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

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

Made available in DSpace on 2022-04-29T08:31:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Acute intermittent hypoxia (AIH) modifies the functioning of the respiratory network, causing respiratory motor facilitation in anesthetized animals and a compensatory increase in pulmonary ventilation in freely behaving animals. However, it is still unclear whether the ventilatory facilitation induced by AIH in unanesthetized animals is associated with changes in the respiratory pattern. We found that Holtzman male rats (80–150 g) exposed to AIH (10 × 6% O2 for 30–40 s every 5 min, n = 9) exhibited a prolonged (30 min) increase in baseline minute ventilation (P < 0.05) compared to control animals (n = 13), combined with the occurrence of late expiratory peak flow events, suggesting the presence of active expiration. The increase in ventilation after AIH was also accompanied by reductions in arterial CO2 and body temperature (n = 5–6, P < 0.05). The systemic treatment with ketanserin (a 5-HT2 receptor antagonist) before AIH prevented the changes in ventilation and active expiration (n = 11) but potentiated the hypothermic response (n = 5, P < 0.05) when compared to appropriate control rats (n = 13). Our findings indicate that the ventilatory long-term facilitation elicited by AIH exposure in unanesthetized rats is linked to the generation of active expiration by mechanisms that may depend on the activation of serotonin receptors. In contrast, the decrease in body temperature induced by AIH may not require 5-HT2 receptor activation. Department of Physiology and Pathology School of Dentistry of Araraquara São Paulo State University (UNESP) Department of Physiology and Pathology School of Dentistry of Araraquara São Paulo State University (UNESP) CAPES: 001 CNPq: 132363/2018-6 FAPESP: 2013/17251-6 FAPESP: 2018/21000-2 CNPq: 310331/2017-0 CNPq: 408950/2018-8 CAPES: 88887.194785/2018-00

Blackburn MB, Andrade MA, Toney GM. Hypothalamic PVN contributes to acute intermittent hypoxia-induced sympathetic but not phrenic long-term facilitation. J Appl Physiol 124: 1233–1243, 2018. First published December 19, 2017; doi: 10.1152/japplphysiol.00743.2017 .— Acute intermittent hypoxia (AIH) repetitively activates the arterial chemoreflex and triggers a progressive increase of sympathetic nerve activity (SNA) and phrenic nerve activity (PNA) referred to as sympathetic and phrenic long-term facilitation (S-LTF and P-LTF), respectively. Neurons of the hypothalamic paraventricular nucleus (PVN) participate in the arterial chemoreflex, but their contribution to AIH-induced LTF is unknown. To determine this, anesthetized rats were vagotomized and exposed to 10 cycles of AIH, each consisting of ventilation for 3 min with 100% O2 followed by 3 min with 15% O2. Before AIH, rats received bilateral PVN injections of artificial cerebrospinal fluid (aCSF; vehicle) or the GABA-A receptor agonist muscimol (100 pmol in 50 nl) to inhibit neuronal activity. Thirty minutes after completing the AIH protocol, during which rats were continuously ventilated with 100% O2, S-LTF and P-LTF were quantified from recordings of integrated splanchnic SNA and PNA, respectively. PVN muscimol attenuated increases of SNA during hypoxic episodes occurring in later cycles (6–10) of AIH ( P < 0.03) and attenuated post-AIH S-LTF ( P < 0.001). Muscimol, however, did not consistently affect peak PNA responses during hypoxic episodes and did not alter AIH-induced P-LTF. These findings indicate that PVN neuronal activity contributes to sympathetic responses during AIH and to subsequent generation of S-LTF. NEW & NOTEWORTHY Neural circuits mediating acute intermittent hypoxia (AIH)-induced sympathetic and phrenic long-term facilitation (LTF) have not been fully elucidated. We found that paraventricular nucleus (PVN) inhibition attenuated sympathetic activation during episodes of AIH and reduced post-AIH sympathetic LTF. Neither phrenic burst patterning nor the magnitude of AIH-induced phrenic LTF was affected. Findings indicate that PVN neurons contribute to AIH-induced sympathetic LTF. Defining mechanisms of sympathetic LTF could improve strategies to reduce sympathetic activity in cardiovascular and metabolic diseases.

