Brainstem Respiratory Network Research Articles

Isoproterenol modulates expiratory activities in the brainstem spinal cord preparation in neonatal mice in vitro

Motor behaviors such as breathing required temporal coordination of different muscle groups to insured efficient ventilation and provide oxygen to the body. This action is the result of interactions between neural networks located within the brainstem. Inspiration and expiration depend at least in part on interactions between two separate oscillators: inspiration is driven by a neural network located in the preBötzinger complex (PreBötC) and active expiration is driven by a network in the parafacial respiratory group (pFRG). Neurons of the pFRG are silent at rest and become active when the respiratory drive increased. This study investigated the temporal coordination between the brainstem respiratory network and the lumbar spinal network that generates spontaneous activities that is different of the induced fictive locomotion. The remaining question is how these activities coordinate early during the development. Results of this study show that brainstem networks contribute to the temporal coordination of the lumbar spontaneous activity during inspiration since lumbar motor activity occurs exclusively during the expiratory time. This study also investigated the role of the β-noradrenergic modulation on the respiratory activities. β-noradrenergic receptors activation increased the frequency of the double bursts and increased expiratory activity at the lumbar level. These results suggest interactions between brainstem and spinal networks and reveal a descending drive that may contribute to the coordination of the respiratory and lumbar spontaneous activities.

Plasticity in the Functional Properties of NMDA Receptors Improves Network Stability during Severe Energy Stress.

Brain energy stress leads to neuronal hyperexcitability followed by a rapid loss of function and cell death. In contrast, the frog brainstem switches into a state of extreme metabolic resilience that allows them to maintain motor function during hypoxia as they emerge from hibernation. NMDA receptors (NMDARs) are Ca2+-permeable glutamate receptors that contribute to the loss of homeostasis during hypoxia. Therefore, we hypothesized that hibernation leads to plasticity that reduces the role of NMDARs within neural networks to improve function during hypoxia. To test this, we assessed a circuit with a large involvement of NMDAR synapses, the brainstem respiratory network of female bullfrogs, Lithobates catesbeianus Contrary to our expectations, hibernation did not alter the role of NMDARs in generating network output, nor did it affect the amplitude, kinetics, and hypoxia sensitivity of NMDAR currents. Instead, hibernation strongly reduced NMDAR Ca2+ permeability and enhanced desensitization during repetitive stimulation. Under severe hypoxia, the normal NMDAR profile caused network hyperexcitability within minutes, which was mitigated by blocking NMDARs. After hibernation, the modified complement of NMDARs protected against hyperexcitability, as disordered output did not occur for at least one hour in hypoxia. These findings uncover state-dependence in the plasticity of NMDARs, whereby multiple changes to receptor function improve neural performance during metabolic stress without interfering with their normal role during healthy conditions.

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.

Exploring sex differences in inhibitory synaptic transmission in the kolliker-fuse of rett syndrome mice

Rett Syndrome (RTT) is a rare neurodevelopmental disorder characterized by severe motor impairments including breathing problems. RTT is caused by mutations in the methyl CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker-Fuse (KF) area of RTT mice, there are fewer GABAergic neurites and injection of a GABA reuptake antagonist alleviates breathing irregularity in vivo. However, no one has examined the physiology of inhibitory synaptic transmission in the KF of RTT mice. In the present work, we tested the hypothesis that inhibitory synaptic transmission was deficient in the respiratory-controlling KF of MeCP2Bird mice. Although girls and women make up the majority of RTT patients, most studies examining synaptic physiology in the brainstem have been performed using male MeCP2-deficient mice. Hemizygous male MeCP2Bird mice lack MeCP2 protein and exhibit rapid onset of severe RTT symptoms which leads to short life spans. Due to partial X-inactivation, MeCP2 expression in heterozygous female MeCP2-deficient mice is mosaic, leading to variable symptom severity and delayed onset, which more closely resembles the human condition. In the present study, we examined inhibitory synaptic transmission in the KF of female heterozygous MeCP2Bird mice compared to male hemizygous MeCP2Bird mice and wild type littermates. Whole-cell voltage-clamp recordings from KF neurons contained in acute brain slices were used to measure inhibitory currents. Most notably, the frequency of spontaneous inhibitory postsynaptic currents is reduced in the KF of male, but not female, MeCP2Bird mice compared to wild type littermates. To better understand this sex difference in inhibitory neurotransmission, we assessed the influence of estrous cycle stage and MeCP2 expression status in the recorded neuron. The outcomes of this research will increase our understanding of synaptic function in respiratory control neurons in female Rett Syndrome mice. This research was supported by the International Rett Syndrome Foundation Basic Research Grant 3608. 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.

