Central Respiratory Control Research Articles

Expression of nischarin, an imidazoline 1 receptor candidate protein, in the ventrolateral medulla of newborn rats

The activation of imidazoline 1 (I1) receptors is suggested to stimulate the respiratory drive in newborn rats. Here, we immunohistochemically examined whether nischarin, an I1 receptor candidate protein, is expressed in the ventrolateral medulla, where cardiorespiratory centers are located. Newborn rats (age, 3–5 days) were deeply anesthetized with isoflurane; the brainstem was dissected, sectioned sagittally, and labeled with nischarin. Nischarin-associated signals were observed broadly throughout the newborn rat brainstem, including at motor nuclei (motor trigeminal nucleus and facial nucleus), sensory nuclei (lateral superior olive, medial and spinal vestibular nuclei, cuneate nucleus, spinal trigeminal nucleus, and solitary nucleus), and the rostral and caudal ventrolateral medullar regions. In particular, the rostral ventrolateral medulla included a layer of aggregated nischarin expression along the ventral surface, and the layer was in close contact with GFAP-positive processes. In addition, some Phox2b-positive neurons were positive for nischarin in the region. Our results reveal nischarin expression in the newborn rat brainstem and suggest that I1 receptor activation at the level of the ventrolateral medulla contributes to central chemoreception and respiratory control in newborn rats.

Slower dispositional respiration is associated with lower pain sensitivity, higher mindfulness and multiple unique neural mechanisms

The relationship between non-volitionally controlled breathing and pain remains unknown. The psychological mechanisms supporting this relationship are also poorly characterized. Anxiety and dispositional mindfulness, two antithetical constructs, are postulated to mediate the relationship between pain and respiration. Anxiety increases breathing and pain. In contrast, dispositional mindfulness is associated with lower anxiety and pain. Importantly, the neural correlates of respiration rate have not been identified. Seventy-four pain-free participants were administered the Five Faceted Mindfulness Questionnaire (FFMQ) and the Spielberger State Anxiety Inventory (SAI) before scanning. Two innocuous (35°C) and noxious (49°C; right calf) stimulation series during pseudo-continuous arterial spin labeling functional MRI and continuous respiratory were then acquired. Subjects were instructed to “remain still and close their eyes” and visual analogue scale (0 = “no pain” – 10 = “worst pain imaginable”) pain intensity ratings were collected after each series. Slower respiration was associated with lower pain intensity ratings during noxious (r = .26, p = .03) and innocuous (r = .28, p = .02) stimulation. Higher mindfulness scores were associated with slower respiration (r = -.28, p = .02) and lower pain (r = -.25, p = .03). State anxiety did not significantly co-vary with pain or respiration (ps > .18). When compared to 35°C, 49°C stimulation produced significant activation in the primary somatosensory cortex corresponding to the stimulation site, bilateral thalamus, secondary somatosensory cortex, anterior/posterior insula, and dorsal anterior cingulate cortex (dACC). Slower respiration was associated with greater activation in the subcallosal cortex, subgenual ACC, right-lateralized dorsolateral prefrontal cortex (dlPFC), putamen, anterior insula, and left caudate. Faster breathing was associated with greater activation in respiratory control centers within the pons, medulla, and cerebellum. These findings suggest that individuals who naturally breathe slower report lower pain and engage multiple unique executive-level processes supporting psychological resilience and self-regulation of affect. This work was supported by the National Center for Complementary and Integrative Health (R21-AT007247, F32-AT006949, K99/R00-AT008238, R01-AT009693; F32 AT010843) and the Mind and Life Institute's Francisco J. Varela Award.

Kv1.1 subunits localize to cardiorespiratory brain networks in mice where their absence induces astrogliosis and microgliosis

Cardiorespiratory collapse following a seizure is a suspected cause of sudden unexpected death in epilepsy (SUDEP), the leading cause of epilepsy-related mortality. In the commonly used Kcna1 gene knockout (Kcna1−/−) mouse model of SUDEP, cardiorespiratory profiling reveals an array of aberrant breathing patterns that could contribute to risk of seizure-related mortality. However, the brain structures mediating these respiratory abnormalities remain unknown. We hypothesize that Kv1.1 deficiency in respiratory control centers of the brain contribute to respiratory dysfunction in Kcna1−/− mice leading to increased SUDEP risk. Thus, in this study, we first used immunohistochemistry to map expression of Kv1.1 protein in cardiorespiratory brain regions of wild-type Kcna1+/+ (WT) mice. Next, GFAP and Iba1 immunostaining was used to test for the presence of astrogliosis and microgliosis, respectively, in the cardiorespiratory centers of Kcna1−/− mice, which could be indicative of seizure-related brain injury that could impair breathing. In WT mice, we detected Kv1.1 protein in all cardiorespiratory centers examined, including the basolateral amygdala, dorsal respiratory group, dorsal motor nucleus of vagus, nucleus ambiguus, ventral respiratory column, and pontine respiratory group, as well as chemosensory centers including the retrotrapezoid and median raphae nuclei. Extensive gliosis was observed in the same areas in Kcna1−/− mice suggesting that seizure-associated brain injury could contribute to respiratory abnormalities.

