Modulation of inspiratory burst duration and frequency by bombesin in vitro.

Mammalian respiratory rhythm-generating circuits in the brainstem are subject to neuromodulation by multiple peptidergic afferent inputs controlling circuit behavior and outputs. Although functionally important, actions of neuropeptide modulators have not been fully characterized. We analyzed at cellular and circuit levels two inspiratory patterns intrinsically generated by the preBötzinger complex (preBötC) and their modulation by the neuropeptides bombesin and substance P (SP) in neonatal rat medullary slices in vitro. We found that, in recordings of hypoglossal nerve and preBötC neuron inspiratory activity, some inspiratory bursts occurring spontaneously under basal conditions have a biphasic shape with longer duration than normal inspiratory bursts and occur at a lower frequency. This biphasic burst pattern has been proposed to represent inspiratory activity underling periodic sighs. Bath-applied bombesin or SP decreased the period and increased the duration of both normal inspiratory and biphasic bursts and their underlying synaptic drives. The ratio of the biphasic long-duration burst period to the normal inspiratory burst period and the ratio of their burst durations remained the same before and after peptidergic modulation. Bombesin increased the frequency of the inspiratory rhythm in a Ca2+-independent manner and the frequency of long-duration bursts in a Ca2+-dependent manner. This finding suggests that period and burst duration coupling are due to intrinsic mechanisms controlling simultaneously timing and burst termination within the inspiratory rhythm-generating network. We propose a model in which signaling cascades activated by bombesin and SP modulate mechanisms controlling inspiratory burst frequency and duration to coordinate preBötC circuit behavioral outputs.

Effects of aconitine on the respiratory activity of brainstem-spinal cord preparations isolated from newborn rats.

Aconitine is a sodium channel opener, but its effects on the respiratory center are not well understood. We investigated the dose-dependent effects of aconitine on central respiratory activity in brainstem-spinal cord preparations isolated from newborn rats. Bath application of 0.5-5 µM aconitine caused an increase in respiratory rhythm and decrease in the inspiratory burst amplitude of the fourth cervical ventral root (C4). Separate application of aconitine revealed that medullary neurons were responsible for the respiratory rhythm increase, and neurons in both the medulla and spinal cord were involved in the decrease of C4 amplitude by aconitine. A local anesthetic, lidocaine (100 µM), or a voltage-dependent sodium channel blocker, tetrodotoxin (0.1 µM), partially antagonized the C4 amplitude decrease by aconitine. Tetrodotoxin treatment tentatively decreased the respiratory rhythm, but lidocaine tended to further increase the rhythm. Treatment with 100 µM riluzole or 100 µM flufenamic acid, which are known to inhibit respiratory pacemaker activity, did not reduce the respiratory rhythm enhanced by aconitine + lidocaine. The application of 1 µM aconitine depolarized the preinspiratory, expiratory, and inspiratory motor neurons. The facilitated burst rhythm of inspiratory neurons after aconitine disappeared in a low Ca2+/high Mg2+ synaptic blockade solution. We showed the dose-dependent effects of aconitine on respiratory activity. The antagonists reversed the depressive effects of aconitine in different manners, possibly due to their actions on different sites of sodium channels. The burst-generating pacemaker properties of neurons may not be involved in the generation of the facilitated rhythm after aconitine treatment.

Activation of respiratory-related bursting in an isolated medullary section from adult bullfrogs.

Breathing is generated by a rhythmic neural circuit in the brainstem, which contains conserved elements across vertebrate groups. In adult frogs, the 'lung area' located in the reticularis parvocellularis is thought to represent the core rhythm generator for breathing. Although this region is necessary for breathing-related motor output, whether it functions as an endogenous oscillator when isolated from other brainstem centers is not clear. Therefore, we generated thick brainstem sections that encompass the lung area to determine whether it can generate breathing-related motor output in a highly reduced preparation. Brainstem sections did not produce activity. However, subsaturating block of glycine receptors reliably led to the emergence of rhythmic motor output that was further enhanced by blockade of GABAA receptors. Output occurred in singlets and multi-burst episodes resembling the intact network. However, burst frequency was slower and individual bursts had longer durations than those produced by the intact preparation. In addition, burst frequency was reduced by noradrenaline and µ-opioids, and increased by serotonin, as observed in the intact network and in vivo. These results suggest that the lung area can be activated to produce rhythmic respiratory-related motor output in a reduced brainstem section and provide new insights into respiratory rhythm generation in adult amphibians. First, clustering breaths into episodes can occur within the rhythm-generating network without long-range input from structures such as the pons. Second, local inhibition near, or within, the rhythmogenic center may need to be overridden to express the respiratory rhythm.