Are thyrotropin-releasing hormone (TRH) and analog taltirelin viable reversal agents of opioid-induced respiratory depression?

Opioid‐induced respiratory depression (OIRD) is a potentially life‐threatening complication of opioid consumption. Apart from naloxone, an opioid antagonist that has various disadvantages, a possible reversal strategy is treatment of OIRD with the hypothalamic hormone and neuromodulator thyrotropin‐releasing hormone (TRH). In this review, we performed a search in electronic databases and retrieved 52 papers on the effect of TRH and TRH‐analogs on respiration and their efficacy in the reversal of OIRD in awake and anesthetized mammals, including humans. Animal studies show that TRH and its analog taltirelin stimulate breathing via an effect at the preBötzinger complex, an important respiratory rhythm generator within the brainstem respiratory network. An additional respiratory excitatory effect may be related to TRH’s analeptic effect. In awake and anesthetized rodents, TRH and taltirelin improved morphine‐ and sufentanil‐induced respiratory depression, by causing rapid shallow breathing. This pattern of breathing increases the work of breathing, dead space ventilation, atelectasis, and hypoxia. In awake and anesthetized humans, a continuous infusion of intravenous TRH with doses up to 8 mg, did not reverse sufentanil‐ or remifentanil‐induced respiratory depression. This is related to poor penetration of TRH into the brain compartment but also other causes are discussed. No human data on taltirelin are available. In conclusion, data from animals and human indicate that TRH is not a viable reversal agent of OIRD in awake or anesthetized humans. Further human studies on the efficacy and safety of TRH’s more potent and longer lasting analog taltirelin are needed as this agent seems to be a more promising reversal drug.

GABAergic and Glycinergic Synaptic Transmission in Respiratory‐Controlling Kolliker‐Fuse Neurons in Female and Male Rett Syndrome Mice

Rett Syndrome (RTT) is a neurodevelopmental disorder characterized by severe motor behavior disturbances including breathing problems. RTT is caused by silencing mutations in the methyl‐CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker‐Fuse (KF) area of RTT mice, there are fewer GABAergic axon terminals and injection of a GABA reuptake antagonist alleviates breathing problems in vivo. However, no one has examined the physiology of GABA or glycine synapses in the KF of RTT mice. In the present work, we tested the hypothesis that GABAergic and glycinergic synapses are deficient in the KF of RTT mice. We performed whole‐cell voltage clamp recordings from KF neurons contained in brain slices of MeCP2‐deficient RTT mice and wild type littermates. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the KF neurons of male RTT mice was reduced compared to wild type (WT) littermates. In contrast, female RTT mice showed no change in sIPSC frequency in the KF compared to control, despite having breathing abnormalities. The amplitude of sIPSCs in the KF of female mice were smaller compared to WT littermates, whereas male RTT mice showed no change in amplitude compared to WT littermates. These results spotlight the importance of studying respiratory controlling KF neurons in both male and female RTT mice, and suggest that reductions in IPSC frequency and amplitude are not sufficient to explain the breathing irregularities observed in RTT in females and males, respectively.

