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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.

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).

Cervical spinal cord injury (cSCI) impairs neural drive to the respiratory muscles, causing life- threatening complications such as respiratory insufficiency and diminished airway protection. Repetitive "low dose" acute intermittent hypoxia (AIH) is a promising strategy to restore motor function in people with chronic SCI. Conversely, "high dose" chronic intermittent hypoxia (CIH; ∼8 h/night), such as experienced during sleep apnea, causes pathology. Sleep apnea, spinal ischemia, hypoxia and neuroinflammation associated with cSCI increase extracellular adenosine concentrations and activate spinal adenosine receptors which in turn constrains the functional benefits of therapeutic AIH. Adenosine 1 and 2A receptors (A

Acute intermittent hypoxia (AIH) elicits long-term facilitation (LTF) of respiration. Although LTF is observed when CO

Moderate acute intermittent hypoxia (mAIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long-term facilitation (pLTF). In middle-aged male and geriatric female rats, mAIH-induced pLTF is attenuated through unknown mechanisms. In young adults, mAIH activates competing intracellular signaling cascades, initiated by serotonin 2 and adenosine 2A (A2A) receptors, respectively. Since spinal A2A receptor inhibition enhances mAIH-induced pLTF, serotonin dominates, and adenosine constrains mAIH-induced plasticity in the daily rest phase, we hypothesized elevated basal adenosine levels in the ventral cervical spinal cord of aged rats shifts this balance, undermining mAIH-induced pLTF. A selective A2A receptor antagonist (MSX-3) or vehicle were delivered intrathecally at C4 in anesthetized young (3-6 months) and aged (20-22 months) Sprague-Dawley rats prior to mAIH (3,5-min episodes; arterial PO2=45-55mmHg). In young males, spinal A2A receptor inhibition enhanced pLTF (119 ± 5%) versus vehicle (55 ± 9%), consistent with prior reports. In old males, pLTF was reduced (25 ± 11%), but A2A receptor inhibition increased pLTF to levels greater than in young males (186 ± 19%). Basal adenosine levels in ventral C3-C5 homogenates are elevated 2-3-fold in old versus young males. These findings advance our understanding of age as a biological variable in phrenic motor plasticity and will help guide translation of mAIH as a therapeutic modality to restore respiratory and non-respiratory movements in older populations afflicted with clinical disorders that compromise movement.

RationaleAcute intermittent hypoxia (AIH) is a promising strategy to induce functional motor recovery following chronic spinal cord injuries and neurodegenerative diseases. Although significant results are obtained, human AIH trials report considerable inter-individual response variability.ObjectivesIdentify individual factors (e.g., genetics, age, and sex) that determine response magnitude of healthy adults to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH).MethodsAssociations of individual factors with the magnitude of AIHH (15, 1-min O2=9.5%, CO2=5% episodes) induced changes in diaphragm motor-evoked potential amplitude (MEP) and inspiratory mouth occlusion pressures (P0.1) were evaluated in 17 healthy individuals (age=27±5 years) compared to Sham. Single nucleotide polymorphisms (SNPs) in genes linked with mechanisms of AIH induced phrenic motor plasticity (BDNF, HTR2A, TPH2, MAOA, NTRK2) and neuronal plasticity (apolipoprotein E,APOE) were tested. Variations in AIHH induced plasticity with age and sex were also analyzed. Additional experiments in humanized (h)ApoEknock-in rats were performed to test causality.ResultsAIHH-induced changes in diaphragm MEP amplitudes were lower in individuals heterozygous forAPOE4(i.e., APOE3/4) alleleversusotherAPOEgenotypes (p=0.048). No significant differences were observed between any other SNPs investigated, notablyBDNFval/met(all p>0.05). Males exhibited a greater diaphragm MEP enhancementversusfemales, regardless of age (p=0.004). Age was inversely related with change in P0.1within the limited age range studied (p=0.007). InhApoE4knock-in rats, AIHH-induced phrenic motor plasticity was significantly lower than hApoE3controls (pConclusionsAPOE4genotype, sex and age are important biological determinants of AIHH-induced respiratory motor plasticity in healthy adults.ADDITION TO KNOWLEDGE BASEAcute intermittent hypoxia (AIH) is a novel rehabilitation strategy to induce functional recovery of respiratory and non-respiratory motor systems in people with chronic spinal cord injury and/or neurodegenerative diseases. Since most AIH trials report considerable inter-individual variability in AIH outcomes, we investigated factors that potentially undermine the response to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH), in healthy humans. We demonstrate that genetics (particularly the lipid transporter,APOE), age and sex are important biological determinants of AIHH-induced respiratory motor plasticity.