Moderate acute intermittent hypoxia (mAIH) elicits a form of respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Preconditioning with modest protocols of chronic intermittent hypoxia enhances pLTF, demonstrating pLTF metaplasticity. Since “low-dose” protocols of repetitive acute intermittent hypoxia (rAIH) show promise as a therapeutic modality to restore respiratory (and nonrespiratory) motor function in clinical disorders with compromised breathing, we tested 1) whether preconditioning with a mild rAIH protocol enhances pLTF and hypoglossal (XII) LTF and 2) whether the enhancement is regulated by glycolytic flux. In anesthetized, paralyzed, and ventilated adult male Lewis rats, mAIH (three 5-min episodes of 10% O2) elicited pLTF (pLTF at 60 min post-mAIH: 49 ± 5% baseline). rAIH preconditioning (ten 5-min episodes of 11% O2/day with 5-min normoxic intervals, 3 times per week, for 4 wk) significantly enhanced pLTF (100 ± 16% baseline). XII LTF was unaffected by rAIH. When glycolytic flux was inhibited by 2-deoxy-d-glucose (2-DG) administered via drinking water (~80 mg·kg−1·day−1), pLTF returned to normal levels (58 ± 8% baseline); 2-DG had no effect on pLTF in normoxia-pretreated rats (59 ± 7% baseline). In ventral cervical (C4/5) spinal homogenates, rAIH increased inducible nitric oxide synthase mRNA vs. normoxic controls, an effect blocked by 2-DG. However, there were no detectable effects of rAIH or 2-DG on several molecules associated with phrenic motor plasticity, including serotonin 2A, serotonin 7, brain-derived neurotrophic factor, tropomyosin receptor kinase B, or VEGF mRNA. We conclude that modest, but prolonged, rAIH elicits pLTF metaplasticity and that a drug known to inhibit glycolytic flux (2-DG) blocks pLTF enhancement.

A sustained elevation in respiratory drive following removal of a stimulus is known as respiratory long-term facilitation (rLTF). This phenomenon has been well demonstrated in humans and other animals following intermittent hypoxia. Potential clinical benefits of enhancing respiratory drive with intermittent hypoxia are likely to be offset by its side effects. However, intermittent negative airway pressure (INAP) has recently been shown in rats to elicit a similar magnitude of rLTF as IH but could have fewer side effects. We investigated whether INAP elicits rLTF in awake humans. 13 healthy male volunteers (20.9 ± 2.8 years) undertook two trials (INAP and Control). In the INAP trial, participants were exposed to one hour of 30-second episodes of negative pressure (-10 cmH 2 O) interspersed by 60-second intervals of atmospheric pressure. In the Control trial, participants breathed at atmospheric pressure for one hour. Changes in ventilation were assessed by comparing ventilation during the final five minutes of a 20-minute baseline period, with the final five minutes of a one-hour recovery period. In the Control trial ventilation increased from 14.7 ± 0.8 L/min during baseline to 16.9 ± 0.9 L/min in recovery (P=0.023). However, in the INAP trial ventilation increased significantly more (P=0.019) between baseline and recovery (14.9 ± 0.9 vs 19.1 ± 0.7 L/min, P=0.002). Data shown as mean ± SEM. In this study INAP elicits rLTF in awake, healthy humans. Further research is required to investigate the responsible mechanisms and determine if this form of rLTF could benefit patients with respiratory insufficiency.

