V2a neurons restore diaphragm function in mice following spinal cord injury.

The specific roles that different types of neurons play in recovery from injury is poorly understood. Here, we show that increasing the excitability of ipsilaterally projecting, excitatory V2a neurons using designer receptors exclusively activated by designer drugs (DREADDs) restores rhythmic bursting activity to a previously paralyzed diaphragm within hours, days, or weeks following a C2 hemisection injury. Further, decreasing the excitability of V2a neurons impairs tonic diaphragm activity after injury as well as activation of inspiratory activity by chemosensory stimulation, but does not impact breathing at rest in healthy animals. By examining the patterns of muscle activity produced by modulating the excitability of V2a neurons, we provide evidence that V2a neurons supply tonic drive to phrenic circuits rather than increase rhythmic inspiratory drive at the level of the brainstem. Our results demonstrate that the V2a class of neurons contribute to recovery of respiratory function following injury. We propose that altering V2a excitability is a potential strategy to prevent respiratory motor failure and promote recovery of breathing following spinal cord injury.

Transformation of Our Understanding of Breathing Control by Molecular Tools.

The rhythmicity of breath is vital for normal physiology. Even so, breathing is enriched with multifunctionality. External signals constantly change breathing, stopping it when under water or deepening it during exertion. Internal cues utilize breath to express emotions such as sighs of frustration and yawns of boredom. Breathing harmonizes with other actions that use our mouth and throat, including speech, chewing, and swallowing. In addition, our perception of breathing intensity can dictate how we feel, such as during the slow breathing of calming meditation and anxiety-inducing hyperventilation. Heartbeat originates from a peripheral pacemaker in the heart, but the automation of breathing arises from neural clusters within the brainstem, enabling interaction with other brain areas and thus multifunctionality. Here, we document how the recent transformation of cellular and molecular tools has contributed to our appreciation of the diversity of neuronal types in the breathing control circuit and how they confer the multifunctionality of breathing.

The Dynamic Basis of Respiratory Rhythm Generation: One Breath at a Time.

Rhythmicity is a universal timing mechanism in the brain, and the rhythmogenic mechanisms are generally dynamic. This is illustrated for the neuronal control of breathing, a behavior that occurs as a one-, two-, or three-phase rhythm. Each breath is assembled stochastically, and increasing evidence suggests that each phase can be generated independently by a dedicated excitatory microcircuit. Within each microcircuit, rhythmicity emerges through three entangled mechanisms: ( a) glutamatergic transmission, which is amplified by ( b) intrinsic bursting and opposed by ( c) concurrent inhibition. This rhythmogenic triangle is dynamically tuned by neuromodulators and other network interactions. The ability of coupled oscillators to reconfigure and recombine may allow breathing to remain robust yet plastic enough to conform to nonventilatory behaviors such as vocalization, swallowing, and coughing. Lessons learned from the respiratory network may translate to other highly dynamic and integrated rhythmic systems, if approached one breath at a time.

Signatures of a magnetic superstructure phase induced by ultrahigh magnetic fields in a breathing pyrochlore antiferromagnet.

The mutual coupling of spin and lattice degrees of freedom is ubiquitous in magnetic materials and potentially creates exotic magnetic states in response to the external magnetic field. Particularly, geometrically frustrated magnets serve as a fertile playground for realizing magnetic superstructure phases. Here, we observe an unconventional two-step magnetostructural transition prior to a half-magnetization plateau in a breathing pyrochlore chromium spinel by means of state-of-the-art magnetization and magnetostriction measurements in ultrahigh magnetic fields available up to 600 T. Considering a microscopic magnetoelastic theory, the intermediate-field phase can be assigned to a magnetic superstructure with a three-dimensional periodic array of 3-up-1-down and canted 2-up-2-down spin molecules. We attribute the emergence of the magnetic superstructure to a unique combination of the strong spin-lattice coupling and large breathing anisotropy.

