Like other brain circuits, the brainstem respiratory network is continually modulated by neurotransmitters that activate slow metabotropic receptors. In many cases, activation of these receptors only subtly modulates the respiratory motor pattern. However, activation of some receptor types evokes the arrest of the respiratory motor pattern as can occur following the activation of μ-opioid receptors. We propose that the varied effects of neuromodulation on the respiratory motor pattern depend on the pattern of neuromodulator receptor expression and their influence on the excitability of their post-synaptic targets. Because a comprehensive characterization of these cellular properties across the respiratory network remains challenging, we test our hypothesis by combining computational modeling with ensemble electrophysiologic recording in the pre-Bötzinger complex (pre-BötC) using high-density multi-electrode arrays (MEA). Our computational model encapsulates the hypothesis that neuromodulatory transmission is organized asymmetrically across the respiratory network to promote rhythm and pattern generation. To test this hypothesis, we increased the strength of subsets of neuromodulatory connections in the model and used selective agonists in situ while monitoring pre-BötC ensemble activities. The in silico simulations of increasing slow inhibition were consistent with experiments examining the effect of systemic administration of the 5HT1aR agonist 8-OH-DPAT. Similarly, the effects of increasing slow excitation in the model were experimentally confirmed in pre-BötC ensemble activities before and after systemic administration of the μ-opioid receptor agonist fentanyl. We conclude that asymmetric neuromodulation can contribute to respiratory rhythm and pattern generation and accounts for its varied effects on breathing.

OBJECTIVE:Desensitization of the larynx significantly impairs swallowing function in patients with tracheostomies. Although it is clinically relevant that adequate airway clearance helps to improve swallowing responsiveness, a robust theoretical proposal has not been provided. Hence, we investigated the impact of prolonged fluid irritation of the trachea and larynx on the swallowing reflex. We additionally analyzed discharge activities of the swallowing-related neurons in presence and absence of airway fluid accumulation. METHODS:We analyzed the likelihood of swallowing evoked by electrical stimulation of the superior laryngeal nerve or oral water injection before and after removal of fluid accumulation in the trachea and larynx using air flow in perfused rats (n = 15). We then transected the superior laryngeal and vagus nerves which convey critical laryngotracheal sensory information. We recorded changes in the neuronal activities in the presence or absence of airway fluid contamination using multi-electrode arrays (n = 8). RESULTS:The frequency of swallows was increased by the removal of airway fluid accumulation and this change lasted at least 60 min. The effects were abolished following the section of either the superior laryngeal or vagus nerve. Specifically, we found that centrally recorded swallowing neurons showed changes in spontaneous discharge activities in the presence or absence of fluid-related laryngotracheal sensory stimulation. CONCLUSION:A diminished likelihood of swallowing caused by prolonged airway irritation can be cautiously improved through airway clearance with respiratory airflow via central and peripheral neuromodulation. Our findings provide a fundamental mechanism to recover impaired laryngotracheal susceptibility for severe dysphagia such as post-tracheostomy dysphagia.

The nucleus tractus solitarius (NTS) contains neurons that relay sensory swallowing commands information from the oropharyngeal cavity and swallowing premotor neurons of the dorsal swallowing group (DSG). However, the spatio-temporal dynamics of the interplay between the sensory relay and the DSG is not well understood. Here, we employed fluorescence imaging after microinjection of the calcium indicator into the NTS in an arterially perfused brainstem preparation of rat (n = 8) to investigate neuronal population activity in the NTS in response to superior laryngeal nerve (SLN) stimulation. Respiratory and swallowing motor activities were determined by simultaneous recordings of phrenic and vagal nerve activity (PNA, VNA). The analysis of SLN stimulation near the threshold triggering a swallowing allowed us to analyze Ca2+ signals related to the sensory relay and the DSG. We show that activation of sensory relay neurons triggers spatially confined Ca2+ signals exclusively unilateral to the stimulated SLN at short latencies (114.3 ± 94.4 ms). However, SLN-evoked swallowing triggered Ca2+ signals bilaterally at longer latencies (200 ± 145.2 ms) and engaged anatomically distributed DSG activity across the dorsal medulla oblongata. The Ca2+ signals originating from the DSG preceded evoked VNA swallow motor bursts, thus the swallowing premotor neurons that drive laryngeal motor pools are located outside the DSG. In conclusion, the study illuminates the spatial–temporal features of sensory-motor integration of swallowing in the NTS and further supports the hypothesis that the NTS harbors swallowing pre-motor neurons that may generate the swallowing motor activity, while first-order pre-motor pools are located outside the DSG.

