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, known as inactivity-induced phrenic motor facilitation (iPMF). Facilitation also occurs transiently in inspiratory intercostal nerve activity following inactivity (iIMF). Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and relevant PKC isoform are unknown. Here, we used RNA interference to test the hypothesis that the atypical PKCζ isoform within phrenic motor neurons is necessary for iPMF, but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural siRNA injections were made in rats to knock down phrenic and intercostal motor neuron PKCζ mRNA (siPKCζ). Control rats received non-targeting siRNA (NTsi) or siPKCθ; PKCθ is required for other forms of respiratory motor plasticity. Phrenic nerve and external intercostal (T2) EMG activity were measured in anesthetized, mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e. neural apnea) from modest hypocapnia, or a similar duration without neural apnea (time control). Phrenic burst amplitude increased from baseline with NTsi (68±10%) and siPKCθ (57±8%) 60 min post-neural apnea versus time controls (-3±3%). In contrast, siPKCζ virtually abolished iPMF (5±4%). While iIMF was transient in all groups, siPKCζ attenuated iIMF 5 min post-neural apnea (50±21%) vs NTsi (97±22%) and siPKCθ (103±20%). Neural inactivity elevated phrenic, but not intercostal responses to hypercapnia--an effect blocked by siPKCζ. We conclude phrenic motor neuron PKCζ is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for transient iIMF.

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.

Cardiorespiratory Responses to Acute Intermittent Hypoxia in Humans With Chronic Spinal Cord Injury.

Brief exposure to repeated episodes of low inspired oxygen, or acute intermittent hypoxia (AIH), is a promising therapeutic modality to improve motor function after chronic, incomplete spinal cord injury (SCI). Although therapeutic AIH is under extensive investigation in persons with SCI, limited data are available concerning cardiorespiratory responses during and after AIH exposure despite implications for AIH safety and tolerability. Thus, we recorded immediate (during treatment) and enduring (up to 30 min post-treatment) cardiorespiratory responses to AIH in 19 participants with chronic SCI (>1 year post-injury; injury levels C1 to T6; American Spinal Injury Association Impairment Scale A to D; mean age = 33.8 ± 14.1 years; 18 males). Participants completed a single AIH (15, 60-sec episodes, inspired O2 ≈ 10%; 90-sec intervals breathing room air) and Sham (inspired O2 ≈ 21%) treatment, in random order. During hypoxic episodes: (1) arterial oxyhemoglobin saturation decreased to 82.1 ± 2.9% (p < 0.001); (2) minute ventilation increased 3.83 ± 2.29 L/min (p = 0.008); and (3) heart rate increased 4.77 ± 6.82 bpm (p = 0.010). Considerable variability in cardiorespiratory responses was found among subjects; some individuals exhibited large hypoxic ventilatory responses (≥0.20 L/min/%, n = 11), whereas others responded minimally (<0.20 L/min/%, n = 8). Apneas occurred frequently during AIH and/or Sham protocols in multiple participants. All participants completed AIH treatment without difficulty. No significant changes in ventilation, heart rate, or arterial blood pressure were found 30 min post-AIH p > 0.05). In conclusion, therapeutic AIH is well tolerated, elicits variable chemoreflex activation, and does not cause persistent changes in cardiorespiratory control/function 30 min post-treatment in persons with chronic SCI.

A Research Protocol to Study the Priming Effects of Breathing Low Oxygen on Enhancing Training-Related Gains in Walking Function for Persons With Spinal Cord Injury: The BO2ST Trial.

