Intrinsic Molecular Proton Sensitivity Underlies GPR4 Effects on Retrotrapezoid Nucleus Neuronal Activation and CO2-Stimulated Breathing.

An interoceptive homeostatic reflex monitors levels of CO2/H+ to maintain blood gas homeostasis and rapidly regulate tissue acid-base balance by driving lung ventilation and CO2 excretion-this CO2-evoked increase in respiration is the hypercapnic ventilatory reflex (HCVR). Retrotrapezoid nucleus (RTN) neurons provide crucial excitatory drive to downstream respiratory rhythm/pattern-generating circuits, and their activity is directly modulated by changes in CO2/H+ RTN neurons express GPR4 and TASK-2, global deletion of which abrogates CO2/H+ activation of RTN neurons and the HCVR. It has not been determined if the intrinsic pH sensitivity of these proton detectors is required for these effects. We used CRISPR/Cas9 genome editing to generate mice with mutations in either of two pH-sensing histidine residues in GPR4 to determine effects on RTN neuronal CO2/H+ sensitivity and the HCVR. In global GPR4(H81F) and GPR4(H167F) mice, CO2-stimulated breathing and CO2-induced RTN neuronal activation were strongly blunted, with no effect on hypoxia-stimulated breathing. In brainstem slices from GPR4(H81F) mice, peak firing of RTN neurons during bath acidification was significantly reduced compared with GPR4 wild-type mice, and a subpopulation of RTN neurons was rendered pH-insensitive, phenocopying previous results from GPR4-deleted mice. These effects were independent of changes in RTN number/distribution, neuronal excitability or transcript levels for GPR4 and TASK-2. CO2-stimulated breathing was reduced to a similar extent in GPR4(H81F) and TASK-2-deleted mice, with combined mutation yielding no additional deficit in the HCVR. Together, these data demonstrate that the intrinsic pH sensitivity of GPR4 is necessary for full elaboration of the HCVR.

Gill ionocyte remodeling mediates blood pH regulation in rockfish (Sebastes diploproa) exposed to environmentally relevant hypercapnia.

Marine fishes excrete excess H+ using basolateral Na+-K+-ATPase (NKA) and apical Na+/H+ exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H+ excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish (Sebastes diploproa) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 µatm Pco2). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1) mRNAs coding for proteins involved in blood acid-base regulation, 2) total NKA and NHE3 protein abundance, and 3) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H+ excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems.NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

Central and peripheral mechanisms underlying respiratory deficits in a mouse model of accelerated senescence.

Aging invariably decreases sensory and motor stimuli and affects several neuronal systems and their connectivity to key brain regions, including those involved in breathing. Nevertheless, further investigation is needed to fully comprehend the link between senescence and respiratory function. Here, we investigate whether a mouse model of accelerated senescence could develop central and peripheral respiratory abnormalities. Adult male Senescence Accelerated Mouse Prone 8 (SAMP8) and the control SAMR1 mice (10 months old) were used. Ventilatory parameters were assessed by whole-body plethysmography, and measurements of respiratory input impedance were performed. SAMP8 mice exhibited a reduction in the density of neurokinin-1 receptor immunoreactivity in the entire ventral respiratory column. Physiological experiments showed that SAMP8 mice exhibited a decreased tachypneic response to hypoxia (FiO2 = 0.08; 10 min) or hypercapnia (FiCO2 = 0.07; 10 min). Additionally, the ventilatory response to hypercapnia increased further due to higher tidal volume. Measurements of respiratory mechanics in SAMP8 mice showed decreased static compliance (Cstat), inspiratory capacity (IC), resistance (Rn), and elastance (H) at different ages (3, 6, and 10 months old). SAMP8 mice also have a decrease in contractile response to methacholine compared to SAMR1. In conclusion, our findings indicate that SAMP8 mice display a loss of the NK1-expressing neurons in the respiratory brainstem centers, along with impairments in both central and peripheral respiratory mechanisms. These observations suggest a potential impact on breathing in a senescence animal model.

