Expiratory Activity Research Articles

Afferent and Efferent Connections of the Postinspiratory Complex (PiCo) Revealed by AAV and Monosynaptic Rabies Viral Tracing.

The control of the respiratory rhythm and airway motor activity is essential for life. Accumulating evidence indicates that the postinspiratory complex (PiCo) is crucial for generating behaviors that occur during the postinspiratory phase, including expiratory laryngeal activity and swallowing. Located in the ventromedial medulla, PiCo is defined by neurons co-expressing two neurotransmitter markers (ChAT and Vglut2/Slc17a6). Here, we mapped the input-output connections of these neurons using viral tracers and intersectional viral-genetic tools. PiCo neurons were specifically targeted by focal injection of a doubly conditional Cre- and FlpO-dependent AAV8 viral marker (AAV8-Con/Fon-TVA-mCherry) into the left PiCo of adult ChatCre/wt: Vglut2FlpO/wt mice, for anterograde axonal tracing. These experiments revealed projections to various brain regions, including the Cu, nucleus of the solitary tract (NTS), Amb, X, XII, Sp5, RMg, intermediate reticular nucleus (IRt), lateral reticular nucleus (LRt), pre-Bötzinger complex (preBötC), contralateral PiCo, laterodorsal tegmental nucleus (LDTg), pedunculopontine tegmental nucleus (PPTg), periaqueductal gray matter (PAG), Kölliker-Fuse (KF), PB, and external cortex of the inferior colliculus (ECIC). A rabies virus (RV) retrograde transsynaptic approach was taken with EnvA-pseudotyped G-deleted (RV-SAD-G-GFP) to similarly target PiCo neurons in ChatCre/wt: Vglut2FlpO/wt mice, following prior injections of helper AAVs (a mixture of AAV-Ef1a-Con/Fon oG and viral vector AAV8-Con/Fon-TVA-mCherry). This combined approach revealed prominent synaptic inputs to PiCo neurons from NTS, IRt, and A1/C1. Although PiCo neurons project axons to the contralateral PiCo area, this approach did not detect direct contralateral connections. We suggest that PiCo serves as a critical integration site, projecting and receiving neuronal connections implicated in breathing, arousal, swallowing, and autonomic regulation.

The pontine Kölliker-Fuse nucleus is important for reduced post-inspiratory airflow elicited by stimulation of the ventral respiratory parafacial region.

Considering that the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) would be an important center in the central nervous system involved in the maintenance and modulation of respiratory activity, we hypothesized that neurons in this nucleus would also be involved in the postinspiratory phase of the respiratory cycle through a connection with the pontine Kölliker-Fuse (KF) region. Here we performed pharmacogenetic manipulation (AAV-hM3D(Gq)-mCherry or AAV-hM4D(Gi)-mCherry) in VGlut2-cre, Ai6 conscious mice to evaluate breathing parameters through whole body plethysmography under baseline conditions (normoxia: FiO2 = 0.21) or under hypercapnia or hypoxia challenges (FiCO2 = 0.07 or FiO2 = 0.08). Under normoxia, selective stimulation of RTN/pFRG resulted in a smaller increase in VE (1,272 ± 102.5, vs. RTN/pFRG stimulation: 1,878 ± 122.1 ml/kg/min), due to a smaller increase in VT (5.4 ± 0.35, vs. RTN/pFRG stimulation: 7.77 ± 0.21 ml/kg) without changing fR in a condition of KF inhibition. However, inhibition of the VGlut2 neurons in the KF did affect the TE1 produced by selective activation of RTN/pFRG (119.9 ± 2.53, vs. RTN/pFRG stimulation: 104 ± 2.46 ms). Both the hypercapnia and hypoxia ventilatory response were reduced after inhibition of VGlut2-expressing KF neurons. Therefore, consistent with anatomical projections RTN/pFRG neurons regulate lung ventilation by controlling all aspects of breathing, i.e breathing frequency, inspiration, postinspiration and active expiration. All the modulation seems to be dependent on the integrity of the glutamatergic neurons in the KF region.

Mapping responses to focal injections of bicuculline in the lateral parafacial region identifies core regions for maximal generation of active expiration

The lateral parafacial area (pFL) is a crucial region involved in respiratory control, particularly in generating active expiration through an expiratory oscillatory network. Active expiration involves rhythmic abdominal (ABD) muscle contractions during late-expiration, increasing ventilation during elevated respiratory demands. The precise anatomical location of the expiratory oscillator within the ventral medulla’s rostro-caudal axis is debated. While some studies point to the caudal tip of the facial nucleus (VIIc) as the oscillator’s core, others suggest more rostral areas. Our study employed bicuculline (a γ-aminobutyric acid type A [GABA-A] receptor antagonist) injections at various pFL sites (–0.2 mm to +0.8 mm from VIIc) to investigate the impact of GABAergic disinhibition on respiration. These injections consistently elicited ABD recruitment, but the response strength varied along the rostro-caudal zone. Remarkably, the most robust and enduring changes in tidal volume, minute ventilation, and combined respiratory responses occurred at more rostral pFL locations (+0.6/+0.8 mm from VIIc). Multivariate analysis of the respiratory cycle further differentiated between locations, revealing the core site for active expiration generation with this experimental approach. Our study advances our understanding of neural mechanisms governing active expiration and emphasizes the significance of investigating the rostral pFL region.

