Breathing Mice Research Articles

Intravital Longitudinal Three-Photon Imaging of Lymphocyte Migration in the Mouse Spleen.

Abstract For the last 20 years, intravital two-photon (2P) imaging has been a powerful tool to visualize lymphocyte dynamics in mouse lymphoid organs such as bone marrows and lymph nodes. Although spleen is a key lymphoid organ for adaptive immunity against blood-derived antigens, the lymphocyte dynamics in the spleen are poorly understood because intravital 2P imaging of spleen is challenging. Visualizing the white pulp in the spleen where most lymphocytes are located requires the excitation light to propagate through the red pulp which contains a lot of blood, causing high light-scattering. In addition, intravital spleen imaging suffers from motion artifact due to the mouse breathing motion. To overcome these challenges, we applied three-photon (3P) microscopy for spleen imaging, which can reduce light scattering and improve image contrast in deeper regions compared to 2P microscopy. We also designed a new implantable, chronic imaging window for reducing spleen motion in our up-light microscope. We could identify white pulp regions by in vivo labeling of the marginal zone of white pulp. Using 3P microscopy at 1300 nm wavelength, we could visualize EGFP+ lymphocytes in white pulps at approximately 400–500 μm depth below the surface while 2P microscopy at 920 nm could only reach imaging depths of 160–250 μm in the same region. We then determined the temperature of the imaging window that is required for maintaining normal lymphocyte motility during imaging by monitoring the lymphocyte motility at different temperatures. This intravital longitudinal 3P imaging technique has the potential to examine lymphocyte dynamics in blood-derived antigen-induced inflammatory process in the spleen.

Orchiectomy exacerbates sleep-disordered breathing induced by intermittent hypoxia in mice

We tested the hypothesis that low testosterone levels alter the regulation of breathing in mice exposed to intermittent hypoxia (IH). We used orchiectomized (ORX) or control (Sham-operated) mice exposed to normoxia or IH (12 h/day, 10 cycles/h, 6% O2) for 14 days. Breathing was measured by whole-body plethysmography to asses the stability of the breathing pattern (frequency distribution of total cycle time - Ttot) and the frequency and duration of spontaneous and post-sigh apneas (PSA). We characterized sighs as inducing one (S1) or more (S2) apnea and determined the sigh parameters (volume, peak inspiratory and expiratory flows, cycle times) associated with PSA. IH increased the frequency and duration of PSA and the proportion of S1 and S2 sighs. The PSA frequency was mostly related to the sigh expiratory time. The effects of IH on PSA frequency were amplified in ORX-IH mice. Our experiments using ORX support the hypothesis that testosterone is involved in the regulation of breathing in mice following IH.

Inhibitory effect of sulfur dioxide inhalation on Hering-Breuer inflation reflex in mice: role of voltage-gated potassium channels.

Slowly adapting receptors (SARs), vagal mechanosensitive receptors located in the lung, play an important role in regulating the breathing pattern and Hering-Breuer inflation reflex (HBIR). Inhalation of high concentration of sulfur dioxide (SO2), a common environmental and occupational air pollutant, has been shown to selectively block the SAR activity in rabbits, but the mechanism underlying this inhibitory effect remained a mystery. We carried out this study to determine if inhalation of SO2 can inhibit the HBIR and change the eupneic breathing pattern, and to investigate further a possible involvement of voltage-gated K+ channels in the inhibitory effect of SO2 on these vagal reflex-mediated responses. Our results showed 1) inhalation of SO2 (600 ppm; 8 min) consistently abolished both the phasic activity of SARs and their response to lung inflation in anesthetized, artificially ventilated mice, 2) inhalation of SO2 generated a distinct inhibitory effect on the HBIR and induced slow deep breathing in anesthetized, spontaneously breathing mice, and these effects were reversible and reproducible in the same animals, 3) This inhibitory effect of SO2 was blocked by pretreatment with 4-aminopyridine (4-AP), a nonselective blocker of voltage-gated K+ channel, and unaffected by pretreatment with its vehicle. In conclusion, this study suggests that this inhibitory effect on the baseline breathing pattern and the HBIR response was primarily mediated through the SO2-induced activation of voltage-gated K+ channels located in the vagal bronchopulmonary SAR neurons.NEW & NOTEWORTHY This study demonstrated that inhaled sulfur dioxide completely and reversibly abolished the activity of vagal bronchopulmonary slowly adapting receptors, significantly inhibited the apneic response to lung inflation, and induced slow deep breathing in anesthetized mice. More importantly, our results further suggested that this inhibitory effect was mediated through an action of sulfur dioxide and its derivatives on the voltage-gated potassium channels expressed in the slowly adapting receptor sensory neurons innervating the lung.