Shape and size of the nasopharyngeal airway is controlled by muscles innervated facial, glossopharyngeal, vagal, and hypoglossal cranial nerves. Contrary to brainstem networks that drive facial, vagal and hypoglossal nerve activities (FNA, VNA, HNA) the discharge patterns and origins of glossopharyngeal nerve activity (GPNA) remain poorly investigated.Here, an in situ perfused brainstem preparation (n=19) was used for recordings of GPNA in relation to phrenic (PNA), FNA, VNA and HNA. Brainstem transections were performed (n=10/19) to explore the role of pontomedullary synaptic interactions in generating GPNA.GPNA generally mirrors FNA and HNA discharge patterns and displays pre-inspiratory activity relative to the PNA, followed by robust inspiratory discharge in coincidence with PNA. Postinspiratory (early expiratory) discharge was, contrary to VNA, generally absent in FNA, GPNA or HNA. As described previously FNA and HNA discharge was virtually eliminated after pontomedullary transection while an apneustic inspiratory motor discharge was maintained in PNA, VNA and GPNA. After brainstem transection GPNA displayed an increased tonic activity starting during mid-expiration and thus developed prolonged pre-inspiratory activity compared to control.In conclusion respiratory GPNA reflects FNA and HNA which implies similar function in controlling upper airway patency during breathing. That GPNA preserved its pre-inspiratory/inspiratory discharge pattern in relation PNA after pontomedullary transection suggest that GPNA premotor circuits may have a different anatomical distribution compared HNA and FNA and thus may therefore hold a unique role in in preserving airway patency.

Breathing is the only vital function that can be volitionally controlled. However, a detailed understanding how volitional (cortical) motor commands can transform vital breathing activity into adaptive breathing patterns that accommodate orofacial behaviors such as swallowing, vocalization or sniffing remains to be developed. Recent neuroanatomical tract tracing studies have identified patterns and origins of descending forebrain projections that target brain nuclei involved in laryngeal adductor function which is critically involved in orofacial behavior. These nuclei include the midbrain periaqueductal gray and nuclei of the respiratory rhythm and pattern generating network in the brainstem, specifically including the pontine Kölliker-Fuse nucleus and the pre-Bötzinger complex in the medulla oblongata. This review discusses the functional implications of the forebrain-brainstem anatomical connectivity that could underlie the volitional control and coordination of orofacial behaviors with breathing.

