Inspiratory Rhythm Generation Research Articles

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.

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.

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.

Inspiratory rhythm generation is stabilized by Ih.

Cellular and network properties must be capable of generating rhythmic activity that is both flexible and stable. This is particularly important for breathing, a rhythmic behavior that dynamically adapts to environmental, behavioral, and metabolic changes from the first to the last breath. The pre-Bötzinger complex (preBötC), located within the ventral medulla, is responsible for producing rhythmic inspiration. Its cellular properties must be tunable, flexible as well as stabilizing. Here, we explore the role of the hyperpolarization-activated, nonselective cation current (Ih) for stabilizing PreBötC activity during opioid exposure and reduced excitatory synaptic transmission. Introducing Ih into an in silico preBötC network predicts that loss of this depolarizing current should significantly slow the inspiratory rhythm. By contrast, in vitro and in vivo experiments revealed that the loss of Ih minimally affected breathing frequency, but destabilized rhythmogenesis through the generation of incompletely synchronized bursts (burstlets). Associated with the loss of Ih was an increased susceptibility of breathing to opioid-induced respiratory depression or weakened excitatory synaptic interactions, a paradoxical depolarization at the cellular level, and the suppression of tonic spiking. Tonic spiking activity is generated by nonrhythmic excitatory and inhibitory preBötC neurons, of which a large percentage express Ih. Together, our results suggest that Ih is important for maintaining tonic spiking, stabilizing inspiratory rhythmogenesis, and protecting breathing against perturbations or changes in network state.NEW & NOTEWORTHY The Ih current plays multiple roles within the preBötC. This current is important for promoting intrinsic tonic spiking activity in excitatory and inhibitory neurons and for preserving rhythmic function during conditions that dampen network excitability, such as in the context of opioid-induced respiratory depression. We therefore propose that the Ih current expands the dynamic range of rhythmogenesis, buffers the preBötC against network perturbations, and stabilizes rhythmogenesis by preventing the generation of unsynchronized bursts.

RNA Interference Via shRNA Decreases the Number of Transcripts of Scn8a in Glutamatergic Interneurons in the preBötC

Rhythmic neural activity that drives the inspiratory phase of the breathing cycle in mammals emanates from glutamatergic interneurons of the pre‐Bötzinger Complex (preBötC). A longstanding question in neuroscience is: what are the underlying mechanisms for inspiratory rhythmogenesis? One proposed mechanism involves the persistent sodium current (INaP), which is the ionic mechanism that underlies the voltage‐dependent bursting‐pacemaker activity of neurons whose rhythmic burst discharges are putatively obligatory for inspiratory rhythm generation. Specifically in brain tissues, the Scn8a gene codes for Nav1.6 ion channels, and those ion channels give rise to INaP.Different genetic tools can help us to understand the role of specific molecules in the generation of motor behaviors, such as breathing. The short hairpin RNA (shRNA) is an artificial RNA molecule, delivered through adeno‐associated virus (AAV), that can inhibit gene expression and promote long‐term knock‐down of the targeted gene.Here we administered Cre‐dependent shRNA to knock‐down Scn8a (thus Nav1.6) in Cre‐expressing glutamatergic neurons in preBötC and quantified mRNA per glutamatergic neuron. We used adult Vglut2Cre mice (10‐14 weeks old) and injected 50 nL of AAVs carrying the shRNA. We used in situ hybridization (RNAscope® Fluorescent Assay) to label mRNA of Scn8a and to identify transduced glutamatergic neurons in the preBötC for RNA quantification at 2 and 6 weeks after injection. Samples were visualized under the Four‐Channel, Upright Confocal Microscope Body Based on Cerna® System (ThorLabs), and images were acquired using the ThorImage®LS Data Acquisition Software, at 40x magnification. The amount of RNA per cell was quantified using the open‐source software Quantitative Pathology(QuPath). We obtained the mean of the number of transcripts per cell ± the standard deviation (SD), and unpaired t‐test was used to evaluate the statistical difference between the shRNA injected mice and their control group (mice whose viral vector contained a non‐targeting shRNA sequence).Our results show that at 2 weeks, the control group had 27.5±9.5 RNA/neuron, whereas the shRNA‐injected had 12.8±3.5, which represents a 53% of decrease in the number of transcripts (P<0.0001). A similar pattern was observed after 6 weeks of the injection in a different cohort, where control animals showed 35.4±6.1 RNA/cells, and the shRNA group only had 24.1±5.2, representing 32% drop in the transcripts for that group (P<0.0001). This initial protocol allowed us to verify that the gene expression has been knocked down at critical time points, encouraging further studies using shRNA to evaluate whether attenuation of Nav1.6 expression in rhythmogenic preBötC neurons impairs breathing, which could resolve the longstanding question regarding the putatively rhythmogenic role of INaP.

