Inspiratory Burst Amplitude Research Articles

Spinal delivery of ampakine CX717 impacts phrenic motor output in adult rats

Throughout the brainstem and spinal cord, α‐amino‐3‐hydroxy‐5‐methy‐4‐isoxazole propionate receptors (AMPARs) play an essential role in respiratory motor control. Ampakines are positive allosteric modulators of AMPARs which can enhance glutamatergic currents. Our laboratory recently reported that intravenous delivery of low‐dose ampakine CX717 can evoke increases in phrenic nerve inspiratory burst amplitude. However, this increased phrenic bursting could reflect an impact of the ampakine on pre‐motor brainstem circuits or a direct action on spinal neurons. In ongoing experiments, we are testing the hypothesis that delivery of ampakines to the cervical spinal cord is sufficient to evoke a sustained increase in phrenic nerve inspiratory burst amplitude. Phrenic motor output was measured in urethane‐anesthetized, mechanically ventilated, vagotomized adult male Sprague‐Dawley rats. Along with amplitude of inspiratory phrenic motor output, arterial blood pressure and end‐tidal CO2 were recorded. For intrathecal delivery of ampakine CX717 (15μl, 500μM, dissolved in 10% HPCD), the spinal cord was exposed via a dorsal approach followed by laminectomy and durotomy to enable drug administration over the mid‐cervical spinal cord. At least 15 min of stable phrenic burst amplitude was recorded as baseline after which intrathecal CX717 was applied. The initial results (n=3 sham, n=3 CX717) indicate that spinally directed CX717 causes a gradual and sustained increase in phrenic inspiratory burst amplitude lasting up to 60 min. Conversely, intrathecal delivery of 10% HPCD (sham) had no consistent impact on phrenic burst amplitude. These preliminary results are consistent with the hypothesis that allosteric modulation of spinal AMPA receptors can increase phrenic motor output. Phrenic motor neurons express AMPA receptors and are a likely candidate for the site of CX717 action.Support or Funding Information5 R01 HL139708 02 (DDF)

Modulation of the tongue muscle activity in response to bronchopulmonary C‐fiber activation following mid‐cervical contused in the rat

The present study was designed to investigate the tongue muscle activity in response to bronchopulmonary C‐fiber activation following mid‐cervical spinal contusion in the rat. The esophageal pressure and electromyogram of extrinsic tongue muscles (genioglossus and hyoglossus) were monitored before and after inhalation of capsaicin (25 and 100 μg/ml) at the acute, subchronic and chronic injured stages after mid‐cervical contusion. The results demonstrated that pre‐inspiratory and inspiratory burst amplitude of tongue muscle were generally enhanced at the acute and subchronic injured stages in mid‐cervical contused animals vs. uninjured animals. Inhalation of capsaicin usually caused a rapid shallow breathing pattern associated with a reduction in pre‐inspiratory and inspiratory activity of the tongue muscle in uninjured animals at the acute injured stage; however, this altered breathing pattern and decreased tongue muscle activity were blunted in contused animals. During the chronic injured stage, the expiratory duration was prolonged to a similar degree in both uninjured and contused animals after inhalation of capsaicin. Notably, inhibition of genioglossus muscle activity during capsaicin inhalation is more severed in chronically contused animals. These results demonstrated that cervical spinal cord injury is associated with alteration of supraspinal respiratory activity, which may modulate upper airway motor outputs and bronchopulmonary C‐fiber mediated respiratory reflexes. The compensatory increase in the tongue muscle activity at the acute injured stage may be benefit to the maintenance of the upper airway patency; however, the susceptible tongue muscle activity during bronchopulmonary C‐fiber activation may increase the risk of airway obstruction during the chronic injured stage.Support or Funding InformationMOST (105‐2628‐B‐110‐002‐MY3 & 108‐2636‐B‐110‐001), Higher Education Sprout Project (07C030111), NSYSU‐KMU Joint Research Project (107‐I001)

Effects of Fluoxetine on Inspiratory Motor Activity and Chemical Control of Breathing in Spontaneously Breathing Urethane‐Anesthetized 6‐OHDA MFB‐lesioned Parkinson’s Disease Rat Model

