Respiratory Motoneurons Research Articles

Spinal TNF-α receptor expression in a rodent model of respiratory motor neuron death

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kills respiratory motor neurons, and mimics aspects of neurodegenerative diseases including breathing deficits. In this model, phrenic long-term facilitation (pLTF) induced by acute intermittent hypoxia is enhanced following 7 days (d) of CTB-SAP vs. control rats, whereas pLTF in 28d CTB-SAP treated rats is comparable to pLTF in controls suggesting that the underlying mechanisms of plasticity at both time points differ. Inflammation is a hallmark of neurodegenerative diseases and is known to prevent pLTF. Furthermore, ketoprofen (NSAID) delivery attenuates pLTF in 7d CTB-SAP-treated rats, but enhances it in 28d CTB-SAP-treated rats; this suggests that inflammation contributes to pLTF in 7d CTB-SAP-treated rats, but undermines pLTF in 28d CTB-SAP-treated rats. In addition, cervical spinal inflammatory marker gene expression ( e.g., TNF-a) is increased in CTB-SAP rats, and pilot data suggest that soluble TNF receptor 1 (sTNFR1) inhibition differentially affects pLTF (Nichols and Smith , ibid). Thus, the goal of the current study is to investigate TNFR1 as a pharmacological target that can modulate inflammation to maximize pLTF and breathing in CTB-SAP rats. We hypothesized that C4 TNFR1 expression would be increased on phrenic motor neurons and glial cells of CTB-SAP rats vs. controls. To address this, we studied TNFR1 expression on phrenic motor neurons and glial cells using immunohistochemistry, confocal microscopy, and ImageJ analysis in male Sprague Dawley rats 7 and 28 days after intrapleural injection of: CTB-SAP or un-conjugated CTB and SAP (control); n=8/group. Our preliminary data (n=4/group) suggest that phrenic TNFR1 expression is increased in 7d CTB-SAP rats vs. controls (p<0.05), but is unaffected in 28d CTB-SAP rats vs. controls (p>0.05). Further data analysis is underway for phrenic as well as glial TNFR1 expression in CTB-SAP rats vs. controls. If C4 TNFR1 expression is increased, this would suggest that TNFR1 is a viable target for therapeutic intervention in CTB-SAP rats, and potentially in patients with breathing deficits due to respiratory motor neuron death. NSF Computational Neuroscience grant, MU CVM COR grant, and Spinal Cord Injury & Disease Research Program grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TNF-a Receptor 1 Activity Is Differentially Required for Phrenic Long-term Facilitation (pLTF) Over the Course of Motor Neuron Death in Adult Rats

