Phrenic Long-term Facilitation Research Articles

Sex hormone supplementation improves breathing and restores respiratory neuroplasticity following C2 hemisection in rats.

In addition to loss of sensory and motor function below the level of the lesion, traumatic spinal cord injury (SCI) may reduce circulating steroid hormones that are necessary for maintaining normal physiological function for extended time periods. For men, who comprise nearly 80% of new SCI cases each year, testosterone is the most abundant circulating sex steroid. SCI often results in significantly reduced testosterone production and may result in chronic low testosterone levels. Testosterone plays a role in respiratory function and the expression of respiratory neuroplasticity. When testosterone levels are low, young adult male rats are unable to express phrenic long-term facilitation (pLTF), an inducible form of respiratory neuroplasticity invoked by acute, intermittent hypoxia (AIH). However, testosterone replacement can restore this respiratory neuroplasticity. Complicating the interpretation of this finding is that testosterone may exert its influence in three possible ways: 1) directly through androgen receptor (AR) activation, 2) through conversion to dihydrotestosterone (DHT) by way of the enzyme 5α-reductase, or 3) through conversion to 17β-estradiol (E2) by way of the enzyme aromatase. DHT signals via AR activation similar to testosterone, but with higher affinity, while E2 activates local estrogen receptors. Evidence to date supports the idea that exogenous testosterone supplementation exerts its influence through estrogen receptor signaling under conditions of low circulating testosterone. Here we explored both recovery of breathing function (measured with whole body barometric plethysmography) and the expression of AIH-induced pLTF in male rats following C2-hemisection SCI. One week post injury, rats were supplemented with either E2 or DHT for 7days. We hypothesized that E2 would enhance ventilation and reveal pLTF following AIH in SCI rats. To our surprise, though E2 did beneficially impact overall breathing recovery following C2-hemisection, both E2 supplementation and DHT restored the expression of AIH-induced pLTF 2weeks post-SCI.

Acute Intermittent Hypoxia with Hypercapnia has Complex effects on Phrenic Long-Term Facilitation that Depend on Delivery in the Rest versus Active Phase in Rats

Exploration of mechanisms giving rise to respiratory motor plasticity, such as phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH), has inspired new therapeutic approaches to treat individuals affected by conditions such as spinal cord injury or other clinical disorders. One key step in the translation between rodent models and humans is the apparent need for hypercapnia (either baseline or within hypoxic episodes) during AIH. Whereas AIH consisting of 15, 1-minute hypoxic episodes (1-minute intervals) elicits robust pLTF in rats, similar protocols have minimal impact in humans—unless PaCO2 levels are increased. Although prior studies report that elevated baseline CO2 actually impairs AIH-induced pLTF in rats, the impact of hypercapnia within hypoxic episodes is not known. Thus, we compared AIH with and without concurrent hypercapnia (AIH versus AIHH) on pLTF in anesthetized rats. Since our laboratory recently reported profound time-of-day effects on pLTF magnitude and/or mechanisms, we also compared AIH versus AIHH-induced pLTF during the daily rest and active phase. We hypothesized that AIHH would enhance pLTF versus AIH in both phases, suggesting it may be a more effective intervention to elicit respiratory motor plasticity and respiratory rehabilitation. pLTF was assessed in anesthetized, paralyzed, vagotomized and ventilated male Sprague-Dawley rats ( n=6 per group) exposed to moderate AIH (arterial PO2 = 40-55mmHg) consisting of: 1) 15, 1-minute hypoxic episodes with concurrent hypercapnia (arterial PCO2 ~50mmHg) or 15, 1-minute episodes of isocapnic hypoxia (baseline arterial PCO2 ~2 mmHg above the recruitment threshold; ~40-42 mmHg). We also compared AIHH versus AIH during the active phase using prior published data (AIHH, n = 4; AIH, n = 7; data from Nair et al., Function, 2023 and Marciante et al., Function, 2023, respectively). Contrary to our hypothesis, pLTF was attenuated 90 minutes post-AIHH (37±8%) versus AIH (83±4%; p<0.001) in the rest phase. In striking contrast, during the active phase, pLTF was greater with AIHH (60±11%) versus AIH (30±4%; P=0.012). These unexpected outcomes demonstrate that time of day (or the rest/active phase) must be considered before adding hypercapnia to an AIH protocol. Although the mechanisms of this difference remain unclear, our results make clear that there is still a great deal to learn about mechanisms of AIH-induced respiratory motor plasticity, including the complex interplay between hypoxia, hypercapnia and their impact on pLTF. Supported by: NIH R01 HL147554 & R01 HL148030 (PI: G. Mitchell). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phrenic Long-term Facilitation Following Severe Acute Intermittent Hypoxia Is Preserved in Geriatric Male Rats