Inhibitory Subpopulations in preBötzinger Complex Play Distinct Roles in Modulating Inspiratory Rhythm and Pattern.

Inhibitory neurons embedded within mammalian neural circuits shape breathing, walking, and other rhythmic motor behaviors. At the core of the neural circuit controlling breathing is the preBötzinger Complex (preBötC), where GABAergic (GAD1/2+) and glycinergic (GlyT2+) neurons are functionally and anatomically intercalated among glutamatergic Dbx1-derived (Dbx1+) neurons that generate rhythmic inspiratory drive. The roles of these preBötC inhibitory neurons in breathing remain unclear. We first characterized the spatial distribution of molecularly defined preBötC inhibitory subpopulations in male and female neonatal double reporter mice expressing either tdTomato or EGFP in GlyT2+, GAD1+, or GAD2+ neurons. We found that the majority of preBötC inhibitory neurons expressed both GlyT2 and GAD2 while a much smaller subpopulation also expressed GAD1. To determine the functional role of these subpopulations, we used holographic photostimulation, a patterned illumination technique, in rhythmically active medullary slices from neonatal Dbx1tdTomato;GlyT2EGFP and Dbx1tdTomato;GAD1EGFP double reporter mice of either sex. Stimulation of 4 or 8 preBötC GlyT2+ neurons during endogenous rhythm prolonged the interburst interval in a phase-dependent manner and increased the latency to burst initiation when bursts were evoked by stimulation of Dbx1+ neurons. In contrast, stimulation of 4 or 8 preBötC GAD1+ neurons did not affect interburst interval or latency to burst initiation. Instead, photoactivation of GAD1+ neurons during the inspiratory burst prolonged endogenous and evoked burst duration and decreased evoked burst amplitude. We conclude that GlyT2+/GAD2+ neurons modulate breathing rhythm by delaying burst initiation while a smaller GAD1+ subpopulation shapes inspiratory patterning by altering burst duration and amplitude.

Orexin Facilitates the Peripheral Chemoreflex via Corticotropin-Releasing Hormone Neurons Projecting to the Nucleus of the Solitary Tract.

We previously showed that orexin neurons are activated by hypoxia and facilitate the peripheral chemoreflex (PCR)-mediated hypoxic ventilatory response (HVR), mostly by promoting the respiratory frequency response. Orexin neurons project to the nucleus of the solitary tract (nTS) and the paraventricular nucleus of the hypothalamus (PVN). The PVN contributes significantly to the PCR and contains nTS-projecting corticotropin-releasing hormone (CRH) neurons. We hypothesized that in male rats, orexin neurons contribute to the PCR by activating nTS-projecting CRH neurons. We used neuronal tract tracing and immunohistochemistry (IHC) to quantify the degree that hypoxia activates PVN-projecting orexin neurons. We coupled this with orexin receptor (OxR) blockade with suvorexant (Suvo, 20 mg/kg, i.p.) to assess the degree that orexin facilitates the hypoxia-induced activation of CRH neurons in the PVN, including those projecting to the nTS. In separate groups of rats, we measured the PCR following systemic orexin 1 receptor (Ox1R) blockade (SB-334867; 1 mg/kg) and specific Ox1R knockdown in PVN. OxR blockade with Suvo reduced the number of nTS and PVN neurons activated by hypoxia, including those CRH neurons projecting to nTS. Hypoxia increased the number of activated PVN-projecting orexin neurons but had no effect on the number of activated nTS-projecting orexin neurons. Global Ox1R blockade and partial Ox1R knockdown in the PVN significantly reduced the PCR. Ox1R knockdown also reduced the number of activated PVN neurons and the number of activated tyrosine hydroxylase neurons in the nTS. Our findings suggest orexin facilitates the PCR via nTS-projecting CRH neurons expressing Ox1R.

Deciphering Mechanisms of Respiratory Fetal-to-Neonatal Transition in Very Preterm Infants.