Brief episodes of low oxygen breathing (therapeutic acute intermittent hypoxia; tAIH) may serve as an effective plasticity-promoting primer to enhance the effects of transcutaneous spinal stimulation-enhanced walking therapy (WALKtSTIM) in persons with chronic (>1 year) spinal cord injury (SCI). Pre-clinical studies in rodents with SCI show that tAIH and WALKtSTIM therapies harness complementary mechanisms of plasticity to maximize walking recovery. Here, we present a multi-site clinical trial protocol designed to examine the influence of tAIH + WALKtSTIM on walking recovery in persons with chronic SCI. We hypothesize that daily (eight sessions, 2 weeks) tAIH + WALKtSTIM will elicit faster, more persistent improvements in walking recovery than either treatment alone. To test our hypothesis, we are conducting a placebo-controlled clinical trial on 60 SCI participants who randomly receive one of three interventions: tAIH + WALKtSTIM; Placebo + WALKtSTIM; and tAIH + WALKtSHAM. Participants receive daily tAIH (fifteen 90-sec episodes at 10% O2 with 60-sec intervals at 21% O2) or daily placebo (fifteen 90-sec episodes at 21% O2 with 60-sec intervals at 21% O2) before a 45-min session of WALKtSTIM or WALKtSHAM. Our primary outcome measures assess walking speed (10-Meter Walk Test), endurance (6-Minute Walk Test), and balance (Timed Up and Go Test). For safety, we also measure pain levels, spasticity, sleep behavior, cognition, and rates of systemic hypertension and autonomic dysreflexia. Assessments occur before, during, and after sessions, as well as at 1, 4, and 8 weeks post-intervention. Results from this study extend our understanding of the functional benefits of tAIH priming by investigating its capacity to boost the neuromodulatory effects of transcutaneous spinal stimulation on restoring walking after SCI. Given that there is no known cure for SCI and no single treatment is sufficient to overcome walking deficits, there is a critical need for combinatorial treatments that accelerate and anchor walking gains in persons with lifelong SCI. Trial Registration: ClinicalTrials.gov, NCT05563103.

Progressive tauopathy disrupts breathing stability and chemoreflexes during presumptive sleep in mice.

Rationale: Although sleep apnea occurs in over 50% of individuals with Alzheimer's Disease (AD) or related tauopathies, little is known concerning the potential role of tauopathy in the pathogenesis of sleep apnea. Here, we tested the hypotheses that, during presumptive sleep, a murine model of tauopathy (rTg4510) exhibits: 1) increased breathing instability; 2) impaired chemoreflex function; and 3) exacerbation of these effects with tauopathy progression. Methods: rTg4510 mice initially develop robust tauopathy in the hippocampus and cortex, and eventually progresses to the brainstem. Type I and II post-sigh apnea, Type III (spontaneous) apnea, sigh, and hypopnea incidence were measured in young adult (5-6 months; n = 10-14/group) and aged (13-15 months; n = 22-24/group) non-transgenic (nTg), monogenic control tetracycline transactivator, and bigenic rTg4510 mice using whole-body plethysmography during presumptive sleep (i.e., eyes closed, curled/laying posture, stable breathing for >200 breaths) while breathing room air (21% O2). Peripheral and central chemoreceptor sensitivity were assessed with transient exposures (5 min) to hyperoxia (100% O2) or hypercapnia (3% and 5% CO2 in 21% O2), respectively. Results: We report significant increases in Type I, II, and III apneas (all p < 0.001), sighs (p = 0.002) and hypopneas (p < 0.001) in aged rTg4510 mice, but only Type III apneas in young adult rTg4510 mice (p < 0.001) versus age-matched nTg controls. Aged rTg4510 mice exhibited profound chemoreflex impairment versus age matched nTg and tTA mice. In rTg4510 mice, breathing frequency, tidal volume and minute ventilation were not affected by hyperoxic or hypercapnic challenges, in striking contrast to controls. Histological examination revealed hyperphosphorylated tau in brainstem regions involved in the control of breathing (e.g., pons, medullary respiratory column, retrotrapezoid nucleus) in aged rTg4510 mice. Neither breathing instability nor hyperphosphorylated tau in brainstem tissues were observed in young adult rTg4510 mice. Conclusion: Older rTg4510 mice exhibit profound impairment in the neural control of breathing, with greater breathing instability and near absence of oxygen and carbon-dioxide chemoreflexes. Breathing impairments paralleled tauopathy progression into brainstem regions that control breathing. These findings are consistent with the idea that tauopathy per se undermines chemoreflexes and promotes breathing instability during sleep.

