Respiratory Modulation Research Articles

Effects of ouabain on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats and in decerebrate and arterially perfused in situ preparation from juvenile rats

The significance of Na/K-ATPase on respiratory rhythm generation is not well understood. We investigated the effects of the Na/K-ATPase blocker, ouabain, on respiratory rhythm. Experiments were performed with brainstem-spinal cord preparation from 0 to 3-day-old Wistar rats and with decerebrate and arterially perfused in situ preparation from juvenile rats (postnatal day 11–13). Newborn rat preparations were superfused at a rate of 3.0ml/min with artificial cerebrospinal fluid, equilibrated with 95% O2 and 5% CO2, pH 7.4, at 26–27°C. Inspiratory activity was monitored from the fourth cervical ventral root (C4). Application of ouabain (15–20min) resulted in a dose-dependent increase in the burst rate of C4 inspiratory activity. After washout, the burst rate further increased to reach quasi-maximum values under each condition (e.g. 183% of control in 1μM, 253% in 10μM, and 303% in 20μM at 30min washout). Inspiratory or pre-inspiratory neurons in the rostral ventrolateral medulla were depolarized. We obtained similar results (i.e. increased phrenic burst rate) in an in situ perfused preparation of juvenile rats. Genes encoding the Na/K-ATPase α subunit were expressed in the region of the parafacial respiratory group (pFRG) in neonatal rats, suggesting that cells (neurons and/or glias) in the pFRG were one of the targets of ouabain. We concluded that Na/K-ATPase activity could be an important factor in respiratory rhythm modulation.

Effects of ubiquinone derivatives on the mitochondrial unselective channel of Saccharomyces cerevisiae.

Ubiquinone derivatives modulate the mammalian mitochondrial Permeability Transition Pore (PTP). Yeast mitochondria harbor a similar structure: the respiration- and ATP-induced Saccharomyces cerevisiae Mitochondrial Unselective Channel ( Sc MUC). Here we show that decylubiquinone, a well-characterized inhibitor of the PTP, suppresses Sc MUC opening in diverse strains and independently of respiratory chain modulation or redox-state. We also found that naturally occurring derivatives such as hexaprenyl and decaprenyl ubiquinones lacked effects on the Sc MUC. The PTP-inactive ubiquinone 5 (Ub5) promoted the Sc MUC-independent activation of the respiratory chain in most strains tested. In an industrial strain however, Ub5 blocked the protection elicited by dUb. The results indicate the presence of a ubiquinone-binding site in the Sc MUC.

Respiratory modulation of sympathetic activity is attenuated in adult rats conditioned with chronic hypobaric hypoxia

Respiratory modulation of sympathetic nerve activity (SNA) depends on numerous factors including prior experience. In our studies, exposing naïve adult, male Sprague-Dawley rats to acute intermittent hypoxia (AIH) enhanced respiratory-modulation of splanchnic SNA (sSNA); whereas conditioning them to chronic hypobaric hypoxia (CHH) attenuated modulation. Further, AIH can evoke increased SNA in the absence phrenic long-term facilitation. We hypothesized that AIH would restore respiratory modulation of SNA in CHH rats. In anesthetized, CHH-conditioned (0.5 atm, 2 wks) rats (n=16), we recorded phrenic and sSNA before during and after AIH (8% O2 for 45s every 5min for 1h). At baseline, sSNA was not modulated with respiration. The sSNA was not recruited during a single brief exposure of hypoxia nor after 10 repetitive exposures. Further, the sSNA chemoresponse was not restored 1h after completing AIH. Thus, CHH-conditioning blocked the short-term plasticity expressed in sympatho-respiratory efferent activities and this was associated with reduced respiratory modulation of sympathetic activity and with attenuation of the sympatho-respiratory chemoresponse.

Effects of bile acids and the bile acid receptor FXR agonist on the respiratory rhythm in the in vitro brainstem medulla slice of neonatal Sprague-Dawley rats.