Mapping responses to focal injections of bicuculline in the lateral parafacial region identifies core regions for maximal generation of active expiration.

The lateral parafacial area (pFL) is a crucial region involved in respiratory control, particularly in generating active expiration through an expiratory oscillatory network. Active expiration involves rhythmic abdominal (ABD) muscle contractions during late-expiration, increasing ventilation during elevated respiratory demands. The precise anatomical location of the expiratory oscillator within the ventral medulla's rostro-caudal axis is debated. While some studies point to the caudal tip of the facial nucleus (VIIc) as the oscillator's core, others suggest more rostral areas. Our study employed bicuculline (a γ-aminobutyric acid type A [GABA-A] receptor antagonist) injections at various pFL sites (-0.2 mm to +0.8 mm from VIIc) to investigate the impact of GABAergic disinhibition on respiration. These injections consistently elicited ABD recruitment, but the response strength varied along the rostro-caudal zone. Remarkably, the most robust and enduring changes in tidal volume, minute ventilation, and combined respiratory responses occurred at more rostral pFL locations (+0.6/+0.8 mm from VIIc). Multivariate analysis of the respiratory cycle further differentiated between locations, revealing the core site for active expiration generation with this experimental approach. Our study advances our understanding of neural mechanisms governing active expiration and emphasizes the significance of investigating the rostral pFL region.

Gastric Pressure Monitoring Unveils Abnormal Patient-Ventilator Interaction Related to Active Expiration: A Retrospective Observational Study.

Patient-ventilator dyssynchrony is frequently observed during assisted mechanical ventilation. However, the effects of expiratory muscle contraction on patient-ventilator interaction are underexplored. The authors hypothesized that active expiration would affect patient-ventilator interaction and they tested their hypothesis in a mixed cohort of invasively ventilated patients with spontaneous breathing activity. This is a retrospective observational study involving patients on assisted mechanical ventilation who had their esophageal pressure (Peso) and gastric pressure monitored for clinical purposes. Active expiration was defined as gastric pressure rise (ΔPgas) greater than or equal to 1.0 cm H2O during expiratory flow without a corresponding change in diaphragmatic pressure. Waveforms of Peso, gastric pressure, diaphragmatic pressure, flow, and airway pressure (Paw) were analyzed to identify and characterize abnormal patient-ventilator interaction. 76 patients were identified with Peso and gastric pressure recordings, of whom 58 demonstrated active expiration with a median ΔPgas of 3.4 cm H2O (interquartile range = 2.4 to 5.3) observed in this subgroup. Among these 58 patients, 23 presented the following events associated with expiratory muscle activity: (1) distortions in Paw and flow that resembled ineffective efforts, (2) distortions similar to autotriggering, (3) multiple triggering, (4) prolonged ventilatory cycles with biphasic inspiratory flow, with a median percentage (interquartile range) increase in mechanical inflation time and tidal volume of 54% (44 to 70%) and 25% (8 to 35%), respectively and (5) breathing exclusively by expiratory muscle relaxation. Gastric pressure monitoring was required to identify the association of active expiration with these events. Respiratory drive, assessed by the rate of inspiratory Peso decrease, was significantly higher in patients with active expiration (median [interquartile range] dPeso/dt: 12.7 [9.0 to 18.5] vs 9.2 [6.8 to 14.2] cmH2O/sec; P < 0.05). Active expiration can impair patient-ventilator interaction in critically ill patients. Without documenting gastric pressure, abnormal patient-ventilator interaction associated with expiratory muscle contraction may be mistakenly attributed to a mismatch between the patient's inspiratory effort and mechanical inflation. This misinterpretation could potentially influence decisions regarding clinical management.

Breath-by-breath Analysis of a Rat Model of Pompe Disease Exposes Deficits in High-frequency Behaviors