Activation of Cold‐Sensitive Afferents Inhibits Aberrant Irritant‐evoked Cardiopulmonary Reflexes in the Spontaneously Hypertensive (SH) Rat

Inhalation of irritants activates airway vagal afferent nerves, evoking cardiopulmonary reflexes. Evidence suggests that cardiopulmonary reflexes evoked by irritant/pollution inhalation are remodeled from bradycardia in normal subjects to tachycardia and ventricular arrhythmia in patients with pre‐existing cardiovascular disease. We have shown similar remodeling in the spontaneously hypertensive (SH) rat: selective activation of airway vagal afferent nerves via inhalation of the TRPA1‐specific agonist allyl isothiocyanate (AITC) evoked parasympathetic‐mediated reflex bradycardia in normotensive wistar‐kyoto rats but evoked sympathetically‐mediated tachycardia and premature ventricular contractions (PVCs) in spontaneously hypertensive (SH) rats.Activation of TRPA1‐expressing airway vagal afferents also evokes cough in some conscious animal models. Cough reflexes have been shown to be inhibited by nasal stimulation with L‐menthol via activation of TRPM8 ion channels expressed on cold‐sensitive trigeminal afferents which regulate brainstem respiratory networks. Here, we investigated the cardiopulmonary reflexes evoked by inhalation of L‐menthol (150mg/ml) alone and in combination with AITC (4.7mg/ml) in SH rats. L‐menthol inhalation alone had no effect on respiration/heart rate, but, when inhaled during AITC inhalation, L‐menthol selectively inhibited the AITC‐evoked tachyarrhythmia and tachypnea. Importantly, the inactive isomer D‐menthol (150mg/ml) failed to affect AITC‐evoked sympathoexcitatory reflexes. In another set of studies, the selective TRPM8 agonist WS‐12 (300mM) also inhibited the AITC‐evoked tachyarrhythmia. Therefore, we conclude that aberrant cardiopulmonary reflexes evoked by irritant inhalation are reduced by activation of menthol‐sensitive pathways within the airways. This data supports the therapeutic potential of menthol treatment against irritant‐evoked cardiopulmonary reflexes.

Potential presynaptic effects of opioids on medullary inspiratory circuits: New insights from a joint neural biomechanical model

Administration of opioids can lead to respiratory depression and eventually death. Opioids can alter the frequency and magnitude of inspiratory motor drive by binding to mu‐opioid receptors on inspiratory neurons located in several areas of the brainstem. Lalley (Am J Physiol: Regul. 285:R1287‐1304, 2003) suggested that some of the effects of fentanyl on bulbospinal inspiratory neuron activity were presynaptic. We tested the feasibility of pre‐ vs post‐synaptic effects of opioids with an integrate‐and‐fire computational neural biomechanical model of the brainstem respiratory network. Our previous simulations utilizing this model involved separately reducing conductance at groups of synapses that excited inspiratory‐augmenting (I‐Aug) or inspiratory‐decrementing (I‐Dec) neuron populations to simulate opioid‐mediated decreases in the presynaptic excitability of excitatory axons to these neurons from inspiratory driver neurons (I‐driver). We found that the presynaptic hypothesis was best supported by a mechanism involving depression of the excitatory synapse between I‐driver and I‐decrementing neurons. The current modeling investigation extends that work by focusing on three inspiratory neuron populations: I‐Aug, I‐Dec, and late inspiratory (late‐I). We separately decreased the conductance of each excitatory synapse to examine how these changes would affect the respiratory pattern. Simulations revealed that systematically decreasing the conductance of synapses from the I‐Aug neuron population to late‐I and I‐ Aug bulbospinal populations by 50% resulted in a decrease in frequency, duration, and amplitude of inspiratory drive. Decreasing the conductance of excitatory synapses from I‐Dec neurons to I‐Aug, and late‐I populations had a limited effect on inspiratory activity. Similar results were observed when the conductance of excitatory synapses was reduced from the late‐I population to its I‐Dec and I‐Aug targets. Overall, these results indicate that a plausible mechanism for respiratory suppression by opioids could include depression of excitatory synaptic actions between selected medullary inspiratory neuron populations.

Advances in Reversal Strategies of Opioid-induced Respiratory Toxicity.

