Respiratory Plasticity Research Articles

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

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated super-oxide dismutase-1 (SOD1G93A), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1G93A (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600μM; 12μL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms preserving/enhancing the capacity for pLTF in MT rats are not known.

Effect of Systemic Application of 5-Hydroxytryptamine on Hypoglossal Nerve Discharge in Anesthetized Rats.

The aim of this study is to investigate whether systemic 5-hydroxytryptamine (5-HT) can promote long-lasting form of respiratory plasticity in vivo via 5-HT2AR-activated protein kinase C (PKC) mechanism. The frequency and peak amplitude of hypoglossal nerve discharges in anesthetized rats were compared before and after intravenous injections of different treatments, including saline, 5-HT, ketanserin tartrate, or staurosporine. The administration of 5-HT at a systemic bolus imposed an initial ephemeral inhibition subsequently followed by striking facilitation, which demonstrates a biphasic manner of hypoglossal nerve output in anesthetized adult rats. The facilitatory stage conformed to the reinforced hypoglossal activity that lasted for more than 60 min after drug administration. The 5-HT evoked biphasic manner of hypoglossal output and hypoglossal nerve activity LTF (hLTF) were 5-HT2A receptor-dependent and coupled to PKC activation. The initial inhibition of hypoglossal activity was associated with nodose ganglion, and the subsequent facilitation was associated with carotid body. The reactive oxygen species (ROS) formation was triggered in the systemic 5-HT2-dependent hLTF model in vivo. The expressions of immunofluorescent histochemistry provide morphological evidence of a 5-HT/5-HT2A receptor coupled to PKC mechanism. In conclusion, systemic 5-HT challenge contributes to long-lasting form of respiratory plasticity and to elicit hLTF or elevated hLTF in animals, which with decreased or even with inhibited peripheral inhibitory activations. The effect of systemic 5-HT was regulated by a 5-HT2AR-activated PKC mechanism.

Graded hypoxia acts through a network of distributed peripheral oxygen chemoreceptors to produce changes in respiratory behaviour and plasticity.

Respiratory behaviour relies critically upon sensory feedback from peripheral oxygen chemoreceptors. During environmental or systemic hypoxia, chemoreceptor input modulates respiratory central pattern generator activity to produce reflex-based increases in respiration and also shapes respiratory plasticity over longer timescales. The best-studied oxygen chemoreceptors are undoubtedly the mammalian carotid bodies; however, questions remain regarding this complex organ's role in shaping respiration in response to varying oxygen levels. Furthermore, many taxa possess distinct oxygen chemoreceptors located within the lungs, airways and cardiovasculature, but the functional advantage of multiple chemoreceptor sites is unclear. In this study, it is demonstrated that a distributed network of peripheral oxygen chemoreceptors exists in Lymnaea stagnalis and significantly modulates aerial respiration. Specifically, Lymnaea breath frequency and duration represent parameters that are shaped by interactions between hypoxic severity and its time-course. Using a combination of behaviour and electrophysiology approaches, the chemosensory pathways underlying hypoxia-induced changes in breath frequency/duration were explored. The current findings demonstrate that breath frequency is uniquely modulated by the known osphradial ganglion oxygen chemoreceptors during moderate hypoxia, while a newly discovered area of pneumostome oxygen chemoreception serves a similar function specifically during more severe hypoxia. Together, these findings suggest that multiple oxygen chemosensory sites, each with their own sensory and modulatory properties, act synergistically to form a functionally distributed network that dynamically shapes respiration in response to changing systemic or environmental oxygen levels. These distributed networks may represent an evolutionarily conserved strategy vis-à-vis respiratory adaptability and have significant implications for the understanding of fundamental respiratory control systems.

Human intermittent hypoxia-induced respiratory plasticity is not caused by inflammation.