Pompe disease (PD) is caused by a loss of function of the enzyme acid-α-glucosidase (GAA) leading to glycogen accumulation, neuromuscular dysfunction, and breathing failure. Here, we characterized longitudinal changes in breathing of GAA null ( Gaa−/−) rats on a breath-by-breath basis using a novel respiratory event detection algorithm. Adult Pompe (n = 5) and Sprague Dawley (n = 5) rats were implanted with chronic diaphragm EMG electrodes. Once a month from 4-10 months of age, full body plethysmography with concurrent EMG was used to record respiratory waveforms under room air and hypoxia. Waveforms were analyzed by the Adjustable Baselines Respiratory Analysis Program (ABRAP) algorithm. An adaptive threshold identified “respiratory events” defined as crossings of a threshold set between the recent maxima and minima of the waveform. After event identification, a range of waveform characteristics are calculated and tagged to that event. Averages of each characteristic were taken for all events and then analyzed based on respiratory rate in breaths/minute (bpm): low (&lt;120 bpm, ‘quiet breathing’), medium (between 120 and 240 bpm) and high (&gt;240 bpm, ‘high frequency sniffng’). In room air, Pompe rats had a lower breathing rate than wildtype rats by month 10 (173 vs 285 bpm, standard error of the difference (SE of diff.) 37, p=0.03). This decrease in frequency was driven by the high rate events as there were no differences in the mean rate of low rate events at month 10 (80 vs 83 bpm, SE of diff. 3) while the frequency of high rate events was significantly lower by 9 months (mo 9: 393 vs 440 bpm, SE of diff. 16, p=0.04, mo 10: 383 vs 440 bpm, SE of diff. 17, p=0.008). Not only was the breathing frequency of high-rate events impaired in Pompe rats, but there was a trend towards decrease in the proportion of events falling in the high rate category indicating less time spent in high rate breathing behaviors (mo 4: 52 vs 59%, SE of diff. 6, mo 10: 25 vs 52%, SE of diff. 9) with a concomitant increase in the proportion of low frequency events. Additionally, there was a main effect of genotype on the latency between diaphragm activation and onset of inspiratory flow during hypoxia with Pompe rats having a longer latency across all months and frequency bands. This difference was most pronounced in the medium (44 vs 29 ms, SE of diff. 4 ms, p=0.003) frequency events. Active expiration was impaired in Pompe animals with peak post-event airflow (maximum positive pressure reached before the next inspiratory effort) blunted across all months. This effect was pronounced in the high frequency events (0.16 mL vs 0.24 mL, SE of diff. 0.01, p=0.0001). These deficits in high frequency events are consistent with progressive neuromuscular diaphragm weakness. We conclude that comprehensive analyses of all respiratory events over extended recording periods enables detection of altered breathing behaviors that may be missed when assessing only short periods of quiet breathing. In turn, this may help uncover a respiratory signature of disease progression in Pompe disease or other neuromuscular conditions. 2R01HD052682-11A1 (DDF, BJB), T32HL134621 (AM), R01HL153102 (ED), SPARC OT2OD023854 (ED), Craig H. Neilsen Pilot Grant (ED). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of glutamatergic interneurons of the lateral parafacial region of mice attenuates the ventilatory response to hypercapnia

The lateral parafacial region (pFL) generates the expiratory motor activity during hypercapnia. It was demonstrated that this region contains excitatory neurons and interneurons (with local projections), with a population of glutamatergic neurons that are silent under resting conditions, but become active at the end of expiration, simultaneously with active expiration, in response to hypercapnia/acidosis. However, the role of pFL glutamatergic interneurons in regulating pulmonary ventilation during hypercapnia is unclear. Herein, we hypothesized that the inhibition of pFL glutamatergic interneurons attenuates the ventilatory responses to hypercapnia. Therefore, we evaluated the effects of the chemogenetic inhibition of pFL glutamatergic interneurons of non-anesthetized mice on ventilatory parameters during resting and hypercapnia. We used genetically modified mice expressing the enzyme cre recombinase in glutamatergic VGLUT2-positive neurons (CEUA 1143/2022), and injected unilaterally in the pFL region a cre-dependent adeno-associated retrograde virus to express the FLP recombinase, followed by a cre- and FLP-dependent virus to express the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (hM4D-Gi) only in pFL glutamatergic interneurons. Using whole-body plethysmography, we measured respiratory frequency (fR), tidal volume (VT), pulmonary ventilation (VE), duration of respiratory cycle, inspiration (DI) and expiration (DE) at normocapnia and hypercapnia (7% CO2) after activating the hM4D-Gi in the pFL glutamatergic interneurons with JHU 37160 (0.1 mg/kg; i.p.). Data are expressed as mean ± standard deviation. By analyzing the expression of mCherry fluorescent protein, we found a significant number of glutamatergic interneurons in the pFL region (68.22 ± 11.7; n=18). At normocapnia, JHU injection did not change the ventilatory parameters of VGLUT2(cre/cre + hM4D-Gi) (n=18) and VGLUT2(cre/cre) (n=12) mice [(ΔfR: -2.4±51 vs 0.2±48 cpm; p=0.88) (ΔVT: -1.3±4 vs -0.2±1 μL·g−1; p=0.39) (ΔVE: -39 ±1198 vs -71±607 μL·g−1·min−1; p=0.72) (ΔCycle duration: 5.3±41 vs -1.9±45 ms; p=0.65) (ΔDI: 6.6±14 vs 6±20 ms; p=0.92) (ΔDE: -1.3±35 vs -1.6±31 ms; p=0.97)]. On the other hand, JHU injection attenuated the hypercapnia-induced ventilatory responses of VGLUT2(cre/cre + hM4D-Gi) (n=18) mice when compared to VGLUT2(cre/cre) (n=9) [(ΔfR: 67±69 vs 157±41 cpm; p=0.0003) (ΔVT: 3.8±5 vs 11.9±4 μL·g−1; p&lt;0.0001) (ΔVE: 2052±2197 vs 5374±1773 μL·g−1·min−1; p&lt;0.0001) (Δcycle duration: -46±50 vs -107±49 ms; p=0.002) (ΔDI: -3.7±16 vs -21±19 ms; p=0.011) (ΔDE: -42±41 vs -86±36 ms; p=0.01)]. In conclusion, our data demonstrate that the pFL glutamatergic interneurons are important for the ventilatory response of mice to hypercapnia. FAPESP, CNPq and CAPES. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Respiration and Cough in Pompe Disease: Results from a neuromechanical computational model of the respiratory control system