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB-SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death, decreased phrenic motor output, and increased microglial cell number. Over the course of motor neuron loss, enhanced A2A receptor-dependent phrenic plasticity is observed early (7d), and then constrained 5-HT receptor-dependent phrenic plasticity is observed later (28d). In addition, COX-1/2-induced inflammation differentially impacts breathing and phrenic plasticity in CTB-SAP rats, but the underlying mechanism by which COX-1/2-induced inflammation impacts breathing and plasticity over the course of motor neuron death has not been studied. We observe that increased cervical neuroinflammatory gene expression ( e.g., tumor necrosis factor-a; TNF-a) is reduced back to control levels in the presence of ketoprofen in CTB-SAP rats, and it is known that TNF-a is involved in phrenic plasticity in naïve rats. Thus, we hypothesized that acute TNF-a receptor inhibition attenuates phrenic plasticity at 7d and enhances it at 28d TNF-a following CTB-SAP-induced neuropathology. Using anesthetized, paralyzed and ventilated CTB-SAP-treated adult Sprague Dawley rats, we studied acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) induced pLTF following acute intrathecal C4 delivery of a TNF-a receptor 1 inhibitor (soluble TNFR1; sTNFR1; 0.13 ug/ul) or vehicle (aCSF) to rats that received bilateral, intrapleural injections of: 1) CTB-SAP (25ug), or 2) un-conjugated CTB and SAP (control) in rats treated 7d or 28d prior. Surprisingly, preliminary results indicate that acute sTNFR1 delivery does not affect pLTF in 7d CTB-SAP-treated rats (n=5; i.e., pLTF still enhanced) but it enhanced pLTF in 28d CTB-SAP-treated rats (n=6; p<0.05) vs. controls (n=6 at 7d; n=7 at 28d) and vs. vehicle treated rats (controls n=6 for 7d and n=9 for 28d; CTB-SAP, n=4 for 7d and 28d each). These preliminary observations suggest that TNF-a does not contribute to pLTF in 7d CTB-SAP-treated rats, whereas it may undermine pLTF in 28d CTB-SAP-treated rats. Studies are underway to assess the cellular source of TNF-a and the impact of chronic sTNFR1 delivery on pLTF, diaphragm activity, and breathing. Since most patients with neuromuscular disorders or neurodegenerative disease develop respiratory insuffciency and ultimately become ventilator dependent, our long-range goal is to develop new strategies to enhance the functional capacity of spared motor neurons and reduce ventilatory compromise in motor neuron loss pathology. These novel strategies, if successful, will identify new therapeutic targets for future, translational studies to enhance breathing in patients with neuromuscular disorders. Supported by a Spinal Cord Injury and Disease Research grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Decreased GABAergic Neurotransmission Promotes Breathing Upon Emergence From Hibernation

The respiratory network must produce reliable output for an animal to survive. Although respiratory motor plasticity is well appreciated, how plasticity mechanisms work to promote robustness following environmental perturbations that disrupt breathing is less clear. During underwater hibernation, respiratory neurons of bullfrogs remain inactive for months, triggering a suite of compensatory mechanisms that aim to maintain respiratory function. We hypothesized that GABAergic inhibition would decrease following hibernation. In control animals, respiratory frequency was under control of GABAAR signaling. Specifically, block of GABAARs dose-dependently decreased respiratory burst frequency in the in vitro brainstem preparation (n=5 preparations). After hibernation, GABAA receptor block had little to no impact on respiratory burst frequency, suggesting the network was no longer reliant on GABAergic inhibition to maintain normal frequency (n=5 preparations). The loss of inhibition was specific to the respiratory network because non-respiratory motor activity and large seizure-like bursts were similarly triggered by GABAA receptor blockade in intact brainstem preparations from controls and hibernators. We next used a semi-intact preparation that maintains functional respiratory-synaptic input onto rhythmic motor neurons to identify compensatory changes in GABAergic tone at the level of the motor neuron. In controls, the firing rate of respiratory motoneurons was constrained by a phasic GABAAR tone because focal application of bicuculline, a GABAAR antagonist, led to an increase in phasic firing frequency (n=12 cells). After hibernation, this bicuculline-mediated increase in phasic firing frequency was attenuated suggesting phasic inhibition onto the motor neuron had decreased (n=9 cells). Brain slice recordings of identified vagal motor neurons revealed that postsynaptic GABAA receptor strength was unchanged following hibernation (n=16 control cells; n=16 hibernation cells). Taken together, our results suggest that GABAergic activity is decreased in premotor networks following hibernation. Despite this dramatic loss of GABAergic function, respiratory output from control preparations was indistinguishable from hibernators. To elucidate the functional impacts of loss of GABAergic activity, we tested the network’s ability to produce output at colder temperatures. In controls, respiratory output slowed as temperature decreased, with most networks nearly silent at 8 degrees Celsius (n=5 preparations). Interestingly, in controls low dose block of GABAA receptors promoted respiratory activity at cold temperatures, with frequency not statistically different than baseline at any temperature tested (n=4 preparations). This suggests that the loss of premotor GABAergic activity we observed could work to promote motor output at colder temperatures, which works adaptively for the animal when emerging from hibernation. NIH R01NS114514 (JS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Generation of human iPSC-derived phrenic-like motor neurons to model respiratory motor neuron degeneration in ALS

