Dysphagia as a Missing Link Between Post-surgical- and Opioid-Related Pneumonia.

PURPOSE: Postoperative pneumonia remains a common complication of surgery, despite increased attention. The purpose of our study was to determine the effects of routine surgery and post-surgical opioid administration on airway protection risk. METHODS: Eight healthy adult cats were evaluated to determine changes in airway protection status and for evidence of dysphagia in two experiments. (1) In four female cats, airway protection status was tracked following routine abdominal surgery (spay surgery) plus low-dose opioid administration (buprenorphine 0.015 mg/kg, IM, q8-12 h; n = 5). (2) Using a cross-over design, four naive cats (2 male, 2 female) were treated with moderate-dose (0.02 mg/kg) or high-dose (0.04 mg/kg) buprenorphine (IM, q8-12 h; n = 5). RESULTS: Airway protection was significantly affected in both experiments, but the most severe deficits occurred post-surgically as 75% of the animals exhibited silent aspiration. CONCLUSION: Oropharyngeal swallow is impaired by the partial mu-opioid receptor agonist buprenorphine, most remarkably in the postoperative setting. These findings have implications for the prevention and management of aspiration pneumonia in vulnerable populations.

Serotonin therapies for opioid-induced disordered swallow and respiratory depression.

Opioids are well-known to cause respiratory depression, but despite clinical evidence of dysphagia, the effects of opioids on swallow excitability and motor pattern are unknown. We tested the effects of the clinically relevant opioid buprenorphine on pharyngeal swallow and respiratory drive in male and female rats. We also evaluated the utility of 5-HT1A agonists (8-OH-DPAT and buspirone) to improve swallowing and breathing following buprenorphine administration. Experiments were performed on 44 freely breathing Sprague-Dawley rats anesthetized with sodium pentobarbital. Bipolar fine wire electrodes were inserted into the mylohyoid, thyroarytenoid, posterior cricoarytenoid, thyropharyngeus, and diaphragm muscles to measure electromyographic (EMG) activity of swallowing and breathing. We evaluated the hypotheses that swallowing varies by stimulus, opioids depress swallowing and breathing, and that 5-HT1A agonists improve these depressions. Our results largely confirmed the following hypotheses: 1) swallow-related EMG activity was larger during swallows elicited by esophageal distension plus oral water infusion than by either stimulus alone. 2) Buprenorphine depressed swallow in both sexes, but females were more susceptible to total swallow suppression. 3) Female animals were also more vulnerable to opioid-induced respiratory depression. 4) 8-OH-DPAT rescued breathing following buprenorphine-induced respiratory arrest, and pretreatment with the partial 5-HT1A agonist buspirone prevented buprenorphine-induced respiratory arrest in female animals. 5) 8-OH-DPAT enhanced mylohyoid and thyropharyngeus EMG amplitude during swallow but did not restore excitability of the swallow pattern generator following total suppression by buprenorphine. Our results highlight sex-specific and behavior-specific effects of buprenorphine and provide preclinical evidence of a 5HT1A agonist for the treatment of respiratory depression and dysphagia.NEW & NOTEWORTHY This is the first study, to our knowledge, to evaluate sex-specific effects of opioid administration on pharyngeal swallow. We expand on a small but growing number of studies that report a lower threshold for opioid-induced respiratory depression in females compared with males, and we are the first to produce this effect with the partial µ-opioid-receptor agonist buprenorphine. This is the first demonstration, to our knowledge, that activation of 5-HT1A receptors can improve swallow and breathing outcomes following systemic buprenorphine administration.

Hypoxia evokes a sequence of raphe-pontomedullary network operations for inspiratory drive amplification and gasping.