Intermittent hypoxia-induced ventilatory neuroplasticity is likely important in obstructive sleep apnea pathophysiology. Although concomitant CO2levels and arousal state critically influence neuroplastic effects of intermittent hypoxia, no studies have investigated intermittent hypercapnic hypoxia effects during sleep in humans. Thus the purpose of this study was to investigate if intermittent hypercapnic hypoxia during sleep induces neuroplasticity (ventilatory long-term facilitation and increased chemoreflex responsiveness) in humans. Twelve healthy males were exposed to intermittent hypercapnic hypoxia (24 × 30 s episodes of 3% CO2and 3.0 ± 0.2% O2) and intermittent medical air during sleep after 2 wk washout period in a randomized crossover study design. Minute ventilation, end-tidal CO2, O2saturation, breath timing, upper airway resistance, and genioglossal and diaphragm electromyograms were examined during 10 min of stable stage 2 sleep preceding gas exposure, during gas and intervening room air periods, and throughout 1 h of room air recovery. There were no significant differences between conditions across time to indicate long-term facilitation of ventilation, genioglossal or diaphragm electromyogram activity, and no change in ventilatory response from the first to last gas exposure to suggest any change in chemoreflex responsiveness. These findings contrast with previous intermittent hypoxia studies without intermittent hypercapnia and suggest that the more relevant gas disturbance stimulus of concomitant intermittent hypercapnia frequently occurring in sleep apnea influences acute neuroplastic effects of intermittent hypoxia. These findings highlight the need for further studies of intermittent hypercapnic hypoxia during sleep to clarify the role of ventilatory neuroplasticity in the pathophysiology of sleep apnea.NEW & NOTEWORTHY Both arousal state and concomitant CO2levels are known modulators of the effects of intermittent hypoxia on ventilatory neuroplasticity. This is the first study to investigate the effects of combined intermittent hypercapnic hypoxia during sleep in humans. The lack of neuroplastic effects suggests a need for further studies more closely replicating obstructive sleep apnea to determine the pathophysiological relevance of intermittent hypoxia-induced ventilatory neuroplasticity.

A progressive and sustained increase in inspiratory-related motor output (“long-term facilitation”) and an augmented ventilatory response to hypoxia occur following acute intermittent hypoxia (AIH). To date, acute plasticity in respiratory motor outputs active in the postinspiratory and expiratory phases has not been studied. The recurrent laryngeal nerve (RLN) innervates laryngeal abductor muscles that widen the glottic aperture during inspiration. Other efferent fibers in the RLN innervate adductor muscles that partially narrow the glottic aperture during postinspiration. The aim of this study was to investigate whether or not AIH elicits a serotonin-mediated long-term facilitation of laryngeal abductor muscles, and if recruitment of adductor muscle activity occurs following AIH. Urethane anesthetized, paralyzed, unilaterally vagotomized, and artificially ventilated adult male Sprague-Dawley rats were subjected to 10 exposures of hypoxia (10% O2 in N2, 45 s, separated by 5 min, n = 7). At 60 min post-AIH, phrenic nerve activity and inspiratory RLN activity were elevated (39 ± 11 and 23 ± 6% above baseline, respectively). These responses were abolished by pretreatment with the serotonin-receptor antagonist, methysergide ( n = 4). No increase occurred in time control animals ( n = 7). Animals that did not exhibit postinspiratory RLN activity at baseline did not show recruitment of this activity post-AIH ( n = 6). A repeat hypoxia 60 min after AIH produced a significantly greater peak response in both phrenic and RLN activity, accompanied by a prolonged recovery time that was also prevented by pretreatment with methysergide. We conclude that AIH induces neural plasticity in laryngeal motoneurons, via serotonin-mediated mechanisms similar to that observed in phrenic motoneurons: the so-called “Q-pathway”. We also provide evidence that the augmented responsiveness to repeat hypoxia following AIH also involves a serotonergic mechanism.

Intermittent hypoxia-induced long-term facilitation (LTF) is variably expressed in the motor output of several inspiratory nerves, such as the phrenic and hypoglossal. Compared to phrenic LTF (pLTF), less is known about hypoglossal LTF (hLTF), although it is often assumed that cellular mechanisms are the same. While fundamental mechanisms appear to be similar, potentially important differences exist in the modulation of pLTF and hLTF. The primary objectives of this paper are to: (1) review similarities and differences in pLTF and hLTF, pointing out knowledge gaps and (2) present new data suggesting that reduced respiratory neural activity elicits differential plasticity in phrenic and hypoglossal output (inactivity-induced phrenic and hypoglossal motor facilitation, iPMF and iHMF), suggesting that these motor pool-specific differences are not unique to LTF. Differences in fundamental mechanisms or modulation of plasticity among motor pools may confer the capacity to mount a complex ventilatory response to specific challenges, particularly in motor pools with different “jobs” in the control of breathing.