Magnitude and Mechanism of Phrenic Long-term Facilitation Shift Between Daily Rest Versus Active Phase.

Plasticity is a fundamental property of the neural system controlling breathing. One key example of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a persistent increase in phrenic nerve activity elicited by acute intermittent hypoxia (AIH). pLTF can arise from distinct cell signaling cascades initiated by serotonin versus adenosine receptor activation, respectively, and interact via powerful cross-talk inhibition. Here, we demonstrate that the daily rest/active phase and the duration of hypoxic episodes within an AIH protocol have profound impact on the magnitude and mechanism of pLTF due to shifts in serotonin/adenosine balance. Using the historical "standard" AIH protocol (3, 5-min moderate hypoxic episodes), we demonstrate that pLTF magnitude is unaffected by exposure in the midactive versus midrest phase, yet the mechanism driving pLTF shifts from serotonin-dominant (midrest) to adenosine-dominant (midactive). This mechanistic "flip" results from combined influences of hypoxia-evoked adenosine release and daily fluctuations in basal spinal adenosine. Since AIH evokes less adenosine with shorter (15, 1-min) hypoxic episodes, midrest pLTF is amplified due to diminished adenosine constraint on serotonin-driven plasticity; in contrast, elevated background adenosine during the midactive phase suppresses serotonin-dominant pLTF. These findings demonstrate the importance of the serotonin/adenosine balance in regulating the amplitude and mechanism of AIH-induced pLTF. Since AIH is emerging as a promising therapeutic modality to restore respiratory and nonrespiratory movements in people with spinal cord injury or ALS, knowledge of how time-of-day and hypoxic episode duration impact the serotonin/adenosine balance and the magnitude and mechanism of pLTF has profound biological, experimental, and translational implications.

APOE4, Age, and Sex Regulate Respiratory Plasticity Elicited by Acute Intermittent Hypercapnic-Hypoxia.

Rationale: Acute intermittent hypoxia (AIH) shows promise for enhancing motor recovery in chronic spinal cord injuries and neurodegenerative diseases. However, human trials of AIH have reported significant variability in individual responses. Objectives: Identify individual factors (eg, genetics, age, and sex) that determine response magnitude of healthy adults to an optimized AIH protocol, acute intermittent hypercapnic-hypoxia (AIHH). Methods: In 17 healthy individuals (age = 27 ± 5 yr), associations between individual factors and changes in the magnitude of AIHH (15, 1-min O2 = 9.5%, CO2 = 5% episodes) induced changes in diaphragm motor-evoked potential (MEP) amplitude and inspiratory mouth occlusion pressures (P0.1) were evaluated. 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)ApoE knock-in rats were performed to test causality. Results: AIHH-induced changes in diaphragm MEP amplitudes were lower in individuals heterozygous for APOE4 (i.e., APOE3/4) compared to individuals with other APOE genotypes (P = 0.048) and the other tested SNPs. Males exhibited a greater diaphragm MEP enhancement versus females, regardless of age (P = 0.004). Additionally, age was inversely related with change in P0.1 (P = 0.007). In hApoE4 knock-in rats, AIHH-induced phrenic motor plasticity was significantly lower than hApoE3 controls (P < 0.05). Conclusions: APOE4 genotype, sex, and age are important biological determinants of AIHH-induced respiratory motor plasticity in healthy adults. Addition to Knowledge Base: 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 disease. Figure 5 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, 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.

Cervical spinal hemisection effects on spinal tissue oxygenation and long-term facilitation of phrenic, renal and splanchnic sympathetic nerve activity.