Hypercapnic signaling influences hypoxic signaling in the control of breathing in C57BL6 mice.

Interactions between hypoxic and hypercapnic signaling pathways, expressed as ventilatory changes occurring during and following a simultaneous hypoxic-hypercapnic gas challenge (HH-C) have not been determined systematically in mice. This study in unanesthetized male C57BL6 mice addressed the hypothesis that hypoxic (HX) and hypercapnic (HC) signaling events display an array of interactions indicative of coordination by peripheral and central respiratory mechanisms. We evaluated the ventilatory responses elicited by hypoxic (HX-C, 10%, O2, 90% N2), hypercapnic (HC-C, 5% CO2, 21%, O2, 90% N2), and HH-C (10% O2, 5%, CO2, 85% N2) challenges to determine whether ventilatory responses elicited by HH-C were simply additive of responses elicited by HX-C and HC-C, or whether other patterns of interactions existed. Responses elicited by HH-C were additive for tidal volume, minute ventilation and expiratory time, among others. Responses elicited by HH-C were hypoadditive of the HX-C and HC-C responses (i.e., HH-C responses were less than expected by simple addition of HX-C and HC-C responses) for frequency of breathing, inspiratory time and relaxation time, among others. In addition, end-expiratory pause increased during HX-C, but decreased during HC-C and HH-C, therefore showing that HC-C responses influenced the HX-C responses when given simultaneously. Return to room-air responses was additive for tidal volume and minute ventilation, among others, whereas they were hypoadditive for frequency of breathing, inspiratory time, peak inspiratory flow, apneic pause, inspiratory and expiratory drives, and rejection index. These data show that HX-C and HH-C signaling pathways interact with one another in additive and often hypoadditive processes.NEW & NOTEWORTHY We present data showing that the ventilatory responses elicited by a hypoxic gas challenge in male C57BL6 mice are markedly altered by coexposure to hypercapnic gas challenge with hypercapnic responses often dominating the hypoxic responses. These data suggest that hypercapnic signaling processes activated within brainstem regions, such as the retrotrapezoid nuclei, may directly modulate the signaling processes within the nuclei tractus solitarius resulting from hypoxic-induced increase in carotid body chemoreceptor input to these nuclei.

Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice

The hypoxic ventilatory response (HVR) is a life-saving reflex, triggered by the activation of chemoreceptor glomus cells in the carotid body (CB) connected with the brainstem respiratory center. The molecular mechanisms underlying glomus cell acute oxygen (O2) sensing are unclear. Genetic disruption of mitochondrial complex I (MCI) selectively abolishes the HVR and glomus cell responsiveness to hypoxia. However, it is unknown what functions of MCI (metabolic, proton transport, or signaling) are essential for O2 sensing. Here we show that transgenic mitochondrial expression of NDI1, a single-molecule yeast NADH/quinone oxidoreductase that does not directly contribute to proton pumping, fully recovers the HVR and glomus cell sensitivity to hypoxia in MCI-deficient mice. Therefore, maintenance of mitochondrial NADH dehydrogenase activity and the electron transport chain are absolutely necessary for O2-dependent regulation of breathing. NDI1 expression also rescues other systemic defects caused by MCI deficiency. These data explain the role of MCI in acute O2 sensing by arterial chemoreceptors and demonstrate the optimal recovery of complex organismal functions by gene therapy.

Simplified Whole Body Plethysmography to Characterize Lung Function During Respiratory Melioidosis

Surrogate animal models of disease are subject to the 3Rs of Responsible Research. There is a frequent revisiting of refinements to animal models to ensure that both animal welfare and scientific insights advance with the availability of new technologies. This article demonstrates the use of Simplified Whole Body Plethysmography (sWBP) to non-invasively study respiratory failure in a model of lethal respiratory melioidosis. sWBP has the sensitivity to detect breathing in mice through the entirety of the course of the disease, allowing for the moribund-associated symptoms (bradypnea and hypopnea) to be measured and potentially used to develop humane endpoint criteria. Some of the benefits of sWBP in the context of respiratory disease are that host breath monitoring comes the closest of any physiologic measurement at assessing dysfunction of the primary infected tissue, namely, the lung. In addition to biological significance, the use of sWBP is rapid and non-invasive, minimizing stress in research animals. This work demonstrates the use of in-house sWBP apparatus to monitor disease throughout the course of respiratory failure in the murine model of respiratory melioidosis.