Hypoxia can trigger a sequence of breathing-related behaviors, from tachypnea to apneusis to apnea and gasping, an autoresuscitative behavior that, via large tidal volumes and altered intrathoracic pressure, can enhance coronary perfusion, carotid blood flow, and sympathetic activity, and thereby coordinate cardiac and respiratory functions. We tested the hypothesis that hypoxia-evoked gasps are amplified through a disinhibitory microcircuit within the inspiratory neuron chain and a distributed efference copy mechanism that generates coordinated gasp-like discharges concurrently in other circuits of the raphe-pontomedullary respiratory network. Data were obtained from 6 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated adult cats. Arterial blood pressure, phrenic nerve activity, end-tidal CO2, and other parameters were monitored. Hypoxia was produced by ventilation with a gas mixture of 5% O2 in nitrogen (N2). Neuron spike trains were recorded at multiple pontomedullary sites simultaneously and evaluated for firing rate modulations and short-time scale correlations indicative of functional connectivity. Experimental perturbations evoked reconfiguration of raphe-pontomedullary circuits during tachypnea, apneusis and augmented bursts, apnea, and gasping. The functional connectivity, altered firing rates, efference copy of gasp drive, and coordinated step increments in blood pressure reported here support a distributed brain stem network model for amplification and broadcasting of inspiratory drive during autoresuscitative gasping that begins with a reduction in inhibition by expiratory neurons and an initial loss of inspiratory drive during hypoxic apnea.

Cough and swallow after laparotomy in anesthetized cats.

An anesthetized cat animal model was used to evaluate changes in cough and swallow after a small midline upper abdominal incision (laparotomy). Two additional conditions were tested: sealing the laparotomy with gentle suctioning via a small cannula, and subsequent closure of the abdominal wall with suture. These abdominal wall manipulations resulted in no changes in the cough reflex, but produced higher motor drive to pharyngeal musculature (thyropharyngeus and geniohyoid muscles) during swallow. Swallow-breathing coordination phase preference shifted towards swallow occurring more during the inspiratory phase. There were no significant changes in cough motor pattern, or cough and swallow number and temporal features. The respiratory changes were limited to reduced inspiratory motor drive to the diaphragm. The results are consistent with an important role of sensory feedback from the abdominal wall in regulation of swallow motor pattern. The level of reflex modulation may depend on the extent of injury and likely on its position in the abdomen.

Serotonin therapies for opioid-induced dysphagia and respiratory depression: sex differences in a rat electromyography model.

Opioids are well-known to cause respiratory depression, but despite clinical evidence of dysphagia, the effects of opioids on swallow excitability and motor pattern are unknown. We sought to test the effects of the clinically-relevant opioid buprenorphine on pharyngeal swallow and respiratory drive in male and female rats. We also evaluated utility of serotonin 5-HT1A agonists (8-OH-DPAT and buspirone) to improve swallowing and breathing outcomes following buprenorphine administration. Experiments were performed on 44 freely breathing Sprague Dawley rats anesthetized with sodium pentobarbital. Bipolar fine wire electrodes were inserted into the mylohyoid, thyroarytenoid, posterior cricoarytenoid, thyropharyngeus and diaphragm muscles to measure electromyographic (EMG) activity of swallowing and breathing behaviors. We evaluated the hypotheses that swallow varies by stimulus, opioids depress swallow and breathing, and that 5-HT1A agonists improve these depressions. Our results largely confirmed the hypotheses: 1) Swallow-related muscle activity was larger during swallows elicited by oral water infusion plus esophageal distension than by either stimulus alone. 2) Buprenorphine depressed swallow in both sexes, but most significantly in females. 3) Female animals were more susceptible to buprenorphine-induced respiratory arrest. 4) 8-OH-DPAT rescued breathing following buprenorphine-induced respiratory arrest, and pre-treatment with the partial 5-HT1A agonist buspirone prevented buprenorphine-induced respiratory arrest in female animals. 5) 8-OH-DPAT enhanced swallow-related mylohyoid drive, but did not restore excitability of the swallow pattern generator following total suppression by buprenorphine. Our results highlight sex-specific and behavior-specific effects of buprenorphine and provide pre-clinical evidence of a 5HT1A agonist for the treatment of respiratory depression and dysphagia.

Synchronization of inspiratory burst onset along the ventral respiratory column in the neonate mouse is mediated by electrotonic coupling.

