Adrenergic α1 receptor activation is sufficient, but not necessary for phrenic long-term facilitation

Acute intermittent hypoxia (AIH; three 5-min hypoxic episodes) causes a form of phrenic motor facilitation (pMF) known as phrenic long-term facilitation (pLTF); pLTF is initiated by spinal activation of Gq protein-coupled 5-HT2 receptors. Because α1 adrenergic receptors are expressed in the phrenic motor nucleus and are also Gq protein-coupled, we hypothesized that α1 receptors are sufficient, but not necessary for AIH-induced pLTF. In anesthetized, paralyzed, and ventilated rats, episodic spinal application of the α1 receptor agonist phenylephrine (PE) elicited dose-dependent pMF (10 and 100 μM, P < 0.05; but not 1 μM). PE-induced pMF was blocked by the α1 receptor antagonist prazosin (1 mM; −20 ± 20% at 60 min, −5 ± 21% at 90 min; n = 6). Although α1 receptor activation is sufficient to induce pMF, it was not necessary for AIH-induced pLTF because intrathecal prazosin (1 mM) did not alter AIH-induced pLTF (56 ± 9% at 60 min, 78 ± 12% at 90 min; n = 9). Intravenous (iv) prazosin (150 μg/kg) appeared to reduce pLTF (21 ± 9% at 60 min, 26 ± 8% at 90 min), but this effect was not significant. Hypoglossal long-term facilitation was unaffected by intrathecal prazosin, but was blocked by iv prazosin (−4 ± 14% at 60 min, −13 ± 18% at 90 min), suggesting different LTF mechanisms in different motor neuron pools. In conclusion, Gq protein-coupled α1 adrenergic receptors evoke pMF, but they are not necessary for AIH-induced pLTF.

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Cervical spinal 5-HT2A and 5-HT2B receptors are both necessary for moderate acute intermittent hypoxia-induced phrenic long-term facilitation

Journal of Applied Physiology ◽

10.1152/japplphysiol.01113.2018 ◽

2019 ◽

Vol 127 (2) ◽

pp. 432-443 ◽

Cited By ~ 5

Author(s):

Arash Tadjalli ◽

Gordon S. Mitchell

Keyword(s):

Receptor Antagonist ◽

Broad Spectrum ◽

Intermittent Hypoxia ◽

Receptor Activation ◽

Mapk Signaling ◽

Receptor Subtypes ◽

Receptor Antagonists ◽

Erk Mapk ◽

Long Term Facilitation

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.

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Phrenic long-term facilitation after acute intermittent hypoxia requires spinal ERK activation but not TrkB synthesis

Journal of Applied Physiology ◽

10.1152/japplphysiol.00098.2012 ◽

2012 ◽

Vol 113 (8) ◽

pp. 1184-1193 ◽

Cited By ~ 43

Author(s):

M. S. Hoffman ◽

N. L. Nichols ◽

P. M. Macfarlane ◽

G. S. Mitchell

Keyword(s):

Intermittent Hypoxia ◽

Serotonin Receptor ◽

Receptor Activation ◽

Motor Nucleus ◽

Small Interfering Rnas ◽

Intrathecal Catheter ◽

New Synthesis ◽

Adenosine 2A Receptor ◽

Long Term Facilitation

Acute intermittent hypoxia (AIH) elicits a form of spinal respiratory plasticity known as phrenic long-term facilitation (pLTF). pLTF requires spinal serotonin receptor-2 activation, the synthesis of new brain-derived neurotrophic factor (BDNF), and the activation of its high-affinity receptor tyrosine kinase, TrkB. Spinal adenosine 2A receptor activation elicits a distinct pathway to phrenic motor facilitation (pMF); this BDNF synthesis-independent pathway instead requires new synthesis of an immature TrkB isoform. Since hypoxia increases extracellular adenosine levels, we tested the hypothesis that new synthesis of TrkB and BDNF contribute to AIH-induced pLTF. Furthermore, given that signaling mechanisms “downstream” from TrkB are unknown in either mechanism, we tested the hypothesis that pLTF requires MEK/ERK and/or phosphatidylinositol 3-kinase (PI3K)/Akt activation. In anesthetized Sprague-Dawley rats, an intrathecal catheter at cervical level 4 was used to deliver drugs near the phrenic motor nucleus. Since pLTF was blocked by spinal injections of small interfering RNAs targeting BDNF mRNA but not TrkB mRNA, only new BDNF synthesis is required for AIH-induced pLTF. Pretreatment with a MEK inhibitor (U0126) blocked pLTF, whereas a PI3K inhibitor (PI-828) had no effect. Thus, AIH-induced pLTF requires MEK/ERK (not PI3K/AKT) signaling pathways. When U0126 was injected post-AIH, pLTF development was halted but not reversed, suggesting that ERK is critical for the development but not maintenance of pLTF. Thus, there are clear mechanistic distinctions between AIH-induced pLTF (i.e., BDNF synthesis and MEK/ERK dependent) versus adenosine 2A receptor-induced pMF (i.e., TrkB synthesis and PI3K/Akt dependent).