Long-Range Respiratory and Theta Oscillation Networks Depend on Spatial Sensory Context.

Neural oscillations can couple networks of brain regions, especially at lower frequencies. The nasal respiratory rhythm, which elicits robust olfactory bulb oscillations, has been linked to episodic memory, locomotion, and exploration, along with widespread oscillatory coherence. The piriform cortex is implicated in propagating the olfactory-bulb-driven respiratory rhythm, but this has not been tested explicitly in the context of both hippocampal theta and nasal respiratory rhythm during exploratory behaviors. We investigated systemwide interactions during foraging behavior, which engages respiratory and theta rhythms. Local field potentials from the olfactory bulb, piriform cortex, dentate gyrus, and CA1 of hippocampus, primary visual cortex, and nasal respiration were recorded simultaneously from male rats. We compared interactions among these areas while rats foraged using either visual or olfactory spatial cues. We found high coherence during foraging compared with home cage activity in two frequency bands that matched slow and fast respiratory rates. Piriform cortex and hippocampus maintained strong coupling at theta frequency during periods of slow respiration, whereas other pairs showed coupling only at the fast respiratory frequency. Directional analysis shows that the modality of spatial cues was matched to larger influences in the network by the respective primary sensory area. Respiratory and theta rhythms also coupled to faster oscillations in primary sensory and hippocampal areas. These data provide the first evidence of widespread interactions among nasal respiration, olfactory bulb, piriform cortex, and hippocampus in awake freely moving rats, and support the piriform cortex as an integrator of respiratory and theta activity.SIGNIFICANCE STATEMENT Recent studies have shown widespread interactions between the nasally driven respiratory rhythm and neural oscillations in hippocampus and neocortex. With this study, we address how the respiratory rhythm interacts with ongoing slow brain rhythms across olfactory, hippocampal, and visual systems in freely moving rats. Patterns of network connectivity change with behavioral state, with stronger interactions at fast and slow respiratory frequencies during foraging as compared with home cage activity. Routing of interactions between sensory cortices depends on the modality of spatial cues present during foraging. Functional connectivity and cross-frequency coupling analyses suggest strong bidirectional interactions between olfactory and hippocampal systems related to respiration and point to the piriform cortex as a key area for mediating respiratory and theta rhythms.

Advancing respiratory-cardiovascular physiology with the working heart-brainstem preparation over 25 years.

Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.

Eupnea and gasping in vivo are facilitated by the activation of 5-HT2A receptors.