Breathing abnormalities are a common non‐motor symptom (NMS) of Parkinson’s Disease (PD), and multiple causes have been proposed, including impaired central respiratory control. While the loss of dopaminergic neurons is the primary pathology of PD, it is becoming better appreciated that serotonergic (5‐HT) dysfunction also plays a role in the development of motor and NMS and complications in PD. To this end, recent studies addressing the role of 5‐HT in PD symptoms have revealed that activation or antagonism of specific 5‐HT receptor subtypes may have therapeutic benefit. 5‐HT neurotransmission is also known to play a critical role in control of breathing; thus, respiratory abnormalities in PD may be further complicated by the progressive 5‐HT dysfunction in PD. Ongoing work in our laboratory has been focused on characterizing the respiratory phenotype of PD using 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced preclinical rat models of PD. Here, we evaluate potential contributions of 5‐HT dysfunction by assessing the effects of acute administration of the selective 5‐HT reuptake inhibitor fluoxetine hydrochloride (FLX) on basal inspiratory motor (diaphragm EMG) activity and hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses in spontaneously breathing urethane‐anesthetized adult female rats 2‐weeks after unilateral medial forebrain bundle (MFB) 6‐OHDA or vehicle/control injection. Basal (BL) diaphragm EMG activity was recorded for ≥30 min, the HVR (12% O2, 90s) and HCVR (7% CO2, 5 min) were assessed, acute FLX (10 mg/kg, iv) was injected and allowed 30 min to exert steady‐state effects, and then the HVR and HCVR were re‐assessed. We found that in both vehicle‐ and 6‐OHDA‐injected rats, administration of FLX produced an initial increase in burst frequency (by ~30%) and amplitude (by ~15%) above BL levels within ~60 s, after which burst frequency attenuated to a new steady‐state level of ~10% above BL levels while burst amplitude returned to slightly below BL levels in vehicle‐injected rats, but attained a new steady‐state level of ~10% above BL levels in 6‐OHDA‐injected rats. The effects of FLX on HVR and HCVR frequency behaviors were negligible in both control‐ and 6‐OHDA‐injected rats although a slight attenuation of the HVR frequency increase (by ~5%) was seen. The effects of FLX on HVR and HCVR amplitude behaviors, while underpowered, showed a trend of slightly enhanced of HVR amplitude (by ~5%) and HCVR amplitude (by ~9%) in 6‐OHDA‐injected rats; however, additional experiments are needed to determine the significance of these observations. While these preliminary observations suggest that (compared to control rats) MFB‐lesioned rats exhibit a difference in FLX‐induced effects on basal inspiratory burst amplitude alterations, acute FLX administration may not be able to fully correct chemical control of breathing deficits. We suggest that additional experiments are needed to better identify specific 5‐HT mechanisms that may contribute to chemical control of breathing deficits in this PD rat model.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson’s Disease Research at Stony Brook University

Acute Fluoxetine Administration Improves Intermittent Hypoxia‐induced Inspiratory Burst Amplitude Deficits During Peak LPS‐induced Neuroinflammatory Phase in Anesthetized Spontaneously Breathing Adult Male Rats

Studies examining the impact of neuroinflammation on various aspects of neural control of breathing typically use systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) and evaluate alterations in ventilatory control during either the induction (~2–4 hr) or resolution (~24 hr) phases of the acute neuroinflammatory response. Changes in LPS‐regulated pro‐inflammatory gene/protein expression, however, have been shown to peak at ~4–6 hrs following LPS exposure, but little is known about the impact of LPS‐induced neuroinflammation on ventilatory control at this time point. Systemic LPS administration has also been shown to increase serotonin (5‐HT) transporter activity and reduce 5‐HT levels. Since numerous inspiratory motor behaviors are 5‐HT‐dependent, including some hypoxic ventilatory behaviors, LPS‐induced changes in 5‐HT could contribute to ventilatory deficits noted in LPS‐induced neuroinflammation studies. To begin to address these issues, we examined the impact of peak phase LPS‐induced neuroinflammation on inspiratory motor behaviors elicited by intermittent hypoxia (IH) in urethane‐anesthetized vagal intact spontaneously breathing adult male Sprague‐Dawley rats in both the absence and presence of acute administration of the SSRI fluoxetine hydrochloride (FLX). Beginning at ~4 hr after systemic LPS (3 mg/kg, ip) or saline (CON) treatment, basal (BL) diaphragm EMG activity was recorded for ~30 min (40% O2), FLX (10 mg/kg) or vehicle (VEH, 0.9% saline) was injected (iv) and allowed 30 min to exert steady‐state effects, and then IH exposure composed of 5 alternating 3‐min episodes of hypoxia (11% O2; 2%CO2) and hyperoxia (40% O2) was delivered, after which the rat was allowed to recover. We found that during the first 30–60s following FLX injection in both CON‐ and LPS‐treated rats, burst amplitude (by ~12%) and frequency (by ~40%) were increased, after which amplitude returned to BL levels and frequency attained a new steady‐state ~10% above BL levels; VEH injection was ineffective in altering either burst amplitude or frequency. As expected during IH exposure in CON‐treated VEH‐injected rats, a robust increase in burst frequency and amplitude were observed with the subsequent hypoxic exposures eliciting progressive augmentation of burst amplitude (PA; IH1, ~45%; IH5, ~65%). In contrast in CON‐treated FLX‐injected rats, IH‐induced amplitude effects were markedly attenuated (IH1‐IH5, ~11–21%), and similar to those seen during IH exposure in LPS‐treated VEH‐injected rats (~7–11%). In LPS‐treated FLX‐injected rats, however, an increase in both burst amplitude and PA (IH1, ~19%; IH5, ~35%) were noted. Similar IH‐induced increases in burst frequency and post hypoxic frequency decline were also observed in both LPS‐ and CON‐treated FLX‐ and VEH‐injected rats. These preliminary observations suggest that acute FLX administration may partially correct the blunted increase in burst amplitude and induce PA in response to IH exposure during peak phase in LPS‐treated rats albeit similar FLX treatment appears to impair IH‐induced amplitude effects in CON‐treated rats.Support or Funding InformationNIH NS101737; SBU Thomas Hartman Center for Parkinson’s Disease Research

Respiratory pattern and phrenic and hypoglossal nerve activity during normoxia and hypoxia in 6-OHDA-induced bilateral model of Parkinson\u2019s disease