Plasticity is a fundamental property of the neuromotor system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity after exposure to intermittent low oxygen or acute intermittent hypoxia (AIH). pLTF can arise from distinct intracellular signaling cascades initiated by serotonin and adenosine; these cascades interact via powerful crosstalk inhibition. We demonstrate the serotonin/adenosine balance varies dramatically with time-of-day and details of the AIH protocol. Using a “standard” AIH protocol, the mechanism driving pLTF shifts from serotonin-dominant, adenosine-constrained during rest, to adenosine-dominant, serotonin-constrained in the active phase. This mechanistic ‘flip’ results from daily changes in basal spinal adenosine levels across time-of-day combined with hypoxia-evoked spinal adenosine release. Since AIH is emerging as a promising therapeutic modality to restore respiratory (and non-respiratory) movements in people with spinal injury or ALS, new knowledge that time-of-day and protocol details impact mechanisms driving pLTF has experimental, biological and translational implications.

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.

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.

We used male BTBR mice carrying the Lep(ob) 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 Lep(ob/ob) (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O(2) 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.

“Low-dose” acute intermittent hypoxia (AIH; 3-15 episodes/day) is emerging as a promising therapeutic strategy to improve motor function after incomplete cervical spinal cord injury (cSCI). Conversely, chronic “high-dose” intermittent hypoxia (CIH; > 80–100 episodes/day) elicits multi-system pathology and is a hallmark of sleep apnea, a condition highly prevalent in individuals with cSCI. Whereas daily AIH (dAIH) enhances phrenic motor plasticity in intact rats, it is abolished by CIH. However, there have been no direct comparisons of prolonged dAIH versus CIH on phrenic motor outcomes after chronic cSCI. Thus, phrenic nerve activity and AIH-induced phrenic long-term facilitation (pLTF) were assessed in anesthetized rats. Experimental groups included: 1) intact rats exposed to 28 days of normoxia (Nx28; 21% O(2); 8 h/day), and three groups with chronic C2 hemisection (C2Hx) exposed to either: 2) Nx28; 3) dAIH (dAIH28; 10, 5-min episodes of 10.5% O(2)/day; 5-min intervals); or 4) CIH (IH28-2/2; 2-min episodes; 2-min intervals; 8 h/day). Baseline ipsilateral phrenic nerve activity was reduced in injured versus intact rats but unaffected by dAIH28 or IH28-2/2. There were no group differences in contralateral phrenic activity. pLTF was enhanced bilaterally by dAIH28 versus Nx28 but unaffected by IH28-2/2. Whereas dAIH28 enhanced pLTF after cSCI, it did not improve baseline phrenic output. In contrast, unlike shorter protocols in intact rats, CIH28-2/2 did not abolish pLTF in chronic C2Hx. Mechanisms of differential responses to dAIH versus CIH are not yet known, particularly in the context of cSCI. Further, it remains unclear whether enhanced phrenic motor plasticity can improve breathing after cSCI.

Moderate acute intermittent hypoxia (mAIH; 35–55 mmHg Pa(O(2))) elicits phrenic long-term facilitation (pLTF) by a mechanism that requires activation of G(q) 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 Pa(O(2))) elicits G(s) protein-coupled adenosine type 2 A receptor-dependent pLTF. Another G(s) 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 G(q) and G(s) 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.