Eupnea and gasping in infancy depend on central nervous system (CNS) serotonin (5-hydroxytryptamine; 5-HT). Although previous in vitro preparations have provided some evidence that 5-HT acts through type 2 A receptors (5-HT2A) to facilitate eupnea and gasping, here the hypothesis addressed is that 5-HT2A receptor activation is necessary for eupnea and the proper generation of gasping in vivo. To test this, we administered 2,5-dimethoxy-4-iodoamphetamine (DOI; 0.25 mg/kg i.p.), a 5-HT2A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.25 mg/kg i.p.), a 5-HT1A agonist, or vehicle (saline) to 7-9-day-old tryptophan hydroxylase 2 knockout (TPH2-/-) mice. A second experiment assessed the effect of MDL-11,939 (MDL; 10 mg/kg i.p.), the specific 5-HT2A antagonist, or vehicle (DMSO) on the gasping of wild-type (TPH2+/+) animals. Drugs were given 15 min prior to five episodes of severe hypoxia that elicited gasping. TPH2-/- breathed more slowly but had the same V̇e and V̇e/V̇o2 compared with TPH2+/+. As previously reported, the gasping of TPH2-/- was significantly delayed (P < 0.001) and occurred at a significantly lower frequency compared with TPH2+/+ (P = 0.04). For both genotypes, DOI hastened eupneic frequency but had no effect on V̇e or V̇e/V̇o2. The gasping of TPH2-/-, although unaffected by 8-OH-DPAT, was indistinguishable from the gasping of TPH2+/+ following DOI. In TPH2+/+, application of MDL led to hypoventilation (P = 0.01), a delay in the appearance of gasping (P = 0.005), and reduced gasp frequency (P = 0.05). These data show that, in vivo, 5-HT2A receptors facilitate both eupnea and gasping. As has been shown in vitro, 5-HT2A probably promotes gasping by exciting hypoxia-resistant pacemaker neurons.NEW & NOTEWORTHY Previous in vitro studies suggest that 5-HT2A receptors contribute to eupnea and are necessary for fictive gasping. The current study shows that the impaired gasping displayed by neonatal TPH2-/- mice, deficient in CNS serotonin, is restored by 5-HT2A receptor activation. Following 5-HT2A blockade, wild-type mice hypoventilated and their gasping resembled that of TPH2-/- mice. This study shows that both eupnea and gasping in vivo rely on the activation of 5-HT2A receptors.

Configuration and dynamics of dominant inspiratory multineuronal activity patterns during eupnea and gasping generation in vitro.

The pre-Bötzinger complex (preBötC), located within the ventral respiratory column, produces inspiratory bursts in varying degrees of synchronization/amplitude. This wide range of population burst patterns reflects the flexibility of the preBötC neurons, which is expressed in variations in the onset/offset times of their activations and their activity during the population bursts, with respiratory neurons exhibiting a large cycle-to-cycle timing jitter both at the population activity onset and at the population activity peak, suggesting that respiratory neurons are stochastically activated before and during the inspiratory bursts. However, it is still unknown whether this stochasticity is maintained while evaluating the coactivity of respiratory neuronal ensembles. Moreover, the preBötC topology also remains unknown. In this study, by simultaneously recording tens of preBötC neurons and using coactivation analysis during the inspiratory periods, we found that the preBötC has a scale-free configuration (mixture of not many highly connected nodes, hubs, with abundant poorly connected elements) exhibiting the rich-club phenomenon (hubs more likely interconnected with each other). PreBötC neurons also produce multineuronal activity patterns (MAPs) that are highly stable and change during the hypoxia-induced reconfiguration. Moreover, preBötC contains a coactivating core network shared by all its MAPs. Finally, we found a distinctive pattern of sequential coactivation of core network neurons at the beginning of the inspiratory periods, indicating that, when evaluated at the multicellular level, the coactivation of respiratory neurons seems not to be stochastic.NEW & NOTEWORTHY By means of multielectrode recordings of preBötC neurons, we evaluated their configuration in normoxia and hypoxia, finding that the preBötC exhibits a scale-free configuration with a rich-club phenomenon. preBötC neurons produce multineuronal activity patterns that are highly stable but change during hypoxia. The preBötC contains a coactivating core network that exhibit a distinctive pattern of coactivation at the beginning of inspirations. These results reveal some network basis of inspiratory rhythm generation and its reconfiguration during hypoxia.

Effects of Cardiac Resynchronization Therapy on Cardio-Respiratory Coupling.

