Inspiratory Rhythm Research Articles

Orexin receptor 2 agonist activates diaphragm and genioglossus muscle through stimulating inspiratory neurons in the pre-Bötzinger complex, and phrenic and hypoglossal motoneurons in rodents.

Orexin-mediated stimulation of orexin receptors 1/2 (OX[1/2]R) may stimulate the diaphragm and genioglossus muscle via activation of inspiratory neurons in the pre-Bötzinger complex, which are critical for the generation of inspiratory rhythm, and phrenic and hypoglossal motoneurons. Herein, we assessed the effects of OX2R-selective agonists TAK-925 (danavorexton) and OX-201 on respiratory function. In in vitro electrophysiologic analyses using rat medullary slices, danavorexton and OX-201 showed tendency and significant effect, respectively, in increasing the frequency of inspiratory synaptic currents of inspiratory neurons in the pre-Bötzinger complex. In rat medullary slices, both danavorexton and OX-201 significantly increased the frequency of inspiratory synaptic currents of hypoglossal motoneurons. Danavorexton and OX-201 also showed significant effect and tendency, respectively, in increasing the frequency of burst activity recorded from the cervical (C3-C5) ventral root, which contains axons of phrenic motoneurons, in in vitro electrophysiologic analyses from rat isolated brainstem-spinal cord preparations. Electromyogram recordings revealed that intravenous administration of OX-201 increased burst frequency of the diaphragm and burst amplitude of the genioglossus muscle in isoflurane- and urethane-anesthetized rats, respectively. In whole-body plethysmography analyses, oral administration of OX-201 increased respiratory activity in free-moving mice. Overall, these results suggest that OX2R-selective agonists enhance respiratory function via activation of the diaphragm and genioglossus muscle through stimulation of inspiratory neurons in the pre-Bötzinger complex, and phrenic and hypoglossal motoneurons. OX2R-selective agonists could be promising drugs for various conditions with respiratory dysfunction.

Inhibitory Subpopulations in preBötzinger Complex Play Distinct Roles in Modulating Inspiratory Rhythm and Pattern.

Inhibitory neurons embedded within mammalian neural circuits shape breathing, walking, and other rhythmic motor behaviors. At the core of the neural circuit controlling breathing is the preBötzinger Complex (preBötC), where GABAergic (GAD1/2+) and glycinergic (GlyT2+) neurons are functionally and anatomically intercalated among glutamatergic Dbx1-derived (Dbx1+) neurons that generate rhythmic inspiratory drive. The roles of these preBötC inhibitory neurons in breathing remain unclear. We first characterized the spatial distribution of molecularly defined preBötC inhibitory subpopulations in male and female neonatal double reporter mice expressing either tdTomato or EGFP in GlyT2+, GAD1+, or GAD2+ neurons. We found that the majority of preBötC inhibitory neurons expressed both GlyT2 and GAD2 while a much smaller subpopulation also expressed GAD1. To determine the functional role of these subpopulations, we used holographic photostimulation, a patterned illumination technique, in rhythmically active medullary slices from neonatal Dbx1tdTomato;GlyT2EGFP and Dbx1tdTomato;GAD1EGFP double reporter mice of either sex. Stimulation of 4 or 8 preBötC GlyT2+ neurons during endogenous rhythm prolonged the interburst interval in a phase-dependent manner and increased the latency to burst initiation when bursts were evoked by stimulation of Dbx1+ neurons. In contrast, stimulation of 4 or 8 preBötC GAD1+ neurons did not affect interburst interval or latency to burst initiation. Instead, photoactivation of GAD1+ neurons during the inspiratory burst prolonged endogenous and evoked burst duration and decreased evoked burst amplitude. We conclude that GlyT2+/GAD2+ neurons modulate breathing rhythm by delaying burst initiation while a smaller GAD1+ subpopulation shapes inspiratory patterning by altering burst duration and amplitude.