Late Onset Pompe Disease (LOPD) is an incurable, rare progressive genetic disease. It is characterized by glycogen build-up, due to mutations in the gene encoding the lysosomal enzyme acid alpha glucosidase, throughout muscle and neural regions. This accumulation is associated with cell death and muscle fiber swelling, eventually leading to respiratory insuffciency and airway clearance (i.e. cough) deficiency. Impairments in effective airway clearance result from the inability to clear normal pulmonary volume. Emerging evidence indicates significant respiratory neuron dysfunction, altering pulmonary function in humans and animal models. It has been suggested that respiratory and airway clearance insuffciency observed in LOPD may be attributed to medullary motoneurons potentially associated with the respiratory and cough pattern generators (Fuller et al., 2013; DeRuisseau et al., 2009). However, the precise neural mechanisms that suppress breathing and cough are not fully understood. One possible target for LOPD respiratory insuffciencies may be reduced medullary inspiratory neuron activity within this network, which could result in decreased phrenic motoneuron output. Using a stochastic neural network simulator that consisted of discrete “integrate and fire” populations, we simulated a systematic decrease in three medullary inspiratory neuron populations: inspiratory driver (I-Driver), inspiratory decrementing (I-Dec), or inspiratory augmenting (I-Aug) neurons. We simulated breathing and three cough trials. By targeting these populations, we expected global decreased neural bursts, suppressed inspiratory motor drive, and/or suppressed expiratory motoneuron output. Simulations indicated that when the I-Dec motoneuron population decreased by 25%, it resulted in apnea and abolished cough. Reducing the I-Aug motoneuron population by 20% resulted in longer inspiratory phase durations (TI) and a decreased respiratory rate. During cough, there was a decrease in the duration and amplitudes of the expiratory motoneuronal activity associated with decreased cough receptor and pulmonary stretch receptor afferent feedback. When the I-Driver motoneuron population was reduced by 25%, TI decreased and I amplitude increased during respiration. During cough, lumbar and phrenic motoneuron discharge duration and amplitude decreased. These results are consistent with previously reported in vivo data. Overall, our simulation data support the plausibility of glycogen storage dysfunction impairing medullary neuronal and premotoneuronal excitability within the inspiratory network. Our results also suggest that some neurons may be more sensitive to glycogen storage than others within the brainstem inspiratory network. Supported by NHLBI L30HL165496, NIH T32 HL 134621 and 3OT2OD023854. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in preBötzinger Complex neuron discharge associated with changes in PCO2 in decerebrate rabbits

Background: Maskrey and Nicol reported a strong increase in minute ventilation with increase in PCO2 (J Physiol 1980, 301: 49-58). We investigated a) whether there was a ceiling to this effect at high CO2 levels and b) whether the change in ventilation was associated with specific changes in respiratory neurons in the preBötzinger complex (preBC). Methods: The study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3-4 kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the C5 rootlet, time averaged and used to calculate inspiratory (TI) and expiratory durations (TE) and peak phrenic activity (PPA). PPA was normalized to the value at PCO2 50mmHg. Individual neurons were recorded in the functionally identified preBC using multichannel electrodes at increasing CO2 levels. Statistical analysis used the Wilcoxon signed-rank test to determine changes in respiratory parameters between CO2 levels and Friedman RM-ANOVA to determine changes in neuronal discharge frequency (Fn). A generalized linear mixed model was used to determine associations between CO2-dependent changes in TI, TE, PPA, and changes in Fn for different neuron types. p&lt;0.05 indicated a significant difference. Results: In 34 animals, the increase in minute ventilation with increasing PCO2 was due mostly to the change in PPA. The increase in respiratory rate was due mostly to a decrease in TE. There was little increase in respiratory output at PCO2 &gt; 50 mmHg. In 209 neurons, increasing PCO2 increased Fn in inspiratory augmenting (I aug, n= 70, p&lt;0.0001 for CO2 30 vs 40 and 40 vs 50mmHg), I parabolic (I para, n=43, p&lt;0.0001 for CO2 40 vs 50 mmHg), pre-inspiratory (pre-I, n=35, p&lt;0.01 for CO2 30 vs 40 mmHg), and expiratory decrementing (E dec, n=44, p&lt;0.01 for CO2 40 vs 50 mmHg) neurons, but not in I dec (n=8) and non-respiratory modulated (NRM, n=9) neurons. The strongest correlation was found between the changes in phase timing and PPA and Fn in I aug and pre-I neurons. Conclusion: We found a ceiling effect to the increase in respiratory output at PCO2 levels &gt; 50 mmHg. Inspiratory preBC neuron subtypes were more affected by the changes in PCO2. NIH R01HL159546. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of somatostatinergic interneurons of the lateral parafacial region reduces the ventilatory response to hypercapnia of mice