Ventilatory instability, reflected by enhanced acute hypoxic (AHVR) and hypercapnic (AHCVR) ventilatory responses is a fundamental component of obstructive sleep apnoea (OSA) pathogenesis. Intermittent hypoxia-induced inflammation is postulated to promote AHVR enhancement in OSA, although the role of inflammation in intermittent hypoxia-induced respiratory changes in humans has not been examined. Thus, this study assessed the role of inflammation in intermittent hypoxia-induced respiratory plasticity in healthy humans.In a double-blind, placebo-controlled, randomised crossover study design, 12 males were exposed to 6 h of intermittent hypoxia on three occasions. Prior to intermittent hypoxia exposures, participants ingested (for 4 days) either placebo or the nonsteroidal anti-inflammatory drugs indomethacin (nonselective cyclooxygenase (COX) inhibitor) and celecoxib (selective COX-2 inhibitor). Pre- and post-intermittent hypoxia resting ventilation, AHVR, AHCVR and serum concentration of the pro-inflammatory cytokine tumour necrosis factor (TNF)-α were assessed.Pre-intermittent hypoxia resting ventilation, AHVR, AHCVR and TNF-α concentrations were similar across all three conditions (p≥0.093). Intermittent hypoxia increased resting ventilation and the AHVR similarly across all conditions (p=0.827), while the AHCVR was increased (p=0.003) and TNF-α was decreased (p=0.006) with only selective COX-2 inhibition.These findings indicate that inflammation does not contribute to human intermittent hypoxia-induced respiratory plasticity. Moreover, selective COX-2 inhibition augmented the AHCVR following intermittent hypoxia exposure, suggesting that selective COX-2 inhibition could exacerbate OSA severity by increasing ventilatory instability.

Intermittent Hypoxia-Induced Spinal Inflammation Impairs Respiratory Motor Plasticity by a Spinal p38 MAP Kinase-Dependent Mechanism

Inflammation is characteristic of most clinical disorders that challenge the neural control of breathing. Since inflammation modulates neuroplasticity, we studied the impact of inflammation caused by prolonged intermittent hypoxia on an important form of respiratory plasticity, acute intermittent hypoxia (three, 5 min hypoxic episodes, 5 min normoxic intervals) induced phrenic long-term facilitation (pLTF). Because chronic intermittent hypoxia elicits neuroinflammation and pLTF is undermined by lipopolysaccharide-induced systemic inflammation, we hypothesized that one night of intermittent hypoxia (IH-1) elicits spinal inflammation, thereby impairing pLTF by a p38 MAP kinase-dependent mechanism. pLTF and spinal inflammation were assessed in anesthetized rats pretreated with IH-1 (2 min hypoxia, 2 min normoxia; 8 h) or sham normoxia and allowed 16 h for recovery. IH-1 (1) transiently increased IL-6 (1.5 ± 0.2-fold; p = 0.02) and inducible nitric oxide synthase (iNOS) (2.4 ± 0.4-fold; p = 0.01) mRNA in cervical spinal homogenates, (2) elicited a sustained increase in IL-1β mRNA (2.4 ± 0.2-fold; p < 0.001) in isolated cervical spinal microglia, and (3) abolished pLTF (-1 ± 5% vs 56 ± 10% in controls; p < 0.001). pLTF was restored after IH-1 by systemic NSAID administration (ketoprofen; 55 ± 9%; p < 0.001) or spinal p38 MAP kinase inhibition (58 ± 2%; p < 0.001). IH-1 increased phosphorylated (activated) p38 MAP kinase immunofluorescence in identified phrenic motoneurons and adjacent microglia. In conclusion, IH-1 elicits spinal inflammation and impairs pLTF by a spinal p38 MAP kinase-dependent mechanism. By targeting inflammation, we may develop strategies to manipulate respiratory motor plasticity for therapeutic advantage when the respiratory control system is compromised (e.g., sleep apnea, apnea of prematurity, spinal injury, or motor neuron disease).