In this study, the effect of cardiac resynchronization therapy (CRT) on the relationship between the cardiovascular and respiratory systems in heart failure subjects was examined for the first time. We hypothesized that alterations in cardio-respiratory interactions, after CRT implantation, quantified by signal complexity, could be a marker of a favorable CRT response. Sample entropy and scaling exponents were calculated from synchronously recorded cardiac and respiratory signals 20 min in duration, collected in 47 heart failure patients at rest, before and 9 months after CRT implantation. Further, cross-sample entropy between these signals was calculated. After CRT, all patients had lower heart rate and CRT responders had reduced breathing frequency. Results revealed that higher cardiac rhythm complexity in CRT non-responders was associated with weak correlations of cardiac rhythm at baseline measurement over long scales and over short scales at follow-up recording. Unlike CRT responders, in non-responders, a significant difference in respiratory rhythm complexity between measurements could be consequence of divergent changes in correlation properties of the respiratory signal over short and long scales. Asynchrony between cardiac and respiratory rhythm increased significantly in CRT non-responders during follow-up. Quantification of complexity and synchrony between cardiac and respiratory signals shows significant associations between CRT success and stability of cardio-respiratory coupling.

Volumetric mapping of the functional neuroanatomy of the respiratory network in the perfused brainstem preparation of rats.

KEY POINTS: The functional neuroanatomy of the mammalian respiratory network is far from being understood since experimental tools that measure neural activity across this brainstem-wide circuit are lacking. Here, we use silicon multi-electrode arrays to record respiratory local field potentials (rLFPs) from 196-364 electrode sites within 8-10 mm3 of brainstem tissue in single arterially perfused brainstem preparations with respect to the ongoing respiratory motor pattern of inspiration (I), post-inspiration (PI) and late-expiration (E2). rLFPs peaked specifically at the three respiratory phase transitions, E2-I, I-PI and PI-E2. We show, for the first time, that only the I-PI transition engages a brainstem-wide network, and that rLFPs during the PI-E2 transition identify a hitherto unknown role for the dorsal respiratory group. Volumetric mapping of pontomedullary rLFPs in single preparations could become a reliable tool for assessing the functional neuroanatomy of the respiratory network in health and disease. ABSTRACT: While it is widely accepted that inspiratory rhythm generation depends on the pre-Bötzinger complex, the functional neuroanatomy of the neural circuits that generate expiration is debated. We hypothesized that the compartmental organization of the brainstem respiratory network is sufficient to generate macroscopic local field potentials (LFPs), and if so, respiratory (r) LFPs could be used to map the functional neuroanatomy of the respiratory network. We developed an approach using silicon multi-electrode arrays to record spontaneous LFPs from hundreds of electrode sites in a volume of brainstem tissue while monitoring the respiratory motor pattern on phrenic and vagal nerves in the perfused brainstem preparation. Our results revealed the expression of rLFPs across the pontomedullary brainstem. rLFPs occurred specifically at the three transitions between respiratory phases: (1) from late expiration (E2) to inspiration (I), (2) from I to post-inspiration (PI), and (3) from PI to E2. Thus, respiratory network activity was maximal at respiratory phase transitions. Spatially, the E2-I, and PI-E2 transitions were anatomically localized to the ventral and dorsal respiratory groups, respectively. In contrast, our data show, for the first time, that the generation of controlled expiration during the post-inspiratory phase engages a distributed neuronal population within ventral, dorsal and pontine network compartments. A group-wise independent component analysis demonstrated that all preparations exhibited rLFPs with a similar temporal structure and thus share a similar functional neuroanatomy. Thus, volumetric mapping of rLFPs could allow for the physiological assessment of global respiratory network organization in health and disease.

Retracted: Control of hypoglossal pre-inspiratory discharge.

