A feature of maternal sleep apnea during gestation causes autism-relevant neuronal and behavioral phenotypes in offspring.

Mounting epidemiologic and scientific evidence indicates that many psychiatric disorders originate from a complex interplay between genetics and early life experiences, particularly in the womb. Despite decades of research, our understanding of the precise prenatal and perinatal experiences that increase susceptibility to neurodevelopmental disorders remains incomplete. Sleep apnea (SA) is increasingly common during pregnancy and is characterized by recurrent partial or complete cessations in breathing during sleep. SA causes pathological drops in blood oxygen levels (intermittent hypoxia, IH), often hundreds of times each night. Although SA is known to cause adverse pregnancy and neonatal outcomes, the long-term consequences of maternal SA during pregnancy on brain-based behavioral outcomes and associated neuronal functioning in the offspring remain unknown. We developed a rat model of maternal SA during pregnancy by exposing dams to IH, a hallmark feature of SA, during gestational days 10 to 21 and investigated the consequences on the offspring's forebrain synaptic structure, synaptic function, and behavioral phenotypes across multiples stages of development. Our findings represent a rare example of prenatal factors causing sexually dimorphic behavioral phenotypes associated with excessive (rather than reduced) synapse numbers and implicate hyperactivity of the mammalian target of rapamycin (mTOR) pathway in contributing to the behavioral aberrations. These findings have implications for neuropsychiatric disorders typified by superfluous synapse maintenance that are believed to result, at least in part, from largely unknown insults to the maternal environment.

Inactivity-induced phrenic motor facilitation requires PKCζ activity within phrenic motor neurons.

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF.NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.

Obstructive Sleep Apnea-Associated Intermittent Hypoxia-Induced Immune Responses in Males, Pregnancies, and Offspring.

Obstructive sleep apnea (OSA), a respiratory sleep disorder associated with cardiovascular diseases, is more prevalent in men. However, OSA occurrence in pregnant women rises to a level comparable to men during late gestation, creating persistent effects on both maternal and offspring health. The exact mechanisms behind OSA-induced cardiovascular diseases remain unclear, but inflammation and oxidative stress play a key role. Animal models using intermittent hypoxia (IH), a hallmark of OSA, reveal several pro-inflammatory signaling pathways at play in males, such as TLR4/MyD88/NF-κB/MAPK, miRNA/NLRP3, and COX signaling, along with shifts in immune cell populations and function. Limited evidence suggests similarities in pregnancies and offspring. In addition, suppressing these inflammatory molecules ameliorates IH-induced inflammation and tissue injury, providing new potential targets to treat OSA-associated cardiovascular diseases. This review will focus on the inflammatory mechanisms linking IH to cardiovascular dysfunction in males, pregnancies, and their offspring. The goal is to inspire further investigations into the understudied populations of pregnant females and their offspring, which ultimately uncover underlying mechanisms and therapeutic interventions for OSA-associated diseases.

Gestational intermittent hypoxia increases FosB-immunoreactive perikaryas in the paraventricular nucleus of the hypothalamus of adult male (but not female) rats.