The lateral parafacial region (pFL), which contains excitatory and inhibitory neurons and interneurons, generates active expiration in response to hypercapnia. Inhibition of inhibitory somatostatinergic (Sst)-positive neurons in parafacial region increases pulmonary ventilation, while hypercapnia inhibits some of them intrinsically. However, the role of pFL Sst-positive interneurons (with local projections) in regulating pulmonary ventilation during hypercapnia is unclear. We hypothesized that the pFL Sst-positive interneurons control the pulmonary ventilation of mice under normocapnia and hypercapnia, as part of a local neural network. Herein, we evaluated the impact of pFL Sst-positive interneurons inhibition on pulmonary ventilation of non-anesthetized mice during normocapnia and hypercapnia. We used genetically modified mice, expressing the cre recombinase in Sst-positive neurons, and injected unilaterally a cre-dependent adeno-associated (AAV) retrograde virus in the pFL region to express the FLP recombinase, followed by a cre- and FLP-dependent AAV to express the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (hM4D-Gi) in the pFL Sst-positive interneurons. We used whole-body plethysmography to measure the respiratory frequency (fR), tidal volume (VT), pulmonary ventilation (VE), as well as the duration of the respiratory cycle, inspiration (DI) and expiration (DE) at normocapnia and hypercapnia (7% CO2) after activation of hM4D-Gi in the pFL Sst-positive interneurons using JHU 37160 (JHU; 0.1 mg/kg; i.p.). Data are expressed as mean ± standard deviation. By analyzing the expression of mCherry fluorescent protein, we found a significant number of Sst-positive interneurons (73.43 ± 10.52) in the pFL region (n=14). JHU administration did not affect the ventilatory parameters of Sst(cre/cre) (n= 9) and Sst(cre/cre + hM4D-Gi) (n= 14) groups at normocapnia [(Δ fR: 6.5 ± 33.56 vs. -14.86 ± 49.03 cpm; p= 0.42) (Δ VT: 0.87 ± 1.15 vs. -0.97 ± 2.58 μL·g−1; p= 0.19) (Δ VE: 235.8 ± 388.8 vs. -315.9 ± 725.1 μL·g−1·min−1; p= 0.16) (Δ duration of the respiratory cycle: -13 ± 45.11 vs. 12.34 ± 53.07 ms; p= 0.39) (Δ DI: 10.36 ± 11.23 vs. 14.7 ± 16.38 ms; p= 0.62) (Δ DE: -23.36 ± 42.42 vs. -2.41± 53.58 ms; p= 0.48)]. On the other hand, JHU administration attenuated the hypercapnia-induced responses of all ventilatory parameters, such as fR (Δ: 44.57 ± 56.66 vs. 126± 21.28 cpm; p= 0.0002), VT (Δ: 1.32 ± 3.35 vs. 4.16 ± 2.56 μL·g−1; p= 0.04), VE (Δ: 948.7 ± 1407.1 vs. 2183 ± 909.4 μL·g−1·min−1; p= 0.03), duration of the respiratory cycle (Δ: -44.57 ± 46.31 vs. -122.1 ± 30.7 ms; p= 0.0002), DI (Δ: 6.79 ± 16.36 vs. -13.03 ± 10.19 ms; p= 0.003) and DE (Δ: -51.37 ± 37.50 vs. -109.1 ± 32.78 ms; p= 0.001) in the Sst(cre/cre + hM4D-Gi) compared with the Sst(cre/cre) group. Thus, we conclude that the pFL Sst-positive interneurons control the ventilatory parameters of mice during hypercapnia, but not during normocapnia. FAPESP, CNPq and CAPES. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypercapnia elicits changes in diaphragm neuromotor control and respiratory timing parameters in awake rats

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals. At baseline, expiration is passive; however, with increased CO2 expiration becomes active, involving the inhibition of postinspiratory DIAm activity. The neuromotor control of the DIAm involves both the recruitment and frequency coding of DIAm motor units (MU’s) and it is not clear which aspect plays a greater role with exposure to hypercapnia. The activity of DIAm MU’s can be split into three phases: recruitment, sustained activity, and derecruitment. The goal of the present study was to evaluate the effects of hypercapnia on the neuromotor control of the DIAm in awake Sprague-Dawley rats. We hypothesized that hypercapnia would lead to increased respiratory drive as estimated by the root-mean-square (RMS) EMG amplitude at the end of the recruitment phase. Accordingly, we implanted chronic fine-wire EMG electrodes into the mid-costal region of the right side of the DIAm in 10 (5 female, 5 male) anesthetized rats. Three days after recovery from surgery, we recorded DIAm EMG in lightly restrained awake rats during normocapnia and hypercapnia (7% CO2, 21% O2, balance N2). We estimated the duration of motor unit recruitment, sustained activity, and derecruitment by assessing the stationarity of DIAm EMG activity and used these durations to determine the RMS EMG amplitude at the completion of recruitment, at the peak, and immediately prior to derecruitment (normalized to the average peak RMS EMG amplitude during normocapnia within each animal). We found that the RMS EMG amplitude at the completion of recruitment was increased from ~75% to 85% with hypercapnia. Peak RMS EMG amplitude during hypercapnia increased from 100% to ~120%, while the amplitude at the onset of derecruitment increased from ~50% to 95%. These data suggest that ~70% of the total RMS EMG peak amplitude can be attributed primarily to motor unit recruitment, while the remaining ~30% can be attributed to frequency modulation during hypercapnia. Importantly the onset-to-offset amplitude difference was ~25% during normocapnia and ~15% during hypercapnia, suggesting that DIAm MU’s were derecruited at higher discharge rates with hypercapnia, likely due to inhibition. We also found that compared to normocapnia, respiratory rate increased from ~85 breaths/min to 125 breaths/min while the burst duration decreased from ~600 ms to ~400 ms during hypercapnia. The duty cycle was maintained around 80% for both, however the duty cycle of the sustained activity duration (i.e. the period during which MU’s are remaining activated) decreased from ~56% to ~28%. Taken together, our data suggest that hypercapnia in awake rats results in an increase in the number of motor units recruited and their discharge rates as well as changes in the phases of DIAm MU activity indicative of active expiration. Funded by NIH HL-146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SubSol-HIe is an AMPK-dependent hypoxia-responsive subnucleus of the nucleus tractus solitarius that coordinates the hypoxic ventilatory response and protects against apnoea in mice