Supplemental Learning in Respiratory Physiology for Healthcare Professionals Towards Successful Treatment of COVID-19.

The immunological and pathophysiological response to COVID-19 can cause severe respiratory impairment affecting gas exchange and lung mechanics. Such was the scale of the respiratory support needed during the first wave of the pandemic, that recruitment of non-respiratory clinical staff was essential to help deal with the growing number of cases. It quickly became apparent that it was vital to rapidly equip these healthcare professionals with appropriate physiological knowledge and practical skills if therapies were to be applied effectively. Furthermore, the unravelling of unusual clinical features of COVID-19, further highlighted a need for knowledge of long-established principles of respiratory physiology. An online digital educational resource, or “respiratory learning tool kit” was developed with interactive material including visualisations, animations, and pathophysiological examples to facilitate understanding. The learning outcomes were centred on physiological principles, essential for understanding the pathophysiology relating to COVID-19, and management and treatment. Topics included principles of gas exchange, gas transport, homeostasis and central control of respiration. These basic physiological principles were linked to pathophysiology and clinical skills around oxygen administration and non-invasive supports such as Continuous Positive Airway Pressure (CPAP). From the degree of engagement and evaluation comments, it was clear that the resource successfully achieved its aim—to increase physiological knowledge and its practical understanding, enabling healthcare professionals to practice with confidence in such an uncertain environment.

Murine-β-coronavirus-induced neuropathogenesis sheds light on CNS pathobiology of SARS-CoV2.

The pandemic caused by SARS-CoV-2 has caused widespread infection and significant mortality across the globe. Combined virology perspective of SARS-CoV-2 with a deep-rooted understanding of pathophysiological and immunological processes underlying the clinical manifestations of COVID-19 is of prime importance. The characteristic symptom of COVID-19 is respiratory distress with diffused alveolar damage, but emerging evidence suggests COVID-19 might also have neurologic consequences. Dysregulated homeostasis in the lungs has proven to be fatal, but one cannot ignore that the inability to breathe might be due to defects in the respiratory control center of the brainstem. While the mechanism of pulmonary distress has been documented in the literature, awareness of neurological features and their pathophysiology is still in the nascent state. This review makes references to the neuro-immune axis and neuro-invasive potential of SARS-CoV and SARS-CoV2, as well as the prototypic H-CoV strains in human brains. Simultaneously, considerable discussion on relevant experimental evidence of mild to severe neurological manifestations of fellow neurotropic murine-β-CoVs (m-CoVs) in the mouse model will help understand the underpinning mechanisms of Neuro-COVID. In this review, we have highlighted the neuroimmunopathological processes in murine CoVs. While MHV infection in mice and SARS-CoV-2 infection in humans share numerous parallels, there are critical differences in viral recognition and viral entry. These similarities are highlighted in this review, while differences have also been emphasized. Though CoV-2 Spike does not favorably interact with murine ACE2 receptor, modification of murine SARS-CoV2 binding domain or development of transgenic ACE-2 knock-in mice might help in mediating consequential infection and understanding human CoV2 pathogenesis in murine models. While a global animal model that can replicate all aspects of the human disease remains elusive, prior insights and further experiments with fellow m-β-CoV-induced cause-effect experimental models and current human COVID-19 patients data may help to mitigate the SARS-CoV-2-induced multifactorial multi-organ failure

Medullary Astrocytes Mediate Irregular Breathing Patterns Generation in Chronic Heart Failure Through Purinergic P2X7 Receptor Signalling