Postnatal Development of the Purinergic Signaling System in the preBötzinger Complex; Implications for the Hypoxic Ventilatory Response

The hypoxic ventilatory response (HVR) comprises a carotid body‐mediated increase in ventilation followed by a secondary, CNS‐mediated decrease, the hypoxic respiratory depression (HRD), that is larger and potentially life‐threatening in newborn mammals. The mechanisms responsible for the greater HRD in newborns are unknown, but purinergic signaling via P2 and P1 receptor (R) mechanisms helps shape this 2° phase. Shortly after the 1° increase in ventilation, astrocytes release ATP in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation), which stimulates breathing via P2Y1Rs and attenuates the HRD. Extracellular ATP (ATPe) is degraded to adenosine (ADOe), which inhibits breathing and is implicated in the HRD. Thus, the impact of ATP on the HVR is determined by the balance between the actions of ATP and ADOe, that could change developmentally and contribute to the greater HRD of newborns. Our goal was to characterize how the many factors that determine the balance between ATP and ADOe signaling change during development, including: P2Y1Rs; ADO A1Rs; ectonucleotidases (ECTOs) that degrade ATPe to ADOe; ADOe clearance by equilibrative nucleoside transporters (ENTs) via transmembrane transport of ADO down its concentration gradient; and ADO kinase (ADK), which is not mature in the cortex until postnatal day (P) 14, but facilitates ADOe clearance by converting ADOi into AMP so that ADOi<ADOe. We used rhythmic preBötC slice preparations to characterize developmental changes in: i) the effects of P2Y1R and ADOR signaling on preBötC rhythm; ii) ECTO activity; and iii) the impact of ENTs and ADK on baseline preBötC rhythm and preBötC responses to ATP and ADO that were locally injected to simulate hypoxia. iv) In vivo head‐out plethysmography was used to assess the role of ADOe clearance mechanisms in shaping the developing HVR.i) The frequency (freq) increase evoked by ATP in the preBötC doubled between P0‐6 and P9‐12, but the P2Y1R agonist MRS 2365‐evoked increase did not change. The A1R inhibition of freq was of similar magnitude but greater duration at P0‐6 compared to P9‐12. ii) ECTO activity, assessed by incubating preBötC tissue in ATP and measuring PO4, was greater at P9‐12. iii) The ADO freq inhibition was 2‐fold longer in ENT knockout mice compared to WT mice at all ages. ADK inhibition (ABT 702) inhibited baseline freq only at P9‐12. iv) Knockout of ENTs blunted the freq response to hypoxia (10% O2) in vivo, but increased the VT response at all ages. Introduction of constitutively active ADK into the brain produced P0‐2 ADKtg mice that responded to hypoxia with an adult‐like, sustained increase in VE/VO2; P0‐2 WT C57BL6 did not respond to hypoxia. This difference disappeared by P6‐8. Surprisingly, the mature HVR of P0‐2 ADKtg mice reflected a greater hypoxic depression of VO2, not a greater increase in VE.Data suggest preBötC sensitivity to P2Y1 and A1Rs does not change, while ECTO activity increases developmentally. ADO actions are prolonged in P0‐6 mice, suggesting that ADOe clearance mechanisms mature postnatally. ENTs appear to play a similar role from birth, which may involve clearing ADOe from the preBötC during hypoxia. ADK maturation also appears important in the development of an adaptive HVR. However, the role of ADK in modulating VE vs metabolic responses to hypoxia remains to be elucidated.