Respiratory disturbances present in Parkinson’s disease (PD) are not well understood. Thus, studies in animal models aimed to link brain dopamine (DA) deficits with respiratory impairment are needed. Adult Wistar rats were lesioned with injection of 6-hydroxydopamine (6-OHDA) into the third cerebral ventricle. Two weeks after hypoxic test was performed in whole-body plethysmography chamber, phrenic (PHR) and hypoglossal (HG) nerve activities were recorded in normoxic and hypoxic conditions in anesthetized, vagotomized, paralyzed and mechanically ventilated rats. The effects of activation and blockade of dopaminergic carotid body receptors were investigated during normoxia in anesthetized spontaneously breathing rats. 6-OHDA injection affected resting respiratory pattern in awake animals: an increase in tidal volume and a decrease in respiratory rate had no effect on minute ventilation. Hypoxia magnified the amplitude and minute activity of the PHR and HG nerve of 6-OHDA rats. The ratio of pre-inspiratory to inspiratory HG burst amplitude was reduced in normoxic breathing. Yet, the ratio of pre-inspiratory time to total time of the respiratory cycle was increased during normoxia. 6-OHDA lesion had no impact on DA and domperidone effects on the respiratory pattern, which indicate that peripheral DA receptors are not affected in this model. Analysis of monoamines confirmed substantial striatal depletion of dopamine, serotonin and noradrenaline (NA) and reduction of NA content in the brainstem. In bilateral 6-OHDA model changes in activity of both nerves: HG (linked with increased apnea episodes) and PHR are present. Demonstrated respiratory effects could be related to specific depletion of DA and NA.

Ampakine pretreatment enables a single brief hypoxic episode to evoke phrenic motor facilitation.

Phrenic long-term facilitation (LTF) is a sustained increase in phrenic motor output occurring after exposure to multiple (but not single) hypoxic episodes. Ampakines are a class of drugs that enhance AMPA receptor function. Ampakines can enhance expression of neuroplasticity, and the phrenic motor system is fundamentally dependent on excitatory glutamatergic currents. Accordingly, we tested the hypothesis that combining ampakine pretreatment with a single brief hypoxic exposure would result in phrenic motor facilitation lasting well beyond the period of hypoxia. Phrenic nerve output was recorded in urethane-anesthetized, ventilated, and vagotomized adult Sprague-Dawley rats. Ampakine CX717 (15 mg/kg iv; n = 8) produced a small increase in phrenic inspiratory burst amplitude and frequency, but values quickly returned to predrug baseline. When CX717 was followed 2 min later by a 5-min exposure to hypoxia (n = 8; ~45 mmHg), a persistent increase in phrenic inspiratory burst amplitude (i.e., phrenic motor facilitation) was observed up to 60 min posthypoxia (103 ± 53% increase from baseline). In contrast, when hypoxia was preceded by vehicle injection (10% 2-hydroxypropyl-β-cyclodextrin; n = 8), inspiratory phrenic bursting was similar to baseline values at 60 min. Additional experiments with another ampakine (CX1739, 15 mg/kg) produced comparable results. We conclude that pairing low-dose ampakine treatment with a single brief hypoxic exposure can evoke sustained phrenic motor facilitation. This targeted approach for enhancing respiratory neuroplasticity may have value in the context of hypoxia-based neurorehabilitation strategies.NEW & NOTEWORTHY A single brief episode of hypoxia (e.g., 3-5 min) does not evoke long-lasting increases in respiratory motor output after the hypoxia is concluded. Ampakines are a class of drugs that enhance AMPA receptor function. We show that pairing low-dose ampakine treatment with a single brief hypoxic exposure can evoke sustained phrenic motor facilitation after the acute hypoxic episode.

Effects of Two‐Hit Lipopolysaccharide (LPS) Administration on Acute Intermittent Hypoxia (AIH)‐Induced Inspiratory Motor Behaviors in Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat

Ongoing studies designed to induce peripheral inflammation, and its accompanying neuroinflammation, have begun to examine various aspects of neural control of breathing. While most studies typically administer the bacterial endotoxin lipopolysaccharide (LPS) systemically or in an airway/lung targeted manner in order to produce models of systemic inflammatory response syndrome (SIRS)/sepsis and acute lung injury (ALI), a ‘two‐hit’ LPS administration protocol in which systemic LPS (ip, 3 mg/kg) is administered ~24 hr prior to an airway/lung targeted (intratracheal (IT); 0.5 mg/kg) LPS injection has been used to better reflect chronic (lung/airway) disease states. One of the most studied respiratory behaviors in which inflammation has been shown to negatively impact breathing is AIH‐induced inspiratory motor (phrenic) long‐term facilitation (pLTF), where in anesthetized, vagotomized, ventilated adult male rats, single‐hit systemic LPS administration attenuates AIH‐induced serotonin2‐dependent pLTF. Here, we examined the impact of two‐hit LPS administration in urethane‐anesthetized vagal intact spontaneously breathing adult male Sprague‐Dawley rats on AIH‐induced inspiratory motor behaviors using AIH composed of five 3‐min episodes of hypoxia (11% O2) separated by 3‐min exposures to hyperoxia (40% O2) with all rats being continuously supplied with 2% CO2. Similar experiments were performed in control rats that were either untreated or received two‐hit saline injections. We found that in control rats, exposure to the repetitive bouts of hypoxia elicited increases in both diaphragm EMG burst frequency (~18% increase) and amplitude (IH1, ~22% to IH5, ~31%) that resulted in progressive augmentation (PA) and LTF inspiratory burst amplitude that persisted for ~75 min post‐AIH (sustained ~12–18% increase above baseline levels); no frequency LTF and a robust post hypoxic frequency decline (PHFD) after each hypoxic exposure and for up to 25 min post‐AIH were also observed. In contrast, in LPS‐treated rats, exposure to the repetitive bouts of hypoxia elicited a more robust increase in diaphragm EMG burst frequency (~25–30% increase) and a blunted or absent amplitude response (IH1, ≤8%). Moreover, in these rats, PA of burst amplitude was absent, inspiratory burst amplitude LTF was highly variable with an overall amplitude increase of ~10% above baseline levels that subsided by 20 min post‐AIH, and PHFD after each hypoxic exposure slightly increased in magnitude and persisted for up to 50 min post‐AIH. These preliminary observations suggest that (compared to control rats) two‐hit LPS administration affects mechanisms underlying multiple components responsible for inspiratory burst frequency and amplitude plasticity in response to hypoxic exposure(s) and following AIH exposure. Additional experiments, including those using other AIH protocols, will be needed to confirm these observations and identify specific mechanisms underlying these differences.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GIRK Channel Activation Contributes to the Muscarinic Modulation of XII Motoneuron Excitability in Neonatal Mice in Vitro