Background: Breathing disorders (BD) are highly prevalent in chronic heart failure (CHF) and are associated with altered central respiratory control. Ample evidence identifies the retrotrapezoid nucleus (RTN) as an important chemosensitivity region for ventilatory control and generation of BD in CHF, however little is known about the cellular mechanisms underlying the RTN/BD relationship. Within the RTN, astrocyte‐mediated purinergic signalling modulates respiration, but the potential contribution of RTN astrocytes to BD in CHF has not been explored. Methods: Selective neuron and/or astrocyte-targeted interventions using either optogenetic and chemogenetic manipulations in the RTN of CHF rats were used to unveil the contribution of the RTN on the development/maintenance of BD, the role played by astrocytes in BD and the molecular mechanism underpinning these alterations. Findings: We showed that episodic photo-stimulation of RTN neurons triggered BD in healthy rats, and that RTN neurons ablation in CHF animals eliminates BD. Also, we found a reduction in astrocytes activity and ATP bioavailability within the RTN of CHF rats, and that chemogenetic restoration of normal RTN astrocyte activity and ATP levels improved breathing regularity in CHF. Importantly, P2X7 receptor (P2X7r) expression was reduced in RTN astrocytes from CHF rats and viral vector-mediated delivery of human P2X7r into astrocytes increases ATP bioavailability and abolished BD. Interpretation: Our results support that RTN astrocytes play a pivotal role on BD generation and maintenance in the setting CHF by a mechanism encompassing P2X7r signalling. Funding: This study was funded in part by the National Research and Development Agency of Chile and the Basal Centre of Excellence in Aging and Regeneration Declaration of Interest: None to declare. Ethical Approval: All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication eighth edition, update 2011). All experimental protocols were approved by the IACUC from the Pontificia Universidad Catolica de Chile (protocol ID #170710022).

Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19.

The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease. Moreover, thromboembolic events throughout the body, including in the CNS, have been described. Given the neurological symptoms observed in a large majority of individuals with COVID-19, SARS-CoV-2 penetrance of the CNS is likely. By various means, we demonstrate the presence of SARS-CoV-2 RNA and protein in anatomically distinct regions of the nasopharynx and brain. Furthermore, we describe the morphological changes associated with infection such as thromboembolic ischemic infarction of the CNS and present evidence of SARS-CoV-2 neurotropism. SARS-CoV-2 can enter the nervous system by crossing the neural-mucosal interface in olfactory mucosa, exploiting the close vicinity of olfactory mucosal, endothelial and nervous tissue, including delicate olfactory and sensory nerve endings. Subsequently, SARS-CoV-2 appears to follow neuroanatomical structures, penetrating defined neuroanatomical areas including the primary respiratory and cardiovascular control center in the medulla oblongata.

Dyspnea and Exercise Limitation in Mild COPD: The Value of CPET

The majority of smokers with chronic obstructive pulmonary disease (COPD) have mild airflow limitation as determined by simple spirometry. Although small airway dysfunction is the hallmark of COPD, many studies attest to complex heterogeneous physiological impairments beyond increased airway resistance. These impairments are related to inflammation of lung parenchyma and its microvasculature, which is obscured by simple spirometry. Recent studies using advanced radiological imaging have highlighted significant structural abnormalities in smokers with relatively preserved spirometry. These important studies have generated considerable interest and have reinforced the pressing need to better understand the physiological consequences of various morphological abnormalities, and their impact on the clinical outcomes and natural history of COPD. The overarching objective of this review is to provide a concise overview of the importance and utility of cardiopulmonary exercise testing (CPET) in clinical and research settings. CPET uniquely allows evaluation of integrated abnormalities of the respiratory, cardio-circulatory, metabolic, peripheral muscle and neurosensory systems during increases in physiologic stress. This brief review examines the results of recent studies in mild COPD that have uncovered consistent derangements in pulmonary gas exchange and development of “restrictive” dynamic mechanics that together contribute to exercise intolerance. We examine the evidence that compensatory increases in inspiratory neural drive from respiratory control centers are required during exercise in mild COPD to maintain ventilation commensurate with increasing metabolic demand. The ultimate clinical consequences of this high inspiratory neural drive are earlier onset of critical respiratory mechanical constraints and increased perceived respiratory discomfort at relatively low exercise intensities.

Volatile Anesthetics Activate a Leak Sodium Conductance in Retrotrapezoid Nucleus Neurons to Maintain Breathing during Anesthesia in Mice.