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Divergent receptor utilization is necessary for phrenic long-term facilitation over the course of motor neuron loss following CTB-SAP intrapleural injections

Journal of Neurophysiology ◽

10.1152/jn.00236.2021 ◽

2021 ◽

Author(s):

Lauren F. Borkowski ◽

Catherine L. Smith ◽

Amy N. Keilholz ◽

Nicole L. Nichols

Keyword(s):

Motor Neuron ◽

Receptor Antagonist ◽

Receptor Activation ◽

Receptor Expression ◽

Male Rats ◽

Neuron Death ◽

Receptor Inhibition ◽

Motor Neuron Death ◽

Long Term Facilitation

Intrapleural injection of cholera toxin B conjugated to saporin (CTB-SAP) mimics respiratory motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) primarily through TrkB and PI3K/Akt-dependent mechanisms (i.e., Gs-pathway, which can be initiated by adenosine 2A (A2A) receptors in naïve rats), while 28d CTB-SAP rats elicit moderate pLTF though BDNF and MEK/ERK-dependent mechanisms (i.e., Gq-pathway, which is typically initiated by serotonin (5-HT) receptors in naïve rats). Here, we tested the hypothesis that pLTF following CTB-SAP is: 1) A2A receptor-dependent at 7d; and 2) 5-HT receptor-dependent at 28d. Adult Sprague Dawley male rats were anesthetized, paralyzed, ventilated, and were exposed to acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) following bilateral, intrapleural injections at 7d and 28d of: 1) CTB-SAP (25 μg), or 2) un-conjugated CTB and SAP (control). Intrathecal C4 delivery included either the: 1) A2A receptor antagonist (MSX-3; 10 μM; 12 μl); or 2) 5-HT receptor antagonist (methysergide; 20 mM; 15 μl). pLTF was abolished with A2A receptor inhibition in 7d, not 28d, CTB-SAP rats vs. controls (p<0.05), while pLTF was abolished following 5-HT receptor inhibition in 28d, not 7d, CTB-SAP rats vs. controls (p<0.05). Additionally, 5-HT2A receptor expression was unchanged in CTB-SAP rats vs. controls, while 5-HT2B receptor expression was decreased in CTB-SAP rats vs. controls (p<0.05). This study furthers our understanding of the contribution of differential receptor activation to pLTF and its implications for breathing following respiratory motor neuron death.

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Selected Contribution: Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia

Journal of Applied Physiology ◽

10.1152/jappl.2001.90.5.2001 ◽

2001 ◽

Vol 90 (5) ◽

pp. 2001-2006 ◽

Cited By ~ 137

Author(s):

D. D. Fuller ◽

A. G. Zabka ◽

T. L. Baker ◽

G. S. Mitchell

Keyword(s):

Receptor Antagonist ◽

Phrenic Nerve ◽

Receptor Activation ◽

Sprague Dawley ◽

Nerve Activity ◽

Sprague Dawley Rats ◽

Episodic Hypoxia ◽

Baseline Levels ◽

Long Term Facilitation

Episodic hypoxia evokes a sustained augmentation of respiratory motor output known as long-term facilitation (LTF). Phrenic LTF is prevented by pretreatment with the 5-hydroxytryptamine (5-HT) receptor antagonist ketanserin. We tested the hypothesis that 5-HT receptor activation is necessary for the induction but not maintenance of phrenic LTF. Peak integrated phrenic nerve activity (∫Phr) was monitored for 1 h after three 5-min episodes of isocapnic hypoxia (arterial Po 2 = 40 ± 2 Torr; 5-min hyperoxic intervals) in four groups of anesthetized, vagotomized, paralyzed, and ventilated Sprague-Dawley rats [ 1) control ( n = 11), 2) ketanserin pretreatment (2 mg/kg iv; n = 7), and ketanserin treatment 0 and 45 min after episodic hypoxia ( n = 7 each)]. Ketanserin transiently decreased ∫Phr, but it returned to baseline levels within 10 min. One hour after episodic hypoxia, ∫Phr was significantly elevated from baseline in control and in the 0- and 45-min posthypoxia ketanserin groups. Conversely, ketanserin pretreatment abolished phrenic LTF. We conclude that 5-HT receptor activation is necessary to initiate (during hypoxia) but not maintain (following hypoxia) phrenic LTF.

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Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

Experimental Neurology ◽

10.1016/j.expneurol.2016.05.012 ◽

2017 ◽

Vol 287 ◽

pp. 130-136 ◽

Cited By ~ 20

Author(s):

Erica A. Dale ◽

Daryl P. Fields ◽

Michael J. Devinney ◽

Gordon S. Mitchell

Keyword(s):

Motor Neuron ◽

Intermittent Hypoxia ◽

Long Term Facilitation

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Intermittent hypoxia and neurorehabilitation

Journal of Applied Physiology ◽

10.1152/japplphysiol.00235.2015 ◽

2015 ◽

Vol 119 (12) ◽

pp. 1455-1465 ◽

Cited By ~ 53

Author(s):

Elisa J. Gonzalez-Rothi ◽

Kun-Ze Lee ◽

Erica A. Dale ◽

Paul J. Reier ◽

Gordon S. Mitchell ◽

...