Rationale: The respiratory mechanisms of a successful transition of preterm infants after birth are largely unknown. Objectives: To describe intrapulmonary gas flows during different breathing patterns directly after birth. Methods: Analysis of electrical impedance tomography data from a previous randomized trial in preterm infants at 26-32 weeks gestational age. Electrical impedance tomography data for individual breaths were extracted, and lung volumes as well as ventilation distribution were calculated for end of inspiration, end of expiratory braking and/or holding maneuver, and end of expiration. Measurements and Main Results: Overall, 10,348 breaths from 33 infants were analyzed. We identified three distinct breath types within the first 10 minutes after birth: tidal breathing (44% of all breaths; sinusoidal breathing without expiratory disruption), braking (50%; expiratory brake with a short duration), and holding (6%; expiratory brake with a long duration). Only after holding breaths did end-expiratory lung volume increase: Median (interquartile range [IQR]) = 2.0 AU/kg (0.6 to 4.3), 0.0 (-1.0 to 1.1), and 0.0 (-1.1 to 0.4), respectively; P < 0.001]. This was mediated by intrathoracic air redistribution to the left and non-gravity-dependent parts of the lung through pendelluft gas flows during braking and/or holding maneuvers. Conclusions: Respiratory transition in preterm infants is characterized by unique breathing patterns. Holding breaths contribute to early lung aeration after birth in preterm infants. This is facilitated by air redistribution during braking/holding maneuvers through pendelluft flow, which may prevent lung liquid reflux in this highly adaptive situation. This study deciphers mechanisms for a successful fetal-to-neonatal transition and increases our pathophysiological understanding of this unique moment in life. Clinical trial registered with www.clinicaltrials.gov (NCT04315636).

Scale Construction of "Breathing" Bi/N-CNSs Quasi-Array Structure with Hierarchical Bi Distribution for Sodium-Ion Battery.

Sodium ion batteries (SIBs) are promising postlithium battery technologies with high safety and low cost. However, their development is hampered by complicated electrode material preparation and unsatisfactory sodium storage performance. Here, a bismuth/N-doped carbon nanosheets (Bi/N-CNSs) composite featuring a quasi-array structure (alternated porous Bi layers and N-CNSs) with hierarchical Bi distribution (large particles of ∼35 nm in Bi layers and ultrafine Bi of ∼8 nm on N-CNSs) is prepared. Bi/N-CNSs delivers an ultralong-lifespan of 26000 cycles at 5 A g-1 and prominent rate capability of 91.5% capacity retention at 100 A g-1. Even at -40 °C, it exhibits a high rate capability of 161 mAh g-1 at 5 A g-1. Notably, the involved preparation method is characterized by a high yield of 14.53 g in a single laboratory batch, which can be further scaled up, and such a method can also be extended to synthesize other metallic-based materials.

Seizures Cause Prolonged Impairment of Ventilation, CO2 Chemoreception and Thermoregulation.