The pontine Kölliker-Fuse nucleus is important for reduced postinspiratory 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 (post-I) 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: [Formula: see text] = 0.21) or under hypercapnia or hypoxia challenges ([Formula: see text] = 0.07 or [Formula: see text] = 0.08). Under normoxia, selective stimulation of RTN/pFRG resulted in a smaller increase in V̇e (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.NEW & NOTEWORTHY Our research reveals specific roles and interactions between the retrotrapezoid nucleus/respiratory parafacial region (RTN/pFRG) and the pontine Kölliker-Fuse (KF) region in controlling respiratory phases. RTN/pFRG neurons are key in regulating all aspects of breathing, including frequency, inspiration, postinspiration, and active expiration. This regulation depends on the functional integrity of glutamatergic neurons in the KF region, aligning with anatomical projections.

Sex and APOE genotype influence respiratory function under hypoxic and hypoxic-hypercapnic conditions.

The apolipoprotein E (APOE) gene has been studied due to its influence on Alzheimer's disease (AD) development and work in an APOE mouse model recently demonstrated impaired respiratory motor plasticity following spinal cord injury (SCI). Individuals with AD often copresent with obstructive sleep apnea (OSA) characterized by cessations in breathing during sleep. Despite the prominence of APOE genotype and sex as factors in AD progression, little is known about the impact of these variables on respiratory control. Ventilation is tightly regulated across many systems, with respiratory rhythm formation occurring in the brainstem but modulated in response to chemoreception. Alterations within these modulatory systems may result in disruptions of appropriate respiratory control and ultimately, disease. Using mice expressing two different humanized APOE alleles, we characterized how sex and the presence of APOE3 or APOE4 influences ventilation during baseline breathing (normoxia) and during respiratory challenges. We show that sex and APOE genotype influence breathing during hypoxic challenge, which may have clinical implications in the context of AD and OSA. In addition, female mice, while responding robustly to hypoxia, were unable to recover to baseline respiratory levels, emphasizing sex differences in disordered breathing.NEW & NOTEWORTHY This study is the first to use whole body plethysmography (WBP) to measure the impact of APOE alleles on breathing under normoxia and during adverse respiratory challenges in a targeted replacement Alzheimer's model. Both sex and genotype were shown to affect breathing under normoxia, hypoxic challenge, and hypoxic-hypercapnic challenge. This work has important implications regarding the impact of genetics on respiratory control as well as applications pertaining to conditions of disordered breathing including sleep apnea and neurotrauma.

Altered ventilatory responses to hypercapnia-hypoxia challenges in a preclinical SUDEP model involve orexin neurons.

Failure to recover from repeated hypercapnia and hypoxemia (HH) challenges caused by severe GCS and postictal apneas may contribute to sudden unexpected death in epilepsy (SUDEP). Our previous studies found orexinergic dysfunction contributes to respiratory abnormalities in a preclinical model of SUDEP, Kcna1-/- mice. Here, we developed two gas challenges consisting of repeated HH exposures and used whole body plethysmography to determine whether Kcna1-/- mice have detrimental ventilatory responses. Kcna1-/- mice exhibited an elevated ventilatory response to a mild repeated hypercapnia-hypoxia (HH) challenge compared to WT. Moreover, 71% of Kcna1-/- mice failed to survive a severe repeated HH challenge, whereas all WT mice recovered. We next determined whether orexin was involved in these differences. Pretreating Kcna1-/- mice with a dual orexin receptor antagonist rescued the ventilatory response during the mild challenge and all subjects survived the severe challenge. In ex vivo extracellular recordings in the lateral hypothalamus of coronal brain slices, we found reducing pH either inhibits or stimulates putative orexin neurons similar to other chemosensitive neurons; however, a significantly greater percentage of putative orexin neurons from Kcna1-/-mice were stimulated and the magnitude of stimulation was increased resulting in augmentation of the calculated chemosensitivity index relative to WT. Collectively, our data suggest that increased chemosensitive activity of orexin neurons may be pathologic in the Kcna1-/- mouse model of SUDEP, and contribute to elevated ventilatory responses. Our preclinical data suggest that those at high risk for SUDEP may be more sensitive to HH challenges, whether induced by seizures or other means; and the depth and length of the HH exposure could dictate the probability of survival.