Sleep specific decreases in airway muscle tone, and particularly the genioglossus muscle of the tongue (primary tongue protruder), have been causally implicated in obstructive sleep apnea. During REM (rapid eye movement) sleep, decreased excitability of the XII motoneurons that innervate the genioglossus muscle may be due to a loss of excitatory noradrenergic modulation, or due to activation of muscarinic acetylcholine receptors that may act via G‐protein coupled inward rectifying potassium (GIRK) channels. GIRK channel activation results in membrane hyperpolarization and thus could mediate a decrease in excitability of XII motoneurons during REM sleep. Here we test the hypothesis that muscarine acts via GIRK channels to inhibit inspiratory burst amplitude of XII motoneurons in vitro. Using immunohistochemical techniques, our preliminary data indicate that XII motoneurons express GIRK1, GIRK2, and GIRK3 isoforms. Next, using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day, P0–5), we investigated the effects of GIRK channel and muscarinic acetycholine receptor activation on XII inspiratory burst characteristics including burst amplitude and burst period. Local application of muscarine (30 s, 100 mM) to the XII motor nucleus had the net effect of increasing XII inspiratory burst amplitude, although local pre‐application of the GIRK channel antagonist Tertiapin Q (TQ, 60 s, 100 nM) led to two different responses. In P0–2 mice (n = 4), muscarine increased burst amplitude to 120% of control (range 105–133%) while pre‐application of TQ followed by local application of muscarine led to a similar 115% (range 104–123%) increase in amplitude, suggesting that muscarine does not activate GIRK channels in P0–2 mice. In P3–5 mice (n = 4), however, local application of muscarine increased burst amplitude to 135% (range 122–156%) of baseline, and pre‐application of TQ to block GIRK channel activation further increased the response to muscarine to 160% (range 130–170%) of baseline amplitude. These results indicate that muscarine acts at GIRK channels to inhibit XII motoneuron excitability in P3–5 mice, but also acts at other channels to enhance XII motoneuron excitability. Preliminary data suggests this effect is specific to muscarinic activation of GIRK channels because bath application of TQ (10 nM) in P3–5 mice resulted in no change in inspiratory burst amplitude (mean: 102% of baseline, range 90–109%, after 30 min in TQ, n = 3). In contrast, bath application of the GIRK channel agonist ML297 (10 mM) resulted burst amplitude decrease to 80–88% of baseline in 2 out of 3 P3–5 neonatal mice and an increase in burst amplitude to 120% of baseline in a third prep. Burst period increased to 179% of baseline (range: 128–263%) indicating that GIRK channels are likely also present in preBötzinger Complex where inspiratory rhythm is generated. These data support the hypothesis that activation of muscarinic receptors in XII motoneurons leads to GIRK channel activation in P3–5 neonatal mice, which may be one mechanism through which muscarine mediates a decrease in XII motoneuron excitability in vivo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Substance P Reshapes Inspiratory and Postinspiratory Activity Generated in Horizontal Brainstem Slices