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Both moderate and severe AIH elicit phenotypically similar pLTF but do so via completely distinct cellular mechanisms. With moderate AIH (mAIH), pLTF arises from a serotonin-dominant, adenosine-constrained mechanism. In aged rats, mAIH-induced pLTF is impaired due to increased basal spinal adenosine levels, which constrain serotonin-dependent pLTF. With severe AIH (sAIH), pLTF arises from an adenosine-dominant, serotonin-constrained mechanism. Since spinal adenosine levels are elevated in aged male rats, we hypothesized that age would actually enhance sAIH-induced- pLTF. Young (~3.5 month) and aged (~20 month) male Sprague Dawley rats were urethane anesthetized, artificially ventilated, vagotomized, paralyzed, and exposed to sAIH (3, 5-minute episodes; arterial Po2 = 25-30 mmHg). Integrated phrenic nerve burst activity was measured before (baseline), during hypoxic episodes and 60 minutes after sAIH. Neither baseline phrenic burst amplitude, the short-term hypoxic phrenic response nor pLTF magnitude (assessed as % change in phrenic burst amplitude from baseline to 60 minutes post-sAIH) were different in aged versus young rats (120 ± 22% vs. 98 ± 17%, respectively). Thus, aged male rats preserve the capacity for adenosine driven phrenic motor plasticity. Although we cannot conclude that sAIH-induced pLTF is actually increased, age effects on adenosine versus serotonin-driven plasticity are clearly different. NIH HL148030. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phrenic Motor Neuron Fractalkine Signaling to Nearby Microglia Regulates Phrenic Long-Term Facilitation

Although microglia are traditionally regarded as innate CNS immune cells, recent studies demonstrate that microglia also play key roles in normal CNS functions. For example, microglia engage in bidirectional communication with other cell types ( e.g., neurons) to regulate diverse processes including neuronal excitability, architecture and synaptic plasticity. Although neuron-microglia interactions regulate hippocampal synaptic plasticity, little is known concerning the role of microglia in regulating respiratory motor plasticity. One well-studied form of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic motor output following acute intermittent hypoxia (AIH). Whereas AIH consisting of moderate hypoxic episodes (mAIH) elicits pLTF by a serotonin-dominant, adenosine-constrained mechanism, severe AIH (sAIH) elicits pLTF by an adenosine-dominant, serotonin-constrained mechanism. Neurons interact with microglia via release of fractalkine (CX3CL1), activating responses from nearby microglia, the only CNS cell-type known to express fractalkine receptors (CX3CR1). Cervical spinal fractalkine protein injections elicit phrenic motor facilitation similar to pLTF and trigger glial ATP/adenosine release, consistent with the hypothesis that neuronal fractalkine release during hypoxia elicits extracellular adenosine accumulation, thereby modulating AIH-induced pLTF. Here, we tested the hypothesis that phrenic motor neuron fractalkine protein underlies the adenosine-constraint to mAIH-induced pLTF, and drives sAIH-induced pLTF. In adult male Sprague Dawley rats, siRNA knockdown of phrenic motor neuron fractalkine mRNA and protein was achieved via intrapleural injections of siRNAs targeting fractalkine mRNA for 3 successive days. Fractalkine knock-down: 1) enhanced serotonin-dependent mAIH-induced pLTF (100±7%; n=6; vs controls: 50±5%; n=7; p<0.001), but 2) attenuated adenosine-dependent, sAIH-induced pLTF (23±12%; n=5; vs controls: 88±7%; n=7; p=0.001). These findings suggest that hypoxia triggers dose-dependent phrenic motor neuron fractalkine release, activating CX3CR1 on nearby microglia. Subsequent microglial ATP release and conversion to adenosine activates phrenic motor neuron adenosine 2A receptors which: 1) undermines mAIH-induced (serotonin-dominant) pLTF; or 2) reaches levels suffcient to drive sAIH-induced (adenosine-dominant) pLTF. These findings increase our understanding of how spinal intercellular interactions regulate AIH-induced phrenic motor plasticity. NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spinal TNF-α receptor expression in a rodent model of respiratory motor neuron death