Functional magnetic resonance imaging (fMRI) suggests that the hypoxic ventilatory response is facilitated by the AMP-activated protein kinase (AMPK), not at the carotid bodies, but within a subnucleus (Bregma -7.5 to -7.1 mm) of the nucleus tractus solitarius that exhibits right-sided bilateral asymmetry. Here, we map this subnucleus using cFos expression as a surrogate for neuronal activation and mice in which the genes encoding the AMPK-α1 (Prkaa1) and AMPK-α2 (Prkaa2) catalytic subunits were deleted in catecholaminergic cells by Cre expression via the tyrosine hydroxylase promoter. Comparative analysis of brainstem sections, relative to controls, revealed that AMPK-α1/α2 deletion inhibited, with right-sided bilateral asymmetry, cFos expression in and thus activation of a neuronal cluster that partially spanned three interconnected anatomical nuclei adjacent to the area postrema: SolDL (Bregma -7.44 mm to -7.48 mm), SolDM (Bregma -7.44 mm to -7.48 mm) and SubP (Bregma -7.48 mm to -7.56 mm). This approximates the volume identified by fMRI. Moreover, these nuclei are known to be in receipt of carotid body afferent inputs, and catecholaminergic neurons of SubP and SolDL innervate aspects of the ventrolateral medulla responsible for respiratory rhythmogenesis. Accordingly, AMPK-α1/α2 deletion attenuated hypoxia-evoked increases in minute ventilation (normalised to metabolism), reductions in expiration time, and increases sigh frequency, but increased apnoea frequency during hypoxia. The metabolic response to hypoxia in AMPK-α1/α2 knockout mice and the brainstem and spinal cord catecholamine levels were equivalent to controls. We conclude that within the brainstem an AMPK-dependent, hypoxia-responsive subnucleus partially spans SubP, SolDM and SolDL, namely SubSol-HIe, and is critical to coordination of active expiration, the hypoxic ventilatory response and defence against apnoea.

Speech-Swallow Dissociation of Velopharyngeal Incompetence with Pseudobulbar Palsy: Evaluation by High-Resolution Manometry.

Patients with pseudobulbar palsy often present with velopharyngeal incompetence. Velopharyngeal incompetence is usually observed during expiratory activities such as speech and/or blowing during laryngoscopy. These patients typically exhibit good velopharyngeal closure during swallowing, which is dissociated from expiratory activities. We named this phenomenon "speech-swallow dissociation" (SSD). SSD on endoscopic findings can help in diagnosing the underlying disease causing dysphagia. This endoscopic finding is qualitative, and the quantitative characteristics of SSD are still unclear. Accordingly, the current study aimed to quantitatively evaluate SSD in patients with pseudobulbar palsy. We evaluated velopharyngeal pressure during swallowing and expiratory activity in 10 healthy subjects and 10 patients with pseudobulbar palsy using high-resolution manometry, and compared the results between the two groups. No significant differences in maximal velopharyngeal contraction pressure (V-Pmax) were observed during dry swallowing between the pseudobulbar palsy group and healthy subjects (190.5mmHg vs. 173.6mmHg; P = 0.583). V-Pmax during speech was significantly decreased in the pseudobulbar palsy group (85.4mmHg vs. 34.5mmHg; P < 0.001). The degree of dissociation of speech to swallowing in V-Pmax, when compared across groups, exhibited a larger difference in the pseudobulbar palsy group, at 52% versus 80% (P = 0.001). Velopharyngeal pressure during blowing was similar to that during speech. Velopharyngeal closure in patients with pseudobulbar palsy exhibited weaker pressure during speech and blowing compared with swallowing, quantitatively confirming the presence of SSD. Pseudobulbar palsy often presents with SSD, and this finding may be helpful in differentiating the etiology of dysphagia.

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

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

Driving pressure of respiratory system and lung stress in mechanically ventilated patients with active breathing

