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.

Inaugural Review Prize 2023: The exercise hyperpnoea dilemma: A 21st-century perspective.

During mild or moderate exercise, alveolar ventilation increases in direct proportion to metabolic rate, regulating arterial CO2 pressure near resting levels. Mechanisms giving rise to the hyperpnoea of exercise are unsettled despite over a century of investigation. In the past three decades, neuroscience has advanced tremendously, raising optimism that the 'exercise hyperpnoea dilemma' can finally be solved. In this review, new perspectives are offered in the hope of stimulating original ideas based on modern neuroscience methods and current understanding. We first describe the ventilatory control system and the challenge exercise places upon blood-gas regulation. We highlight relevant system properties, including feedforward, feedback and adaptive (i.e., plasticity) control of breathing. We then elaborate a seldom explored hypothesis that the exercise ventilatory response continuously adapts (learns and relearns) throughout life and ponder if the memory 'engram' encoding the feedforward exercise ventilatory stimulus could reside within the cerebellum. Our hypotheses are based on accumulating evidence supporting the cerebellum's role in motor learning and the numerous direct and indirect projections from deep cerebellar nuclei to brainstem respiratory neurons. We propose that cerebellar learning may be obligatory for the accurate and adjustable exercise hyperpnoea capable of tracking changes in life conditions/experiences, and that learning arises from specific cerebellar microcircuits that can be interrogated using powerful techniques such as optogenetics and chemogenetics. Although this review is speculative, we consider it essential to reframe our perspective if we are to solve the till-now intractable exercise hyperpnoea dilemma.

Mild inflammation impairs acute intermittent hypoxia-induced phrenic long-term facilitation by a spinal adenosine-dependent mechanism.

Inflammation undermines neuroplasticity, including serotonin-dependent phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH: 3, 5-min episodes, arterial Po2: 40-50 mmHg; 5-min intervals). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist, lipopolysaccharide (LPS; 100 µg/kg, ip), abolishes mAIH-induced pLTF by unknown mechanisms. In the central nervous system, neuroinflammation primes glia, triggering ATP release and extracellular adenosine accumulation. As spinal adenosine 2 A (A2A) receptor activation impairs mAIH-induced pLTF, we hypothesized that spinal adenosine accumulation and A2A receptor activation are necessary in the mechanism whereby LPS impairs pLTF. We report that 24 h after LPS injection in adult male Sprague Dawley rats: 1) adenosine levels increase in ventral spinal segments containing the phrenic motor nucleus (C3-C5; P = 0.010; n = 7/group) and 2) cervical spinal A2A receptor inhibition (MSX-3, 10 µM, 12 µL intrathecal) rescues mAIH-induced pLTF. In LPS vehicle-treated rats (saline, ip), MSX-3 enhanced pLTF versus controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; P = 0.002; n = 6/group). In LPS-treated rats, pLTF was abolished as expected (4 ± 6% baseline; n = 6), but intrathecal MSX-3 restored pLTF to levels equivalent to MSX-3-treated control rats (120 ± 14% baseline; P < 0.001; n = 6; vs. LPS controls with MSX-3: P = 0.539). Thus, inflammation abolishes mAIH-induced pLTF by a mechanism that requires increased spinal adenosine levels and A2A receptor activation. As repetitive mAIH is emerging as a treatment to improve breathing and nonrespiratory movements in people with spinal cord injury or ALS, A2A inhibition may offset undermining effects of neuroinflammation associated with these neuromuscular disorders.NEW & NOTEWORTHY Mild inflammation undermines motor plasticity elicited by mAIH. In a model of mAIH-induced respiratory motor plasticity (phrenic long-term facilitation; pLTF), we report that inflammation induced by low-dose lipopolysaccharide undermines mAIH-induced pLTF by a mechanism requiring increased cervical spinal adenosine and adenosine 2 A receptor activation. This finding advances the understanding of mechanisms impairing neuroplasticity, potentially undermining the ability to compensate for the onset of lung/neural injury or to harness mAIH as a therapeutic modality.

BDNF-induced phrenic motor facilitation shifts from PKCθ to ERK dependence with mild systemic inflammation.