NEW FINDINGS: What is the topic of this review? This review explores the modulatory role of lung vagal afferents and intra-neuraxial and carotid body chemoreceptors upon hypoglossal pre-inspiratory activity. What advances does it highlight? Pre-inspiratory activity manifesting in hypoglossal neural efferent discharge may be potentiated by mechanical interruption of vagal continuity and challenge with administration of a hypoxic and/or hypercapnic gas mixture and attenuated by static and/or dynamic pulmonary stretch. Differential excitability of, or premotoneuronal volleys exhibiting distinct spatiotemporal patterns of discharge arriving at, motoneurons residing within the hypoglossal motor nucleus may emergently generate phase-spanning pre-inspiratory inspiratory activity of hypoglossal neural efferent discharge manifest at the population level. ABSTRACT: The hypoglossal nerve (XII) innervates muscles mediating excursive movements of the tongue. The population discharge of hypoglossalmotoneuronal axons constituting the hypoglossal nerve precedes and extends through the inspiratory epoch. The epoch subtended between the onsets of hypoglossal and phrenic neural discharge constitutes so-called pre-inspiration. Hypoglossal pre-inspiratory neural discharge serendipitously displaces the tongue along a tensor reducing upper airway resistance anticipative of succeeding inspiratory efforts. Hypoglossal motoneurons exhibiting discharge onset during pre-inspiration experience successive hyperpolarization of membrane voltage and attenuation of unitary spiking frequency, although a subset may, paradoxically and state-dependently, exhibit depolarization of membrane voltage and augmentation of neuronal spiking frequency, by dynamic stretch placed upon the alveolar walls and interstitium. Marked static elevation of positive-end expiratory pressure may induce hypoglossal bursting decoupled from phasic rhythmic phrenic discharge. Augmentation of the amplitude and/or duration of hypoglossal inspiratory discharge during successive pre-inspiratory and inspiratory epochs by inhalation of a hypoxic and/or hypercapnic gas mixture remains restrained in the presence of intact vagal inputs and is potentiated by interruptions of vagal continuity. Unravelling the mechanisms underlying the genesis of pre-inspiratory activity will inform our understanding of respiratory rhythm generation and pattern shaping. In the present work, I seek to explore the mechanisms underlying modulation of hypoglossal pre-inspiratory discharge by hypercapnia, hypoxia and static and dynamic lung stretch placed upon hypoglossal pre-inspiratory activity, the mechanisms underlying the generation of hypoglossal pre-inspiratory activity, and the extent of microanatomical and functional overlap between propriobulbar interneuronal microcircuits generating hypoglossal pre-inspiratory activity and propriobulbar interneuronal microcircuit oscillators generating pre-inspiratory activity inaugurally inducing respiratory rhythmic activity and thus use experimental data from previous work and that developed by other investigators to explore the modulatory role of lung vagal afferents and intra-neuraxial and carotid body chemoreceptors upon hypoglossal pre-inspiratory activity.

Multiscale Entropy Analysis: Application to Cardio-Respiratory Coupling.

It is known that in pathological conditions, physiological systems develop changes in the multiscale properties of physiological signals. However, in real life, little is known about how changes in the function of one of the two coupled physiological systems induce changes in function of the other one, especially on their multiscale behavior. Hence, in this work we aimed to examine the complexity of cardio-respiratory coupled systems control using multiscale entropy (MSE) analysis of cardiac intervals MSE (RR), respiratory time series MSE (Resp), and synchrony of these rhythms by cross multiscale entropy (CMSE) analysis, in the heart failure (HF) patients and healthy subjects. We analyzed 20 min of synchronously recorded RR intervals and respiratory signal during relaxation in the supine position in 42 heart failure patients and 14 control healthy subjects. Heart failure group was divided into three subgroups, according to the RR interval time series characteristics (atrial fibrillation (HFAF), sinus rhythm (HFSin), and sinus rhythm with ventricular extrasystoles (HFVES)). Compared with healthy control subjects, alterations in respiratory signal properties were observed in patients from the HFSin and HFVES groups. Further, mean MSE curves of RR intervals and respiratory signal were not statistically different only in the HFSin group (p = 0.43). The level of synchrony between these time series was significantly higher in HFSin and HFVES patients than in control subjects and HFAF patients (p < 0.01). In conclusion, depending on the specific pathologies, primary alterations in the regularity of cardiac rhythm resulted in changes in the regularity of the respiratory rhythm, as well as in the level of their asynchrony.

The rhythm of memory: how breathing shapes memory function.