Neural bases for the genesis and CO2 therapy of periodic Cheyne-Stokes breathing in neonatal male connexin-36 knockout mice.

Periodic Cheyne-Stokes breathing (CSB) oscillating between apnea and crescendo-decrescendo hyperpnea is the most common central apnea. Currently, there is no proven therapy for CSB, probably because the fundamental pathophysiological question of how the respiratory center generates this form of breathing instability is still unresolved. Therefore, we aimed to determine the respiratory motor pattern of CSB resulting from the interaction of inspiratory and expiratory oscillators and identify the neural mechanism responsible for breathing regularization induced by the supplemental CO2 administration. Analysis of the inspiratory and expiratory motor pattern in a transgenic mouse model lacking connexin-36 electrical synapses, the neonatal (P14) Cx36 knockout male mouse, with a persistent CSB, revealed that the reconfigurations recurrent between apnea and hyperpnea and vice versa result from cyclical turn on/off of active expiration driven by the expiratory oscillator, which acts as a master pacemaker of respiration and entrains the inspiratory oscillator to restore ventilation. The results also showed that the suppression of CSB by supplemental 12% CO2 in inhaled air is due to the stabilization of coupling between expiratory and inspiratory oscillators, which causes the regularization of respiration. CSB rebooted after washout of CO2 excess when the inspiratory activity depressed again profoundly, indicating that the disability of the inspiratory oscillator to sustain ventilation is the triggering factor of CSB. Under these circumstances, the expiratory oscillator activated by the cyclic increase of CO2 behaves as an "anti-apnea" center generating the crescendo-decrescendo hyperpnea and periodic breathing. The neurogenic mechanism of CSB identified highlights the plasticity of the two-oscillator system in the neural control of respiration and provides a rationale base for CO2 therapy.

A Silicosis Mouse Model Established by Repeated Inhalation of Crystalline Silica Dust.

Silicosis can be caused by exposure to respiratory crystalline silica dust (CSD) in an industrial environment. The pathophysiology, screening, and treatment of silicosis in humans have all been extensively studied using the mouse silicosis model. By repeatedly making mice inhale CSD into their lungs, the mice can mimic the clinical symptoms of human silicosis. This methodology is practical and efficient in terms of time and output and does not cause mechanical injury to the upper respiratory tract due to surgery. Furthermore, this model can successfully mimic acute/chronic transformation process of silicosis. The main procedures were as follows. The sterilized 1-5 µm CSD powder was fully ground, suspended in saline, and dispersed in an ultrasonic water bath for 30 min. Mice under isoflurane-induced anesthesia switched from shallow rapid breathing to deep, slow aspiration for approximately 2 s. The mouse was placed in the palm of a hand, and the thumb tip gently touched the lip edge of the mouse's jaw to straighten the airway. After each exhalation, the mice breathed in the silica suspension drop by drop through one nostril, completing the process within 4-8 s. After the mice's breathing had stabilized, their chest was stroked and caressed to prevent the inhaled CSD from being coughed up. The mice were then returned to the cage. In conclusion, this model can quantify CSD along the typical physiological passage of tiny particles into the lung, from the upper respiratory tract to the terminal bronchioles and alveoli. It can also replicate the recurrent exposure of employees due to work. The model can be performed by one person and does not need expensive equipment. It conveniently and effectively simulates the disease features of human silicosis with high repeatability.

Optogenetic activation of the tongue in spontaneously breathing mice

Inadequate tongue muscle activation contributes to dysarthria, dysphagia, and obstructive sleep apnea. Thus, treatments which increase tongue muscle activity have potential clinical benefit. We hypothesized that lingual injection of an adeno-associated virus (AAV) encoding channelrhodopsin-2 (ChR2) would enable light-induced activation of tongue motor units during spontaneous breathing. An AAV serotype 9 vector (pACAGW-ChR2-Venus-AAV9, 8.29 × 1011 vg) was injected to the posterior tongue in adult C57BL/6J mice. After 12 weeks, mice were anesthetized and posterior tongue electromyographic (EMG) activity was recorded during spontaneous breathing; a light source was positioned near the injection site. Light-evoked EMG responses increased with the intensity and duration of pulses. Stimulus trains (250 ms) evoked EMG bursts that were comparable to endogenous (inspiratory) tongue muscle activation. Histology confirmed lingual myofiber transgene expression. We conclude that intralingual AAV9-ChR2 delivery enables light evoked lingual EMG activity. These proof-of-concept studies lay the groundwork for clinical application of this novel approach to lingual therapeutics.