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kills respiratory motor neurons, and mimics aspects of neurodegenerative diseases including breathing deficits. In this model, phrenic long-term facilitation (pLTF) induced by acute intermittent hypoxia is enhanced following 7 days (d) of CTB-SAP vs. control rats, whereas pLTF in 28d CTB-SAP treated rats is comparable to pLTF in controls suggesting that the underlying mechanisms of plasticity at both time points differ. Inflammation is a hallmark of neurodegenerative diseases and is known to prevent pLTF. Furthermore, ketoprofen (NSAID) delivery attenuates pLTF in 7d CTB-SAP-treated rats, but enhances it in 28d CTB-SAP-treated rats; this suggests that inflammation contributes to pLTF in 7d CTB-SAP-treated rats, but undermines pLTF in 28d CTB-SAP-treated rats. In addition, cervical spinal inflammatory marker gene expression ( e.g., TNF-a) is increased in CTB-SAP rats, and pilot data suggest that soluble TNF receptor 1 (sTNFR1) inhibition differentially affects pLTF (Nichols and Smith , ibid). Thus, the goal of the current study is to investigate TNFR1 as a pharmacological target that can modulate inflammation to maximize pLTF and breathing in CTB-SAP rats. We hypothesized that C4 TNFR1 expression would be increased on phrenic motor neurons and glial cells of CTB-SAP rats vs. controls. To address this, we studied TNFR1 expression on phrenic motor neurons and glial cells using immunohistochemistry, confocal microscopy, and ImageJ analysis in male Sprague Dawley rats 7 and 28 days after intrapleural injection of: CTB-SAP or un-conjugated CTB and SAP (control); n=8/group. Our preliminary data (n=4/group) suggest that phrenic TNFR1 expression is increased in 7d CTB-SAP rats vs. controls (p<0.05), but is unaffected in 28d CTB-SAP rats vs. controls (p>0.05). Further data analysis is underway for phrenic as well as glial TNFR1 expression in CTB-SAP rats vs. controls. If C4 TNFR1 expression is increased, this would suggest that TNFR1 is a viable target for therapeutic intervention in CTB-SAP rats, and potentially in patients with breathing deficits due to respiratory motor neuron death. NSF Computational Neuroscience grant, MU CVM COR grant, and Spinal Cord Injury & Disease Research Program grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

N-Methyl-D-aspartate receptor (NMDAR) activity in the nucleus tractus solitarii (nTS) is required for the full expression of phrenic long-term facilitation after acute intermittent hypoxia

Exposure to acute intermittent hypoxia (AIH) induces a persistent elevation of phrenic nerve activity during the normoxic period, or phrenic long-term facilitation (pLTF). We have previously shown that nTS activity is necessary for both the development and maintenance of pLTF. However, the underlying cellular mechanisms and pathways are not yet clear. Hypoxia induces the release of glutamate from chemoafferents in the nTS, where it binds to AMPA and NMDA receptors. Previous studies have shown that activation of NMDARs increases neuronal calcium influx and triggers long-term potentiation. The present study aimed to test the role of NMDARs in pLTF and, in particular, to determine whether activation of these receptors in the nTS is required for hypoxia-induced pLTF. Mean arterial pressure (MAP) and splanchnic sympathetic and phrenic nerve activity (sSNA and PhrNA) were recorded in anesthetized, vagotomized, neuromuscularly blocked, and artificially ventilated male Sprague-Dawley rats. Animals were exposed to 10 bouts of 10% O2 (45 sec, every 5 min; AIH). Time controls (TC) were exposed to a single hypoxia bout. Parameters were measured for an additional 1 hour after the end of AIH and compared to their initial baseline (Bsl) values or TC. The NMDAR antagonist MK-801 (3 mg kg (-1), i.v.) was injected systemically, or AP5 (10 mM, 60 nl) nanoinjected bilaterally into the nTS, after the development of AIH-induced pLTF. To test whether induction of AIH or blockade of NMDARs alters neuronal function of the nTS, nTS neuronal Ca2+ responses to AIH and MK-801 were measured by fiber photometry in 3 rats previously nanoinjected with a Ca2+ indicator GCaMP-expressing virus in the nTS. Minute PhrNA (MinPhrNA, frequency x amplitude) increased 1hr after AIH (% of Bsl, 438 ± 184%; mean ± SE; p <0.01; t-test) and was significantly greater than TC (74 ± 15%Bsl p <0.01; t-test), indicating pLTF. AIH also increased nTS neuronal Ca2+. Systemic blockade of NMDARs after the development of pLTF decreased MAP, sSNA, and nTS Ca2+ and eliminated pLTF, consistent with others. Bilateral nTS nanoinjection of AP5 reduced but did not abolish pLTF (before AP5: 449 ± 109% vs. after AP5: 371 ± 107%Bsl; p <0.05; paired t-test), increased sSNA (before AP5: 192 ± 31% vs. after AP5: 219 ± 42%; p= 0.08; paired t-test) but did not alter MAP. nTS AP5 after TC had no effect on MAP, MinPhrNA (before AP5: 99 ± 46% vs. after AP5: 103 ± 43%; p = 0.44; t-test) or sSNA (before AP5: 244 ± 92% vs. after AP5: 245 ± 81%; =0.94; paired t-test). Altogether, these results show that systemic blockade of NMDARs eliminates pLTF, and our data suggest that nTS NMDARs are critical components for the maintenance of AIH-induced neuroplasticity. R01-HL-98602. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ventilatory long-term facilitation following acute intermittent hypoxia in unanesthetized rats: influence of short hypoxic episodes and changes in metabolic rate

