filtering of afferent inputs through short-term depression (STD) of neurotransmission, has been identified in the vagal-carotid control of the expiratory off-switch [3]. The differentiator response is characterized by: 1) a habituation-like adaptation to the vagal Hering-Breuer (HB) reflex lengthening or carotid chemore-flex shortening of expiratory duration; 2) secondary adaptive effects in the form of pontine-mediated and NMDA receptor-dependent post-stimulus rebound and recovery of these vagal or carotid [4](or hypoxia [5]) mediated responses. To elucidate the mechanism of the habituation of the HB reflex, we obtained extracellular field potentials (FPs) in the medial and commissural regions of the nucleus tractus solitarius (NTS) evoked by electrical vagal stimulation (1 min) in urethane-anesthetized, paralyzed and mechanically ventilated rats (n =17) in vivo. FPs from vagal A-fibers (stimulus current, 20–70 μA, response latency, 4–5 ms) revealed a stimulus frequency-dependent (20–80 Hz) STD similar to the synaptic accommodation observed in vitro [6] and the habituation of HB reflex in vivo [4]. FPs from C-fibers (150–350 μA, 20–45 ms) also demonstrated a frequency-dependent (5–20 Hz) STD during afferent activation but their amplitude remained depressed for >50 s post-stimulation. Microinjection of the NMDA receptor antagonist D-APV abolished the short-term memory following C-fiber activation but not the frequency-dependent STD with A- or C-fibers. The characteristics of the vagal A-and C-fiber mediated responses are reminiscent of the type I and type II NTS neurons found in vitro [7]. To elucidate the mechanism of the pontine-mediated adaptation of the HB reflex, we obtained extracellular recordings of pontine neuronal activity simultaneously with phrenic nerve recording during electrical vagal stimulation (1.5×T, 80 Hz, 1 min). Neurons with tonic and respiratory modulated activity were found within or near the parabrachialis (PB) and Kolliker-Fuse (KF) complex. Most tonic neurons in KF (n = 11) were depressed during and/or shortly after vagal stimulation that elicited the characteristic biphasic adaptation of the HB reflex in the phrenic activity, whereas most tonic neurons in medial PB (n = 22) were not affected. Activity of respiratory modulated neurons (n = 4) recorded in KF remained in phase with phrenic activity during vagal stimulation. Results showed that vagal input induced STD of tonic neurotransmission in KF. These findings support a dual-process nonassociative learning model of integral-differential calculus computations in the brain [8] through activity-dependent STP and STD of neurotransmission in primary and secondary afferent pathways.

Responses of simultaneously recorded respiratory-related medullary neurons to stimulation of multiple sensory modalities.

This study addresses the hypothesis that multiple afferent systems share elements of a distributed brain stem network that modulates the respiratory motor pattern. Data were collected from 18 decerebrate, bilaterally vagotomized, paralyzed, artificially ventilated cats. Up to 28 neurons distributed in the rostral and caudal ventral respiratory group, nucleus tractus solitarius, and raphe obscurus were recorded simultaneously with microelectrode arrays. Phases of the respiratory cycle and inspiratory drive were assessed from integrated efferent phrenic nerve activity. Carotid chemoreceptors were stimulated by injection of CO2-saturated saline solution via the external carotid artery. Baroreceptors were stimulated by increased blood pressure secondary to inflation of an embolectomy catheter in the descending aorta. Cutaneous nociceptors were stimulated by pinching a footpad. Four hundred seventy-four neurons were tested for respiratory modulated firing rates and responses; 403 neurons were tested with stimulation of all 3 modalities. Chemoreceptor stimulation and pinch, perturbations that tend to increase respiratory drive, caused similar responses in 52 neurons; 28 responded oppositely. Chemoreceptor and baroreceptor stimulation resulted in similar primary responses in 45 neurons; 48 responded oppositely. Similar responses to baroreceptor stimulation and pinch were recorded for 38 neurons; opposite effects were measured in 26 neurons. Among simultaneously recorded neurons, distinct combinations of firing rate changes were evoked in response to stimulation of the different modalities. The results show a functional convergence of information from carotid chemoreceptors, baroreceptors, and cutaneous nociceptors on respiratory-modulated neurons distributed in the medulla. The data are consistent with the hypothesis that brain stem neurons have overlapping memberships in multifunctional groups that influence the respiratory motor pattern.

Ventrolateral medullary respiratory network and a model of cough motor pattern generation.