The triple oscillator hypothesis posits that breathing is generated by three rhythmogenic microcircuits contained within the ventrolateral medulla. The preBötzinger Complex (preBötC) generates the inexorable inspiratory phase of breathing, whereas the conditional postinspiratory and active expiratory phases of breathing are generated by the Postinspiratory Complex (PiCo) and lateral parafacial respiratory group (PFL), respectively. Neuromodulators including norepinephrine, opioids, and somatostatin are known to differentially modulate the activity of each rhythmic circuit, likely contributing to the independence of each breathing phase and the flexibility of the respiratory rhythm. However, whether the functional boundaries of each of these microcircuits are static or can change dynamically based on the neuromodulatory state of the network is unknown. The neuromodulator Substance P (SP) facilitates breathing frequency in vivo and also in medullary slices that isolate the preBötC in vitro. Using a neonatal mouse horizontal brainstem slice preparation that preserves the rostrocaudal extent of the ventral medulla, we tested the hypothesis that SP differentially modulates bursting activity in the preBötC and PiCo and explored how SP may reshape the distribution of inspiratory activity within the wider medullary network. Rhythmic bursting activity was simultaneously recorded from the preBötC and PiCo (n=5). As expected, recordings from the PiCo, located ~500μm rostral to the preBötC, revealed a slow postinspiratory rhythm with robust bursts occurring after only ~1/12 preBötC bursts under control conditions. Following introduction of 0.5–1.0μM SP into the bath superfusion solution, PiCo bursts decreased in amplitude (~40%) and accelerated such that they occurred 1:1 with preBötC bursts without changing the time interval between peak preBötC and PiCo activity. In some PiCo recordings, we noted a small amount of inspiratory activity synchronized with preBötC bursts that was eliminated in the presence of SP. To test whether SP alters the spatial distribution of inspiratory activity along the ventral medulla, we simultaneously recorded inspiratory population activity generated at the preBötC and 400–600μm rostral (n=8). Following application of 1μM SP frequency was increased (~90%) and inspiratory burst amplitude was reduced by ~20% at the preBötC, whereas burst amplitude decreased by ~50% in rostral recordings. Based on these preliminary experiments, we hypothesize that SP reshapes breathing activity generated within the medulla by facilitating postinspiratory activity and reducing the extent of inspiratory activity rostral of the preBötC to shrink the size of the active inspiratory network. Differential neuromodulatory properties and dynamic functional boundaries of the rhythmogenic circuits that generate breathing may contribute to the remarkable flexibility and robustness of this vital physiological behavior.Support or Funding InformationNIH F32 HL134207NIH R01 HL126523This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of Two‐Hit Lipopolysaccharide (LPS) Administration on 8‐OH DPAT‐induced Inspiratory Burst Amplitude and Frequency Changes in Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat with Prior Single‐Bout Acute Intermittent Hypoxia Exposure

Inflammation has been suggested to alter a variety of physiological processes by its effects on serotonergic (5‐HT) neurotransmission. In the respiratory neural control system, LPS‐induced neuroinflammation has been shown to affect multiple aspects of central ventilatory control, including serotonin2 (5‐HT2)‐dependent inspiratory motor behaviors. While numerous studies have focused on the role of 5‐HT2A receptors in neural control of breathing, the roles of other 5‐HT receptors are beginning to be appreciated. To this end, activation of 5‐HT1A receptors has been shown to exert marked effects on inspiratory motor activity; however, the impact of LPS‐induced neuroinflammation on 5‐HT1A receptor‐mediated inspiratory motor behaviors remains to be determined. To begin to address this issue, we studied the effects of systemic application of the 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐npropylamino) tetralin (8‐OH DPAT) on basal inspiratory (diaphragm) EMG burst amplitude and frequency in spontaneously breathing urethane‐anesthetized adult male Sprague‐Dawley rats following a ‘two‐hit’ lipopolysaccharide (LPS) administration protocol in which systemic LPS (3 mg/kg, ip) was administered ~24 hr prior to an intratracheal LPS (0.5 mg/kg, IT) injection. Similar 8‐OH DPAT experiments were performed in control rats that were either untreated or received two‐hit saline injections. All rats used for this study had also undergone an acute intermittent hypoxia (AIH) exposure protocol that was completed at least 90 min prior to administration of 8‐OH DPAT. For this study, we compare diaphragm EMG burst activities before (baseline, BL) systemic administration of 8‐OH DPAT (0.3 mg/kg, iv) to those obtained at various time points for the 30 min period immediately following administration. We found that in both control and LPS‐treated rats, administration of 8‐OH DPAT similarly affected burst frequency but differentially affected burst amplitude. In both groups of rats, 8‐OH DPAT injection produced an ~50% increase in burst frequency above BL levels within 30–60s, but by 5 min post injection, this increase was attenuated to an ~30% increase above BL levels that persisted for the remainder of the 30 min recording period. While 8‐OH DPAT injection produced an initial ~15% increase in burst amplitude above BL levels within 60s in both groups of rats, a further increase in burst amplitude up to ~30% above BL was noted by 15 min post injection in control rats, and this increase persisted for the remainder of the 30 min recording period while in LPS‐treated rats, a slight attenuation of the burst amplitude increase was noted. These preliminary observations suggest that (compared to control rats) two‐hit LPS administration may differentially affect mechanisms by which 5‐HT1A receptor activation modulates inspiratory burst frequency and amplitude. Additional experiments will be needed to identify specific mechanisms underlying these differences.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute Fluoxetine (FLX) Administration Improves Inspiratory Motor Activity Response to Acute Single Bout Combined Hypoxic‐Hypercapnic Exposure in Urethane‐anesthetized Spontaneously Breathing Adult Male Sprague‐Dawley Rat at 24‐hr Post‐Lipopolysaccharide (LPS) Injection