Astrocytes mediate Short‐term Plasticity of Breathing

Respiratory plasticity contributes to stabilization of ventilation. However, its cellular mechanism has not been clarified. We hypothesized that astrocytes mediate respiratory plasticity, based on the discovery that astrocytes are actively involved in neural plasticity in various brain functions. To examine our hypothesis, we analyzed the effects of ONO‐2506, an inhibitor of astrocytic activation, on post‐hypoxic short‐term potentiation of breathing. Ventilatory parameters of unanesthetized adult mice were measured by whole body plethysmography (n=11). To test the hypoxic response, mice breathed room air, then hypoxic gas (7% O2, 93% N2), and again room air. After recording control hypoxic response with recovery time course, low and high doses of ONO‐2506 was ip injected. Although ONO‐2506 little affected ventilatory augmentation during hypoxic loading, it suppressed post‐stimulus potentiation of breathing. High dose ONO‐2506 completely eliminated post‐stimulus potentiation. We conclude that post‐stimulus potentiation of breathing is mediated by astrocytes. We propose a model to explain the mechanism of post‐hypoxic potentiation. Hypoxia stimulates the respiratory neuronal network through the excitation of the carotid body. Neurotransmitters spilled from excited synapses activate nearby astrocytes. These activated astrocytes persistently excite respiratory neuronal network by continuously releasing gliotransmitters, and short‐term respiratory plasticity emerges.

Sex Differences in Microglial Responses to Chronic Intermittent Hypoxia

Chronic intermittent hypoxia is a hallmark of obstructive sleep apnea (OSA) that causes neuroinflammation and impairs one form of respiratory motor plasticity, phrenic nerve long‐term facilitation (pLTF). Interestingly, the prevalence of OSA is lower in women than in men, although this difference greatly decreases after menopause, suggesting that ovarian hormones may play a protective role. Although estrogen is well known to be anti‐inflammatory following ischemic/hypoxic injury, little is known about its ability to control neuroinflammation as it impacts respiratory neuroplasticity, or in response to inflammation induced by aspects of sleep disordered breathing. Our pilot data suggest that compared to males, microglia (CNS resident immune cells) from females display reduced inflammatory activities as evidenced by reduced cytokine expression (interleukins (IL)‐1b and ‐6), and decreased upregulation of surface CD14 levels. We evaluated differences in estrogen receptor alpha (ERa) expression in male and female microglia because ERa is well‐known to mediate anti‐inflammatory effects of estrogen in the CNS. Although we found no detectable differences in basal ERa expression, we did find that CIH reduced ERa in male microglia, but not in female. These preliminary observations suggest that males may not receive the same benefit of ERa‐mediated anti‐inflammatory activities that females do. Current studies are aimed at understanding the role of gonadal estrogen in creating these sexual dimorphisms in microglial neuroinflammatory responses to CIH. Results from this work will provide significant insight into mechanisms that can be exploited to minimize destabilization of respiratory plasticity in both sexes. (Supported by NIH R01 HL111598).

Phrenic Long‐term Facilitation (pLTF) Following Acute Intermittent Hypoxia is NADPH Oxidase Independent in a Rat Model (SOD1G93A) of ALS

Acute intermittent hypoxia (AIH; 3, 5‐min episodes, 11% O2) elicits a form of respiratory plasticity known as pLTF. In normal rats, pLTF requires NADPH oxidase activity and superoxide formation. Since enhanced pLTF is observed in SOD1G93A rats at disease end‐stage (Nichols et al., 2011) despite increased superoxide dismutase‐1 expression and decreased superoxide accumulation in phrenic motor neurons (Satriotomo et al., 2008), we tested the hypothesis that AIH‐induced pLTF is NADPH oxidase independent in anesthetized,paralyzed and ventilated end‐stage SOD1G93A rats (versus age‐matched, wild‐type littermates). Following intrathecal injections of vehicle (control) or an NADPH oxidase inhibitor (apocynin; 600μM; 12μL), pLTF was abolished in wild‐type (7±3%, n = 4; controls: 40±4%, n=5; p&lt;0.05), but not end‐stage SOD1G93A rats (78±15%, n = 4; controls: 92±18%, n=4; p&gt;0.05). Since pLTF is no longer dependent on NADPH oxidase activity in end‐stage SOD1G93A rats, we suggest that key molecular targets of reactive oxygen species that normally constrain pLTF (e.g. protein phosphatases) are down‐regulated with disease progression in ALS.[Supported by NIH K99 HL119606, R01 HL080209 and R01 HL069064]

Unilateral Denervation of the Diaphragm Muscle Increases Central Drive Only During Ventilatory Behaviors

