Respiratory Central Pattern Generator Research Articles

Back Stimulation with Gaussian Frequency Biphasic Burst Stimulation for Swallowing Motor Activation

Aspiration of food containing bacteria or saliva into the trachea, leading to bacterial lung infection, is termed aspiration pneumonia. In the United States, aspiration pneumonia results in over 57,000 deaths annually, constituting 2.3% of the total mortality. Similarly, according to data from 2021 in Japan, more than 49,000 individuals succumbed to aspiration pneumonia, accounting for approximately 3.4% of the total mortality, making it the sixth leading cause of death in Japan. Notably, the risk of mortality due to aspiration pneumonia can be mitigated through early detection and prevention of swallowing disorders. Consequently, this research focuses on rehabilitation strategies for swallowing disorders. Swallowing is a coordinated motor function, orchestrated by a program involving multiple muscles under the directives of the Swallow Central Pattern Generator (CPG) located in the medulla. Thus, activation of the Swallowing CPG is pivotal for effective swallowing rehabilitation. To this end, we propose a novel method to activate the Swallow CPG by electrical stimulating of the back. The back has been recognized as a site that activates the expiratory phase of respiration, where the swallow reflex is often triggered. Moreover, given the adjacency of the Swallowing CPG and the Respiratory CPG, we postulate that stimulating the back to activate the expiratory phase can lead to enhanced activation of swallowing movements. Methods: Surface electrodes were placed on the back (T9~T10) and neck of adult rats. Initially, burst stimuli of two polarities were randomly generated at frequencies of either 30 Hz or 300 Hz in accordance with a Gaussian distribution at one of the two sites. The electric stimuli were applied for 120 seconds, three times at each location. To evaluate the impact of stimulus frequency and location on swallowing movements, we compared four groups (n=4 in each group). Following stimulation at one site, identical waveforms of stimulation were administered to both the back and neck for 120 seconds, three times. Swallowing was induced by water injection into the mouth after each stimulation. Electromyography (EMG) of three swallow-related muscles (mylohyoid, thyroarytenoid, and thyropharyngeus) was recorded and their amplitudes compared, both before stimulation (control) and after six consecutive stimulations. Results: It was observed that stimulation of both the back and neck resulted in larger maximum muscle potentials in all measured cases. Furthermore, the lower frequency of 30 Hz yielded larger muscle potentials than 300 Hz. Statistical analysis revealed that back stimulation led to early activation of the thyropharyngeus muscle compared to neck stimulation. Conclusion: The discovery that burst electrical stimulation with Gaussian frequency applied to the back activates swallow muscle groups suggests a potential novel swallow therapy option. This work was supported by NIH grants HL 111215, HL 103415 and OT20D001983, the Craig H. Neilsen Foundation Pilot Research Grant 546714, Kentucky Spinal Cord and Head Injury Research Trust, the Commonwealth of Kentucky Challenge for Excellence, and Grant-in-Aid for JSPS Fellows Number JP23KJ0966. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lumbar epidural electrical stimulation enhances respiration in mice