Opioids may produce life-threatening respiratory depression and death from their actions at the opioid receptors within the brainstem respiratory neuronal network. Since there is an increasing number of conditions where the administration of the opioid receptor antagonist naloxone is inadequate or undesired, there is an increased interest in the development of novel reversal and prevention strategies aimed at providing efficacy close to that of the opioid receptor antagonist naloxone but with fewer of its drawbacks such as its short duration of action and lesser ability to reverse high-affinity opioids, such as carfentanil, or drug combinations. To give an overview of this highly relevant topic, the authors systematically discuss predominantly experimental pharmacotherapies, published in the last 5 yr, aimed at reversal of opioid-induced respiratory depression as alternatives to naloxone. The respiratory stimulants are discussed based on their characteristics and mechanism of action: nonopioid controlled substances (e.g., amphetamine, cannabinoids, ketamine), hormones (thyrotropin releasing hormone, oxytocin), nicotinic acetylcholine receptor agonists, ampakines, serotonin receptor agonists, antioxidants, miscellaneous peptides, potassium channel blockers acting at the carotid bodies (doxapram, ENA001), sequestration techniques (scrubber molecules, immunopharmacotherapy), and opioids (partial agonists/antagonists). The authors argue that none of these often still experimental therapies are sufficiently tested with respect to efficacy and safety, and many of the agents presented have a lesser efficacy at deeper levels of respiratory depression, i.e., inability to overcome apnea, or have ample side effects. The authors suggest development of reversal strategies that combine respiratory stimulants with naloxone. Furthermore, they encourage collaborations between research groups to expedite development of viable reversal strategies of potent synthetic opioid-induced respiratory depression.

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.

Opioids and obstructive sleep apnea.

Freire C, Sennes LU, Polotsky VY. Opioids and obstructive sleep apnea. J Clin Sleep Med. 2022;18(2):647-652.

The mechanisms of neuroplasticity during acclimatization to‐ and deacclimatization from chronic hypercapnia are fundamentally different

A hallmark of respiratory-related diseases, such as chronic obstructive pulmonary disease (COPD), is chronic hypercapnia (CH). Common therapeutic intervention for patients experiencing CH is mechanical ventilation to restore blood-gas homeostasis. While many neurophysiological consequences of CH have been characterized, little is known about the effects reversing CH. The primary goal of the present study was to test the hypothesis that abrupt deacclimatization from CH would significantly alter markers of neuroplasticity and tryptophan metabolism, but not levels of excitatory neuromodulators. Utilizing our goat model of increased inspired CO2 (InCO2)-induced CH, adult female goats were exposed to an InCO2 of 6% for 30 days (d) followed by a return to room air for 24 hours (h). Following 24h of recovery, goats were euthanized, and brainstems were rapidly extracted. Tissue punches of key nuclei throughout the brainstem respiratory network (hypoglossal motor nucleus (XII), nucleus tractus solitarius (NTS)/dorsal motor nucleus of the vagus (DMV), ventral respiratory column (VRC), medullary raphe (MR), ventrolateral medulla (VLM), retrotrapezoid nucleus (RTN), and cuneate nucleus (CN)) were obtained and used for either western blot or HPLC analysis. Changes in glutamatergic signaling, neuroinflammation, tryptophan metabolism, and neuromodulators concentration, following recovery from CH, were assessed. Following 24h of recovery, there were no significant differences in AMPA or NMDA receptor expression or phosphorylation, compared to room air control goats, across all nuclei investigated. Similarly, there were no significant differences in the inflammatory cytokine IL1B following 24h recovery across all nuclei investigated, compared to room air control goats. However, there was significantly (P<0.05) lower expression of key enzymes of tryptophan metabolism (Indolamine 2,3-dioxygenase (IDO); Tryptophan Hydroxylase (TPH) and neuronal markers (NeuN) compared to control within the rostral VLM and MR. Within the solitary complex (NTS & DMV), there was significantly (P>0.05) greater concentrations in norepinephrine (+132%±49), serotonin (+348%±142), and dopamine (+86%±53) at 24h of recovery compared to 30d of CH. We conclude that although specific markers of neuroplasticity and neuroinflammation were unchanged from control during deacclimatization, there were brainstem-site dependent changes in excitatory neuromodulators during deacclimatization from CH. These data suggest that the mechanisms of neuroplasticity during acclimatization to- and deacclimatization from- CH are fundamentally different.