The aim was to investigate the effect of propofol anesthesia on the phrenic long-term facilitation (LTF) in rats. We hypothesized that there would be a significant attenuation of LTF during propofol compared with urethane anesthesia. Fourteen adult, male, anesthetized, vagotomized, paralyzed, and mechanically ventilated Sprague-Dawley rats (7 per group), were exposed to the acute intermittent hypoxia (AIH) protocol. Peak phrenic nerve amplitude (PNA), burst frequency (f), and breathing rhythm parameters (Ti, Te, Ttot) were analyzed during the first hypoxia (TH1), as well as at 15 (T15), 30 (T30), and 60 minutes (T60) after the final hypoxic episode, and compared to the baseline values. In propofol-anesthetized rats no significant changes of PNA were recorded after the last hypoxic episode, i.e. no LTF was induced. There was a significant increase of PNA (59.4 ± 6.6%, p

Phrenic long-term facilitation (pLTF) is a form of hypoxia-induced spinal respiratory motor plasticity that requires new synthesis of brain derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, tropomyosin receptor kinase B (TrkB). Since the cellular location of relevant TrkB receptors is not known, we utilized intrapleural siRNA injections to selectively knock down TrkB receptor protein within phrenic motor neurons. TrkB receptors within phrenic motor neurons are necessary for BDNF-dependent acute intermittent hypoxia-induced pLTF, demonstrating that phrenic motor neurons are a critical site of respiratory motor plasticity.

Phrenic long-term facilitation (pLTF) is a persistent increase in phrenic nerve activity after acute intermittent hypoxia (AIH). Distinct cell-signaling cascades give rise to pLTF depending on the severity of hypoxemia within hypoxic episodes. Moderate AIH (mAIH; three 5-min episodes, PaO2 ∼35–55 mmHG) elicits pLTF by a serotonin (5-HT)-dependent mechanism that requires new synthesis of brain-derived neurotrophic factor (BDNF), activation of its high-affinity receptor (TrkB), and ERK MAPK signaling. In contrast, severe AIH (sAIH; three 5-min episodes, PaO2 ∼25–30 mmHG) elicits pLTF by an adenosine-dependent mechanism that requires new TrkB synthesis and Akt signaling. Although both mechanisms require spinal protein synthesis, the newly synthesized proteins are distinct, as are the neurochemicals inducing plasticity (serotonin vs. adenosine). In many forms of neuroplasticity, new protein synthesis requires translational regulation via mammalian target of rapamycin (mTOR) signaling. Since Akt regulates mTOR activity, we hypothesized that mTOR activity is necessary for sAIH- but not mAIH-induced pLTF. Phrenic nerve activity in anesthetized, paralyzed, and ventilated rats was recorded before, during, and 60 min after mAIH or sAIH. Rats were pretreated with intrathecal injections of 20% DMSO (vehicle controls) or rapamycin (0.1 mM, 12 μl), a selective mTOR complex 1 inhibitor. Consistent with our hypothesis, rapamycin blocked sAIH- but not mAIH-induced pLTF. Thus spinal mTOR activity is required for adenosine-dependent (sAIH) but not serotonin-dependent (mAIH) pLTF, suggesting that distinct mechanisms regulate new protein synthesis in these forms of spinal neuroplasticity.