Characterizing Muscarinic Receptor Subtype Roles in Inspiratory Burst Modulation of Hypoglossal Motoneurons in Mice

Loss of tongue muscle (innervated by hypoglossal (XII) motoneurons) tone is responsible for airway obstruction during sleep. Contributing factors include loss of noradrenergic drive and activation of muscarinic acetylcholine receptors (mAChRs). While noradrenergic effects are well studied, less is known about muscarinic effects. In mice, XII motoneurons express four mAChRs; M1, M3, and M5 are excitatory while M2 is inhibitory. We hypothesize that M2 is upregulated with postnatal maturation whereas M1, M3, and M5 are downregulated. We therefore expect that activation of M1, M3, and M5 receptors will potentiate inspiratory bursting early in postnatal development whereas activation of M2 receptors will inhibit inspiratory bursting later in postnatal development. To test this, we utilized rhythmic medullary slice preparations from mice at postnatal days 0-5 (P0-5) and P9-14, which contained preBötzinger Complex (site of inspiratory rhythm generation) and XII motoneurons. Blocking M1 receptors locally with pirenzepine (10 μM & 100 μM) decreased the muscarinic potentiation of inspiratory bursting in P0-5 mice at 100 μM (168±23% at 10 μM and 129±16% at 100 μM in muscarine [p<0.05] vs 183±34% muscarine (100 μM) control response, n=9). Activating M1 receptors locally with cevimeline (1 mM) for 30s or 60s did not mediate potentiation of inspiratory bursting as seen with muscarine (116±7% at 30s and 117±7% at 60s vs 181±40% control muscarine response [n=6, p<0.05]). Bath application of M2 antagonist methoctramine (MTH) in P0-5 mice at 1 μM had no effect on muscarine-potentiation of inspiratory bursting (207±109% at 1 μM MTH in muscarine vs 212±84% muscarine control response, n=9). Preliminary analysis of the P9-14 age group using 1 μM MTH suggests there may be an increase in muscarine-mediated potentiation of inspiratory bursting with M2 blockade (170±56% at 1 μM MTH in muscarine vs 152±56% muscarine control response, n=6, p>0.5). Bath application of M3 receptor antagonist 4-DAMP in P0-5 mice at 10 nM had no effect on muscarine-potentiated inspiratory bursting (172±12% vs 178±30% muscarine control), whereas application at 100 nM decreased muscarine-mediated inspiratory burst potentiation compared to control (133±12%, n=4, p<0.05). However, bath application with another M3 receptor antagonist, J Fumarate 104129, had no significant effect on muscarinic modulation of inspiratory bursting in P0-5 mice (188±46% at 0.6 nM [n=6], 185±32% at 6 nM [n=6], and 144±24% at 60 nM [n=4] vs 176±34% muscarine control [n=6]). Overall, our data partially supports a role of M1 receptors contributing to muscarinic potentiation of inspiratory bursting in XII motoneurons, with the contributions of M2 and M3 appearing insignificant early in postnatal life. Preliminary analysis suggests that M2 has a larger contribution later in maturation. Future research will evaluate the contribution of muscarinic receptor subtypes to inspiratory burst modulation later in postnatal life. NIH/NHLBI Funding: R15HL148870. 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.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic–cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Human IPSC-Derived PreBötC-Like Neurons and Development of an Opiate Overdose and Recovery Model.

Opioid overdose is the leading cause of drug overdose lethality, posing an urgent need for investigation. The key brain region for inspiratory rhythm regulation and opioid-induced respiratory depression (OIRD) is the preBötzinger Complex (preBötC) and current knowledge has mainly been obtained from animal systems. This study aims to establish a protocol to generate human preBötC neurons from induced pluripotent cells (iPSCs) and develop an opioid overdose and recovery model utilizing these iPSC-preBötC neurons. A de novo protocol to differentiate preBötC-like neurons from human iPSCs is established. These neurons express essential preBötC markers analyzed by immunocytochemistry and demonstrate expected electrophysiological responses to preBötC modulators analyzed by patch clamp electrophysiology. The correlation of the specific biomarkers and function analysis strongly suggests a preBötC-like phenotype. Moreover, the dose-dependent inhibition of these neurons' activity is demonstrated for four different opioids with identified IC50's comparable to the literature. Inhibition is rescued by naloxone in a concentration-dependent manner. This iPSC-preBötC mimic is crucial for investigating OIRD and combating the overdose crisis and a first step for the integration of a functional overdose model into microphysiological systems.