HYPOTHESES: Moderate acute intermittent hypoxia (mAIH) elicits plasticity in both respiratory (phrenic long-term facilitation; pLTF) and sympathetic nerve activity (sympLTF) in rats. Although mAIH produces pLTF in normal rats, inconsistent results are reported after cervical spinal cord injury (cSCI), possibly due to greater spinal tissue hypoxia below the injury site. There are no reports concerning cSCI effects on sympLTF. Since mAIH is being explored as a therapeutic modality to restore respiratory and non-respiratory movements in humans with chronic SCI, both effects are important. To understand cSCI effects on mAIH-induced pLTF and sympLTF, partial or complete C2 spinal hemisections (C2Hx) were performed and, 2 weeks later, we assessed: 1) ipsilateral cervical spinal tissue oxygen tension; 2) ipsilateral & contralateral pLTF; and 3) ipsilateral sympLTF in splanchnic and renal sympathetic nerves. METHODS: Male Sprague-Dawley rats were studied intact, or after partial (single slice) or complete C2Hx (slice with ∼1 mm aspiration). Two weeks post-C2Hx, rats were anesthetized and prepared for recordings of bilateral phrenic nerve activity and spinal tissue oxygen pressure (PtO2). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats. RESULTS: Spinal PtO2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO2 was positively correlated with mean arterial pressure. Complete C2Hx impaired ipsilateral but not contralateral pLTF; with partial C2Hx, ipsilateral pLTF was unaffected. In intact rats, mAIH elicited splanchnic and renal sympLTF. Complete C2Hx had minimal impact on baseline ipsilateral splanchnic or renal sympathetic nerve activity and renal, but not splanchnic, sympLTF remained intact. CONCLUSION: Greater tissue hypoxia likely impairs pLTF and splanchnic sympLTF post-C2Hx, although renal sympLTF remains intact. Increased sympathetic nerve activity post-mAIH may have therapeutic benefits in individuals living with chronic SCI since anticipated elevations in systemic blood pressure may mitigate hypotension characteristic of people living with SCI.

Increased spinal adenosine impairs phrenic long-term facilitation in aging rats.

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. Spinal A2A receptor inhibition enhances mAIH-induced pLTF, meaning, serotonin dominates, and adenosine constrains mAIH-induced plasticity in the daily rest phase. Thus, 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 was delivered intrathecally at C4 in anesthetized young (3-6 mo) and aged (20-22 mo) Sprague-Dawley rats before mAIH (3,5-min episodes; arterial Po2 = 45-55 mmHg). In young males, spinal A2A receptor inhibition enhanced pLTF (119 ± 5%) vs. vehicle (55 ± 9%), consistent with prior reports. In old males, pLTF was reduced to 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 two- to threefold in old vs. 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 nonrespiratory movements in older populations afflicted with clinical disorders that compromise movement.NEW & NOTEWORTHY Advanced age undermines respiratory motor plasticity, specifically phrenic long-term facilitation (pLTF) following moderate acute intermittent hypoxia (mAIH). We report that spinal adenosine increases in aged male rats, undermining mAIH-induced pLTF via adenosine 2A (A2A) receptor activation, an effect reversed by selective spinal adenosine 2A receptor inhibition. These findings advance our understanding of mechanisms that impair neuroplasticity, and the ability to compensate for the onset of lung or neural injury with age, and may guide efforts to harness mAIH as a treatment for clinical disorders that compromise breathing and other movements.

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.

APOE4, Age & Sex Regulate Respiratory Plasticity Elicited By Acute Intermittent Hypercapnic-Hypoxia.

