Persistent Glossopharyngeal Nerve Respiratory Discharge Patterns after Ponto-Medullary Transection.

Shape and size of the nasopharyngeal airway is controlled by muscles innervated facial, glossopharyngeal, vagal, and hypoglossal cranial nerves. Contrary to brainstem networks that drive facial, vagal and hypoglossal nerve activities (FNA, VNA, HNA) the discharge patterns and origins of glossopharyngeal nerve activity (GPNA) remain poorly investigated. Here, an in situ perfused brainstem preparation (n=19) was used for recordings of GPNA in relation to phrenic (PNA), FNA, VNA and HNA. Brainstem transections were performed (n=10/19) to explore the role of pontomedullary synaptic interactions in generating GPNA. GPNA generally mirrors FNA and HNA discharge patterns and displays pre-inspiratory activity relative to the PNA, followed by robust inspiratory discharge in coincidence with PNA. Postinspiratory (early expiratory) discharge was, contrary to VNA, generally absent in FNA, GPNA or HNA. As described previously FNA and HNA discharge was virtually eliminated after pontomedullary transection while an apneustic inspiratory motor discharge was maintained in PNA, VNA and GPNA. After brainstem transection GPNA displayed an increased tonic activity starting during mid-expiration and thus developed prolonged pre-inspiratory activity compared to control. In conclusion respiratory GPNA reflects FNA and HNA which implies similar function in controlling upper airway patency during breathing. That GPNA preserved its pre-inspiratory/inspiratory discharge pattern in relation PNA after pontomedullary transection suggest that GPNA premotor circuits may have a different anatomical distribution compared HNA and FNA and thus may therefore hold a unique role in in preserving airway patency.

Peritoneal sepsis caused by Escherichia coli triggers brainstem inflammation and alters the function of sympatho-respiratory control circuits.

BACKGROUND: Sepsis has a high mortality rate due to multiple organ failure. However, the influence of peripheral inflammation on brainstem autonomic and respiratory circuits in sepsis is poorly understood. Our working hypothesis is that peripheral inflammation affects central autonomic circuits and consequently contributes to multiorgan failure in sepsis. METHODS: In an Escherichia coli (E. coli)-fibrin clot model of peritonitis, we first recorded ventilatory patterns using plethysmography before and 24 h after fibrin clot implantation. To assess whether peritonitis was associated with brainstem neuro-inflammation, we measured cytokine and chemokine levels in Luminex assays. To determine the effect of E. coli peritonitis on brainstem function, we assessed sympatho-respiratory nerve activities at baseline and during brief (20 s) hypoxemic ischemia challenges using in situ-perfused brainstem preparations (PBPs) from sham or infected rats. PBPs lack peripheral organs and blood, but generate vascular tone and in vivo rhythmic activities in thoracic sympathetic (tSNA), phrenic and vagal nerves. RESULTS: Respiratory frequency was greater (p < 0.001) at 24 h post-infection with E. coli than in the sham control. However, breath-by-breath variability and total protein in the BALF did not differ. IL-1ß (p < 0.05), IL-6 (p < 0.05) and IL-17 (p < 0.04) concentrations were greater in the brainstem of infected rats. In the PBP, integrated tSNA (p < 0.05) and perfusion pressure were greater (p < 0.001), indicating a neural-mediated pathophysiological high sympathetic drive. Moreover, respiratory frequency was greater (p < 0.001) in PBPs from infected rats than from sham rats. Normalized phase durations of inspiration and expiration were greater (p < 0.009, p < 0.015, respectively), but the post-inspiratory phase (p < 0.007) and the breath-by-breath variability (p < 0.001) were less compared to sham PBPs. Hypoxemic ischemia triggered a biphasic response, respiratory augmentation followed by depression. PBPs from infected rats had weaker respiratory augmentation (p < 0.001) and depression (p < 0.001) than PBPs from sham rats. In contrast, tSNA in E. coli-treated PBPs was enhanced throughout the entire response to hypoxemic ischemia (p < 0.01), consistent with sympathetic hyperactivity. CONCLUSION: We show that peripheral sepsis caused brainstem inflammation and impaired sympatho-respiratory motor control in a single day after infection. We conclude that central sympathetic hyperactivity may impact vital organ systems in sepsis.