The fatal motor neuron (MN) disease Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive MN degeneration. Phrenic MNs (phMNs) controlling the activity of the diaphragm are prone to degeneration in ALS, leading to death by respiratory failure. Understanding of the mechanisms of phMN degeneration in ALS is limited, mainly because human experimental models to study phMNs are lacking. Here we describe a method enabling the derivation of phrenic-like MNs from human iPSCs (hiPSC-phMNs) within 30 days. This protocol uses an optimized combination of small molecules followed by cell-sorting based on a cell-surface protein enriched in hiPSC-phMNs, and is highly reproducible using several hiPSC lines. We show further that hiPSC-phMNs harbouring ALS-associated amplification of the C9orf72 gene progressively lose their electrophysiological activity and undergo increased death compared to isogenic controls. These studies establish a previously unavailable protocol to generate human phMNs offering a disease-relevant system to study mechanisms of respiratory MN dysfunction.

Respiratory therapies for Amyotrophic Lateral Sclerosis: A state of the art review.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative condition noteworthy for upper and lower motor neuron death. Involvement of respiratory motor neuron pools leads to progressive pathology. These impairments include decreases in neural activation and muscle coordination, progressive airway obstruction, weakened airway defenses, restrictive lung disease, increased risk of pulmonary infections, and weakness and atrophy of respiratory muscles. These neural, airway, pulmonary, and neuromuscular changes deteriorate integrated respiratory-related functions including sleep, cough, swallowing, and breathing. Ultimately, respiratory complications account for a large portion of morbidity and mortality in ALS. This state-of-the-art review highlights applications of respiratory therapies for ALS, including lung volume recruitment, mechanical insufflation-exsufflation, non-invasive ventilation, and respiratory strength training. Therapeutic acute intermittent hypoxia, an emerging therapeutic tool for inducing respiratory plasticity will also be introduced. A focus on emerging evidence and future work underscores the common goal to continue to improve survival for patients living with ALS.

Effects of acute intermittent hypoxia on P0.1 in humans with spinal cord injury

Spinal cord injury (SCI) disrupts neural pathways to respiratory motor neurons, diminishing respiratory drive and breathing function. Breathing impairment remains a leading cause of illness and death after SCI. Therapeutic acute intermittent hypoxia (tAIH; 15, 1-minute episodes of 9% inspired O 2 , 1.5 min normoxic intervals), a promising strategy to restore breathing function after SCI, promotes spinal neuroplasticity in rodent models. However, in humans, respiratory drive is unchanged after a single tAIH session; thus we tested the hypothesis that tAIH on five successive days increases neuromechanical respiratory drive in humans living with chronic SCI. Sixteen participants with chronic SCI (1 female; 33.5 + 15.3 years of age; 1-28 years post-SCI; 11 cervical, 5 thoracic; C1-T6 AIS A-D) were studied; their respiratory function was 52% ± 19% of age predicted expiratory pressure generation, and 123% ± 32% for inspiratory pressure. Five days of tAIH or Sham treatment were delivered in a randomized, cross-over design (minimum 3 weeks apart). Airway occlusion pressure in the first 0.1s of inspiration (P0.1), a surrogate for neuromechanical respiratory drive prior to within-breath sensory feedback, was collected at baseline and at 1, 3, and 7-days post-treatment. Repeated measures ANOVA revealed no significant effect of time (F(3)=.49; P=.69) or group (F(3)=1.33; P=.27). Despite the lack of significant differences in this preliminary analysis, P0.1 increased by 25% on day 3 versus 0.8% after Sham. Because of this change, pairwise comparisons were conducted, revealing a near significant increase in P0.1, 3 days post tAIH (MD=-.17; P =.06). Thus, although P0.1, a surrogate for neuromechanical respiratory drive, did not significantly increase after 5 days of AIH in people with SCI, the marginally significant change at day 3 suggests further investigation is warranted. Next steps include assessment in a larger sample, investigation of biomarkers for treatment effects, and examination of combinatorial interventions including task specific training and/or mild CO 2 elevations to enhance AIH-induced neuroplasticity in the drive to breathe. DoD SCIRP Clinical Trial Award W81XWH-17-SCIRP-CTA; The Craig H. Neilsen Foundation (JW); NIH T32 HL134621 (MM); and the UF McKnight Brain Institute. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute Intermittent Hypoxia and Respiratory Strength Training to Improve Respiratory Function in Individuals with Chronic Spinal Cord Injury: Preliminary Outcomes