Breathing is a singularly robust behavior, yet this motor pattern is continuously modulated at slow and fast timescales to maintain blood-gas homeostasis, while intercalating orofacial behaviors. This functional multiplexing goes beyond the rhythmogenic function that is typically ascribed to medullary respiration-modulated networks and may explain lack of progress in identifying the mechanism and constituents of the respiratory rhythm generator. By recording optically along the ventral respiratory column in medulla, we found convergent evidence that rhythmogenic function is distributed over a dispersed and heterogeneous network that is synchronized by electrotonic coupling across a neuronal syncytium. First, high-speed recordings revealed that inspiratory onset occurred synchronously along the column and did not emanate from a rhythmogenic core. Second, following synaptic isolation, synchronized stationary rhythmic activity was detected along the column. This activity was attenuated following gap junction blockade and was silenced by tetrodotoxin. The layering of syncytial and synaptic coupling complicates identification of rhythmogenic mechanism, while enabling functional multiplexing.

Deglutition and the Regulation of the Swallow Motor Pattern.

Despite centuries of investigation, questions and controversies remain regarding the fundamental genesis and motor pattern of swallow. Two significant topics include inspiratory muscle activity during swallow (Schluckatmung, i.e., "swallow-breath") and anatomical boundaries of the swallow pattern generator. We discuss the long history of reports regarding the presence or absence of Schluckatmung and the possible advantages of and neural basis for such activity, leading to current theories and novel experimental directions.

The influence of CO2 on spatiotemporal features of mechanically induced cough in anesthetized cats.

Effective cough requires a significant increase in lung volume used to produce the shear forces on the airway to clear aspirated material. This increase in tidal volume during cough, along with an increase in tidal frequency during bouts of paroxysmal cough produces profound hyperventilation and thus reduces arterial CO2. While there are several reports in the literature regarding the effects of hypercapnia, hyperoxia, and hypoxia on cough, there is little research quantifying the effects of hypocapnia on the cough reflex. We hypothesized that decreased CO2 would enhance coughing. In 12 spontaneously breathing adult male cats, we compared bouts of prolonged mechanically stimulated cough, in which cough induced hyperventilation (CHV) was allowed to occur, with isocapnic cough trials where we maintained eupneic end-tidal CO2 by adding CO2 to the inspired gas. Isocapnia slightly increased cough number and decreased esophageal pressures with no change in EMG magnitudes or phase durations. The cough-to-eupnea transition was also analyzed between CHV, isocapnia, and a third group of animals that were mechanically hyperventilated to apnea. The transition to eupnea was highly sensitive to added CO2, and CHV apneas were much shorter than those produced by mechanical hyperventilation. We suggest that the cough pattern generator is relatively insensitive to CHV. In the immediate post-cough period, the appearance of breathing while CO2 is very low suggests a transient reduction in apneic threshold following a paroxysmal cough bout.

Laryngeal and swallow dysregulation following acute cervical spinal cord injury.

Laryngeal function is vital to airway protection. Although swallow is mediated by the brainstem, the mechanism underlying the increased risk of dysphagia after cervical spinal cord injury (SCI) is unknown. We hypothesized that: 1) loss of descending phrenic drive affects swallow and breathing differently, and 2) loss of ascending spinal afferent information alters swallow regulation. We recorded electromyograms (EMGs) from upper airway and chest wall muscles in freely breathing pentobarbital-anesthetized cats and rats. Laryngeal abductor activity during inspiration increased about twofold following C2 lateral hemisection. Ipsilateral to the injury, the crural diaphragm EMG amplitude was reduced during breathing (62 ± 25% change postinjury), but no animal had complete termination of activity; 75% of animals had increased contralateral diaphragm recruitment, but this did not reach significance. During swallow, laryngeal adductor and pharyngeal constrictor muscles increased activity, and diaphragm activity was bilaterally suppressed. This was unexpected because of the ipsilateral-specific response during breathing. Swallow-breathing coordination was disrupted by injury, and more swallows occurred during early expiration. Finally, to determine if the chest wall is a major source of feedback for laryngeal regulation, we performed T1 total transections in rats. As in the C2 lateral hemisection, inspiratory laryngeal recruitment was the first feature noted after injury. In contrast to the C2 lateral hemisection, diaphragmatic drive increased after T1 transection. Overall, we found that SCI alters laryngeal drive during swallow and breathing, and alters swallow-related diaphragm activity. Our results show behavior-specific effects, suggesting that swallow is affected more than breathing is by SCI, and emphasizing the need for additional studies on the effect of ascending afferents from the spinal cord on laryngeal function.NEW & NOTEWORTHY This is the first manuscript to determine the impact of cSCI on laryngeal and swallow function, and to describe a possible mechanism for dysphagia and altered airway protection after injury.