Background Sepsis has a high mortality rate due to multiple organ failure. However, the influence of peripheral inflammation on brainstem autonomic and respiratory circuits in sepsis is poorly understood. Our working hypothesis is that peripheral inflammation affects central autonomic circuits and consequently contributes to multiorgan failure in sepsis. Methods In an Escherichia coli ( E. coli )–fibrin clot model of peritonitis, we first recorded ventilatory patterns using plethysmography before and 24 h after fibrin clot implantation. To assess whether peritonitis was associated with brainstem neuro-inflammation, we measured cytokine and chemokine levels in Luminex assays. To determine the effect of E. coli peritonitis on brainstem function, we assessed sympatho-respiratory nerve activities at baseline and during brief (20 s) hypoxemic ischemia challenges using in situ-perfused brainstem preparations (PBPs) from sham or infected rats. PBPs lack peripheral organs and blood, but generate vascular tone and in vivo rhythmic activities in thoracic sympathetic (tSNA), phrenic and vagal nerves. Results Respiratory frequency was greater ( p < 0.001) at 24 h post-infection with E. coli than in the sham control. However, breath-by-breath variability and total protein in the BALF did not differ. IL-1β ( p < 0.05), IL-6 ( p < 0.05) and IL-17 ( p < 0.04) concentrations were greater in the brainstem of infected rats. In the PBP, integrated tSNA ( p < 0.05) and perfusion pressure were greater ( p < 0.001), indicating a neural-mediated pathophysiological high sympathetic drive. Moreover, respiratory frequency was greater ( p < 0.001) in PBPs from infected rats than from sham rats. Normalized phase durations of inspiration and expiration were greater ( p < 0.009, p < 0.015, respectively), but the post-inspiratory phase ( p < 0.007) and the breath-by-breath variability ( p < 0.001) were less compared to sham PBPs. Hypoxemic ischemia triggered a biphasic response, respiratory augmentation followed by depression. PBPs from infected rats had weaker respiratory augmentation ( p < 0.001) and depression ( p < 0.001) than PBPs from sham rats. In contrast, tSNA in E. coli -treated PBPs was enhanced throughout the entire response to hypoxemic ischemia ( p < 0.01), consistent with sympathetic hyperactivity. Conclusion We show that peripheral sepsis caused brainstem inflammation and impaired sympatho-respiratory motor control in a single day after infection. We conclude that central sympathetic hyperactivity may impact vital organ systems in sepsis.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

The mammalian three-phase respiratory motor pattern of inspiration, post-inspiration and expiration is expressed in spinal and cranial motor nerve discharge and is generated by a distributed ponto-medullary respiratory pattern generating network. Respiratory motor pattern generation depends on a rhythmogenic kernel located within the pre-Bötzinger complex (pre-BötC). In the present study, we tested the effect of unilateral and bilateral inactivation of the pre-BötC after local microinjection of the GABAA receptor agonist isoguvacine (10 mM, 50 nl) on phrenic (PNA), hypoglossal (HNA) and vagal nerve (VNA) respiratory motor activities in an in situ perfused brainstem preparation of rats. Bilateral inactivation of the pre-BötC triggered cessation of phrenic (PNA), hypoglossal (HNA) and vagal (VNA) nerve activities for 15-20 min. Ipsilateral isoguvacine injections into the pre-BötC triggered transient (6-8 min) cessation of inspiratory and post-inspiratory VNA (p < 0.001) and suppressed inspiratory HNA by - 70 ± 15% (p < 0.01), while inspiratory PNA burst frequency increased by 46 ± 30% (p < 0.01). Taken together, these observations confirm the role of the pre-BötC as the rhythmogenic kernel of the mammalian respiratory network in situ and highlight a significant role for the pre-BötC in the transmission of vagal inspiratory and post-inspiratory pre-motor drive to the nucleus ambiguus.

Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.

The mammalian three-phase respiratory motor pattern of inspiration, post-inspiration and expiration is expressed in spinal and cranial motor nerve activities. This pattern is generated by a hierarchical brainstem-wide pre-motor network. However, the local rhythmogenic circuit of the pre-Bötzinger complex (pre-BötC) was established as the ‘noeud vitale’ necessary and sufficient to initiate inspiration. In present study, we tested the effect of unilateral and bilateral inactivation of the pre-BötC (microinjection of the GABA A receptor agonist isoguvacine 10 mM, 50 nl) on respiratory motor activities in an in situ perfused brainstem preparation of rat. As expected, bilateral inactivation of the pre-BötC triggered cessation of phrenic (PNA), hypoglossal (HNA) and vagal (VNA) nerve activities for 15-20 min. Partial recovery from bilateral isoguvacine injection was characterized by erratic activity in all recorded motor nerves reminiscent to patterns observed after disturbed excitatory-inhibitory balance within the local pre-BötC circuit. Surprisingly, ipsilateral isoguvacine injections into the pre-BötC triggered transient (6-8 min) cessation of inspiratory and post-inspiratory VNA (p<0.001) and suppressed inspiratory HNA by -70 ± 15% (p<0.01), while inspiratory PNA burst frequency increased by 46 ± 30% (p<0.01). Taken together, these observations confirm the role of the pre-BötC as the ‘noeud vitale’ of the mammalian respiratory network in situ and highlight a significant role for the pre-BötC in the expression of vagal post-inspiratory and inspiratory activity.