Volatile anesthetics moderately depress respiratory function at clinically relevant concentrations. Phox2b-expressing chemosensitive neurons in the retrotrapezoid nucleus, a respiratory control center, are activated by isoflurane, but the underlying mechanisms remain unclear. The hypothesis of this study was that the sodium leak channel contributes to the volatile anesthetics-induced modulation of retrotrapezoid nucleus neurons and to respiratory output. The contribution of sodium leak channels to isoflurane-, sevoflurane-, and propofol-evoked activity of Phox2b-expressing retrotrapezoid nucleus neurons and respiratory output were evaluated in wild-type and genetically modified mice lacking sodium leak channels (both sexes). Patch-clamp recordings were performed in acute brain slices. Whole-body plethysmography was used to measure the respiratory activity. Isoflurane at 0.42 to 0.50 mM (~1.5 minimum alveolar concentration) increased the sodium leak channel-mediated holding currents and conductance from -75.0 ± 12.9 to -130.1 ± 34.9 pA (mean ± SD, P = 0.002, n = 6) and 1.8 ± 0.5 to 3.6 ± 1.0 nS (P = 0.001, n = 6), respectively. At these concentrations, isoflurane increased activity of Phox2b-expressing retrotrapezoid nucleus neurons from 1.1 ± 0.2 to 2.8 ± 0.2 Hz (P < 0.001, n = 5), which was eliminated by bath application of gadolinium or genetic silencing of sodium leak channel. Genetic silencing of sodium leak channel in the retrotrapezoid nucleus resulted in a diminished ventilatory response to carbon dioxide in mice under control conditions and during isoflurane anesthesia. Sevoflurane produced an effect comparable to that of isoflurane, whereas propofol did not activate sodium leak channel-mediated holding conductance. Isoflurane and sevoflurane increase neuronal excitability of chemosensitive retrotrapezoid nucleus neurons partly by enhancing sodium leak channel conductance. Sodium leak channel expression in the retrotrapezoid nucleus is required for the ventilatory response to carbon dioxide during anesthesia by isoflurane and sevoflurane, thus identifying sodium leak channel as a requisite determinant of respiratory output during anesthesia of volatile anesthetics.

An Algorithm to Predict Life-Threatening Complications using Heart Rate Variability and the Circadian Heart Rate Difference with a Special Interest on Covid-19, Sudep Children with Congenital Heart Disease and Obesity

Corona virus disease (COVID-19) has been declared as a pandemic by the WHO with a global mortality rate of about 3.4%. More recently the neuroinvasive potential of SARS-CoV2 was emphasized as a potential cause for respiratory failure. Such pathophysiology has been investigated in sudden unexplained death in epilepsy (SUDEP) including functional neuroimaging that demonstrates alterations to networks involved in central autonomic and respiratory control located in the brainstem. For risk stratification in these patients, one method may be heart rate and heart rate variability (HRV) monitoring. Method: For a better understanding, we compare HRV monitoring in two cases; 1.) Twenty Holter ECGs of a boy with generalized tonic-clonic seizures up to his dead at the age of 10.5 years with special interest on an acute respiratory failure at the age of 5.4 years. 2.) Thirty one Holter ECGs of a 58-year old pediatric cardiologist who survived an infection with COVID-19. During his disease 24-hour Holter electrocardiography (ECG) was performed continuously over 10 days. Moreover, 24-hour Holter ECGs from the last 10 years were available. The derived algorithm that depends on the global heart rate variability and circadian heart rate difference was proofed in 151 healthy children, 26 children with a fatal outcome or transplantation, 151 patients with operated congenital heart disease, 130 obese children and healthy adult data from literature. Results: In both cases we observe a decline of the global heart rate variability SDNN together with a loss of the circadian heart rate difference. The derived algorithm differentiate healthy children from children with a fatal outcome. The algorithm identify 7.3% of 151 patients with operated congenital heart disease and 5.4% of children with obesity as candidates for COVID-19 complications. Conclusions: A sudden decline of HRV together with a loss of the circadian heart rate difference may indicate a life-threatening complication in critical illness.