Role of NaV1.6-mediated persistent sodium current and bursting-pacemaker properties in breathing rhythm generation

Inspiration is the inexorable active phase of breathing. The brainstem pre-Bötzinger complex (preBötC) gives rise to inspiratory neural rhythm, but its underlying cellular and ionic bases remain unclear. The long-standing "pacemaker hypothesis" posits that the persistent Na+ current (INaP) that gives rise to bursting-pacemaker properties in preBötC interneurons is essential for rhythmogenesis. We tested the pacemaker hypothesis by conditionally knocking out and knocking down the Scn8a (Nav1.6 [voltage-gated sodium channel 1.6]) gene in core rhythmogenic preBötC neurons. Deleting Scn8a substantially decreases the INaP and abolishes bursting-pacemaker activity, which slows inspiratory rhythm invitro and negatively impacts the postnatal development of ventilation. Diminishing Scn8a via genetic interference has no impact on breathing in adult mice. We argue that the Scn8a-mediated INaP is not obligatory but that it influences the development andrhythmic function of the preBötC. The ubiquity of the INaP in respiratory brainstem interneurons could underlie breathing-related behaviors such as neonatal phonation or rhythmogenesis in different physiological conditions.

Hippocampal ensemble dynamics and memory performance are modulated by respiration during encoding

During offline brain states, such as sleep and memory consolidation, respiration coordinates hippocampal activity. However, the role of breathing during online memory traces remains unclear. Here, we show that respiration can be recruited during online memory encoding. Optogenetic manipulation was used to control activation of the primary inspiratory rhythm generator PreBötzinger complex (PreBötC) in transgenic mice. When intermittent PreBötC-induced apnea covered the object exploration time during encoding, novel object detection was impaired. Moreover, the mice did not exhibit freezing behavior during presentation of fear-conditioned stimuli (CS+) when PreBötC-induced apnea occurred at the exact time of encoding. This apnea did not evoke changes in CA3 cell ensembles between presentations of CS+ and conditioned inhibition (CS−), whereas in normal breathing, CS+ presentations produced dynamic changes. Our findings demonstrate that components of central respiratory activity (e.g., frequency) during online encoding strongly contribute to shaping hippocampal ensemble dynamics and memory performance.

Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry.

The pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH. We utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC. We found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC. We provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

Chronic pulmonary fibrosis alters the functioning of the respiratory neural network.

Some patients with idiopathic pulmonary fibrosis present impaired ventilatory variables characterised by low forced vital capacity values associated with an increase in respiratory rate and a decrease in tidal volume which could be related to the increased pulmonary stiffness. The lung stiffness observed in pulmonary fibrosis may also have an effect on the functioning of the brainstem respiratory neural network, which could ultimately reinforce or accentuate ventilatory alterations. To this end, we sought to uncover the consequences of pulmonary fibrosis on ventilatory variables and how the modification of pulmonary rigidity could influence the functioning of the respiratory neuronal network. In a mouse model of pulmonary fibrosis obtained by 6 repeated intratracheal instillations of bleomycin (BLM), we first observed an increase in minute ventilation characterised by an increase in respiratory rate and tidal volume, a desaturation and a decrease in lung compliance. The changes in these ventilatory variables were correlated with the severity of the lung injury. The impact of lung fibrosis was also evaluated on the functioning of the medullary areas involved in the elaboration of the central respiratory drive. Thus, BLM-induced pulmonary fibrosis led to a change in the long-term activity of the medullary neuronal respiratory network, especially at the level of the nucleus of the solitary tract, the first central relay of the peripheral afferents, and the Pre-Bötzinger complex, the inspiratory rhythm generator. Our results showed that pulmonary fibrosis induced modifications not only of pulmonary architecture but also of central control of the respiratory neural network.