Compensatory respiratory plasticity is critical to preserve breathing capacity after phrenic denervation (DNV), when the contribution of the ipsilateral phrenic motor neuron (PhrMn) pool is abolished and the force generating capacity of the diaphragm muscle (DIAm) is compromised. In rats, ventilation is maintained after DNV, and this is most likely mediated by an increase in central drive to the contralateral PhrMn pool. The extent of central drive to PhrMns was estimated by measuring the root mean square (RMS) of DIAm EMG at 75ms (RMS75) after the onset of activity. Transdiaphragmatic pressure (Pdi) was measured to estimate DIAm force. We hypothesized that after unilateral DNV central drive to the contralateral PhrMn pool increases across motor behaviors. Immediately following DNV, there was a significant increase in RMS75 during ventilatory behaviors (eupnea, and hypoxia‐hypercapnia). RMS75 did not change during non‐ventilatory behaviors (airway occlusion). These results indicate that after DNV there is: 1) an increase in central drive to the contralateral PhrMn pool during ventilatory behaviors such that Pdi was maintained; 2) central drive to the contralateral PhrMn pool was unchanged during more forceful (greater than ~50% Pdimax) non‐ventilatory behaviors, suggesting that central drive was near maximum. These results highlight the compensatory plasticity in the neural control of the DIAm and the importance of measuring multiple motor behaviors.

Mid‐Cervical Spinal Neuron Activity during Intermittent Hypoxia

The cervical spinal cord contains a complex propriospinal network, and cervical spinal interneurons show both inspiratory‐ and expiratory‐related discharge patterns. However, the role of cervical interneurons in spinal respiratory plasticity is unknown. We hypothesize that a stimulus that induces plasticity in phrenic motor output – intermittent hypoxia (IH) ‐ also triggers plasticity of cervical interneuron activity. Bilateral phrenic motor output was recorded in anesthetized, ventilated adult rats and a multi‐electrode array used to record mid‐cervical spinal neuron activity during baseline, 3, 5‐min hypoxic episodes (11% O2) separated by 5‐min of baseline oxygen (50% O2), and for 60‐min after hypoxia. During baseline (i.e. hyperoxia, normocapnia), four basic interneuron discharge patterns have been observed: 1) phasic inspiratory, 2) phasic expiratory, 3) tonic, or 4) silent. IH altered the firing rate of most (&gt;90%) phasic and tonic neurons, and some tonic neurons shifted to an inspiratory firing pattern. IH also triggered recruitment of previously “silent” neurons. Evaluation of the eta squared (h2) statistic, which estimates the strength of respiratory‐related discharge, indicated increased respiratory modulation in most neurons after IH. These preliminary data suggest that mid‐cervical spinal interneurons express IH‐induced plasticity consistent with the hypothesis that IH promotes synaptic coupling between phrenic motor neurons and interneurons. Support: NIH 1R01NS080180‐01A1 (DDF), State of Florida Brain and Spinal Cord Injury Research Program (DDF, PJR).

Serotonin Receptor Expression on Astrocytes Near Spinal Phrenic Motor Circuitry

Spinal 5‐HT2 and 5‐HT7 receptor activation both trigger respiratory motor plasticity expressed as long‐lasting phrenic motor facilitation, although via distinct cellular mechanisms. Although most studies of serotonin‐dependent spinal respiratory plasticity have focused on motoneurons, 5‐HT receptors are also expressed in glia. Although little is known concerning the function of glia in spinal respiratory plasticity, glia play important roles in in other models of neuroplasticity. Here we present preliminary results concerning 5‐HT receptor expression on GFAP‐positive astrocytes near spinal phrenic motor circuitry. Using immunofluorescence and pseudorabies virus as a retrograde transynaptic tracer from the diaphragm of adult rats, we identified phrenic motoneurons, pre‐phrenic cervical interneurons and nearby GFAP‐positive astrocytes. Initial results indicate 5‐HT2A and 5‐HT7 receptor co‐localization in GFAP positive cells in the vicinity of phrenic‐associated neurons in the cervical gray matter, raising the possibility that spinal astrocytes play an important role in 5‐HT‐dependent phrenic motor plasticity.