Moderate acute intermittent hypoxia (mAIH) elicits a form of phrenic motor plasticity known as phrenic long-term facilitation (pLTF), which requires spinal 5-HT2 receptor activation, ERK/MAP kinase signaling, and new brain-derived neurotrophic factor (BDNF) synthesis. New BDNF protein activates TrkB receptors that normally signal through PKCθ to elicit pLTF. Phrenic motor plasticity elicited by spinal drug administration (e.g., BDNF) is referred to by a more general term: phrenic motor facilitation (pMF). Although mild systemic inflammation elicited by a low lipopolysaccharide (LPS) dose (100 µg/kg; 24 h prior) undermines mAIH-induced pLTF upstream from BDNF protein synthesis, it augments pMF induced by spinal BDNF administration through unknown mechanisms. Here, we tested the hypothesis that mild inflammation shifts BDNF/TrkB signaling from PKCθ to alternative pathways that enhance pMF. We examined the role of three known signaling pathways associated with TrkB (MEK/ERK MAP kinase, PI3 kinase/Akt, and PKCθ) in BDNF-induced pMF in anesthetized, paralyzed, and ventilated Sprague Dawley rats 24 h post-LPS. Spinal PKCθ inhibitor (TIP) attenuated early BDNF-induced pMF (≤30 min), with minimal effect 60-90 min post-BDNF injection. In contrast, MEK inhibition (U0126) abolished BDNF-induced pMF at 60 and 90 min. PI3K/Akt inhibition (PI-828) had no effect on BDNF-induced pMF at any time. Thus, whereas BDNF-induced pMF is exclusively PKCθ-dependent in normal rats, MEK/ERK is recruited by neuroinflammation to sustain, and even augment downstream plasticity. Because AIH is being developed as a therapeutic modality to restore breathing in people living with multiple neurological disorders, it is important to understand how inflammation, a common comorbidity in many traumatic or degenerative central nervous system disorders, impacts phrenic motor plasticity.NEW & NOTEWORTHY We demonstrate that even mild systemic inflammation shifts signaling mechanisms giving rise to BDNF-induced phrenic motor plasticity. This finding has important experimental, biological, and translational implications, particularly since BDNF-dependent spinal plasticity is being translated to restore breathing and nonrespiratory movements in diverse clinical disorders, such as spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS).

Acute intermittent hypercapnic-hypoxia elicits central neural respiratory motor plasticity in humans.

Acute intermittent hypoxia (AIH) elicits long-term facilitation (LTF) of respiration. Although LTF is observed when CO2 is elevated during AIH in awake humans, the influence of CO2 on corticospinal respiratory motor plasticity is unknown. Thus, we tested the hypotheses that acute intermittent hypercapnic-hypoxia (AIHH): (1) enhances cortico-phrenic neurotransmission (reflecting volitional respiratory control); and (2) elicits ventilatory LTF (reflecting automatic respiratory control). Eighteen healthy adults completed four study visits. Day 1 consisted of anthropometry and pulmonary function testing. On Days 2, 3 and 4, in a balanced alternating sequence, participants received: AIHH, poikilocapnic AIH, and normocapnic-normoxia (Sham). Protocols consisted of 15, 60 s exposures with 90 s normoxic intervals. Transcranial (TMS) and cervical (CMS) magnetic stimulation were used to induce diaphragmatic motor-evoked potentials and compound muscle action potentials, respectively. Respiratory drive was assessed via mouth occlusion pressure (P0.1 ), and minute ventilation measured at rest. Dependent variables were assessed at baseline and 30-60 min after exposures. Increases in TMS-evoked diaphragm potential amplitudes were observed following AIHH vs. Sham (+28 ± 41%, P = 0.003), but not after AIH. No changes were observed in CMS-evoked diaphragm potential amplitudes. Mouth occlusion pressure also increased after AIHH (+21 ± 34%, P = 0.033), but not after AIH. Ventilatory LTF was not observed after any treatment. We demonstrate that AIHH elicits central neural mechanisms of respiratory motor plasticity and increases resting respiratory drive in awake humans. These findings may have important implications for neurorehabilitation after spinal cord injury and other neuromuscular disorders compromising breathing. KEY POINTS: The occurrence of respiratory long-term facilitation following acute exposure to intermittent hypoxia is believed to be dependent upon CO2 regulation - mechanisms governing the critical role of CO2 have seldom been explored. We tested the hypothesis that acute intermittent hypercapnic-hypoxia (AIHH) enhances cortico-phrenic neurotransmission in awake healthy humans. The amplitude of diaphragmatic motor-evoked potentials induced by transcranial magnetic stimulation was increased after AIHH, but not the amplitude of compound muscle action potentials evoked by cervical magnetic stimulation. Mouth occlusion pressure (P0.1 , an indicator of neural respiratory drive) was also increased after AIHH, but not tidal volume or minute ventilation. Thus, moderate AIHH elicits central neural mechanisms of respiratory motor plasticity, without measurable ventilatory long-term facilitation in awake humans.