The independent effects of hydrostatic pressure and hypercapnic breathing during water immersion on ventilatory sensitivity and cerebrovascular reactivity.

Head-out water immersion (HOWI) induces ventilatory and hemodynamic changes, which may be a result of hydrostatic pressure, augmented arterial CO2 tension, or a combination of both. We hypothesized that the hydrostatic pressure and elevated CO2 tension that occur during HOWI will contribute to an augmented ventilatory sensitivity to CO2 and an attenuated cerebrovascular reactivity to CO2 during water immersion. Twelve subjects [age: 24 ± 3 yr, body mass index (BMI): 25 ± 3 kg/m2] completed HOWI, waist water immersion with CO2 (WWI + CO2), and WWI, where a rebreathing test was conducted at baseline, 10, 30, and 60 min, and postimmersion. End-tidal pressure of carbon dioxide ([Formula: see text]), minute ventilation, expired gases, blood pressure, heart rate, and middle cerebral artery blood velocity were recorded continuously. [Formula: see text] increased throughout all visits (P ≤ 0.011), was similar during HOWI and WWI + CO2 (P ≥ 0.264), and was greater during WWI + CO2 versus WWI at 10, 30, and 60 min (P < 0.001). When HOWI vs. WWI + CO2 were compared, the change in ventilatory sensitivity to CO2 was different at 10 (0.59 ± 0.34 vs. 0.06 ± 0.23 L/min/mmHg; P < 0.001), 30 (0.58 ± 0.46 vs. 0.15 ± 0.25 L/min/mmHg; P < 0.001), and 60 min (0.63 ± 0.45 vs. 0.16 ± 0.34 L/min/mmHg; P < 0.001), whereas there were no differences between conditions for cerebrovascular reactivity to CO2 (P ≥ 0.163). When WWI + CO2 versus WWI were compared, ventilatory sensitivity to CO2 was not different between conditions (P ≥ 0.642), whereas the change in cerebrovascular reactivity to CO2 was different at 30 min (-0.56 ± 0.38 vs. -0.30 ± 0.25 cm/s/mmHg; P = 0.010). These data indicate that during HOWI, ventilatory sensitivity to CO2 increases due to the hydrostatic pressure, whereas cerebrovascular reactivity to CO2 decreases due to the combined effects of immersion.NEW & NOTEWORTHY Although not fully elucidated, the ventilatory and hemodynamic alterations during water immersion appear to be a result of the combined effects of immersion (i.e., elevated [Formula: see text], central hypervolemia, increased cerebral perfusion, increased work of breathing, etc.). Our findings demonstrate that an augmented ventilatory sensitivity to CO2 during immersion may be due to the hydrostatic pressure across the chest wall, whereas an attenuated cerebrovascular reactivity to CO2 may be due to the combined effects of immersion.

Initiation of noninvasive ventilation in patients with amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Noninvasive ventilation (NIV) has been shown to improve survival and symptom burden in patients with amyotrophic lateral sclerosis (ALS). However, limited data exist regarding the clinical and physiological parameters at the time of NIV initiation. This study aimed to describe the clinical characteristics and respiratory physiological markers in a cohort of ALS patients with chronic respiratory failure. METHODS: This is a single-center retrospective cohort study of patients with ALS assessed for NIV initiation between February 2012 and January 2021. NIV was initiated based on insurance eligibility criteria: daytime hypercapnia, defined by partial pressure of carbon dioxide (PaCO2) >45 mm Hg using diurnal transcutaneous CO2 (TcCO2) as a surrogate, a maximal inspiratory pressure (MIP) <60 cmH2O or forced vital capacity (FVC) <50% predicted normal. RESULTS: We identified 335 patients with ALS and chronic respiratory failure referred to an outpatient home ventilation clinic for NIV initiation. The mean age was 64 years ±11; 151 (45%) were female, 326 (97%) were white, and 100 (29%) had bulbar-onset ALS. At the time of NIV initiation, the mean FVC was 64% ± 19%, the mean MIP; 41 cmH2O ± 17, and diurnal TcCO2; 40 ± 6 mmHg. The most common reasons for NIV initiation were MIP <60 cmH2O (58%) and multiple concomitant indications (28%). Within 1 year of NIV initiation, 126 (37%) patients were deceased. DISCUSSION: We found that impairment in inspiratory force was the most common reason for NIV initiation and often preceded significant declines in FVC.