Intrahepatic cholestasis of pregnancy is always accompanied by adverse fetal outcomes such as malfunctions of respiration. Farnesoid X receptor (FXR) plays a critical role in the homeostasis of bile acids. Thus, we are determined to explore the effects of farnesoid X receptor (FXR) and five bile acids on respiratory rhythm generation and modulation of neonatal rats. Spontaneous periodic respiratory-related rhythmical discharge activity (RRDA) was recorded from hypoglossal nerves during the perfusion of modified Krebs solution. Group 1–6 was each given GW4064 and five bile acids of chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), cholic acid (CA) as well as ursodeoxycholic acid (UDCA) at different concentrations to identify their specific functions on respiratory rhythm modulations. Group 7 was applied to receive FXR blocker Z-guggulsterone and Z-guggulsterone with the above bile acids separately to explore the role of FXR in the respiratory rhythm modulation. Group 8 was given dimethyl sulfoxide (DMSO) as controls. Apart from UDCA, CDCA, DCA LCA and CA all exerted effects on RRDA recorded from hypoglossal nerves in a concentration-dependent manner. Respiratory cycle (RC), Inspiratory time (TI), Expiratory Time (TE) and Integral Amplitude (IA) were influenced and such effects could be reversed by Z-guggulsterone. FXR may contribute to the effects on the modulation of respiratory rhythm exerted by bile acids.

Intrinsic chemosensitivity of rostral ventrolateral medullary sympathetic premotor neurons in the in situ arterially perfused preparation of rats.

Brainstem hypoperfusion is a major excitant of sympathetic activity triggering hypertension, but the exact mechanisms involved remain incompletely understood. A major source of excitatory drive to preganglionic sympathetic neurons originates from the ongoing activity of premotor neurons in the rostral ventrolateral medulla (RVLM sympathetic premotor neurons). The chemosensitivity profile of physiologically characterized RVLM sympathetic premotor neurons during hypoxia and hypercapnia remains unclear. We examined whether physiologically characterized RVLM sympathetic premotor neurons can sense brainstem ischaemia intrinsically. We addressed this issue in a unique in situ arterially perfused preparation before and after a complete blockade of fast excitatory and inhibitory synaptic transmission. During hypercapnic hypoxia, respiratory modulation of RVLM sympathetic premotor neurons was lost, but tonic firing of most RVLM sympathetic premotor neurons was elevated. After blockade of fast excitatory and inhibitory synaptic transmission, RVLM sympathetic premotor neurons continued to fire and exhibited an excitatory firing response to hypoxia but not hypercapnia. This study suggests that RVLM sympathetic premotor neurons can sustain high levels of neuronal discharge when oxygen is scarce. The intrinsic ability of RVLM sympathetic premotor neurons to maintain responsivity to brainstem hypoxia is an important mechanism ensuring adequate arterial pressure, essential for maintaining cerebral perfusion in the face of depressed ventilation and/or high cerebral vascular resistance.

Importance of vertical integration in teaching and assessment of physiological concepts

Abstract Objectives Under the existing system of undergraduate curriculum, physiology teaching was done in a traditional non-case based manner. The objective of this research was to develop and implement a respiratory module which would incorporate vertically integrated learning and assessment using multiple teaching methodologies. Methods A respiratory modular team finalized the clinical cases, themes, relevant objectives, learning strategies, time table and assessment tools. The exam results of this Vertically Integrated Respiratory Module were compared with the results of traditionally delivered Introductory Module for the same group of students. Students' feedback questionnaire was also administered at the end of module. Results 99% of the students responded that integrated approach makes learning and understanding easy. 90 % of the students hoped that this mode of teaching would be helpful to them in future clinical years and 92% of the students favored the continuation of this integrated approach in future. Comparison of the results indicated that the passing percentage and the mean score (89% and 68.5) was higher in the Vertically Integrated Respiratory Module as compared to the non case based Introductory Module (70% and 61.5). Conclusion Based on the students feedback we may conclude that teaching physiology via vertically integrated approach may be superior than traditional non case based teaching, because it enhances students interest and facilitates meaningful and deep learning.

