Site For Respiratory Rhythm Generation Research Articles

Maternal Methadone Destabilizes Neonatal Breathing and Desensitizes Neonates to Opioid-Induced Respiratory Frequency Depression.

Pregnant women and developing infants are understudied populations in the opioid crisis, despite the rise in opioid use during pregnancy. Maternal opioid use results in diverse negative outcomes for the fetus/newborn, including death; however, the effects of perinatal (maternal and neonatal) opioids on developing respiratory circuitry are not well understood. Given the profound depressive effects of opioids on central respiratory networks controlling breathing, we tested the hypothesis that perinatal opioid exposure impairs respiratory neural circuitry, creating breathing instability. Our data demonstrate maternal opioids increase apneas and destabilize neonatal breathing. Maternal opioids also blunted opioid-induced respiratory frequency depression acutely in neonates; a unique finding since adult respiratory circuity does not desensitize to opioids. This desensitization normalized rapidly between postnatal days 1 and 2 (P1 and P2), the same age quantal slowing emerged in respiratory rhythm. These data suggest significant reorganization of respiratory rhythm generating circuits at P1–2, the same time as the preBötzinger Complex (key site of respiratory rhythm generation) becomes the dominant respiratory rhythm generator. Thus, these studies provide critical insight relevant to the normal developmental trajectory of respiratory circuits and suggest changes to mutual coupling between respiratory oscillators, while also highlighting how maternal opioids alter these developing circuits. In conclusion, the results presented demonstrate neurorespiratory disruption by maternal opioids and blunted opioid-induced respiratory frequency depression with neonatal opioids, which will be important for understanding and treating the increasing population of neonates exposed to gestational opioids.

Drowning a frog respiratory oscillator in a wash of network excitability

To be part of a respiratory rhythm generating circuit, a brainstem nuclei must (1) have phasic activity timed to the rhythm (correlation), (2) be able to speed the rhythm when excited (sufficiency), and (3) be able to slow the rhythm or terminate it when inhibited (necessity). A discrete brainstem nucleus passing these tests is currently considered a critical site for respiratory rhythm generation (an oscillator) if the nuclei continues to be rhythmic when physically isolated.Compelling and accumulating evidence in support of oscillators has been found in many vertebrates including lampreys, bullfrogs, turtles, birds, mice and rats. In bullfrogs and rodents, three putative oscillators have been discovered: the lung power stroke, lung priming and buccal oscillators in frogs; and in mammals the para Facial Respiratory Group (pFRG), Post‐Inspiratory Complex (PICO), and the PreBötzinger Complex (PreBötC). As the buccal oscillator in frogs and the PreBötC in mammals are located in the same brainstem rhombomere, r7, they may be homologous. Identification of multiple ‐‐ possibly homomogous ‐‐ oscillators in different species has led to the Oscillator Hypothesis which, in its strongest form, posits that respiratory rhythm is produced solely by brainstem oscillators and phase of motor bursts is determine by their interaction.However, almost complete (90%) destruction of the putative PreBötC in goats over several days was insufficient to terminate breathing. One explanations for this observation is a network architecture in which respiratory oscillators are no more than isolatable units in a much larger rhythmogenic network. To test if this is the case in frogs, we (a) located the frog buccal oscillator using necessity and sufficiency tests in an isolated hemisected brainstem preparation; (b) increased the excitability of the hemisection which increases the frequency and amplitude of the buccal motor pattern; and (c) again tested for necessity. We found that with network excitation, the necessity of the buccal oscillator disappeared, replaced by distributed rhythmogenesis in the surrounding premotor network.These data suggest that the Oscillator Hypothesis may need to be revised in frogs and possibly other vertebrates; the existence of the buccal oscillator as a discrete site of rhythm generation appears to be state dependent and may only be demonstrable in some conditions.Support or Funding InformationThis work was supported by NSERCThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glial TLR4 signaling does not contribute to opioid-induced depression of respiration.