Long-term facilitation (LTF), a form of serotonin-dependent respiratory plasticity induced by intermittent hypoxia, decreases with increasing age or following gonadectomy in male Sprague-Dawley (SD) rats. Ageing is accompanied by decreasing levels of testosterone, which in turn influences serotonergic function. In addition, LTF in young male rats differs among strains. Thus, we tested the hypothesis that LTF is similar in middle-aged and gonadectomized young male rats of an inbred rat strain commonly used in studies on ageing (F344) by comparison with SD rats. We further tested whether the magnitude of LTF correlates with circulating serum levels of testosterone and/or progesterone. Young and middle-aged intact and young gonadectomized (GDX) male Fischer 344 rats were anaesthetized, neuromuscularly blocked and ventilated. Integrated phrenic and hypoglossal (XII) nerve activities were measured before, during and 60 min following three 5-min episodes of isocapnic hypoxia. LTF was observed in phrenic motor output in young and middle-aged intact and young GDX rats. In contrast, XII LTF was observed only in young intact rats. In middle-aged and young GDX rats, XII LTF was significantly lower than in young intact rats (P < 0.05). Furthermore, XII LTF was positively correlated with the testosterone/progesterone ratio. These data show that serotonin-dependent plasticity in upper airway respiratory output is similar in F344 and SD rat strains. Furthermore, LTF is similarly impaired in middle-aged and gonadectomized male rats, suggesting that gonadal hormones play an important role in modulating the capacity for neuroplasticity in upper airway motor control.

Episodic hypoxia elicits a long-lasting augmentation of phrenic inspiratory activity known as long-term facilitation (LTF). We investigated the respective contributions of carotid chemoafferent neuron activation and hypoxia to the expression of LTF in urethane-anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats. One hour after three 5-min isocapnic hypoxic episodes [arterial Po 2(PaO2 ) = 40 ± 5 Torr], integrated phrenic burst amplitude was greater than baseline in both carotid-denervated ( n = 8) and sham-operated ( n = 7) rats ( P < 0.05), indicating LTF. LTF was reduced in carotid-denervated rats relative to sham ( P < 0.05). In this and previous studies, rats were ventilated with hyperoxic gas mixtures (inspired oxygen fraction = 0.5) under baseline conditions. To determine whether episodic hyperoxia induces LTF, phrenic activity was recorded under normoxic (PaO2 = 90–100 Torr) conditions before and after three 5-min episodes of isocapnic hypoxia (PaO2 = 40 ± 5 Torr; n = 6) or hyperoxia (PaO2 > 470 Torr; n= 6). Phrenic burst amplitude was greater than baseline 1 h after episodic hypoxia ( P < 0.05), but episodic hyperoxia had no detectable effect. These data suggest that hypoxia per se initiates LTF independently from carotid chemoafferent neuron activation, perhaps through direct central nervous system effects.

Amyotrophic lateral sclerosis (ALS) is a devastating disease leading to progressive motor neuron degeneration and death by ventilatory failure. In a model of ALS (SOD1G93A), pLTF following acute intermittent hypoxia is enhanced at disease end-stage by unknown mechanisms (Nichols et al., ibid, 2010). Intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kills respiratory motor neurons and mimics motor neuron death observed in ALS rats (Nichols et al., ibid, 2013). This CTB-SAP model allows us to study the impact of respiratory motor neuron death on breathing without many complications attendant to ALS. Here, we tested the hypothesis that phrenic motor neuron death is sufficient to enhance pLTF. pLTF was assessed in anesthetized, paralyzed and ventilated Sprague Dawley rats 7 and 28 days following bilateral intrapleural injections of: 1) CTB-SAP (25μg), or 2) un-conjugated CTB and SAP (control). CTB-SAP enhanced pLTF at 7 (CTB-SAP: 145±22%, n=10 vs. Control: 54±5%; ...

Acute intermittent hypoxia (AIH) triggers a form of respiratory plasticity known as long-term facilitation (LTF), which is manifested as a progressive increase in respiratory motor activity that lasts for minutes to hours after the hypoxic stimulus is removed. Respiratory LTF has been reported in numerous animal models, but it appears to be influenced by a variety of factors (e.g., species, age, and gender). While most studies focusing on respiratory LTF have been conducted in adult (including young adult) rat preparations, little is known about the influence of postnatal maturation on AIH-induced respiratory LTF. To begin to address this issue, we examined diaphragm EMG activity in response to and at 5-min intervals for 60 min following three 5-min episodes of hypoxia (8% O2) in urethane-anesthetized spontaneously breathing P14-P15 neonatal rats (n=15). For these experiments, the hypoxic episodes were separated by hyperoxia (40% O2), and all rats were continuously supplied with ~4% CO2. During the AIH trials, burst frequency was increased by ~20-90% above baseline in each of the rats examined while changes in burst amplitude were highly variable. Following the AIH episodes, respiratory LTF was characterized by predominantly an increase in burst frequency (fLTF) ranging from ~10% to 55%, with most rats exhibiting a 20-40% increase. In seven rats, however, an increase in amplitude (ampLTF) (~10%, n=3; ~20%, n=3; ~30%, n=1) was also noted. These data suggest that in contrast to observations in anesthetized ventilated adult rats, in anesthetized spontaneously breathing P14-P15 neonatal rats, respiratory LTF is dominated by fLTF, not ampLTF.