Dynamics of ventilatory pattern variability and Cardioventilatory Coupling during systemic inflammation in rats.

Introduction: Biometrics of common physiologic signals can reflect health status. We have developed analytics to measure the predictability of ventilatory pattern variability (VPV, Nonlinear Complexity Index (NLCI) that quantifies the predictability of a continuous waveform associated with inhalation and exhalation) and the cardioventilatory coupling (CVC, the tendency of the last heartbeat in expiration to occur at preferred latency before the next inspiration). We hypothesized that measures of VPV and CVC are sensitive to the development of endotoxemia, which evoke neuroinflammation. Methods: We implanted Sprague Dawley male rats with BP transducers to monitor arterial blood pressure (BP) and recorded ventilatory waveforms and BP simultaneously using whole-body plethysmography in conjunction with BP transducer receivers. After baseline (BSLN) recordings, we injected lipopolysaccharide (LPS, n = 8) or phosphate buffered saline (PBS, n =3) intraperitoneally on 3 consecutive days. We recorded for 4-6 h after the injection, chose 3 epochs from each hour and analyzed VPV and CVC as well as heart rate variability (HRV). Results: First, the responses to sepsis varied across rats, but within rats the repeated measures of NLCI, CVC, as well as respiratory frequency (fR), HR, BP and HRV had a low coefficient of variation, (<0.2) at each time point. Second, HR, fR, and NLCI increased from BSLN on Days 1-3; whereas CVC decreased on Days 2 and 3. In contrast, changes in BP and the relative low-(LF) and high-frequency (HF) of HRV were not significant. The coefficient of variation decreased from BSLN to Day 3, except for CVC. Interestingly, NLCI increased before fR in LPS-treated rats. Finally, we histologically confirmed lung injury, systemic inflammation via ELISA and the presence of the proinflammatory cytokine, IL-1ß, with immunohistochemistry in the ponto-medullary respiratory nuclei. Discussion: Our findings support that NLCI reflects changes in the rat's health induced by systemic injection of LPS and reflected in increases in HR and fR. CVC decreased over the course to the experiment. We conclude that NLCI reflected the increase in predictability of the ventilatory waveform and (together with our previous work) may reflect action of inflammatory cytokines on the network generating respiration.

Cannabinoid Receptor mRNA Expression in Central and Peripheral Tissues in a Rodent Model of Peritonitis.

Introduction: Our laboratory investigates changes in the respiratory pattern during systemic inflammation in various rodent models. The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates inflammation. Inflammation not only affects cannabinoid (CB) 1 and CB2 receptor gene expression (Cnr1 and Cnr2), but also increases the predictability of the ventilatory pattern. Objectives: Our primary objective was to track ventilatory pattern variability and transcription of Cnr1 and Cnr2 mRNA, and of Il1b, Il6, and tumor necrosis factor-alpha (Tnfa) mRNAs at multiple time points in central and peripheral tissues during systemic inflammation induced by peritonitis. Methods: In male Sprague Dawley rats (n=24), we caused peritonitis by implanting a fibrin clot containing either 0 or 25×106 Escherichia coli intraperitoneally. We recorded breathing with whole-animal plethysmography at baseline and 1 h before euthanasia. We euthanized the rats at 3, 6, or 12 h after inoculation and harvested the pons, medulla, lung, and heart for gene expression analysis. Results: With peritonitis, Cnr1 mRNA more than Cnr2 mRNA was correlated to Il1b, Il6, and Tnfa mRNAs in medulla, pons, and lung and changed oppositely in the pons, medulla, and lung. These changes were associated with increased predictability of ventilatory pattern. Specifically, nonlinear complexity index correlated with increased Cnr1 mRNA in the pons and medulla, and coefficient of variation for cycle duration correlated with Cnr1 and Cnr2 mRNAs in the lung. Conclusion: The mRNAs for ECS receptors varied with time during the central and peripheral inflammatory response to peritonitis. These changes occurred in the brainstem, which contains the network that generates breathing pattern and thus, may participate in ventilatory pattern changes during systemic inflammation.

Heartbeats entrain breathing via baroreceptor-mediated modulation of expiratory activity.