Changes in cerebral vascular reactivity following mild repetitive head injury in awake rats: modeling the human experience.

The changes in brain function in response to mild head injury are usually subtle and go undetected. Physiological biomarkers would aid in the early diagnosis of mild head injury. In this study we used hypercapnia to follow changes in cerebral vascular reactivity after repetitive mild head injury. We hypothesized head injury would reduce vascular reactivity. Rats were maintained on a reverse light-dark cycle and head impacted daily at 24 h intervals over three days. All head impacts were delivered while rats were fully awake under red light illumination. There was no neuroradiological evidence of brain damage. After the 3rd impact rats were exposed to 5% CO2 and imaged for changes in BOLD signal. All imaging was done while rats were awake without the confound of anesthesia. The data were registered to a 3D MRI rat atlas with 171 segmented brain areas providing site specific information on vascular reactivity. The changes in vascular reactivity were not uniform across the brain. The prefrontal cortex, somatosensory cortex and basal ganglia showed the hypothesized decrease in vascular reactivity while the cerebellum, thalamus, brainstem, and olfactory system showed an increase in BOLD signal to hypercapnia.

Association Between Dyscapnia, Ventilatory Variables, and Mortality in Patients With Acute Respiratory Distress Syndrome-A Retrospective Cohort Study.

Background: This study aimed to investigate the associations between dyscapnia, ventilatory variables, and mortality. We hypothesized that the association between mechanical power or ventilatory ratio and survival is mediated by dyscapnia. Methods: Patients with moderate or severe acute respiratory distress syndrome (ARDS), who received mechanical ventilation within the first 48 h after admission to the intensive care unit for at least 48 h, were included in this retrospective single-center study. Values of arterial carbon dioxide (PaCO2) were categorized into "hypercapnia" (PaCO2 ≥ 50 mm Hg), "normocapnia" (PaCO2 36-49 mmHg), and "hypocapnia" (PaCO2 ≤ 35 mm Hg). We used path analyses to assess the associations between ventilatory variables (mechanical power and ventilatory ratio) and mortality, where hypocapnia or hypercapnia were included as mediating variables. Results: Between December 2017 and April 2021, 435 patients were included. While there was a significant association between mechanical power and hypercapnia (BEM = 0.24 [95% CI: 0.15; 0.34], P < .01), there was no significant association between mechanical power or hypercapnia and ICU mortality. The association between mechanical power and intensive care unit (ICU) mortality was fully mediated by hypocapnia (BEM = -0.10 [95% CI: -0.19; 0.00], P = .05; BMO = 0.38 [95% CI: 0.13; 0.63], P < .01). Ventilatory ratio was significantly associated with hypercapnia (B = 0.23 [95% CI: 0.14; 0.32], P < .01). There was no significant association between ventilatory ratio, hypercapnia, and mortality. There was a significant effect of ventilatory ratio on mortality, which was fully mediated by hypocapnia (BEM = -0.14 [95% CI: -0.24; -0.05], P < .01; BMO = 0.37 [95% CI: 0.12; 0.62], P < .01). Conclusion: In mechanically ventilated patients with moderate or severe ARDS, the association between mechanical power and mortality was fully mediated by hypocapnia. Likewise, there was a mediating effect of hypocapnia on the association between ventilatory ratio and ICU mortality. Our results indicate that the debate on dyscapnia and outcome after ARDS should consider the impact of ventilatory variables.