5-HT1A Receptor Agonist Treatment Partially Ameliorates Rett Syndrome Phenotypes in mecp2-Null Mice by Rescuing Impairment of Neuron Transmission and the CREB/BDNF Signaling Pathway.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene that encodes methyl CpG-binding protein 2 (MECP2) and is characterized by the loss of acquired motor and language skills, stereotypic movements, respiratory abnormalities and autistic features. There has been no effective treatment for this disorder until now. In this study, we used a Mecp2-null (KO) mouse model of RTT to investigate whether repeated intraperitoneal treatment with the 5-HT1A receptor agonist tandospirone could improve the RTT phenotype. The results showed that administration of tandospirone significantly extended the lifespan of Mecp2-KO mice and obviously ameliorated RTT phenotypes, including general condition, hindlimb clasping, gait, tremor and breathing in Mecp2-KO mice. Tandospirone treatment significantly improved the impairment in GABAergic, glutaminergic, dopaminergic and serotoninergic neurotransmission in the brainstem of Mecp2-KO mice. Decreased dopaminergic neurotransmission in the cerebellum of Mecp2-KO mice was also significantly increased by tandospirone treatment. Moreover, RNA-sequencing analysis found that tandospirone modulates the RTT phenotype, partially through the CREB1/BDNF signaling pathway in Mecp2-KO mice. These findings provide a new option for clinical treatment.

Comparison of Wound Healing Effect of Skin Micrograft Impregnated into Two Kinds of Artificial Dermis in a Murine Wound Model.

Skin MG was prepared from the skin of C57BL/6JJcl mice using Rigeneracons and administered onto Pelnac Gplus and Integra sheets. The amount of MG suspension impregnated in Pelnac Gplus and Integra was evaluated. Pelnac Gplus and Integra sheets combined with MG were applied to murine defects, and wound area, neoepithelium length, granulation tissue formation, and newly formed capillaries were compared with the control groups on days 7 and 14. The weight percentage of the MG absorbed by Pelnac Gplus and Integra was 88.8% ± 3.5% and 28.2% ± 7.0%, respectively (P < 0.05). In the in vivo experiment, the area of newly formed granulation tissue and both the number and area of newly formed capillaries in the PelnacG + MG group were significantly larger than those in the control group at 14 days after implantation (P < 0.05). Skin MG was successfully impregnated into Pelnac Gplus by simple administration but not into Integra. Administration of skin MG into the Pelnac Gplus promoted granulation formation and angiogenesis. Pelnac Gplus was more suitable than Integra in the combination therapy.

The role of adenosine in alcohol-induced respiratory suppression

Alcohol-related poisoning is the foremost cause of death resulting from excessive acute alcohol consumption. Respiratory failure is crucial to the pathophysiology of fatal alcohol poisoning. Alcohol increases accumulation of extracellular adenosine. Adenosine suppresses breathing. The goal of this investigation was to test the hypothesis that adenosine signaling contributes to alcohol-induced respiratory suppression. In the first experiment, the breathing of mice was monitored following an injection of the non-selective adenosine receptor antagonist caffeine (40 mg/kg), alcohol (5 g/kg), or alcohol and caffeine combined. Caffeine reduced alcohol-induced respiratory suppression suggesting that adenosine contributes to the effects of alcohol on breathing. The second experiment utilized the same experimental design, but with the blood brain barrier impermeant non-selective adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT, 60 mg/kg) instead of caffeine. 8-SPT did not reduce alcohol-induced respiratory suppression suggesting that adenosine is contributing to alcohol-induced respiratory suppression in the central nervous system. The third and fourth experiments used the same experimental design as the first, but with the selective A1 receptor antagonist DPCPX (1 mg/kg) and the selective A2A receptor antagonist istradefylline (3.3 mg/kg). Istradefylline, but not DPCPX, reduced alcohol-induced respiratory suppression indicating an A2A receptor mediated effect. In the fifth experiment, alcohol-induced respiratory suppression was evaluated in Adk+/- mice which have impaired adenosine metabolism. Alcohol-induced respiratory suppression was exacerbated in Adk+/- mice. These findings indicate that adenosinergic signaling contributes to alcohol-induced respiratory suppression. Improving our understanding of how alcohol affects breathing may lead to better treatment strategies and better outcomes for patients with severe alcohol poisoning.

Genetic variation in HIF-2α attenuates ventilatory sensitivity and carotid body growth in chronic hypoxia in high-altitude deer mice.

