Orexin receptor 2 agonist activates diaphragm and genioglossus muscle through stimulating inspiratory neurons in the pre-Bötzinger complex, and phrenic and hypoglossal motoneurons in rodents.

Orexin-mediated stimulation of orexin receptors 1/2 (OX[1/2]R) may stimulate the diaphragm and genioglossus muscle via activation of inspiratory neurons in the pre-Bötzinger complex, which are critical for the generation of inspiratory rhythm, and phrenic and hypoglossal motoneurons. Herein, we assessed the effects of OX2R-selective agonists TAK-925 (danavorexton) and OX-201 on respiratory function. In in vitro electrophysiologic analyses using rat medullary slices, danavorexton and OX-201 showed tendency and significant effect, respectively, in increasing the frequency of inspiratory synaptic currents of inspiratory neurons in the pre-Bötzinger complex. In rat medullary slices, both danavorexton and OX-201 significantly increased the frequency of inspiratory synaptic currents of hypoglossal motoneurons. Danavorexton and OX-201 also showed significant effect and tendency, respectively, in increasing the frequency of burst activity recorded from the cervical (C3-C5) ventral root, which contains axons of phrenic motoneurons, in in vitro electrophysiologic analyses from rat isolated brainstem-spinal cord preparations. Electromyogram recordings revealed that intravenous administration of OX-201 increased burst frequency of the diaphragm and burst amplitude of the genioglossus muscle in isoflurane- and urethane-anesthetized rats, respectively. In whole-body plethysmography analyses, oral administration of OX-201 increased respiratory activity in free-moving mice. Overall, these results suggest that OX2R-selective agonists enhance respiratory function via activation of the diaphragm and genioglossus muscle through stimulation of inspiratory neurons in the pre-Bötzinger complex, and phrenic and hypoglossal motoneurons. OX2R-selective agonists could be promising drugs for various conditions with respiratory dysfunction.

Comparative electrophysiological characterization of ammodytoxin A, a ß-neurotoxin from the nose-horned viper venom, and its enzymatically inactive mutant.

Presynaptic- or ß-neurotoxicity of secreted phospholipases A2 (sPLA2) is a complex process. For full expression of ß-neurotoxicity, the enzymatic activity of the toxin is essential. However, it has been shown that not all toxic effects of a ß-neurotoxin depend on its enzymatic activity, for example, the inhibition of mitochondrial cytochrome c oxidase. The main objective of this study was to verify whether it is possible to observe and study the phospholipase-independent actions of ß-neurotoxins by a standard ex vivo twitch-tension experimental approach. To this end, we compared the effects of a potent snake venom ß-neurotoxin, ammodytoxin A (AtxA), and its enzymatically inactive mutant AtxA(D49S) on muscle contraction of the mouse phrenic nerve-hemidiaphragm preparation. While AtxA significantly affected the amplitude of the indirectly evoked isometric muscle contraction, the resting tension of the neuromuscular (NM) preparation, the amplitude of the end-plate potential (EPP), the EPP half decay time and the resting membrane potential, AtxA(D49S) without enzymatic activity did not. From this, we can conclude that the effects of AtxA independent of enzymatic activity cannot be studied with classical electrophysiological measurements on the isolated NM preparation. Our results also suggest that the inhibition of cytochrome c oxidase activity by AtxA is not involved in the rapid NM blockade by this ß-neurotoxin, but that its pathological consequences are rather long-term. Interestingly, in our experimental setup, AtxA upon direct stimulation reduced the amplitude of muscle contraction and induced contracture of the hemidiaphragm, effects that could be interpreted as myotoxic.

Effects of exercise and doxorubicin on acute diaphragm neuromuscular transmission failure.