Rationale: Acute 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. Objectives: Identify 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). Methods: Associations of individual factors with the magnitude of AIHH (15, 1-min O 2 =9.5%, CO 2 =5% episodes) induced changes in diaphragm motor-evoked potential amplitude (MEP) and inspiratory mouth occlusion pressures (P 0.1 ) 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, HTR 2A , TPH 2 , MAOA, NTRK 2 ) 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 ) ApoE knock-in rats were performed to test causality. Results: AIHH-induced changes in diaphragm MEP amplitudes were lower in individuals heterozygous for APOE 4 ( i.e., APOE 3/4 ) allele versus other APOE genotypes (p=0.048). No significant differences were observed between any other SNPs investigated, notably BDNFval/met ( all p>0.05 ). Males exhibited a greater diaphragm MEP enhancement versus females, regardless of age (p=0.004). Age was inversely related with change in P 0.1 within the limited age range studied (p=0.007). In hApoE 4 knock-in rats, AIHH-induced phrenic motor plasticity was significantly lower than hApoE 3 controls (p<0.05). Conclusions: APOE 4 genotype, sex and age are important biological determinants of AIHH-induced respiratory motor plasticity in healthy adults. ADDITION TO KNOWLEDGE BASE: Acute 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.

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).

Intermittent Hypoxia Differentially Regulates Adenosine Receptors in Phrenic Motor Neurons with Spinal Cord Injury.

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 (A1, A2A) compete to determine net cAMP signaling and likely the tAIH efficacy with chronic cSCI. Since cSCI and intermittent hypoxia may regulate adenosine receptor expression in phrenic motor neurons, we tested the hypotheses that: 1) daily AIH (28 days) downregulates A2A and upregulates A1 receptor expression; 2) CIH (28 days) upregulates A2A and downregulates A1 receptor expression; and 3) cSCI alters the impact of CIH on adenosine receptor expression. Daily AIH had no effect on either adenosine receptor in intact or injured rats. However, CIH exerted complex effects depending on injury status. Whereas CIH increased A1 receptor expression in intact (not injured) rats, it increased A2A receptor expression in spinally injured (not intact) rats. The differential impact of CIH reinforces the concept that the injured spinal cord behaves in distinct ways from intact spinal cords, and that these differences should be considered in the design of experiments and/or new treatments for chronic cSCI.

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.

Dose-dependent phosphorylation of endogenous Tau by intermittent hypoxia in rat brain.

Intermittent hypoxia, or intermittent low oxygen interspersed with normal oxygen levels, has differential effects that depend on the "dose" of hypoxic episodes (duration, severity, number per day, and number of days). Whereas "low dose" daily acute intermittent hypoxia (dAIH) elicits neuroprotection and neuroplasticity, "high dose" chronic intermittent hypoxia (CIH) similar to that experienced during sleep apnea elicits neuropathology. Sleep apnea is comorbid in >50% of patients with Alzheimer's disease-a progressive, neurodegenerative disease associated with brain amyloid and chronic Tau dysregulation (pathology). Although patients with sleep apnea present with higher Tau levels, it is unknown if sleep apnea through attendant CIH contributes to onset of Tau pathology. We hypothesized CIH characteristic of moderate sleep apnea would increase dysregulation of phosphorylated Tau (phospho-Tau) species in Sprague-Dawley rat hippocampus and prefrontal cortex. Conversely, we hypothesized that dAIH, a promising neurotherapeutic, has minimal impact on Tau phosphorylation. We report a dose-dependent intermittent hypoxia effect, with region-specific increases in 1) phospho-Tau species associated with human Tauopathies in the soluble form and 2) accumulated phospho-Tau in the insoluble fraction. The latter observation was particularly evident with higher CIH intensities. This important and novel finding is consistent with the idea that sleep apnea and attendant CIH have the potential to accelerate the progression of Alzheimer's disease and/or other Tauopathies.NEW & NOTEWORTHY Sleep apnea is highly prevalent in people with Alzheimer's disease, suggesting the potential to accelerate disease onset and/or progression. These studies demonstrate that intermittent hypoxia (IH) induces dose-dependent, region-specific Tau phosphorylation, and are the first to indicate that higher IH "doses" elicit both endogenous, (rat) Tau hyperphosphorylation and accumulation in the hippocampus. These findings are essential for development and implementation of new treatment strategies that minimize sleep apnea and its adverse impact on neurodegenerative diseases.