0049 Orexin 2 receptor agonist activates pre-Bötzinger complex neurons, phrenic motoneurons and the diaphragm in rodents

Abstract Introduction Neurons in the pre-Bötzinger complex are critical for the generation of inspiratory rhythm and regulation of activity of phrenic motoneurons, which control the activity of the diaphragm. Dysregulation of this neuronal network and resulting dysfunction of the diaphragm is associated with central sleep apnea (CSA). Orexins by stimulating both orexin 1 receptor (OX1R) and/or orexin 2 receptor (OX2R) can activate these neurons and the diaphragm. In this study, we assessed effects of OX2R-selective agonists on the activity of neurons in the pre-Bötzinger complex, phrenic motoneurons, and the diaphragm. Methods OX2R-selective agonists danavorexton (TAK-925) and OX-201 (a tool compound with longer plasma half-life than danavorexton in rodents) were used. OX2R and OX1R agonistic activities were examined by calcium mobilization assay in hOX2R/CHO-K1 cells and hOX1R/CHO-K1 cells, respectively. Activity of neurons in the pre-Bötzinger complex and phrenic motoneurons was investigated by electrophysiological recording in rat tissues. Activity of the diaphragm was evaluated by electromyogram recording in anesthetized rats. Respiratory activity was evaluated by whole-body plethysmography recording in mice. Results Danavorexton and OX-201 showed potent OX2R agonistic activity with 50% effective concentration (EC50) values of 5.5 nM and 8.0 nM, respectively. Danavorexton and OX-201 had >5,000-fold and 1,000-fold OX2R selectivity over OX1R, respectively. In in vitro electrophysiological analyses, danavorexton and OX-201 increased the burst frequency of neurons in the pre-Bötzinger complex (EC50: 300 nM and 760 nM, respectively) and phrenic motoneurons (EC50: 1.6 µM and not determined, respectively). Intravenous administration of OX-201 at 1 and 3 mg/kg increased burst frequency of the diaphragm in anesthetized rats and oral administration at 3 mg/kg increased respiratory activity in conscious mice. Conclusion OX2R-selective agonists may enhance respiratory function via activation of the diaphragm through stimulation of neurons in the pre-Bötzinger complex and phrenic motoneurons. OX2R agonists may have therapeutic potential in patients with CSA if enhancement of respiratory function by OX2R agonists can be observed at non-arousal concentrations. Further preclinical and clinical studies are worth conducting in patients with CSA. Support (if any) This work was conducted by Takeda Pharmaceutical Company Limited.

0048 Orexin 2 receptor agonist activates pre-Bötzinger complex neurons, hypoglossal motoneurons and the genioglossus muscle in rodents

Abstract Introduction Neurons in the pre-Bötzinger complex are critical for the generation of inspiratory rhythm and control the activity of the genioglossus muscle via activation of hypoglossal motoneurons. Activation of the genioglossus muscle causes anterior movement of the tongue and widens the upper airway. As obstructive sleep apnea (OSA) is associated with obstruction of the upper airway, activation of this neuronal pathway may be a novel therapeutic approach. Orexins have been shown to activate these neurons by stimulation of both orexin 1 receptor (OX1R) and/or orexin 2 receptor (OX2R). In this study, we assessed effects of OX2R-selective agonists on activity of neurons in the pre-Bötzinger complex, hypoglossal motoneurons, and the genioglossus muscle. Methods OX2R-selective agonists danavorexton (TAK-925) and OX-201 (a tool compound with longer plasma half-life than danavorexton in rodents) were used. Arousal effects were investigated by electroencephalogram/electromyogram recording in C57BL/6J mice and OX2R knockout mice. Activity of neurons in the pre-Bötzinger complex and hypoglossal motoneurons was investigated by electrophysiological recording in rat tissues. Activity of the genioglossus muscle was evaluated by electromyogram recording in anesthetized rats. Results Oral administration of OX-201 increased wakefulness time during sleep phase in C57BL6J mice but not in OX2R knockout mice. In in vitro electrophysiological analyses, danavorexton, and OX-201 increased the burst frequency of neurons in the pre-Bötzinger complex (EC50: 300 nM and 760 nM, respectively) and hypoglossal motoneurons (EC50: 610 nM and 260 nM, respectively). Intravenous administration of OX-201 at 1 mg/kg increased burst amplitude of the genioglossus muscle in anesthetized rats. Conclusion OX2R-selective agonists may enhance the respiratory function via activation of the genioglossus muscle through stimulation of neurons in the pre-Bötzinger complex and hypoglossal motoneurons. OX2R agonists may have therapeutic potential in patients with OSA if enhancement of respiratory function by OX2R agonists can be observed at non-arousal concentrations. Further preclinical and clinical studies are worth conducting in patients with OSA. Support (if any) This work was conducted by Takeda Pharmaceutical Company Limited.