1. Intermittent hypoxia elicits long-term facilitation (LTF) of phrenic motor output in anaesthetized rats. We tested the hypothesis that an equal cumulative duration of continuous hypoxia would not elicit phrenic LTF. 2. Integrated phrenic nerve activity was recorded in urethane-anaesthetized, vagotomized, paralysed and ventilated rats exposed to: (1) 3 X 3 min hypoxic episodes (inspired O2 fraction (FI, O2) = 0.11) separated by 5 min hyperoxia (FI,O2 = 0.5; n = 6), (2) 9 min continuous hypoxia (n = 6), or (3) 20 min continuous hypoxia (n = 7). Isocapnia was maintained throughout the protocol. 3. Consistent with previous studies, phrenic amplitude was significantly elevated for at least 1 h following intermittent hypoxia (78 +/- 15% 60 min post-hypoxia; P0.05) with an associated increase in burst frequency (11 +/- 2.1 bursts min-1; P0.05). In contrast, 9 or 20 min continuous hypoxia did not elicit LTF of either phrenic amplitude (4.7 +/- 5.1 and 10.1 +/- 10.2% 60 min post-hypoxia, respectively; P0.05) or frequency (4.6 +/- 1.3 and 5.1 +/- 2 bursts min-1 60 min post-hypoxia, respectively; P0.05). 4. The results indicate that hypoxia-induced long-term facilitation of phrenic motor output is sensitive to the pattern of hypoxic exposure in anaesthetized rats.

The aim was to investigate whether intravenous infusion of remifentanil would depress phrenic long term facilitation (pLTF) evoked by acute intermittent hypoxia (AIH) in adult, male, urethane anaesthetized Sprague-Dawley rats, bilaterally vagotomized, paralyzed and mechanically ventilated. The experimental group received a remifentanil infusion (0.5 μg/kg/min i.v., n=12), whereas the control group (n=6) received saline. Rats were exposed to AIH protocol. Phrenic nerve amplitude (PNA), burst frequency (f) and breathing rhythm parameters (Ti, Te, Ttot) were analyzed during 5 hypoxias and at 15, 30, and 60 minutes after the final hypoxia, and compared to baseline values. At the end of the experiment, the infusion of remifentanil was stopped and phrenic nerve activity was compared to baseline values prior to remifentanil infusion. In the control group, peak phrenic nerve activity (pPNA) significantly increased at 60 min (T60, increase by 138.8±28.3%, p=0.006) after the last hypoxic episode compared to baseline values, i.e. pLTF was induced. In remifentanil treated rats, there were no significant changes in peak phrenic nerve activity at T60 compared to baseline values (decrease by 5.3±16.5%, p>0.05), i.e. pLTF was abolished. Fifteen minutes following cessation of remifentanil infusion, pPNA increased by 93.2plusmn ; 40.2% (p