Getting jittery about the mechanism of hypertension in sleep apnoea.

Sleep apnoea kills. The prevalence of the sleep-related breathing disorder is high and apparently on the rise. Growing too is the alarming list of associated co-morbidities. What is crystal clear is the causal relationship between sleep apnoea and hypertension. In obstructive sleep apnoea (OSA), recurrent airway obstruction during the night gives rise to repeated bouts of hypoxaemia, with resultant elevated sympathetic nervous activity and pressor responses. The pernicious spillover of heightened sympathetic tone persists during the day when patients do not experience obstructive airway events; thus, sympathetic overactivity is strongly implicated in the manifestation of the daytime hypertensive phenotype in OSA (Hedner et al. 1988). Mechanisms of sympathoexcitation and the subsequent development of hypertension remain elusive. It is established that presympathetic neurones of the rostral ventrolateral medulla show activity entrained to the respiratory cycle. Enhanced respiratory–sympathetic coupling is observed in several animal models of neurogenic hypertension (Moraes et al. 2012, 2014). Importantly, elevated respiratory–sympathetic coupling has been shown to present before the onset of high blood pressure, suggesting a causal influence. Of interest, hyperexcitability in pre- and postinspiratory neurones of the medullary respiratory network has been identified as a key driver of enhanced sympathetic activity in the spontaneously hypertensive rat (Moraes et al. 2014), which is very exciting indeed. Arguing against this potential unifying hypothesis, however, are data that appear to show no increase in the respiratory modulation of sympathetic vasoconstrictor drive in human patients with essential hypertension (Fatouleh & Macefield, 2011). What about OSA? Perturbation in the central control of breathing is a feature of the syndrome. Perhaps enhanced respiratory–sympathetic coupling drives aberrant sympathetic outflow in OSA patients? In this issue of Experimental Physiology, Fatouleh et al. (2014) have the nerve to suggest otherwise! Into the air; and what seemed corporal melted As breath into the wind. Would they had stay'd! So that's that? Not quite: sapere vedere. Time to catch your breath! To explore further the temporal profile of the respiratory modulation of sympathetic nervous output, correlations were constructed allowing a comprehensive analysis of the normalized timing of MSNA activity relative to the respective respiratory cycles for each test subject. This approach revealed a greater respiratory modulation of MSNA in OSA patients, with lower mean MSNA activity during inspiration and expiration, but elevated activity in the postinspiratory phase in comparison to control subjects. In short, whilst the magnitude of the respiratory modulation does not differ between OSA patients and control subjects, a temporal jitter exists such that respiratory–sympathetic coupling is de facto more pronounced in OSA patients. Of note, CPAP treatment had mixed effects on the syncopated respiratory-related rhythm of MSNA in OSA patients (though the authors contend that it was largely reversed by treatment). Of particular interest, CPAP did not completely reverse MSNA levels to control. The relevance of this finding lies in the recognized failure of CPAP to lower blood pressure in OSA patients, including those with confirmed compliant CPAP usage, such as the patients in the study by Fatouleh et al. (2014). And breathe short-winded accents of new broils To be commenced in strands afar remote Those battles lie ahead, but the pressure is rising. We will remain sympathetic to the cause. But should we continue to look for inspiration or simply get to the heart of the matter? Readers are invited to give their opinion on this article. To submit a comment, go to: http://ep.physoc.org/letters/submit/expphysiol;99/10/1283. None declared. None declared.

Increasing signal processing sophistication in the calculation of the respiratory modulation of the photoplethysmogram (DPOP).