Caffeine Enhances Intermittent Hypoxia-Induced Gains in Walking Function for People with Chronic Spinal Cord Injury.

Incomplete spinal cord injury (iSCI) often results in lifelong walking impairments that limit functional independence. Thus, treatments that trigger enduring improvement in walking after iSCI are in high demand. Breathing brief episodes of low oxygen (i.e., acute intermittent hypoxia, AIH) enhances breathing and walking function in rodents and humans with chronic iSCI. Pre-clinical studies found that AIH also causes the accumulation of extracellular adenosine that undermines AIH-induced functional plasticity. Pharmacologically blocking adenosine A2a receptors (A2aR) prior to AIH resulted in a dramatic improvement in motor facilitation in rodents with iSCI; however, a similar beneficial effect in humans is unclear. Thus, we conducted a double-blind, placebo-controlled, crossover randomized study to test the hypothesis that a non-selective A2aR antagonist (i.e., caffeine) enhances AIH-induced effects on walking function in people with chronic (≥1yr) iSCI. We enrolled 12 participants to receive daily (5 days) caffeine or placebo (4 mg/kg) 30 min before breathing 15, 1.5-min low oxygen (AIH; FIO2 = 0.10) or SHAM (FIO2 = 0.21) episodes with 1-min intervals. We quantified walking function as the change in the 10-meter walk test (speed) and 6-min walk test (endurance) relative to baseline, on Day 5 post-intervention, and on follow-up Days 12 and 19. Participants walked faster (Day 19; p < 0.001) and farther (Day 19; p = 0.012) after caffeine+AIH and the boost in speed persisted more than after placebo+AIH or caffeine+SHAM (Day 19; p < 0.05). These results support our hypothesis that a caffeine pre-treatment to AIH training shows promise as a strategy to augment walking speed in persons with chronic iSCI.

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.

Therapeutic acute intermittent hypoxia: A translational roadmap for spinal cord injury and neuromuscular disease.

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 and respiratory muscle recruitment in people with amyotrophic lateral sclerosis: A preliminary study.

Respiratory failure is the main cause of death in amyotrophic lateral sclerosis (ALS). Since no effective treatments to preserve independent breathing are available, there is a critical need for new therapies to preserve or restore breathing ability. Since acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity in rodent ALS models, and may restore breathing ability in people with ALS, we performed a proof-of-principle study to investigate this possibility in ALS patients. Quiet breathing, sniff nasal inspiratory pressure (SNIP) and maximal inspiratory pressure (MIP) were tested in 13 persons with ALS and 10 age-matched controls, before and 60 min post-AIH (15, 1 min episodes of 10% O2, 2 min normoxic intervals) or sham AIH (continuous normoxia). The root mean square (RMS) of the right and left diaphragm, 2nd parasternal, scalene and sternocleidomastoid muscles were monitored. A vector analysis was used to calculate summated vector magnitude (Mag) and similarity index (SI) of collective EMG activity during quiet breathing, SNIP and MIP maneuvers. AIH facilitated tidal volume and minute ventilation (treatment main effects: p < 0.05), and Mag (ie. collective respiratory muscle activity; p < 0.001) during quiet breathing in ALS and control subjects, but there was no effect on SI during quiet breathing. SNIP SI decreased in both groups post-AIH (p < 0.005), whereas Mag was unchanged (p = 0.09). No differences were observed in SNIP or MIP post AIH in either group. Discomfort was not reported during AIH by any subject, nor were adverse events observed. Thus, AIH may be a safe way to increase collective inspiratory muscle activity during quiet breathing in ALS patients, although a single AIH presentation was not sufficient to significantly increase peak inspiratory pressure generation. These preliminary results provide evidence that AIH may improve breathing function in people with ALS, and that future studies of prolonged, repetitive AIH protocols are warranted.

Daily acute intermittent hypoxia enhances serotonergic innervation of hypoglossal motor nuclei in rats with and without cervical spinal injury.

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.