Opioids activate glia in the central nervous system in part by activating the toll-like receptor 4 (TLR4)/myeloid differentiation 2 (MD2) complex. TLR4/MD2-mediated activation of glia by opioids compromises their analgesic actions. Glial activation is also hypothesized as pivotal in opioid-mediated reward and tolerance and as a contributor to opioid-mediated respiratory depression. We tested the contribution of TLR4 to opioid-induced respiratory depression using rhythmically active medullary slices that contain the pre-Bötzinger Complex (preBötC, an important site of respiratory rhythm generation) and adult rats in vivo. Injection with DAMGO (μ-opioid receptor agonist; 50 μM) or bath application of DAMGO (500 nM) or fentanyl (1 μM) slowed frequency recorded from XII nerves to 40%, 40%, or 50% of control, respectively. This DAMGO-mediated frequency inhibition was unaffected by preapplication of lipopolysaccharides from Rhodobacter sphaeroides (a TLR4 antagonist, 2,000 ng/ml) or (+)naloxone (1-10 μM, a TLR4-antagonist). Bath application of (-)naloxone (500 nM; a TLR4 and μ-opioid antagonist), however, rapidly reversed the opioid-mediated frequency decrease. We also compared the opioid-induced respiratory depression in slices in vitro in the absence and presence of bath-applied minocycline (an inhibitor of microglial activation) and in slices prepared from mice injected (ip) 18 h earlier with minocycline or saline. Minocycline had no effect on respiratory depression in vitro. Finally, the respiratory depression evoked in anesthetized rats by tail vein infusion of fentanyl was unaffected by subsequent injection of (+)naloxone, but completely reversed by (-)naloxone. These data indicate that neither activation of microglia in preBötC nor TLR4/MD2-activation contribute to opioid-induced respiratory depression.

GABAergic and glycinergic inputs modulate rhythmogenic mechanisms in the lamprey respiratory network

We have previously shown that GABA and glycine modulate respiratory activity in the in vitro brainstem preparations of the lamprey and that blockade of GABAA and glycine receptors restores the respiratory rhythm during apnoea caused by blockade of ionotropic glutamate receptors. However, the neural substrates involved in these effects are unknown. To address this issue, the role of GABAA, GABAB and glycine receptors within the paratrigeminal respiratory group (pTRG), the proposed respiratory central pattern generator, and the vagal motoneuron region was investigated both during apnoea induced by blockade of glutamatergic transmission and under basal conditions through microinjections of specific antagonists. The removal of GABAergic, but not glycinergic transmission within the pTRG, causes the resumption of rhythmic respiratory activity during apnoea, and reveals the presence of a modulatory control of the pTRG under basal conditions. A blockade of GABAA and glycine receptors within the vagal region strongly increases the respiratory frequency through disinhibition of neurons projecting to the pTRG from the vagal region. These neurons were retrogradely labelled (neurobiotin) from the pTRG. Intense GABA immunoreactivity is observed both within the pTRG and the vagal area, which corroborates present findings. The results confirm the pTRG as a primary site of respiratory rhythm generation, and suggest that inhibition modulates the activity of rhythm-generating neurons, without any direct role in burst formation and termination mechanisms.

Initiating inspiration outside the medulla does produce eupneic breathing

ARTERIAL PCO2 IS DICTATED by the ratio between the level of pulmonary gas exchange and alveolar ventilation (VA), not minute ventilation. If so, how could isocapnia be maintained if only the magnitude (tidal volume, VT) and duration (TI and TTOT) of the neural output produced by the central pattern generator of breathing (CPG) can be altered? In other words, what is the strategy used by the respiratory control system to match its output to the level of pulmonary gas exchange to keep arterial blood gas homeostasis if this neural output con- tributes to dead space ventilation? In this Viewpoint, we will show that when the spontaneous breathing is replaced by a cortically triggered breathing pattern at various breathing frequencies (we will name this new rhythm an "ecto-rhythm"), the relationship between TTOT and VT has a positive intercept that averages the pulmonary dead space. As a result, the coupling between ventilation and the pulmonary gas exchange becomes possible (17). We will present the idea that whenever isocapnia is maintained during spontaneous or automatic breathing, the magnitude and dura- tion of breath cycles must display the same relationship as during an ecto-rhythm (15). Conversely, we will discuss the implications of the observation that breathing can display the same essential regulatory properties whether generated outside or within the pontomedullary CPG area (7). Ecto-rhythm, breathing pattern, and gas exchange: the re-

Commentaries on Viewpoint: Central chemoreception is a complex system function that involves multiple brain stem sites

to the editor: Amniote vertebrates adjust the acid-base status of the blood through ventilatory alterations of PaCO2 and, to some degree, by renal modulation of bicarbonate concentration. Receptor systems for the ventilatory acid-base regulation has been studied mainly in mammals, in which the