The primary hypothesis of this study was that the cough motor pattern is produced, at least in part, by the medullary respiratory neuronal network in response to inputs from "cough" and pulmonary stretch receptor relay neurons in the nucleus tractus solitarii. Computer simulations of a distributed network model with proposed connections from the nucleus tractus solitarii to ventrolateral medullary respiratory neurons produced coughlike inspiratory and expiratory motor patterns. Predicted responses of various "types" of neurons (I-DRIVER, I-AUG, I-DEC, E-AUG, and E-DEC) derived from the simulations were tested in vivo. Parallel and sequential responses of functionally characterized respiratory-modulated neurons were monitored during fictive cough in decerebrate, paralyzed, ventilated cats. Coughlike patterns in phrenic and lumbar nerves were elicited by mechanical stimulation of the intrathoracic trachea. Altered discharge patterns were measured in most types of respiratory neurons during fictive cough. The results supported many of the specific predictions of our cough generation model and suggested several revisions. The two main conclusions were as follows: 1) The Bötzinger/rostral ventral respiratory group neurons implicated in the generation of the eupneic pattern of breathing also participate in the configuration of the cough motor pattern. 2) This altered activity of Bötzinger/rostral ventral respiratory group neurons is transmitted to phrenic, intercostal, and abdominal motoneurons via the same bulbospinal neurons that provide descending drive during eupnea.

Respiratory activity in the facial nucleus in an in vitro brainstem of tadpole, Rana catesbeiana.

1. In studies of the central neural control of breathing, little advantage has been taken of comparative approaches. We have developed an in vitro brainstem preparation using larval Rana catesbeiana which generates two rhythmic neural activities characteristic of lung and gill ventilation. Based on the pattern of the facial (VII) nerve activity both lung and gill rhythm-related respiratory cycles were divided into three distinct phases. The purpose of this study was to characterize and classify membrane potential trajectories of respiratory motoneurons in the VII nucleus at intermediate stages (XII-XVII) of development. 2. Seventy-five respiratory-modulated neurons were recorded intracellularly within the facial motor nucleus region. Their resting membrane potential was between -40 and -80 mV. Sixty of them were identified as VII motoneurons and fifteen were non-antidromically activated. Membrane potentials of fifty-six of the seventy-five neurons were modulated with both lung (5-27 mV) and gill rhythms (3-15 mV) and the remaining nineteen neurons had only a modulation with lung rhythmicity (6-23 mV). No cells with gill modulation alone were observed. 3. All of the cells modulated with lung rhythmicity had only phase-bound depolarizing or hyperpolarizing membrane potential swings which could be categorized into four distinct patterns. In contrast, of the fifty-six cells modulated with gill rhythmicity, thirty-two were phasically depolarized during distinct phases of the gill cycle (four patterns were distinguished), whereas the remaining twenty-four were phase spanning with two distinct patterns. The magnitudes of lung and gill modulations were proportionally related to each other in the cells modulated with both rhythms. 4. In all sixteen neurons studied, a reduction or a reversal of phasic inhibitory inputs during a portion of the lung or gill respiratory cycle was observed following a negative current or chloride ion (Cl-) injection. The phasic membrane resistance modulation in relation to the gill rhythm was analysed in six neurons and a relative decrease in the somatic membrane resistance (0.7-8.1 M omega) was detected during the periods of hyperpolarization. 5. We propose that, at these intermediate stages of development: (a) both gill and lung respiratory oscillations in motoneurons are generated by respiratory premotor neurons having only a few distinct activity patterns; (b) these patterns delineate distinct portions of the centrally generated respiratory cycles; and (c) phasic synaptic inhibition, mediated by Cl-, contributes to shaping the membrane potential trajectories of respiratory motoneurons.

Respiratory-modulated and phrenic afferent-driven neurons in the cervical spinal cord (C4-C6) of the fluorocarbon-perfused guinea pig.

The potential contributions of cervical spinal interneurons to the neural control of respiration have been investigated by extracellularly recording the patterns of activity of neurons in the C4-C6 spinal cord during fictive respiration in the fluorocarbon-perfused, adult guinea pig. Two types of neurons were recorded: respiratory-modulated neurons, whose activity was modulated with respiration, and phrenic-driven neurons, which were excited by electrical stimulation of the phrenic nerve. Respiratory-modulated neurons (n = 20) could be divided into inspiratory, expiratory, and phase-spanning neurons, based on their patterns of activity during fictive respiration. Respiratory-modulated neurons showed varying dependencies on the type of breathing; during spontaneous augmented breaths, one-half exhibited patterns of activity that were significantly different to those seen during normal, fictive respiration, whereas the other half of the respiratory-modulated neurons showed similar patterns of activity during both normal and augmented breaths. Phrenic-driven neurons (n = 22) could be divided into short-latency (7-12 ms), moderate-latency (12-25 ms), and inhibited neurons, but were only occasionally and weakly modulated with respiration. The results suggest that respiratory-modulated C4-C6 spinal neurons may contribute to the neural control of respiration, with different subpopulations specialized for different types of respiratory tasks, and that phrenic-driven neurons may be interposed in sensory or reflex pathways, such as the spinothalamic tract or phrenic-to-phrenic inhibitory reflex.