The mammalian olfactory bulb displays a prominent respiratory rhythm, which is linked to the sniff cycle and is driven by sensory input from olfactory receptors in the nasal sensory epithelium. In rats and mice, respiratory frequencies occupy the same band as the hippocampal θ-rhythm, which has been shown to be a key player in memory processes. Hippocampal and olfactory bulb rhythms were previously found to be uncorrelated except in specific odor-contingency learning circumstances. However, many recent electrophysiological studies in both rodents and humans reveal a surprising cycle-by-cycle influence of nasal respiration on neuronal activity throughout much of the cerebral cortex beyond the olfactory system, including the prefrontal cortex, hippocampus, and subcortical structures. In addition, respiratory phase has been shown to influence higher-frequency oscillations associated with cognitive functions, including attention and memory, such as the power of γ-rhythms and the timing of hippocampal sharp wave ripples. These new findings support respiration's role in cognitive function, which is supported by studies in human subjects, in which nasal respiration has been linked to memory processes. Here, we review recent reports from human and rodent experiments that link respiration to the modulation of memory function and the neurophysiological processes involved in memory in rodents and humans. We argue that respiratory influence on the neuronal activity of two key memory structures, the hippocampus and prefrontal cortex, provides a potential neuronal mechanism behind respiratory modulation of memory.

Effects of eugenol on respiratory burst generation in newborn rat brainstem-spinal cord preparations.

Eugenol is contained in several plants including clove and is used as an analgesic drug. In the peripheral and central nervous systems, this compound modulates neuronal activity through action on voltage-gated ionic channels and/or transient receptor potential channels. However, it is unknown whether eugenol exerts any effects on the respiratory center neurons in the medulla. We examined the effects of eugenol on respiratory rhythm generation in the brainstem-spinal cord preparation from newborn rat (P0-P3). The preparations were superfused by artificial cerebrospinal fluid at 25-26 °C, and inspiratory C4 ventral root activity was monitored. Membrane potentials of respiratory neurons were recorded in the parafacial region of the rostral ventrolateral medulla. Bath application of eugenol (0.5-1 mM) decreased respiratory rhythm accompanied by strong inhibition of the burst activity of pre-inspiratory neurons. After washout, respiratory rhythm partly recovered, but the inspiratory burst duration was extremely shortened, and this continued for more than 60 min after washout. The shortening of C4 inspiratory burst by eugenol was not reversed by capsazepine (TRPV1 antagonist) or HC-030031 (TRPA1 antagonist), whereas the depression was partially blocked by GABAA antagonist bicuculline and glycine antagonist strychnine or GABAB antagonist phaclofen. A spike train of action potentials in respiratory neurons induced by depolarizing current pulse was depressed by application of eugenol. Eugenol decreased the negative slope conductance of pre-inspiratory neurons, suggesting blockade of persistent Na+ current. These results suggest that changes in both membrane excitability and synaptic connections are involved in the shortening of respiratory neuron bursts by eugenol.

Local cortical activity of distant brain areas can phase-lock to the olfactory bulb's respiratory rhythm in the freely behaving rat.

An important unresolved question about neural processing is the mechanism by which distant brain areas coordinate their activities and relate their local processing to global neural events. A potential candidate for the local-global integration are slow rhythms such as respiration. In this study, we asked if there are modulations of local cortical processing that are phase-locked to (peripheral) sensory-motor exploratory rhythms. We studied rats on an elevated platform where they would spontaneously display exploratory and rest behaviors. Concurrent with behavior, we monitored whisking through electromyography and the respiratory rhythm from the olfactory bulb (OB) local field potential (LFP). We also recorded LFPs from dorsal hippocampus, primary motor cortex, primary somatosensory cortex, and primary visual cortex. We defined exploration as simultaneous whisking and sniffing above 5 Hz and found that this activity peaked at ~8 Hz. We considered rest as the absence of whisking and sniffing, and in this case, respiration occurred at ~3 Hz. We found a consistent shift across all areas toward these rhythm peaks accompanying behavioral changes. We also found, across areas, that LFP gamma (70-100 Hz) amplitude could phase-lock to the animal's OB respiratory rhythm, a finding indicative of respiration-locked changes in local processing. In a subset of animals, we also recorded the hippocampal theta activity and found that occurred at frequencies overlapped with respiration but was not spectrally coherent with it, suggesting a different oscillator. Our results are consistent with the notion of respiration as a binder or integrator of activity between brain regions.

Effects of a TRPV1 agonist capsaicin on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats.