Spinal cord epidural electrical stimulation (EES) has emerged as a novel approach to modulate and interrogate the neural circuit(s) underlying sensorimotor functions in the past few decades. Recent studies have revealed that cervical EES can regulate respiration in both human subjects and animal models. Furthermore, coupled cervical and lumbar stimulations significantly increased the rhythmic motor outputs in both the cervical and lumbar spinal cord. We hypothesize that the lumbar and cervical coupling is underpinned by cervico-lumbar synaptic connections, and thus, EES at level of the lumbar spinal cord alone may also facilitate respiration via the cervico-lumbar circuit. In this study, we applied EES to the dorsal surface of the lumbar spinal cord between levels 1 and 6 (L1-6) in anesthetized mice. We found that rostral lumbar (L1-2) EES at a stimulation intensity of 0.5 mA elicited significantly higher respiratory frequency, tidal volume, and minute ventilation responses compared to the other lumbar levels (L3-4 and L5-6) tested at intensities ranging from 0.1-1.0 mA. The increased respiratory activity elicited by lumbar stimulation persisted even after the stimulation ceased. Using the optimal stimulation parameters that we identified, we further investigated the underlying mechanism of the lumbar EES-induced respiratory response by examining the neuronal activation patterns. Using immunostaining, we detected c-fos signals, a marker of neuronal activity, in the cervical spinal cord and brainstem after EES at L1-2 using 0.5 mA at 30Hz. This indicates that focal lumbar stimulation elicits remote neuronal activation in cervical spinal cord and brainstem, regions that contain respiration-related neural circuits. These findings suggest that lumbar EES may facilitate respiration by recruiting paraspinal respiratory circuits and potentially supra-spinal respiratory central pattern generator(s). This study provides insights into the development of novel approaches to restore normal respiratory patterns for patients with respiratory disorders, particularly spinal cord injuries. This work was supported by The Louis and Harold Price Foundation, H & H Evergreen Foundation, Jonathan and Susan Dolgen Foundation, the National Institute of Drug Abuse (R01DA047637), and the National Institute of Neurological Disorders and Stroke (UH3NS119772) in the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Novel role for non-invasive neuromodulation techniques in central respiratory dysfunction.

Respiration is a crucial steady-state function of human life. Central nervous system injury can damage the central respiratory pattern generator (CRPG) or interrupt its outflow, leading to central respiratory paralysis and dysfunction, which can endanger the patient's life. At present, there is no effective means to reverse this process. Commonly used non-invasive neuromodulation techniques include repetitive transcranial magnetic stimulation (rTMS), transcranial direct current stimulation (tDCS) and so forth, which have been widely applied in nervous system diseases and their various secondary symptoms, but rarely in respiratory function. Clinical and animal studies have confirmed that TMS is also suitable for investigating the excitability and plasticity of ascending corticospinal respiratory pathways. In addition, although rTMS and tDCS differ in their respective mechanisms, both can regulate respiratory networks in healthy individuals and in diseased states. In this review, we provide an overview of the physiology of respiration, the use of TMS to assess the excitability of corticophrenic pathways in healthy individuals and in central respiratory disorders, followed by an overview of the animal and clinical studies of rTMS, tDCS and so forth in regulating respiratory circuits and the possible mechanisms behind them. It was found that the supplementary motor area (SMA) and the phrenic motor neuron (PMN) may be key regulatory areas. Finally, the challenges and future research directions of neuroregulation in respiratory function are proposed. Through understanding how neuromodulation affects the respiratory neural circuit non-invasively, we can further explore the therapeutic potential of this neuromodulation strategy, so as to promote the recovery of respiratory function after central nervous system diseases or injury.

Early development of respiratory motor circuits in larval zebrafish (Danio rerio).

Rhythm-generating circuits in the vertebrate hindbrain form synaptic connections with cranial and spinal motor neurons, to generate coordinated, patterned respiratory behaviors. Zebrafish provide a uniquely tractable model system to investigate the earliest stages in respiratory motor circuit development in vivo. In larval zebrafish, respiratory behaviors are carried out by muscles innervated by cranial motor neurons-including the facial branchiomotor neurons (FBMNs), which innervate muscles that move the jaw, buccal cavity, and operculum. However, it is unclear when FBMNs first receive functional synaptic input from respiratory pattern-generating neurons, and how the functional output of the respiratory motor circuit changes across larval development. In the current study, we used behavior and calcium imaging to determine how early FBMNs receive functional synaptic inputs from respiratory pattern-generating networks in larval zebrafish. Zebrafish exhibited patterned operculum movements by 3days postfertilization (dpf), though this behavior became more consistent at 4 and 5dpf. Also by 3dpf, FBMNs fell into two distinct categories ("rhythmic" and "nonrhythmic"), based on patterns of neural activity. These two neuron categories were arranged differently along the dorsoventral axis, demonstrating that FBMNs have already established dorsoventral topography by 3dpf. Finally, operculum movements were coordinated with pectoral fin movements at 3dpf, indicating that the operculum behavioral pattern was driven by synaptic input. Taken together, this evidence suggests that FBMNs begin to receive initial synaptic input from a functional respiratory central pattern generator at or prior to 3dpf. Future studies will use this model to study mechanisms of normal and abnormal respiratory circuit development.