Differential effects of acute cerebellectomy on cough in spontaneously breathing cats.

The role of the cerebellum in controlling the cough motor pattern is not well understood. We hypothesized that cerebellectomy would disinhibit motor drive to respiratory muscles during cough. Cough was induced by mechanical stimulation of the tracheobronchial airways in anesthetized, spontaneously breathing adult cats (8 male, 1 female), and electromyograms (EMGs) were recorded from upper airway, chest wall, and abdominal respiratory muscles. Cough trials were performed before and at two time points after total cerebellectomy (10 minutes and >1 hour). Unlike a prior report in paralyzed, decerebrated, and artificially ventilated animals, we observed that cerebellectomy had no effect on cough frequency. After cerebellectomy, thoracic inspiratory muscle EMG magnitudes increased during cough (diaphragm EMG increased by 14% at 10 minutes, p = 0.04; parasternal by 34% at 10 minutes and by 32% at >1 hour, p = 0.001 and 0.03 respectively). During cough at 10 minutes after cerebellectomy, inspiratory esophageal pressure was increased by 44% (p = 0.004), thyroarytenoid (laryngeal adductor) muscle EMG amplitude increased 13% (p = 0.04), and no change was observed in the posterior cricoarytenoid (laryngeal abductor) EMG. Cough phase durations did not change. Blood pressure and heart rate were reduced after cerebellectomy, and respiratory rate also decreased due to an increase in duration of the expiratory phase of breathing. Changes in cough-related EMG magnitudes of respiratory muscles suggest that the cerebellum exerts inhibitory control of cough motor drive, but not cough number or phase timing in response to mechanical stimuli in this model early after cerebellectomy. However, results varied widely at >1 hour after cerebellectomy, with some animals exhibiting enhancement or suppression of one or more components of the cough motor behavior. These results suggest that, while the cerebellum and behavior-related sensory feedback regulate cough, it may be difficult to predict the nature of the modulation based on total cerebellectomy.

Rapid activation of esophageal mechanoreceptors alters the pharyngeal phase of swallow: Evidence for inspiratory activity during swallow.

Swallow is a complex behavior that consists of three coordinated phases: oral, pharyngeal, and esophageal. Esophageal distension (EDist) has been shown to elicit pharyngeal swallow, but the physiologic characteristics of EDist-induced pharyngeal swallow have not been specifically described. We examined the effect of rapid EDist on oropharyngeal swallow, with and without an oral water stimulus, in spontaneously breathing, sodium pentobarbital anesthetized cats (n = 5). Electromyograms (EMGs) of activity of 8 muscles were used to evaluate swallow: mylohyoid (MyHy), geniohyoid (GeHy), thyrohyoid (ThHy), thyropharyngeus (ThPh), thyroarytenoid (ThAr), cricopharyngeus (upper esophageal sphincter: UES), parasternal (PS), and costal diaphragm (Dia). Swallow was defined as quiescence of the UES with overlapping upper airway activity, and it was analyzed across three stimulus conditions: 1) oropharyngeal water infusion only, 2) rapid esophageal distension (EDist) only, and 3) combined stimuli. Results show a significant effect of stimulus condition on swallow EMG amplitude of the mylohyoid, geniohyoid, thyroarytenoid, diaphragm, and UES muscles. Collectively, we found that, compared to rapid cervical esophageal distension alone, the stimulus condition of rapid distension combined with water infusion is correlated with increased laryngeal adductor and diaphragm swallow-related EMG activity (schluckatmung), and post-swallow UES recruitment. We hypothesize that these effects of upper esophageal distension activate the brainstem swallow network, and function to protect the airway through initiation and/or modulation of a pharyngeal swallow response.

Evidence of intermediate reticular formation involvement in swallow pattern generation, recorded optically in the neonate rat sagittally sectioned hindbrain.