NEW FINDINGS: Cardio-ventilatory coupling refers to the onset of inspiration occurring at a preferential latency following the last heartbeat (HB) in expiration. According to the cardiac-trigger hypothesis, the pulse pressure initiates an inspiration via baroreceptor activation. However, the central neural substrate mediating this coupling remains undefined. Using a combination of animal data, human data and mathematical modelling, this study tests the hypothesis that the HB, by way of pulsatile baroreflex activation, controls the initiation of inspiration that occurs through a rapid neural activation loop from the carotid baroreceptors to Bötzinger complex expiratory neurons. ABSTRACT: Cardio-ventilatory coupling refers to a heartbeat (HB) occurring at a preferred latency prior to the next breath. We hypothesized that the pressure pulse generated by a HB activates baroreceptors that modulate brainstem expiratory neuronal activity and delay the initiation of inspiration. In supine male subjects, we recorded ventilation, electrocardiogram and blood pressure during 20-min epochs of baseline, slow-deep breathing and recovery. In in situ rodent preparations, we recorded brainstem activity in response to pulses of perfusion pressure. We applied a well-established respiratory network model to interpret these data. In humans, the latency between a HB and onset of inspiration was consistent across different breathing patterns. In in situ preparations, a transient pressure pulse during expiration activated a subpopulation of expiratory neurons normally active during post-inspiration, thus delaying the next inspiration. In the model, baroreceptor input to post-inspiratory neurons accounted for the effect. These studies are consistent with baroreflex activation modulating respiration through a pauci-synaptic circuit from baroreceptors to onset of inspiration.

Traube-Hering waves are formed by interaction of respiratory sinus arrhythmia and pulse pressure modulation in healthy men.

Excessive blood pressure variation is linked to the development of hypertension and other diseases. This study assesses the relative role of respiratory sinus arrhythmia (RSA) and pulse pressure (PP) on the amplitude and timing of blood pressure variability with respiration [Traube-Hering (TH) waves]. We analyzed respiratory, electrocardiogram, and blood pressure traces from healthy, supine male subjects (n = 10, mean age = 26.7 ± 1.4) during 20-min epochs of resting, slow deep breathing (SDB), and recovery. Across all epochs, blood pressure and heart rate (HR) were modulated with respiration and the magnitude of RSA; TH waves increased during SDB. The data were deconstructed using a simple mathematical model of blood pressure to dissect the relative roles of RSA and PP on TH waves. We constructed the time series of the R-wave peaks and compared the recorded TH waves with that predicted by the model. Given that cardiac output is determined by both heart rate and stroke volume, it was surprising that the magnitude of the TH waves could be captured by only HR modulation. However, RSA alone did not accurately predict the timing of TH waves relative to the respiratory cycle. Adding respiratory modulation of PP to the model corrected the phase shift showing the expected pattern of BP rising during inspiration with the peak of the TH wave during early expiration. We conclude that short-term variability of blood pressure referred to as TH waves has at least two independent mechanisms whose interaction forms their pattern: RSA and respiratory-driven changes in PP.NEW & NOTEWORTHY Variability in blood pressure has become an important metric to consider as more is learned about the link between excessive blood pressure variability and adverse health outcomes. In this study using slow deep breathing in human subjects, we found that heart rate and pulse pressure variations have comparable effects on the amplitude of blood pressure waves, and it is the common action of the two that defines the phase relationship between respiration and blood pressure oscillations.

Brainstem inflammation modulates the ventilatory pattern and its variability after acute lung injury in rodents.