AbstractRespiration and swallowing are vital orofacial motor behaviours that require the coordination of the activity of two brainstem central pattern generators (r-CPG, sw-CPG). Here, we use computational modelling to further elucidate the neural substrate for breathing-swallowing coordination. We progressively construct several computational models of the breathing-swallowing circuit, starting from two interacting half-centre oscillators for each CPG. The models are based exclusively on neuronal nodes with spike-frequency adaptation, having a parsimonious description of intrinsic properties. These basic models undergo a stepwise integration of synaptic connectivity between central sensory relay, sw- and r-CPG neuron populations to match experimental data obtained in a perfused brainstem preparation. In the model, stimulation of the superior laryngeal nerve (SLN, 10s) reliably triggers sequential swallowing with concomitant glottal closure and suppression of inspiratory activity, consistent with the motor pattern in experimental data. Short SLN stimulation (100ms) evokes single swallows and respiratory phase resetting yielding similar experimental and computational phase response curves. Subsequent phase space analysis of model dynamics provides further understanding of SLN-mediated respiratory phase resetting. Consistent with experiments, numerical circuit-busting simulations show that deletion of ponto-medullary synaptic interactions triggers apneusis and eliminates glottal closure during sequential swallowing. Additionally, systematic variations of the synaptic strengths of distinct network connections predict vulnerable network connections that can mediate clinically relevant breathing-swallowing disorders observed in the elderly and patients with neurodegenerative disease. Thus, the present model provides novel insights that can guide future experiments and the development of efficient treatments for prevalent breathing-swallowing disorders.Key pointsThe coordination of breathing and swallowing depends on synaptic interactions between two functionally distinct central pattern generators (CPGs) in the dorsal and ventral brainstem.We model both CPGs as half-centre oscillators with spike-frequency adaptation to identify the minimal connectivity sufficient to mediate physiologic breathing-swallowing interactions.The resultant computational model(s) can generate sequential swallowing patterns including concomitant glottal closure during simulated 10s stimulation of the superior laryngeal nerve (SLN) consistent with experimental data.In silico, short (100 ms) SLN stimulation triggers a single swallow which modulates the respiratory cycle duration consistent with experimental recordings.By varying the synaptic connectivity strengths between the two CPGs and the sensory relay neurons, and by inhibiting specific nodes of the network, the model predicts vulnerable network connections that may mediate clinically relevant breathing-swallowing disorders.

Cardio-respiratory coupling is reflected as respiratory sinus arrhythmia (RSA) and inspiratory-related bursting of sympathetic nerve activity. Inspiratory-related inhibitory and/or postinspiratory-related excitatory drive of cardiac vagal motoneurons (CVMs) can generate RSA. Since respiratory oscillations may depend on synaptic inhibition, we investigated the effects of blocking glycinergic neurotransmission (systemic and local application of the glycine receptor (GlyR) antagonist, strychnine) on the expression of the respiratory motor pattern, RSA and sympatho-respiratory coupling. We recorded heart-rate, phrenic, recurrent laryngeal and thoracic sympathetic nerve activities (PNA, RLNA, t-SNA) in a working-heart-brainstem preparation of rats, and show that systemic strychnine (50–200 ​nM) abolished RSA and triggered a shift of postinspiratory RLNA into inspiration, while t-SNA remained unchanged. Bilateral strychnine microinjection into the ventrolateral medullary area containing CVMs and laryngeal motoneurons (LMNs) of the nucleus ambiguus (NA/CVLM), the nucleus tractus solitarii, pre-Bötzinger Complex, Bötzinger Complex or Kölliker-Fuse nuclei revealed that only NA/CVLM strychnine microinjections mimicked the effects of systemic application. In all other target nuclei, except the Bötzinger Complex, GlyR-blockade attenuated the inspiratory-tachycardia of the RSA to a similar degree while evoking only a modest change in respiratory motor patterning, without changing the timing of postinspiratory-RLNA, or t-SNA. Thus, glycinergic inhibition at the motoneuronal level is involved in the generation of RSA and the separation of inspiratory and postinspiratory bursting of LMNs. Within the distributed ponto-medullary respiratory pre-motor network, local glycinergic inhibition contribute to the modulation of RSA tachycardia, respiratory frequency and phase duration but, surprisingly it had no major role in the mediation of respiratory-sympathetic coupling.