1237 Positional central sleep apnea in a child: not always obstructive

Abstract Introduction Spinal cord compression at the craniocervical junction can cause central sleep apnea (CSA) in children, due to mechanical disruption of respiratory control centers. Polysomnography (PSG) pre/post surgical decompression is indicated to evaluate treatment response. Report of Case A 5-year-old with Wolf-Hirschhorn syndrome, developmental delay, partial agenesis of the corpus callosum, hypotonia, incomplete segmentation of C1 with subluxation leading to stenosis, cleft palate, and obstructive sleep apnea (OSA) presented for post-surgical PSG. Prior PSGs revealed oAHI 55/hr and 7/hr, cAHI 3/hr and 2/hr at 1 and 10 months of age, respectively. At 46 months, patient underwent suboccipital decompression at C1 for severe craniocervical stenosis. Post-operative PSG revealed emergence of CSA with cAHI 8/hr and stability of OSA with an oAHI 8/hr. Positional analysis revealed worsening in lateral position (lateral cAHI 10.6/hr, supine cAHI 4.6/hr). Central events were up to 33 seconds long with nadir desaturation of 76% in lateral position compared to 20 seconds and nadir 88% in supine position. Head CT and MRI showed incomplete ossification of the C1 vertebra with subluxation of the left lateral mass leading to absence of CSF flow at the craniocervical junction. Repeat decompression and fixation is planned by neurosurgery; in the interim supine sleep was recommended and family declined BiPAP. Conclusion In our case, CSA worsened with left lateral positioning and improved with supine positioning. Lateral positioning likely increased subluxation of the left lateral mass seen on CT/MRI, and eased subluxation in supine sleep causing position-dependent dynamic impingement of the respiratory control centers at the level of subluxation. The absence of findings prior to decompression may reflect increased instability after decompression or increased stenosis with growth. Our case highlights the importance of positional analysis for central as well as the more conventional obstructive sleep apnea to understand pathogenesis and guide therapy.

Motor Neuron Pathology Leads to Respiratory Deficiency in Optn−/− Mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease with no current cure. Respiratory failure is the leading cause of death in ALS. Death occurs3–5 years after diagnosis when patients with ALS ultimately succumb to inadequate ventilation, hypoxia, and respiratory failure. Several genes are associated with ALS. One of these genes encodes optineurin (OPTN) which is associated with neurodegeneration in both ALS and glaucoma. Optineurin has multiple roles in various biochemical pathways such as regulation of inflammation and autophagy. However, the exact mechanism by which loss of OPTN results in progressive neural degeneration and respiratory failure in ALS is still unclear.In order to understand the impact of OPTN deficiency on respiratory function, our goal was to study the respiratory pathophysiology in the Optn−/− mouse. The hypothesis driving this work is that the Optn−/− mouse has respiratory insufficiency due to degeneration of the respiratory motor unit – motor neuron, nerve, neuromuscular junction and muscle. Physiological, histological and molecular outcome measures were used to assess the impact of OPTN on the respiratory system. We used whole body plethysmography (WBP) to assess breathing at baseline and during a respiratory challenge with hypercapnic and hypoxic conditions (FiCO2: 0.07, FiO2: 0.10; nitrogen balance). During the respiratory challenge, compared to the WT mice, Optn−/− mice had significantly lower tidal volume, minute ventilation, peak inspiratory flow, and peak expiratory flow starting at 6 months of age indicating weakened muscle strength. Furthermore, throughout the challenge period Optn−/− mice spent a greater amount of time in apnea indicating pathology in the respiratory control centers. The weakness and pathology in control of breathing during the respiratory challenge progresses as the mice age. At 1 year of age, Optn−/− mice have stunted growth accompanied by reduced diaphragm size compared to WT mice. Postmortem immunohistochemical studies of the medulla reveal Optn−/− mice have fewer hypoglossal motor neurons. Finally, the hypoglossal (XII) nerves of Optn−/− mice had significantly reduced g‐ratio which indicates pathology and decompaction of myelin sheaths. Within the XII nerve, there are an increase in both the number of mitochondria and area of mitochondria. These along with elevated levels of LC3‐II to LC3‐I indicate reduced mitophagy. In conclusion, the Optn−/− ALS mouse model displays motor neuron pathology, increased nerve demyelination and aberrant control of breathing leading to respiratory insufficiency.

Automated Detection of Onset and Offset of Multiunit Inspiratory Burst Activity Using a Threshold and Window Independent Approach

