Spinal nNOS regulates phrenic motor facilitation by a 5-HT2B receptor- and NADPH oxidase-dependent mechanism

Abstract

Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF) by a mechanism that requires spinal serotonin (5-HT) receptor activation and NADPH oxidase (NOX) activity. Here, we investigated whether: 1) spinal nitric oxide synthase (NOS) activity is necessary for AIH-induced pLTF; 2) episodic exogenous nitric oxide (NO) is sufficient to elicit phrenic motor facilitation (pMF) without AIH (i.e. pharmacologically); and 3) NO-induced pMF requires spinal 5-HT2B receptor and NOX activation. In anesthetised, mechanically ventilated adult male rats, AIH (3×5min episodes; 10% O2; 5min) elicited a progressive increase in the amplitude of integrated phrenic nerve bursts (i.e. pLTF), which lasted 60 min post-AIH (45.1 ± 8.6% baseline). Pre-treatment with intrathecal (i.t.) injections of a neuronal NOS inhibitor (nNOS-inhibitor-1) near the phrenic motor nucleus attenuated pLTF (14.7 ± 2.5%), whereas an inducible NOS (iNOS) inhibitor (1400W) had no effect (56.3 ± 8.0%). Episodic i.t. injections (3×5μl volume; 5mins) of a NO donor (sodium nitroprusside; SNP) elicited pMF similar in time-course and magnitude (40.4 ± 6.0%, 60 min post-injection) to AIH-induced pLTF. SNP-induced pMF was blocked by a 5-HT2B receptor antagonist (SB206553), a superoxide dismutase mimetic (MnTMPyP), and two NOX inhibitors (apocynin and DPI). Neither pLTF nor pMF were affected by pre-treatment with a PKG inhibitor (KT-5823). Thus, spinal nNOS activity is necessary for AIH-induced pLTF, and episodic spinal NO is sufficient to elicit pMF by a mechanism that requires 5-HT2B receptor activation and NOX-derived ROS formation, which indicates AIH (and NO) elicits spinal respiratory plasticity by a nitrergic-serotonergic mechanism.

Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS

“…Both increase in the cerebrospinal fluid of MT mice (Liu et al, 1999) and may enable pLTF in MT rats. Further, since spinal nitric oxide is necessary and sufficient for pLTF in normal rats (MacFarlane et al, 2014), nitric oxide synthase upregulation with increased nitric oxide production in spared phrenic motor neurons could contribute to pLTF in MT rats. Preliminary data from our laboratory suggest that hydrogen peroxide, hydroxyl radical and nitric oxide formation/production are increased in putative phrenic motor neurons in end-stage MT rats (Satriotomo et al, 2008).…”

Spinal metaplasticity in respiratory motor control

“…For example, serotonin receptor antagonists applied to the C3–C5 cervical spine region during AIH abolishes pLTF; demonstrating that spinal serotonin receptor activation in the immediate vicinity of the phrenic motor nucleus is necessary for pLTF (Fuller et al, 2001 ; Baker-Herman and Mitchell, 2002 ). AIH-induced pLTF is also abolished by cervical spinal pre-treatment with siRNAs targeting BDNF mRNA (Baker-Herman et al, 2004 ) and neuronal nitric oxide synthase (nNOS; MacFarlane et al, 2014 ). Conversely, activation of cervical spinal serotonin type 2A, 2B and 7 receptors (MacFarlane and Mitchell, 2009 ; Hoffman and Mitchell, 2011 ; MacFarlane et al, 2011 ), TrkB receptors (the high affinity brain derived neurotrophic factor (BDNF) receptor; Baker-Herman et al, 2004 ), and nNOS (MacFarlane et al, 2014 ) can all give rise to phenotypically similar pMF in the absence of hypoxia (i.e., mimicking pLTF).…”

“…AIH-induced pLTF is also abolished by cervical spinal pre-treatment with siRNAs targeting BDNF mRNA (Baker-Herman et al, 2004 ) and neuronal nitric oxide synthase (nNOS; MacFarlane et al, 2014 ). Conversely, activation of cervical spinal serotonin type 2A, 2B and 7 receptors (MacFarlane and Mitchell, 2009 ; Hoffman and Mitchell, 2011 ; MacFarlane et al, 2011 ), TrkB receptors (the high affinity brain derived neurotrophic factor (BDNF) receptor; Baker-Herman et al, 2004 ), and nNOS (MacFarlane et al, 2014 ) can all give rise to phenotypically similar pMF in the absence of hypoxia (i.e., mimicking pLTF). Collectively, these experiments reveal important aspects of the signaling pathway giving rise to AIH-induced pLTF, but also identify important signaling checkpoints that are independently sufficient for pMF.…”

Enhancement of phrenic long-term facilitation following repetitive acute intermittent hypoxia is blocked by the glycolytic inhibitor 2-deoxyglucose

“…pLTF metaplasticity may arise from amplification of the same elements that give rise to mAIHinduced pLTF in normal rats. Basic pLTF mechanisms, including spinal serotonin (5-HT) 2 receptor activation (6,20,32,39), downstream signaling via ERK MAP kinases, TrkB, and protein kinase C (9,12,29), new BDNF protein synthesis (5), reactive oxygen species formation (38,41), and neuronal nitric oxide (NO) synthase (nNOS) activity (40), have been reviewed in recent years (7,13,15,42,48). In support of the idea that these molecules play an important role in rAIH-induced pLTF metaplasticity, rAIH increases BDNF protein expression in ventral cervical segments containing the phrenic motor nucleus (64) and within phrenic motor neurons per se (37,58).…”