Sleep-disordered breathing is a respiratory disorder commonly experienced by pregnant women. The recurrent hypoxaemic events associated with sleep-disordered breathing have deleterious consequences for the mother and fetus. Adult male (but not female) rats born to dams subjected to gestational intermittent hypoxia (GIH) have a higher resting blood pressure than control animals and show behavioural/neurodevelopmental disorders. The origin of this persistent, sex-specific effect of GIH in offspring is unknown, but disruption of the neuroendocrine stress pathways is a key mechanism by which gestational stress increases disease risk in progeny. Using FosB immunolabelling as a chronic marker of neuronal activation, we determined whether GIH augments basal expression of FosB in the perikaryas of cells in the paraventricular nucleus of the hypothalamus (PVN), a key structure in the regulation of the stress response and blood pressure. From gestational day 10, female rats were subjected to GIH for 8 h/day (light phase) until the day before delivery (gestational day 21); GIH consisted of 2 min hypoxic bouts (10.5% O2 ) alternating with normoxia. Control rats were exposed to intermittent normoxia over the same period (GNX). At adulthood (10-15 weeks), the brains of male and female rats were harvested for FosB immunohistochemistry. In males, GIH augmented PVN FosB labelling density by 30%. Conversely, PVN FosB density in GIH females was 28% lower than that of GNX females. We conclude that GIH has persistent and sex-specific impacts on the development of stress pathways, thereby offering a plausible mechanism by which GIH can disturb neural development and blood pressure homeostasis in adulthood. NEW FINDINGS: What is the central question of this study? In pregnant women, sleep apnoea increases the risk of disease for the offspring at various life stages. Given that gestational stress disrupts the programming of the stress pathways, we determined whether exposing female rats to gestational intermittent hypoxia (GIH) activates hypothalamic neurons regulating the stress response in adult rats. What is the main finding and its importance? Using FosB immunolabelling as a marker of marker of neuronal activation, we showed that GIH augmented basal activation of the paraventricular nucleus of the hypothalamus in males, but not females. Disruption of the stress pathways is a new hypothesis to explain the persistent and sex-specific impacts of GIH on offspring health.

Silent hypoxaemia in COVID-19 patients.

The clinical presentation of COVID-19 due to infection with SARS-CoV-2 is highly variable with the majority of patients having mild symptoms while others develop severe respiratory failure. The reason for this variability is unclear but is in critical need of investigation. Some COVID-19 patients have been labelled with 'happy hypoxia', in which patient complaints of dyspnoea and observable signs of respiratory distress are reported to be absent. Based on ongoing debate, we highlight key respiratory and neurological components that could underlie variation in the presentation of silent hypoxaemia and define priorities for subsequent investigation.

Sex- and Region-Specific Differences in the Transcriptomes of Rat Microglia from the Brainstem and Cervical Spinal Cord.

The neural control system underlying breathing is sexually dimorphic with males being more vulnerable to dysfunction. Microglia also display sex differences, and their role in the architecture of brainstem respiratory rhythm circuitry and modulation of cervical spinal cord respiratory plasticity is becoming better appreciated. To further understand the molecular underpinnings of these sex differences, we performed RNA sequencing of immunomagnetically isolated microglia from brainstem and cervical spinal cord of adult male and female rats. We used various bioinformatics tools (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome, STRING, MAGICTRICKS) to functionally categorize identified gene sets, as well as to pinpoint common transcriptional gene drivers that may be responsible for the observed transcriptomic differences. We found few sex differences in the microglial transcriptomes derived from the brainstem, but several hundred genes were differentially expressed by sex in cervical spinal microglia. Comparing brainstem and spinal microglia within and between sexes, we found that the major factor guiding transcriptomic differences was central nervous system (CNS) location rather than sex. We further identified key transcriptional drivers that may be responsible for the transcriptomic differences observed between sexes and CNS regions; enhancer of zeste homolog 2 emerged as the predominant driver of the differentially downregulated genes. We suggest that functional gene alterations identified in metabolism, transcription, and intercellular communication underlie critical microglial heterogeneity and sex differences in CNS regions that contribute to respiratory disorders categorized by dysfunction in neural control. These data will also serve as an important resource data base to advance our understanding of innate immune cell contributions to sex differences and the field of respiratory neural control. SIGNIFICANCE STATEMENT: The contributions of central nervous system (CNS) innate immune cells to sexually dimorphic differences in the neural circuitry controlling breathing are poorly understood. We identify key transcriptomic differences, and their transcriptional drivers, in microglia derived from the brainstem and the C3-C6 cervical spinal cord of healthy adult male and female rats. Gene alterations identified in metabolism, gene transcription, and intercellular communication likely underlie critical microglial heterogeneity and sex differences in these key CNS regions that contribute to the neural control of breathing.

Competing mechanisms of plasticity impair compensatory responses to repetitive apnoea.