In animal models, inspiratory electromyogram (EMG) or neurogram (ENG) multiunit activity recorded with various electrode configurations are commonly used for assessing multiple aspects of central respiratory network control. The cyclic inspiratory electrogram ‘bursts’ are complex, and generally characterized by an augmenting fast‐oscillatory amplitude pattern that begins to decrement after peak activity. Accurate automated capture of the bounds of these bursts in time would allow for in‐depth analysis of intra‐burst features, but is difficult to achieve due to high stochasticity of the underlying network interactions, particularly at the decrementing phase of the burst. Common approaches to estimate these bounds often use threshold and/or window‐based smoothing or binning methods that minimize signal variance around the overall trend. These approaches rely on conservation of burst‐to‐burst characteristics to maintain accuracy and consistency; however, features underlying the onset and/or offset of the burst may be obscured and not be adequately captured. To overcome this limitation, we present an unbiased method for automated estimation of burst bounds that is not dependent on thresholding or windowing methods, and assess the capabilities of this method on rectified diaphragm EMG and phrenic ENG time‐series bursts recorded from urethane anesthetized Sprague‐Dawley rats. For our method, each burst is initially identified and approached using an arbitrary time point between adjacent bursts that contains the burst onset and extends toward the center of the burst. A multidimensional time‐invariant distribution is then achieved using time‐delay embedding of all amplitude values that occur at time‐series slope inversions. The variability and magnitude of the distribution are grouped using spectral clustering to identify potential timings for transitions from non‐burst to burst activity, and the maximum second differential of a running variance is identified as the most likely transition and established as the burst onset. The entire process is then repeated from an arbitrary point containing the burst offset to similarly identify the most likely offset transition, after which the next burst is identified and evaluated. This process is then repeated for each burst in the time‐series. The algorithm was evaluated by comparing automated and manual selection of burst bounds in both phrenic nerve ENG and diaphragm EMG recordings. Preliminary comparisons show a mean difference of inspiratory burst onset: 9.0±6.2 ms, offset: 16.6±8.7 ms, and duration: 24.2±11.8 ms in phrenic ENG, and a mean difference of inspiratory burst onset: 7.2±2.8 ms, offset: 36.4±7.3 ms, and duration 37.4±5.8 ms in diaphragm EMG, which is similar to that observed for manual bounds selection between individuals. This algorithm rapidly processes data (approximately 25 bursts/s) and maintains accuracy despite changes in burst amplitude and/or frequency; thus, it presents an efficient and consistent method for high throughput burst identification that may facilitate burst analysis for long duration time series data.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson’s Disease Research at Stony Brook University

Estrus Cycle Stages Differentially Alter Ventilatory Pattern Variability (VPV) in Septic Female Rats

The sex hormones, estrogen and progesterone, affect central respiratory control. Progesterone, increases respiratory frequency (fR). Sepsis, a disease process caused by a dysregulated systemic inflammatory response, also affects respiratory control that can be quantified by Ventilatory Pattern Variability (VPV). We hypothesized that natural hormone fluctuations during the female estrus cycle affect VPV and its differential control during sepsis. The estrus cycle of female Sprague Dawley rats (n = 19) was assessed by collecting, hematoxylin/eosin staining, and identifying vaginal epithelial cells. Proestrus, in which progesterone peaks, was determined by the absence of both leukocytes and cornified multinucleated epithelial cells. Rats were inoculated with fibrin pellets containing 0 (D0) or 100x 10^ 6 (D100) E. coli cells during proestrus. Before and 12 h after inoculation, we recorded the ventilatory pattern in conscious unrestrained rats using whole‐body plethysmography. VPV [coefficient of variation (CV) for the duration of the respiratory cycle (CV‐Ttot), autocorrelation function (AC), mutual information (MI), sample entropy (SampEn), and nonlinear complexity index (NLCI)] was compared across four experimental groups: 1) D0 proestrus rats (n = 3), 2) D100 proestrus rats (n = 7), 3) D0 non‐proestrus rats (n = 4), and 4) D100 non‐proestrus rats (n = 5) using one‐way analysis of variance (ANOVA). In the non‐proestrus groups, SampEn was greater in D0 than D100 (1.31±0.011 vs 1.14±0.002 Nats, p = 0.006), but in proestrus rats, SampEn did not vary with inoculation (D0, 1.26±0.003 vs D100, 1.26±0.004 Nats, NS). However, CV‐Ttot, AC and MI trended toward but did not reach significance given the low n. These preliminary data suggest that a decrease in VPV due to septicemia occurs in nonproestrus stages, whereas did not occur in proestrus.Support or Funding InformationFunded By: VA Research Service (I01BX004197); NIH (U01 EB021960)

Effects of Fluoxetine on Inspiratory Motor Activity and Chemical Control of Breathing in Spontaneously Breathing Urethane‐Anesthetized 6‐OHDA MFB‐lesioned Parkinson’s Disease Rat Model