Swallow is a primitive behavior regulated by medullary networks, responsible for movement of food/liquid from the oral cavity to the esophagus. To investigate how functionally heterogeneous networks along the medullary intermediate reticular formation (IRt) and ventral respiratory column (VRC) control swallow, we electrically stimulated the nucleus tractus solitarius to induce fictive swallow between inspiratory bursts, with concurrent optical recordings using a synthetic Ca2+ indicator in the neonatal sagittally sectioned rat hindbrain (SSRH) preparation. Simultaneous recordings from hypoglossal nerve rootlet (XIIn) and ventral cervical spinal root C1-C2 enabled identification of the system-level correlates of 1) swallow (identified as activation of the XIIn but not the cervical root) and 2) Breuer-Hering expiratory reflex (BHE; lengthened expiration in response to stimuli during expiration). Optical recording revealed reconfiguration of respiration-modulated networks in the ventrolateral medulla during swallow and the BHE reflex. Recordings identified novel spatially compact networks in the IRt near the facial nucleus (VIIn) that were active during fictive swallow, suggesting that the swallow network is not restricted to the caudal medulla. These findings also establish the utility of using this in vitro preparation to investigate how functionally heterogeneous medullary networks interact and reconfigure to enable a repertoire of orofacial behaviors.NEW & NOTEWORTHY For the first time, medullary networks that control breathing and swallow are recorded optically. Episodic swallows are induced via electrical stimulation along the dorsal medulla, in and near the NTS, during spontaneously occurring fictive respiration. These findings establish that networks regulating both orofacial behaviors and breathing are accessible for optical recording at the surface of the sagittally sectioned rodent hindbrain preparation.

Sex-specific vagal and spinal modulation of breathing with chest compression.

Lung volume is modulated by sensory afferent feedback via vagal and spinal pathways. The purpose of this study was to systematically alter afferent feedback with and without a mechanical challenge (chest compression). We hypothesized that manipulation of afferent feedback by nebulization of lidocaine, extra-thoracic vagotomy, or lidocaine administration to the pleural space would produce differential effects on the motor pattern of breathing during chest compression in sodium pentobarbital anesthetized rats (N = 43). Our results suggest that: 1) pulmonary stretch receptors are not the sole contributor to breathing feedback in adult male and female rats; 2) of our manipulations, chest compression had the largest effect on early expiratory diaphragm activity ("yield"); 3) reduction of spinally-mediated afferent feedback modulates breathing patterns most likely via inhibition; and 4) breathing parameters demonstrate large sex differences. Compared to males, female animals had lower respiratory rates (RR), which were further depressed by vagotomy, while chest compression increased RR in males, and decreased yield in females without changing RR. Collectively, our results suggest that balance between tonic vagal inhibition and spinal afferent feedback maintains breathing characteristics, and that it is important to specifically evaluate sex differences when studying control of breathing.

Sex-specific vagal and spinal modulation of swallow and its coordination with breathing.

Swallow-breathing coordination is influenced by changes in lung volume, which is modulated by feedback from both vagal and spinal sensory afferents. The purpose of this study was to manipulate feedback from these afferents, with and without a simultaneous mechanical challenge (chest compression), in order to assess the influence of each sensory pathway on swallow in rats. We hypothesized that manipulation of afferent feedback would shift the occurrence of swallow toward the inspiratory phase of breathing. Afferent feedback was perturbed by lidocaine nebulization, extra-thoracic vagotomy, or lidocaine administration to the pleural space in sodium pentobarbital anesthetized rats (N = 43). These different afferent perturbations were performed both in control conditions (no chest compression), and with chest compression. Manipulating pulmonary stretch receptor-mediated volume feedback in male animals decreased swallow occurrence. Female rats appear to rely more on spinal afferent feedback, as swallow occurrence shifted to late expiration with chest compression and vagotomy or lidocaine injections. Results suggest that sex-specific mechanisms modulate swallow-breathing coordination, and that vagal feedback is inhibitory to swallow-related muscles, while spinal feedback from pleural afferents has excitatory effects. This study supports the theory that a balance of vagal and spinal afferent feedback is necessary to maintain an optimal swallow pattern and swallow-breathing coordination.