The heat-sensitive transient receptor potential vanilloid 1 (TRPV1) channels are expressed in the peripheral and central nervous systems. However, there is no report on how the activation of TRPV1 causes the modulation of neuronal activity in the medullary respiratory center. We examined effects of capsaicin, a specific agonist of TRPV1 channels, on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats. Capsaicin induced a biphasic response in the respiratory rhythm (a transient decrease followed by an increase in the C4 rate). The second-phase excitatory effect (but not the initial inhibitory effect) in the biphasic response was partly blocked by capsazepine or AMG9810 (TRPV1 antagonists). Capsaicin caused strong desensitization. After its washout, the strength of C4 burst inspiratory activity was augmented once per four to five respiratory cycles. The preinspiratory and inspiratory neurons showed tonic firings due to membrane depolarization during the initial inhibitory phase. In the presence of TTX, capsaicin increased the fluctuation of the membrane potential of the CO2-sensitive preinspiratory neurons in the parafacial respiratory group (pFRG), accompanied by slight depolarization. The C4 inspiratory activity did not stop, even 60-90 min after the application of 50/100 µM capsaicin. Voltage-sensitive dye imaging demonstrated that the spatiotemporal pattern of the respiratory rhythm generating networks after application of capsaicin (50 µM, 70-90 min) was highly similar to the control. A histochemical analysis using TRPV1 channel protein antibodies and mRNA demonstrated that the TRPV1 channel-positive cells were widely distributed in the reticular formation of the medulla, including the pFRG. Our results showed that the application of capsaicin in the medulla has various influences on the respiratory center: transient inhibitory and subsequent excitatory effects on the respiratory rhythm and periodical augmentation of the inspiratory burst pattern. The effects of capsaicin were partially blocked by TRPV1 antagonists but could be also induced at least partially via the non-specific action. Our results also suggested a minor contribution of the TRPV1 channels to central chemoreception.

Lethal avian influenza A (H5N1) virus induces ataxic breathing in mice with apoptosis of pre-Botzinger complex neurons expressing neurokinin-1 receptor.

Lethal influenza A (H5N1) induces respiratory failure in humans. Although it also causes death at 7 days postinfection (dpi) in mice, the development of the respiratory failure and the viral impact on pre-Botzinger complex (PBC) neurons expressing neurokinin 1 receptor (NK1R), which is the respiratory rhythm generator, have not been explored. Body temperature, weight, ventilation, and arterial blood pH and gases were measured at 0, 2, 4, and 6 dpi in control, lethal HK483, and nonlethal HK486 viral-infected mice. Immunoreactivities (IR) of PBC NK1R, H5N1 viral nucleoprotein (NP), and active caspase-3 (CASP3; a marker for apoptosis) were detected at 6 dpi. HK483, but not HK486, mice showed the following abnormalities: 1) gradual body weight loss and hypothermia; 2) tachypnea at 2-4 dpi and ataxic breathing with long-lasting apneas and hypercapnic hypoxemia at 6 dpi; and 3) viral replication in PBC NK1R neurons with NK1R-IR reduced by 75% and CASP3-IR colabeled at 6 dpi. Lethal H5N1 viral infection causes tachypnea at the early stage and ataxic breathing and apneas (hypercapnic hypoxemia) leading to death at the late stage. Its replication in the PBC induces apoptosis of local NK1R neurons, contributing to ataxic breathing and respiratory failure.

Eupnea, tachypnea, and autoresuscitation in a closed-loop respiratory control model.