The height and LnLF power cover the most of sex differences during orthostasis

The sex differences in autonomic regulation of cardiovascular and respiratory system, as one of the key physiological mechanisms of disease, till now remain underexplored. We hypothesized that cardiovascular and respiratory systems share the same autonomic mechanisms, represented by heart rate variability (HRV) indexes, opposing the effect of orthostasis, which are partially different between sexes.The 147 participants, 78 men and 69 women were recruited from the student population of local University for the research study. The low and high frequency heart rate variability (LF and HF HRV) was computed from ECG recording in the sitting position for 5 minutes of resting and slow breathing (6 breaths per minute) stages. The sitting position was used according to requirement of spirometry system. The tidal volume (TV) and respiratory rate (RR) were recorded for one minute at the end of each stage. Systolic and diastolic blood pressure (SBP and DBP) were measured with automatic digital sphygmomanometer (OMRON EVOLV, Japan). Two‐way repeated measures MANOVA and MANCOVA (SPSS 22) were used to test for effects of orthostasis and sex on HR, SBP, DBP, HRV variables, TV, RR, and MV. Multiple stepwise regression analysis was used to determine predictors of cardiovascular and respiratory variables. Written informed consent was obtained from each participant.In the current study we found significant main effects of orthostasis on HR, DBP, LnLF, LnHF (P<0.001 for each), and significant main effects of sex on HR, SBP, LnLF, TV, and MV (for HR P=0.004, for LnLF P=0.006, for the rest P<0.001). Follow the significant sex by orthostasis interaction we found that RR increased only for men upon standing but not for women.Multiple stepwise linear regression analysis revealed that when adjusting for potentially confounding variables, the change of cardiovagal‐mediated LnHF power (Figure 1A) was an independent predictor of HR and RR response to orthostasis (Table 1, Models 1 and 2). Women was an independent predictor of dRR, while Women by dLnLF interaction determined dHR, showing that LnLF, indicating according to our hypothesis, baroreflex‐mediated sympathoinhibition, contribute to HR response in women (Fig. 1B).The BMI by LnLF power interaction was a negative predictor of resting RR (Table 1, Model 3). It is suggested that baroreflex‐mediated sympathoinhibition contribute to respiratory central pattern generator particularly in people with increased BMI. The same interaction was a positive predictor of TV (Table 1, Model 4) indicating that increased time of respiratory cycle accompines greater TV.The comparisons of the similar models with and without dummy variables indicating “Women”, revealed that the dummy variable replaced “Height” or “LnLF”, suggesting that differences in both independent variables relate to mechanisms controlling cardiorespiratory system specific to female gender.In conclusion, cardiovagal‐mediated LnHF HRV power controls both HR and RR and determines their response to orthostasis. The differences in height and LnLF power cover the most of the sex difference in mechanisms controlling cardiovascular and respiratory variables and their response to orthostasis.

Chapter 7 - Breathing during sleep

The depth, rate, and regularity of breathing change following transition from wakefulness to sleep. Interactions between sleep and breathing involve direct effects of the central mechanisms that generate sleep states exerted at multiple respiratory regulatory sites, such as the central respiratory pattern generator, respiratory premotor pathways, and motoneurons that innervate the respiratory pump and upper airway muscles, as well as effects secondary to sleep-related changes in metabolism. This chapter discusses respiratory effects of sleep as they occur under physiologic conditions. Breathing and central respiratory neuronal activities during nonrapid eye movement (NREM) sleep and REM sleep are characterized in relation to activity of central wake-active and sleep-active neurons. Consideration is given to the obstructive sleep apnea syndrome because in this common disorder, state-dependent control of upper airway patency by upper airway muscles attains high significance and recurrent arousals from sleep are triggered by hypercapnic and hypoxic episodes. Selected clinical trials are discussed in which pharmacological interventions targeted transmission in noradrenergic, serotonergic, cholinergic, and other state-dependent pathways identified as mediators of ventilatory changes during sleep. Central pathways for arousals elicited by chemical stimulation of breathing are given special attention for their important role in sleep loss and fragmentation in sleep-related respiratory disorders.