Doxorubicin (DOX) is a highly effective anthracycline antibiotic used to treat a wide variety of cancers including breast cancer, leukemia and lymphoma. Unfortunately, clinical use of DOX is limited due to adverse off-target effects resulting in fatigue, respiratory muscle weakness and dyspnea. The diaphragm is the primary muscle of inspiration and respiratory insufficiency is likely the result of both muscle weakness and neural impairment. However, the contribution of neuropathology to DOX-induced respiratory muscle dysfunction is unclear. We hypothesized that diaphragm weakness following acute DOX exposure is associated with neurotoxicity and that exercise preconditioning is sufficient to improve diaphragm muscle contractility by maintaining neuromuscular integrity. Adult female Sprague-Dawley rats were randomized into four experimental groups: 1) sedentary-saline, 2) sedentary-DOX, 3) exercise-saline or 4) exercise-DOX. Endurance exercise preconditioning consisted of treadmill running for 1 h/day at 30 m/min for 10 days. Twenty-four hours after the last bout of exercise, animals were treated with DOX (20 mg/kg, I.P.) or saline (equal volume). Our results demonstrate that 48-h following DOX administration diaphragm muscle specific force is reduced in sedentary-DOX rats in response to both phrenic nerve and direct diaphragm stimulation. Importantly, endurance exercise preconditioning in DOX-treated rats attenuated the decrease in diaphragm contractile function, reduced neuromuscular transmission failure and altered phrenic nerve morphology. These changes were associated with an exercise-induced reduction in circulating biomarkers of inflammation, nerve injury and reformation. Therefore, the results are consistent with exercise preconditioning as an effective way of reducing respiratory impairment via preservation of phrenic-diaphragm neuromuscular conduction.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high-affinity full-length tropomyosin-related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth, we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal phrenic MN (PhMN) survival. In the present study, we used a 1NMPP1-sensitive TrkBF616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MN (PhMN) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, whereas DIAm strips were frozen at ∼1.5× resting length, cryosectioned, and stained with hematoxylin and eosin to assess CSA. We observed an ∼34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1-treated TrkBF616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling.NEW & NOTEWORTHY During early postnatal development, BDNF/TrkB signaling promotes PhMN survival. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact PhMN size. Inhibition of BDNF/TrkB signaling in early postnatal development does not impact the number or CSA of DIAm fibers.

Dyspnea induced by hemidiaphragmatic paralysis after ultrasound-guided supraclavicular brachial plexus block in a morbidly obese patient.

RATIONALE: Hemidiaphragmatic paralysis (HDP) is a frequent complication of the brachial plexus block, caused by unintentional blockade of ipsilateral phrenic nerve. HDP did not rise enough alarm and attention to most anesthesiologists, because most patients with no coexisting comorbid diseases are asymptomatic and able to tolerate it. However, it may cause severe respiratory complication for patients with preexisting compromised cardiorespiratory function. PATIENT CONCERNS: A 67-year-old woman with morbidly obesity was planned to receive opening reduction and internal fixation of right humeral shaft fracture under regional anesthesia considering less respiratory and cardiovascular system interference compared with general anesthesia. DIAGNOSES: After ultrasound guided supraclavicular brachial plexus block, the patient developed severe hypoxia and hypercapnia.Unintentional block of phrenic nerve and diaphragm paralysis was diagnosed by diaphragm ultrasound, which was considered as the main reason of severe hypoxia. INTERVENTIONS: It led to a conversion from regional anesthesia to general anesthesia with endotracheal intubation for patient's safety and smooth operation. OUTCOMES: The unintentional phrenic nerve block leads to a prolonged ventilation time, length of stay in intensive care unit and length of stay in hospital. LESSONS: This case report highlights the risk of diaphragm paralysis in morbidly obese patients. Though new diaphragm sparing brachial plexus block (BPB) methods were developed intended to reduce the risk of HDP, no approaches could absolutely spare phrenic nerve involvement. Therefore, clinicians should always consider the risk of HDP associated with BPBs. For each individual, a detailed preoperative evaluation and sufficient preparation are paramount to avoid serious complications.

Spinally delivered ampakine CX717 increases phrenic motor output in adult rats.

Ampakines are synthetic molecules that allosterically modulate AMPA-type glutamate receptors. We tested the hypothesis that delivery of ampakines to the intrathecal space could stimulate neural drive to the diaphragm. Ampakine CX717 (20 mM, dissolved in 10 % HPCD) or an HPCD vehicle solution were delivered via a catheter placed in the intrathecal space at the fourth cervical segment in urethane-anesthetized, mechanically ventilated adult male Sprague-Dawley rats. The electrical activity of the phrenic nerve was recorded for 60-minutes following drug application. Intrathecal application of CX717 produced a gradual and sustained increase in phrenic inspiratory burst amplitude (n = 10). In contrast, application of HPCD (n = 10) caused no sustained change in phrenic motor output. Phrenic burst rate, heart rate, and mean arterial pressure were similar between CX717 and HPCD treated rats. We conclude that intrathecally delivered ampakines can modulate phrenic motor output. This approach may have value for targeted induction of spinal neuroplasticity in the context of neurorehabiliation.