Upregulation of breathing rate during running exercise by central locomotor circuits in mice

While respiratory adaptation to exercise is compulsory to cope with the increased metabolic demand, the neural signals at stake remain poorly identified. Using neural circuit tracing and activity interference strategies in mice, we uncover here two systems by which the central locomotor network can enable respiratory augmentation in relation to running activity. One originates in the mesencephalic locomotor region (MLR), a conserved locomotor controller. Through direct projections onto the neurons of the preBötzinger complex that generate the inspiratory rhythm, the MLR can trigger a moderate increase of respiratory frequency, prior to, or even in the absence of, locomotion. The other is the lumbar enlargement of the spinal cord containing the hindlimb motor circuits. When activated, and through projections onto the retrotrapezoid nucleus (RTN), it also potently upregulates breathing rate. On top of identifying critical underpinnings for respiratory hyperpnea, these data also expand the functional implication of cell types and pathways that are typically regarded as “locomotor” or “respiratory” related.

Cholinergic projections to the preBötzinger complex.

Rhythmic inspiratory activity is generated in the preBötzinger complex (preBötC), a neuronal network located bilaterally in the ventrolateral medulla. Cholinergic neurotransmission affects respiratory rhythmogenic neurons and inhibitory glycinergic neurons in the preBötC. Acetylcholine has been extensively investigated given that cholinergic fibers and receptors are present and functional in the preBötC, are important in sleep/wake cycling, and modulate inspiratory frequency through its action on preBötC neurons. Despite its role in modulating inspiratory rhythm, the source of acetylcholine input to the preBötC is not known. In the present study, we used retrograde and anterograde viral tracing approaches in transgenic mice expressing Cre-recombinase driven by the choline acetyltransferase promoter to identify the source of cholinergic inputs to the preBötC. Surprisingly, we observed very few, if any, cholinergic projections originating from the laterodorsal and pedunculopontine tegmental nuclei (LDT/PPT), two main cholinergic, state-dependent systems long hypothesized as the main source of cholinergic inputs to the preBötC. On the contrary, we identified glutamatergic and GABAergic/glycinergic neurons in the PPT/LDT that send projections to the preBötC. Although these neurons contribute minimally to the direct cholinergic modulation of preBötC neurons, they could be involved in state-dependent regulation of breathing. Our data also suggest that the source of cholinergic inputs to the preBötC appears to originate from cholinergic neurons in neighboring regions of the medulla, the intermediate reticular formation, the lateral paragigantocellularis, and the nucleus of the solitary tract.