Chapter 1 - Respiratory rhythm and pattern generation: Brainstem cellular and circuit mechanisms

Breathing movements in mammals are driven by rhythmic neural activity automatically generated within spatially and functionally organized brainstem neural circuits comprising the respiratory central pattern generator (CPG). This chapter reviews up-to-date experimental information and theoretical studies of the cellular and circuit mechanisms of respiratory rhythm and pattern generation operating within critical components of this CPG in the lower brainstem. Over the past several decades, there have been substantial advances in delineating the spatial architecture of essential medullary regions and their regional cellular and circuit properties required to understand rhythm and pattern generation mechanisms. A fundamental concept is that the circuits in these regions have rhythm-generating capabilities at multiple cellular and circuit organization levels. The regional cellular properties, circuit organization, and control mechanisms allow flexible expression of neural activity patterns for a repertoire of respiratory behaviors under various physiologic conditions that are dictated by requirements for homeostatic regulation and behavioral integration. Many mechanistic insights have been provided by computational modeling studies driven by experimental results and have advanced understanding in the field. These conceptual and theoretical developments are discussed.

The art of facilitation: a delicate hormonal balance!

The art of facilitation: a delicate hormonal balance!

Expiratory abdominal muscle nerve is active at flexor phase, while inspiratory phrenic nerve is not active during locomotion evoked by 5-HT and NMDA in the neonatal rat

Abdominal muscles are involved in respiration and locomotion. In the isolated pons-spinal cord-rib attached preparation from neonatal rat, the phrenic nerve and abdominal muscles show inspiratory and expiratory activity, respectively. Using this preparation, we investigated whether the bath application of NMDA and 5-HT could evoke locomotor activities in the fourth cervical ventral root (C4VR), phrenic nerve, and abdominal muscle nerve (ilioinguinal nerve, IIG-n). We also observed rib and abdominal muscle movements visually. The phrenic nerve and C4VR showed inspiratory activity consistently under the control conditions, whereas IIG-n showed expiratory activity only at the beginning of the experiment. During the chemically-induced locomotion, both C4VR and IIG-n showed locomotor activity, and IIG-n in particular showed flexor activity. During the flexor activity, lateral bending of the rib cage to the recording site was observed. The phrenic nerve showed weak or no apparent locomotor activity. We concluded that the central pattern generator (CPG) for locomotion provides stronger excitatory synaptic inputs to C4 motoneurons innervating neck and shoulder muscles than the inputs to the phrenic motoneurons. Thus, the locomotor CPG provides a suitable amount of inputs to the functionally proper motoneurons. This preparation will be useful to explore how the respiratory and locomotor CPGs select proper motoneurons to give synaptic inputs and are coordinated with each other.

A CORDIC based real-time implementation and analysis of a respiratory central pattern generator

Central pattern generators (CPGs) are dedicated neural circuits which can generate rhythmic motor patterns even in absence of sensory input with extraordinary robustness and flexibility. In this paper, a biologically realistic model of a respiratory CPG with four neurons is implemented on a reconfigurable Field-Programmable Gate Array (FPGA) system. Considering the limitations of hardware resources, we first propose a modified respiratory CPG model with Coordinate Rotation Digital Computer (CORDIC) algorithm to save limited resources and reduce complexity. And then, all the multipliers are replaced with a method which is appropriate and effective for hardware implementation to avoid the use of the area-intensive multipliers. The implementation results show that rhythmic oscillations are successfully generated by the respiratory CPG network and the resource utilization is greatly reduced, which shows the potential for building large-scale spiking neural networks. The proposed high-performance and real-time implementation of the respiratory CPG network on the FPGA system can speed up the process to gain new insights into the respiratory network and can also be developed into applications for respiratory rhythm generation and modulation.

Baclofen destabilises breathing during sleep in healthy humans: A randomised, controlled, double-blind crossover trial.