Effective non-invasive ventilation reduces muscle sympathetic nerve activity in patients with stable hypercapnic COPD.

Increased sympathetic drive is of prognostic significance in chronic obstructive pulmonary disease (COPD) but its determinants remain poorly understood. One potential mechanism may be chemoreflex-mediated adrenergic stimulation caused by sustained hypercapnia. This study determined the impact of non-invasive ventilation (NIV) on muscle sympathetic nerve activity (MSNA) in patients with stable hypercapnic COPD. Ten patients (age 70 ± 7 years, GOLD stage 3-4) receiving long-term NIV (mean inspiratory positive airway pressure 21 ± 7 cmH2O) underwent invasive MSNA measurement via the peroneal nerve during spontaneous breathing and NIV. Compared with spontaneous breathing, NIV significantly reduced hypercapnia (PaCO2 51.5 ± 6.9 vs 42.6 ± 6.1 mmHg, p < 0.0001) along with the burst rate (64.4 ± 20.9 vs 59.2 ± 19.9 bursts/min, p = 0.03) and burst incidence (81.7 ± 29.3 vs 74.1 ± 26.9 bursts/100 heartbeats, p = 0.04) of MSNA. This shows for the first time that correcting hypercapnia with NIV decreases MSNA in COPD.

Arterial blood gas analysis or venous blood gas analysis for adult hospitalised patients with respiratory presentations: a systematic review.

BACKGROUND: Identification of hypoxaemia and hypercapnia is essential for the diagnosis and treatment of acute respiratory failure. While arterial blood gas (ABG) analysis is standard for PO2 and PCO2 measurement, venous blood gas (VBG) analysis is increasingly used as an alternative. Previous systematic reviews established that VBG reporting of PO2 and PCO2 is less accurate, but the impacts on clinical management and patient outcomes are unknown. AIMS: This study aimed to systematically review available evidence of the clinical impacts of using ABGs or VBGs and examine the arteriovenous difference in blood gas parameters. METHODS: A comprehensive search of the MEDLINE, Embase and Cochrane Library databases since inception was conducted. Included studies were prospective or cross-sectional studies comparing peripheral ABG to peripheral VBG in adult non-critical care inpatients presenting with respiratory symptoms. RESULTS: Of 15 119 articles screened, 15 were included. No studies were found that examined clinical impacts resulting from using VBG compared to ABG. Included studies focused on the agreement between ABG and VBG measurements of pH, PO2, PCO2 and HCO3 -. Due to the heterogeneity of the included studies, qualitative evidence synthesis was performed. While the arteriovenous difference in pH and HCO3 - was generally predictable, the difference in PO2 and PCO2 was more significant and less predictable. CONCLUSIONS: Our study reinforces the notion that VBG is not comparable to ABG for physiological measurements. However, a key revelation from our research is the significant lack of data regarding the clinical implications of using VBG instead of ABG, a common scenario in clinical practice. This highlights a critical knowledge gap.

Nocturnal urination is associated with the presence of higher ventilatory chemosensitivity in patients with obstructive sleep apnea.

PURPOSE: Chemosensitivity is an essential part of the pathophysiological mechanisms of obstructive sleep apnea (OSA). This study aims to use the rebreathing method to assess hypercapnic ventilatory response (HCVR) and analyze the association between chemosensitivity and certain symptoms in patients with OSA. METHODS: A total of 104 male patients with diagnosed OSA were enrolled. The HCVR was assessed using rebreathing methods under hypoxia exposure to reflect the overall chemosensitivity. Univariate and multivariate linear regression were used to explore the association with chemosensitivity. Participants were enrolled in the cluster analysis using certain symptoms, basic characteristics, and polysomnographic indices. RESULTS: At similar baseline values, the high chemosensitivity group (n = 39) demonstrated more severe levels of OSA and nocturnal hypoxia than the low chemosensitivity group (n = 65). After screening the possible associated factors, nocturnal urination, rather than OSA severity, was found to be positively associated with the level of chemosensitivity. Cluster analysis revealed three distinct groups: Cluster 1 (n = 32, 34.0%) held younger, obese individuals with nocturnal urination, elevated chemosensitivity level, and very severe OSA; Cluster 2 (41, 43.6%) included middle-aged overweighted patients with nocturnal urination, increased chemosensitivity level, but moderate-severe OSA; and Cluster 3 (n = 21, 22.3%) contained middle-aged overweighted patients without nocturnal urination, with a lowered chemosensitivity level and only moderate OSA. CONCLUSION: The presence of nocturnal urination in male patients with OSA may be a sign of higher levels of ventilatory chemosensitivity, requiring early therapy efforts independent of AHI levels.