The gene encoding HIF-2α, Epas1, has experienced a history of natural selection in many high-altitude taxa, but the functional role of mutations in this gene is still poorly understood. We investigated the influence of the high-altitude variant of Epas1 in North American deer mice (Peromyscus maniculatus) on the control of breathing and carotid body growth during chronic hypoxia. We created hybrids between high- and low-altitude populations of deer mice to disrupt linkages between genetic loci so that the physiological effects of Epas1 alleles (Epas1H and Epas1L , respectively) could be examined on an admixed genomic background. In general, chronic hypoxia (4weeks at 12kPa O2 ) enhanced ventilatory chemosensitivity (assessed as the acute ventilatory response to hypoxia), increased total ventilation and arterial O2 saturation during progressive poikilocapnic hypoxia, and increased haematocrit and blood haemoglobin content across genotypes. However, the effects of chronic hypoxia on ventilatory chemosensitivity were attenuated in mice that were homozygous for the high-altitude Epas1 allele (Epas1H/H ). Carotid body growth and glomus cell hyperplasia, which was strongly induced in Epas1L/L mice in chronic hypoxia, was not observed in Epas1H/H mice. Epas1 genotype also modulated the effects of chronic hypoxia on metabolism and body temperature depression in hypoxia, but had no effects on haematological traits. These findings confirm the important role of HIF-2α in modulating ventilatory sensitivity and carotid body growth in chronic hypoxia, and show that genetic variation in Epas1 is responsible for evolved changes in the control of breathing and metabolism in high-altitude deer mice. KEY POINTS: High-altitude natives of many species have experienced natural selection on the gene encoding HIF-2α, Epas1, including high-altitude populations of deer mice. HIF-2α regulates ventilation and carotid body growth in hypoxia, and so the genetic variants in Epas1 in high-altitude natives may underlie evolved changes in control of breathing. Deer mice from controlled crosses between high- and low-altitude populations were used to examine the effects of Epas1 genotype on an admixed genomic background. The high-altitude variant was associated with reduced ventilatory chemosensitivity and carotid body growth in chronic hypoxia, but had no effects on haematology. The results help us better understand the genetic basis for the unique physiological phenotype of high-altitude natives.

Sampling and Analysis of Low-Molecular-Weight Volatile Metabolites in Cellular Headspace and Mouse Breath.

Volatile compounds, abundant in breath, can be used to accurately diagnose and monitor a range of medical conditions. This offers a noninvasive, low-cost approach with screening applications; however, the uptake of this diagnostic approach has been limited by conflicting published outcomes. Most published reports rely on large scale screening of the public, at single time points and without reference to ambient air. Here, we present a novel approach to volatile sampling from cellular headspace and mouse breath that incorporates multi-time-point analysis and ambient air subtraction revealing compound flux as an effective proxy of active metabolism. This approach to investigating breath volatiles offers a new avenue for disease biomarker discovery and diagnosis. Using gas chromatography mass spectrometry (GC/MS), we focus on low molecular weight, metabolic substrate/by-product compounds and demonstrate that this noninvasive technique is sensitive (reproducible at ~1 µg cellular protein, or ~500,000 cells) and capable of precisely determining cell type, status and treatment. Isolated cellular models represent components of larger mammalian systems, and we show that stress- and pathology-indicative compounds are detectable in mice, supporting further investigation using this methodology as a tool to identify volatile targets in human patients.

Carotid body responses to O2 and CO2 in hypoxia-tolerant naked mole rats.

Naked mole rats (NMRs) exhibit blunted hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). The mechanism(s) underlying these responses are largely unknown. We hypothesized that attenuated carotid body (CB) sensitivity to hypoxia and hypercapnia contributes to the near absence of ventilatory responses to hypoxia and CO2 in NMRs. We measured ex vivo CB sensory nerve activity, phrenic nerve activity (an estimation of ventilation), and blood gases in urethane-anesthetized NMRs and C57BL/6 mice breathing normoxic, hypoxic, or hypercapnic gases. CB morphology, carbon monoxide, and H2 S levels were also determined. Relative to mice, NMRs had blunted CB and HVR. Morphologically, NMRs have larger CBs, which contained more glomus cells than in mice. Furthermore, NMR glomus cells form a dispersed pattern compared to a clustered pattern in mice. Hemeoxygenase (HO)-1 mRNA was elevated in NMR CBs, and an HO inhibitor increased CB sensitivity to hypoxia in NMRs. This increase was blocked by an H2 S synthesis inhibitor, suggesting that interrupted gas messenger signaling contributes to the blunted CB responses and HVR in NMRs. Regarding hypercapnia, CB and ventilatory responses to CO2 in NMRs were larger than in mice. Carbonic anhydrase (CA)-2 mRNA is elevated in NMR CBs, and a CA inhibitor blocked the augmented CB response to CO2 in NMRs, indicating CA activity regulates augmented CB response to CO2 . Consistent with our hypothesis, impaired CB responses to hypoxia contribute in part to the blunted HVR in NMRs. Conversely, the HCVR and CB are more sensitive to CO2 in NMRs.