Periodic breathing is frequent in patients with severe heart failure. Apart from being an indicator of severity, periodic breathing has its own deleterious consequences (sleep-related oxygen desaturations, sleep fragmentation), which justifies attempts to correct it irrespective of the underlying disease. Animal models and human data suggest that baclofen can reconfigure respiratory central pattern generators. We hypothesised that baclofen, a GABAB agonist, may thus be able to correct periodic breathing in humans. Healthy volunteers were exposed to hypoxia during sleep. Participants who developed periodic breathing (n = 14 [53 screened]) were randomly assigned to double-blind oral baclofen (progressively increased to 60 mg/d) or placebo. The primary outcome was the coefficient of variation (CoVar) of respiratory cycle total time considered as an indicator of breathing irregularity. Secondary outcomes included the CoVar of tidal volume, apnoea-hypopnoea index, sleep fragmentation index and ventilatory complexity (noise limit). The analysis was conducted in 9 subjects after exclusion of incomplete datasets. CoVar of respiratory cycle total time significantly increased with baclofen during non-rapid eye movement sleep (median with placebo 56.00% [37.63-78.95]; baclofen 85.42% [68.37-86.40], P = .020; significant difference during the N1-N2 phases of sleep but not during the N3 phase). CoVar of tidal volume significantly increased during N1-N2 sleep. The apnoea-hypopnoea index, sleep fragmentation index and ventilatory complexity were not significantly different between placebo and baclofen. Baclofen did not stabilise breathing in our model. On the contrary, it increased respiratory variability. Baclofen should probably not be used in patients with or at risk of periodic breathing.

Inhibitory control of active expiration by the Bötzinger complex in rats.

Contraction of abdominal muscles at the end of expiration during metabolic challenges (such as hypercapnia and hypoxia) improves pulmonary ventilation. The emergence of this active expiratory pattern requires the recruitment of the expiratory oscillator located on the ventral surface of the medulla oblongata. Here we show that an inhibitory circuitry located in the Bötzinger complex is an important source of inhibitory drive to the expiratory oscillator. This circuitry, mediated by GABAergic and glycinergic synapses, provides expiratory inhibition that restrains the expiratory oscillator under resting condition and regulates the formation of abdominal expiratory activity during active expiration. By combining experimental and modelling approaches, we propose the organization and connections within the respiratory network that control the changes in the breathing pattern associated with elevated metabolic demand. The expiratory neurons of the Bötzinger complex (BötC) provide inhibitory inputs to the respiratory network, which, during eupnoea, are critically important for respiratory phase transition and duration control. Here, we investigated how the BötC neurons interact with the expiratory oscillator located in the parafacial respiratory group (pFRG) and control the abdominal activity during active expiration. Using the decerebrated, arterially perfused in situ preparations of juvenile rats, we recorded the activity of expiratory neurons and performed pharmacological manipulations of the BötC and pFRG during hypercapnia or after the exposure to short-term sustained hypoxia - conditions that generate active expiration. The experimental data were integrated in a mathematical model to gain new insights into the inhibitory connectome within the respiratory central pattern generator. Our results indicate that the BötC neurons may establish mutual connections with the pFRG, providing expiratory inhibition during the first stage of expiration and receiving excitatory inputs during late expiration. Moreover, we found that application of GABAergic and glycinergic antagonists in the BötC caused opposing effects on abdominal expiratory activity, suggesting complex inhibitory circuitry within the BötC. Using mathematical modelling, we propose that the BötC network organization and its interactions with the pFRG restrain abdominal activity under resting conditions and contribute to abdominal expiratory pattern formation during active expiration observed during hypercapnia or after the exposure to short-term sustained hypoxia.

Neurons in the Intermediate Reticular Nucleus Coordinate Postinspiratory Activity, Swallowing, and Respiratory-Sympathetic Coupling in the Rat.