Swallow Motor Pattern Is Modulated by Fixed or Stochastic Alterations in Afferent Feedback.

Afferent feedback can appreciably alter the pharyngeal phase of swallow. In order to measure the stability of the swallow motor pattern during several types of alterations in afferent feedback, we assessed swallow during a conventional water challenge in four anesthetized cats, and compared that to swallows induced by fixed (20 Hz) and stochastic (1-20Hz) electrical stimulation applied to the superior laryngeal nerve. The swallow motor patterns were evaluated by electromyographic activity (EMG) of eight muscles, based on their functional significance: laryngeal elevators (mylohyoid, geniohyoid, and thyrohyoid); laryngeal adductor (thyroarytenoid); inferior pharyngeal constrictor (thyropharyngeus); upper esophageal sphincter (cricopharyngeus); and inspiratory activity (parasternal and costal diaphragm). Both the fixed and stochastic electrical stimulation paradigms increased activity of the laryngeal elevators, produced short-term facilitation evidenced by increasing swallow durations over the stimulus period, and conversely inhibited swallow-related diaphragm activity. Both the fixed and stochastic stimulus conditions also increased specific EMG amplitudes, which never occurred with the water challenges. Stochastic stimulation increased swallow excitability, as measured by an increase in the number of swallows produced. Consistent with our previous results, changes in the swallow motor pattern for pairs of muscles were only sometimes correlated with each other. We conclude that alterations in afferent feedback produced particular variations of the swallow motor pattern. We hypothesize that specific SLN feedback might modulate the swallow central pattern generator during aberrant feeding conditions (food/liquid entering the airway), which may protect the airway and serve as potentially important clinical diagnostic indicators.

Buccal rhythmogenesis and CO2 sensitivity in Lithobates catesbeianus tadpole brainstems across metamorphosis.

Bullfrog tadpoles ventilate both the buccal cavity and lung. In isolated brainstems, the midbrain/pons influences CO2 responsiveness and timing of lung ventilatory bursting, depending on larval development. However, little is known about midbrain/pons influences on buccal burst patterns. As such, we investigated how removal of this region affects buccal burst shape and CO2 responsiveness across development. We measured facial nerve activity in brainstems isolated from tadpoles during early and late developmental stages, under normal and elevated levels of CO2. Brainstems were either left intact or transected by removing the midbrain/pons. In late stage preparations, buccal burst pattern differed between intact and reduced preparations, and bursts were responsive to elevated CO2 in these reduced preparations. These results suggest the midbrain/pons affects tadpole buccal burst pattern and CO2 responsiveness, perhaps similar to its influences on lung ventilation.

The rostral medulla of bullfrog tadpoles contains critical lung rhythmogenic and chemosensitive regions across metamorphosis.

The development of amphibian breathing provides insight into vertebrate respiratory control mechanisms. Neural oscillators in the rostral and caudal medulla drive ventilation in amphibians, and previous reports describe ventilatory oscillators and CO2 sensitive regions arise during different stages of amphibian metamorphosis. However, inconsistent findings have been enigmatic, and make comparisons to potential mammalian counterparts challenging. In the current study we assessed amphibian central CO2 responsiveness and respiratory rhythm generation during two different developmental stages. Whole-nerve recordings of respiratory burst activity in cranial and spinal nerves were made from intact or transected brainstems isolated from tadpoles during early or late stages of metamorphosis. Brainstems were transected at the level of the trigeminal nerve, removing rostral structures including the nucleus isthmi, midbrain, and locus coeruleus, or transected at the level of the glossopharyngeal nerve, removing the putative buccal oscillator and caudal medulla. Removal of caudal structures stimulated the frequency of lung ventilatory bursts and revealed a hypercapnic response in normally unresponsive preparations derived from early stage tadpoles. In preparations derived from late stage tadpoles, removal of rostral or caudal structures reduced lung burst frequency, while CO2 responsiveness was retained. Our results illustrate that structures within the rostral medulla are capable of sensing CO2 throughout metamorphic development. Similarly, the region controlling lung ventilation appears to be contained in the rostral medulla throughout metamorphosis. This work offers insight into the consistency of rhythmic respiratory and chemosensitive capacities during metamorphosis.