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.

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the central nervous system (CNS) such that most therapeutics lack efficacy against brain tumors or neurological disorders due to their inability to cross the BBB. Therefore, developing new drug delivery platforms to facilitate drug transport to the CNS and understanding their mechanism of transport are crucial for the efficacy of therapeutics. Here, we report (i) carbon dots prepared from glucose and conjugated to fluorescein (GluCD-F) cross the BBB in zebrafish and rats without the need of an additional targeting ligand and (ii) uptake mechanism of GluCDs is glucose transporter-dependent in budding yeast. Glucose transporter-negative strain of yeast showed undetectable GluCD accumulation unlike the glucose transporter-positive yeast, suggesting glucose-transporter-dependent GluCD uptake. We tested GluCDs' ability to cross the BBB using both zebrafish and rat models. Following the injection to the heart, wild-type zebrafish showed GluCD-F accumulation in the central canal consistent with the transport of GluCD-F across the BBB. In rats, following intravenous administration, GluCD-F was observed in the CNS. GluCD-F was localized in the gray matter (e.g. ventral horn, dorsal horn, and middle grey) of the cervical spinal cord consistent with neuronal accumulation. Therefore, neuron targeting GluCDs hold tremendous potential as a drug delivery platform in neurodegenerative disease, traumatic injury, and malignancies of the CNS., Glucose-based carbon dots (GluCDs) can cross blood–brain barrier in zebrafish and rat after intravenous injections and accumulate in neurons in rat CNS. Cell uptake of GluCDs involve glucose transporter proteins in a budding yeast model.

The diaphragmatic motor-evoked potential (MEP) induced by transcranial magnetic stimulation (TMS) permits electrophysiological assessment of the cortico-diaphragmatic pathway. Despite the value of TMS for investigating diaphragm motor integrity in health and disease, reliability of the technique has not been established. The study aim was to determine within- and between-session reproducibility of surface electromyogram recordings of TMS-evoked diaphragm potentials. Fifteen healthy young adults participated (6 females, age = 29 ± 7 yr). Diaphragm activation was determined by gradually increasing the stimulus intensity from 60 to 100% of maximal stimulator output (MSO). A minimum of seven stimulations were performed at each intensity. A second block of stimuli was delivered 30 min later for within-day comparisons, and a third block was performed on a separate day for between-day comparisons. Reliability of diaphragm MEPs was assessed at 100% MSO using intraclass correlation coefficients (ICC) and 95% limits of agreement (LOA). MEP latency (ICC = 0.984, P < 0.001), duration (ICC = 0.958, P < 0.001), amplitude (ICC = 0.950, P < 0.001), and area (ICC = 0.956, P < 0.001) were highly reproducible within-day. Between-day reproducibility was good to excellent for all MEP characteristics (latency ICC = 0.953, P < 0.001; duration ICC = 0.879, P = 0.002; amplitude ICC = 0.789, P = 0.019; area ICC = 0.815, P = 0.012). Data revealed less precision between-day versus within-day, as evidenced by wider LOA for all MEP characteristics. Large within- and between-subject variability in MEP amplitude and area was observed. In conclusion, TMS is a reliable means of inducing diaphragm potentials in most healthy individuals. NEW & NOTEWORTHY Transcranial magnetic stimulation (TMS) is a noninvasive technique to assess neural impulse conduction along the cortico-diaphragmatic pathway. The reliability of diaphragm motor-evoked potentials (MEP) induced by TMS is unknown. Notwithstanding large variability in MEP amplitude, we found good-to-excellent reproducibility of all MEP characteristics (latency, duration, amplitude, and area) both within- and between-day in healthy adult men and women. Our findings support the use of TMS and surface EMG to assess diaphragm activation in humans.

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.