The Role of the Ih Current in Rhythmic Stability of the PreBötzinger Complex

The generation of rhythmicity is a fundamental property of the nervous system. Common amongst all rhythmogenic networks is the ability to reconfigure and quickly adapt to changes in metabolic, environmental, and behavioral demands. Yet, this flexibility, and plasticity must be counter‐balanced by the need to maintain stability. A group of neurons collectively known as the preBötzinger complex (preBötC) within the ventral respiratory column of the medulla assemble in a network that is both necessary and sufficient to maintain inspiratory rhythm generation. Multiple ionic conductances contribute to rhythm generation. One known for its role in the generation of rhythmic activity across multiple neural networks is the hyperpolarization activated cation current (Ih). The Ih­ current is a voltage‐dependent mixed cationic current that is activated upon phasic hyperpolarization of neurons. The role of Ih in rhythmogenesis within the preBötC is inconclusive. Some reports find that blocking Ih has minimal effect, while others conclude that this increases the rate of rhythmic bursting throughout the network. To date, Ih was specifically studied in rhythmically active neurons with unknown transmitter phenotype. Herein we sought to determine the distribution of Ih amongst the population of tonically and rhythmically active excitatory and inhibitory neurons throughout the preBötC. Additionally, we tested whether removing Ih­ in‐vitro and in‐vivo renders the network more susceptible to perturbations. Using in‐vitro patch clamping we found that Ih showed greatest expression amongst excitatory (87% of DBX1+), and inhibitory (89% of VGAT+) tonically active cell populations. Removing Ih using ZD7288 silenced nearly all tonically spiking neurons. This led to a minimal effect on the rhythmic activity of fully synchronized population bursts, but significantly increased the incidence of less synchronized burstlet activity. Moreover, removing Ih and silencing tonic activity within the network rendered the preBötC more susceptible to suppression following opioid (DAMGO) and CNQX administration. This effect was recapitulated in‐vivo with microdialysis of ZD7288 into the preBötC, which caused normally well‐tolerated doses of morphine to become lethal via terminal apnea. In summary, Ihplays a complex role within the rhythm generating network of the preBötC. Under optimal conditions, Ih is not necessary for maintaining fully synchronized bursting within the network. Yet, Ih becomes increasingly important during perturbations that challenge network function, suggesting this ionic conductance contributes to maintaining the robustness of breathing during everchanging physiological and environmental demands.

Opto‐ and Chemogenetic Dissection of Ictal Apnea Neural Circuitry

Sudden Unexpected Death in Epilepsy (SUDEP) is defined as the sudden, unexpected and unexplained death of a person with epilepsy and accounts for between 8 and 17% of epilepsy‐related deaths, rising to 50% for patients with refractory epilepsy. In a mouse model of SUDEP, we have recently shown that death is due to seizure‐induced respiratory arrest. In addition, apnea is initiated during the tonic phase and tonic respiratory muscle contraction is a possible mechanism of apnea. In the present study, we explore 1) whether tonic activity of the inspiratory rhythm generator in the brainstem and/or 2) upper motor neuron activity in the motor cortex drives ictal apnea.We recorded video, electrocorticogram (ECoG), electrocardiogram (ECG), and breathing via whole body plethysmography in adult mice carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D; “D/+ mice”) during audiogenic seizures. This mutation was identified from a patient that died from SUDEP and D/+ mice have severe convulsive seizures with apnea and many suffer seizure‐induced death.To test the hypothesis that tonic activity from the inspiratory oscillator results in apnea, we implanted fiberoptic ferrules bilaterally into the Bötzinger Complex (BötC) of mice that express Channelrhodopsin2 (ChR2) under the vesicular GABA transporter (VGAT; “VGAT‐ChR2” mice) that were crossed with D/+ mice (Fig. 1A & B). The goal of the experiment is to photostimulate BötC during ictal apnea to inhibit tonic inspiratory activity and produce expiration (Fig. 1C). Seizures were evoked using a 15 kHz pure tone, as we have done before (Fig. 1D). Trains of light pulses (50 ms pulses, 5 mW of 473 nm light) were evoked repetitively during ictal apnea (Fig. 1E & F). However, this did not recover normal breathing rhythm and apnea duration was no different for any photostimulation paradigm versus control (p = 0.7892, F = 0.1747, One‐Way ANOVA; Fig. 1G). Although breathing was not affected during seizures, the effects on baseline breathing were substantial; for example, inspiration was inhibited for a full 10 second photostimulation train (Fig. 1H).To test the necessity of ictal activity from upper motor neurons in the motor cortex are required for generating ictal apnea, we expressed iDREADD receptors in cortical excitatory neurons of D/+ mice and injected CNO i.p. prior to inducing seizures with a 15 kHz pure tone (Fig. 2A). Under control conditions, seizures presented with the usual tonic phase apnea and spike wave discharges (SWDs) in the motor cortex (Fig. 2B). CNO was able to robustly inhibit the SWDs, but the tonic phase and apnea were not affected (Figs. 2C‐E). The effect of 3 mg/kg CNO on ECoG power was significantly greater than the effect on apnea (p = 0.0059, paired t‐test).In sum, we found that the core inspiratory oscillator circuitry in the brainstem is likely bypassed to create tonic inspiratory activity. Furthermore, inhibition of cortical upper motor neurons has no effect on apnea. Thus, our interpretation is that other pools of upper motor neurons must drive the tonic inspiratory activity and apnea.