5‐HT7 receptors expressed in the mouse parafacial region are not required for respiratory chemosensitivity

A brainstem homeostatic system senses CO2/H+ to regulate ventilation, blood gases and acid‐base balance. Neurons of the retrotrapezoid nucleus (RTN) and medullary raphe are both implicated in this mechanism as respiratory chemosensors, but recent pharmacological work suggested that the CO2/H+‐sensitivity of RTN neurons is mediated indirectly – by raphe‐derived serotonin acting on 5‐HT7 receptors. To investigate this further, we characterized Htr7 transcript expression in phenotypically identified RTN neurons using multiplex single cell qRT‐PCR and RNAscope. Although present in multiple neurons in the parafacial region of the ventrolateral medulla, Htr7 expression was undetectable in most RTN neurons (Nmb+/Phox2b+) concentrated in the densely packed cell group ventrolateral to the facial nucleus. Where detected, Htr7 expression was modest and often associated with RTN neurons that extend dorsolaterally to partially encircle the facial nucleus. These more dorsolateral Nmb+/Htr7+ neurons tended to express high levels of Nmb and low levels of the intrinsic RTN proton detectors, Gpr4 and Kcnk5. In mouse brainstem slices, CO2‐stimulated firing in RTN neurons was mostly unaffected by a 5‐HT7 receptor antagonist, SB269970 (n=11/12). At the whole animal level, microinjection of SB269970 into the RTN of conscious mice blocked respiratory stimulation by co‐injected LP‐44, a 5‐HT7 receptor agonist, but had no effect on CO2‐stimulated breathing in those same mice. We conclude that Htr7 is expressed by a minor subset of RTN neurons with a molecular profile distinct from the established chemoreceptors and that 5‐HT7 receptors have negligible effects on CO2‐evoked firing activity in RTN neurons or on CO2‐stimulated breathing in mice.

Vitamin D Supplementation Partially Rescues Breathing Deficits in Mecp2 Hemizygous Null Mice, a Model of Rett Syndrome

Rett syndrome (RTT) is a severe X‐linked progressive neurodevelopmental disorder affecting ~1 in 10,000 females. It is the second most common genetic cause of cognitive disability in girls, after Down syndrome. Girls with this devastating disorder develop relatively normally for 6‐18 months, after which they undergo a period of rapid regression. They lose many motor skills, the growth of their brain slows, and they develop repetitive, autistic behaviors and seizures. Autonomic disruptions are a leading cause of early death in RTT, and rescue of breathing deficits is considered a primary goal in the development of therapeutic interventions. We initiated a proof‐of‐concept study by investigating male Mecp2 hemizygous null mice as they have the strongest phenotype which occurs at an early age; male Mecp2‐null (KO) mice have also been characterized thoroughly in the literature. We tested the hypothesis that Vitamin D supplementation (VitD) would ameliorate, or partially rescue breathing and cardiovascular deficits in Mecp2 KO mice. Mice were tested at 4 weeks of age (PRE), then placed on a control (1 IU Vitamin D/g) or VitD (50 IU Vitamin D/g) diet, and re‐tested at 8 weeks of age (POST). To examine our hypothesis, unrestrained barometric plethysmography was used to quantify breathing frequency (breaths/min), tidal volume (VT; mL/breath), VT/inspiratory time (VT/Ti; mL/sec), minute ventilation (VE; mL/min/g) and the ventilatory equivalent for CO2 (VE/VCO2) in both wildtype (WT; n=6) and Mecp2 KO (n=5) littermates. Data Sciences International Ponemah software was used to analyze flow tracings during exposure to air (20.93% O2, balanced N2). The MouseOx Plus by STARR Life Sciences was utilized to collect heart rate (HR) and oxygen saturation in freely moving conscious mice. Data are expressed as mean ± S.D., WT vs. KO. In the PRE group, Mecp2 KO mice had dysregulated breathing and HR as previously demonstrated in the literature (data not shown). In the POST VitD diet groups, VT (0.24±0.02 vs 0.15±0.06 mL/breath; p=0.01) and VT/Ti remained lower (2.085±0.313 vs. 1.291±0.583 mL/sec; p=0.017) in Mecp2 KO. However, breathing frequency (166±21 vs. 166±19 breaths/min), VE/g (1.07±0.12 vs. 0.99±0.57 mL/min/g), ratio of VE/VCO2 (112±47 vs. 103±46) and HR (582±84 vs. 532±59 beats/min) were similar between WT and KO. The number of oxygen desaturations were not significant between WT and KO, but the values were variable across groups and included a number of desaturations (12±7 vs. 5±7 desaturations/30 minutes), similar to findings we have previously described in young control mice. These data reveal a partial rescue of breathing and HR deficits in Mecp2 KO mice following 4 weeks of VitD. The reduced VT/Ti suggests an attenuated neural drive to breath in Mecp2 KO vs. WT that persists after VitD. Current studies are examining the responses to hypoxia and hypercapnia and will identify more information about oxygen and carbon dioxide sensing in these mice following control and VitD diets.