KEY POINTS: Compared with sham rats, rats a week after acute lung injury (ALI) express more pro-inflammatory cytokines in their brainstem respiratory control nuclei, exhibit a higher respiratory frequency (fR) and breathe with a more predictable pattern. These characteristics of the respiratory pattern persist in in situ preparations even after minimizing pulmonary and chemo-afferent inputs. Interleukin (IL)-1ß microinjected in the nucleus tractus solitarii increases fR and the predictability of the ventilatory pattern similar to rats with ALI. Intracerebroventricular infusion of indomethacin, an anti-inflammatory drug, mitigates the effect of ALI on fR and ventilatory pattern variability. We conclude that changes in the ventilatory pattern after ALI result not only from sensory input due to pulmonary damage and dysfunction but also from neuro-inflammation. ABSTRACT: Acute lung injury (ALI) increases respiratory rate (fR) and ventilatory pattern variability (VPV), but also evokes peripheral and central inflammation. We hypothesized that central inflammation has a role in determining the ventilatory pattern after ALI. In rat pups, we intratracheally injected either bleomycin to induce ALI or saline as a sham control. One week later, we recorded the ventilatory pattern of the rat pups using flow-through plethysmography, then formed in situ preparations from these pups and recorded their 'fictive' patterns from respiratory motor nerves. Compared with the ventilatory pattern of the sham rat pups, injured rat pups had increased fR and predictability. Surprisingly, the fictive patterns of the in situ preparations from ALI pups retained these characteristics despite removing their lungs to eliminate pulmonary sensory inputs and perfusing them with hyperoxic artificial cerebral spinal fluid to minimize peripheral chemoreceptor input. Histological processing revealed increased immunoreactivity of the pro-inflammatory cytokine Interleukin-1ß (IL-1ß) in the nucleus tractus solitarii (nTS) from ALI but not sham rats. In subsequent experiments, we microinjected IL-1ß in the nTS bilaterally in anaesthetized naïve adult rats, which increased fR and predictability of ventilatory pattern variability (VPV) after 2 h. Finally, we infused indomethacin intracerebroventricularly during the week of survival after ALI. This did not affect sham rats, but mitigated changes in fR and VPV in ALI rats. We conclude that neuro-inflammation has an essential role in determining the ventilatory pattern of ALI rats.

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.

Peripheral-to-central immune communication at the area postrema glial-barrier following bleomycin-induced sterile lung injury in adult rats.

The pathways for peripheral-to-central immune communication (P â†’ C I-comm) following sterile lung injury (SLI) are unknown. SLI evokes systemic and central inflammation, which alters central respiratory control and viscerosensory transmission in the nucleus tractus solitarii (nTS). These functional changes coincide with increased interleukin-1 beta (IL-1ß) in the area postrema, a sensory circumventricular organ that connects P â†’ C I-comm to brainstem circuits that control homeostasis. We hypothesize that IL-1ß and its downstream transcriptional target, cyclooxygenase-2 (COX-2), mediate P â†’ C I-comm in the nTS. In a rodent model of SLI induced by intratracheal bleomycin (Bleo), the sigh frequency and duration of post-sigh apnea increased in Bleo- compared to saline- treated rats one week after injury. This SLI-dependent change in respiratory control occurred concurrently with augmented IL-1ß and COX-2 immunoreactivity (IR) in the funiculus separans (FS), a barrier between the AP and the brainstem. At this barrier, increases in IL-1ß and COX-2 IR were confined to processes that stained for glial fibrillary acidic protein (GFAP) and that projected basolaterally to the nTS. Further, FS radial-glia did not express TNF-α or IL-6 following SLI. To test our hypothesis, we blocked central COX-1/2 activity by intracerebroventricular (ICV) infusion of Indomethacin (Ind). Continuous ICV Ind treatment prevented Bleo-dependent increases in GFAP + and IL-1ß + IR, and restored characteristics of sighs that reset the rhythm. These data indicate that changes in sighs following SLI depend partially on activation of a central COX-dependent P â†’ C I-comm via radial-glia of the FS.

Increasing Local Excitability of Brainstem Respiratory Nuclei Reveals a Distributed Network Underlying Respiratory Motor Pattern Formation.

The core circuit of the respiratory central pattern generator (rCPG) is located in the ventrolateral medulla, especially in the pre-Bötzinger complex (pre-BötC) and the neighboring Bötzinger complex (BötC). To test the hypothesis that this core circuit is embedded within an anatomically distributed pattern-generating network, we investigated whether local disinhibition of the nucleus tractus solitarius (NTS), the Kölliker-Fuse nuclei (KFn), or the midbrain periaqueductal gray area (PAG) can similarly affect the respiratory pattern compared to disinhibition of the pre-BötC/BötC core. In arterially-perfused brainstem preparations of rats, we recorded the three-phase respiratory pattern (inspiration, post-inspiration and late-expiration) from phrenic and vagal nerves before and after bilateral microinjections of the GABA(A)R antagonist bicuculline (50 nl, 10 mM). Local disinhibition of either NTS, pre-BötC/BötC, or KFn, but not PAG, triggered qualitatively similar disruptions of the respiratory pattern resulting in a highly significant increase in the variability of the respiratory cycle length, including inspiratory and expiratory phase durations. To quantitatively analyze these motor pattern perturbations, we measured the strength of phase synchronization between phrenic and vagal motor outputs. This analysis showed that local disinhibition of all brainstem target nuclei, but not the midbrain PAG, significantly decreased the strength of phase synchronization. The convergent perturbations of the respiratory pattern suggest that the rCPG expands rostrally and dorsally from the designated core but does not include higher mid-brain structures. Our data also suggest that excitation-inhibition balance of respiratory network synaptic interactions critically determines the network dynamics that underlie vital respiratory rhythm and pattern formation.