Carotid chemoreceptors tune breathing via multipath routing: reticular chain and loop operations supported by parallel spike train correlations.

We tested the hypothesis that carotid chemoreceptors tune breathing through parallel circuit paths that target distinct elements of an inspiratory neuron chain in the ventral respiratory column (VRC). Microelectrode arrays were used to monitor neuronal spike trains simultaneously in the VRC, peri-nucleus tractus solitarius (p-NTS)-medial medulla, the dorsal parafacial region of the lateral tegmental field (FTL-pF), and medullary raphe nuclei together with phrenic nerve activity during selective stimulation of carotid chemoreceptors or transient hypoxia in 19 decerebrate, neuromuscularly blocked, and artificially ventilated cats. Of 994 neurons tested, 56% had a significant change in firing rate. A total of 33,422 cell pairs were evaluated for signs of functional interaction; 63% of chemoresponsive neurons were elements of at least one pair with correlational signatures indicative of paucisynaptic relationships. We detected evidence for postinspiratory neuron inhibition of rostral VRC I-Driver (pre-Bötzinger) neurons, an interaction predicted to modulate breathing frequency, and for reciprocal excitation between chemoresponsive p-NTS neurons and more downstream VRC inspiratory neurons for control of breathing depth. Chemoresponsive pericolumnar tonic expiratory neurons, proposed to amplify inspiratory drive by disinhibition, were correlationally linked to afferent and efferent "chains" of chemoresponsive neurons extending to all monitored regions. The chains included coordinated clusters of chemoresponsive FTL-pF neurons with functional links to widespread medullary sites involved in the control of breathing. The results support long-standing concepts on brain stem network architecture and a circuit model for peripheral chemoreceptor modulation of breathing with multiple circuit loops and chains tuned by tegmental field neurons with quasi-periodic discharge patterns. NEW & NOTEWORTHY We tested the long-standing hypothesis that carotid chemoreceptors tune the frequency and depth of breathing through parallel circuit operations targeting the ventral respiratory column. Responses to stimulation of the chemoreceptors and identified functional connectivity support differential tuning of inspiratory neuron burst duration and firing rate and a model of brain stem network architecture incorporating tonic expiratory "hub" neurons regulated by convergent neuronal chains and loops through rostral lateral tegmental field neurons with quasi-periodic discharge patterns.

Functional connectivity in raphé-pontomedullary circuits supports active suppression of breathing during hypocapnic apnea.

Hyperventilation is a common feature of disordered breathing. Apnea ensues if CO2 drive is sufficiently reduced. We tested the hypothesis that medullary raphé, ventral respiratory column (VRC), and pontine neurons have functional connectivity and persistent or evoked activities appropriate for roles in the suppression of drive and rhythm during hyperventilation and apnea. Phrenic nerve activity, arterial blood pressure, end-tidal CO2, and other parameters were monitored in 10 decerebrate, vagotomized, neuromuscularly-blocked, and artificially ventilated cats. Multielectrode arrays recorded spiking activity of 649 neurons. Loss and return of rhythmic activity during passive hyperventilation to apnea were identified with the S-transform. Diverse fluctuating activity patterns were recorded in the raphé-pontomedullary respiratory network during the transition to hypocapnic apnea. The firing rates of 160 neurons increased during apnea; the rates of 241 others decreased or stopped. VRC inspiratory neurons were usually the last to cease firing or lose rhythmic activity during the transition to apnea. Mayer wave-related oscillations (0.04-0.1 Hz) in firing rate were also disrupted during apnea. Four-hundred neurons (62%) were elements of pairs with at least one hyperventilation-responsive neuron and a correlational signature of interaction identified by cross-correlation or gravitational clustering. Our results support a model with distinct groups of chemoresponsive raphé neurons contributing to hypocapnic apnea through parallel processes that incorporate disfacilitation and active inhibition of inspiratory motor drive by expiratory neurons. During apnea, carotid chemoreceptors can evoke rhythm reemergence and an inspiratory shift in the balance of reciprocal inhibition via suppression of ongoing tonic expiratory neuron activity.