Sudden unexpected death in epilepsy (SUDEP) has been linked to respiratory dysfunction, but the mechanisms underlying this association remain unclear. Here we found that both focal and generalized convulsive seizures (GCSs) in epilepsy patients caused a prolonged decrease in the hypercapnic ventilatory response (HCVR; a measure of respiratory CO2 chemoreception). We then studied Scn1a R1407X/+ (Dravet syndrome; DS) and Scn8a N1768D/+ (D/+) mice of both sexes, two models of SUDEP, and found that convulsive seizures caused a postictal decrease in ventilation and severely depressed the HCVR in a subset of animals. Those mice with severe postictal depression of the HCVR also exhibited transient postictal hypothermia. A combination of blunted HCVR and abnormal thermoregulation is known to occur with dysfunction of the serotonin (5-hydroxytryptamine; 5-HT) system in mice. Depleting 5-HT with para-chlorophenylalanine (PCPA) mimicked seizure-induced hypoventilation, partially occluded the postictal decrease in the HCVR, exacerbated hypothermia, and increased postictal mortality in DS mice. Conversely, pretreatment with the 5-HT agonist fenfluramine reduced postictal inhibition of the HCVR and hypothermia. These results are consistent with the previous observation that seizures cause transient impairment of serotonergic neuron function, which would be expected to inhibit the many aspects of respiratory control dependent on 5-HT, including baseline ventilation and the HCVR. These results provide a scientific rationale to investigate the interictal and/or postictal HCVR as noninvasive biomarkers for those at high risk of seizure-induced death, and to prevent SUDEP by enhancing postictal 5-HT tone.SIGNIFICANCE STATEMENT There is increasing evidence that seizure-induced respiratory dysfunction contributes to the pathophysiology of sudden unexpected death in epilepsy (SUDEP). However, the cellular basis of this dysfunction has not been defined. Here, we show that seizures impair CO2 chemoreception in some epilepsy patients. In two mouse models of SUDEP we found that generalized convulsive seizures impaired CO2 chemoreception, and induced hypothermia, two effects reported with serotonergic neuron dysfunction. The defects in chemoreception and thermoregulation were exacerbated by chemical depletion of serotonin and reduced with fenfluramine, suggesting that seizure-induced respiratory dysfunction may be due to impairment of serotonin neuron function. These findings suggest that impaired chemoreception because of transient inhibition of serotonergic neurons may contribute to the pathophysiology of SUDEP.

Neuromedin B-Expressing Neurons in the Retrotrapezoid Nucleus Regulate Respiratory Homeostasis and Promote Stable Breathing in Adult Mice.

Respiratory chemoreceptor activity encoding arterial Pco2 and Po2 is a critical determinant of ventilation. Currently, the relative importance of several putative chemoreceptor mechanisms for maintaining eupneic breathing and respiratory homeostasis is debated. Transcriptomic and anatomic evidence suggests that bombesin-related peptide Neuromedin-B (Nmb) expression identifies chemoreceptor neurons in the retrotrapezoid nucleus (RTN) that mediate the hypercapnic ventilatory response, but functional support is missing. In this study, we generated a transgenic Nmb-Cre mouse and used Cre-dependent cell ablation and optogenetics to test the hypothesis that RTN Nmb neurons are necessary for the CO2-dependent drive to breathe in adult male and female mice. Selective ablation of ∼95% of RTN Nmb neurons causes compensated respiratory acidosis because of alveolar hypoventilation, as well as profound breathing instability and respiratory-related sleep disruption. Following RTN Nmb lesion, mice were hypoxemic at rest and were prone to severe apneas during hyperoxia, suggesting that oxygen-sensitive mechanisms, presumably the peripheral chemoreceptors, compensate for the loss of RTN Nmb neurons. Interestingly, ventilation following RTN Nmb -lesion was unresponsive to hypercapnia, but behavioral responses to CO2 (freezing and avoidance) and the hypoxia ventilatory response were preserved. Neuroanatomical mapping shows that RTN Nmb neurons are highly collateralized and innervate the respiratory-related centers in the pons and medulla with a strong ipsilateral preference. Together, this evidence suggests that RTN Nmb neurons are dedicated to the respiratory effects of arterial Pco2/pH and maintain respiratory homeostasis in intact conditions and suggest that malfunction of these neurons could underlie the etiology of certain forms of sleep-disordered breathing in humans.SIGNIFICANCE STATEMENT Respiratory chemoreceptors stimulate neural respiratory motor output to regulate arterial Pco2 and Po2, thereby maintaining optimal gas exchange. Neurons in the retrotrapezoid nucleus (RTN) that express the bombesin-related peptide Neuromedin-B are proposed to be important in this process, but functional evidence has not been established. Here, we developed a transgenic mouse model and demonstrated that RTN neurons are fundamental for respiratory homeostasis and mediate the stimulatory effects of CO2 on breathing. Our functional and anatomic data indicate that Nmb-expressing RTN neurons are an integral component of the neural mechanisms that mediate CO2-dependent drive to breathe and maintain alveolar ventilation. This work highlights the importance of the interdependent and dynamic integration of CO2- and O2-sensing mechanisms in respiratory homeostasis of mammals.