Intermittent hypoxia elicits protocol-dependent effects on hypoglossal (XII) motor plasticity. Whereas low-dose, acute intermittent hypoxia (AIH) elicits serotonin-dependent plasticity in XII motor neurons, high-dose, chronic intermittent hypoxia (CIH) elicits neuroinflammation that undermines AIH-induced plasticity. Preconditioning with repeated AIH and mild CIH enhance AIH-induced XII motor plasticity. Since intermittent hypoxia pre-conditioning could enhance serotonin-dependent XII motor plasticity by increasing serotonergic innervation density of the XII motor nuclei, we tested the hypothesis that 3 distinct intermittent hypoxia protocols commonly studied to elicit plasticity (AIH) or simulate aspects of sleep apnea (CIH) differentially affect XII serotonergic innervation. Sleep apnea and associated CIH are common in people with cervical spinal injuries and, since repetitive AIH is emerging as a promising therapeutic strategy to improve respiratory and non-respiratory motor function after spinal injury, we also tested the hypotheses that XII serotonergic innervation is increased by repetitive AIH and/or CIH in rats with cervical C2 hemisections (C2Hx). Serotonergic innervation was assessed via immunofluorescence in male Sprague Dawley rats, with and without C2Hx (beginning 8 weeks post-injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5-min 10.5% O2 episodes per day; 5-min normoxic intervals); 3) mild CIH (5-min 10.5% O2 episodes; 5-min intervals; 8 h/day); and 4) moderate CIH (2-min 10.5% O2 episodes; 2-min intervals; 8 h/day). Daily AIH, but neither CIH protocol, increased the area of serotonergic immunolabeling in the XII motor nuclei in both intact and injured rats. C2Hx per se had no effect on XII serotonergic innervation density. Thus, daily AIH may increases XII serotonergic innervation and function, enhancing the capacity for serotonin-dependent, AIH-induced plasticity in upper airway motor neurons. Such effects may preserve upper airway patency and/or swallowing ability in people with cervical spinal cord injuries and other clinical disorders that compromise breathing and airway defense.

We review progress towards greater mechanistic understanding and clinical translation of a strategy to improve respiratory and non-respiratory motor function in people with neuromuscular disorders, therapeutic acute intermittent hypoxia (tAIH). In 2016 and 2020, workshops to create and update a "road map to clinical translation" were held to help guide future research and development of tAIH to restore movement in people living with chronic, incomplete spinal cord injuries. After briefly discussing the pioneering, non-targeted basic research inspiring this novel therapeutic approach, we then summarize workshop recommendations, emphasizing critical knowledge gaps, priorities for future research effort, and steps needed to accelerate progress as we evaluate the potential of tAIH for routine clinical use. Highlighted areas include: 1) greater mechanistic understanding, particularly in non-respiratory motor systems; 2) optimization of tAIH protocols to maximize benefits; 3) identification of combinatorial treatments that amplify plasticity or remove plasticity constraints, including task-specific training; 4) identification of biomarkers for individuals most/least likely to benefit from tAIH; 5) assessment of long-term tAIH safety; and 6) development of a simple, safe and effective device to administer tAIH in clinical and home settings. Finally, we update ongoing clinical trials and recent investigations of tAIH in SCI and other clinical disorders that compromise motor function, including ALS, multiple sclerosis, and stroke.