Acute morphine blocks spinal respiratory motor plasticity via long-latency mechanisms that require toll-like receptor 4 signalling.

KEY POINTS: While respiratory complications following opioid use are mainly mediated via activation of mu opioid receptors, long-latency off-target signalling via innate immune toll-like receptor 4 (TLR4) may impair other essential elements of breathing control such as respiratory motor plasticity. In adult rats, pre-treatment with a single dose of morphine blocked long-term facilitation (LTF) of phrenic motor output via a long-latency TLR4-dependent mechanism. In the phrenic motor nucleus, morphine triggered TLR4-dependent activation of microglial p38 MAPK - a key enzyme that orchestrates inflammatory signalling and is known to undermine phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. Therefore, we suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy. ABSTRACT: Opioid-induced respiratory dysfunction is a significant public health burden. While respiratory effects are mediated via mu opioid receptors, long-latency off-target opioid signalling through innate immune toll-like receptor 4 (TLR4) may modulate essential elements of breathing control, particularly respiratory motor plasticity. Plasticity in respiratory motor circuits contributes to the preservation of breathing in the face of destabilizing influences. For example, respiratory long-term facilitation (LTF), a well-studied model of respiratory motor plasticity triggered by acute intermittent hypoxia, promotes breathing stability by increasing respiratory motor drive to breathing muscles. Some forms of respiratory LTF are exquisitely sensitive to inflammation and are abolished by even a mild inflammation triggered by TLR4 activation (e.g. via systemic lipopolysaccharides). Since opioids induce inflammation and TLR4 activation, we hypothesized that opioids would abolish LTF through a TLR4-dependent mechanism. In adult Sprague Dawley rats, pre-treatment with a single systemic injection of the prototypical opioid agonist morphine blocks LTF expression several hours later in the phrenic motor system - the motor pool driving diaphragm muscle contractions. Morphine blocked phrenic LTF via TLR4-dependent mechanisms because pre-treatment with (+)-naloxone - the opioid inactive stereoisomer and novel small molecule TLR4 inhibitor - prevented impairment of phrenic LTF in morphine-treated rats. Morphine triggered TLR4-dependent activation of microglial p38 MAPK within the phrenic motor system - a key enzyme that orchestrates inflammatory signalling and undermines phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. We suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy by restoring endogenous mechanisms of plasticity within respiratory motor circuits.

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.

The influence of intermittent hypoxia, obesity, and diabetes on male genitourinary anatomy and voiding physiology.

We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.

Mechanisms of severe acute intermittent hypoxia-induced phrenic long-term facilitation.