Plasticity is a hallmark feature of the neural system controlling breathing. Acute intermittent hypoxia (AIH) elicits plasticity in multiple respiratory motor pools ( e.g., phrenic, intercostal, XII, etc.) and has emerged as a promising therapeutic approach to restore lost respiratory (and non-respiratory) function in people with spinal cord injury and other clinical disorders that compromise movement. In anesthetized rats during the rest phase, AIH-induced phrenic long-term facilitation is enhanced when the AIH consists of short (1-min) versus longer (5-min) moderate hypoxic episodes (PaO2 ~40-50 mmHg), even with the same cumulative duration of hypoxia (15-min). However, since respiratory plasticity is influenced by a variety of behavioral and physiological factors such as sensory feedback, lung/chest wall mechanics and arousal state, the effectiveness of specific AIH protocols may differ when implemented in unanesthetized rats. Since it is unknown how hypoxic episode duration impacts ventilatory long-term facilitation (vLTF) in unanesthetized rats during the rest phase, we initially tested the hypothesis that short (1-min) hypoxic episodes (FIO2 = 0.09) elicit vLTF in young male Sprague-Dawley rats via whole-body plethysmography; metabolic CO2 production was also assessed. Following 15, 1-min hypoxic episodes, significant vLTF was observed, and this effect was more pronounced when ventilation was normalized to metabolic rate ( i.e. VE/VCO2; p < 0.05). AIH-induced vLTF was accompanied by hypometabolism and a post-hypoxic temperature decrease (p < 0.01), consistent with previous work. Additional studies will be performed to compare these results with “traditional” AIH protocols ( i.e., 5-min hypoxic episodes), and across the rats’ rest versus active phase. In perspective, these results set the stage for future investigations concerning the optimization of AIH protocols in unanesthetized rodent models, including rodent models of injury or disease. NIH HL148030 & 147554 (Mitchell). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

APOE Genotype Differentially Regulates Respiratory Motor Plasticity in the Active vs Rest phase

Acute intermittent hypoxia (AIH) elicits respiratory motor plasticity and is emerging as a promising strategy to improve breathing and other (non-respiratory) movements in clinical disorders that end life due to respiratory insuffciency ( e.g., spinal cord injury & ALS). AIH-induced phrenic long-term facilitation (pLTF) is the most commonly studied form of respiratory motor plasticity and presents as a persistent increase in phrenic nerve activity after AIH. Specific factors ( e.g. aging, inflammation, time of day) undermine AIH-induced pLTF. However, little information is available concerning the role of specific genes that regulate AIH-induced phrenic motor plasticity. Apolipoproteins (ApoE) are highly expressed in the central nervous system and function as lipid carriers. The APOE gene is tri-allelic ( E2, E3 and E4); ApoE2 and E3 proteins are associated with neuroprotective and neuro-neutral phenotypes, respectively. ApoE4 impairs synaptic plasticity and is implicated in neurodegeneration, particularly Alzheimer’s Disease. Indeed, we recently discovered that specific APOE alleles predict human expression of mAIH-induced plasticity in cortico-spinal pathways to the phrenic/diaphragm motor system. Initial experiments using human APOE4 knock-in rats demonstrate a causal effect in limiting phrenic motor plasticity (vs APOE3) during the active phase. However, the impact of APOE genotype on respiratory plasticity across the daily rest/active cycle is unknown. We tested the hypothesis that APOE4 undermines AIH-induced pLTF vsAPOE3 in a time-of-day-dependent manner. Anesthetized, vagotomized, paralyzed and ventilated adult (3-4 months) male Sprague-Dawley rats with knock-in humanized (h) APOE3 and h APOE4 were presented with AIH (15,1 min hypoxic episodes; arterial PO2 = 40-50mmHg). During the active phase, AIH-induced pLTF was significantly greater in h APOE3 (33±11%) vs h APOE4 rats (-26±7%; p<0.001; both n=6). In contrast, rest phase AIH-induced pLTF was similar in both h APOE3 and h APOE4 rats (121±12% and 122±5%, respectively; both n=7). These experiments demonstrate a causal relationship whereby APOE genotype and time-of-day interact to undermine AIH-induced pLTF in a manner that is unique to the active phase in rats. Since AIH has emerged as a promising neurotherapeutic to preserve/restore breathing in clinical disorders such as spinal cord injury and neuromuscular disease, understanding how genetic factors and time-of-day of AIH delivery impact phrenic motor plasticity will help identify individuals most/least likely to respond to treatment and reveal new targets for precision interventions of AIH. NIH R01HL148030 (GSM), NIH R01HL149800 (GSM), NIH T32HL134621-5 (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Temporal Dynamics and Therapeutic Implications of Phrenic Long-Term Facilitation in Respiratory Control.

Temporal Dynamics and Therapeutic Implications of Phrenic Long-Term Facilitation in Respiratory Control.

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.

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

HypothesesModerate 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. MethodsMale 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. ResultsSpinal 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. ConclusionGreater 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.