DPOP (∆POP or Delta-POP) is a non-invasive parameter which measures the strength of respiratory modulations present in the pulse oximetry photoplethysmogram (pleth) waveform. It has been proposed as a non-invasive surrogate parameter for pulse pressure variation (PPV) used in the prediction of the response to volume expansion in hypovolemic patients. Many groups have reported on the DPOP parameter and its correlation with PPV using various semi-automated algorithmic implementations. The study reported here demonstrates the performance gains made by adding increasingly sophisticated signal processing components to a fully automated DPOP algorithm. A DPOP algorithm was coded and its performance systematically enhanced through a series of code module alterations and additions. Each algorithm iteration was tested on data from 20 mechanically ventilated OR patients. Correlation coefficients and ROC curve statistics were computed at each stage. For the purposes of the analysis we split the data into a manually selected ‘stable’ region subset of the data containing relatively noise free segments and a ‘global’ set incorporating the whole data record. Performance gains were measured in terms of correlation against PPV measurements in OR patients undergoing controlled mechanical ventilation. Through increasingly advanced pre-processing and post-processing enhancements to the algorithm, the correlation coefficient between DPOP and PPV improved from a baseline value of R = 0.347 to R = 0.852 for the stable data set, and, correspondingly, R = 0.225 to R = 0.728 for the more challenging global data set. Marked gains in algorithm performance are achievable for manually selected stable regions of the signals using relatively simple algorithm enhancements. Significant additional algorithm enhancements, including a correction for low perfusion values, were required before similar gains were realised for the more challenging global data set.

Slow and deep respiration suppresses steady-state sympathetic nerve activity in patients with chronic heart failure: from modeling to clinical application.

Influences of slow and deep respiration on steady-state sympathetic nerve activity remain controversial in humans and could vary depending on disease conditions and basal sympathetic nerve activity. To elucidate the respiratory modulation of steady-state sympathetic nerve activity, we modeled the dynamic nature of the relationship between lung inflation and muscle sympathetic nerve activity (MSNA) in 11 heart failure patients with exaggerated sympathetic outflow at rest. An autoregressive exogenous input model was utilized to simulate entire responses of MSNA to variable respiratory patterns. In another 18 patients, we determined the influence of increasing tidal volume and slowing respiratory frequency on MSNA; 10 patients underwent a 15-min device-guided slow respiration and the remaining 8 had no respiratory modification. The model predicted that a 1-liter, step increase of lung volume decreased MSNA dynamically; its nadir (-33 ± 22%) occurred at 2.4 s; and steady-state decrease (-15 ± 5%), at 6 s. Actually, in patients with the device-guided slow and deep respiration, respiratory frequency effectively fell from 16.4 ± 3.9 to 6.7 ± 2.8/min (P < 0.0001) with a concomitant increase in tidal volume from 499 ± 206 to 1,177 ± 497 ml (P < 0.001). Consequently, steady-state MSNA was decreased by 31% (P < 0.005). In patients without respiratory modulation, there were no significant changes in respiratory frequency, tidal volume, and steady-state MSNA. Thus slow and deep respiration suppresses steady-state sympathetic nerve activity in patients with high levels of resting sympathetic tone as in heart failure.

Increased intrinsic excitability of muscle vasoconstrictor preganglionic neurons may contribute to the elevated sympathetic activity in hypertensive rats.