KEY POINTS: Intermittent reductions in respiratory neural activity, a characteristic of many ventilatory disorders, leads to inadequate ventilation and arterial hypoxia. Both intermittent reductions in respiratory neural activity and intermittent hypoxia trigger compensatory enhancements in inspiratory output when experienced separately, forms of plasticity called inactivity-induced inspiratory motor facilitation (iMF) and long-term facilitation (LTF), respectively. Reductions in respiratory neural activity that lead to moderate, but not mild, arterial hypoxia occludes plasticity expression, indicating that concurrent induction of iMF and LTF impairs plasticity through cross-talk inhibition of their respective signalling pathways. Moderate hypoxia undermines iMF by enhancing NR2B-containing NMDA receptor signalling, which can be rescued by exogenous retinoic acid, a molecule necessary for iMF. These data suggest that in ventilatory disorders characterized by reduced inspiratory motor output, such as sleep apnoea, endogenous mechanisms of compensatory plasticity may be impaired, and that exogenously activating respiratory plasticity may be a novel strategy to improve breathing. ABSTRACT: Many forms of sleep apnoea are characterized by recurrent reductions in respiratory neural activity, which leads to inadequate ventilation and arterial hypoxia. Both recurrent reductions in respiratory neural activity and hypoxia activate mechanisms of compensatory plasticity that augment inspiratory output and lower the threshold for apnoea, inactivity-induced inspiratory motor facilitation (iMF) and long-term facilitation (LTF), respectively. However, despite frequent concurrence of reduced respiratory neural activity and hypoxia, mechanisms that induce and regulate iMF and LTF have only been studied separately. Here, we demonstrate that recurrent reductions in respiratory neural activity ('neural apnoea') accompanied by cessations in ventilation that result in moderate (but not mild) hypoxaemia do not elicit increased inspiratory output, suggesting that concurrent induction of iMF and LTF occludes plasticity. A key role for NMDA receptor activation in impairing plasticity following concurrent neural apnoea and hypoxia is indicated since recurrent hypoxic neural apnoeas triggered increased phrenic inspiratory output in rats in which spinal NR2B-containing NMDA receptors were inhibited. Spinal application of retinoic acid, a key molecule necessary for iMF, bypasses NMDA receptor-mediated constraints, thereby rescuing plasticity following hypoxic neural apnoeas. These studies raise the intriguing possibility that endogenous mechanisms of compensatory plasticity may be impaired in some individuals with sleep apnoea, and that exogenously activating pathways giving rise to respiratory plasticity may be a novel pharmacological strategy to improve breathing.

Intermittent apnea elicits inactivity-induced phrenic motor facilitation via a retinoic acid- and protein synthesis-dependent pathway.

Respiratory motoneuron pools must provide rhythmic inspiratory drive that is robust and reliable, yet dynamic enough to respond to respiratory challenges. One form of plasticity that is hypothesized to contribute to motor output stability by sensing and responding to inadequate respiratory neural activity is inactivity-induced phrenic motor facilitation (iPMF), an increase in inspiratory output triggered by a reduction in phrenic synaptic inputs. Evidence suggests that mechanisms giving rise to iPMF differ depending on the pattern of reduced respiratory neural activity (i.e., neural apnea). A prolonged neural apnea elicits iPMF via a spinal TNF-α-induced increase in atypical PKC activity, but little is known regarding mechanisms that elicit iPMF following intermittent neural apnea. We tested the hypothesis that iPMF triggered by intermittent neural apnea requires retinoic acid and protein synthesis. Phrenic nerve activity was recorded in urethane-anesthetized and -ventilated rats treated intrathecally with an inhibitor of retinoic acid synthesis (4-diethlyaminobenzaldehyde, DEAB), a protein synthesis inhibitor (emetine), or vehicle (artificial cerebrospinal fluid) before intermittent (5 episodes, ~1.25 min each) or prolonged (30 min) neural apnea. Both DEAB and emetine abolished iPMF elicited by intermittent neural apnea but had no effect on iPMF elicited by a prolonged neural apnea. Thus different patterns of reduced respiratory neural activity elicit phenotypically similar iPMF via distinct spinal mechanisms. Understanding mechanisms that allow respiratory motoneurons to dynamically tune their output may have important implications in the context of respiratory control disorders that involve varied patterns of reduced respiratory neural activity, such as central sleep apnea and spinal cord injury.NEW & NOTEWORTHY We identify spinal retinoic acid and protein synthesis as critical components in the cellular cascade whereby repetitive reductions in respiratory neural activity elicit rebound increases in phrenic inspiratory activity.