Mechanisms of opioid-induced respiratory depression.

Opioid-induced respiratory depression (OIRD), the primary cause of opioid-induced death, is the neural depression of respiratory drive which, together with a decreased level of consciousness and obstructive sleep apnea, cause ventilatory insufficiency. Variability of responses to opioids and individual differences in physiological and neurological states (e.g., anesthesia, sleep-disordered breathing, concurrent drug administration) add to the risk. Multiple sites can independently exert a depressive effect on breathing, making it unclear which sites are necessary for the induction of OIRD. The generatorof inspiratoryrhythm is the preBötzinger complex (preBötC) in the ventrolateral medulla. Other important brainstem respiratory centres includethe pontine Kölliker-Fuse and adjacent parabrachial nuclei (KF/PBN) in the dorsal lateral pons, and the dorsal respiratory group in the medulla. Deletion of μ opioid receptors from neurons showed that the preBötC and KF/PBN contribute to OIRD with the KF as a respiratory modulator and the preBötC as inspiratory rhythm generator. Glutamatergic neurons expressing NK-1R and somatostatin involved in the autonomic function of breathing, and modulatory signal pathways involvingGIRK and KCNQ potassium channels, remain poorly understood. Reversal of OIRD has relied heavily on naloxone which also reverses analgesia but mismatches between the half-lives of naloxone and opioids can make it difficult to clinically safely avoid OIRD. Maternal opioid use, which is rising, increases apneas and destabilizes neonatal breathing but opioid effects on maternal and neonatal respiratory circuits in neonatal abstinence syndrome (NAS) are not well understood. Methadone, administered to alleviate symptoms of NAS in humans, desensitizes rats to RD.

Perinatal inflammation and gestational intermittent hypoxia disturbs respiratory rhythm generation and long-term facilitation in vitro: Partial protection by acute minocycline

Perinatal inflammation triggers breathing disturbances early in life and affects the respiratory adaptations to challenging conditions, including the generation of amplitude long-term facilitation (LTF) by acute intermittent hypoxia (AIH). Some of these effects can be avoided by anti-inflammatory treatments like minocycline. Since little is known about the effects of perinatal inflammation on the inspiratory rhythm generator, located in the preBötzinger complex (preBötC), we tested the impact of acute lipopolysaccharide (LPS) systemic administration (sLPS), as well as gestational LPS (gLPS) and gestational chronic IH (gCIH), on respiratory rhythm generation and its long-term response to AIH in a brainstem slice preparation from neonatal mice. We also evaluated whether acute minocycline administration could influence these effects. We found that perinatal inflammation induced by sLPS or gLPS, as well as gCIH, modulate the frequency, signal-to-noise ratio and/or amplitude (and their regularity) of the respiratory rhythm recorded from the preBötC in the brainstem slice. Moreover, all these perinatal conditions inhibited frequency LTF and amplitude long-term depression (LTD); gCIH even induced frequency LTD of the respiratory rhythm after AIH. Some of these alterations were not observed in slices pre-treated in vitro with minocycline, when compared with slices obtained from naïve pups, suggesting that ongoing inflammatory conditions affect respiratory rhythm generation and its plasticity. Thus, it is likely that alterations in the inspiratory rhythm generator and its adaptive responses could contribute to the respiratory disturbances observed in neonates that suffered from perinatal inflammatory challenges.