Hypertension is associated with pathologically increased sympathetic drive to the vasculature. This has been attributed to increased excitatory drive to sympathetic preganglionic neurons (SPN) from brainstem cardiovascular control centers. However, there is also evidence supporting increased intrinsic excitability of SPN. To test this hypothesis, we made whole cell recordings of muscle vasoconstrictor-like (MVClike) SPN in the working-heart brainstem preparation of spontaneously hypertensive (SH) and normotensive Wistar-Kyoto (WKY) rats. The MVClike SPN have a higher spontaneous firing frequency in the SH rat (3.85 ± 0.4 vs. 2.44 ± 0.4 Hz in WKY; P = 0.011) with greater respiratory modulation of their activity. The action potentials of SH SPN had smaller, shorter afterhyperpolarizations (AHPs) and showed diminished transient rectification indicating suppression of an A-type potassium conductance (IA). We developed mathematical models of the SPN to establish if changes in their intrinsic properties in SH rats could account for their altered firing. Reduction of the maximal conductance density of IA by 15–30% changed the excitability and output of the model from the WKY to a SH profile, with increased firing frequency, amplified respiratory modulation, and smaller AHPs. This change in output is predominantly a consequence of altered synaptic integration. Consistent with these in silico predictions, we found that intrathecal 4-aminopyridine (4-AP) increased sympathetic nerve activity, elevated perfusion pressure, and augmented Traube-Hering waves. Our findings indicate that IA acts as a powerful filter on incoming synaptic drive to SPN and that its diminution in the SH rat is potentially sufficient to account for the increased sympathetic output underlying hypertension.

Estimating the baroreflex and respiratory modulation of peripheral vascular resistance.

The peripheral vascular resistance (RPV) control is known to be largely sympathetically-mediated; thus assessment of the RPV control would allow us to infer valuable information regarding sympathetic nervous activity. The linear and 2nd-order nonlinear minimal models were used to capture the influences of blood pressure (baroreflex) and respiration (respiratory-coupling) on fluctuations of RPV. To validate the minimal models, they were applied on the "data" generated by the simulation model developed in our previous study. This study demonstrated that the linear minimal model was able to recover the "true" (simulated) kernels. The nonlinear model was able to detect the increase in nonlinearity in the system. The system gains derived from the estimated kernels showed strong relationship with the simulation gains, suggesting that the system gains could be employed as potential biomarkers of autonomic function. These results also showed that the nonlinear model had sufficient sensitivity to detect the difference in autonomic reactivity between subjects with mild and severe metabolic syndrome and obstructive sleep apnea syndrome exposed to orthostatic stress.

The midbrain periaqueductal grey has no role in the generation of the respiratory motor pattern, but provides command function for the modulation of respiratory activity

It has previously been shown that stimulation of cell-columns in the periaqueductal grey (PAG) triggers site-specific cardiorespiratory effects. These are believed to facilitate changes in behaviour through coordinated changes in autonomic outflow. Here, we investigated whether PAG-evoked respiratory commands can be studied in situ using the decerebrate perfused brainstem preparation. Phrenic, vagus and abdominal iliohypogastric nerves were recorded before and after microinjection of l-glutamate (30–50nl, 10mM) or isoguvacine (GABA-receptor agonist, 30–50nl, 10mM) into the PAG. l-glutamate microinjection triggered a range of site-specific respiratory modulations (n=17 preparations). Subsequent microinjection of isoguvacine into the same PAG sites had no effect on the baseline respiratory motor pattern or rhythm. We conclude that while the PAG has no function in respiratory pattern generation, PAG-evoked respiratory modulations can be evoked in situ in the absence of higher brain centres and while homeostatic parameters that may affect respiratory drive are held static.

Respiratory modulation of muscle sympathetic nerve activity in obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is associated with elevated muscle sympathetic nerve activity (MSNA) during normoxic daytime wakefulness, leading to hypertension. We tested the hypothesis that respiratory–sympathetic coupling, postulated to be the underlying cause of neurogenic hypertension, is increased in OSA. Muscle sympathetic nerve activity, blood pressure, ECG and respiration were recorded in 21 normotensive control subjects and 21 newly diagnosed patients with OSA before and after 6 and 12 months of treatment with continuous positive airway pressure. Muscle sympathetic nerve activity was recorded via tungsten microelectrodes inserted percutaneously into the peroneal nerve. Cardiac and respiratory modulation of MSNA was quantified from the cross-correlation histograms constructed between the sympathetic spikes and either ECG or respiration. Muscle sympathetic nerve activity was significantly elevated in newly diagnosed OSA patients compared with control subjects (53 ± 2 versus 28 ± 2 bursts min−1). There was a significant fall in MSNA after 6 months of continuous positive airway pressure (37 ± 2 bursts min−1), with no further change after 12 months (37 ± 2 bursts min−1). There were no significant differences in the magnitude of respiratory modulation of MSNA between the OSA patients and control subjects (40 ± 3.1 versus 39 ± 3.4%). However, when considering the normalized temporal profile there were changes in the respiratory patterning of MSNA in OSA, with more activity occurring in postinspiration and less in inspiration and expiration. This was largely reversed following long-term continuous positive airway pressure.