Point:Counterpoint: Medullary pacemaker neurons are essential for both eupnea and gasping in mammals vs. medullary pacemaker neurons are essential for gasping, but not eupnea, in mammals

to the editor: Implicit in the commentaries concerning the possible role of medullary pacemakers in the neurogenesis of eupnea and gasping is one critical and recurring question: How are in vitro rhythms related to these classical patterns of automatic ventilatory activity? Suzue ([5][1]) considered

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Bötzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal

The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal

The following letters are in response to the Point:Counterpoint series “The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal” that appears in this issue. To the Editor : Unique data from three of our past

The Last Word: Point:Counterpoint authors respond to commentaries on “The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBötC) is the primary site of respiratory rhythm generation in the mammal”

To the Editor : We hypothesize that respiratory rhythm originates from two interacting generators; the preBotzinger complex (preBotC) and the retrotrapezoid nucleus (RTN)/parafacial respiratory group (pFRG). Some discussants view our hypothesis as too simplistic and proposed concepts of state-

Novel Data Supporting the Two Respiratory Rhythm Oscillator Hypothesis. Focus on “Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats”

Among the most rewarding moments in scientific discovery are when the pieces of a puzzle provided by different laboratories start to fall into place, forming a coherent and new view of the world. Several years ago, a consensus was emerging that respiratory rhythm in mammals arose from a single

Postnatal Development Differentially Affects Voltage-Activated Calcium Currents in Respiratory Rhythmic Versus Nonrhythmic Neurons of the Pre-Bötzinger Complex

The mammalian respiratory network reorganizes during early postnatal life. We characterized the postnatal developmental changes of calcium currents in neurons of the pre-Bötzinger complex (pBC), the presumed site for respiratory rhythm generation. The pBC contains not only respiratory rhythmic (R) but also nonrhythmic neurons (nR). Both types of neurons express low- and high-voltage-activated (LVA and HVA) calcium currents. This raises the interesting issue: do calcium currents of the two co-localized neuron types have similar developmental profiles? To address this issue, we used the whole cell patch-clamp technique to compare in transverse slices of mice LVA and HVA calcium current amplitudes of the two neuron populations (R and nR) during the first and second postnatal week (P0-P16). The amplitude of HVA currents did not significantly change in R pBC-neurons (P0-P16), but it significantly increased in nR pBC-neurons during P8-P16. The dehydropyridine (DHP)-sensitive current amplitudes did not significantly change during the early postnatal development, suggesting that the observed amplitude changes in nR pBC-neurons are caused by (DHP) insensitive calcium currents. The ratio between HVA calcium current amplitudes dramatically changed during early postnatal development: At P0-P3, current amplitudes were significantly larger in R pBC-neurons, whereas at P8-P16, current amplitudes were significantly larger in nR pBC-neurons. Our results suggest that calcium currents in pBC neurons are differentially altered during postnatal development and that R pBC-neurons have fully expressed calcium currents early during postnatal development. This may be critical for stable respiratory rhythm generation in the underlying rhythm generating network.

Regulation of respiratory neuron development by neurotrophic and transcriptional signaling mechanisms

Functionally diverse populations of respiratory neurons appear to be targets of common neurotrophic and transcriptional signaling pathways. For example, peripheral chemoafferent neurons and noradrenergic neurons in the pontine A5 cell group both require co-signaling by brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF) for survival, growth and/or phenotypic differentiation. Moreover, these same cell groups are dependent on the Phox2 family of transcription factors for early cell type specification. In addition, BDNF and its receptor, TrkB, are expressed in the pre-Botzinger complex (pBC), a critical site for respiratory rhythm generation, and exogenous BDNF can modulate the activity of pBC neurons. This convergence of BDNF, GDNF and Phox2 dependencies may help to explain how mutations in each of these pathways can result in human developmental disorders of breathing.

Pontine influences on respiratory control in ectothermic and heterothermic vertebrates

Respiratory rhythm generators appear both evolutionarily and developmentally as paired segmental rhythm generators in the reticular formation, associated with the motor nuclei of cranial nerves V, VII, IX, X, and XII. Those associated with the Vth and VIIth motor nuclei are "pontine" in origin and in fishes that employ a buccal suction/force pump for breathing the primary pair of respiratory rhythm generators are associated with the trigeminal nuclei. In amphibians, while the basic respiratory pump remains the same, the dominant site of respiratory rhythm generation has been assumed by the facial, glossopharyngeal and vagal motor nuclei. In reptiles, birds and mammals, in general there is a switch to an aspiration pump driven by thoraco-lumbar muscles innervated by spinal nerves. In these groups, the critical sites necessary for respiratory rhythmogenesis now sit near the ponto-medullary border, in the parafacial region (which may underlie expiratory-dominated, intercostal-abdominal breathing in non-mammalian tetrapods) and in a more caudal region, the preBotzinger complex (which may underlie inspiratory-dominated diaphragmatic breathing in mammals).