Acute intermittent hypoxia (AIH) elicits phrenic motor plasticity via multiple distinct cellular mechanisms. With moderate AIH, phrenic motor facilitation (pMF) requires Gq protein-coupled serotonin type 2 receptor activation, ERK MAP kinase activity, and new synthesis of brain-derived neurotrophic factor. In contrast, severe AIH elicits pMF by an adenosine-dependent mechanism that requires exchange protein activated by cAMP, Akt, and mammalian target of rapamycin (mTOR) activity, followed by new tyrosine receptor kinase B protein synthesis; this same pathway is also initiated by Gs protein-coupled serotonin 7 receptors (5-HT7). Because the metabolic sensor AMP-activated protein kinase (AMPK) inhibits mTOR-dependent protein synthesis, and mTOR signaling is necessary for 5-HT7 but not 5-HT2 receptor-induced pMF, we hypothesized that spinal AMPK activity differentially regulates pMF elicited by these distinct receptor subtypes. Serotonin type 2A receptor [5-HT2A; (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride] or 5-HT7 (AS-19) receptor agonists were administered intrathecally at C4 (3 injections, 5-min intervals) while recording integrated phrenic nerve activity in anesthetized, vagotomized, paralyzed, and ventilated rats. Consistent with our hypothesis, spinal AMPK activation with 2-deoxyglucose or metformin blocked 5-HT7, but not 5-HT2A receptor-induced pMF; in both cases, pMF inhibition was reversed by spinal administration of the AMPK inhibitor compound C. Thus, AMPK differentially regulates cellular mechanisms of serotonin-induced phrenic motor plasticity. NEW & NOTEWORTHY Spinal AMP-activated protein kinase (AMPK) overactivity, induced by local 2-deoxyglucose or metformin administration, constrains serotonin 7 (5-HT7) receptor-induced (but not serotonin type 2A receptor-induced) respiratory motor facilitation, indicating that metabolic challenges might regulate specific forms of respiratory motor plasticity. Pharmacological blockade of spinal AMPK activity restores 5-HT7 receptor-induced respiratory motor facilitation in the presence of either 2-deoxyglucose or metformin, showing that AMPK is an important regulator of 5-HT7 receptor-induced respiratory motor plasticity.

Serotonin (5-HT) is a key regulator of spinal respiratory motor plasticity. For example, spinal 5-HT receptor activation is necessary for the induction of phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity triggered by moderate acute intermittent hypoxia (mAIH). mAIH-induced pLTF is blocked by cervical spinal application of the broad-spectrum 5-HT-receptor antagonist, methysergide. However, methysergide does not allow distinctions between the relative contributions of different 5-HT receptor subtypes. Intravenous administration of the Gq protein-coupled 5-HT2A/2C receptor antagonist ketanserin blocks mAIH-induced pLTF when administered before, but not after, mAIH; thus, 5-HT2 receptor activation is necessary for the induction but not maintenance of mAIH-induced pLTF. However, systemic ketanserin administration does not identify the site of the relevant 5-HT2A/2C receptors. Furthermore, this approach does not differentiate between the roles of 5-HT2A versus 5-HT2C receptors, nor does it preclude involvement of other Gq protein-coupled metabotropic 5-HT receptors capable of eliciting long-lasting phrenic motor facilitation, such as 5-HT2B receptors. Here we tested the hypothesis that mAIH-induced pLTF requires cervical spinal 5-HT2 receptor activation and determined which 5-HT2 receptor subtypes are involved. Anesthetized, paralyzed, and ventilated adult male Sprague Dawley rats were pretreated intrathecally with cervical (~C3-C5) spinal injections of subtype selective 5-HT2A/2C, 5-HT2B, or 5-HT2C receptor antagonists before mAIH. Whereas cervical spinal 5-HT2C receptor inhibition had no impact on mAIH-induced pLTF, pLTF was no longer observed after pretreatment with either 5-HT2A/2C or 5-HT2B receptor antagonists. Furthermore, spinal pretreatment with an MEK/ERK MAPK inhibitor blocked phrenic motor facilitation elicited by intrathecal injections of 5-HT2A but not 5-HT2B receptor agonists. Thus, mAIH-induced pLTF requires concurrent cervical spinal activation of both 5-HT2A and 5-HT2B receptors. However, these distinct receptor subtypes contribute to phrenic motor facilitation via distinct downstream signaling cascades that differ in their requirement for ERK MAPK signaling. The demonstration that both 5-HT2A and 5-HT2B receptors make unique contributions to mAIH-induced pLTF advances our understanding of mechanisms that underlie 5-HT-induced phrenic motor plasticity. NEW & NOTEWORTHY Moderate acute intermittent hypoxia (mAIH) triggers a persistent enhancement in phrenic motor output, an effect termed phrenic long-term facilitation (pLTF). mAIH-induced pLTF is blocked by cervical spinal application of the broad-spectrum serotonin (5-HT) receptor antagonist methysergide, demonstrating the need for spinal 5-HT receptor activation. However, the exact type of 5-HT receptors required for initiation of pLTF remains unknown. To the best of our knowledge, the present study is the first to demonstrate that 1) spinal coactivation of two distinct Gq protein-coupled 5-HT2 receptor subtypes is necessary for mAIH-induced pLTF, and 2) these receptors contribute to pLTF via cascades that differ in their requirement for ERK MAPK signaling.