Breathing results from sequential recruitment of muscles in the expiratory, inspiratory, and postinspiratory (post-I) phases of the respiratory cycle. Here we investigate whether neurons in the medullary intermediate reticular nucleus (IRt) are components of a central pattern generator (CPG) that generates post-I activity in laryngeal adductors and vasomotor sympathetic nerves and interacts with other members of the central respiratory network to terminate inspiration. We first identified the region of the (male) rat IRt that contains the highest density of lightly cholinergic neurons, many of which are glutamatergic, which aligns well with the putative postinspiratory complex in the mouse (Anderson et al., 2016). Acute bilateral inhibition of this region reduced the amplitudes of post-I vagal and sympathetic nerve activities. However, although associated with reduced expiratory duration and increased respiratory frequency, IRt inhibition did not affect inspiratory duration or abolish the recruitment of post-I activity during acute hypoxemia as predicted. Rather than representing an independent CPG for post-I activity, we hypothesized that IRt neurons may instead function as a relay that distributes post-I activity generated elsewhere, and wondered whether they could be a site of integration for para-respiratory CPGs that drive the same outputs. Consistent with this idea, IRt inhibition blocked rhythmic motor and autonomic components of fictive swallow but not swallow-related apnea. Our data support a role for IRt neurons in the transmission of post-I and swallowing activity to motor and sympathetic outputs, but suggest that other mechanisms also contribute to the generation of post-I activity.SIGNIFICANCE STATEMENT Interactions between multiple coupled oscillators underlie a three-part respiratory cycle composed from inspiratory, postinspiratory (post-I), and late-expiratory phases. Central post-I activity terminates inspiration and activates laryngeal motoneurons. We investigate whether neurons in the intermediate reticular nucleus (IRt) form the central pattern generator (CPG) responsible for post-I activity. We confirm that IRt activity contributes to post-I motor and autonomic outputs, and find that IRt neurons are necessary for activation of the same outputs during swallow, but that they are not required for termination of inspiration or recruitment of post-I activity during hypoxemia. We conclude that this population may not represent a distinct CPG, but instead may function as a premotor relay that integrates activity generated by diverse respiratory and nonrespiratory CPGs.

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.

Increasing Local Excitability of Brainstem Respiratory Nuclei Reveals a Distributed Network Underlying Respiratory Motor Pattern Formation.

The core circuit of the respiratory central pattern generator (rCPG) is located in the ventrolateral medulla, especially in the pre-Bötzinger complex (pre-BötC) and the neighboring Bötzinger complex (BötC). To test the hypothesis that this core circuit is embedded within an anatomically distributed pattern-generating network, we investigated whether local disinhibition of the nucleus tractus solitarius (NTS), the Kölliker-Fuse nuclei (KFn), or the midbrain periaqueductal gray area (PAG) can similarly affect the respiratory pattern compared to disinhibition of the pre-BötC/BötC core. In arterially-perfused brainstem preparations of rats, we recorded the three-phase respiratory pattern (inspiration, post-inspiration and late-expiration) from phrenic and vagal nerves before and after bilateral microinjections of the GABA(A)R antagonist bicuculline (50 nl, 10 mM). Local disinhibition of either NTS, pre-BötC/BötC, or KFn, but not PAG, triggered qualitatively similar disruptions of the respiratory pattern resulting in a highly significant increase in the variability of the respiratory cycle length, including inspiratory and expiratory phase durations. To quantitatively analyze these motor pattern perturbations, we measured the strength of phase synchronization between phrenic and vagal motor outputs. This analysis showed that local disinhibition of all brainstem target nuclei, but not the midbrain PAG, significantly decreased the strength of phase synchronization. The convergent perturbations of the respiratory pattern suggest that the rCPG expands rostrally and dorsally from the designated core but does not include higher mid-brain structures. Our data also suggest that excitation-inhibition balance of respiratory network synaptic interactions critically determines the network dynamics that underlie vital respiratory rhythm and pattern formation.

Resetting the Respiratory Rhythm with a Spinal Central Pattern Generator.