Hypercapnia and lung function parameters in chronic obstructive pulmonary disease.

BACKGROUND: In advanced chronic obstructive pulmonary disease (COPD), hypercapnia may occur due to severe bronchial obstruction with lung hyperinflation. Non-invasive ventilation (NIV) provides the standard of care intended to achieve physiological PCO2 levels, thereby reducing overall mortality. The present study aimed to evaluate pulmonary function parameters derived from spirometry (forced vital capacity [FVC], forced expiratory volume in 1 s [FEV1]), body plethysmography (residual volume [RV], total lung capacity [TLC]), and lung diffusion capacity for carbon monoxide (single-breath method [DCO-SB], alveolar-volume corrected values [DCO-VA]) as predictors of chronic hypercapnia in patients with advanced COPD. METHODS: This monocentric, retrospective observational study included 423 COPD patients. Receiver operating characteristic (ROC) curve analysis and cross-validation were used to assess lung function parameters' diagnostic accuracy for predicting chronic hypercapnia, with the resulting performance expressed as area under the ROC curve (AUROC). We performed univariable and multivariable binary logistic regression analysis to determine if these parameters were independently associated with chronic hypercapnia, with probabilities reported as odds ratios [OR] with 95% confidence intervals [95%CI]. RESULTS: FVC% (AUROC 0.77 [95%CI 0.72-0.81], P < 0.01) and FEV1% (AURIC 0.75 [95%CI 0.70-0.79], P < 0.01) exhibited reasonable accuracy in the prediction of chronic hypercapnia, whereas lung diffusion capacity performed poorly (AUROC 0.64 [95%CI 0.58-0.71] for DCO-SB%, P < 0.01). FVC% (OR 0.95 [95%CI 0.93-0.97], P < 0.01) and FEV1% (OR 0.97 [95%CI 0.94-0.99], P = 0.029) were the only parameters associated independently with chronic hypercapnia in logistic regression analysis. FVC and FEV1 thresholds that best separated hypercapnic from normocapnic subjects reached 56% and 33% of predicted values. CONCLUSIONS: Routinely collected pulmonary function parameters, particularly FVC% and FEV1%, may predict chronic hypercapnia during COPD progression.

Early fMRI responses to somatosensory and optogenetic stimulation reflect neural information flow.

Blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) has been widely used to localize brain functions. To further advance understanding of brain functions, it is critical to understand the direction of information flow, such as thalamocortical versus corticothalamic projections. For this work, we performed ultrahigh spatiotemporal resolution fMRI at 15.2 T of the mouse somatosensory network during forepaw somatosensory stimulation and optogenetic stimulation of the primary motor cortex (M1). Somatosensory stimulation induced the earliest BOLD response in the ventral posterolateral nucleus (VPL), followed by the primary somatosensory cortex (S1) and then M1 and posterior thalamic nucleus. Optogenetic stimulation of excitatory neurons in M1 induced the earliest BOLD response in M1, followed by S1 and then VPL. Within S1, the middle cortical layers responded to somatosensory stimulation earlier than the upper or lower layers, whereas the upper cortical layers responded earlier than the other two layers to optogenetic stimulation in M1. The order of early BOLD responses was consistent with the canonical understanding of somatosensory network connections and cannot be explained by regional variabilities in the hemodynamic response functions measured using hypercapnic stimulation. Our data demonstrate that early BOLD responses reflect the information flow in the mouse somatosensory network, suggesting that high-field fMRI can be used for systems-level network analyses.