Acute intermittent hypoxia (AIH) elicits distinct mechanisms of phrenic motor plasticity initiated by brainstem neural network activation versus local (spinal) tissue hypoxia. With moderate AIH (mAIH), hypoxemia activates the carotid body chemoreceptors and (subsequently) brainstem neural networks associated with the peripheral chemoreflex, including medullary raphe serotonergic neurons. Serotonin release and receptor activation in the phrenic motor nucleus then elicits phrenic long-term facilitation (pLTF). This mechanism is independent of tissue hypoxia, since electrical carotid sinus nerve stimulation elicits similar serotonin-dependent pLTF. In striking contrast, severe AIH (sAIH) evokes a spinal adenosine-dependent, serotonin-independent mechanism of pLTF. Spinal tissue hypoxia per se is the likely cause of sAIH-induced pLTF, since local tissue hypoxia elicits extracellular adenosine accumulation. Thus, any physiological condition exacerbating spinal tissue hypoxia is expected to shift the balance towards adenosinergic pLTF. However, since these mechanisms compete for dominance due to mutual cross-talk inhibition, the transition from serotonin to adenosine dominant pLTF is rather abrupt. Any factor that compromises spinal cord circulation will limit oxygen availability in spinal cord tissue, favoring a shift in the balance towards adenosinergic mechanisms. Such shifts may arise experimentally from treatments such as carotid denervation, or spontaneous hypotension or anemia. Many neurological disorders, such as spinal cord injury or stroke compromise local circulatory control, potentially modulating tissue oxygen, adenosine levels and, thus, phrenic motor plasticity. In this brief review, we discuss the concept that local (spinal) circulatory control and/or oxygen delivery regulates the relative contributions of distinct pathways to phrenic motor plasticity.

After spinal cord injury (SCI) respiratory complications are a leading cause of morbidity and mortality. Acute intermittent hypoxia (AIH) triggers spinal respiratory motor plasticity in rodent models, and repetitive AIH may have the potential to restore breathing capacity in those with SCI. As an initial approach to provide proof of principle for such effects, we tested single-session AIH effects on breathing function in adults with chronic SCI. 17 adults (13 males; 34.1±14.5 years old; 13 motor complete SCI; >6 months post injury) completed two randomly ordered sessions, AIH versus sham. AIH consisted of 15, 1-minute episodes (hypoxia: 10.3% O(2); sham: 21% O(2)) interspersed with room air breathing (1.5 minutes, 21% oxygen); no attempt was made to regulate arterial CO(2) levels. Blood oxygen saturation (SpO(2)), maximal inspiratory and expiratory pressures (MIP; MEP), forced vital capacity (FVC), and mouth occlusion pressure within 0.1s (P(0.1)) were assessed. Outcomes were compared using nonparametric Wilcoxon’s tests, or a 2×2 ANOVA. Baseline SpO(2) was 97.2 ± 1.3% and was unchanged during sham experiments. During hypoxic episodes, SpO(2) decreased to 84.7 ± 0.9%, and returned to baseline levels during normoxic intervals. Outcomes were unchanged from baseline post-sham. Greater increases in MIP were evident post AIH vs. sham (median values; +10.8 cmH(2)O vs. −2.6 cmH(2)O respectively, 95% confidence interval (−18.7) – (−4.3), p=.006) with a moderate Cohen’s effect size (0.68). P(0.1), MEP and FVC did not change post-AIH. A single AIH session increased maximal inspiratory pressure generation, but not other breathing functions in adults with SCI. Reasons may include greater spared innervation to inspiratory versus expiratory muscles or differences in the capacity for AIH-induced plasticity in inspiratory motor neuron pools. Based on our findings, the therapeutic potential of AIH on breathing capacity in people with SCI warrants further investigation.