Ventilatory pattern variability as a biometric for severity of acute lung injury in rats.

We hypothesize that ventilatory pattern variability (VPV) varies with the magnitude of acute lung injury (ALI). In adult male rats, we instilled a low- or high- dose of bleomycin or saline (PBS) intratracheally. While representative samples of pulmonary tissue indicated graded lung injury, coefficient of variation (CV) of TTOT did not differ among the 3 groups. Broncho-alveolar lavage fluid (BALF), respiratory rate (fR), mutual information were greater in ALI than sham rats; but did not differ between bleomycin doses. However, nonlinear complexity index (NLCI), which is the difference in sample entropy between original and surrogate data sets was greater for high- versus low- dose; but did not differ between low-dose and sham groups. Further, NLCI correlated to an injury index based on protein concentration of BALF and failure to gain weight. Finally, Receiver Operator Curves (ROCs) indicated that both mutual information and NLCI had greater sensitivity and specificity than fR and CVTTOT in identifying ALI. Thus, nonlinear analyses of VPV can distinguish ALI and out performs fR as a biometric.

Rapid and robust restoration of breathing long after spinal cord injury.

There exists an abundance of barriers that hinder functional recovery following spinal cord injury, especially at chronic stages. Here, we examine the rescue of breathing up to 1.5 years following cervical hemisection in the rat. In spite of complete hemidiaphragm paralysis, a single injection of chondroitinase ABC in the phrenic motor pool restored robust and persistent diaphragm function while improving neuromuscular junction anatomy. This treatment strategy was more effective when applied chronically than when assessed acutely after injury. The addition of intermittent hypoxia conditioning further strengthened the ventilatory response. However, in a sub-population of animals, this combination treatment caused excess serotonergic (5HT) axon sprouting leading to aberrant tonic activity in the diaphragm that could be mitigated via 5HT2 receptor blockade. Through unmasking of the continuing neuroplasticity that develops after injury, our treatment strategy ensured rapid and robust patterned respiratory recovery after a near lifetime of paralysis.

Periodicity: A Characteristic of Heart Rate Variability Modified by the Type of Mechanical Ventilation After Acute Lung Injury.

We present a novel approach to quantify heart rate variability (HRV) and the results of applying this approach to synthetic and original data sets. Our approach evaluates the periodicity of heart rate by calculating the transform of Relative Shannon Entropy, the maximum value of the RR interval periodogram, and the maximum, mean values, and sample entropy of the autocorrelation function. Synthetic data were generated using a Van der Pol oscillator; and the original data were electrocardiogram (ECG) recordings from anesthetized rats after acute lung injury while on biologically variable (BVV) or continuous mechanical ventilation (CMV). Analysis of the synthetic data revealed that our measures were correlated highly to the bandwidth of the oscillator and assessed periodicity. Then, applying these analytical tools to the ECGs determined that the heart rate (HR) of BVV group had less periodicity and higher variability than the HR of the CMV group. Quantifying periodicity effectively identified a readily apparent difference in HRV during BVV and CMV that was not identified by power spectral density measures during BVV and CMV. Cardiorespiratory coupling is the probable mechanism for HRV increasing during BVV and becoming periodic during CMV. Thus, the absence or presence of periodicity in ventilation determined HRV, and this mechanism is distinctly different from the cardiorespiratory uncoupling that accounts for the loss of HRV during sepsis.

Lung-injury depresses glutamatergic synaptic transmission in the nucleus tractus solitarii via discrete age-dependent mechanisms in neonatal rats.