Nonsteroidal anti-inflammatory drug (ketoprofen) delivery differentially impacts phrenic long-term facilitation in rats with motor neuron death induced by intrapleural CTB-SAP injections.

Intrapleural injections of cholera toxin B conjugated to saporin (CTB-SAP) selectively eliminates respiratory (e.g., phrenic) motor neurons, and mimics motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Additionally, microglial density increases in the phrenic motor nucleus following CTB-SAP. This CTB-SAP rodent model allows us to study the impact of motor neuron death on the output of surviving phrenic motor neurons, and the underlying mechanisms that contribute to enhancing or constraining their output at 7 days (d) or 28d post-CTB-SAP injection. 7d CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) through the Gs-pathway (inflammation-resistant in naïve rats), while pLTF is elicited though the Gq-pathway (inflammation-sensitive in naïve rats) in control and 28d CTB-SAP rats. In 7d and 28d male CTB-SAP rats and controls, we evaluated the effect of cyclooxygenase-1/2 enzymes on pLTF by delivery of the nonsteroidal anti-inflammatory drug, ketoprofen (IP), and we hypothesized that pLTF would be unaffected by ketoprofen in 7d CTB-SAP rats, but pLTF would be enhanced in 28d CTB-SAP rats. In anesthetized, paralyzed and ventilated rats, pLTF was surprisingly attenuated in 7d CTB-SAP rats and enhanced in 28d CTB-SAP rats (both p < 0.05) following ketoprofen delivery. Additionally in CTB-SAP rats: 1) microglia were more amoeboid in the phrenic motor nucleus; and 2) cervical spinal inflammatory-associated factor expression (TNF-α, BDNF, and IL-10) was increased vs. controls in the absence of ketoprofen (p < 0.05). Following ketoprofen delivery, TNF-α and IL-10 expression was decreased back to control levels, while BDNF expression was differentially affected over the course of motor neuron death in CTB-SAP rats. This study furthers our understanding of factors (e.g., cyclooxygenase-1/2-induced inflammation) that contribute to enhancing or constraining pLTF and its implications for breathing following respiratory motor neuron death.

New Modified Recombinant Botulinum Neurotoxin Type F with Enhanced Potency.

Botulinum neurotoxins (BoNTs) are notorious toxins and powerful agents and can be lethal, causing botulism, but they are also widely used as therapeutics, particularly to treat neuromuscular disorders. As of today, the commercial BoNT treatments available are from native A or B serotypes. Serotype F has shown efficacy in a clinical trial but has scarcely been used, most likely due to its medium duration of effect. Previously, the uniqueness of the light chain of the F7 subtype was identified and reported, showing an extended interaction with its substrates, VAMPs 1, 2 and 3, and a superior catalytic activity compared to other BoNT/F subtypes. In order to more extensively study the properties of this neurotoxin, we engineered a modified F7 chimera, mrBoNT/F7-1, in which all the regions of the neurotoxin were identical to BoNT/F7 except the activation loop, which was the activation loop from BoNT/F1. Use of the activation loop from BoNT/F1 allowed easier post-translational proteolytic activation of the recombinant protein without otherwise affecting its properties. mrBoNT/F7-1 was expressed, purified and then tested in a suite of in vitro and in vivo assays. mrBoNT/F7-1 was active and showed enhanced potency in comparison to both native and recombinant BoNT/F1. Additionally, the safety profile remained comparable to BoNT/F1 despite the increased potency. This new modified recombinant toxin F7 could be further exploited to develop unique therapeutics to address unmet medical needs.

Evaluation of Protection by Caffeic Acid, Chlorogenic Acid, Quercetin and Tannic Acid against the In Vitro Neurotoxicity and In Vivo Lethality of Crotalus durissus terrificus (South American Rattlesnake) Venom.