Optogenetic activation of enkephalinergic neurons reveals excitatory effects in the respiratory rhythm generating network

The primary cause of death during opioid overdose is respiratory depression. Opioid-induced respiratory depression (OIRD) is characterized by a pronounced decrease in the frequency and regularity of inspiratory efforts. This rhythm suppression is caused, in part, by activation of mu-opioid receptors (MORs) in the preBötzinger Compex (preBötC), a region in the ventral medulla that generates respiratory rhythm. In response to exogenous opioids, MOR-expressing preBötC neurons undergo modest hyperpolarization and glutamatergic synaptic transmission is suppressed via effects on presynaptic release. While MOR activation often leads to [LG1] destabilization and cessation of the inspiratory rhythm, under conditions of elevated network excitability it can also stabilize rhythm generation. In addition to MOR-expressing neurons, the preBötC also contains neurons that express enkephalin, an endogenous ligand for the MOR encoded by the Penk gene. Yet, little is known about how enkephalinergic neurons and endogenous MOR signaling affect respiratory rhythm generation. We hypothesized that activation of Penk neurons in the preBötC would suppress rhythm generation which under conditions of high excitability stabilizes network activity. To test this hypothesis, we optogenetically stimulated enkephalinergic preBötC neurons while recording respiratory activityin brainstem slices from PenkCre; Rosa26Chr2:YFP neonatal mice. To our surprise, optogenetic activation of Penk neurons strongly excited, rather than suppressed, the preBötC network. Specifically, brief 200-ms light pulses evoked inspiratory bursts with a high probability and low latency, consistent with the effects of activating glutamatergic preBötC neurons. Further, prolonged 30-Hz or continuous stimulations of Penk neurons increased the frequency of the inspiratory rhythm and these effects persisted following blockade of MORs with Naloxone. Based on the initial results, we predict that Penk neurons predominantly affect preBötC activity through glutamatergic signaling. The co-release of enkephalin may serve as a negative feedback mechanism that regulates the presynaptic release of glutamate to protect against network over-excitation. Understanding the role of endogenous MOR signaling in this vital respiratory network may inspire strategies to prevent respiratory depression during administration of exogenous opioids. R00 HL145004 (Baertsch) This is the full abstract presented at the American Physiology Summit 2023 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.

Scaling down synaptic strength results in changes to the pattern of fictive breathing in a model of the brainstem respiratory network: medullary and pontine sources

Opioids remain a primary vehicle for pain management prescribed by clinicians. However, opioid-induced respiratory depression (OIRD) can cause apneas and be fatal at elevated doses. Two sites of action for these opioids have been suggested in the brainstem, one in the pons and the other in the preBötzinger Complex of the medulla. Additionally, there is electrophysiological evidence suggesting that an inward-rectifying K + current is opened upon μ-opioid receptor (μOR) activation causing neuronal hyperpolarizations. Activation of μORs may cause depletion of glutamate release from excitatory neurons. Our previous simulation efforts have demonstrated the plausibility of presynaptic actions of opioids among inspiratory neurons of the medulla and pons in depressing inspiratory drive or eliciting apnea within specific and singular synaptic connections. Here, we tested the effects of diminishing the strength of excitatory synaptic currents among several different classes of respiratory neurons in a large-scale model of the brainstem respiratory network and whether it would replicate OIRD. We simulated the simultaneous actions of μOR on multiple presynaptic sites among the medullary inspiratory network. We began by scaling down the synaptic conductances of target populations of medullary inspiratory neurons. The core of the model is made up of neurons that produce the fundamental inspiratory rhythm (“I-Driver” neurons), as well as neurons that normally have an inspiratory decrementing (“I-Dec”) or inspiratory augmenting (“I-Aug”) firing pattern and various expiratory phase firing patterns (“E-Dec,” “E-Aug,” “E-Tonic”). I-Driver neurons are analogous to medullary preBötzinger Complex neurons that putatively express μORs. In our model, these neurons project back onto other I-Driver neurons to promote recurrent excitation but also project onto I-Aug and I-Dec populations. Decreasing the synaptic strength from I-Driver neurons to these populations collectively slows fictive breathing until extended apnea occurs. To further understand how this apnea develops we systematically scaled down the synaptic strengths of I-Driver to each of the other specific populations and found that the excitatory connection from I-Driver to I-Dec populations is most influential in producing apnea. However, we also found that excitatory drive from tonic, non-respiratory modulated (NRM) neurons in the pons to the medulla could influence this pattern by grading down synaptic strength. In addition to any direct hyperpolarizing effects of opioids on μOR-expressing neurons, these data support the plausibility that OIRD could arise from changes due to glutamate release. This work was supported by the National Institutes of Health grant 1R01HL155721-01, 1R01HL163008, and T32HL134621. This is the full abstract presented at the American Physiology Summit 2023 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.