Transition periods (TPs) are brief stages in CNS development where neural circuits can exhibit heightened vulnerability to pathologic conditions such as injury or infection. This susceptibility is due in part to specialized mechanisms of synaptic plasticity, which may become activated by inflammatory mediators released under pathologic conditions. Thus, we hypothesized that the immune response to lung injury (LI) mediated synaptic changes through plasticity-like mechanisms that depended on whether LI occurred just before or after a TP. We studied the impact of LI on brainstem 2nd-order viscerosensory neurons located in the nucleus tractus solitarii (nTS) during a TP for respiratory control spanning (postnatal day (P) 11-15). We injured the lungs of Sprague-Dawley rats by intratracheal instillation of Bleomycin (or saline) just before (P9-11) or after (P17-19) the TP. A week later, we prepared horizontal slices of the medulla and recorded spontaneous and evoked excitatory postsynaptic currents (sEPSCs/eEPSCs) in vitro from neurons in the nTS that received monosynaptic glutamatergic input from the tractus solitarii (TS). In rats injured before the TP (pre-TP), neurons exhibited blunted sEPSCs and TS-eEPSCs compared to controls. The decreased TS-eEPSCs were mediated by differences in postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid receptors (AMPAR). Specifically, compared to controls, LI rats had more Ca2+-impermeable AMPARs (CI-AMPARs) as indicated by: 1) the absence of current-rectification, 2) decreased sensitivity to polyamine, 1-Naphthyl-acetyl-spermine-trihydrochloride (NASPM) and 3) augmented immunoreactive staining for the CI-AMPAR GluA2. Thus, pre-TP-LI acts postsynaptically to blunt glutamatergic transmission. The neuroimmune response to pre-TP-LI included microglia hyper-ramification throughout the nTS. Daily intraperitoneal administration of minocycline, an inhibitor of microglial/macrophage function prevented hyper-ramification and abolished the pre-TP-LI evoked synaptic changes. In contrast, rat-pups injured after the TP (post-TP) exhibited microglia hypo-ramification in the nTS and had increased sEPSC amplitudes/frequencies, and decreased TS-eEPSC amplitudes compared to controls. These synaptic changes were not associated with changes in CI-AMPARs, and instead involved greater TS-evoked use-dependent depression (reduced paired pulse ratio), which is a hallmark of presynaptic plasticity. Thus we conclude that LI regulates the efficacy of TS → nTS synapses through discrete plasticity-like mechanisms that are immune-mediated and depend on whether the injury occurs before or after the TP for respiratory control.

Phasic inhibition as a mechanism for generation of rapid respiratory rhythms.

Central neural networks operate continuously throughout life to control respiration, yet mechanisms regulating ventilatory frequency are poorly understood. Inspiration is generated by the pre-Bötzinger complex of the ventrolateral medulla, where it is thought that excitation increases inspiratory frequency and inhibition causes apnea. To test this model, we used an in vitro optogenetic approach to stimulate select populations of hindbrain neurons and characterize how they modulate frequency. Unexpectedly, we found that inhibition was required for increases in frequency caused by stimulation of Phox2b-lineage, putative CO2-chemosensitive neurons. As a mechanistic explanation for inhibition-dependent increases in frequency, we found that phasic stimulation of inhibitory neurons can increase inspiratory frequency via postinhibitory rebound. We present evidence that Phox2b-mediated increases in frequency are caused by rebound excitation following an inhibitory synaptic volley relayed by expiration. Thus, although it is widely thought that inhibition between inspiration and expiration simply prevents activity in the antagonistic phase, we instead propose a model whereby inhibitory coupling via postinhibitory rebound excitation actually generates fast modes of inspiration.

Bifurcation of the respiratory response to lung inflation in anesthetized dogs.

Numerous studies have demonstrated the effect of lung volume on prolongation of duration of expiration (TE) with limited understanding of the TE shortening and termination of expiration as observed in newborn. In 14 dogs, the effects of varied onset of lung inflation during expiration on the TE were evaluated. When lung inflation was applied in the first part of expiration (20-60% of TE) TE was lengthened. However, in the second portion (60-80% of TE) of expiration, lung inflation either terminated or prolonged TE; whereas in the last portion of expiration (80-90% of TE), lung inflation tended to terminate expiration prematurely. The effects were abolished after bilateral vagotomy. We postulate that prolongation of TE relates to the Breuer-Hering inflation reflex, which increases the time needed for a passive expiration; whereas the ability to shorten TE could relate to Head's paradoxical reflex acting to initiate inspiration or to activate inspiratory motor activity to brake expiratory flow as occurs in the newborn.