Spinal adenosine 2A receptor inhibition restores phrenic long-term facilitation during mild systemic inflammation

Inflammation undermines several forms of neuroplasticity, including phrenic long-term facilitation (pLTF), a form of respiratory motor plasticity elicited by moderate acute intermittent hypoxia (mAIH; 3, 5-min episodes, 5 min intervals; arterial PO2 ~ 40–50 mmHg). Mild inflammation elicited by a low dose of the TLR-4 receptor agonist lipopolysaccharide (LPS; 100 mg/kg, i.p.) abolishes mAIH-induced pLTF by a mechanism that requires spinal p38 MAP kinase and protein phosphatase activity. Neuroinflammation activates microglia and other cells in the CNS, elevating extracellular levels of adenosine—a purine nucleoside known to suppress mAIH-induced pLTF. We hypothesized basal adenosine levels in ventral spinal C 3 -C 5 segments are increased in adult male Sprague-Dawley rats 24 hours post-LPS administration, thereby undermining mAIH-induced pLTF via adenosine 2A receptor (A 2A ) activation. Thus, we predicted that cervical intrathecal administration of the A 2A receptor inhibitor MSX-3 (10 μM, 12 μL) would rescue mAIH-induced pLTF in rats pre-treated with LPS. As predicted, LPS significantly increased basal ventral C 3 -C 5 adenosine levels (p=0.010 vs controls; n=7 per group). In control rats (saline injection, i.p.), MSX-3 enhanced pLTF versus vehicle controls (LPS: 110 ± 16% baseline; controls: 53 ± 6%; p=0.002; each n=6). As expected, mAIH-induced pLTF was abolished in LPS-treated rats (4 ± 6% baseline; n=6). MSX-3 restored mAIH-induced pLTF to levels equivalent to MSX-3 treated control rats (120 ± 14%; p<0.001; n=6; versus LPS controls with MSX-3: p=0.539). Thus, mild LPS-induced inflammation abolishes mAIH-induced pLTF by a mechanism associated with increased spinal adenosine levels and A 2A receptor activation. As we move mAIH forward as a therapeutic modality to improve breathing and non-respiratory movements in people with spinal cord injury or ALS, pretreatment with A 2A receptor inhibitors may offset the undermining effects of systemic inflammation associated with these severe neuromuscular disorders. NIH HL147554, HL148030, T32HL134621 (ABM) and the UF McKnight Brain Institute This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pattern of Acute Intermittent Hypoxia Protocol Impacts Phrenic Long-term Facilitation

An important model of respiratory motor plasticity is phrenic long-term facilitation (pLTF), a prolonged increase in phrenic nerve activity following intermittent exposure to low oxygen (acute intermittent hypoxia, AIH). pLTF arises from distinct intracellular signaling cascades initiated by serotonin 2 and adenosine 2A (A 2A ) receptor activation, respectively, that interact via powerful crosstalk inhibition. Studies of AIH-induced motor plasticity typically use moderate AIH protocols consisting of 3, 5 min hypoxic episodes in rodent models, but 15, 1 min hypoxic episodes in humans. Since certain factors ( e.g. severity or duration of hypoxia) influence pLTF expression, it is essential to understand the influence of AIH protocol details on motor plasticity. Here we tested the hypothesis that serotonin-dominant plasticity is enhanced when the duration of hypoxic episodes in the AIH protocol is shorter due to less hypoxia-evoked adenosine release, eliminating adenosine constraint and revealing enhanced plasticity. Anesthetized, vagotomized, paralyzed and ventilated male Sprague-Dawley rats were exposed to different AIH protocols with the same cumulative duration (15 min) and severity (arterial Po2 = 40-55mmHg) of hypoxic episodes: 1) 3, 5 min episodes with 5 min intervals (3x5); and 2) 15, 1 min hypoxic episodes with 1 min intervals (15x1). Consistent with prior reports, 3x5 AIH elicits ~60% pLTF (56±5%; n=7). In contrast, 15x1 AIH elicits significantly greater pLTF (139±14%; n=7; p<0.001). To determine if this effect is due to differences in hypoxia evoked-adenosine release in the ventral C 4 spinal cord, tissue oxygen pressure and spinal adenosine concentration were measured with an oxygen optode, and adenosine probe, respectively. Although tissue oxygen pressure between protocols was not different, hypoxia-evoked adenosine release was substantially greater with the 3x5 vs. 15x1 AIH protocols (p<0.001; both n=3). To verify pLTF magnitude results from differential spinal A 2A receptor activation, rats were pretreated with a selective A 2A receptor antagonist, MSX-3 (10uM; 12 uL) delivered intrathecally at C 4 ; with the 3x5 AIH protocol, pLTF was enhanced as expected (141±15%; p<0.001; n=7). However, MSX-3 had no effect on pLTF elicited by the 15x1 AIH protocol (122±11%; n=7; p=0.931). Thus, the mechanism driving pLTF is serotonin-dominant and adenosine-constrained with 3x5 AIH, but adenosine- unconstrained with the 15x1 AIH protocol. We conclude that, by shortening hypoxic episodes from 5 to 1 min (with the same cumulative hypoxic duration), reduced hypoxia-evoked adenosine release reveals the full extent of serotonin-driven plasticity. AIH is emerging as a promising therapeutic modality to restore respiratory and non-respiratory movements in people with spinal injury and ALS. Thus, knowledge that the AIH protocol can be optimized by reducing the length of individual hypoxic episodes has important experimental, biological and translational implications. Supported by: NIH R01HL148030, R01HL149800 & T32HL134621 (ABM), and UF Brain Spinal Cord Research Trust Fund (BSCIRTF) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Shorter hypoxic and inter-hypoxic intervals enhance acute intermittent hypoxia-induced phrenic long-term facilitation