Depletion of substance P and glutamate by capsaicin blocks respiratory rhythm in neonatal rat in vitro.

The specific role of the neuromodulator substance P (SP) and its target, the neurokinin 1 receptor (NK1R), in the generation and regulation of respiratory activity is not known. The preBötzinger complex (preBötC), an essential site for respiratory rhythm generation, contains glutamatergic NK1R-expressing neurones that are strongly modulated by exogenously applied SP or acute pharmacological blockade of NK1Rs. We investigated the effects of capsaicin, which depletes neuropeptides (including SP) and glutamate from presynaptic terminals, on respiratory motor output in medullary slice preparations of neonatal rat that generate respiratory-related activity. Bath application of capsaicin slowed respiratory motor output in a dose- and time-dependent manner. Respiratory rhythm could be restored by bath application of SP or glutamate transporter blockers. Capsaicin also evoked dose-dependent glutamate release and depleted SP in fibres within the preBötC. Our results suggest that depletion of SP (or other peptides) and/or glutamate by capsaicin causes a cessation of respiratory rhythm in neonatal rat slices.

RT-PCR reveals muscarinic acetylcholine receptor mRNA in the pre-Bötzinger complex.

Muscarinic receptors mediate the postsynaptic excitatory effects of acetylcholine (ACh) on inspiratory neurons in the pre-Bötzinger complex (pre-BötC), the hypothesized site for respiratory rhythm generation. Because pharmacological tools for identifying the subtypes of the muscarinic receptors that underlie these effects are limited, we probed for mRNA for these receptors in the pre-BötC. We used RT-PCR to determine the expression of muscarinic receptor subtypes in tissue punches of the pre-BötC taken from rat medullary slices. Cholinergic receptor subtype M(2) and M(3) mRNAs were observed in the first round of PCR amplification. All five subtypes, M(1)-M(5), were observed in the second round of amplification. Our results suggest that the majority of muscarinic receptor subtypes in the pre-BötC are M(2) and M(3), with minor expression of M(1), M(4), and M(5).

Differential expression of connexin26 and connexin32 in the pre-Bötzinger complex of neonatal and adult rat.

The pre-Bötzinger complex (pre-BötC) is hypothesized to be the site for respiratory rhythm generation in mammals. Studies examining the cellular mechanisms mediating rhythm generation have focused on the role of chemically mediated synaptic interactions; however, electrotonic synaptic interactions (i.e., electrotonic coupling), which occur by means of gap junctions, may also play a role. Here, we used immunoblot and immunohistochemical analyses to determine whether the pre-BötC contains the gap junction proteins necessary for electrotonic communication and whether the presence and distribution of these gap junction proteins show a developmental change in expression. We found that both connexin26 (Cx26) and connexin32 (Cx32) were expressed in pre-BötC neurons of neonatal and adult rats; however, the relative amounts and their distribution varied by age. Cx26 labeling was seen in a high proportion of pre-BötC neurons in neonatal rats < or = 7 days postnatal (P7) but declined with increasing age. In contrast, Cx32 labeling was sparse in pre-BötC neurons of neonatal rats < or = P7, but increased with increasing age; the highest proportion was seen in adult rats. These data suggest the potential for gap junctional communication in the pre-BötC of both neonatal and adult rats, and we propose that the gap junction proteins Cx26 and Cx32 form the neuroanatomic substrate for this gap junctional communication, which may be important in the synchronization of neural activity generating respiratory rhythm.

Identification of two types of inspiratory pacemaker neurons in the isolated respiratory neural network of mice.

In the respiratory network of mice, we characterized with the whole cell patch-clamp technique pacemaker properties in neurons discharging in phase with inspiration. The respiratory network was isolated in a transverse brain stem slice containing the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. After blockade of respiratory network activity with 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), 18 of 52 inspiratory neurons exhibited endogenous pacemaker activity, which was voltage dependent, could be reset by brief current injections and could be entrained by repetitive stimuli. In the pacemaker group (n = 18), eight neurons generated brief bursts (0.43 +/- 0.03 s) at a relatively high frequency (1.05 +/- 0.12 Hz) in CNQX. These bursts resembled the bursts that these neurons generated in the intact network during the interval between two inspiratory bursts. Cadmium (200 microM) altered but did not eliminate this bursting activity, while 0.5 microM tetrodotoxin suppressed bursting activity. Another set of pacemaker neurons (10 of 18) generated in CNQX longer bursts (1.57 +/- 0.07 s) at a lower frequency (0.35 +/- 0.01 Hz). These bursts resembled the inspiratory bursts generated in the intact network in phase with the population activity. This bursting activity was blocked by 50-100 microM cadmium or 0.5 microM tetrodotoxin. We conclude that the respiratory neural network contains pacemaker neurons with two types of bursting properties. The two types of pacemaker activities might have different functions within the respiratory network.