A Framework for Patient State Tracking by Classifying Multiscalar Physiologic Waveform Features.

OBJECTIVE: state-of-the-art algorithms that quantify nonlinear dynamics in physiologic waveforms are underutilized clinically due to their esoteric nature. We present a generalizable framework for classifying multiscalar waveform features, designed for patient-state tracking directly at the bedside. METHODS: an artificial neural network classifier was designed to evaluate multiscale waveform features against a fingerprint database of multifractal synthetic time series. The results are mapped into a physiologic state space for near real-time patient-state tracking. RESULTS: the framework was validated on cardiac beat-to-beat dynamics processed with the multiscale entropy algorithm, and assessed using PhysioNet databases. We then applied our algorithm to predict 28-day mortality for sepsis patients, and found it had greater prognostic accuracy than standard clinical severity scores. CONCLUSION: we developed a novel framework to classify multiscale features of beat-to-beat dynamics, and performed an initial clinical validation to demonstrate that our approach generates a robust quantification of a patient's state, compatible with real-time bedside implementations. SIGNIFICANCE: the framework generates meaningful and actionable patient-specific information, and could facilitate the dissemination of a new class of "always-on" diagnostic tools.

Kölliker-Fuse nuclei regulate respiratory rhythm variability via a gain-control mechanism.

Respiration varies from breath to breath. On the millisecond timescale of spiking, neuronal circuits exhibit variability due to the stochastic properties of ion channels and synapses. Does this fast, microscopic source of variability contribute to the slower, macroscopic variability of the respiratory period? To address this question, we modeled a stochastic oscillator with forcing; then, we tested its predictions experimentally for the respiratory rhythm generated by the in situ perfused preparation during vagal nerve stimulation (VNS). Our simulations identified a relationship among the gain of the input, entrainment strength, and rhythm variability. Specifically, at high gain, the periodic input entrained the oscillator and reduced variability, whereas at low gain, the noise interacted with the input, causing events known as "phase slips", which increased variability on a slow timescale. Experimentally, the in situ preparation behaved like the low-gain model: VNS entrained respiration but exhibited phase slips that increased rhythm variability. Next, we used bilateral muscimol microinjections in discrete respiratory compartments to identify areas involved in VNS gain control. Suppression of activity in the nucleus tractus solitarii occluded both entrainment and amplification of rhythm variability by VNS, confirming that these effects were due to the activation of the Hering-Breuer reflex. Suppressing activity of the Kölliker-Fuse nuclei (KFn) enhanced entrainment and reduced rhythm variability during VNS, consistent with the predictions of the high-gain model. Together, the model and experiments suggest that the KFn regulates respiratory rhythm variability via a gain control mechanism.

C57BL/6J mouse apolipoprotein A2 gene is deterministic for apnea.

RATIONALE: Brainstem apolipoprotein AII (apoa2) mRNA expression correlates with apnea in breathing present in the adult C57Bl/6J (B6) sleep apnea model. OBJECTIVES: To test the hypothesis that the B6 apoa2 gene contributes to the trait, we performed plethysmographic testing in apoa2 knock out (KO: -/-) mice, an in situ brainstem-spinal cord preparation comparing KO to WT (+/+) mice, and B6xDBA recombinant inbred strains (RISs). MEASUREMENTS AND MAIN RESULTS: Apoa2 WT do, but KO and heterozygote (+/-) mice do not exhibit apnea during post-hypoxic breathing, measured in vivo. In the in situ model, pauses and instability in fictive phrenic bursting are substantially reduced in KO vs. WT preparations. In 24 RISs, apnea number in vivo was higher in strains with B6 apoa2 than with DBA apoa2 alleles. CONCLUSIONS: The B6 apoa2 polymorphism is directly involved in breath production, and its identification suggests a novel pathway influencing risk for adult sleep apnea.