Long term facilitation is a form of respiratory plasticity that is induced following exposures to the repeated episodes of intermittent hypoxia. It represents one of the most studied model of respiratory plasticity and a model of obstructive sleep apnea as well. A variety of different variables could affect manifestation of LTF and the choice of anesthetic might have a great impact. Over the past 30 years in an animal model of paralyzed, bilaterally vagotomized, mechanically ventilated rats, urethane was used as an anesthetic of choice because of its minimal effects on respiratory, cardiovascular, and autonomic nervous system. Adult, male, anesthetized, bilaterally vagotomized, paralyzed and mechanically ventilated Sprague Dawley rats were exposed to an acute intermittent hypoxia (AIH) protocol consisting of 5 hypoxic exposures (9% O2 in air) lasting for 3 min each, separated by hyperoxic (50% O2 in air) 3 minutes periods. Peak phrenic nerve activity, respiratory frequency, and respiratory rhythm parameters were analyzed during hypoxia, as well as at 15, 30, and 60 min after the end of the last hypoxic episode. We investigated effects of volatile anesthetics (sevoflurane and isoflurane), opioid anesthetics (remifentanil) and propofol on the manifestation of phrenic LTF and on hypoxic ventilatory response. Results of our research do highlight the potential caveats that might be associated with the selection of a given anesthetic regime to examine phrenic LTF.

Sustained augmentation of phrenic nerve activity evoked by acute intermittent hypoxia (AIH) represents form of central nervous system plasticity, known as phrenic long term facilitation (pLTF). Previous studies have shown that pLTF is serotonin (5-HT) dependent, and systemic application of selective and non- selective 5-HT receptor antagonists could impair induction and preservation of pLTF. Microinjections of non-selective 5-HT receptor antagonist methysergide into the raphe nuclei prevented identification of specific 5-HT receptor subtype involved in modulation of pLTF at supraspinal level. Therefore, present study was preformed to determine whether blockade of 5-HT1A receptors in caudal raphe region influences induction of pLTF. Our hypothesis was that microinjections of selective 5-HT1A receptor antagonist WAY- 100635 into the caudal raphe nucleus could prevent induction of phrenic LTF after exposure to AIH. Adult, male, urethane- anesthetized, vagotomized, paralyzed and mechanically ventilated Sprague-Dawley rats were exposed to AIH protocol. Experimental group of animals (n=7) received microinjection of WAY- 100635 (1mM, 20±5 nl) into the caudal raphe nucleus, whereas control group (n=7) received microinjection of 0.9% saline (20±5 nl) into the same site. Peak phrenic nerve activity (pPNA), burst frequency (f), and respiratory rhythm parameters were analyzed during five hypoxic episodes (TH1-5), as well as at 15 (T15), 30 (T30), and 60 (T60) minutes after the end of the last hypoxic episode. In the control group, pPNA was elevated from baseline (to 172.9±17.5%, P=0.008) at 60 minutes after episodic hypoxia, indicating pLTF. Microinjections of WAY-100635 into the raphe nuclei prior to hypoxic stimulation attenuated pLTF. There was no significant decrease of pPNA from baseline (-15.4±16.9%) at T60. Microinjections of selective 5-HT1A receptor antagonist WAY-100635 into the raphe nuclei prior to episodic hypoxia exposure prevented induction of pLTF, indicating that 5-HT1A receptor activation at supraspinal level is important for induction of pLTF.

This study examined whether time of day and repeated exposure to intermittent hypoxia have an impact on the hypoxic ventilatory response (HVR) and ventilatory long-term facilitation (vLTF). Thirteen participants with sleep apnea were exposed to twelve 4-min episodes of isocapnic hypoxia followed by a 30-min recovery period each day for 10 days. On days 1 (initial day) and 10 (final day) participants completed the protocol in the evening (PM); on the remaining days the protocol was completed in the morning (AM). The HVR was increased in the morning compared with evening on the initial (AM 0.83 ± 0.08 vs. PM 0.64 ± 0.11 l·min−1·%SaO2−1; P ≤ 0.01) and final days (AM 1.0 ± 0.08 vs. PM 0.81 ± 0.09 l·min−1·%SaO2−1; P ≤ 0.01, where %SaO2 refers to percent arterial oxygen saturation). Moreover, the magnitude of the HVR was enhanced following daily exposure to intermittent hypoxia in the morning (initial day 0.83 ± 0.08 vs. final day 1.0 ± 0.08 l·min−1·%SaO2−1; P ≤ 0.03) and evening (initial day 0.64 ± 0.11 vs. final day 0.81 ± 0.09 l·min−1·%SaO2−1; P ≤ 0.03). vLTF was reduced in the morning compared with the evening on the initial (AM 19.03 ± 0.35 vs. PM 22.30 ± 0.49 l/min; P ≤ 0.001) and final (AM 20.54 ± 0.32 vs. PM 23.11 ± 0.54 l/min; P ≤ 0.01) days. Following daily exposure to intermittent hypoxia, vLTF was enhanced in the morning (initial day 19.03 ± 0.35 vs. final day 20.54 ± 0.32 l/min; P ≤ 0.01). We conclude that the HVR is increased while vLTF is decreased in the morning compared with the evening in individuals with sleep apnea and that the magnitudes of these phenomena are enhanced following daily exposure to intermittent hypoxia.