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.

Differential Respiratory Effects of µ‐opioid Receptor Activation within the preBötzinger Complex and Neighbouring Respiration‐related Regions in the Rabbit

It is well known that opioids induce respiratory depression by activating µ-opioid receptors (MORs). Although MORs are expressed throughout the entire brainstem respiratory network, the effects of their activation within the different respiration-related regions are still unclear. We investigated the contribution of the preBötzinger Complex (preBötC) and the neighbouring Bötzinger Complex (BötC) and inspiratory portion of the ventral respiratory group (iVRG) to the MOR-induced respiratory depression in anesthetized, vagotomized, paralyzed and artificially ventilated adult rabbits making use of microinjections (30-50 nl) of the MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO). Dose-dependent effects were observed. DAMGO microinjected at 0.1 mM into the preBötC and the BötC caused progressive reductions in peak phrenic amplitude associated with tonic phrenic activity and patterns of breathing displaying irregularities that were more marked in the preBötC (ataxic breathing). Increases or no changes in respiratory frequency accompanied these responses in the preBötC and the BötC, respectively. Tonic apneic effects developed at 0.5 mM. DAMGO microinjected into the iVRG provoked decreases in the amplitude and frequency of phrenic bursts at 0.1 mM and apnea at 0.5 mM. DAMGO-induced increases in the variability of peak phrenic amplitude and time components of the breathing pattern were more pronounced after microinjections into the preBötC. Local application of 5 mM naloxone reversed the apneic effects in all instances. The results provide further insides into the role of MORS in the genesis of respiratory depression induced by systemically administered opioids. They suggest that various areas of the respiratory network differentially contribute to the opioid-induced respiratory disorders and support the view that the preBötC may play a prominent role.

The Role of Noradrenergic and Glutamatergic Signaling in Noradrenergic Neurons in Respiratory Control

Central noradrenergic (NA) neurons are a critical component of the brainstem respiratory network that maintains blood CO2 and O2 levels through respiratory chemoreflexes. NA dysfunction is implicated in numerous developmental respiratory disorders such as the fatal Sudden Infant Death Syndrome (SIDS), Rett Syndrome, and Congenital Central Hypoventilation Syndrome (CCHS), in which abnormal chemosensory function is implicated. However, which NA neurons modulate respiratory chemoreflexes and the underlying mechanisms are still not fully understood. Previous studies in our lab utilizing intersectional chemogenetic (hM4D and hM3D DREADD mediated) inhibition and excitation identified multiple rhombomere (transient genetically-defined hindbrain segments arising during early development) specific NA populations that play a role in the adult hypercapnic chemoreflex. NA neurons are typically thought to communicate with synaptic partners through noradrenaline as their primary neurotransmitter. However, the excitatory neurotransmitter, glutamate, is also expressed in subpopulations of NA neurons including those derived from rhombomeres playing a role in the hypercapnic reflex. Prior studies have implicated NA derived glutamatergic signaling in respiratory control. Thus, we seek to determine the relative roles of noradrenergic and glutamatergic signaling in rhombomeric NA populations that are involved in adult respiratory chemoreflexes. To determine the requirement of noradrenergic signaling in the hypercapnic reflex, we aim to conditionally ablate noradrenergic signaling in rhombomeric NA sub-populations by crossing rhombomere specific Credrivers with the THflox/flox(tyrosine hydroxylase) allele to remove dopamine and downstream (nor) adrenaline production in targeted neurons. We also aim to determine if the glutamate producing NA neurons represented a larger functional cohort in breathing control. We therefore used our intersectional strategy to target and chemogenetically activate (hM3D) or inhibite (hM4De) glutamate co-expressing NA neurons (defined by partially overlapping Vglut2Cre; DBHFLPo activity) which are expected to comprise a super-set of rhombomere specific NA neurons that play a role in respiratory chemoreflexes. Whole-body barometric plethysmography was used to measure the respiratory function of the mice under room air (21%O2,79%N2) and hypercapnia (21%O2, 74%N2, 5% CO2). Our preliminary data suggests that removing noradrenergic signaling in a key rhombomeric NA subpopulation does not inhibit the hypercapnic reflex while DREADD mediated inhibition does reduce the hypercapnic reflex. Acute inhibition of glutamate-expressing NA neurons shows a reduction in the hypercapnic ventilatory reflex while acute activation of these same neurons shows a significantly enhanced reflex. Altogether, these preliminary data suggest that glutamate production may delineate a functionally specific NA subpopulation in the hypercapnic reflex and offers additional clues to the respective roles of noradrenaline and glutamatergic signaling in the noradrenergic system.