LPS‐induced neuroinflammation has been suggested to alter serotonergic (5‐HT) neurotransmission by acting on the serotonin transporter to alter serotonin levels. In the respiratory neural control system, LPS‐induced neuroinflammation has been shown to affect multiple aspects of central ventilatory control, including 5‐HT‐dependent inspiratory motor behaviors. Ongoing work in our laboratory has been examining the impact of LPS‐induced neuroinflammation on neural control of breathing, including 5‐HT‐mediated inspiratory motor behaviors. Here, we examine the effects of acute systemic administration of the selective serotonin reuptake inhibitor (SSRI) fluoxetine hydrochloride (FLX) on basal inspiratory (diaphragm) EMG activity and the maximal chemoreceptor stimulation ventilatory response (MCVR) elicited by an acute single bout of combined hypoxic‐hypercapnic exposure in spontaneously breathing urethane‐anesthetized adult male Sprague‐Dawley rats 24‐hr after systemic LPS (3 mg/kg, ip) or vehicle (0.9% saline, ip) administration. For these experiments, basal diaphragm EMG activity was recorded for 30 min under baseline (BL) conditions (40% O2), FLX (10 mg/kg, iv) was then administered and allowed to exert its effects for 30 min, after which a 3 min MCVR (12% O2, 7%CO2) challenge was performed, followed by 30 min recovery; control experiments using vehicle (VEH; 0.9% saline) instead of FLX were also performed. We found that FLX and VEH administration were ineffective in producing significant changes in inspiratory burst amplitude or frequency in either 24‐hr post‐LPS‐ or saline‐treated rats although during the first 30–60s following FLX injection, a small transient increase (~10–15%) in burst frequency was noted for both groups. In 24‐hr post‐LPS‐ and saline‐treated rats, for the MCVR following VEH injection, most rats exhibited a sustained increase in burst amplitude (≥30% above BL) and a robust increase in burst frequency (~15–30%) that gradually returned to BL levels before the end of the challenge. In 24‐hr post‐LPS and saline‐treated rats, for the MCVR following FLX injection, a robust and maintained increase in burst amplitude (40–50% above FLX level) was also observed; however, marked differences were noted for the MCVR frequency behavior. In this case, in 24‐hr post‐saline‐treated rats, the increase in burst frequency degraded as the challenge progressed (40% above FLX level during min 1 but 97% of FLX level during min 3) while in 24‐hr post‐LPS‐treated rats, the increase in burst frequency remained relatively stable (~25% above FLX level during min 1 and ~20% above FLX level during min 3). These preliminary observations suggest that (compared to control rats), acute FLX administration following systemic LPS‐induced neuroinflammation may be capable of improving inspiratory motor deficits in hypoxic‐hypercapnic chemical control of breathing.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of glycinergic inhibition on respiratory rhythmic hypoglossal bursting in the rat.

The hypoglossal nerve displays respiratory rhythmic bursting and is composed of preinspiratory and inspiratory activity which is important in maintaining upper airway patency. The present study was designed to examine the modulatory role of glycinergic inhibition in respiratory rhythmic hypoglossal bursting. The activity of the phrenic nerve, as well as the medial and lateral branches of the hypoglossal nerve, was recorded simultaneously in urethane-anesthetized and mechanically ventilated adult rats in response to moderate and high levels of sustained lung inflation. The results demonstrated that inspiratory activity of the phrenic nerve gradually reduced with increasing lung inflation. The burst amplitude and discharge onset of the hypoglossal nerve branches were enhanced during moderate lung inflation but inhibited by high levels of lung inflation. These lung volume-mediated respiratory reflexes were abolished following a bilateral cervical vagotomy. In addition, intravenous administration of a glycine receptor antagonist (strychnine, 1 μmole/kg) attenuated preceding onset of rhythmic hypoglossal bursting but enhanced inspiratory hypoglossal burst amplitude during the baseline. Moreover, both excitatory and inhibitory effects of lung inflation on hypoglossal nerve activity were attenuated following a glycine transmission blockade. These results suggest that glycinergic inhibition modulated rhythmic hypoglossal bursting and was involved in mediating lung volume-induced respiratory reflexes.

Excitatory Modulation of the preBötzinger Complex Inspiratory Rhythm Generating Network by Endogenous Hydrogen Sulfide.

Hydrogen Sulfide (H2S) is one of three gasotransmitters that modulate excitability in the CNS. Global application of H2S donors or inhibitors of H2S synthesis to the respiratory network has suggested that inspiratory rhythm is modulated by exogenous and endogenous H2S. However, effects have been variable, which may reflect that the RTN/pFRG (retrotrapezoid nucleus, parafacial respiratory group) and the preBötzinger Complex (preBötC, critical for inspiratory rhythm generation) are differentially modulated by exogenous H2S. Importantly, site-specific modulation of respiratory nuclei by H2S means that targeted, rather than global, manipulation of respiratory nuclei is required to understand the role of H2S signaling in respiratory control. Thus, our aim was to test whether endogenous H2S, which is produced by cystathionine-β-synthase (CBS) in the CNS, acts specifically within the preBötC to modulate inspiratory activity under basal (in vitro/in vivo) and hypoxic conditions (in vivo). Inhibition of endogenous H2S production by bath application of the CBS inhibitor, aminooxyacetic acid (AOAA, 0.1–1.0 mM) to rhythmic brainstem spinal cord (BSSC) and medullary slice preparations from newborn rats, or local application of AOAA into the preBötC (slices only) caused a dose-dependent decrease in burst frequency. Unilateral injection of AOAA into the preBötC of anesthetized, paralyzed adult rats decreased basal inspiratory burst frequency, amplitude and ventilatory output. AOAA in vivo did not affect the initial hypoxia-induced (10% O2, 5 min) increase in ventilatory output, but enhanced the secondary hypoxic respiratory depression. These data suggest that the preBötC inspiratory network receives tonic excitatory modulation from the CBS-H2S system, and that endogenous H2S attenuates the secondary hypoxic respiratory depression.

Power spectral analysis of hypoglossal nerve activity during intermittent hypoxia-induced long-term facilitation in mice.