Systemic envenomation by Crotalus durissus terrificus (South American rattlesnake) can cause coagulopathy, rabdomyolysis, acute kidney injury, and peripheral neuromuscular blockade, the latter resulting in flaccid paralysis. Previous studies have shown that plant products such as tannic acid and theaflavin can protect against the neuromuscular blockade caused by C. d. terrificus venom in vitro. In this work, we used mouse-isolated phrenic nerve-diaphragm preparations to examine the ability of caffeic acid, chlorogenic acid, and quercetin to protect against C. d. terrificus venom-induced neuromuscular blockade in vitro. In addition, the ability of tannic acid to protect against the systemic effects of severe envenomation was assessed in rats. Preincubation of venom with caffeic acid (0.5 mg/mL), chlorogenic acid (1 mg/mL), or quercetin (0.5 mg/mL) failed to protect against venom (10 µg/mL)-induced neuromuscular blockade. In rats, venom (6 mg kg-1, i.p.) caused death in ~8 h, which was prevented by preincubation of venom with tannic acid or the administration of antivenom 2 h post-venom, whereas tannic acid given 2 h post-venom prolonged survival (~18.5 h) but did not prevent death. Tannic acid (in preincubation protocols or given 2 h post-venom) had a variable effect on blood creatinine and urea and blood/urine protein levels and prevented venom-induced leukocytosis. Tannic acid attenuated the histological lesions associated with renal damage in a manner similar to antivenom. The protective effect of tannic acid appeared to be mediated by interaction with venom proteins, as assessed by SDS-PAGE. These findings suggest that tannic acid could be a potentially useful ancillary treatment for envenomation by C. d. terrificus.

Ampakine pretreatment enables a single hypoxic episode to produce phrenic motor facilitation with no added benefit of additional episodes.

Repeated short episodes of hypoxia produce a sustained increase in phrenic nerve output lasting well beyond acute intermittent hypoxia (AIH) exposure (i.e., phrenic long-term facilitation; pLTF). Pretreatment with ampakines, drugs which allosterically modulate AMPA receptors, enables a single brief episode of hypoxia to produce pLTF, lasting up to 90 min after hypoxia. Here, we tested the hypothesis that ampakine pretreatment would enhance the magnitude of pLTF evoked by repeated bouts of hypoxia. Phrenic nerve output was recorded in urethane-anesthetized, mechanically ventilated, and vagotomized adult male Sprague-Dawley rats. Initial experiments demonstrated that ampakine CX717 (15 mg/kg iv) caused an acute increase in phrenic nerve inspiratory burst amplitude reaching 70 ± 48% baseline (BL) after 2 min (P = 0.01). This increased bursting was not sustained (2 ± 32% BL at 60 min, P = 0.9). When CX717 was delivered 2 min before a single episode of isocapnic hypoxia (5 min, [Formula: see text] = 44 ± 9 mmHg), facilitation of phrenic nerve burst amplitude occurred (96 ± 62% BL at 60 min, P < 0.001). However, when CX717 was given 2 min before three, 5-min hypoxic episodes ([Formula: see text] = 45 ± 6 mmHg) pLTF was attenuated and did not reach statistical significance (24 ± 29% BL, P = 0.08). In the absence of CX717 pretreatment, pLTF was observed after three (74 ± 33% BL at 60 min, P < 0.001) but not one episode of hypoxia (1 ± 8% BL at 60 min, P = 0.9). We conclude that pLTF is not enhanced when ampakine pretreatment is followed by repeated bouts of hypoxia. Rather, the combination of ampakine and a single hypoxic episode appears to be ideal for producing sustained increase in phrenic motor output.NEW & NOTEWORTHY Pretreatment with ampakine CX717 created conditions that enabled an acute bout of moderate hypoxia to evoke phrenic motor facilitation, but this response was not observed when ampakine pretreatment was followed by intermittent hypoxia. Thus, in anesthetized and spinal intact rats, the combination of ampakine and one bout of hypoxia appears ideal for triggering respiratory neuroplasticity.

Effects of sevoflurane and adenosine receptor antagonist on the sugammadex-induced recovery from rocuronium-induced neuromuscular blockade in rodent phrenic nerve-hemidiaphragm tissue specimens.