Microcircuit Synchronization and Heavy-Tailed Synaptic Weight Distribution Augment preBötzinger Complex Bursting Dynamics.

The preBötzinger Complex (preBötC) encodes inspiratory time as rhythmic bursts of activity underlying each breath. Spike synchronization throughout a sparsely connected preBötC microcircuit initiates bursts that ultimately drive the inspiratory motor patterns. Using minimal microcircuit models to explore burst initiation dynamics, we examined the variability in probability and latency to burst following exogenous stimulation of a small subset of neurons, mimicking experiments. Among various physiologically plausible graphs of 1000 excitatory neurons constructed using experimentally determined synaptic and connectivity parameters, directed Erdos-Rényi graphs with a broad (lognormal) distribution of synaptic weights best captured the experimentally observed dynamics. preBötC synchronization leading to bursts was regulated by the efferent connectivity of spiking neurons that are optimally tuned to amplify modest preinspiratory activity through input convergence. Using graph-theoretic and machine learning-based analyses, we found that input convergence of efferent connectivity at the next-nearest neighbor order was a strong predictor of incipient synchronization. Our analyses revealed a crucial role of synaptic heterogeneity in imparting exceptionally robust yet flexible preBötC attractor dynamics. Given the pervasiveness of lognormally distributed synaptic strengths throughout the nervous system, we postulate that these mechanisms represent a ubiquitous template for temporal processing and decision-making computational motifs.SIGNIFICANCE STATEMENT Mammalian breathing is robust, virtually continuous throughout life, yet is inherently labile: to adapt to rapid metabolic shifts (e.g., fleeing a predator or chasing prey); for airway reflexes; and to enable nonventilatory behaviors (e.g., vocalization, breathholding, laughing). Canonical theoretical frameworks-based on pacemakers and intrinsic bursting-cannot account for the observed robustness and flexibility of the preBötzinger Complex rhythm. Experiments reveal that network synchronization is the key to initiate inspiratory bursts in each breathing cycle. We investigated preBötC synchronization dynamics using network models constructed with experimentally determined neuronal and synaptic parameters. We discovered that a fat-tailed (non-Gaussian) synaptic weight distribution-a manifestation of synaptic heterogeneity-augments neuronal synchronization and attractor dynamics in this vital rhythmogenic network, contributing to its extraordinary reliability and responsiveness.

The Impact of Inflammation on Thermal Hyperpnea: Relevance for Heat Stress and Febrile Seizures.

Extreme heat caused by climate change is increasing the transmission of infectious diseases, resulting in a sharp rise in heat-related illness and mortality. Understanding the mechanistic link between heat, inflammation, and disease is thus important for public health. Thermal hyperpnea, and consequent respiratory alkalosis, is crucial in febrile seizures and convulsions induced by heat stress in humans. Here, we address what causes thermal hyperpnea in neonates and how it is affected by inflammation. Transient receptor potential cation channel subfamily V member 1 (TRPV1), a heat-activated channel, is sensitized by inflammation and modulates breathing and thus may play a key role. To investigate whether inflammatory sensitization of TRPV1 modifies neonatal ventilatory responses to heat stress, leading to respiratory alkalosis and an increased susceptibility to hyperthermic seizures, we treated neonatal rats with bacterial LPS, and breathing, arterial pH, in vitro vagus nerve activity, and seizure susceptibility were assessed during heat stress in the presence or absence of a TRPV1 antagonist (AMG-9810) or shRNA-mediated TRPV1 suppression. LPS-induced inflammatory preconditioning lowered the threshold temperature and latency of hyperthermic seizures. This was accompanied by increased tidal volume, minute ventilation, expired CO2, and arterial pH (alkalosis). LPS exposure also elevated vagal spiking and intracellular calcium concentrations in response to hyperthermia. TRPV1 inhibition with AMG-9810 or shRNA reduced the LPS-induced susceptibility to hyperthermic seizures and altered the breathing pattern to fast shallow breaths (tachypnea), making each breath less efficient and restoring arterial pH. These results indicate that inflammation exacerbates thermal hyperpnea-induced respiratory alkalosis associated with increased susceptibility to hyperthermic seizures, primarily mediated by TRPV1 localized to vagus neurons.