Synaptic activities of the periaqueductal gray (PAG) can modulate or appropriate the respiratory motor activities in the context of behavior and emotion via descending projections to nucleus retroambiguus. However, alternative anatomical pathways for the mediation of PAG-evoked respiratory modulation via core nuclei of the brainstem respiratory network remains only partially described. We injected the retrograde tracer Cholera toxin subunit B (CT-B) in the pontine Kölliker-Fuse nucleus (KFn, n =5), medullary Bötzinger (BötC, n =3) and pre-Bötzinger complexes (pre-BötC; n =3), and the caudal raphé nuclei ( n =3), and quantified the ascending and descending connectivity of the PAG. CT-B injections in the KFn, pre-BötC, and caudal raphé, but not in the BötC, resulted in CT-B-labeled neurons that were predominantly located in the lateral and ventrolateral PAG columns. In turn, CT-B injections into the lateral and ventrolateral PAG columns ( n =4) yield the highest numbers of CT-B-labeled neurons in the KFn and far fewer numbers of labeled neurons in the pre-BötC and caudal raphé. Analysis of the relative projection strength revealed that the KFn shares the densest reciprocal connectivity with the PAG (ventrolateral and lateral columns, in particular). Overall, our data imply that the PAG may engage a distributed respiratory rhythm and pattern generating network beyond the nucleus retroambiguus to mediate downstream modulation of breathing. However, the reciprocal connectivity of the KFn and PAG suggests specific roles for synaptic interaction between these two nuclei that are most likely related to the regulation of upper airway patency during vocalization or other volitional orofacial behaviors. Highlights The lateral and ventrolateral PAG project to the primary respiratory network. The Kölliker-Fuse nucleus shares the densest reciprocal connectivity with the PAG. The Bötzinger complex appears to have very little connectivity with the PAG.