Phrenic long-term facilitation (pLTF) is a form of respiratory motor plasticity evoked by repeated exposure to low oxygen (acute intermittent hypoxia; AIH). AIH has emerged as a promising therapeutic approach to restore lost respiratory and non-respiratory function in people with spinal cord injury and other clinical disorders that compromise movement. Optimal AIH protocols ( e.g. episode severity/duration and number of hypoxic episodes) to elicit plasticity (and therapeutic benefit) have not yet been established. Three, 5-minute moderate hypoxic episodes with 5-minute intervals have been used most often to study pLTF. New data suggest that the same cumulative duration of hypoxia ( i.e. 15 min), when presented in shorter episodes, enhances pLTF by minimizing hypoxia-evoked adenosine release. Although the “standard” AIH protocol can be improved by reducing the duration of hypoxic episodes, little attention has yet been given to the duration of inter-hypoxic intervals. Thus, we began investigations concerning the impact of interval duration and episode number in moderate AIH protocols (mAIH; arterial PO2 = 40-55mmHg). pLTF was assessed in anesthetized, paralyzed, vagotomized and ventilated male Sprague-Dawley rats exposed to mAIH consisting of: 1) 3, 1 minute hypoxic episodes (3x1) with either 5-, 2- or 1-minute inter-hypoxic intervals; or 2) 2, 5-minute hypoxic episodes with a single 15-minute interval, compared with the conventional protocol (3, 5 minute hypoxic episodes, 5 minute intervals). mAIH consisting of 3x1, 5-min intervals elicit similar pLTF versus 3, 5 min episodes with 5-min intervals (64 ± 7% and 64% ± 1%, respectively; both n=3) at 60 minutes post-hypoxia. Thus, shorter intervals in an otherwise similar mAIH protocol consisting of 3x1 minute hypoxic episodes significantly enhance pLTF (1-minute: 146 ± 15%, n=5; 2 minute: 92 ± 17%, n = 3). Two, 5-minute hypoxic episodes with a single 15-minute inter-hypoxic interval still elicited pLTF of 51 ± 26% (n=4). These preliminary findings suggest both the hypoxic episode and interval duration are important determinants of mAIH-induced pLTF. Optimizing AIH-induced motor plasticity is necessary as we harness its therapeutic potential to treat severe neuromuscular disorders that compromise breathing. Supported by: NIH HL147554, HL148030, T32HL134621 (ABM) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Impact of Acute Hyperbaric Oxygen Treatment on Phrenic Motor Output in Adult Male Rats

Hyperbaric oxygen (HBO) therapy involves exposure to 100% oxygen delivered above one atmosphere absolute (ATA), typically for ≤1 hour. HBO therapy is commonly used for promoting wound healing and treating decompression sickness. HBO treatment radically increases arterial oxygen content and can stimulate production of anti‐oxidants and suppress inflammation. These and other HBO‐induced responses could potentially impact the neural regulation of breathing. Here we investigated if exposure to HBO (100% O2, 2 ATA, 1 hr) impacts baseline phrenic motor output or the ability to express phrenic long term facilitation induced by acute intermittent hypoxia (AIH). Adult male Sprague‐Dawley rats were treated with HBO (n=8) or sham treatment (n=7, room air, 1 ATA), and then immediately urethane‐anesthetized and mechanically ventilated. Rats were vagotomized and phrenic nerve output was recorded using a suction electrode. On average, 86±23 min elapsed from the end to HBO treatment to the initiation of phrenic recordings. After establishing the CO2 apneic and recruitment thresholds, a stable baseline was maintained at 2 mmHg above of recruitment threshold for 15‐20 min. Rats were then exposed to AIH which consisted of three 5‐min episodes of moderate hypoxia (arterial partial pressure of O2, PaO2, approximately 45 mmHg) separated by 5‐min intervals of baseline O2conditions. Arterial samples were drawn at baseline, during hypoxia and every 20 min following AIH for blood‐gas analysis. Phrenic activity was recorded for 60 min after AIH. An unpaired two‐tailed t‐test was applied to compare baseline (BL) parameters between groups. Analysis of long term facilitation of phrenic burst amplitude after AIH (expressed as %BL) was done using two‐way repeated measures ANOVA. The HBO treatment did not affect baseline parameters including phrenic burst amplitude (p=0.34), burst frequency (p=0.48), mean arterial blood pressure (p=0.66), PaO2 (p=0.86) or PaCO2 (p=0.27). Phrenic LTF tended to be greater in the HBO group (burst amplitude 81±50 %BL at 60 min) as compared to control (43±9 %BL at 60 min). However, this difference was not statistically significant (treatment effect, P=0.16). Our preliminary conclusion is that a single session of HBO at 2 ATA for 1 hour has no detectable impact on baseline phrenic motor output recorded approximately 90 minutes after end of the HBO treatment. Additional experiments are needed before drawing a firm conclusion regarding the impact of HBO on phrenic long‐term facilitation.