An optimized method for estimating the tidal volume from intracardiac or body surface electrocardiographic signals: implications for estimating minute ventilation.

The ability to accurately monitor tidal volume (TV) from electrocardiographic (ECG) signals holds significant promise for improving diagnosis treatment across a variety of clinical settings. The objective of this study was to develop a novel method for estimating the TV from ECG signals. In 10 mechanically ventilated swine, we collected intracardiac electrograms from catheters in the coronary sinus (CS), left ventricle (LV), and right ventricle (RV), as well as body surface electrograms, while TV was varied between 0 and 750 ml at respiratory rates of 7-14 breaths/min. We devised an algorithm to determine the optimized respirophasic modulation of the amplitude of the ECG-derived respiratory signal. Instantaneous measurement of respiratory modulation showed an absolute error of 72.55, 147.46, 85.68, 116.62, and 50.89 ml for body surface, CS, LV, RV, and RV-CS leads, respectively. Minute TV estimation demonstrated a more accurate estimation with an absolute error of 69.56, 153.39, 79.33, 122.16, and 48.41 ml for body surface, CS, LV, RV, and RV-CS leads, respectively. The RV-CS and body surface leads provided the most accurate estimations that were within 7 and 10% of the true TV, respectively. Finally, the absolute error of the bipolar RV-CS lead was significantly lower than any other lead configuration (P < 0.0001). In conclusion, we have demonstrated that ECG-derived respiratory modulation provides an accurate estimation of the TV using intracardiac or body surface signals, without the need for additional hardware.

Impact of lower body negative pressure induced hypovolemia on peripheral venous pressure waveform parameters in healthy volunteers

Lower body negative pressure (LBNP) creates a reversible hypovolemia by sequestrating blood volume in the lower extremities. This study sought to examine the impact of central hypovolemia on peripheral venous pressure (PVP) waveforms in spontaneously breathing subjects. With IRB approval, 11 healthy subjects underwent progressive LBNP (baseline, −30, −75, and −90 mmHg or until the subject became symptomatic). Each was monitored for heart rate (HR), finger arterial blood pressure (BP), a chest respiratory band and PVP waveforms which are generated from a transduced upper extremity intravenous site. The first subject was excluded from PVP analysis because of technical errors in collecting the venous pressure waveform. PVP waveforms were analyzed to determine venous pulse pressure, mean venous pressure, pulse width, maximum and minimum slope (time domain analysis) together with cardiac and respiratory modulations (frequency domain analysis). No changes of significance were found in the arterial BP values at −30 mmHg LBNP, while there were significant reductions in the PVP waveforms time domain parameters (except for 50% width of the respiration induced modulations) together with modulation of the PVP waveform at the cardiac frequency but not at the respiratory frequency. As the LBNP progressed, arterial systolic BP, mean BP and pulse pressure, PVP parameters and PVP cardiac modulation decreased significantly, while diastolic BP and HR increased significantly. Changes in hemodynamic and PVP waveform parameters reached a maximum during the symptomatic phase. During the recovery phase, there was a significant reduction in HR together with a significant increase in HR variability, mean PVP and PVP cardiac modulation. Thus, in response to mild hypovolemia induced by LBNP, changes in cardiac modulation and other PVP waveform parameters identified hypovolemia before detectable hemodynamic changes.