There is evidence that a variety of central and afferent stimuli, including swallowing, can produce phase resetting in the respiratory rhythmicity. Also, there are reports about the intrinsic linkage between locomotion and respiration. However, little is known about the interaction between the central pattern generators (CPGs) for scratching and respiration. The present study aims to examine whether the activation of scratching CPG produces phase resetting of the respiratory rhythm. We employed decerebrate cats to apply brief tactile stimuli to the pinna during the inspiratory-expiratory transition. We observed that those stimuli to the pinna not eliciting fictive scratching did not reset the respiratory rhythm. However, when the pinna stimuli elicited fictive scratching, then the respiratory rhythm exhibited a significant phase resetting. We also found interneurons in the medulla oblongata exhibiting phase resetting related to scratching-CPG episodes. This second finding suggests that this type of resetting involves brainstem components of the respiratory CPG. These results shed new light on the resetting action from a spinal CPG on the respiratory rhythm.

Activation of Phox2b-Expressing Neurons in the Nucleus Tractus Solitarii Drives Breathing in Mice.

The nucleus tractus solitarii (NTS) is implicated in the control of breathing, but the neuronal phenotype and circuit mechanism involved in such a physiological function remain incompletely understood. This study focused on the respiratory role of paired-like homeobox 2b gene (Phox2b)-expressing NTS neurons and sought to determine whether selective stimulation of this set of neurons activates breathing in male mice. A Cre-dependent vector encoding a Gq-coupled human M3 muscarinic receptor (hM3Dq) was microinjected into the NTS of Phox2b-Cre transgenic mice. The hM3Dq-transduced neurons were pharmacologically activated in conscious mice while respiratory effects were measured by plethysmography. We demonstrate that chemogenetic stimulation of Phox2b-expressing NTS neurons significantly increased baseline minute volume via an increase in respiratory frequency rather than tidal volume. Chemogenetic stimulation also synergized with moderate CO2 stimulation to enhance pulmonary ventilatory response. Selective ablation of Phox2b-expressing NTS neurons notably attenuated a hypercapnic ventilatory response. Moreover, histological evidence revealed that stimulation of Phox2b-expressing NTS neurons increased neuronal activity of the preBötzinger complex. Finally, we presented the neuroanatomical evidence of direct projection of Phox2b-expressing NTS neurons to putative respiratory central pattern generator. Overall, these findings suggest that selective activation of Phox2b-expressing NTS neurons potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator and this group of neurons is also required for the hypercapnic ventilatory response.SIGNIFICANCE STATEMENT The nucleus tractus solitarii (NTS) has been implicated in the control of breathing. The paired-like homeobox 2b gene (Phox2b) is the disease-defining gene for congenital central hypoventilation syndrome and is restrictively present in brainstem nucleus, including the NTS. Using a chemogenetic approach, we demonstrate herein that selective stimulation of Phox2b-expressing NTS neurons vigorously potentiates baseline pulmonary ventilation via an excitatory drive to respiratory central pattern generator in rodents. Genetic ablation of these neurons attenuates the hypercapnic ventilatory response. We also suggest that a fraction of Phox2b-expressing neurons exhibit CO2 sensitivity and presumably function as central respiratory chemoreceptors. The methodology is expected to provide a future applicability to the patients with sleep-related hypoventilation or apnea.

Interaction between the retrotrapezoid nucleus and the parafacial respiratory group to regulate active expiration and sympathetic activity in rats.

The retrotrapezoid nucleus (RTN) contains chemosensitive cells that distribute CO2-dependent excitatory drive to the respiratory network. This drive facilitates the function of the respiratory central pattern generator (rCPG) and increases sympathetic activity. It is also evidenced that during hypercapnia, the late-expiratory (late-E) oscillator in the parafacial respiratory group (pFRG) is activated and determines the emergence of active expiration. However, it remains unclear the microcircuitry responsible for the distribution of the excitatory signals to the pFRG and the rCPG in conditions of high CO2. Herein, we hypothesized that excitatory inputs from chemosensitive neurons in the RTN are necessary for the activation of late-E neurons in the pFRG. Using the decerebrated in situ rat preparation, we found that lesions of neurokinin-1 receptor-expressing neurons in the RTN region with substance P-saporin conjugate suppressed the late-E activity in abdominal nerves (AbNs) and sympathetic nerves (SNs) and attenuated the increase in phrenic nerve (PN) activity induced by hypercapnia. On the other hand, kynurenic acid (100 mM) injections in the pFRG eliminated the late-E activity in AbN and thoracic SN but did not modify PN response during hypercapnia. Iontophoretic injections of retrograde tracer into the pFRG of adult rats revealed labeled phox2b-expressing neurons within the RTN. Our findings are supported by mathematical modeling of chemosensitive and late-E populations within the RTN and pFRG regions as two separate but interacting populations in a way that the activation of the pFRG late-E neurons during hypercapnia require glutamatergic inputs from the RTN neurons that intrinsically detect changes in CO2/pH.