Ventral and dorsal cervical spinal cord electrical epidural stimulation facilitated respiration differently in the presence of opioids in human via different neural circuits

Opioid overdose is one of the leading causes of overdose related deaths in the United States of America, accounting for 70.6% of overdose fatalities in 2019 alone. A leading cause of death among opioid overdoses is respiratory depression. Epidural electrical stimulation (EES) emerges as a novel approach of facilitating rhythmic motor activities such as locomotion and respiration. Previous studies conducted in humans and rodent models demonstrated respiratory augmentations induced by EES delivered to the dorsal cervical spinal cord. Importantly, EES at the dorsal cervical spinal cord opposes opioid-induced respiratory depression in human. To reveal the mechanism underpins how cervical EES modulate the respiratory neural circuit, we conducted this comparative study between dorsal cervical EES to the ventral EES in patients with opioid-induced respiratory suppression or depression. We hypothesize that the ventral cervical EES activates the local motor neuronal pools while the dorsal EES recruits both sensory and motor cervical circuits as well as accesses supraspinal structures such as medulla. We recruited and consented 25 patients who underwent anterior (ventral) cervical spinal cord surgery and compared the effect of ventral cervical EES to to the effect of dorsal cervical ESS in a dataset that we collected from 18 patients undergoing cervical spinal surgery. In the 25 patients, the EES was deliver to the ventral surface of spinal cord ranging from cervical level 3 to 7 (C3 to C7) for no more than 90 seconds at the optimal intensity ranging from 0.5 mA to 5 mA with stimulation frequencies 5 Hz or 30 Hz. In both the dorsal and ventral EES groups, the subjects were anesthetized with low dose or high dose remifentanil that partially or completely depressed voluntary respiration. We observed three main differences between the dorsal and ventral cervical EEST in regulating respiration in the presence of remifentanil. First, dorsal cervical EES induced the resetting of inspiratory rhythm while ventral cervical EES did not. Second, dorsal but not ventral cervical EES induced longer lasting respiratory modulation after the stimulation stopped. Third, dorsal cervical EES induced both frequency and amplitude changes of respiration while ventral cervical EES modulated the amplitude of the respiration more significantly. These observations suggest that the dorsal and ventral cervical EES facilitate respiration via different neural circuits. This work was supported by the National Institute of Drug Abuse in the National Institutes of Health: R01 DA047637, Louis and Harold Price Foundation, H & H Evergreen Foundation, and J. Yang Family Foundation. This is the full abstract presented at the American Physiology Summit 2023 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.

A carotid body-brainstem neural circuit mediates sighing in hypoxia

Increased ventilation is a critical process that occurs when the body responds to a hypoxic environment. Sighs are long, deep breaths that prevent alveolar collapse, and their frequency is significantly increased by hypoxia. In this study, we first show that sighing is induced by hypoxia as a function of increased hypoxic severity and that hypoxia-induced sighing is capable of increasing the oxygen saturation in a mouse model. We next found that the gastrin-releasing peptide (Grp) expressing neurons in the nucleus of the solitary tract (NTS) are important in mediating hypoxia-induced sighing. Retrograde tracing from these Grp neurons reveals their direct afferent input from the petrosal ganglion neurons that innervate the carotid body, the major peripheral chemoreceptor that senses blood oxygen. Acute hypoxia preferentially activates these Grp neuronsin the NTS. Photoactivation of these neurons through their projections in the inspiratory rhythm generator in the ventral medulla induces sighing, whereas genetic ablation or chemogenetic silencing of these neurons specifically diminishes the sighs, but not other respiratory responses, induced by hypoxia. Finally, the mice with reduced sighing in hypoxia exhibit an elevated heart-rate increase, which may compensate for maintaining the blood oxygen level. Therefore, we identified a neural circuit that connects the carotid body to the breathing control center in the ventral medulla with a specific function for hypoxia-induced sighing, which restores the oxygen level.