Reciprocal connectivity of the periaqueductal gray with the ponto-medullary respiratory network in rat

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

Forebrain projection neurons target functionally diverse respiratory control areas in the midbrain, pons, and medulla oblongata.

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

The Mesencephalic Periaqueductal Gray, a Further Structure Involved in Breathing Failure Underlying Sudden Infant Death Syndrome.

The aim of this study was to investigate the involvement of the periaqueductal gray (PAG), an area of gray matter surrounding the cerebral aqueduct of Sylvius, in the pathogenetic mechanism of SIDS, a syndrome frequently ascribed to arousal failure from sleep. We reconsidered the same samples of brainstem, more precisely midbrain specimens, taken from a large series of sudden infant deaths, namely 46 cases aged from 1 to about 7 months, among which 26 SIDS and 20 controls, in which we already highlighted significant developmental alterations of the substantia nigra, another mesencephalic structure with a critical role in breath and awakening regulation. Specific histological and immunohistochemical methods were applied to examine the PAG cytoarchitecture and the expression of the tyrosine hydroxylase, a marker of catecholaminergic neurons. Hypoplasia of the PAG subnucleus medialis was observed in 65% of SIDS but never in controls; tyrosine hydroxylase expression was significantly higher in controls than in SIDS. A significant correlation was found between these findings and those related to the substantia nigra, demonstrating a link between these neuronal centers and the brainstem respiratory network and a common involvement in the sleep-arousal phase failure leading to SIDS.

Seizure-related apneas have an inconsistent linkage to amygdala seizure spread.

Sudden unexpected death in epilepsy (SUDEP) is a frequent cause of death in epilepsy. Respiratory dysfunction is implicated as a critical factor in SUDEP pathophysiology. Human studies have shown that electrical stimulation of the amygdala resulted in apnea, indicating that the amygdala has a role in respiration control. Unilateral amygdala stimulation resulted in immediate onset of respiratory dysfunction occurring only during nose breathing. In small numbers of patients, some but not all spontaneous seizures resulted in apnea occurring shortly after seizure spread to the amygdala. With this study we aimed to determine whether seizure onset or spread to the amygdala was necessary and sufficient to cause apnea. We investigated the temporal relationship between apnea/hypopnea (AH) onset and initial seizure involvement within the amygdala in patients with implanted depth electrodes. Data from 17 patients (11 female) with 47 seizures were analyzed. With seven seizures (three patients), AH preceded amygdala seizure involvement by 2 to 55seconds. There was no AH with four seizures (three patients) that involved the amygdala. With eight seizures (four patients) AH occurred within 2seconds following amygdala seizure onset. With 28 seizures, AH started >2seconds after amygdala seizure onset (range 3-158seconds). Following seizure onset, there was a significant difference between AH onset time and amygdala seizure onset (P<.001). The mean ± standard deviation (SD) AH onset was 27.8±41.06seconds, and the mean time to amygdala involvement was 8.83±20.19seconds. There is a wide range of AH onset times relative to amygdala seizure involvement. With some seizures, amygdala seizure involvement occurs without AH. With other seizures, AH precedes amygdala seizures, suggesting that, with spontaneous seizures, involvement of the amygdala may not be crucial to induction of AH with all seizures. Other pathophysiology impacting brainstem respiratory networks may be of greater relevance to seizure-triggered apneas.