The role of neuronal excitation and inhibition in the pre-Bötzinger complex on the cough reflex in the cat.

Brainstem respiratory neuronal network significantly contributes to cough motor pattern generation. Neuronal populations in the pre-Bötzinger complex (PreBötC) represent a substantial component for respiratory rhythmogenesis. We studied the role of PreBötC neuronal excitation and inhibition on mechanically induced tracheobronchial cough in 15 spontaneously breathing, pentobarbital anesthetized adult cats (35 mg/kg, iv initially). Neuronal excitation by unilateral microinjection of glutamate analog d,l-homocysteic acid resulted in mild reduction of cough abdominal electromyogram (EMG) amplitudes and very limited temporal changes of cough compared with effects on breathing (very high respiratory rate, high amplitude inspiratory bursts with a short inspiratory phase, and tonic inspiratory motor component). Mean arterial blood pressure temporarily decreased. Blocking glutamate-related neuronal excitation by bilateral microinjections of nonspecific glutamate receptor antagonist kynurenic acid reduced cough inspiratory and expiratory EMG amplitude and shortened most cough temporal characteristics similarly to breathing temporal characteristics. Respiratory rate decreased and blood pressure temporarily increased. Limiting active neuronal inhibition by unilateral and bilateral microinjections of GABAA receptor antagonist gabazine resulted in lower cough number, reduced expiratory cough efforts, and prolongation of cough temporal features and breathing phases (with lower respiratory rate). The PreBötC is important for cough motor pattern generation. Excitatory glutamatergic neurotransmission in the PreBötC is involved in control of cough intensity and patterning. GABAA receptor-related inhibition in the PreBötC strongly affects breathing and coughing phase durations in the same manner, as well as cough expiratory efforts. In conclusion, differences in effects on cough and breathing are consistent with separate control of these behaviors.NEW & NOTEWORTHY This study is the first to explore the role of the inspiratory rhythm and pattern generator, the pre-Bötzinger complex (PreBötC), in cough motor pattern formation. In the PreBötC, excitatory glutamatergic neurotransmission affects cough intensity and patterning but not rhythm, and GABAA receptor-related inhibition affects coughing and breathing phase durations similarly to each other. Our data show that the PreBötC is important for cough motor pattern generation, but cough rhythmogenesis appears to be controlled elsewhere.

P2Y 1 receptor modulation of Ih in the preBötzinger inspiratory rhythm generator may underlie the ATP‐mediated attenuation of the hypoxic respiratory depression