BackgroundDuring control mechanical ventilation (CMV), the driving pressure of the respiratory system (ΔPrs) serves as a surrogate of transpulmonary driving pressure (ΔPlung). Expiratory muscle activity that decreases end-expiratory lung volume may impair the validity of ΔPrs to reflect ΔPlung. This prospective observational study in patients with acute respiratory distress syndrome (ARDS) ventilated with proportional assist ventilation (PAV+), aimed to investigate: (1) the prevalence of elevated ΔPlung, (2) the ΔPrs-ΔPlung relationship, and (3) whether dynamic transpulmonary pressure (Plungsw) and effort indices (transdiaphragmatic and respiratory muscle pressure swings) remain within safe limits.MethodsThirty-one patients instrumented with esophageal and gastric catheters (n = 22) were switched from CMV to PAV+ and respiratory variables were recorded, over a maximum of 24 h. To decrease the contribution of random breaths with irregular characteristics, a 7-breath moving average technique was applied. In each patient, measurements were also analyzed per deciles of increasing lung elastance (Elung). Patients were divided into Group A, if end-inspiratory transpulmonary pressure (PLEI) increased as Elung increased, and Group B, which showed a decrease or no change in PLEI with Elung increase.ResultsIn 44,836 occluded breaths, ΔPlung ≥ 12 cmH2O was infrequently observed [0.0% (0.0–16.9%) of measurements]. End-expiratory lung volume decrease, due to active expiration, was associated with underestimation of ΔPlung by ΔPrs, as suggested by a negative linear relationship between transpulmonary pressure at end-expiration (PLEE) and ΔPlung/ΔPrs. Group A included 17 and Group B 14 patients. As Elung increased, ΔPlung increased mainly due to PLEI increase in Group A, and PLEE decrease in Group B. Although ΔPrs had an area receiver operating characteristic curve (AUC) of 0.87 (95% confidence intervals 0.82–0.92, P < 0.001) for ΔPlung ≥ 12 cmH2O, this was due exclusively to Group A [0.91 (0.86–0.95), P < 0.001]. In Group B, ΔPrs showed no predictive capacity for detecting ΔPlung ≥ 12 cmH2O [0.65 (0.52–0.78), P > 0.05]. Most of the time Plungsw and effort indices remained within safe range.ConclusionIn patients with ARDS ventilated with PAV+, injurious tidal lung stress and effort were infrequent. In the presence of expiratory muscle activity, ΔPrs underestimated ΔPlung. This phenomenon limits the usefulness of ΔPrs as a surrogate of tidal lung stress, regardless of the mode of support.

Expiratory Muscle Activity Counteracts Positive End-Expiratory Pressure and Is Associated with Fentanyl Dose in Patients with Acute Respiratory Distress Syndrome.

Rationale: Hypoxemia during mechanical ventilation might be worsened by expiratory muscle activity, which reduces end-expiratory lung volume through lung collapse. A proposed mechanism of benefit of neuromuscular blockade in acute respiratory distress syndrome (ARDS) is the abolition of expiratory efforts. This may contribute to the restoration of lung volumes. The prevalence of this phenomenon, however, is unknown. Objectives: To investigate the incidence and amount of end-expiratory lung impedance (EELI) increase after the administration of neuromuscular blocking agents (NMBAs), clinical factors associated with this phenomenon, its impact on regional lung ventilation, and any association with changes in pleural pressure. Methods: We included mechanically ventilated patients with ARDS monitored with electrical impedance tomography (EIT) who received NMBAs in one of two centers. We measured changes in EELI, a surrogate for end-expiratory lung volume, before and after NMBA administration. In an additional 10 patients, we investigated the characteristic signatures of expiratory muscle activity depicted by EIT and esophageal catheters simultaneously. Clinical factors associated with EELI changes were assessed. Measurements and Main Results: We included 46 patients, half of whom showed an increase in EELI of >10% of the corresponding Vt (46.2%; IQR, 23.9-60.9%). The degree of EELI increase correlated positively with fentanyl dosage and negatively with changes in end-expiratory pleural pressures. This suggests that expiratory muscle activity might exert strong counter-effects against positive end-expiratory pressure that are possibly aggravated by fentanyl. Conclusions: Administration of NMBAs during EIT monitoring revealed activity of expiratory muscles in half of patients with ARDS. The resultant increase in EELI had a dose-response relationship with fentanyl dosage. This suggests a potential side effect of fentanyl during protective ventilation.

Respiratory-resolved five-dimensional flow cardiovascular magnetic resonance : In-vivo validation and respiratory-dependent flow changes in healthy volunteers and patients with congenital heart disease

BackgroundThis study aimed to validate respiratory-resolved 5D flow MRI against real-time 2D phase contrast MRI, assess the impact of number of respiratory states, and measure the impact of respiration on hemodynamics in congenital heart disease (CHD) patients. MethodsRespiratory-resolved 5D flow MRI derived net and peak flow measurements were compared to real-time 2D phase contrast MRI derived measurements in 10 healthy volunteers. Pulmonary to systemic flow ratios (Qp:Qs) were measured in 19 CHD patients and aortopulmonary collateral burden was measured in 5 Fontan patients. Additionally, the impact of number of respiratory states on measured respiratory-driven net flow changes was investigated in 10 healthy volunteers and 19 CHD patients (shunt physiology, n=11, single ventricle disease (SVD), n=8). ResultsThere was good agreement between 5D flow MRI and real-time 2D phase contrast derived net and peak flow. Respiratory driven changes had good correlation (rho=0.64, p<0.001). In healthy volunteers, fewer than four respiratory states reduced measured respiratory driven flow changes in veins (5.2mL/cycle, p<0.001) and arteries (1.7mL/cycle, p=0.05). Respiration drove substantial venous net flow changes in SVD (64% change) and shunt patients (57% change). Respiration had significantly greater impact in SVD patients compared to shunt patients in the right and left pulmonary arteries (46% vs 15%, p=0.003 & 59% vs 20%, p=0.002). Qp:Qs varied by 37±24% over respiration in SVD patients and 12±20% in shunt patients. Aortopulmonary collateral burden varied by 118±84% over respiration in Fontan patients. The smallest collateral burden was measured during active inspiration in all patients and the greatest burden was during active expiration in 4 of 5 patients. Reduced respiratory resolution blunted measured flow changes in the caval veins of shunt and SVD patients (p<0.005). ConclusionsRespiratory-resolved 5D flow MRI measurements agree with real-time 2D phase contrast. Venous measurements are sensitive to number of respiratory states, whereas arterial measurements are more robust. Respiration has substantial impact on caval vein flow, Qp:Qs, and collateral burden in CHD patients.