Disruption of BMP4 signaling is associated with laryngeal birth defects in a mouse model.

Laryngeal birth defects are considered rare, but they can be life-threatening conditions. The BMP4 gene plays an important role in organ development and tissue remodeling throughout life. Here we examined its role in laryngeal development complementing similar efforts for the lung, pharynx, and cranial base. Our goal was to determine how different imaging techniques contribute to a better understanding of the embryonic anatomy of the normal and diseased larynx in small specimens. Contrast-enhanced micro CT images of embryonic larynx tissue from a mouse model with Bmp4 deletion informed by histology and whole-mount immunofluorescence were used to reconstruct the laryngeal cartilaginous framework in three dimensions. Laryngeal defects included laryngeal cleft, laryngeal asymmetry, ankylosis and atresia. Results implicate BMP4 in laryngeal development and show that the 3D reconstruction of laryngeal elements provides a powerful approach to visualize laryngeal defects and thereby overcoming shortcomings of 2D histological sectioning and whole mount immunofluorescence.

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

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

A narrative review of periodic breathing during sleep at high altitude: From acclimatizing lowlanders to adapted highlanders.

Periodic breathing during sleep at high altitude is almost universal among sojourners. Here, in the context of acclimatization and adaptation, we provide a contemporary review on periodic breathing at high altitude, and explore whether this is an adaptive or maladaptive process. The mechanism(s), prevalence and role of periodic breathing in acclimatized lowlanders at high altitude are contrasted with the available data from adapted indigenous populations (e.g. Andean and Tibetan highlanders). It is concluded that (1) periodic breathing persists with acclimatization in lowlanders and the severity is proportional to sleeping altitude; (2) periodic breathing does not seem to coalesce with poor sleep quality such that, with acclimatization, there appears to be a lengthening of cycle length and minimal impact on the average sleeping oxygen saturation; and (3) high altitude adapted highlanders appear to demonstrate a blunting of periodic breathing, compared to lowlanders, comprising a feature that withstands the negative influences of chronic mountain sickness. These observations indicate that periodic breathing persists with high altitude acclimatization with no obvious negative consequences; however, periodic breathing is attenuated with high altitude adaptation and therefore potentially reflects an adaptive trait to this environment.

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.

Descending motor drive does not interact with muscle metaboreflex for ventilation regulation during rhythmic exercise in healthy humans.

The muscle metaboreflex effect on pulmonary ventilation (V̇E) regulation is more apparent during rhythmic exercise than rest, possibly because this reflex interacts with other mechanisms regulating V̇E during voluntary contractions, such as central command. Therefore, we tested whether one part of central command, the descending component of motor execution (i.e., descending motor drive), and the muscle metaboreflex interact synergistically to regulate V̇E. Thirteen healthy adults (9 men) completed four experiments in random order under isocapnia. The muscle metaboreflex was activated by rhythmic handgrip exercise at 60% maximal voluntary contraction (MVC) force with the dominant hand. Then, the muscle metaboreflex remained active during a 4-minute recovery period via post-exercise circulatory occlusion (PECO), or it was inactivated, maintaining free blood flow to the dominant upper limb. During the last 2-minutes of the handgrip exercise recovery, participants either performed rhythmic voluntary plantar flexion with the dominant leg at 30% MVC torque to generate descending motor drive or the dominant leg's calf muscles were involuntarily activated by electrical stimulation at a similar torque level (i.e., without descending motor drive). V̇E increased to a similar level during handgrip exercise in all conditions (≈22 L/min, P = 0.364). PECO maintained V̇E elevated above recovery with free blood flow (≈17 L/min vs. ≈13 L/min, P = 0.009). However, voluntary and involuntary plantar flexion with or without PECO evoked similar V̇E responses (∆ ≈ 4 L/min, P = 0.311). Therefore, an interaction between descending motor drive and muscle metaboreflex is not ubiquitous for V̇E regulation during rhythmic exercise.