How sensory information influences the dynamics of rhythm generation varies across systems, and general principles for understanding this aspect of motor control are lacking. Determining the origin of respiratory rhythm generation is challenging because the mechanisms in a central circuit considered in isolation may be different from those in the intact organism. We analyze a closed-loop respiratory control model incorporating a central pattern generator (CPG), the Butera-Rinzel-Smith (BRS) model, together with lung mechanics, oxygen handling, and chemosensory components. We show that 1) embedding the BRS model neuron in a control loop creates a bistable system; 2) although closed-loop and open-loop (isolated) CPG systems both support eupnea-like bursting activity, they do so via distinct mechanisms; 3) chemosensory feedback in the closed loop improves robustness to variable metabolic demand; 4) the BRS model conductances provide an autoresuscitation mechanism for recovery from transient interruption of chemosensory feedback; and 5) the in vitro brain stem CPG slice responds to hypoxia with transient bursting that is qualitatively similar to in silico autoresuscitation. Bistability of bursting and tonic spiking in the closed-loop system corresponds to coexistence of eupnea-like breathing, with normal minute ventilation and blood oxygen level and a tachypnea-like state, with pathologically reduced minute ventilation and critically low blood oxygen. Disruption of the normal breathing rhythm, through either imposition of hypoxia or interruption of chemosensory feedback, can push the system from the eupneic state into the tachypneic state. We use geometric singular perturbation theory to analyze the system dynamics at the boundary separating eupnea-like and tachypnea-like outcomes.NEW & NOTEWORTHY A common challenge facing rhythmic biological processes is the adaptive regulation of central pattern generator (CPG) activity in response to sensory feedback. We apply dynamical systems tools to understand several properties of a closed-loop respiratory control model, including the coexistence of normal and pathological breathing, robustness to changes in metabolic demand, spontaneous autoresuscitation in response to hypoxia, and the distinct mechanisms that underlie rhythmogenesis in the intact control circuit vs. the isolated, open-loop CPG.

The Onset of the Fetal Respiratory Rhythm: An Emergent Property Triggered by Chemosensory Drive?

The mechanisms responsible for the onset of respiratory activity during fetal life are unknown. The onset of respiratory rhythm may be a consequence of the genetic program of each of the constituents of the respiratory network, so they start to interact and generate respiratory cycles when reaching a certain degree of maturation. Alternatively, generation of cycles might require the contribution of recently formed sensory inputs that will trigger oscillatory activity in the nascent respiratory neural network. If this hypothesis is true, then sensory input to the respiratory generator must be already formed and become functional before the onset of fetal respiration. In this review, we evaluate the timing of the onset of the respiratory rhythm in comparison to the appearance of receptors, neurotransmitter machinery, and afferent projections provided by two central chemoreceptive nuclei, the raphe and locus coeruleus nuclei.

Testing the hypothesis of neurodegeneracy in respiratory network function with a priori transected arterially perfused brain stem preparation of rat.

Degeneracy of respiratory network function would imply that anatomically discrete aspects of the brain stem are capable of producing respiratory rhythm. To test this theory we a priori transected brain stem preparations before reperfusion and reoxygenation at 4 rostrocaudal levels: 1.5 mm caudal to obex (n = 5), at obex (n = 5), and 1.5 (n = 7) and 3 mm (n = 6) rostral to obex. The respiratory activity of these preparations was assessed via recordings of phrenic and vagal nerves and lumbar spinal expiratory motor output. Preparations with a priori transection at level of the caudal brain stem did not produce stable rhythmic respiratory bursting, even when the arterial chemoreceptors were stimulated with sodium cyanide (NaCN). Reperfusion of brain stems that preserved the pre-Bötzinger complex (pre-BötC) showed spontaneous and sustained rhythmic respiratory bursting at low phrenic nerve activity (PNA) amplitude that occurred simultaneously in all respiratory motor outputs. We refer to this rhythm as the pre-BötC burstlet-type rhythm. Conserving circuitry up to the pontomedullary junction consistently produced robust high-amplitude PNA at lower burst rates, whereas sequential motor patterning across the respiratory motor outputs remained absent. Some of the rostrally transected preparations expressed both burstlet-type and regular PNA amplitude rhythms. Further analysis showed that the burstlet-type rhythm and high-amplitude PNA had 1:2 quantal relation, with burstlets appearing to trigger high-amplitude bursts. We conclude that no degenerate rhythmogenic circuits are located in the caudal medulla oblongata and confirm the pre-BötC as the primary rhythmogenic kernel. The absence of sequential motor patterning in a priori transected preparations suggests that pontine circuits govern respiratory pattern formation.