Sevoflurane affects on the A1 receptor in the central nervous system and potentiates the action of neuromuscular blocking agents. In the present study, we investigated whether sevoflurane (SEVO) has the ability to potentiate the neuromuscular blocking effect of rocuronium and if the specific antagonist of adenosine receptor (SLV320) can reverse this effect. In this study, phrenic nerve-hemidiaphragm tissue specimens were obtained from 40 Sprague-Dawley (SD) rats. The specimens were immersed in an organ bath filled with Krebs buffer and stimulated by a train-of-four (TOF) pattern using indirect supramaximal stimulation at 20 s intervals. The specimens were randomly allocated to control, 2-chloroadenosine (CADO), SEVO, or SLV320 + SEVO groups. In the CADO and SLV320 + SEVO groups, CADO and SLV320 were added to the organ bath from the start to a concentration of 10 µM and 10 nM, respectively. We then proceeded with rocuronium-induced blockade of >95% depression of the first twitch tension of TOF (T1) and TOF ratio (TOFR). In the SEVO and SLV320 + SEVO groups, SEVO was added to the Krebs buffer solution to concentration of 400-500 µM for 10 min. Sugammadex-induced T1 and TOFR recovery was monitored for 30 min until >95% of T1 and >0.9 of TOFR were confirmed, and the recovery pattern was compared by plotting these data. T1 recovery in the SEVO and CADO groups was significantly delayed compared with the control and SLV320 + SEVO groups (p < .05). In conclusion, sevoflurane affects on the A1 receptor at the neuromuscular junction and delays sugammadex-induced recovery from neuromuscular blockade.

Thyroid hormones during the perinatal period are necessary to respiratory network development of newborn rats.

Thyroid hormones (THs) are essential for foetal brain development. Because the gestating mother is the main source of THs to the foetus, maternal hypothyroidism and/or premature birth compromise neurological outcomes in the offspring. Respiratory instability and recurrent apneas due to immaturity of the respiratory control network are major causes of morbidity in infants. Inadequate TH supply may be sufficient to delay perinatal maturation of the respiratory control system; however, this hypothesis remains untested. To address this issue, maternal hypothyroidism was induced by adding methimazole (MMI; 0.02% w/v) to the drinking water of pregnant dams from conception to postpartum day 4 (P4). The effect of TH supplementation on respiratory function was tested by injecting levothyroxine (L-T4) in newborns at P1. Respiratory function was assessed by plethysmography (in vivo) and recording of phrenic output from medullary preparations (in vitro). By comparison with controls, TH deficiency increased the frequency of apneas and decreased basal ventilation in vivo and prevented the age-dependent increase in phrenic burst frequency normally observed in vitro. The effects of TH deficiency on GABAergic modulation of respiratory activity were measured by bath application of muscimol (GABAA agonist) or bicuculline (GABAA antagonist). The phrenic burst frequency responses to GABAergic agents were consistently greater in preparations from TH deficient pups. L-T4 supplementation reversed part of the respiratory anomalies related to MMI treatment in vitro. We conclude that TH deficiency during the perinatal period is sufficient to delay maturation of the respiratory control network development. Excessive GABAergic inhibition may contribute to this effect.

Effects of acetylcholine on hypoglossal and C4 nerve activity in brainstem-spinal cord preparations from newborn rat.

Effects of acetylcholine (ACh) on respiratory activity have been an intriguing theme especially in relation to central chemoreception and the control of hypoglossal nerve activity. We studied the effects of ACh on hypoglossal and phrenic (C4) nerve activities and inspiratory and pre-inspiratory neurons in the rostral ventrolateral medulla in brainstem-spinal cord preparations from newborn rats. ACh application increased respiratory rhythm, decreased inspiratory hypoglossal and C4 nerve burst amplitude, and enhanced pre-inspiratory hypoglossal activity. ACh induced membrane depolarization of pre-inspiratory neurons that might be involved in facilitation of respiratory rhythm by ACh. Effects of ACh on hypoglossal and C4 nerve activity were partially reversed by a nicotinic receptor blocker, mecamylamine. Further application of a muscarinic receptor antagonist, oxybutynin, resulted in slight increase of hypoglossal (but not C4) burst amplitude. Thus, ACh induced different effects on hypoglossal and C4 nerve activity in the brainstem-spinal cord preparation.

The Influence of Silver Nanoparticles Against Toxic Effects of Philodryas olfersii Venom.