Chemogenetic activation of glutamatergic preBötzinger Complex neurons increases breathing in mice

ObjectiveRespiratory rhythm is generated by complex neural circuits in the medulla. At the core of these circuits is the preBötzinger Complex (preBötC), a collection of cells generating respiratory rhythm in the ventral part of the medulla. In addition to its role in generating respiratory rhythm, the preBötC also plays a significant role in opioid‐induced respiratory depression. The preBötC is made up of a heterogenous group of neurons, with a large portion being glutamatergic and expressing µ‐opioid receptors. A marker for these glutamatergic neurons is vesicular glutamate transporter 2 (vGLUT2). Here we propose that targeting vGLUT2‐expressing preBötC neurons using chemogenetics can stimulate respiratory rhythm and potentially alleviate opioid‐induced respiratory depression.HypothesisActivation of vGLUT2 preBötC neurons stimulates breathing and alleviates respiratory depression by fentanyl in mice.MethodsUsing a cre‐lox recombination approach, we expressed the excitatory hM3Dq (a modified version of the excitatory M3 muscarinic receptor) into vGLUT2 preBötC neurons. To this aim, we injected into the preBötC recombinant adeno‐associated virus transcriptionally activated by Cre‐recombinase containing hM3Dq and mCherry DNA flanked by lox sites in vglut2‐IRES‐Cre male mice (Jackson Labs). Four weeks after adeno‐associated virus, mice were anesthetized with isoflurane and respiratory activity was measured through electrodes implanted into the diaphragm and the genioglossus muscle. Mice were given consecutive intramuscular injections of saline and the hM3Dq ligand, clozapine‐N‐oxide (CNO; 0.5 and 1 mg/kg), while respiratory responses were monitored. Brains were collected for histological analysis with expression of hM3Dq determined by expression of mCherry.ResultsInjection of 0.5mg/kg CNO elicited a 24.55% increase in respiratory rate 8 minutes following CNO injection. Injection of 1mg/kg CNO destabilized breathing within 6‐25 minutes following injection. Histology confirmed expression of mCherry in the region of the preBötC. Further studies will determine whether stimulation of vGLUT2 preBötC neurons can prevent or reverse respiratory rate depression elicited by fentanyl.ConclusionsOur preliminary data showed that stimulation of glutamatergic preBötC neurons can increase respiratory rate in anesthetized conditions. Further studies will determine whether stimulation of glutamatergic preBötC cells also increases respiratory rate in freely‐behaving mice and whether the glutamatergic preBötC neurons are viable targets for the development of preventive drug therapies for respiratory depression during opioid overdose.

Treatment of Sleep Disordered Breathing with Leptin Loaded Extracellular Vesicles

IntroductionObstructive sleep apnea (OSA) is the most common type of sleep disordered breathing (SDB), characterized by recurrent periods of upper airway obstruction during sleep leading to increase in morbidity and mortality. The prevalence of OSA exceeds 50% in individuals with obesity and 10‐20% also develop hypercapnia and hypoventilation during sleep which characterizes obesity hypoventilation syndrome (OHS). There is no effective pharmacotherapy for SDB. Leptin, an adipocyte derived hormone, can stimulate breathing and is a potential therapeutic candidate. However, diet‐induced obesity is associated with impairment of leptin transport across the blood‐brain barrier. Previous studies have shown that extracellular vesicles (EVs) naturally derived from macrophages can penetrate the BBB and be used for drug delivery. The objective of this study was to determine if EVs overcome the BBB and treat SDB in DIO mice.MethodsTo examine the ability of EVs to cross the BBB, lean (n=5) and DIO (n=5) C57BL/6J mice were injected with fluorescent EVs or saline into the lateral tail vein. After 4h EV biodistribution was evaluated by Bioimaging and Infrared Spectroscopy (IVIS). Sleep studies were performed in a plethysmography chamber in a separate subgroup of male, DIO (n=10) and lean (n=10) mice. Mice received saline, empty exosomes, free leptin, or leptin‐loaded exosomes in a crossover manner. Similar design was used to determine energy expenditure in a Comprehensive Laboratory Animal Monitoring System (CLAMS).ResultsWe demonstrated that EVs accumulated in the brain and the transport across the BBB was 2‐times more efficient in DIO when compared to lean mice (p&lt;0.005). In DIO mice, leptin EVs induced 1.7‐2.2‐fold increases in minute ventilation and 1.5‐2.0‐fold increases in maximal inspiratory flow during flow‐limited and non‐flow limited breaths (p&lt;0.05). In contrast, free leptin had no effect. There was no effect of leptin EVs on metabolism. These findings suggest that leptin EVs mitigated upper airway obstruction and improved control of breathing in DIO mice. Lean mice did not present significant sleep disordered breathing and no differences were observed between groups.ConclusionWe demonstrated that EVs overcome the BBB and that leptin loaded EVs treat SDB in DIO mice.