Does spinal direct current stimulation enhance synaptic inputs to phrenic motoneurons in humans? A double-blind, sham controlled, randomized crossover study

Background Pilot studies have evidenced the possibility of altering corticospinal projections to phrenic motoneurons (PMNs) and modifying respiratory behavior using repetitive transcranial magnetic stimulation (rTMS). Similar evidence lacks regarding induced neuroplasticity at the spinal level. Objective This study was designed to investigate the immediate and long-lasting effects of a single session of transcutaneous spinal direct current stimulation (tsDCS), a non-invasive way of modulating spinal cord functions, on corticophrenic excitability in healthy humans. Methods Using a double-blind, controlled, randomized crossover approach, we examined the effects of anodal, cathodal, and sham tsDCS delivered to C3–C5 on: – diaphragm motor-evoked potentials (DiMEP) elicited by transcranial magnetic stimulation (TMS); – resting motor threshold (rMT) of the diaphragm and short interval intracortical inhibition (sICI) thought to reflect GABAergic inhibition in the motor cortex; – spontaneous ventilation, as measured by respiratory inductance plethysmography and a measure of ventilatory mathematical complexity (noise limit). Results and conclusions Both anodal and cathodal tsDCS induced a progressive increase of DiMEP amplitude during stimulation that persisted for at least 20 min after current offset. They also lowered rMT. sICI and noise limit were unchanged, suggesting that enhanced excitability was spinal rather than cortical or medullary. Cathodal tsDCS induced a persistent increase in tidal volume. tsDCS can therefore result in polarity-dependent breathing pattern changes, which opens the perspective of harnessing respiratory plasticity as a therapeutic tool in disorders altering the central ventilatory command or its spinal transmission.

Intermittent reductions in respiratory neural activity elicit spinal TNF-α-independent, atypical PKC-dependent inactivity-induced phrenic motor facilitation.

In many neural networks, mechanisms of compensatory plasticity respond to prolonged reductions in neural activity by increasing cellular excitability or synaptic strength. In the respiratory control system, a prolonged reduction in synaptic inputs to the phrenic motor pool elicits a TNF-α- and atypical PKC-dependent form of spinal plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although iPMF may be elicited by a prolonged reduction in respiratory neural activity, iPMF is more efficiently induced when reduced respiratory neural activity (neural apnea) occurs intermittently. Mechanisms giving rise to iPMF following intermittent neural apnea are unknown. The purpose of this study was to test the hypothesis that iPMF following intermittent reductions in respiratory neural activity requires spinal TNF-α and aPKC. Phrenic motor output was recorded in anesthetized and ventilated rats exposed to brief intermittent (5, ∼1.25 min), brief sustained (∼6.25 min), or prolonged sustained (30 min) neural apnea. iPMF was elicited following brief intermittent and prolonged sustained neural apnea, but not following brief sustained neural apnea. Unlike iPMF following prolonged neural apnea, spinal TNF-α was not required to initiate iPMF during intermittent neural apnea; however, aPKC was still required for its stabilization. These results suggest that different patterns of respiratory neural activity induce iPMF through distinct cellular mechanisms but ultimately converge on a similar downstream pathway. Understanding the diverse cellular mechanisms that give rise to inactivity-induced respiratory plasticity may lead to development of novel therapeutic strategies to treat devastating respiratory control disorders when endogenous compensatory mechanisms fail.

Respiratory function after selective respiratory motor neuron death from intrapleural CTB–saporin injections