Central Respiration and Mechanical Ventilation in the Gating of Swallow With Breathing.

Swallow-breathing coordination safeguards the lower airways from tracheal aspiration of bolus material as it moves through the pharynx into the esophagus. Impaired movements of the shared muscles or structures of the aerodigestive tract, or disruptions in the interaction of brainstem swallow and respiratory central pattern generators (CPGs) result in dysphagia. To maximize lower airway protection these CPGs integrate respiratory rhythm generation signals and vagal afferent feedback to synchronize swallow with breathing. Despite extensive study, the roles of central respiratory activity and vagal feedback from the lungs as key elements for effective swallow-breathing coordination remain unclear. The effect of altered timing of bronchopulmonary vagal afferent input on swallows triggered during electrical stimulation of the superior laryngeal nerves or by injection of water into the pharyngeal cavity was studied in decerebrate, paralyzed, and artificially ventilated cats. We observed two types of single swallows that produced distinct effects on central respiratory-rhythm across all conditions: post-inspiratory type swallows disrupted central-inspiratory activity without affecting expiration, whereas expiratory type swallows prolonged expiration without affecting central-inspiratory activity. Repetitive swallows observed during apnea reset the E2 phase of central respiration and produced facilitation of swallow motor output nerve burst durations. Moreover, swallow initiation was negatively modulated by vagal feedback and was reset by lung inflation. Collectively, these findings support a novel model of reciprocal inhibition between the swallow CPG and inspiratory or expiratory cells of the respiratory CPG where lung distension and phases of central respiratory activity represent a dual peripheral and central gating mechanism of swallow-breathing coordination.

New insights into a decerebrate feline model of swallow‐breathing coordination

In the medulla, the swallow and respiratory central pattern generators (CPGs) interact to coordinate swallow initiation and permit swallow execution during the expiratory phase of breathing. Disruption of swallow‐breathing coordination can result in dysphagia, which is a major cause of morbidity and mortality in many neuro‐muscular diseases, especially stroke. Despite extensive study, the precise mechanisms of swallow‐breathing coordination are not known. We tested the hypothesis that both central inspiratory activity and vagal feedback from the lungs are key elements for effective swallow‐breathing coordination. Swallows were triggered by electrical stimulation of the superior laryngeal nerves (SLN) or by injection of water into the pharyngeal cavity in decerebrate, paralyzed and artificially ventilated cats while motor activities were recorded from phrenic, hypoglossal, lumbar and vagal nerves. Mechanical ventilation was either triggered by central inspiratory activity or set independently from the phrenic activity to alter the timing of bronchopulmonary vagal afferent input and allow analysis of its influence on swallow‐breathing coordination. We observed two types of swallows that produced opposite effects on central respiratory‐rhythm resetting across all conditions: post‐Inspiratory type swallows disrupted central I activity without affecting expiration, whereas expiratory type swallows prolonged expiration without affecting central inspiratory activity. Repetitive swallows observed during apnea reset the E2 phase of central respiration and produced facilitation of swallow motor output. Moreover, swallow initiation was negatively modulated by vagal feedback and was reset by lung inflation. Collectively, these findings support a reciprocal inhibition between central inspiration and the swallow CPG and they are consistent with a role for pulmonary stretch receptors in the coordination between brainstem CPGs for breathing and swallowing. They may also prove relevant for healthcare management of dysphagia.Support or Funding InformationOT2OD023854‐01, HL109025This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.