Angiotensin II increases respiratory rhythmic activity in the preBötzinger complex without inducing astroglial calcium signaling.

Angiotensin II (Ang II) is the primary modulator of the renin-angiotensin system and has been widely studied for its effect on the cardiovascular system. While a few studies have also indicated an involvement of Ang II in the regulation of breathing, very little is known in this regard and its effect on brainstem respiratory regions such as the preBötzinger complex (preBötC), the kernel for inspiratory rhythm generation, has not been investigated yet. This study reports that Ang II temporarily increases phrenic nerve activity in the working heart-brainstem preparation, indicating higher central respiratory drive. Previous studies have shown that the carotid body is involved in mediating this effect and we revealed that the preBötC also plays a part, using acute slices of the brainstem. It appears that Ang II is increasing the respiratory drive in an AT1R-dependent manner by optimizing the interaction of inhibitory and excitatory neurons of the preBötC. Thus, Ang II-mediated effects on the preBötC are potentially involved in dysregulating breathing in patients with acute lung injury.

µ-Opioid Receptor Activation Reduces Glutamate Release in the PreBötzinger Complex in Organotypic Slice Cultures.

The inspiratory rhythm generator, located in the brainstem preBötzinger complex (preBötC), is dependent on glutamatergic signaling and is affected profoundly by opioids. Here, we used organotypic slice cultures of the newborn mouse brainstem of either sex in combination with genetically encoded sensors for Ca2+, glutamate, and GABA to visualize Ca2+, glutamatergic and GABAergic signaling during spontaneous rhythm and in the presence of DAMGO. During spontaneous rhythm, the glutamate sensor SF-iGluSnFR.A184S revealed punctate synapse-like fluorescent signals along dendrites and somas in the preBötC with decay times that were prolonged by the glutamate uptake blocker (TFB-TBOA). The GABA sensor iGABASnFR showed a more diffuse fluorescent signal during spontaneous rhythm. Rhythmic Ca2+- and glutamate transients had an inverse relationship between the spontaneous burst frequency and the burst amplitude of the Ca2+ and glutamate signals. A similar inverse relationship was observed when bath applied DAMGO reduced spontaneous burst frequency and increased the burst amplitude of Ca2+, glutamate, and GABA transient signals. However, a hypoxic challenge reduced both burst frequency and Ca2+ transient amplitude. Using a cocktail that blocked glutamatergic, GABAergic, and glycinergic transmission to indirectly measure the release of glutamate/GABA in response to an electrical stimulus, we found that DAMGO reduces the release of glutamate in the preBötC but has no effect on GABA release. This suggest that the opioid mediated slowing of respiratory rhythm involves presynaptic reduction of glutamate release, which would impact the ability of the network to engage in recurrent excitation, and may result in the opioid-induced slowing of inspiratory rhythm.SIGNIFICANCE STATEMENT Opioids slow down breathing rhythm by affecting neurons in the preBötzinger complex (preBötC) and other brainstem regions. Here, we used cultured slices of the preBötC to better understand this effect by optically recording Ca2+, glutamate, and GABA transients during preBötC activity. Spontaneous rhythm showed an inverse relationship between burst frequency and burst amplitude in the Ca2+ and glutamate signals. Application of the opioid DAMGO slowed the rhythm, with a concomitant increase in Ca2+, glutamate, and GABA signals. When rhythm was blocked pharmacologically, DAMGO reduced the presynaptic release of glutamate, but not GABA. These data suggest the mechanism of action of opioids involves presynaptic reduction of glutamate release, which may play an important role in the opioid-induced slowing of inspiratory rhythm.