Disinhibition does not play a role in endomorphin-2-induced changes in inspiratory motoneuron output produced by in vitro neonatal rat preparations.

Low level activation of mu-opioid receptors (MORs) in neonatal rat brainstem-spinal cord preparations increases inspiratory burst amplitude recorded on cervical spinal roots. We tested whether: (1) MOR activation with an endogenous ligand, such as endomorphin-2, increases inspiratory burst amplitude, (2) disinhibition of GABAergic or glycinergic inhibitory synaptic transmission is involved, and (3) inflammation alters endomorphin-2 effects. Using neonatal rat (P0-P3) brainstem-spinal cord preparations, bath-applied endomorphin-2 (10-200 nM) increased inspiratory burst amplitude and decreased burst frequency. Blockade of GABAA receptors (picrotoxin), glycine receptors (strychnine), or both (picrotoxin and strychnine) did not abolish endomorphin-2-induced effects. In preparations isolated from neonatal rats injected 3 h previously with lipopolysaccharide (LPS, 0.1 mg/kg), endomorphin-2 continued to decrease burst frequency but abolished the burst amplitude increase. Collectively, these data indicate that disinhibition of inhibitory synaptic transmission is unlikely to play a role in endomorphin-2-induced changes in inspiratory motor output, and that different mechanisms underlie the endomorphin-2-induced increases in inspiratory burst amplitude and decreases in burst frequency.

Gestational Intermittent Hypoxia Enhances Mammary Stem Cells and Alters Tumor Phenotype in Adult Female Offspring.

An adverse perinatal environment can increase long-term cancer risk, although the precise nature of associated perinatal triggers remain unknown. Sleep apnea is a common condition during pregnancy, characterized by recurrent cessations in breathing during sleep, and the potential consequences of sleep apnea during pregnancy as it relates to breast cancer risk in offspring have not been explored. To model sleep apnea, Sprague-Dawley dams were exposed during gestation to nightly intermittent hypoxia (GIH) or normoxia (GNx), and the mammary glands of female offspring were examined. GIH offspring demonstrated increased epithelial stem and progenitor cell populations, which are associated with diminished transforming growth factor beta (TGFß) activity. Elevations in adipose tissue stem cells in the mammary gland were also identified in GIH offspring. In aging females, mammary tumors formed in GIH offspring. These tumors displayed a dramatic increase in stroma compared to tumors from GNx offspring, as well as distinct patterns of expression of stem cell-related pathways. Together, these results suggest that exposure to sleep apnea during pregnancy leads to lasting changes in the mammary glands of female offspring. Increased stem and progenitor cell populations as a result of GIH exposure could enhance long-term breast cancer risk, as well as alter the clinical behavior of resulting breast tumors.

Microglia regulate motor neuron plasticity via reciprocal fractalkine/adenosine signaling.

Microglia are innate CNS immune cells that play key roles in supporting key CNS functions including brain plasticity. We now report a previously unknown role for microglia in regulating neuroplasticity within spinal phrenic motor neurons, the neurons driving diaphragm contractions and breathing. We demonstrate that microglia regulate phrenic long-term facilitation (pLTF), a form of respiratory memory lasting hours after repetitive exposures to brief periods of low oxygen (acute intermittent hypoxia; AIH) via neuronal/microglial fractalkine signaling. AIH-induced pLTF is regulated by the balance between competing intracellular signaling cascades initiated by serotonin vs adenosine, respectively. Although brainstem raphe neurons release the relevant serotonin, the cellular source of adenosine is unknown. We tested a model in which hypoxia initiates fractalkine signaling between phrenic motor neurons and nearby microglia that triggers extracellular adenosine accumulation. With moderate AIH, phrenic motor neuron adenosine 2A receptor activation undermines serotonin-dominant pLTF; in contrast, severe AIH drives pLTF by a unique, adenosine-dominant mechanism. Phrenic motor neuron fractalkine knockdown, cervical spinal fractalkine receptor inhibition on nearby microglia, and microglial depletion enhance serotonin-dominant pLTF with moderate AIH but suppress adenosine-dominant pLTF with severe AIH. Thus, microglia play novel functions in the healthy spinal cord, regulating hypoxia-induced neuroplasticity within the motor neurons responsible for breathing.