Respiratory resetting induced by spinal cord stimulation in the cat.

Electrical stimulation (50-150 microA, 0.5-ms duration, 3-300 Hz) was performed within three different regions (lateral, ventrolateral, and ventral) of the C2-C3 spinal cord of decerebrate, vagotomized, paralyzed, and artificially ventilated cats. Spinal cord stimulation sites were located by inserting monopolar or bipolar stimulating electrodes either at the dorsolateral sulcus or at least 1 mm medial or lateral to the sulcus. With stimulation at each site, alterations in respiratory rhythm, orthodromic phrenic nerve responses, and antidromic activation of medullary respiratory-modulated neurons were examined. Phrenic nerve responses to cervical spinal cord stimulation consisted of an early excitation (2-4 ms) and/or a late excitation (4-8 ms). Stimulation of the lateral region evoked the greatest amplitude early response and stimulation of the ventrolateral region produced the greatest late excitation. All three stimulus sites elicited antidromic activation of some respiratory-modulated neurons in the dorsal (DRG) and ventral respiratory groups (VRG). The lateral region was the least effective resetting site, and it had the highest incidence of antidromic activation of both DRG and VRG neurons. The ventrolateral region of the cervical spinal cord was the most effective resetting site, but it had the lowest incidence of antidromic activation of DRG respiratory-modulated neurons. In addition, resetting responses were observed with spinal cord stimulation at similar sites in the thoracic and lumbar spinal cord regions thought to be devoid of inspiratory bulbospinal axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitation of dorsal and ventral respiratory group neurons by phrenic nerve afferents.

The projections of phrenic nerve afferents to neurons in the dorsal (DRG) and ventral (VRG) respiratory group were studied in anesthetized, paralyzed, and vagotomized cats. Extracellular recordings of neuronal responses to vagal nerve and cervical phrenic nerve stimulation (CPNS) indicated that about one-fourth of the DRG respiratory-modulated neurons were excited by phrenic nerve afferents with an onset latency of approximately 20 ms. In addition, non-respiratory-modulated neurons within the DRG were recruited by CPNS. Although some convergence of vagal and phrenic afferent input was observed, most neurons were affected by only one type of afferent. In contrast to the DRG, only 3 out of 28 VRG respiratory-modulated neurons responded to CPNS. A second study determined that most of these neuronal responses were due to activation of diaphragmatic afferents since 90% of the DRG units activated by CPNS were also excited at a longer latency by thoracic phrenic nerve stimulation. The difference in onset latency of neuronal excitation indicates an afferent peripheral conduction velocity of about 10 m/s, which suggests that they are predominately small myelinated fibers (group III) making paucisynaptic connections with DRG neurons. Decerebration, decerebellation, and bilateral transection of the dorsal columns at C2 do not abolish the neuronal responses to cervical PNS.

Activity of bulbar respiratory modulated neurons and restart of respiration after hypocapnic apnea in rabbits.

The activity of respiratory modulated neurons at the end of the apneic pause and during restart of respiration and the diaphragmatic mass activity were examined and both were compared to quiet respiration. Thresholds of mutual inhibition of neurons are unevenly distributed within various phase types of neurons.

Inhibitory action of vagal nerve and spinal cord afferents on discharge pattern of bulbar respiratory modulated neurons.

Single vagal nerve stimuli delivered during burst discharge of respiratory modulated neurons entailed lengthening of an interspike interval in 37% of all units tested. Occasionally stimuli delivered to the spinal cord caused lengthening of interspike intervals in respiratory modulated neurons and in other bulbar units the discharge of which was not modulated with respiration. Vagal stimuli often entailed lengthening of the following interspike interval, while the interval during which stimuli were applied remained unaltered; latencies suggested that inhibitory pathways were polysynaptic. In expiratory-inspiratory (EI) and inspiratory (I) neurons, lengthenings of intervals roughly doubled those noted in inspiratory-expiratory (IE) and expiratory (E) units. Lengthening of intervals depended on stimulus incidence within the intervals and was greatest when stimuli were delivered during the late part of the intervals. In EI and I units, lengthening of intervals occurred mainly during the middle part of burst discharge, in IE and E neurons, however, towards the end of the burst. Conditioning of discharge of respiratory modulated neurons could be demonstrated.

Time course of excitatory and inhibitory states of bulbar respiratory modulated neurons.

In respiratory modulated neurons of rabbits, vagally mediated inhibition is not bound to resting membrane potential oscillations. Latency of spinally evoked antidromical spike invasion, however, is shorter and threshold voltage is lower during the shift of membrane potential towards depolarization accompanying burst discharge.