Moderate acute intermittent hypoxia (mAIH; 35-55 mmHg PaO2) elicits phrenic long-term facilitation (pLTF) by a mechanism that requires activation of Gq protein-coupled serotonin type 2 receptors, MEK/ERK MAP kinase, and NADPH oxidase activity and is constrained by cAMP-PKA signaling. In contrast, severe AIH (sAIH; 25-35 mmHg PaO2) elicits Gs protein-coupled adenosine type 2 A receptor-dependent pLTF. Another Gs protein-coupled receptor, serotonin 7 receptors, elicits phrenic motor facilitation (pMF) by a mechanism that requires exchange protein activated by cyclic AMP (EPAC) and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) activation and is constrained by NADPH oxidase activity. Here, we tested the hypothesis that the same downstream signaling mechanisms giving rise to serotonin 7 (vs. serotonin 2) receptor-induced pMF underlie sAIH-induced pLTF. In anesthetized rats, sAIH-induced pLTF was compared after pretreatment with intrathecal (C4) injections of inhibitors for: 1) EPAC (ESI-05); 2) MEK/ERK (UO126); 3) PKA (KT-5720); 4) PI3K/Akt (PI828); and 5) NADPH oxidase (apocynin). In partial agreement with our hypothesis, sAIH-induced pLTF was abolished by ESI-05 and PI828 and marginally enhanced by apocynin but, surprisingly, was abolished by UO126 and attenuated by KT-5720. Mechanisms of sAIH-induced pLTF reflect elements of both Gq and Gs pathways to pMF, likely as a consequence of the complex, cross-talk interactions between them.NEW & NOTEWORTHY Distinct mechanisms give rise to pLTF induced by moderate and severe AIH. We demonstrate that, unlike moderate AIH, severe AIH-induced pLTF requires EPAC and PI3K/Akt and is marginally constrained by NADPH oxidase activity. Surprisingly, sAIH-induced pLTF requires MEK/ERK activity similar to moderate AIH-induced pLTF and is reduced by PKA inhibition. We suggest sAIH-induced pLTF arises from complex interactions between dominant mechanisms characteristic of moderate versus severe AIH-induced pLTF.

Daily acute intermittent hypoxia enhances phrenic motor output and stimulus-evoked phrenic responses in rats.

Plasticity is a hallmark of the respiratory neural control system. Phrenic long-term facilitation (pLTF) is one form of respiratory plasticity characterized by persistent increases in phrenic nerve activity following acute intermittent hypoxia (AIH). Although there is evidence that key steps in the cellular pathway giving rise to pLTF are localized within phrenic motor neurons (PMNs), the impact of AIH on the strength of breathing-related synaptic inputs to PMNs remains unclear. Furthermore, the functional impact of AIH is enhanced by repeated/daily exposure to AIH (dAIH). Here, we explored the effects of AIH versus 2 wk of dAIH preconditioning on spontaneous and evoked phrenic responses in anesthetized, paralyzed, and mechanically ventilated rats. Evoked phrenic potentials were elicited by respiratory cycle-triggered lateral funiculus stimulation at the C2 spinal level delivered before and 60 min post-AIH (or the equivalent in time controls). Charge-balanced biphasic pulses (100 µs/phase) of progressively increasing intensity (100-700 µA) were delivered during the inspiratory and expiratory phases of the respiratory cycle. Although robust pLTF (∼60% from baseline) was observed after a single exposure to moderate AIH (3 × 5 min; 5-min intervals), there was no effect on evoked phrenic responses, contrary to our initial hypothesis. However, in rats preconditioned with dAIH, baseline phrenic nerve activity and evoked responses were increased, suggesting that repeated exposure to AIH enhances functional synaptic strength when assessed using this technique. The impact of daily AIH preconditioning on synaptic inputs to PMNs raises interesting questions that require further exploration.NEW & NOTEWORTHY Two weeks of daily acute intermittent hypoxia (dAIH) preconditioning enhanced stimulus-evoked phrenic responses to lateral funiculus stimulation (targeting respiratory bulbospinal projection to phrenic motor neurons). Furthermore, dAIH preconditioning enhanced baseline phrenic motor output responses to maximal chemoreflex activation in intact rats.

Systemic inflammation suppresses spinal respiratory motor plasticity via mechanisms that require serine/threonine protein phosphatase activity.

BACKGROUND: Inflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system-a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)-a key enzyme necessary for the expression of phrenic motor plasticity. METHODS: To study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons. RESULTS: We confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 µg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators. CONCLUSIONS: This study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.

Circadian clock genes and respiratory neuroplasticity genes oscillate in the phrenic motor system.