Respiratory neuroplasticity: Mechanisms and translational implications of phrenic motor plasticity.

Widespread appreciation that neuroplasticity is an essential feature of the neural system controlling breathing has emerged only in recent years. In this chapter, we focus on respiratory motor plasticity, with emphasis on the phrenic motor system. First, we define related but distinct concepts: neuromodulation and neuroplasticity. We then focus on mechanisms underlying two well-studied models of phrenic motor plasticity: (1) phrenic long-term facilitation following brief exposure to acute intermittent hypoxia; and (2) phrenic motor facilitation after prolonged or recurrent bouts of diminished respiratory neural activity. Advances in our understanding of these novel and important forms of plasticity have been rapid and have already inspired translation in multiple respects: (1) development of novel therapeutic strategies to preserve/restore breathing function in humans with severe neurological disorders, such as spinal cord injury and amyotrophic lateral sclerosis; and (2) the discovery that similar plasticity also occurs in nonrespiratory motor systems. Indeed, the realization that similar plasticity occurs in respiratory and nonrespiratory motor neurons inspired clinical trials to restore leg/walking and hand/arm function in people living with chronic, incomplete spinal cord injury. Similar application may be possible to other clinical disorders that compromise respiratory and non-respiratory movements.

Gestational Intermittent Hypoxia Programs Hypertensive Response in Female Rat Offspring: Impact of Ovaries.

Obstructive sleep apnea (OSA) is a chronic condition frequently observed in pregnant women. We have shown that gestational intermittent hypoxia (GIH), a hallmark of OSA, leads to sex-specific impairment in the endothelium-dependent relaxation response and an increase in blood pressure in adult male but not female rat offspring. The present study tested the hypothesis that functional ovaries normalize GIH-induced hypertensive response in female offspring. Experiments were done in female offspring of pregnant rats exposed to normoxia or GIH (FIO2 21-10.5% from gestational days 10 to 21). Ovariectomy and sham surgery were performed at 5 weeks of age. Pups born to GIH dams were significantly smaller than the controls, but they exhibited catch-up growth and were similar to controls by 5 weeks of age. Ovariectomy significantly exacerbated bodyweight gain to a similar extent in both control and GIH offspring. Marked increases in blood pressure were observed in pre-pubertal GIH offspring compared to controls; however, after puberty, blood pressure in GIH offspring progressively decreased and became normotensive at adulthood. Ovariectomy led to the maintenance of higher blood pressure in post-pubertal GIH offspring with no significant effect in controls. Vascular contractile and relaxation responses were not affected in the GIH and control offspring; however, ovariectomy selectively decreased endothelium-dependent relaxation response along with a decrease in endothelial nitric oxide synthase expression in the GIH offspring. These findings suggest that functional ovaries are crucial in protecting females against GIH-mediated endothelial dysfunction and hypertension in adulthood.

Mu-opioid receptor-dependent transformation of respiratory motor pattern in neonates in vitro.