We thank Dr Swen Hülsmann for his thoughtful and supportive letter to the editor regarding our recent publication (Dhingra et al. 2020). Dr Hülsmann (2021) noted the absence of a discussion of the role of the recently described post-inspiratory complex (PiCo) in the generation of the post-inspiratory phase of the breathing pattern. Indeed, post-inspiratory control of glottal adduction is an essential element controlling expiratory airflow during the eupneic three-phase breathing pattern and during both expulsive (coughing, sneezing, etc.) and non-respiratory behaviours (swallowing, vocalisation, etc.); and loss of airway-protective post-inspiratory glottal constriction, for instance during swallowing, may result in aspiration pneumonia in neurodegenerative diseases (Dutschmann et al. 2014; Anderson et al. 2016). In our study, it was striking that respiratory local field potentials (LFPs) occurring at the transition from inspiration (I) to post-inspiration (PI) were the most widely distributed of any respiratory phase transition. In general, LFPs are thought to largely reflect the summation of synchronous synaptic conductances because of their long-time constants. Therefore, our observation suggests that synaptic conductances within many respiratory network areas were maximal precisely at the I–PI transition. This observation certainly negates the conceptual model put forward in the ‘triple-oscillator hypothesis’ that interactions between only the pre-Bötzinger complex, PiCo and parafacial respiratory group/retro-trapezoid nucleus account for the three-phase respiratory motor pattern (Anderson & Ramirez, 2017). This conclusion is further underscored by our previous publication, which demonstrated at the motor output level that perfused brainstem preparations containing all elements of the ‘triple-oscillator hypothesis’, but lacking pontine components of the network, are insufficient to generate eupnea, and instead produce a pathologic apneustic inspiratory motor pattern expressed synchronously on vagal, hypoglossal and phrenic nerves (Jones & Dutschmann, 2016). As noted in Dr Hülsmann’s letter, Anderson et al. (2016) has 100+ citations. Yet, to our knowledge, only one of those studies sought to confirm its findings (Toor et al. 2019). Together, these two papers fail to demonstrate that the PiCo is necessary for the generation of the eupneic post-inspiratory motor pattern under intact network conditions, especially when compared with similar perturbations of the activity of the Kölliker–Fuse nuclei (KFn). Inhibition of the KFn with isoguvacine completely ablates post-inspiratory laryngeal, cervical vagal nerve activity (cVNA) (Dutschmann & Herbert, 2006; Dutschmann et al. 2021), whereas inhibition of the PiCo with isoguvacine only reduces the peak amplitude of cVNA (Toor et al. 2019). Modulation of KFn activity with DAMGO, a μ-opioid receptor agonist, also results in a complete ablation of post-inspiratory cVNA (Levitt et al. 2015), whereas modulation of PiCo activity with DAMGO only reduces the amplitude of cVNA (Anderson et al. 2016). Clearly, the pons is necessary for the expression of post-inspiratory cVNA, and hence the three-phase respiratory motor pattern, and its absence disrupts PI more than any medullary counterpart including the PiCo. On the other hand, our recent work suggests to us that focusing on the pons, medulla or any particular respiratory area in isolation might also be short-sighted since local modulation of excitability within any key respiratory network area is sufficient to severely disrupt the three-phase respiratory motor pattern (Dhingra et al. 2019a,b), and since LFPs associated with the inspiratory off-switch (e.g. I–PI transition) can be detected in areas even we did not previously consider (Dhingra et al. 2020). Alternatively, Toor et al. (2019) convincingly demonstrated that the PiCo is necessary for sequential swallowing in vivo and may relate more to the ventral swallowing group (for review, see Jean, 2001). This interpretation is consistent with the fact that breathing and swallowing are coupled via the post-inspiratory phase (Dick et al. 1993; Oku, 2020). Indeed, resuscitation of the eupneic three-phase respiratory motor pattern in the perfused brainstem preparation begins first with swallowing and subsequently with swallowterminated inspiratory bursts (Bautista et al. 2014). Further, inhibition of the KFn unmasks swallow-terminated inspiratory motor patterns (Bautista & Dutschmann, 2014; Bautista et al. 2014). Perhaps the primary physiological role of the PiCo is in swallowing? In our paper, we did not include a horizontal view of the dataset since this would not allow the reader to differentiate LFPs arising from dorsal versus ventral aspects of the volume. Instead, for completeness, we chose to provide the cycle-triggered average respiratory LFPs of all electrode sites in a representative recording in Fig. 2 (Dhingra et al. 2020). Examining the medial averaged LFP traces at the level of the facial nerve (Fig. 2F and G in Dhingra et al. 2020), there were some small amplitude LFPs at the I–PI transition. Perhaps these reflected the engagement of the PiCo in the transition from I to PI? However, as discussed above and in our paper, the striking feature of the dataset as a whole was the widespread engagement of respiratory network areas in the collective transition to PI, rather than the dominance of any one area. Taken together, this feature of our dataset and the absence of any other substantial evidence supporting either the PiCo as the sole respiratory area necessary for PI or the triple-oscillator hypothesis led to our decision to forgo a discussion of these latter topics. Instead, our discussion focused on what respiratory LFPs might reflect, how respiratory LFPs might arise and the broad features of their spatio-temporal patterning. In conclusion, understanding the network-level mechanisms underlying respiratory motor pattern formation in the intact brainstem clearly remains an open challenge for the field. We hope that others will see our efforts to develop an approach to map respiratory LFPs at the spatial scale of the intact network in single perfused brainstem preparations as a first step toward the goal of providing our field with the appropriate experimental tools necessary to tackle this outstanding challenge.