Mechanisms underlying regulation of respiratory pattern by nicotine in preBötzinger complex.

Cholinergic neurotransmission plays a role in regulation of respiratory pattern. Nicotine from cigarette smoke affects respiration and is a risk factor for sudden infant death syndrome (SIDS) and sleep-disordered breathing. The cellular and synaptic mechanisms underlying this regulation are not understood. Using a medullary slice preparation from neonatal rat that contains the preBötzinger Complex (preBötC), the hypothesized site for respiratory rhythm generation, and generates respiratory-related rhythm in vitro, we examined the effects of nicotine on excitatory neurotransmission affecting inspiratory neurons in preBötC and on the respiratory-related motor activity from hypoglossal nerve (XIIn). Microinjection of nicotine into preBötC increased respiratory frequency and decreased the amplitude of inspiratory bursts, whereas when injected into XII nucleus induced a tonic activity and an increase in amplitude but not in frequency of inspiratory bursts from XIIn. Bath application of nicotine (0.2--0.5 microM, approximately the arterial blood nicotine concentration immediately after smoking a cigarette) increased respiratory frequency up to 280% of control in a concentration-dependent manner. Nicotine decreased the amplitude to 82% and increased the duration to 124% of XIIn inspiratory bursts. In voltage-clamped preBötC inspiratory neurons (including neurons with pacemaker properties), nicotine induced a tonic inward current of -19.4 +/- 13.4 pA associated with an increase in baseline noise. Spontaneous excitatory postsynaptic currents (sEPSCs) present during the expiratory period increased in frequency to 176% and in amplitude to 117% of control values; the phasic inspiratory drive inward currents decreased in amplitude to 66% and in duration to 89% of control values. The effects of nicotine were blocked by mecamylamine (Meca). The inspiratory drive current and sEPSCs were completely eliminated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence or absence of nicotine. In the presence of tetrodotoxin (TTX), low concentrations of nicotine did not induce any tonic current or any increase in baseline noise, nor affect the input resistance in inspiratory neurons. In this study, we demonstrated that nicotine increased respiratory frequency and regulated respiratory pattern by modulating the excitatory neurotransmission in preBötC. Activation of nicotinic acetylcholine receptors (nAChRs) enhanced the tonic excitatory synaptic input to inspiratory neurons including pacemaker neurons and at the same time, inhibited the phasic excitatory coupling between these neurons. These mechanisms may account for the cholinergic regulation of respiratory frequency and pattern.

Unstable breathing rhythms and quasiperiodicity in the pre-Bötzinger complex.

Breathing in neonatal mammals is notoriously unstable, which can impair normal ventilatory function. Do unstable breathing patterns arise from deterministic mechanisms in the central nervous system? To address this question we examined unstable respiratory patterns using in vitro preparations from neonatal and embryonic rodents that isolate the brain stem kernel containing networks that generate and control breathing’. We also analyzed patterns of ventilation in neonates in vivo and in simulations of rhythm generation using a mathematical model of the kernel2. In all of these studies inspiratory rhythms showed evidence of quasiperiodicity, and in some cases the “quasiperiodic transition to chaos” identified in oscillatory dynamical systems by Ruelle and Takens3. In the quasiperiodic regime the inspiratory pattern undergoes amplitude modulation that becomes increasingly disordered and unstable as neuronal excitability is elevated, and leads to chaos-like states of irregular respiratory rhythm. We found that quasiperiodic activity is an intrinsic property of the developing respiratory network that originates in the pre-Botzinger complex (pre-BotC), the proposed site for respiratory rhythm generation in mammals4. Electrophysiological and computational analyses showed that quasiperiodicity can arise in the pre-BotC from rhythm-generating neurons with intrinsic oscillatory bursting-pacemaker properties5, thus we identify a potential mechanism for the onset of deterministic respiratory instability in immature mammals.