Endogenous opioid peptides activating mu-opioid receptors (MORs) are part of an intricate neuromodulatory system that coordinates and optimizes respiratory motor output to maintain blood-gas homeostasis. MOR activation is typically associated with respiratory depression but also has excitatory effects on breathing and respiratory neurons. We hypothesized that low level MOR activation induces excitatory effects on the respiratory motor pattern. Thus, low concentrations of an MOR agonist drug (DAMGO, 10-200 nM) were bath-applied to neonatal rat brainstem-spinal cord preparations while recording inspiratory-related motor output on cervical spinal roots (C4-C5). Bath-applied DAMGO (50-200 nM) increased inspiratory motor burst amplitude by 40-60% during (and shortly following) drug application with decreased burst frequency and minute activity. Reciprocal changes in inspiratory burst amplitude and frequency were balanced such that 20 min after DAMGO (50-200 nM) application, minute activity was unaltered compared to pre-DAMGO levels. The DAMGO-induced inspiratory burst amplitude increase did not require crossed cervical spinal pathways, was expressed on thoracic ventral spinal roots (T4-T8) and remained unaltered by riluzole pretreatment (blocks persistent sodium currents associated with gasping). Split-bath experiments showed that the inspiratory burst amplitude increase was induced only when DAMGO was bath-applied to the brainstem and not the spinal cord. Thus, MOR activation in neonates induces a respiratory burst amplitude increase via brainstem-specific mechanisms. The burst amplitude increase counteracts the expected MOR-dependent frequency depression and may represent a new mechanism by which MOR activation influences respiratory motor output.

Gestational Intermittent Hypoxia Induces Sex-Specific Impairment in Endothelial Mechanisms and Sex Steroid Hormone Levels in Male Rat Offspring.

Obstructive sleep apnea (OSA) is highly prevalent during gestation and is linked with adverse fetal outcomes. We examined whether gestational intermittent hypoxia (GIH), the main feature of OSA, leads to sex-specific alterations in cardiovascular function and vascular mechanisms in the offspring. Pregnant rats exposed to intermittent hypoxia or ambient air from gestation days 10 to 21 and their offspring were used for the study. GIH exposure did not affect water and food intake in dams. Compared to controls, the male and female offspring born to GIH dams were smaller in weight by 14% and 12%, respectively, and exhibited catch-up growth. Cardiac function was not affected in either GIH males or females. At 12 weeks of age, blood pressure was increased in GIH males, but not GIH females, compared to their control counterparts. While mesenteric arterial contractile responses to phenylephrine and endothelin were unaffected in GIH males and females, relaxation response to acetylcholine was reduced in GIH males but not GIH females. Relaxation to sodium nitroprusside was unaffected in both GIH males and females. Total eNOS expression was not affected, but phospho(Ser1177)-eNOS levels were decreased in GIH males. eNOS expression and its phosphorylation status were unaffected in GIH females. Serum testosterone and estradiol levels were higher in GIH males but were unaltered in GIH females. Together, these findings suggest that GIH leads to a sex-specific increase in blood pressure in adult male offspring with blunted endothelium-mediated relaxation, decreased eNOS activity, and elevated sex steroid hormone levels.

Retinoic acid receptor alpha activation is necessary and sufficient for plasticity induced by recurrent central apnea.

Reductions in respiratory-related synaptic inputs to inspiratory motor neurons initiate a form of plasticity that proportionally enhances inspiratory motor output, even in the absence of changing blood gases. This form of plasticity is known as inactivity-induced inspiratory motor facilitation (iMF). iMF triggered by brief, recurrent reductions in respiratory neural activity requires local retinoic acid (RA) synthesis, but receptor subtypes activated by RA are unknown. To test the hypothesis that retinoic acid receptor alpha (RARα) is necessary for iMF, RAR subtype-specific inhibitors were delivered intrathecally above the phrenic motor pool in urethane-anesthetized, ventilated rats before 5, ∼1 min central apneas (without hypoxia; separated by 5 min) while monitoring phrenic inspiratory output. Pretreatment with a spinal RARα inhibitor impaired the capacity for recurrent central apnea to trigger long-lasting increases in phrenic inspiratory output, but plasticity was expressed in rats pretreated with an RARß/γ inhibitor. Intrathecal RA application in the absence of reduced respiratory neural activity elicited an increase in phrenic inspiratory output, which was prevented by pretreatment with an RARα inhibitor. These data indicate that spinal RARα activation is necessary for iMF triggered by recurrent reductions in respiratory neural activity, and that RARα activation in/near the phrenic motor pool in the absence of respiratory neural activity deprivation is sufficient to elicit phrenic inspiratory motor facilitation. Understanding cellular cascades underlying plasticity induced by reductions in respiratory neural activity may define avenues for pharmacological intervention in disorders in which endogenous compensatory mechanisms that defend ongoing inspiratory motor output are impaired.NEW & NOTEWORTHY Local mechanisms near phrenic motor neurons respond to reductions in respiratory-related synaptic inputs by triggering a chemoreflex-independent, proportional enhancement in inspiratory output, a form of plasticity called inactivity-induced inspiratory motor facilitation (iMF). Here, we show that activation of spinal retinoic acid receptor alpha (RARα) is necessary to trigger phrenic iMF, and that spinal RARα activation in the absence of respiratory neural activity deprivation is sufficient to elicit phrenic inspiratory facilitation.

Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats.

Neuroplasticity is a fundamental property of the respiratory control system, enabling critical adaptations in breathing to meet the challenges, but little is known whether neonates express neuroplasticity similar to adults. We tested the hypothesis that, similar to adults, tyrosine receptor kinase B (TrkB) or adenosine A2a receptor activation in neonates are independently sufficient to elicit respiratory motor facilitation, and that co-induction of TrkB and A2a receptor-dependent plasticity undermines respiratory motor facilitation. TrkB receptor activation with 7,8-dihydroxyflavone (DHF) in neonatal brainstem-spinal cord preparations induced a long-lasting increase in respiratory motor output in 55 % of preparations, whereas adenosine A2a receptor activation with CGS21680 only sporadically induced respiratory motor plasticity. CGS21680 and DHF co-application prevented DHF-dependent respiratory motor facilitation, whereas co-application of MSX-3 (adenosine A2a receptor antagonist) and DHF more rapidly induced respiratory motor plasticity. Collectively, these data suggest that mechanisms underlying respiratory neuroplasticity may be only partially operational in early neonatal life, and that adenosine A2a receptor activation undermines TrkB-induced respiratory plasticity.

Respiratory frequency plasticity during development.

Respiratory frequency plasticity is a long-lasting increase in breathing frequency due to a perturbation. Mechanisms underlying respiratory frequency are poorly understood, and there is little evidence of frequency plasticity in neonates. This hybrid review/research article discusses available literature regarding frequency plasticity and highlights potential research opportunities. Also, we include data demonstrating a model of frequency plasticity using isolated neonatal rat brainstem-spinal cord preparations. Specifically, substance P (SubP) application induced a long-lasting (>60 min) increase in spontaneous respiratory motor burst frequency, particularly in brainstem-spinal cords with the pons attached; there were no male/female differences. SubP-induced frequency plasticity is dependent on the application pattern, such that intermittent (rather than sustained) SubP applications induce more frequency plasticity. SubP-induced frequency plasticity was blocked by a neurokinin-1 receptor antagonist. Thus, the newborn rat respiratory control system has the capacity to express frequency plasticity. Identifying mechanisms that induce frequency plasticity may lead to novel methods to safely treat breathing disorders in premature and newborn infants.

Cervical spinal contusion alters Na+-K+-2Cl- and K+-Cl- cation-chloride cotransporter expression in phrenic motor neurons.

Spinal chloride-dependent synaptic inhibition is critical in regulating breathing and requires neuronal chloride gradients established by cation-chloride cotransporters Na+-K+-2Cl- (NKCC1) and K+-Cl- (KCC2). Spinal transection disrupts NKCC1/KCC2 balance, diminishing chloride gradients in neurons below injury, contributing to spasticity and chronic pain. It is not known if similar disruptions in NKCC1/KCC2 balance occur in respiratory motor neurons after incomplete cervical contusion (C2SC). We hypothesized that C2SC disrupts NKCC1/KCC2 balance in phrenic motor neurons. NKCC1 and KCC2 immunoreactivity was assessed in CtB-positive phrenic motor neurons. Five weeks post-C2SC: 1) neither membrane-bound nor cytosolic NKCC1 expression were significantly changed, although the membrane/cytosolic ratio increased, consistent with net chloride influx; and 2) both membrane and cytosolic KCC2 expression increased, although the membrane/cytosolic ratio decreased, consistent with net chloride efflux. Thus, contrary to our original hypothesis, complex shifts in NKCC1/KCC2 balance occur post-C2SC. The functional significance of these changes remains unclear.