Keyword(s):

Intermittent Hypoxia ◽

Serotonin Receptor ◽

De Novo ◽

Spinal Injury ◽

Human Subjects ◽

Receptor Activation ◽

Long Term Facilitation ◽

Traditional Activity ◽

De Novo Protein Synthesis

In recent years, it has become clear that brief, repeated presentations of hypoxia [i.e., acute intermittent hypoxia (AIH)] can boost the efficacy of more traditional therapeutic strategies in certain cases of neurologic dysfunction. This hypothesis derives from a series of studies in animal models and human subjects performed over the past 35 yr. In 1980, Millhorn et al. (Millhorn DE, Eldridge FL, Waldrop TG. Respir Physiol 41: 87-103, 1980) showed that electrical stimulation of carotid chemoafferent neurons produced a persistent, serotonin-dependent increase in phrenic motor output that outlasts the stimulus for more than 90 min (i.e., a “respiratory memory”). AIH elicits similar phrenic “long-term facilitation” (LTF) by a mechanism that requires cervical spinal serotonin receptor activation and de novo protein synthesis. From 2003 to present, a series of studies demonstrated that AIH can induce neuroplasticity in the injured spinal cord, causing functional recovery of breathing capacity after cervical spinal injury. Subsequently, it was demonstrated that repeated AIH (rAIH) can induce recovery of limb function, and the functional benefits of rAIH are greatest when paired with task-specific training. Since uncontrolled and/or prolonged intermittent hypoxia can elicit pathophysiology, a challenge of intermittent hypoxia research is to ensure that therapeutic protocols are well below the threshold for pathogenesis. This is possible since many low dose rAIH protocols have induced functional benefits without evidence of pathology. We propose that carefully controlled rAIH is a safe and noninvasive modality that can be paired with other neurorehabilitative strategies including traditional activity-based physical therapy or cell-based therapies such as intraspinal transplantation of neural progenitors.

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IL-1 receptor activation undermines respiratory motor plasticity after systemic inflammation

Journal of Applied Physiology ◽

10.1152/japplphysiol.01051.2017 ◽

2018 ◽

Vol 125 (2) ◽

pp. 504-512 ◽

Cited By ~ 7

Author(s):

Austin D. Hocker ◽

Adrianne G. Huxtable

Keyword(s):

Systemic Inflammation ◽

Interleukin 1 ◽

Treatment Strategies ◽

Receptor Activation ◽

Inflammatory Signaling ◽

Ventilatory Control ◽

Respiratory Plasticity ◽

Long Term Facilitation ◽

Dose Dependent

Inflammation undermines respiratory motor plasticity, yet we are just beginning to understand the inflammatory signaling involved. Because interleukin-1 (IL-1) signaling promotes or inhibits plasticity in other central nervous system regions, we tested the following hypotheses: 1) IL-1 receptor (IL-1R) activation after systemic inflammation is necessary to undermine phrenic long-term facilitation (pLTF), a model of respiratory motor plasticity induced by acute intermittent hypoxia (AIH), and 2) spinal IL-1β is sufficient to undermine pLTF. pLTF is significantly reduced 24 h after lipopolysaccharide (LPS; 100 μg/kg ip, 12 ± 18%, n = 5) compared with control (57 ± 25%, n = 6) and restored by peripheral IL-1R antagonism (63 ± 13%, n = 5, AF-12198, 0.5 mg/kg ip, 24 h). Furthermore, acute, spinal IL-1R antagonism (1 mM AF-12198, 15 μl it) restored pLTF (53 ± 15%, n = 4) compared with LPS-treated rats (11 ± 10%; n = 5), demonstrating IL-1R activation is necessary to undermine pLTF after systemic inflammation. However, in healthy animals, pLTF persisted after spinal, exogenous recombinant rat IL-1β (rIL-1β) (1 ng ± AIH; 66 ± 26%, n = 3, 10 ng ± AIH; 102 ± 49%, n = 4, 100 ng + AIH; 93 ± 51%, n = 3, 300 ng ± AIH; 37 ± 40%, n = 3; P < 0.05 from baseline). In the absence of AIH, spinal rIL-1β induced progressive, dose-dependent phrenic amplitude facilitation (1 ng; −3 ± 5%, n = 3, 10 ng; 8 ± 22%, n = 3, 100 ng; 31 ± 12%, P < 0.05, n = 4, 300 ng; 51 ± 17%, P < 0.01 from baseline, n = 4). In sum, IL-1R activation, both systemically and spinally, undermines pLTF after LPS-induced systemic inflammation, but IL-1R activation is not sufficient to abolish plasticity. Understanding the inflammatory signaling inhibiting respiratory plasticity is crucial to developing treatment strategies utilizing respiratory plasticity to promote breathing during ventilatory control disorders.NEW & NOTEWORTHY This study gives novel insights concerning mechanisms by which systemic inflammation undermines respiratory motor plasticity. We demonstrate that interleukin-1 signaling, both peripherally and centrally, undermines respiratory motor plasticity. However, acute, exogenous interleukin-1 signaling is not sufficient to undermine respiratory motor plasticity.

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