PURPOSE: A silver nanoparticle obtained by reducing salts with solid dispersion of curcumin (130 nm, 0.081 mg mL-1) was used to counteract against the toxic - edematogenic, myotoxic, and neurotoxic - effects of Philodryas olfersii venom. METHODS: The edematogenic effect was evaluated by plasma extravasation in rat dorsal skin after injection of 50 µg per site of venom alone or preincubated with 1, 10, and 100 µL of AgNPs; the myotoxicity was evaluated by measuring the creatine kinase released into the organ-bath before the treatment and at the end of each experiment; and neurotoxicity was evaluated in chick biventer cervicis using the conventional myographic technique, face to the exogenous acetylcholine (ACh) and potassium chloride (KCl) added into the bath before the treatment and after each experiment. Preliminarily, a concentration-response curve of AgNPs was carried out to select the concentration to be used for neutralizing assays, which consists of neutralizing the venom-induced neuromuscular paralysis and edema by preincubating AgNPs with venom for 30 min. RESULTS: The P. olfersii venom-induced edema (n=6) and a complete neuromuscular blockade (n=4) that includes the total and unrecovered block of ACh and KCl contractures. AgNPs produced a concentration-dependent decrease the venom-induced edema (n=6) from 223.3% to 134.4% and to 100.5% after 10 and 100 µL AgNPs-preincubation, respectively. The preincubation of venom with AgNPs (1 µL; n=6) was able to maintain 46.5 ± 10.9% of neuromuscular response under indirect stimuli, 39.2 ± 9.7% of extrinsic nicotinic receptors functioning in absence of electrical stimulus and 28.3 ± 8.1% of responsiveness to potassium on the sarcolemmal membrane. The CK release was not affected by any experimental protocol which was like control. CONCLUSION: AgNPs interact with constituents of P. olfersii venom responsible for the edema-forming activity and neuromuscular blockade, but not on the sarcolemma membrane-acting constituents. The protective effect of the studied AgNPs on avian preparation points out to molecular targets as intrinsic and extrinsic nicotinic receptors.

High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks.

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABAA and GABAB receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

The pontine Kölliker-Fuse nucleus gates facial, hypoglossal, and vagal upper airway related motor activity.

The pontine Kölliker-Fuse nucleus (KFn) is a core nucleus of respiratory network that mediates the inspiratory-expiratory phase transition and gates eupneic motor discharges in the vagal and hypoglossal nerves. In the present study, we investigated whether the same KFn circuit may also gate motor activities that control the resistance of the nasal airway, which is of particular importance in rodents. To do so, we simultaneously recorded phrenic, facial, vagal and hypoglossal cranial nerve activity in an in situ perfused brainstem preparation before and after bilateral injection of the GABA-receptor agonist isoguvacine (50-70 nl, 10 mM) into the KFn (n = 11). Our results show that bilateral inhibition of the KFn triggers apneusis (prolonged inspiration) and abolished pre-inspiratory discharge of facial, vagal and hypoglossal nerves as well as post-inspiratory discharge in the vagus. We conclude that the KFn plays a critical role for the eupneic regulation of naso-pharyngeal airway patency and the potential functions of the KFn in regulating airway patency and orofacial behavior is discussed.

Methocarbamol blocks muscular Nav 1.4 channels and decreases isometric force of mouse muscles.

BACKGROUND: The muscle relaxant methocarbamol is widely used for the treatment of muscle spasms and pain syndromes. To elucidate molecular mechanisms of its action, we studied its influence on neuromuscular transmission, on isometric muscle force, and on voltage-gated Na+ channels. METHODS: Neuromuscular transmission was investigated in murine diaphragm-phrenic nerve preparations and muscle force studied on mouse soleus muscles. Nav 1.4 channels and Nav 1.7 channels were functionally expressed in eukaryotic cell lines. RESULTS: Methocarbamol, at 2 mM, decreased the decay of endplate currents, slowed the decay of endplate potentials and reduced tetanic force of soleus muscles. The drug reversibly inhibited current flow through muscular Nav 1.4 channels, while neuronal Nav 1.7 channels were unaffected. CONCLUSIONS: The study provides evidence for peripheral actions of methocarbamol on skeletal muscle. Muscular Na+ channels are a molecular target of methocarbamol. Since Nav 1.7 currents were unaffected, methocarbamol is unlikely to exert its analgesic effect by directly blocking Nav 1.7 channels.

Suppression of phrenic nerve activity as a potential predictor of imminent sudden unexpected death in epilepsy (SUDEP).