Power spectral analyses of electrical signals from respiratory nerves reveal prominent oscillations above the primary rate of breathing. Acute exposure to intermittent hypoxia can induce a form of neuroplasticity known as long-term facilitation (LTF), in which inspiratory burst amplitude is persistently elevated. Most evidence indicates that the mechanisms of LTF are postsynaptic and also that high-frequency oscillations within the power spectrum show coherence across different respiratory nerves. Since the most logical interpretation of this coherence is that a shared presynaptic mechanism is responsible, we hypothesized that high-frequency spectral content would be unchanged during LTF. Recordings of inspiratory hypoglossal (XII) activity were made from anesthetized, vagotomized, and ventilated 129/SVE mice. When arterial O2 saturation (SaO2) was maintained >96%, the XII power spectrum and burst amplitude were unchanged for 90 min. Three, 1-min hypoxic episodes (SaO2 = 50 ± 10%), however, caused a persistent (>60 min) and robust (>400% baseline) increase in burst amplitude. Spectral analyses revealed a rightward shift of the signal content during LTF, with sustained increases in content above ∼125 Hz following intermittent hypoxia and reductions in power at lower frequencies. Changes in the spectral content during LTF were qualitatively similar to what occurred during the acute hypoxic response. We conclude that high-frequency content increases during XII LTF in this experimental preparation; this may indicate that intermittent hypoxia-induced plasticity in the premotor network contributes to expression of XII LTF.

Activation of the hypothalamic paraventricular nucleus by forebrain hypertonicity selectively increases tonic vasomotor sympathetic nerve activity.

We recently reported that mean arterial pressure (MAP) is maintained in water-deprived rats by an irregular tonic component of vasomotor sympathetic nerve activity (SNA) that is driven by neuronal activity in the hypothalamic paraventricular nucleus (PVN). To establish whether generation of tonic SNA requires time-dependent (i.e., hours or days of dehydration) neuroadaptive responses or can be abruptly generated by even acute circuit activation, forebrain sympathoexcitatory osmosensory inputs to PVN were stimulated by infusion (0.1 ml/min, 10 min) of hypertonic saline (HTS; 1.5 M NaCl) through an internal carotid artery (ICA). Whereas isotonic saline (ITS; 0.15 M NaCl) had no effect (n = 5), HTS increased (P < 0.001; n = 6) splanchnic SNA (sSNA), phrenic nerve activity (PNA), and MAP. Bilateral PVN injections of muscimol (n = 6) prevented HTS-evoked increases of integrated sSNA and PNA (P < 0.001) and attenuated the accompanying pressor response (P < 0.01). Blockade of PVN NMDA receptors with d-(2R)-amino-5-phosphonovaleric acid (AP5; n = 6) had similar effects. Analysis of respiratory rhythmic bursting of sSNA revealed that ICA HTS increased mean voltage (P < 0.001) without affecting the amplitude of inspiratory or expiratory bursts. Analysis of cardiac rhythmic sSNA likewise revealed that ICA HTS increased mean voltage. Cardiac rhythmic sSNA oscillation amplitude was also increased, which is consistent with activation of arterial baroreceptor during the accompanying pressor response. Increased mean sSNA voltage by HTS was blocked by prior PVN inhibition (muscimol) and blockade of PVN NMDA receptors (AP5). We conclude that even acute glutamatergic activation of PVN (i.e., by hypertonicity) is sufficient to selectively increase a tonic component of vasomotor SNA.

Hypoxia triggers short term potentiation of phrenic motoneuron discharge after chronic cervical spinal cord injury

Repeated exposure to hypoxia can induce spinal neuroplasticity as well as respiratory and somatic motor recovery after spinal cord injury (SCI). The purpose of the present study was twofold: to define the capacity for a single bout of hypoxia to trigger short-term plasticity in phrenic output after cervical SCI and to determine the phrenic motoneuron (PhrMN) bursting and recruitment patterns underlying the response. Hypoxia-induced short term potentiation (STP) of phrenic motor output was quantified in anesthetized rats 11weeks following lateral spinal cord hemisection at C2 (C2Hx). A 3-min hypoxic episode (12–14% O2) always triggered STP of inspiratory burst amplitude, the magnitude of which was greater in phrenic bursting ipsilateral vs. contralateral to C2Hx. We next determined if STP could be evoked in recruited (silent) PhrMNs ipsilateral to C2Hx. Individual PhrMN action potentials were recorded during and following hypoxia using a “single fiber” approach. STP of bursting activity did not occur in cells initiating bursting at inspiratory onset, but was robust in recruited PhrMNs as well as previously active cells initiating bursting later in the inspiratory effort. We conclude that following chronic C2Hx, a single bout of hypoxia triggers recruitment of PhrMNs in the ipsilateral spinal cord with bursting that persists beyond the hypoxic exposure. The results provide further support for the use of short bouts of hypoxia as a neurorehabilitative training modality following SCI.

P2Y1 receptor-mediated potentiation of inspiratory motor output in neonatal rat in vitro.