Intermittent hypoxia (IH) can induce a persistent increase in neural drive to the respiratory muscles known as long-term facilitation (LTF). LTF of phrenic inspiratory activity is often studied in anesthetized animals after phrenicotomy (PhrX), with subsequent recordings being made from the proximal stump of the phrenic nerve. However, severing afferent and efferent axons in the phrenic nerve has the potential to alter the excitability of phrenic motoneurons, which has been hypothesized to be an important determinant of phrenic LTF. Here we test the hypothesis that acute PhrX influences immediate and long-term phrenic motor responses to hypoxia. Phrenic neurograms were recorded in anesthetized, ventilated, and vagotomized adult male rats with intact phrenic nerves or bilateral PhrX. Data were obtained before (i.e., baseline), during, and after three 5-min bouts of isocapnic hypoxia. Inspiratory burst amplitude during hypoxia (%baseline) was greater in PhrX than in phrenic nerve-intact rats ( P < 0.001). Similarly, burst amplitude 55 min after IH was greater in PhrX than in phrenic nerve-intact rats (175 ± 9 vs. 126 ± 8% baseline, P < 0.001). In separate experiments, phrenic bursting was recorded before and after PhrX in the same animal. Afferent bursting that was clearly observable in phase with lung deflation was immediately abolished by PhrX. The PhrX procedure also induced a form of facilitation as inspiratory burst amplitude was increased at 30 min post-PhrX ( P = 0.01 vs. pre-PhrX). We conclude that, after PhrX, axotomy of phrenic motoneurons and, possibly, removal of phrenic afferents result in increased phrenic motoneuron excitability and enhanced LTF following IH.

Plasticity is a hallmark of neural systems, including the neural system controlling breathing (Mitchell and Johnson, 2003). Despite its biological and potential clinical significance, our understanding of mechanisms giving rise to any form of respiratory plasticity remains incomplete. Here we discuss recent advances in our understanding of cellular mechanisms giving rise to phrenic long-term facilitation (pLTF), a long-lasting increase in phrenic motor output induced by acute intermittent hypoxia (AIH). Recently, we have come to realize that multiple, distinct mechanisms are capable of giving rise to long-lasting phrenic motor facilitation (PMF); we use PMF as a general term that includes AIH-induced pLTF. It is important to begin an appreciation and understanding of these diverse pathways. Hence, we introduce a nomenclature based on upstream steps in the signaling cascade leading to PMF. Two pathways are featured here: the “Q” and the “S” pathways, named because they are induced by metabotropic receptors coupled to Gq and Gs proteins, respectively. These pathways appear to interact in complex and interesting ways, thus providing a range of potential responses in the face of changing physiological conditions or the onset of disease.

Sleep-disordered breathing is a frequent condition across the age spectrum in the clinical setting, and is characterized by recurring episodes of hypoxia during sleep, as well as by disruption of sleep integrity. It has become clear that this highly prevalent condition leads to substantial morbidities primarily affecting the central nervous and cardiovascular systems and is also associated with marked alterations in respiratory patterning. Substantial advances have occurred in the last decade on our understanding of the pathophysiological role played intermittent hypoxia in these altered phenotypes. In this chapter, the current conceptual frameworks on the mechanisms underlying the consequences of episodic hypoxemia during sleep will be reviewed, with particular attention to cognitive deficits and altered neural control of breathing. When appropriate, differences in the response patterns as dictated by developmental stages at which intermittent hypoxia occurs will also be addressed.