Aging Impairs Phrenic Long‐Term Facilitation in Rats by an Adenosine‐Dependent Mechanism

Phrenic long‐term facilitation (pLTF) is a form of respiratory plasticity elicited by acute intermittent hypoxia (AIH). AIH activates competing intracellular signaling cascades, initiated by phrenic motor neuron serotonin 2 and adenosine 2A (A2A) receptors, respectively. With moderate AIH (mAIH), the serotonin‐dependent Q pathway to phrenic motor facilitation dominates, with an adenosine constraint. Consequently, spinal A2Areceptor inhibition enhances mAIH‐induced pLTF. In middle‐aged male, and geriatric acyclic female rats, pLTF is attenuated through unknown mechanisms. We hypothesize that basal spinal adenosine levels are elevated in aged rats, thereby undermining mAIH‐induced pLTF. Thus, we predict that spinal A2A receptor inhibition in aged male rats will restore pLTF to levels equal to young male rats with A2A receptor inhibition. A selective A2A receptor antagonist (MSX‐3; 10 uM, 12 uL) or vehicle (0.9% sterile saline) were delivered intrathecally (C4) in anesthetized, vagotomized, paralyzed and ventilated young (3‐6 months) and aged (20‐22 months) male Sprague‐Dawley rats prior to mAIH (3, 5 min hypoxic episodes; arterial Po2= 45‐55 mmHg). In young males, mAIH elicits ~ 60% pLTF at 60 min post‐AIH. Spinal A2A receptor inhibition significantly enhanced mAIH‐induced pLTF in young males (119 + 5%; n = 6) vs vehicle controls (55 + 9%; n = 6; p &lt; 0.001) and time controls (12 + 4%; n = 6; p &lt; 0.001). In aged males, A2A receptor inhibition enhanced mAIH‐induced pLTF to a greater extent (186 + 19%; n = 7) vs vehicle controls (25 + 11%; n = 7; p &lt; 0.001) and time controls (3 + 5%; n = 4; p &lt; 0.001). Measured basal adenosine levels in ventral C3‐C5 homogenates were significantly elevated in aged vs young males (p = 0.008). Preliminary data suggest aged female rats (16‐18 months) also have enhanced mAIH‐induced pLTF with A2A receptor inhibition (173+ 24%; n = 3) vs vehicle controls (53 + 7%; n = 3) and time controls (5 + 17%; n = 2) in persistent estrus. These findings advance our understanding of mechanisms whereby age undermines mAIH‐induced phrenic motor plasticity. Understanding age and sex effects on AIH‐induced respiratory motor plasticity is crucial as we harness therapeutic AIH to treat severe neuromuscular disorders that compromise breathing.

Daily ketoprofen restores AIH‐induced phrenic long‐term facilitation after prolonged chronic intermittent hypoxia in spinal intact and chronically injured rats