Impact of Serotonergic 5HT1A and 5HT2A Receptor Activation on the Respiratory Response to Hypercapnia in a Rat Model of Parkinson's Disease.

In Parkinson's disease (PD), along with typical motor dysfunction, abnormal breathing is present; the cause of which is not well understood. The study aimed to analyze the effects of stimulation of the serotonergic system with 5-HT1A and 5-HT2A agonists in a model of PD induced by injection of 6-hydroxydopamine (6-OHDA). To model PD, bilateral injection of 6-OHDA into both striata was performed in male Wistar rats. Respiratory disturbances in response to 7% hypercapnia (CO2 in O2) in the plethysmographic chamber before and after stimulation of the serotonergic system and the incidence of apnea were studied in awake rats 5 weeks after 6-OHDA or vehicle injection. Administration of 6-OHDA reduced the concentration of serotonin (5-HT), dopamine (DA) and norepinephrine (NA) in the striatum and the level of 5-HT in the brainstem of treated rats, which have been associated with decreased basal ventilation, impaired respiratory response to 7% CO2 and increased incidence of apnea compared to Sham-operated rats. Intraperitoneal (i.p.) injection of the 5-HT1AR agonist 8-OH-DPAT and 5-HT2AR agonist NBOH-2C-CN increased breathing during normocapnia and hypercapnia in both groups of rats. However, it restored reactivity to hypercapnia in 6-OHDA group to the level present in Sham rats. Another 5-HT2AR agonist TCB-2 was only effective in increasing normocapnic ventilation in 6-OHDA rats. Both the serotonergic agonists 8-OH-DPAT and NBOH-2C-CN had stronger stimulatory effects on respiration in PD rats, compensating for deficits in basal ventilation and hypercapnic respiration. We conclude that serotonergic stimulation may have a positive effect on respiratory impairments that occur in PD.

A5 noradrenergic neurons and breathing control in neonate rats.

The pontine A5 noradrenergic group contributes to the maturation of the respiratory system before birth in rats. These neurons are connected to the neural network responsible for respiratory rhythmogenesis. In the present study, we investigated the participation of A5 noradrenergic neurons in neonates (P7-8 and P14-15) in the control of ventilation during hypoxia and hypercapnia in in vivo experiments using conjugated saporin anti-dopamine beta-hydroxylase (DßH-SAP) to specifically ablate noradrenergic neurons. Thus, DßH-SAP (420 ng/µL) or saporin (SAP, control) was injected into the A5 region of neonatal male Wistar rats. Hypoxia reduced respiratory variability in control animals; however, A5 lesion prevented this effect in P7-8 rats. Our data suggest that noradrenergic neurons of the A5 region in neonate rats do not participate in the control of ventilation under baseline and hypercapnic conditions, but exert an inhibitory modulation on breathing variability under hypoxic challenge in early life (P7-8).

Carotid body chemosensitivity is not attenuated during cold water diving.