Time-restricted feeding reveals a role for neural respiratory clocks in optimizing daily ventilatory-metabolic coupling in mice.

The master circadian clock, located in the suprachiasmatic nuclei (SCN), organizes the daily rhythm in minute ventilation (V̇e). However, the extent that the daily rhythm in V̇e is secondary to SCN-imposed O2 and CO2 cycles (i.e., metabolic rate) or driven by other clock mechanisms remains unknown. Here, we experimentally shifted metabolic rate using time-restricted feeding (without affecting light-induced synchronization of the SCN) to determine the influence of metabolic rate in orchestrating the daily V̇e rhythm. Mice eating predominantly at night exhibited robust daily rhythms in O2 consumption (V̇o2), CO2 production (V̇co2), and V̇e with similar peak times (approximately ZT18) that were consistent with SCN organization. However, feeding mice exclusively during the day separated the relative timing of metabolic and ventilatory rhythms, resulting in an approximately 8.5-h advance in V̇co2 and a disruption of the V̇e rhythm, suggesting opposing circadian and metabolic influences on V̇e. To determine if the molecular clock of cells involved in the neural control of breathing contributes to the daily V̇e rhythm, we examined V̇e in mice lacking BMAL1 in Phox2b-expressing respiratory cells (i.e., BKOP mice). The ventilatory and metabolic rhythms of predominantly night-fed BKOP mice did not differ from wild-type mice. However, in contrast to wild-type mice, exclusive day feeding of BKOP mice led to an unfettered daily V̇e rhythm with a peak time aligning closely with the daily V̇co2 rhythm. Taken together, these results indicate that both daily V̇co2 changes and intrinsic circadian time-keeping within Phox2b respiratory cells are predominant orchestrators of the daily rhythm in ventilation.NEW & NOTEWORTHY The master circadian clock organizes the daily rhythm in ventilation; however, the extent that this rhythm is driven by SCN regulation of metabolic rate versus other clock mechanisms remains unknown. We report that metabolic rate alone is insufficient to explain the daily oscillation in ventilation and that neural respiratory clocks within Phox2b-expressing cells additionally optimize breathing. Collectively, these findings advance our mechanistic understanding of the circadian rhythm in ventilatory control.

Identification of eupneic breathing using machine learning.

The diaphragm muscle (DIAm) is the primary inspiratory muscle in mammals. In awake animals, considerable heterogeneity in the electromyographic (EMG) activity of the DIAm reflects varied ventilatory and nonventilatory behaviors. Experiments in awake animals are an essential component to understanding the neuromotor control of breathing, which has especially begun to be appreciated within the last decade. However, insofar as the intent is to study the control of breathing, it is paramount to identify DIAm EMG activity that in fact reflects breathing. Current strategies for doing so in a reproducible, reliable, and efficient fashion are lacking. In the present article, we evaluated DIAm EMG from awake animals using hierarchical clustering across four-dimensional feature space to classify eupneic breathing. Our model, which can be implemented with automated threshold of the clustering dendrogram, successfully identified eupneic breathing with high F1 score (0.92), specificity (0.70), and accuracy (0.88), suggesting that it is a robust and reliable tool for investigating the neural control of breathing.NEW & NOTEWORTHY The heterogeneity of diaphragm muscle (DIAm) activity in awake animals reflects real motor behavior diversity but makes assessments of eupneic breathing challenging. The present article uses an unsupervised machine learning model to identify eupneic breathing amidst a deluge of different DIAm electromyography (EMG) burst patterns in awake rats. This technique offers a scalable and reliable tool that improves efficiency of DIAm EMG analysis and minimizes potential sources of bias.