Specific Respiratory Neuron Types Have Increased Excitability That Drive Presympathetic Neurones in Neurogenic Hypertension

A major aspect of hypertension is excessive sympathetic activity but the reasons for this remain elusive. Sympathetic tone is increased in the spontaneously hypertensive (SH) rat reflecting, in part, enhanced respiratory-sympathetic coupling. We aimed to identify which respiratory cells might have altered properties. Using the working heart-brain stem preparation, we monitored simultaneously sympathetic and respiratory nerve activity in combination with intracellular recordings of physiologically characterized medullary presympathetic or respiratory neurons. In SH rats, respiratory modulation of both inspiratory and postinspiratory phases of sympathetic activity was larger relative to Wistar rats. An additional burst of sympathetic activity in the preinspiratory phase was also present in SH rats. After synaptic isolation of rostral medullary presympathetic neurons, there was no difference in their excitability compared with neurons in Wistar rats. Rather, both pre-Bötzinger preinspiratory and Bötzinger postinspiratory neurons had increased neuronal excitability in SH rats relative to Wistar rats; this was attributed to higher input resistance/reduced leak current in preinspiratory neurons and reduced calcium activated potassium conductance in postinspiratory neurons. Thus, the respiratory network of the SH rat is reconfigured to a pattern dominated by heightened excitability of preinspiratory and postinspiratory neurons. These neurons both provide augmented excitatory synaptic drive to rostral medullary presympathetic neurons contributing to excessive sympathetic nerve activity associated with hypertension in the in situ SH rat. Our data indicate selective modulation of potassium conductances in 2 subsets of respiratory neurons contributing to neurogenic hypertension.

The preBötzinger complex as a hub for network activity along the ventral respiratory column in the neonate rat

In vertebrates, respiratory control is ascribed to heterogeneous respiration-modulated neurons along the Ventral Respiratory Column (VRC) in medulla, which includes the preBötzinger Complex (preBötC), the putative respiratory rhythm generator. Here, the functional anatomy of the VRC was characterized via optical recordings in the sagittaly sectioned neonate rat hindbrain, at sampling rates permitting coupling estimation between neuron pairs, so that each neuron was described using unitary, neuron-system, and coupling attributes. Structured coupling relations in local networks, significantly oriented coupling in the peri-inspiratory interval detected in pooled data, and significant correlations between firing rate and expiratory duration in subsets of neurons revealed network regulation at multiple timescales. Spatially averaged neuronal attributes, including coupling vectors, revealed a sharp boundary at the rostral margin of the preBötC, as well as other functional anatomical features congruent with identified structures, including the parafacial respiratory group and the nucleus ambiguus. Cluster analysis of attributes identified two spatially compact, homogenous groups: the first overlapped with the preBötC, and was characterized by strong respiratory modulation and dense bidirectional coupling with itself and other groups, consistent with a central role for the preBötC in respiratory control; the second lay between preBötC and the facial nucleus, and was characterized by weak respiratory modulation and weak coupling with other respiratory neurons, which is congruent with cardiovascular regulatory networks that are found in this region. Other groups identified using cluster analysis suggested that networks along VRC regulated expiratory duration, and the transition to and from inspiration, but these groups were heterogeneous and anatomically dispersed. Thus, by recording local networks in parallel, this study found evidence for respiratory regulation at multiple timescales along the VRC, as well as a role for the preBötC in the integration of functionally disparate respiratory neurons.

Lingual muscle activity across sleep-wake States in rats with surgically altered upper airway.