Circadian rhythms are endogenous and entrainable daily patterns of physiology and behavior. Molecular mechanisms underlie circadian rhythms, characterized by an ~24-h pattern of gene expression of core clock genes. Although it has long been known that breathing exhibits circadian rhythms, little is known concerning clock gene expression in any element of the neuromuscular system controlling breathing. Furthermore, we know little concerning gene expression necessary for specific respiratory functions, such as phrenic motor plasticity. Thus, we tested the hypotheses that transcripts for clock genes (Bmal1, Clock, Per1, and Per2) and molecules necessary for phrenic motor plasticity (Htr2a, Htr2b, Bdnf, and Ntrk2) oscillate in regions critical for phrenic/diaphragm motor function via RT-PCR. Tissues were collected from male Sprague-Dawley rats entrained to a 12-h light-dark cycle at 4 zeitgeber times (ZT; n = 8 rats/group): ZT5, ZT11, ZT17, and ZT23; ZT0 = lights on. Here, we demonstrate that 1) circadian clock genes (Bmal1, Clock, Per1, and Per2) oscillate in regions critical for phrenic/diaphragm function, including the caudal medulla, ventral C3-C5 cervical spinal cord, and diaphragm; 2) the clock protein BMAL1 is localized within CtB-labeled phrenic motor neurons; 3) genes necessary for intermittent hypoxia-induced phrenic/diaphragm motor plasticity (Htr2b and Bdnf) oscillate in the caudal medulla and ventral C3-C5 spinal cord; and 4) there is higher intensity of immunofluorescent BDNF protein within phrenic motor neurons at ZT23 compared with ZT11 (n = 11 rats/group). These results suggest local circadian clocks exist in the phrenic motor system and confirm the potential for local circadian regulation of neuroplasticity and other elements of the neural network controlling breathing.

Synergy between Acute Intermittent Hypoxia and Task-Specific Training.

Acute intermittent hypoxia (AIH) and task-specific training (TST) synergistically improve motor function after spinal cord injury; however, mechanisms underlying this synergistic relation are unknown. We propose a hypothetical working model of neural network and cellular elements to explain AIH-TST synergy. Our goal is to forecast experiments necessary to advance our understanding and optimize the neurotherapeutic potential of AIH-TST.

Protein kinase Cδ constrains the S-pathway to phrenic motor facilitation elicited by spinal 5-HT7 receptors or severe acute intermittent hypoxia.

KEY POINTS: Concurrent 5-HT2A (Q pathway) and 5-HT7 (S pathway) serotonin receptor activation cancels phrenic motor facilitation due to mutual cross-talk inhibition. Spinal protein kinase Cδ (PKCδ) or protein kinase A inhibition restores phrenic motor facilitation with concurrent Q and S pathway activation, demonstrating a key role for these kinases in cross-talk inhibition. Spinal PKCδ inhibition enhances adenosine-dependent severe acute intermittent hypoxia-induced phrenic long-term facilitation (S pathway), consistent with relief of cross-talk inhibition. ABSTRACT: Intermittent spinal serotonin receptor activation elicits long-lasting phrenic motor facilitation (pMF), a form of respiratory motor plasticity. When activated alone, spinal Gq protein-coupled serotonin 2A receptors (5-HT2A ) initiate pMF by a mechanism that requires ERK-MAP kinase signalling and new BDNF protein synthesis (Q pathway). Spinal Gs protein-coupled serotonin 7 (5-HT7 ) and adenosine 2A (A2A ) receptor activation also elicits pMF, but via distinct mechanisms (S pathway) that require Akt signalling and new TrkB protein synthesis. Although studies have shown inhibitory cross-talk interactions between these competing pathways, the underlying cellular mechanisms are unknown. We propose the following hypotheses: (1) concurrent 5-HT2A and 5-HT7 activation undermines pMF; (2) protein kinase A (PKA) and (3) NADPH oxidase mediate inhibitory interactions between Q (5-HT2A ) and S (5-HT7 ) pathways. Selective 5-HT2A (DOI hydrochloride) and 5HT7 (AS-19) agonists were administered intrathecally at C4 (three injections, 5-min intervals) in anaesthetized, vagotomized and ventilated male rats. With either spinal 5-HT2A or 5-HT7 activation alone, phrenic amplitude progressively increased (pMF). In contrast, concurrent 5-HT2A and 5-HT7 activation failed to elicit pMF. The 5-HT2A -induced Q pathway was restored by inhibiting PKA activity (Rp-8-Br-cAMPS). NADPH oxidase inhibition did not prevent cross-talk inhibition. Therefore, we investigated alternative mechanisms to explain Q to S pathway inhibition. Spinal protein kinase C (PKC) inhibition with Gö6983 or PKCδ peptide inhibitor restored the 5-HT7 -induced S pathway to pMF, revealing PKCδ as the relevant isoform. Spinal PKCδ inhibition enhanced the S pathway-dependent form of pMF elicited by severe acute intermittent hypoxia. We suggest that powerful constraints between 5-HT2A and 5-HT7 or A2A receptor-induced pMF are mediated by PKCδ and PKA, respectively.