Tonic carotid body (CB) activity is reduced during exposure to cold and hyperoxia. We tested the hypotheses that cold water diving lowers CB chemosensitivity and augments CO2 retention more than thermoneutral diving. Thirteen subjects [age: 26 ± 4 yr; body mass index (BMI): 26 ± 2 kg/m2) completed two 4-h head-out water immersion protocols in a hyperbaric chamber (1.6 ATA) in cold (15°C) and thermoneutral (25°C) water. CB chemosensitivity was assessed with brief hypercapnic ventilatory response ([Formula: see text]) and hypoxic ventilatory response ([Formula: see text]) tests before dive, 80 and 160 min into the dive (D80 and D160, respectively), and immediately after and 60 min after dive. Data are reported as an absolute mean (SD) change from predive. End-tidal CO2 pressure increased during both the thermoneutral water dive [D160: +2 (3) mmHg; P = 0.02] and the cold water dive [D160: +1 (2) mmHg; P = 0.03]. Ventilation increased during the cold water dive [D80: 4.13 (4.38) and D160: 7.75 (5.23) L·min-1; both P < 0.01] and was greater than the thermoneutral water dive at both time points (both P < 0.01). [Formula: see text] was unchanged during the dive (P = 0.24) and was not different between conditions (P = 0.23). [Formula: see text] decreased during the thermoneutral water dive [D80: -3.45 (3.61) and D160: -2.76 (4.04) L·min·mmHg-1; P < 0.01 and P = 0.03, respectively] but not the cold water dive. However, [Formula: see text] was not different between conditions (P = 0.17). In conclusion, CB chemosensitivity was not attenuated during the cold stress diving condition and does not appear to contribute to changes in ventilation or CO2 retention.

Orexin contributes to eupnea within a critical period of postnatal development.

Orexin neurons are active in wakefulness and mostly silent in sleep. In adult rats and humans, orexin facilitates the hypercapnic ventilatory response but has little effect on resting ventilation. The influence of orexin on breathing in the early postnatal period, and across states of vigilance, have not been investigated. This is relevant as the orexin system may be impaired in Sudden Infant Death Syndrome (SIDS) cases. We addressed three hypotheses: 1) orexin provides a drive to breathe in infancy; 2) the effect of orexin depends on stage of postnatal development; and 3) orexin has a greater influence on breathing in wakefulness compared with sleep. Whole body plethysmography was used to monitor breathing of infant rats at three ages: postnatal days (P) 7-8, 12-14, and 17-19. Respiratory variables were analyzed in wakefulness (W), quiet sleep (QS), and active sleep (AS), following suvorexant (5 mg/kg ip), a dual orexin receptor antagonist, or vehicle (DMSO). Effects of suvorexant on ventilatory responses to graded hypercapnia ([Formula: see text] = 0.02, 0.04, 0.06), hypoxia ([Formula: see text] = 0.10), and hyperoxia ([Formula: see text] = 1.0) at P12-14 were also tested. At P12-14, but not at other ages, suvorexant significantly reduced respiratory frequency in all states, reduced the ventilatory equivalent in QW and QS, and increased [Formula: see text] to ∼5 mmHg. Suvorexant had no effect on ventilatory responses to graded hypercapnia or hypoxia. Hyperoxia eliminated the effects of suvorexant on respiratory frequency at P12-14. Our data suggest that orexin preserves eupneic frequency and ventilation in rats, specifically at ∼2 wk of age, perhaps by facilitating tonic peripheral chemoreflex activity.

Quantitative myocardial first-pass perfusion imaging of CO2 -induced vasodilation in rats.

Inducible hypercapnia is an alternative for increasing the coronary blood flow necessary to facilitate the quantification of myocardial blood flow during hyperemia. The current study aimed to quantify the pharmacokinetic effect of a CO2 gas challenge on myocardial perfusion in rats using high-resolution, first-pass perfusion CMR and compared it with pharmacologically induced hyperemia using regadenoson. A dual-contrast, saturation-recovery, gradient-echo sequence with a Cartesian readout was used on a small-animal 9.4-T scanner; additional cine images during hyperemia/rest were recorded with an ultrashort echo time sequence. The mean myocardial blood flow value at rest was 6.1 ± 1.4 versus 13.9 ± 3.7 and 14.3 ± 4 mL/g/min during vasodilation with hypercapnia and regadenoson, respectively. Accordingly, the myocardial flow reserve value was 2.6 ± 1.1 for the gas challenge and 2.5 ± 1.4 for regadenoson. During hyperemia with both protocols, a significantly increased cardiac output was found. It was concluded that hypercapnia leads to significantly increased coronary flow and yields similar myocardial flow reserves in healthy rats as compared with pharmacological stimulation. Accordingly, inducible hypercapnia can be selected as an alternative stressor in CMR studies of myocardial blood flow in small animals.