Amyotrophic lateral sclerosis (ALS) causes progressive motor neuron degeneration, paralysis and death by ventilatory failure. In rodent ALS models: 1) breathing capacity is preserved until late in disease progression despite major respiratory motor neuron death, suggesting unknown forms of compensatory respiratory plasticity; and 2) spinal microglia become activated in association with motor neuron cell death. Here, we report a novel experimental model to study the impact of respiratory motor neuron death on compensatory responses without many complications attendant to spontaneous motor neuron disease. In specific, we used intrapleural injections of cholera toxin B fragment conjugated to saporin (CTB–SAP) to selectively kill motor neurons with access to the pleural space. Motor neuron survival, CD11b labeling (microglia), ventilatory capacity and phrenic motor output were assessed in rats 3–28days after intrapleural injections of: 1) CTB–SAP (25 and 50μg), or 2) unconjugated CTB and SAP (i.e. control; (CTB+SAP). CTB–SAP elicited dose-dependent phrenic and intercostal motor neuron death; 7days post-25μg CTB–SAP, motor neuron survival approximated that in end-stage ALS rats (phrenic: 36±7%; intercostal: 56±10% of controls; n=9; p<0.05). CTB–SAP caused minimal cell death in other brainstem or spinal cord regions. CTB–SAP: 1) increased CD11b fractional area in the phrenic motor nucleus, indicating microglial activation; 2) decreased breathing during maximal chemoreceptor stimulation; and 3) diminished phrenic motor output in anesthetized rats (7days post-25μg, CTB–SAP: 0.3±0.07V; CTB+SAP: 1.5±0.3; n=9; p<0.05). Intrapleural CTB–SAP represents a novel, inducible model of respiratory motor neuron death and provides an opportunity to study compensation for respiratory motor neuron loss.

Intermittent hypercapnia enhances CO2 responsiveness and overcomes serotonergic dysfunction

Serotonergic dysfunction compromises ventilatory chemosensitivity and may enhance vulnerability to pathologies such as the Sudden Infant Death Syndrome (SIDS). We have shown raphé contributions to central chemosensitivity involving serotonin (5-HT)-and γ-aminobutyric acid (GABA)-mediated mechanisms. We tested the hypothesis that mild intermittent hypercapnia (IHc) induces respiratory plasticity, due in part to strengthening of GABA mechanisms. Rat pups were IHc-pretreated (eight consecutive cycles; 5min 5% CO2 – air, 10min air) or constant normocapnia-pretreated as a control, each day for 5 consecutive days beginning at P12. We subsequently assessed CO2 responsiveness using the in situ perfused brainstem preparation. Hypercapnic responses were determined with and without pharmacological manipulation. Results show IHc-pretreatment induces plasticity sufficient for responsiveness despite removal of otherwise critical ketanserin-sensitive mechanisms. Responsiveness following IHc-pretreatment was absent if ketanserin was combined with GABAergic antagonism, indicating that plasticity depends on GABAergic mechanisms. We propose that IHc-induced plasticity could reduce the severity of reflex dysfunctions underlying pathologies such as SIDS.

Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia (1091.2)

Plasticity is an important aspect of the neural control of breathing. One well‐studied form of respiratory plasticity is phrenic long‐term facilitation (pLTF) induced by acute intermittent, but not sustained hypoxia. pLTF pattern sensitivity partially reflects differential regulation by okadaic acid‐sensitive protein phosphatases (PP) during intermittent vs. sustained hypoxia (Wilkerson et al., J Neurosci, 2008), although the relevant phosphatase has not been identified. Here, we show that pLTF is revealed after sustained hypoxia (25 min, 10.5% O2) in rats pre‐treated with bilateral intrapleural injections of small interfering RNAs (siRNAs, Accell‐modified, Thermoscientific; 30 μl/side, 3.33 μM, 3 days) targeting PP1 mRNA (40±14% baseline, n=4). siRNAs targeting PP2A (10±13%, n=3), PP5 (12±13%, n=4) and non‐targeting siRNAs (‐1±11%, n=5) had no effect. siRNAs had no effect on the response during hypoxia. Preliminary immunohistochemistry confirms a small knockdown of targeted phosphatases within the phrenic motor nucleus. PP1 appears to be the relevant okadaic acid‐sensitive phosphatase regulating pLTF.Grant Funding Source: Supported by: HL111598, HL080209, Francis Family Foundation (AGH).