Opioid‐Induced Respiratory Depression (OIRD) in C57BL/6J (B6) Mice is Mitigated by Prefrontal Cortex Neostigmine Administration

Opioids diminish the wakefulness stimulus for breathing in humans (10.1152/physiol.00043.2020) and mice (10.1152/jn.00017.2021). The neuronal networks underlying the wakefulness stimulus remain poorly understood. Cholinergic transmission in the prefrontal cortex (PFC) promotes cortical EEG activation (10.1152/jn.2002.87.6.2817), increases wakefulness (10.3389/fnins.2020.567849), and stimulates breathing (10.1096/fasebj.2021.35.S1.01735). This study is using whole‐body plethysmography (WBP) to test the hypothesis that microinjection of the acetylcholinesterase inhibitor neostigmine (Neo) into the PFC attenuates OIRD caused by systemic fentanyl administration in male B6 mice. All procedures followed the FASEB Statement of Principles for the use of Animals in Research and Education. Previous studies of the dose‐dependent effects of fentanyl in B6 mice used CO2 to stimulate breathing (10.1038/npp.2015.201). Therefore, we first quantified the dose‐dependent effects of systemic fentanyl administration on B6 mice breathing room air. Mice (n=12) received a 0.3 mL intraperitoneal (IP) injection of saline (control) and 8 increasing doses of fentanyl (0.001 to 3 mg/kg), each separated by one half‐log unit. Repeated measures one‐way ANOVA showed that fentanyl caused a dose‐dependent decrease (F(2.47; 27.12)= 20.30, p&lt;0.0001) in minute ventilation (V̇E) quantified as mL/min/g body weight. Additional mice (n=4) were implanted with a 26‐gauge guide cannula aimed for the PFC at coordinates (mm) A/P = 1.94; M/L = 0.3; D/V = 2.5 relative to bregma (histology pending). Seven to ten days after surgery, WBP was used to evaluate the respiratory effects of 0.1 mg/kg IP fentanyl in combination with unilateral PFC microinjection of saline (control) or 0.016 nmol/50nL Neo (4.8 ng). Breathing was compared among three conditions: 1=(IP saline+PFC saline), 2=(IP fentanyl+PFC saline), and 3=(IP fentanyl+PFC Neo). Centrally administered drugs rapidly diffuse away from a microinjection site. Accordingly, V̇E was quantified during the initial 10 min after PFC microinjection. Comparison between conditions 1 and 2 showed that IP fentanyl caused a 19.7% decrease in V̇E. Comparing conditions 2 and 3 showed that IP fentanyl+PFC Neo caused a 44.3% increase in V̇E. Regression analyses revealed that PFC Neo delayed the onset of OIRD. During the initial 5 min after PFC Neo, time accounted for 84% of the variance in V̇E compared to 60% of the variance after PFC saline. During the 6 to 10 min after PFC injection, time explained only 27% of the variance in V̇E, likely due to Neo diffusing away from the PFC. The slope of the minute ventilation line (V̇E=(0.03)(min)+3.2) increased after IP fentanyl + PFC Neo and decreased (V̇E=(‐0.12)(min)+3.8) after IP fentanyl + PFC saline. The slopes of these two functions were significantly different (p&lt;0.01). For up to 8 min, 4.8 ng of Neo delivered into the PFC diminished OIRD caused by 0.1 mg/kg IP fentanyl. These findings support our working hypothesis that enhancing cholinergic transmission in the PFC promotes to the wakefulness stimulus for breathing and mitigates OIRD.