Mechanisms of Enhanced Phrenic Long-Term Facilitation in SOD1G93A Rats.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease, causing muscle paralysis and death from respiratory failure. Effective means to preserve/restore ventilation are necessary to increase the quality and duration of life in ALS patients. At disease end-stage in a rat ALS model (SOD1G93A ), acute intermittent hypoxia (AIH) restores phrenic nerve activity to normal levels via enhanced phrenic long-term facilitation (pLTF). Mechanisms enhancing pLTF in end-stage SOD1G93A rats are not known. Moderate AIH-induced pLTF is normally elicited via cellular mechanisms that require the following: Gq-protein-coupled 5-HT2 receptor activation, new BDNF synthesis, and MEK/ERK signaling (the Q pathway). In contrast, severe AIH elicits pLTF via a distinct mechanism that requires the following: Gs-protein-coupled adenosine 2A receptor activation, new TrkB synthesis, and PI3K/Akt signaling (the S pathway). In end-stage male SOD1G93A rats and wild-type littermates, we investigated relative Q versus S pathway contributions to enhanced pLTF via intrathecal (C4) delivery of small interfering RNAs targeting BDNF or TrkB mRNA, and MEK/ERK (U0126) or PI3 kinase/Akt (PI828) inhibitors. In anesthetized, paralyzed and ventilated rats, moderate AIH-induced pLTF was abolished by siBDNF and UO126, but not siTrkB or PI828, demonstrating that enhanced pLTF occurs via the Q pathway. Although phrenic motor neuron numbers were decreased in end-stage SOD1G93A rats (∼30% survival; p < 0.001), BDNF and phosphorylated ERK expression were increased in spared phrenic motor neurons (p < 0.05), consistent with increased Q-pathway contributions to pLTF. Our results increase understanding of respiratory plasticity and its potential to preserve/restore breathing capacity in ALS.SIGNIFICANCE STATEMENT Since neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), end life via respiratory failure, the ability to harness respiratory motor plasticity to improve breathing capacity could increase the quality and duration of life. In a rat ALS model (SOD1G93A ) we previously demonstrated that spinal respiratory motor plasticity elicited by acute intermittent hypoxia is enhanced at disease end-stage, suggesting greater potential to preserve/restore breathing capacity. Here we demonstrate that enhanced intermittent hypoxia-induced phrenic motor plasticity results from amplification of normal cellular mechanisms versus addition/substitution of alternative mechanisms. Greater understanding of mechanisms underlying phrenic motor plasticity in ALS may guide development of new therapies to preserve and/or restore breathing in ALS patients.

Phrenic motor neuron adenosine 2A receptors elicit phrenic motor facilitation.

KEY POINTS: Although adenosine 2A (A2A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A2A receptor siRNA injections. A2A receptor siRNA injections selectively knocked down A2A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced phrenic motor facilitation. Upregulation of A2A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury. ABSTRACT: Cervical spinal adenosine 2A (A2A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A2A receptors for pMF are unknown. This is an important question since the physiological outcome of A2A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A2A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A2A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A2A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A2A receptor knock-down was verified by a ∼45% decrease in A2A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P < 0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A2A receptor agonist CGS21680 was greatly attenuated in siA2A (21%) versus siNT treated rats (147%; P < 0.01). There were no significant effects of siA2A on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A2A receptors relevant to A2A receptor-induced pMF.

Systemic inflammation inhibits serotonin receptor 2-induced phrenic motor facilitation upstream from BDNF/TrkB signaling.