Breathing abnormalities are a common non‐motor symptom (NMS) of Parkinson’s Disease (PD), and multiple causes have been proposed, including impaired central respiratory control. While the loss of dopaminergic neurons is the primary pathology of PD, it is becoming better appreciated that serotonergic (5‐HT) dysfunction also plays a role in the development of motor and NMS and complications in PD. To this end, recent studies addressing the role of 5‐HT in PD symptoms have revealed that activation or antagonism of specific 5‐HT receptor subtypes may have therapeutic benefit. 5‐HT neurotransmission is also known to play a critical role in control of breathing; thus, respiratory abnormalities in PD may be further complicated by the progressive 5‐HT dysfunction in PD. Ongoing work in our laboratory has been focused on characterizing the respiratory phenotype of PD using 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced preclinical rat models of PD. Here, we evaluate potential contributions of 5‐HT dysfunction by assessing the effects of acute administration of the selective 5‐HT reuptake inhibitor fluoxetine hydrochloride (FLX) on basal inspiratory motor (diaphragm EMG) activity and hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses in spontaneously breathing urethane‐anesthetized adult female rats 2‐weeks after unilateral medial forebrain bundle (MFB) 6‐OHDA or vehicle/control injection. Basal (BL) diaphragm EMG activity was recorded for ≥30 min, the HVR (12% O2, 90s) and HCVR (7% CO2, 5 min) were assessed, acute FLX (10 mg/kg, iv) was injected and allowed 30 min to exert steady‐state effects, and then the HVR and HCVR were re‐assessed. We found that in both vehicle‐ and 6‐OHDA‐injected rats, administration of FLX produced an initial increase in burst frequency (by ~30%) and amplitude (by ~15%) above BL levels within ~60 s, after which burst frequency attenuated to a new steady‐state level of ~10% above BL levels while burst amplitude returned to slightly below BL levels in vehicle‐injected rats, but attained a new steady‐state level of ~10% above BL levels in 6‐OHDA‐injected rats. The effects of FLX on HVR and HCVR frequency behaviors were negligible in both control‐ and 6‐OHDA‐injected rats although a slight attenuation of the HVR frequency increase (by ~5%) was seen. The effects of FLX on HVR and HCVR amplitude behaviors, while underpowered, showed a trend of slightly enhanced of HVR amplitude (by ~5%) and HCVR amplitude (by ~9%) in 6‐OHDA‐injected rats; however, additional experiments are needed to determine the significance of these observations. While these preliminary observations suggest that (compared to control rats) MFB‐lesioned rats exhibit a difference in FLX‐induced effects on basal inspiratory burst amplitude alterations, acute FLX administration may not be able to fully correct chemical control of breathing deficits. We suggest that additional experiments are needed to better identify specific 5‐HT mechanisms that may contribute to chemical control of breathing deficits in this PD rat model.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson’s Disease Research at Stony Brook University

The Respiratory Phenotype of Pompe Disease Mouse Models.

Pompe disease is a glycogen storage disease caused by a deficiency in acid α-glucosidase (GAA), a hydrolase necessary for the degradation of lysosomal glycogen. This deficiency in GAA results in muscle and neuronal glycogen accumulation, which causes respiratory insufficiency. Pompe disease mouse models provide a means of assessing respiratory pathology and are important for pre-clinical studies of novel therapies that aim to treat respiratory dysfunction and improve quality of life. This review aims to compile and summarize existing manuscripts that characterize the respiratory phenotype of Pompe mouse models. Manuscripts included in this review were selected utilizing specific search terms and exclusion criteria. Analysis of these findings demonstrate that Pompe disease mouse models have respiratory physiological defects as well as pathologies in the diaphragm, tongue, higher-order respiratory control centers, phrenic and hypoglossal motor nuclei, phrenic and hypoglossal nerves, neuromuscular junctions, and airway smooth muscle. Overall, the culmination of these pathologies contributes to severe respiratory dysfunction, underscoring the importance of characterizing the respiratory phenotype while developing effective therapies for patients.

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

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

Muscarinic Modulation of Morphologically Identified Glycinergic Neurons in the Mouse PreBötzinger Complex.