Key points The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem‐wide circuit are lacking. Here, we use silicon multi‐electrode arrays to record respiratory local field potentials (rLFPs) from 196–364 electrode sites within 8–10 mm3 of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post‐inspiration (PI) and late‐expiration (E2). rLFPs peaked specifically at the three respiratory phase transitions, E2–I, I–PI and PI–E2. We show, for the first time, that only the I–PI transition engages a brainstem‐wide network, and that rLFPs during the PI–E2 transition identify a hitherto unknown role for the dorsal respiratory group. Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease. AbstractWhile it is widely accepted that inspiratory rhythm generation depends on the pre‐Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network. We developed an approach using silicon multi‐electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically at the three transitions between respiratory phases: (1) from late expiration (E2) to inspiration (I), (2) from I to post‐inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions. Spatially, the E2–I, and PI–E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post‐inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group‐wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thus share a similar functional neuroanatomy. Thus, volumetric mapping of rLFPs could allow for the physiological assessment of global respiratory network organization in health and disease.

Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuraxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer Cholera toxin subunit B (CT-B) in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-Bötzinger complex (pre-BötC) or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely-labeled neurons in the forebrain 12-14 days post-injection. Overall, our data reveals that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g. sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain.

A very recent epidemiological study provides preliminary evidence that living in habitats located at 2500 m above sea level (mass) might protect from the development of severe respiratory symptoms following infection with the novel SARS-CoV-2 virus. This epidemiological finding raises the question of whether physiological mechanisms underlying the acclimatization to high altitude identifies therapeutic targets for the effective treatment of severe acute respiratory syndrome pivotal to the reduction of global mortality during the COVID-19 pandemic. This article compares the symptoms of acute mountain sickness (AMS) with those of SARS-CoV-2 infection and explores overlapping patho-physiological mechanisms of the respiratory system including impaired oxygen transport, pulmonary gas exchange and brainstem circuits controlling respiration. In this context, we also discuss the potential impact of SARS-CoV-2 infection on oxygen sensing in the carotid body. Finally, since erythropoietin (EPO) is an effective prophylactic treatment for AMS, this article reviews the potential benefits of implementing FDA-approved erythropoietin-based (EPO) drug therapies to counteract a variety of acute respiratory and non-respiratory (e.g. excessive inflammation of vascular beds) symptoms of SARS-CoV-2 infection.

Breathing can be voluntarily modulated via descending inputs from the forebrain to evoke respiratory‐related behaviours, such as vocalization, sniffing, swallowing or breath‐holding. Such behaviors require controlled laryngeal adduction and thus, are conducted during the post‐inspiratory phase of respiratory cycle. However, descending pathways that connect forebrain regions with primary post‐inspiratory control areas such as the pontine Kölliker‐Fuse nucleus (KF) and the medullary Bötzinger complex (BötC) remain to be identified. Here, we investigated the topography of forebrain descending projection neurons to a variety of bulbar respiratory nuclei.We locally microinjected the conventional retrograde tracer cholera toxin subunit B (CT‐B, 100–150nL) into the BötC, KF, the pre‐Bötzinger complex (pre‐BötC), the midline raphé nuclei and the midbrain periaqueductal gray (PAG). Twelve days after unilateral CT‐B injections, brains were sectioned (40μm) and immunohistochemically stained with an anti‐CT‐B antibody. The strength of descending projections was qualitatively assessed: as strong (+++), moderate (++) or weak (+) numbers of CT‐B labeled cell bodies.Retrogradely labelled neurons after unilateral injections into the lateral PAG confirmed the predominantly ipsilateral location of strong and moderate descending projection neurons in the cingulate (+++), pre‐limbic (+++), ectorhinal (++), motor (+++) and insular (++) cortices, the lateral septum (++), amygdala (+++) and hypothalamus (+++). In comparison, retrogradely labeled neurons after unilateral KF injection were also found ipsilaterally in the motor (++), prelimbic (++) and insular cortices (+++), the amygdala (++) and hypothalamus (+++). However, amongst all analysed descending target areas, only the KF receives substantial inputs from the ectorhinal (+++) and endopiriform (++) cortices. In addition, the medullary BötC receives weaker inputs from prelimbic (+) and insular (+) cortices and receives moderate inputs from the amygdala and hypothalamus. Descending projection neurons to the pre‐BötC were in accordance with the literature: motor (+) and insular (+) cortices, amygdala (+++) and hypothalamus (++). Finally, descending inputs to the medullary raphé obscurus and raphé magnus nuclei also arose from motor, prelimbic and insular cortices, amygdala and hypothalamus. However, these projections were significantly weaker compared to KF or PAG.The results suggest that descending forebrain projections into respiratory control areas are organized in general pathways that originate from motor, prelimbic and insular cortices as well as the amygdala and hypothalamus. However, only the KF, a key area for the gating of post‐inspiratory activity and respiratory plasticity, receives projections arising from the endopiriform and ectorhinal cortex. The functional implications of these descending control pathways need to be explored in future studies.Support or Funding InformationMelbourne Research Scholarship (University of Melbourne) [181858] to PT‐B.