Although systemic inflammation induced by even a low dose of lipopolysaccharide (LPS, 100 µg/kg) impairs respiratory motor plasticity, little is known concerning cellular mechanisms giving rise to this inhibition. Phrenic motor facilitation (pMF) is a form of respiratory motor plasticity elicited by pharmacological agents applied to the cervical spinal cord, or by acute intermittent hypoxia (AIH; 3, 5-min hypoxic episodes); when elicited by AIH, pMF is known as phrenic long-term facilitation (pLTF). AIH consisting of moderate hypoxic episodes (mAIH, arterial Po2 = 35-55 mmHg) elicits pLTF via the Q pathway to pMF, a mechanism that requires spinal serotonin (5HT2) receptor activation and new brain-derived neurotrophic factor (BDNF) protein synthesis. Although mild systemic inflammation attenuates mAIH-induced pLTF via spinal p38 MAP kinase activation, little is known concerning how p38 MAP kinase activity inhibits the Q pathway. Here, we confirmed that 24 h after a low LPS dose (100 µg/kg ip), mAIH-induced pLTF is greatly attenuated. Similarly, pMF elicited by intrathecal cervical injections of 5HT2A (DOI; 100 µM; 3 × 6 µl) or 5HT2B receptor agonists (BW723C86; 100 µM; 3 × 6 µl) is blocked 24 h post-LPS. When pMF was elicited by intrathecal BDNF (100 ng, 12 µl), pMF was actually enhanced 24 h post-LPS. Thus 5HT2A/2B receptor-induced pMF is impaired downstream from 5HT2 receptor activation, but upstream from BDNF/TrkB signaling. Mechanisms whereby LPS augments BDNF-induced pMF are not yet known. NEW & NOTEWORTHY These experiments give novel insights concerning mechanisms whereby systemic inflammation undermines serotonin-dependent, spinal respiratory motor plasticity, yet enhances brain-derived neurotrophic factor (BDNF)/TrkB signaling in phrenic motor neurons. These insights may guide development of new strategies to elicit functional recovery of breathing capacity in patients with respiratory impairment by reducing (or bypassing) the impact of systemic inflammation characteristic of clinical disorders that compromise breathing.

Cross-talk inhibition between 5-HT2B and 5-HT7 receptors in phrenic motor facilitation via NADPH oxidase and PKA.

Intermittent spinal serotonin receptor activation elicits phrenic motor facilitation (pMF), a form of spinal respiratory motor plasticity. Episodic activation of either serotonin type 2 (5-HT2) or type 7 (5-HT7) receptors elicits pMF, although they do so via distinct cellular mechanisms known as the Q (5-HT2) and S (5-HT7) pathways to pMF. When coactivated, these pathways interact via mutual cross-talk inhibition. Although we have a rudimentary understanding of mechanisms mediating cross-talk interactions between spinal 5-HT2 subtype A (5-HT2A) and 5-HT7 receptor activation, we do not know if similar interactions exist between 5-HT2 subtype B (5-HT2B) and 5-HT7 receptors. We confirmed that either spinal 5-HT2B or 5-HT7 receptor activation alone elicits pMF and tested the hypotheses that 1) concurrent activation of both receptors suppresses pMF due to cross-talk inhibition; 2) 5-HT7 receptor inhibition of 5-HT2B receptor-induced pMF requires protein kinase A (PKA) activity; and 3) 5-HT2B receptor inhibition of 5-HT7 receptor-induced pMF requires NADPH oxidase (NOX) activity. Selective 5-HT2B and 5-HT7 receptor agonists were administered intrathecally at C4 (3 injections, 5-min intervals) to anesthetized, paralyzed, and ventilated rats. Whereas integrated phrenic nerve burst amplitude increased after selective spinal 5-HT2B or 5-HT7 receptor activation alone (i.e., pMF), pMF was no longer observed with concurrent 5-HT2B and 5-HT7 receptor agonist administration. With concurrent receptor activation, pMF was rescued by inhibiting either NOX or PKA activity, demonstrating their roles in cross-talk inhibition between these pathways to pMF. This report demonstrates cross-talk inhibition between 5-HT2B- and 5-HT7 receptor-induced pMF and that NOX and PKA activity are necessary for that cross-talk inhibition.

Sustained Hypoxia Elicits Competing Spinal Mechanisms of Phrenic Motor Facilitation.