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in patients with refractory epilepsy. Centrally-mediated respiratory dysfunction has been identified as one of the principal mechanisms responsible for SUDEP. Seizures generate a surge in adenosine release. Elevated adenosine levels suppress breathing. Insufficient metabolic clearance of a seizure-induced adenosine surge might be a precipitating factor in SUDEP. In order to deliver targeted therapies to prevent SUDEP, reliable biomarkers must be identified to enable prompt intervention. Because of the integral role of the phrenic nerve in breathing, we hypothesized that suppression of phrenic nerve activity could be utilized as predictive biomarker for imminent SUDEP. We used a rat model of kainic acid-induced seizures in combination with pharmacological suppression of metabolic adenosine clearance to trigger seizure-induced death in tracheostomized rats. Recordings of EEG, blood pressure, and phrenic nerve activity were made concomitant to the seizure. We found suppression of phrenic nerve burst frequency to 58.9% of baseline (p < 0.001, one-way ANOVA) which preceded seizure-induced death; importantly, irregularities of phrenic nerve activity were partly reversible by the adenosine receptor antagonist caffeine. Suppression of phrenic nerve activity may be a useful biomarker for imminent SUDEP. The ability to reliably detect the onset of SUDEP may be instrumental in the timely administration of potentially lifesaving interventions.

The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity.

The extraordinarily potent clostridial neurotoxins (CNTs) comprise tetanus neurotoxin (TeNT) and the seven established botulinum neurotoxin serotypes (BoNT/A-G). They are composed of four structurally independent domains: the roles of the catalytically active light chain, the translocation domain HN, and the C-terminal receptor binding domain HCC are largely resolved, but that of the HCN domain sandwiched between HN and HCC has remained unclear. Here, mutants of BoNT/A, BoNT/B, and TeNT were generated by deleting their HCN domains or swapping HCN domains between each other. Both deletion and replacement of TeNT HCN domain by HCNA and HCNB reduced the biological activity similarly, by ~95%, whereas BoNT/A and B deletion mutants displayed >500-fold reduced activity in the mouse phrenic nerve hemidiaphragm assay. Swapping HCN domains between BoNT/A and B hardly impaired their biological activity, but substitution with HCNT did. Binding assays revealed that in the absence of HCN, not all receptor binding sites are equally well accessible. In conclusion, the presence of HCN is vital for CNTs to exert their neurotoxicity. Although structurally similar, the HCN domain of TeNT cannot equally substitute those of BoNT and vice versa, leaving the possibility that HCNT plays a different role in the intoxication mechanism of TeNT.

Ampakines stimulate phrenic motor output after cervical spinal cord injury.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors increases phrenic motor output. Ampakines are a class of drugs that are positive allosteric modulators of AMPA receptors. We hypothesized that 1) ampakines can stimulate phrenic activity after incomplete cervical spinal cord injury (SCI), and 2) pairing ampakines with brief hypoxia could enable sustained facilitation of phrenic bursting. Phrenic activity was recorded ipsilateral (IL) and contralateral (CL) to C2 spinal cord hemisection (C2Hx) in anesthetized adult rats. Two weeks after C2Hx, ampakine CX717 (15 mg/kg, i.v.) increased IL (61 ± 46% baseline, BL) and CL burst amplitude (47 ± 26%BL) in 8 of 8 rats. After 90 min, IL and CL bursting remained above baseline (BL) in 7 of 8 rats. Pairing ampakine with a single bout of acute hypoxia (5-min, arterial partial pressure of O2 ~ 50 mmHg) had a variable impact on phrenic bursting, with some rats showing a large facilitation that exceeded the response of the ampakine alone group. At 8 weeks post-C2Hx, 7 of 8 rats increased IL (115 ± 117%BL) and CL burst amplitude (45 ± 27%BL) after ampakine. The IL burst amplitude remained above BL for 90-min in 7 of 8 rats; CL bursting remained elevated in 6 of 8 rats. The sustained impact of ampakine at 8 weeks was not enhanced by hypoxia exposure. Intravenous vehicle (10% 2-Hydroxypropyl-ß-cyclodextrin) did not increase phrenic bursting at either time point. We conclude that ampakines effectively stimulate neural drive to the diaphragm after cervical SCI. Pairing ampakines with a single hypoxic exposure did not consistently enhance phrenic motor facilitation.