Acute intermittent hypoxia (AIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long‐term facilitation (pLTF). In recent years, this form of plasticity has been leveraged for therapeutic benefit to restore respiratory (and non‐respiratory) motor function after spinal cord injury (SCI). While therapeutic AIH shows tremendous promise as a non‐invasive and easy‐to administer rehabilitation strategy, its efficacy may be undermined by factors such as inflammation (Huxtable et al., 2011). For example, a single night of chronic intermittent hypoxia (CIH) simulating that experienced by an individual with moderate sleep apnea (2 min hypoxic episodes; 2 min intervals; 8 hrs) abolishes AIH‐induced pLTF through a mechanism that requires systemic inflammation and spinal p38 MAP kinase activity (Huxtable et al., 2015). Similarly, prolonged CIH (7‐28 days) also abolishes pLTF (Gonzalez‐Rothi et al., 2021; El Chami and colleagues, unpublished), though the underlying mechanisms have not yet been defined. Since CIH induces time‐dependent cascades of distinct spinal inflammatory gene expression (Smith et al., 2013), we hypothesize that prolonged CIH (versus a single night) blunts pLTF through an inflammation‐dependent mechanism, although the specific pathways may differ with cumulative/repeated exposures. Further, since respiratory insufficiency is a leading cause of morbidity and mortality following cervical SCI and sleep disordered breathing/sleep apnea are highly prevalent (nearly 80% in cervical SCI), it is important to understand how untreated sleep apnea may interact with and/or undermine the efficacy of therapeutic interventions like AIH in chronic SCI. Thus, we are exploring the impact of anti‐inflammatory administration on AIH‐induced pLTF in both intact rats and rats with chronic (12 wks) C3/C4 mid‐cervical spinal cord contusion, preconditioned with 7 days of moderate CIH. In intact rats, daily administration of the non‐steroidal anti‐inflammatory drug, ketoprofen rescued expression of pLTF following prolonged CIH (70.4% increase from baseline, p=0.0039 vs. vehicle). Interestingly, a single dose of ketoprofen delivered just prior to pLTF assessments did not restore expression of plasticity (19.5% increase, p=0.8842 vs. vehicle), suggesting a shift in the underlying mechanisms by which CIH impacts phrenic motor plasticity that occurs with cumulative exposure over time. Similarly, in rats with chronic cSCI, daily ketoprofen rescued expression of pLTF following prolonged CIH (62.9% increase from baseline, p=0.0348 vs. vehicle). Upcoming experiments will explore the role of p38 MAP kinase specifically after prolonged CIH. Improved understanding of factors that undermine plasticity, (e.g., CIH, inflammation), will enable us to identify strategies to mitigate deleterious effects and optimize functional benefits, which is essential for effectively translating promising interventions like therapeutic AIH to clinical practice.

Acute Intermittent Hypoxia Preconditioning Elicits Age and Sex‐Dependent Changes in Molecules Known to Regulate Phrenic Motor Plasticity

Acute intermittent hypoxia (AIH) elicits a form of respiratory motor plasticity known as phrenic long‐term facilitation (pLTF); preconditioning with daily AIH (dAIH) enhances pLTF, demonstrating metaplasticity. Two additional factors impacting pLTF expression are age and sex: whereas moderate AIH‐induced pLTF decreases with age in males, it increases in middle aged females. Unfortunately, little is known concerning mechanisms of dAIH preconditioning or age‐dependent sexual dimorphism in pLTF. Moderate AIH elicits 2 distinct signaling cascades in phrenic motor neurons initiated by 5HT2A/2B receptors (Q pathway) versus adenosine 2A or 5HT7 receptors (S pathway), respectively. The Q and S pathways interact via mutual cross‐talk inhibition, a powerful regulator of pLTF expression. To begin exploring the hypotheses that dAIH preconditioning and age/sex effects on pLTF can be accounted for by shifts in molecules regulating the Q and S pathways, we assessed mRNA of key molecules known to regulate pLTF in ventral cervical spinal homogenates from segments containing the phrenic motor nucleus from young (3 month) and middle‐aged (12 month) male and female Sprague‐Dawley rats. Rats (n=8/group) were exposed to sham or dAIH for 14 days (15, 1 min hypoxic episodes/day), and sacrificed 24 hours after the last AIH exposure. Tissues were harvested, preserved in RNA stabilizing solution (&lt;10 min), and stored (‐80°C) until mRNA extraction. mRNA levels of molecules that regulate pLTF were assessed via RT PCR, including: serotonin 2A (Htr2a) &amp; 2B (Htr2b); adenosine 2A (Adora2a); exchange protein activated by cAMP (Epac); PKA catalytic subunit (Prkaa1); PKA regulatory subunit (Prkar1a); fractalkine (Cx3cl1); phosphodiesterase type 4 (Pde4b); Gp91 and p47(NOX genes); and the Pkcδisoform. We also quantified protein expression in phrenic motor neurons (via immunofluorescence) for BDNF and adenosine‐1A &amp; ‐2A receptors in rats fixed with 4% paraformaldehyde (n=4/group; analysis via MATLAB codes to quantify optical density in labeled motor neurons). We found significantly more Pde4b, Adora2a, and Pkcδ mRNA in young and older females versus age matched males; Epacwas also elevated, but only in young females (p&lt;0.001). p47 was increased in older rats of both sexes (p&lt;0.01). p47, Cx3cl1, Adora2a and Pkcδ mRNA were reduced by dAIH in older females (p&lt;0.05), but not other groups. There were age‐dependent increases in adenosine 1A (p &lt;0.0001) and 2A (p &lt;0.0001) receptors, and BDNF (p&lt;0.001) protein post‐AIH in both sexes. Thus, there are age, sex and dAIH preconditioning effects on molecules known to regulate the Q and S pathways to phrenic motor facilitation. These novel findings advance our understanding of pLTF, and inform translational research concerning the therapeutic potential of dAIH to treat breathing deficits in individuals of different ages or sex.