PreBötzinger complex inspiratory rhythm-generating networks are excited by metabotropic purinergic receptor subtype 1 (P2Y1R) activation. Despite this, and the fact that inspiratory MNs express P2Y1Rs, the role of P2Y1Rs in modulating motor output is not known for any MN pool. We used rhythmically active brainstem-spinal cord and medullary slice preparations from neonatal rats to investigate the effects of P2Y1R signalling on inspiratory output of phrenic and XII MNs that innervate diaphragm and airway muscles, respectively. MRS2365 (P2Y1R agonist, 0.1 mm) potentiated XII inspiratory burst amplitude by 60 ± 9%; 10-fold higher concentrations potentiated C4 burst amplitude by 25 ± 7%. In whole-cell voltage-clamped XII MNs, MRS2365 evoked small inward currents and potentiated spontaneous EPSCs and inspiratory synaptic currents, but these effects were absent in TTX at resting membrane potential. Voltage ramps revealed a persistent inward current (PIC) that was attenuated by: flufenamic acid (FFA), a blocker of the Ca(2+)-dependent non-selective cation current ICAN; high intracellular concentrations of BAPTA, which buffers Ca(2+) increases necessary for activation of ICAN; and 9-phenanthrol, a selective blocker of TRPM4 channels (candidate for ICAN). Real-time PCR analysis of mRNA extracted from XII punches and laser-microdissected XII MNs revealed the transcript for TRPM4. MRS2365 potentiated the PIC and this potentiation was blocked by FFA, which also blocked the MRS2365 potentiation of glutamate currents. These data suggest that XII MNs are more sensitive to P2Y1R modulation than phrenic MNs and that the P2Y1R potentiation of inspiratory output occurs in part via potentiation of TRPM4-mediated ICAN, which amplifies inspiratory inputs.

Consequences of gestational stress on GABAergic modulation of respiratory activity in developing newborn pups

The GABAergic system modulates respiratory activity and undergoes substantial changes during early life. Because this maturation process is sensitive to stress, we tested the hypothesis that gestational stress (GS) alters development of GABAergic modulation of respiratory control in rat pups. The respiratory responses to the selective GABAA receptor agonist muscimol were compared between pups born to dams subjected to GS (bright light and predator odor; 20min/day from G9 to G19) or maintained under standard (control) conditions. Respiratory activity was measured on 1 and 4 days old pups of both sexes using in vivo (whole body plethysmography) and in vitro (isolated brainstem-spinal cord preparation) approaches. In intact pups, muscimol injection (0.75mg/kg; i.p.) depressed minute ventilation; this response was less in GS pups, and at P4, muscimol augmented minute ventilation in GS females. Bath application of muscimol (0.01–0.5μM) onto brainstem preparations decreased inspiratory (C4) burst frequency and amplitude in a dose-dependent manner; the responsiveness decreased with age. However, GS had limited effects on these results. We conclude that the results obtained in vivo are consistent with our hypothesis and show that GS delays maturation of GABAergic modulation of respiratory activity. The differences in the results observed between experimental approaches (in vivo versus in vitro) indicate that the effect of prenatal stress on maturation of GABAergic modulation of respiratory control mainly affects the peripheral/metabolic components of the respiratory control system.

Intrapleural Administration of AAV9 Improves Neural and Cardiorespiratory Function in Pompe Disease

Pompe disease is a neuromuscular disease resulting from deficiency in acid α-glucosidase (GAA), results in cardiac, skeletal muscle, and central nervous system (CNS) pathology. Enzyme replacement therapy (ERT) has been shown to partially correct cardiac and skeletal muscle dysfunction. However, ERT does not cross the blood-brain barrier and progressive CNS pathology ensues. We tested the hypothesis that intrapleural administration of recombinant adeno-associated virus (rAAV9)-GAA driven by a cytomegalovirus (CMV) or desmin (DES) promoter would improve cardiac and respiratory function in Gaa(-/-) mice through a direct effect and retrograde transport to motoneurons. Cardiac magnetic resonance imaging revealed significant improvement in ejection fraction in rAAV9-GAA-treated animals. Inspiratory phrenic and diaphragm activity was examined at baseline and during hypercapnic respiratory challenge. Mice treated with AAV9 had greater relative inspiratory burst amplitude during baseline conditions when compared with Gaa(-/-). In addition, efferent phrenic burst amplitude was significantly correlated with diaphragm activity in both AAV9-DES and AAV9-CMV groups but not in Gaa(-/-). This is the first study to indicate improvements in cardiac, skeletal muscle, and respiratory neural output following rAAV administration in Pompe disease. These results further implicate a role for the CNS in Pompe disease pathology and the critical need to target the neurologic aspects in developing therapeutic strategies.

Sympathetic network drive during water deprivation does not increase respiratory or cardiac rhythmic sympathetic nerve activity

Effects of water deprivation on rhythmic bursting of sympathetic nerve activity (SNA) were investigated in anesthetized, bilaterally vagotomized, euhydrated (control) and 48-h water-deprived (WD) rats (n = 8/group). Control and WD rats had similar baseline values of mean arterial pressure, heart rate, end-tidal CO2, and central respiratory drive. Although integrated splanchnic SNA (sSNA) was greater in WD rats than controls (P < 0.01), analysis of respiratory rhythmic bursting of sSNA revealed that inspiratory rhythmic burst amplitude was actually smaller (P < 0.005) in WD rats (+68 ± 6%) than controls (+208 ± 20%), and amplitudes of the early expiratory (postinspiratory) trough and late expiratory burst of sSNA were not different between groups. Further analysis revealed that water deprivation had no effect on either the amplitude or periodicity of the cardiac rhythmic oscillation of sSNA. Collectively, these data indicate that the increase of sSNA produced by water deprivation is not attributable to either increased respiratory or cardiac rhythmic burst discharge. Thus the sympathetic network response to acute water deprivation appears to differ from that of chronic sympathoexcitation in neurogenic forms of arterial hypertension, where increased respiratory rhythmic bursting of SNA and baroreflex adaptations have been reported.