Enhanced phrenic long‐term facilitation in rats with motor neuron death from intrapleural CTB‐saporin injections (713.1)

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%; n=15; p&lt;0.05), but not 28 days post‐injection (CTB‐SAP: 61±14%, n=11 vs. Control: 37±9; n=5; p&gt;0.05). Thus, pLTF at 7 (not 28) days post‐CTB‐SAP more closely resembles pLTF in end‐stage ALS rats, suggesting that processes unique to the period of active motor neuron death enhance pLTF. This project increases our understanding of respiratory plasticity and its implications for breathing in motor neuron disease.[Supported by NIH HL080209, HL69024 and the Francis Families Foundation]Grant Funding Source: Supported by NIH HL080209, HL69024 and the Francis Families Foundation

Inactivity‐induced phrenic motor facilitation does not require new protein synthesis (713.8)

Reduced respiratory neural activity elicits a TNFα‐ and atypical PKC‐dependent form of spinal plasticity in phrenic motor output termed inactivity‐induced phrenic motor facilitation (iPMF), a long‐lasting increase in phrenic nerve burst amplitude apparent once respiratory neural activity is restored. Many forms of neuroplasticity require protein synthesis for long‐term expression, including the most widely studied form of spinal respiratory plasticity, phrenic long‐term facilitation (pLTF; Baker‐Herman and Mitchell, 2002) elicited following acute intermittent hypoxia. We hypothesized that iPMF would also require de novo protein synthesis. Briefly, Sprague Dawley rats were urethane anesthetized, vagotomized, paralyzed, and prepared for electrophysiological recording. Rats received intrathecal injection (C4) of vehicle (aCSF; n=5) or the protein synthesis inhibitor emetine (n=6; 1ug/kg; Baker‐Herman and Mitchell, 2002) ~15 min prior to a prolonged (30min) period of neural apnea. As expected, in aCSF treated rats, phrenic nerve amplitude was significantly increased compared to baseline 5 min following reversal of neural apnea (53±11, %baseline) and was maintained for &gt;60 min (54±16, %baseline), indicating long‐lasting iPMF. Contrary to our hypothesis, rats receiving emetine also expressed long‐lasting iPMF such that phrenic nerve burst amplitude was significantly increased from baseline at both 5 (41±8, %baseline) and 60 min (42±12,%baseline) following neural apnea (p&lt;0.05). Importantly, phrenic nerve burst amplitude in rats receiving intrathecal emetine was not significantly different from rats receiving aCSF at any time point following neural apnea (p&gt;0.05). Together, these data suggest that iPMF is a form of long‐lasting plasticity that does not require new protein synthesis.Grant Funding Source: Supported by NIH HL105511

Neither serotonin nor adenosine-dependent mechanisms preserve ventilatory capacity in ALS rats

In rats over-expressing SOD1G93A, ventilation is preserved despite significant loss of respiratory motor neurons. Thus, unknown forms of compensatory respiratory plasticity may offset respiratory motor neuron cell death. Although mechanisms of such compensation are unknown, other models of respiratory motor plasticity may provide a conceptual guide. Multiple cellular mechanisms give rise to phrenic motor facilitation; one mechanism requires spinal serotonin receptor and NADPH oxidase activity whereas another requires spinal adenosine receptor activation. Here, we studied whether these mechanisms contribute to compensatory respiratory plasticity in SOD1G93A rats. Using plethysmography, we assessed ventilation in end-stage SOD1G93A rats after: (1) serotonin depletion with parachlorophenylalanine (PCPA), (2) serotonin (methysergide) and A2A (MSX-3) receptor inhibition, (3) NADPH oxidase inhibition (apocynin), and (4) combined treatments. The ability to increase ventilation was not decreased by individual or combined treatments; thus, these mechanisms do not maintain breathing capacity at end-stage motor neuron disease. Possible mechanisms giving rise to enhanced breathing capacity with combined treatment in end-stage SOD1G93A rats are discussed.

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

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 anesthetized, mechanically ventilated adult male rats, AIH (3×5-min episodes; 10% O2; 5min) elicited a progressive increase in the amplitude of integrated phrenic nerve bursts (i.e. pLTF), which lasted 60min 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; 5min) of a NO donor (sodium nitroprusside; SNP) elicited pMF similar in time-course and magnitude (40.4±6.0%, 60min 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 was affected by pre-treatment with a protein kinase G (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.