Obstructive sleep apnea (OSA) patients have increased upper airway muscle activity, including such lingual muscles as the genioglossus (GG), geniohyoid (GH), and hyoglossus (HG). This adaptation partially protects their upper airway against obstructions. Rodents are used to study the central neural control of sleep and breathing but they do not naturally exhibit OSA. We investigated whether, in chronically instrumented, behaving rats, disconnecting the GH and HG muscles from the hyoid (H) apparatus would result in a compensatory increase of other upper airway muscle activity (electromyogram, EMG) and/or other signs of upper airway instability. We first determined that, in intact rats, lingual (GG and intrinsic) muscles maintained stable activity levels when quantified based on 2 h-long recordings conducted on days 6 through 22 after instrumentation. We then studied five rats in which the tendons connecting the GH and HG muscles to the H apparatus were experimentally severed. When quantified across all recording days, lingual EMG during slow-wave sleep (SWS) was modestly but significantly increased in rats with surgically altered upper airway [8.6 ± 0.7% (SE) vs. 6.1 ± 0.7% of the mean during wakefulness; p = 0.012]. Respiratory modulation of lingual EMG occurred mainly during SWS and was similarly infrequent in both groups, and the incidence of sighs and central apneas also was similar. Thus, a weakened action of selected lingual muscles did not produce sleep-disordered breathing but resulted in a relatively elevated activity in other lingual muscles during SWS. These results encourage more extensive surgical manipulations with the aim to obtain a rodent model with collapsible upper airway.

The Kölliker‐Fuse nucleus drives respiratory modulation of cardiac vagal tone (686.15)

Cardiac vagal tone is a major determinant of health, but its origin is unknown. It is present and well developed in the decerebrate perfused working heart‐brainstem preparation (WHBP) of rat, indicating that it arises from within the brain stem. Direct recordings from the cardiac vagal branch in the WHBP (juvenile rats day 17‐25, n=10) showed spike activity that peaked in late inspiration/postinspiration, correlating with the bradycardia of respiratory sinus arrhythmia (RSA). Bilateral inhibition of neurons in the Kölliker‐Fuse nucleus (KF) of the pons by microinjections of the GABAA receptor agonist, isoguvacine (10 mM, 40‐60nl), abolished the post‐inspiratory peak but had little effect on mean cardiac vagal nerve activity. That activity assumed a tonic pattern, interrupted by brief pauses around the onset of inspiration. RSA was abolished, and heart rate rose modestly (~ 20 beats/min). Transection of the caudal pons at the level of the facial nuclei substantially reduced mean cardiac vagal activity (50‐80%) and raised heart rate substantially (~ 50 beats/min). These results indicate that cardiac vagal tone is distinct from RSA, though both depend on supramedullary connections. Drive from the KF is responsible for RSA, while the origins of most cardiac vagal tone remain to be determined.Grant Funding Source: ARC

A Direct and Nonredundant Role for Thymic Stromal Lymphopoietin on Antiviral CD8 T Cell Responses in the Respiratory Mucosa

Mucosally produced thymic stromal lymphopoietin (TSLP) regulates Th2 responses by signaling to dendritic cells and CD4 T cells. Activated CD8 T cells express the TSLP receptor (TSLPR), yet a direct role for TSLP in CD8 T cell immunity in the mucosa has not been described. Because TSLP shares signaling components with IL-7, a cytokine important for the development and survival of memory CD8 T cells in systemic infection models, we hypothesized that TSLP spatially and nonredundantly supports the development of these cells in the respiratory tract. In this study, we demonstrate that influenza infection induces the early expression of TSLP by lung epithelial cells with multiple consequences. The global loss of TSLP responsiveness in TSLPR(-/-) mice enhanced morbidity and delayed viral clearance. Using a competitive adoptive transfer system, we demonstrate that selective loss of TSLPR signaling on antiviral CD8 T cells decreases their accumulation specifically in the respiratory tract as early as day 8 after infection, primarily due to a proliferation deficiency. Importantly, the subsequent persistence of memory cells derived from this pool was also qualitatively and quantitatively affected. In this regard, the local support of antiviral CD8 T cells by TSLP is well suited to the mucosa, where responses must be tempered to prevent excessive inflammation. Taken together, these data suggest that TSLP uniquely participates in local immunity in the respiratory tract and modulation of TSLP levels may promote long-term CD8 T cell immunity in the mucosa when other prosurvival signals are limiting.