Ab. No. 160 Design and Develop a Low-Cost Biofeedback Respiratory Device to Improve Lung Function in Pulmonary Patients

Introduction: Over the past years in medical field, biofeedback (BF) therapy programs have gained lot of importance in patient education. Using BF promises to overcome challenges that is faced in routine teaching methods such as, low focus during therapy and the difficulty of transferring it into everyday practice. The innovation of this proposal is biofeedback with good visual feedback in the form of a game. By this method, the patient would be made aware of his or her reactions through the modification of features in the game and may learn to control the physiological parameters using the feedback provided by the visual input and succeed in learning. Methods: The therapeutic instrument with a biofeedback intended device which contains a mouthpiece (disposable), filter (mesh), and a steel tube which would be created using hardware and coding. The device will have games that focuses on inspiratory and expiratory training activities and this would help to improve adherence. Result and Conclusion: Major application in pulmonary rehabilitation to train the patient effectively to improve lung function thereby reduce symptoms and to improve the quality of life of patient, for home-based exercise program, for research and clinical need. Pulmonary dysfunction causes many symptoms that affects the day today life of the patients, improving the respiratory muscle function reduces such symptoms and improves quality of life of the pulmonary patients. Exercise compliance of the patients is a challenging factor often faced by therapists and proposed device would help in improving the exercise adherence as its interesting and innovative.

Divergent expiratory braking activity of costal and crural diaphragm

BackgroundThere is increasing clinical interest in understanding the contribution of the diaphragm in early expiration, especially during mechanical ventilation. However, current experimental evidence is limited, so essential activity of the diaphragm during expiration and diaphragm segmental differences in expiratory activity, are unknown. ObjectivesTo determine if: 1) the diaphragm is normally active into expiration during spontaneous breathing and hypercapnic ventilation, 2) expiratory diaphragmatic activity is distributed equally among the segments of the diaphragm, costal and crural. MethodsIn 30 spontaneously breathing male and female canines, awake without confounding anesthetic, we measured directly both inspiratory and expiratory electrical activity (EMG), and corresponding mechanical shortening, of costal and crural diaphragm, during room air and hypercapnia. ResultsDuring eupnea, costal and crural diaphragm are active into expiration, showing significant and distinct expiratory activity, with crural expiratory activity greater than costal, for both magnitude and duration. This diaphragm segmental difference diverged further during progressive hypercapnic ventilation: crural expiratory activity progressively increased, while costal expiratory activity disappeared. ConclusionThe diaphragm is not passive during expiration. During spontaneous breathing, expiratory activity -“braking”- of the diaphragm is expressed routinely, but is not equally distributed. Crural muscle “braking” is greater than costal muscle in magnitude and duration.With increasing ventilation during hypercapnia, expiratory activity -“braking”- diverges notably. Crural expiratory activity greatly increases, while costal expiratory “braking” decreases in magnitude and duration, and disappears.Thus, diaphragm expiratory "braking" action represents an inherent, physiological function of the diaphragm, distinct for each segment, expressing differing neural activation.

Exploring the role of the Kölliker-Fuse nucleus in breathing variability by mathematical modelling.

The Kölliker-Fuse nucleus (KF), which is part of the parabrachial complex, participates in the generation of eupnoea under resting conditions and the control of active abdominal expiration when increased ventilation is required. Moreover, dysfunctions in KF neuronal activity are believed to play a role in the emergence of respiratory abnormalities seen in Rett syndrome (RTT), a progressive neurodevelopmental disorder associated with an irregular breathing pattern and frequent apnoeas. Relatively little is known, however, about the intrinsic dynamics of neurons within the KF and how their synaptic connections affect breathing pattern control and contribute to breathing irregularities. In this study, we use a reduced computational model to consider several dynamical regimes of KF activity paired with different input sources to determine which combinations are compatible with known experimental observations. We further build on these findings to identify possible interactions between the KF and other components of the respiratory neural circuitry. Specifically, we present two models that both simulate eupnoeic as well as RTT-like breathing phenotypes. Using nullcline analysis, we identify the types of inhibitory inputs to the KF leading to RTT-like respiratory patterns and suggest possible KF local circuit organizations. When the identified properties are present, the two models also exhibit quantal acceleration of late-expiratory activity, a hallmark of active expiration featuring forced exhalation, with increasing inhibition to KF, as reported experimentally. Hence, these models instantiate plausible hypotheses about possible KF dynamics and forms of local network interactions, thus providing a general framework as well as specific predictions for future experimental testing. KEY POINTS: The Kölliker-Fuse nucleus (KF), a part of the parabrachial complex, is involved in regulating normal breathing and controlling active abdominal expiration during increased ventilation. Dysfunction in KF neuronal activity is thought to contribute to respiratory abnormalities seen in Rett syndrome (RTT). This study utilizes computational modelling to explore different dynamical regimes of KF activity and their compatibility with experimental observations. By analysing different model configurations, the study identifies inhibitory inputs to the KF that lead to RTT-like respiratory patterns and proposes potential KF local circuit organizations. Two models are presented that simulate both normal breathing and RTT-like breathing patterns. These models provide testable hypotheses and specific predictions for future experimental investigations, offering a general framework for understanding KF dynamics and potential network interactions.