Premature infants often stop breathing briefly (apnea) because the brainstem respiratory network is still immature (apnea of prematurity, AOP). During these apneas, oxygen levels fall (hypoxia) triggering a biphasic hypoxic ventilatory response comprising an initial increase in ventilation followed by a centrally mediated secondary depression that can be life-threatening. Caffeine is used to stimulate breathing in AOP. However, ~20% of these infants do not respond to caffeine. Thus, alternate therapies are needed. Recent data suggest that during hypoxia, ATP is released in the preBötzinger Complex (preBötC, inspiratory rhythm generator) where it acts via P2Y1 receptors (Rs) to excite inspiratory neurons, increase ventilation and attenuate the secondary hypoxic depression. A mechanistic understanding of this P2Y1R excitation is lacking. Preliminary whole-cell analysis of inspiratory preBötC neurons in vitro revealed P2Y1R currents that reverse near -40 mV and increase in amplitude with neuron hyperpolarization. Based on these data, we tested the hypotheses that the increase in inspiratory frequency evoked by ATP in the preBötC in vitro and the ATP-mediated increase in ventilation during hypoxia in vivo reflects P2Y1R potentiation of the hyperpolarization-activated inward current, Ih (carried by cyclic nucleotide–gated, HCN, channels). Application of MRS2365 (P2Y1R agonist, 100 µM) to Ih-expressing inspiratory preBötC neurons in rhythmic brainstem slices from neonatal rat potentiated Ih by 32±6% (at -100 mV, n=18) and depolarized voltage-dependence of Ih activation by ~10 mV. The MRS2365 inward current and its potentiation of Ih were blocked by the Ih blocker, ZD7288 (100 μM). ZD7288 also attenuated the MRS2365-evoked frequency increase by 60-90%. Ih currents in preBötC neurons were sensitive to ZD7288 (100 µM) but not ivabradine (30 µM), suggesting HCN2 but not HCN1,4 subunit involvement. In paralyzed, anesthetized, vagotomized ventilated adult rats, bilateral microinfusion of ZD7288 (250 µM, but not 100 µM) into the preBötC increased the secondary hypoxic (10% O2) depression by 68±23% (n=6) compared to rats that received aCSF microinfusion (n=4). This effect was regionally specific. If the bilateral microinfusions of ZD7288 were not both in the preBötC (e.g., 1 in preBötC and the other in BötC or rVRG), ZD7288 had no effect on the hypoxic ventilatory response. Finally, in anesthetized, vagotomized, spontaneously-breathing adult rats, unilateral reverse microdialysis of ZD7288 (100 µM) in the preBötC area (n=13) increased the hypoxic frequency depression by 20±6% (compared to aCSF, n=6). These data suggest that the P2Y1R excitation of the preBötC network is produced, in part, via potentiation of Ih in a subgroup of inspiratory neurons, and that potentiation of Ih in the preBötC attenuates the hypoxic ventilatory depression in adult rats. Data also identify the HCN2 subunit as a potential target for new strategies to counteract respiratory depression in AOP.

Neuronal Mediators of the Frequency Increase Evoked by Activation of P2Y 1 Receptors in the preBötzinger Complex Inspiratory Rhythm Generating Network in Vitro

The ventilatory response to acute hypoxia (HVR) comprises an initial, carotid body-mediated increase in breathing followed by a centrally-mediated secondary respiratory depression when ventilation falls to a new steady state. Recent evidence suggests that ATP is released from astrocytes in the preBötzinger Complex (preBötC; site of inspiratory rhythm generation) during the secondary phase where it acts via P2Y1 receptors (Rs) to excite preBötC neurons, increase breathing frequency and attenuate the secondary respiratory depression. The preBötC network comprises a heterogenous population of excitatory and inhibitory inspiratory neurons that can be further classified based on expression of transcription factors, receptors and transmitters, discharge pattern and membrane properties. An important goal is to establish the neuronal phenotype(s) that underlie the P2Y1R frequency increase. Perhaps surprisingly, preBötC frequency increases in response to optogenetic stimulation of excitatory OR inhibitory preBötC neurons. Thus, the objective of this study was to determine if the P2Y1R-mediated frequency increase results from activation of excitatory (glutamatergic) or inhibitory (GABA/glycinergic) preBötC inspiratory neurons. Analysis of P2Y1R and somatostatin (SST) immunolabeling in the medulla of neonatal vGLUT2TdTomato and GLYT2EGFP reporter mice showed that P2Y1R immunolabeled neurons are concentrated in a discrete region of the ventral respiratory column (VRC) that features strong SST immunolabeling (a preBötC marker). P2Y1R+ neurons were sparse in all other regions of the VRC. In addition, 95% of P2Y1R+ neurons were glutamatergic (VGLUT2+), but <10% of glutamatergic neurons were P2Y1R+. None of the GLYT2+ (glycinergic) preBötC neurons were P2Y1R+. Whole-cell analyses of neurons in rhythmic slices from vGLUT2 mice showed that all VGLUT2+ inspiratory preBötC neurons excited by the P2Y1R agonist MRS2365 (100 µM) were glutamatergic, but only1/3 of VGLUT2+ neurons were P2Y1R sensitive. In contrast, calcium imaging of rhythmic slices identified subpopulations (4/16) of glutamatergic and non glutamatergic inspiratory neurons that in TTX were P2Y1R sensitive. Finally, we demonstrated that the frequency increase evoked by MRS2365 (100 µM) in the preBötC was unaffected by block of inhibitory synaptic transmission via GABAA (bicuculline, 20 µM) and glycine (strychnine, 20 µM) receptor antagonists. In summary, we found that P2Y1R-expressing neurons are concentrated in the preBötC and are primarily glutamatergic. P2Y1R-sensitive neurons are primarily glutamatergic, but a subpopulation is nonglutamatergic. These data, combined with the observation that the P2Y1R-evoked excitation of the preBötC does not depend on synaptic inhibition, suggest that the P2Y1R-mediated excitation of the preBötC is primarily achieved through activation of excitatory, glutamatergic neurons.