UNLABELLED: Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal motor plasticity. Competing mechanisms give rise to phrenic motor facilitation (pMF; a general term including pLTF) depending on the severity of hypoxia within episodes. In contrast, moderate acute sustained hypoxia (mASH) does not elicit pMF. By varying the severity of ASH and targeting competing mechanisms of pMF, we sought to illustrate why moderate AIH (mAIH) elicits pMF but mASH does not. Although mAIH elicits serotonin-dependent pLTF, mASH does not; thus, mAIH-induced pLTF is pattern sensitive. In contrast, severe AIH (sAIH) elicits pLTF through adenosine-dependent mechanisms, likely from greater extracellular adenosine accumulation. Because serotonin- and adenosine-dependent pMF interact via cross talk inhibition, we hypothesized that pMF is obscured because the competing mechanisms of pMF are balanced and offsetting during mASH. Here, we demonstrate the following: (1) blocking spinal A2A receptors with MSX-3 reveals mASH-induced pMF; and (2) sASH elicits A2A-dependent pMF. In anesthetized rats pretreated with intrathecal A2A receptor antagonist injections before mASH (PaO2 = 40-54 mmHg) or sASH (PaO2 = 25-36 mmHg), (1) mASH induced a serotonin-dependent pMF and (2) sASH induced an adenosine-dependent pMF, which was enhanced by spinal serotonin receptor inhibition. Thus, competing adenosine- and serotonin-dependent mechanisms contribute differentially to pMF depending on the pattern/severity of hypoxia. Understanding interactions between these mechanisms has clinical relevance as we develop therapies to treat severe neuromuscular disorders that compromise somatic motor behaviors, including breathing. Moreover, these results demonstrate how competing mechanisms of plasticity can give rise to pattern sensitivity in pLTF. SIGNIFICANCE STATEMENT: Intermittent hypoxia elicits pattern-sensitive spinal plasticity and improves motor function after spinal injury or during neuromuscular disease. Specific mechanisms of pattern sensitivity in this form of plasticity are unknown. We provide evidence that competing mechanisms of phrenic motor facilitation mediated by adenosine 2A and serotonin 2 receptors are differentially expressed, depending on the pattern/severity of hypoxia. Understanding how these distinct mechanisms interact during hypoxic exposures differing in severity and duration will help explain interesting properties of plasticity, such as pattern sensitivity, and may help optimize therapies to restore motor function in patients with neuromuscular disorders that compromise movement.

Spinal BDNF-induced phrenic motor facilitation requires PKCθ activity.

Spinal brain-derived neurotrophic factor (BDNF) is necessary and sufficient for certain forms of long-lasting phrenic motor facilitation (pMF). BDNF elicits pMF by binding to its high-affinity receptor, tropomyosin receptor kinase B (TrkB), on phrenic motor neurons, potentially activating multiple downstream signaling cascades. Canonical BDNF/TrkB signaling includes the 1) Ras/RAF/MEK/ERK MAP kinase, 2) phosphatidylinositol 3-kinase (PI3K)/Akt, and 3) PLCγ/PKC pathways. Here we demonstrate that spinal BDNF-induced pMF requires PLCγ/PKCθ in normal rats but not MEK/ERK or PI3K/Akt signaling. Cervical intrathecal injections of MEK/ERK (U0126) or PI3K/Akt (PI-828; 100 µM, 12 µl) inhibitor had no effect on BDNF-induced pMF (90 min after BDNF; U0126 + BDNF: 59 ± 14%, PI-828 + BDNF: 59 ± 8%, inhibitor vehicle + BDNF: 56 ± 7%; all P ≥ 0.05). In contrast, PKCθ inhibition with theta inhibitory peptide (TIP; 0.86 mM, 12 µl) prevented BDNF-induced pMF (90 min after BDNF; TIP + BDNF: -2 ± 2%; P ≤ 0.05 vs. other groups). Thus BDNF-induced pMF requires downstream PLCγ/PKCθ signaling, contrary to initial expectations.NEW AND NOTEWORTHY We demonstrate that BDNF-induced pMF requires downstream signaling via PKCθ but not MEK/ERK or PI3K/Akt signaling. These data are essential to understand the sequence of the cellular cascade leading to BDNF-dependent phrenic motor plasticity.