The cholinergic system plays an essential role in central respiratory control, but the underlying mechanisms remain elusive. We used whole-cell recordings in brainstem slices from juvenile mice expressing enhanced green fluorescent protein (EGFP) under the control of the glycine transporter type 2 (GlyT2) promoter, to examine muscarinic modulation of morphologically identified glycinergic neurons in the preBötzinger complex (preBötC), an area critical for central inspiratory rhythm generation. Biocytin-filled reconstruction of glycinergic neurons revealed that the majority of them had few primary dendrites and had axons arborized within their own dendritic field. Few glycinergic neurons had axon collaterals extended towards the premotor/motor areas or ran towards the contralateral preBötC, and had more primary dendrites and more compact dendritic trees. Spontaneously active glycinergic neurons fired regular spikes, or less frequently in a “burst-like” pattern at physiological potassium concentration. Muscarine suppressed firing in the majority of regular spiking neurons via M2 receptor activation while enhancing the remaining neurons through M1 receptors. Interestingly, rhythmic bursting was augmented by muscarine in a small group of glycinergic neurons. In contrast to its heterogeneous modulation of glycinergic neuronal excitability, muscarine generally depressed inhibitory and excitatory synaptic inputs onto both glycinergic and non-glycinergic preBötC neurons, with a stronger effect on inhibitory input. Notably, presynaptic muscarinic attenuation of excitatory synaptic input was dependent on M1 receptors in glycinergic neurons and on M2 receptors in non-glycinergic neurons. Additional field potential recordings of excitatory synaptic potentials in the M2 receptor knockout mice indicate that glycinergic and non-glycinergic neurons contribute equally to the general suppression by muscarine of excitatory activity in preBötC circuits. In conclusion, our data show that preBötC glycinergic neurons are morphologically heterogeneous, and differ in the properties of synaptic transmission and muscarinic modulation in comparison to non-glycinergic neurons. The dominant and cell-type-specific muscarinic inhibition of synaptic neurotransmission and spiking may contribute to central respiratory disturbances in high cholinergic states.

Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Despite the prevalence of CO2 retention in human disease, little is known about the adaptive neurobiological effects of chronic hypercapnia. We have recently shown 30-d exposure to increased inspired CO2 (InCO2) leads to a steady-state ventilation that exceeds the level predicted by the sustained acidosis and the acute CO2/H+ chemoreflex, suggesting plasticity within respiratory control centers. Based on data showing brainstem changes in aminergic and inflammatory signaling during carotid body denervation-induced hypercapnia, we hypothesized chronic hypercapnia per se will lead to similar changes. We found that: 1) increased InCO2 increased IL-1β in the medullary raphe (MR), ventral respiratory column, and cuneate nucleus after 24 h, but not after 30 d of hypercapnia; 2) the number of serotonergic and total neurons were reduced within the MR and ventrolateral medulla following 30 d of increased InCO2; 3) markers of tryptophan metabolism were altered following 24 h, but not 30 d of InCO2; and 4) there were few changes in brainstem amine levels following 24 h or 30 d of increased InCO2. We conclude that these changes may contribute to initiating or maintaining respiratory neuroplasticity during chronic hypercapnia but alone do not account for ventilatory acclimatization to chronic increased InCO2.-Burgraff, N. J., Neumueller, S. E., Buchholz, K. J., LeClaire, J., Hodges, M. R., Pan, L., Forster, H. V. Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Centrally and peripherally injected nesfatin-1-evoked respiratory responses

Nesfatin-1, which is an anorexiogenic peptide, plays a crucial role as a neurotransmitter and/or neuromodulator in the central nervous system for cardiovascular control and energy balance etc. It is expressed abundantly in multiple brain nuclei including central respiratory control areas such as nucleus tractus solitarius, nucleus ambiguous, dorsal vagal complex, dorsal motor nucleus of the vagus nerve, and hypothalamus. To date, no previous studies have been found to report nesfatin-1-evoked respiratory effects. Therefore, the present study was designed to investigate the possible impacts of centrally and/or peripherally injected nesfatin-1 on respiratory parameters in either 12h-fasted or fed-ad libitum rats.Intracerebroventricular (ICV) administration of nesfatin-1 provoked significant hyperventilation by increasing tidal volume (TV), respiratory rate (RR) and respiratory minute ventilation (RMV) in both the 12h-fasted and the fed-ad libitum Spraque Dawley rats in dose- and time- dependent manner. Moreover, the hyperventilatory effects of centrally injected nesfatin-1 were more potent in the fed-ad libitum rats. Intravenous injection of nesfatin-1 induced a significant rise in RR and RMV, but not in TV, in the fed-ad libitum rats.In conclusion, these findings plainly report that both centrally and/or peripherally injected nesfatin-1 induces significant hyperventilatory effects in the 12h-fasted and the fed-ad libitum rats. These hyperventilatory effects of nesfatin-1 might show a discrepancy according to the food intake of the rats and the delivery method of the peptide.