Limited Capacity for Clearance of Adenosine from the preBötzinger Complex Inspiratory Network Contributes to the Profound Depression of Breathing by Hypoxia in the Very Young

Breathing in premature infants often stops briefly (apnea) because the brainstem network that controls breathing is immature (apnea of prematurity, AOP). Apneas cause hypoxia, triggering the hypoxic ventilatory response (HVR), which comprises an initial increase in ventilation followed by a centrally mediated 2° decrease. In adults, breathing remains above baseline during this 2° phase, but in the very young it falls below baseline leading to a life-threatening feedback loop where apnea causes hypoxia, respiratory depression, further hypoxia and so on. Understanding the cause of this powerful hypoxic inhibition of breathing in the very young, a major question in perinatal physiology, could inform new therapies for AOP. The transmitter ATP is released in the preBötzinger Complex (preBötC, site of inspiratory rhythm generation) during hypoxia where it increases breathing and attenuates the 2° depression. However, extracellular ATP (ATPe) is degraded to adenosine (ADO), which inhibits breathing and is implicated in AOP; e.g., caffeine blocks ADOe actions and is the first line treatment for AOP. We hypothesize that the greater 2° inhibition of breathing in prematurity is due to an immature system for clearing ADOe. ADOe-mediated inhibition stops with its removal from the extracellular space, which depends on i) equilibrative nucleoside transporters (ENTs) that move ADOe across cell membranes down its concentration gradient, and ii) ADO kinase (ADK), an enzyme that keeps the level of ADO inside cells lower than outside so ENTs clear ADOe. How ENT activity develops in the preBötC is unknown. However, the form of ADK that influences ADOe in the brain is minimally functional until 2 weeks of age in rodents. To assess the importance of ADOe clearance in the development of the HVR, we used plethysmography to measure the HVR (10% O2) in 0-56 day old (P0-14) wild-type (WT), ENT knockout (KO), and ADKtg mice engineered to have functional ADK throughout life. In WT and ENT KO neonates (P0-2), ventilation fell below baseline after ~7 min in hypoxia, but the hypoxia-evoked frequency increase was markedly reduced in the ENT KO. Strikingly, P0-2 ADKtg mice showed the mature breathing response; ventilation remained above baseline levels throughout hypoxia. At P12 in WT and ENT KO, the HVR had still not matured; ventilation fell below baseline during the 2° phase, yet it remained above baseline in P12 ADKtg mice. To assess the importance of ENT and ADK specifically in the preBötC, we isolated the preBötC network in rhythmic brain slices from P0-12 mice. The ADO inhibition of preBötC frequency lasted twice as long in P0-12 ENT KO mice compared to WT. Conversely, the decrease in basal frequency caused by ADK inhibition (ABT-702) increased with age (P0-2, 15%; P9-11, 23%). These data suggest that i) ENTs are important for ADO clearance in the preBötC after birth and ii) low ADK activity at birth is a major contributor to the greater hypoxic respiratory depression in early development.