Spinal cord injuries (SCI) disrupt neural pathways between the brain and spinal cord, causing impairment of motor function and loss of independent mobility. Spontaneous plasticity in spared neural pathways improves function but is often insufficient to restore normal function. One unique approach to augment plasticity in spinal synaptic pathways is acute intermittent hypoxia (AIH), meaning brief exposure to mild bouts of low oxygen, interspersed with normoxia. While the administration of AIH elicits rapid plasticity and enhances volitional somatic motor output in the lower-limbs of people with incomplete SCI, it is not known if AIH-induced neuroplasticity is equally prevalent in spinal motor pathways regulating upper-extremity motor-function. In addition, how long the motor effects are retained following AIH has not yet been established. The goal of this research was to investigate changes in hand strength and upper-limb function elicited by episodic hypoxia, and to establish how long these effects were sustained in persons with incomplete cervical SCI. We conducted a randomized, blinded, placebo-controlled and cross-over design study consisting of a single AIH or sham AIH session in 14 individuals with chronic, incomplete cervical SCI. In a subset of six participants, we also performed a second protocol to determine the cumulative effects of repetitive AIH (i.e., two consecutive days). In both protocols, hand dynamometry and clinical performance tests were performed pre- and post-exposure. We found that a single AIH session enhanced bilateral grip and pinch strength, and that this effect peaked ~3 h post-intervention. The strength change was substantially higher after AIH versus sham AIH. These findings demonstrate the potential of AIH to improve upper-extremity function in persons with chronic SCI, although follow-up studies are needed to investigate optimal dosage and duration of effect.

An important model of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic burst amplitude following acute intermittent hypoxia (AIH). Moderate AIH elicits pLTF by a serotonin-dependent mechanism known as the Q pathway to phrenic motor facilitation. In contrast, severe AIH (greater hypoxemia) increases spinal adenosine accumulation and activates phrenic motor neuron adenosine 2A receptors, thereby initiating a distinct mechanism of plasticity known as the S pathway. Since the Q and S pathways interact via mutual cross-talk inhibition, the balance between spinal serotonin release and adenosine accumulation is an important pLTF regulator. Spinal injury decreases spinal tissue oxygen pressure (PtO(2)) caudal to injury. Since AIH is being explored as a neurotherapeutic to restore breathing ability after cervical spinal injury, we tested the hypothesis that decreased PtO(2) in the phrenic motor nucleus after C2 spinal hemisection (C2Hx) undermines moderate AIH-induced pLTF, likely due to shifts in the adenosine/serotonin balance. We recorded C3/4 ventral cervical PtO(2) with an optode, and bilateral phrenic nerve activity in anesthetized, paralyzed and ventilated rats, with and without C2Hx. In intact rats, PtO(2) was lower during severe versus moderate AIH as expected. In chronic C2Hx rats (> 8 weeks post-injury), PtO(2) was lower during baseline and moderate hypoxic episodes, approaching severe AIH levels in intact rats. After C2Hx, pLTF was blunted ipsilateral, but observed contralateral to injury. We conclude that C2Hx compromises PtO(2) near the phrenic motor nucleus and undermines pLTF, presumably due to a shift in the serotonin versus adenosine balance during hypoxic episodes. These findings have important implications for optimizing AIH protocols in our efforts to restore breathing ability with therapeutic AIH in people with chronic cervical spinal injury.

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.

Acute intermittent hypoxia (AIH) and task-specific training (TST) synergistically improve motor function after spinal cord injury; however, mechanisms underlying this synergistic relationship 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.