The neuropeptide, relaxin‐3, is expressed by pontine nucleus incertus (NI) neurons. Relaxin‐3 and synthetic agonist peptides modulate arousal and cognitive processes via activation of the rela xin‐ family peptide 3 receptor (RXFP3). We recently demonstrated that a double‐chain RXFP3 peptidomimetic (RXFP3‐A2) in the nucleus of the solitary tract (NTS) triggered a mild stimulation of respiration and augmented the chemoreceptor reflex in an in situ perfused brainstem preparation ( Furuya et al., 2020). In the present study, we assessed the central respiratory effects of systemic application and local microinjection into the NTS, Kölliker‐Fuse nucleus (KF) or NI of a single chain RXFP3 peptidomimetic (B18) in the perfused brainstem preparation.Systemic application of B18 (2 μM) triggered a dose‐dependent increase in respiratory rate by 22 ± 8%. At this concentration of B18, the NaCN‐evoked (0.1% w/v, 100 μl, bolus injection) tachycardia of the arterial chemoreceptor reflex was augmented by 95 ± 14% compared to control (p<0.001, n=4). Local microinjections into the NTS also increased respiratory frequency (28 ± 5%, p<0.05, n=6) and enhanced the NaCN‐evoked tachycardia by 59%. Microinjections into the KF only triggered a mild increase in respiratory frequency (18 ± 7%, p<0.05, n=6) but had no effect on the NaCN‐evoked chemoreceptor reflex. Finally, microinjections into the NI (n=6) had no effect on either stationary breathing activity, or on the chemoreceptor reflex. We conclude that relaxin‐3 neurons target RXFP3 in respiratory control areas and acts as a general respiratory stimulant, causing mild increases in respiratory frequency. Importantly, RXFP3 stimulation significantly enhanced the respiratory response of arterial chemoreceptor reflex, implicating a major neuromodulatory role in this specific reflex pathway.

The perfused working heart brainstem preparation of rodents has become a widely used tool to study brainstem function. Here, we adapt this experimental technique for newborn guinea pigs (postnatal day 7-14) to develop a tool that enables investigation of airway defense mechanisms not observed in other rodents. The perfused guinea pig brainstem preparation generates a stable eupnea-like motor pattern recorded from the phrenic, recurrent laryngeal and intercostal nerves and basic cardio-respiratory reflexes, including the arterial chemoreceptor, the baroreceptor reflex. In addition a fictive laryngeal cough reflex can be reliably elicited after mechanical stimulation of the trachea. Single unit recordings within the ponto-medullary respiratory column show robust central respiratory neuronal activity. Additionally, as in other species ponto-medullary transection of the brainstem produces apneusis. The latter suggests that the preparation fully preserves ponto-medullary synaptic connectivity that is required for eupnea-like respiratory rhythm and pattern formation and the mediation of various cardio-respiratory reflexes. We conclude that this novel research tool provides an alternative to established rat and mouse preparations and may become a experimental tool for the investigation of central mechanisms that mediate laryngeal cough.