Spinal cord injury (SCI) disrupts neural pathways to respiratory motor neurons, causing paralysis and breathing impairment, a major cause of illness and death in SCI. Acute intermittent hypoxia (AIH) is a promising therapeutic modality that elicits spinal synaptic plasticity, improving breathing in rodent models of SCI. AIH also elicits plasticity in limb motor systems in both rodent models and humans with chronic SCI, where functional gains are greatest when paired with task-specific training. The impact of similar combinatorial treatments on breathing ability is unknown. Here, we report preliminary breathing outcomes in seventeen adults with chronic SCI who received AIH and task-specific training (respiratory strength training; RST). Seventeen males with chronic SCI (33.7 ± 15 yrs of age; 1-28 yrs post-SCI; 12 cervical, 5 thoracic injuries; C1-T6 AIS A-D). Respiratory function was 52% ± 19% of the age-predicted normative function for expiratory pressure generation and 123% ± 32% of normative values for inspiratory function. Participants completed four random ordered 5-day protocols of AIH, Sham, RST, and AIH+RST, >3 weeks apart. AIH consisted of 15, 1-minute episodes of breathing a hypoxic gas mixture (9% oxygen), interspersed with 1.5-minute episodes of breathing room air (21% oxygen). Thirty minutes after AIH, RST involved both inspiratory and expiratory training; 4 sets of 6-12 breaths with the pressure threshold device set to 80% of each individual’s maximum. Sham treatments replicated AIH but used room air. A linear mixed model was used to test for differences in maximal inspiratory pressure (MIP), maximal expiratory pressure (MEP), and forced vital capacity (FVC) prior to and 1 day post-intervention. MIP increased 10±15.6% (pre 120±34cmH20, post 129±36 cmH20) 1 day after RST (p=0.001) and 6±10.5% (pre 123±34cmH20; post 131±34cmH20) 1 day after AIH+RST (p=0.04), but did not change after AIH or Sham. MIP increased by >10% in 5/17 participants following AIH+RST and 9/17 participants following RST. Neither MEP nor FVC changed following any block. Although there were no significant differences between AIH+RST and RST intervention blocks, there was a medium effect size (0.44). Since RST and AIH+RST preliminary outcomes suggest functional respiratory benefit, future steps will determine if the combinatorial treatment elicits greater gains. Examination of biomarkers associated with outcomes will provide insight into outcome variability. Department of Defense CDMRP W81XWH1810718 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuron populations use variable combinations of short-term feedback mechanisms to stabilize firing rate.

Neurons tightly regulate firing rate and a failure to do so leads to multiple neurological disorders. Therefore, a fundamental question in neuroscience is how neurons produce reliable activity patterns for decades to generate behavior. Neurons have built-in feedback mechanisms that allow them to monitor their output and rapidly stabilize firing rate. Most work emphasizes the role of a dominant feedback system within a neuronal population for the control of moment-to-moment firing. In contrast, we find that respiratory motoneurons use 2 activity-dependent controllers in unique combinations across cells, dynamic activation of an Na+ pump subtype, and rapid potentiation of Kv7 channels. Both systems constrain firing rate by reducing excitability for up to a minute after a burst of action potentials but are recruited by different cellular signals associated with activity, increased intracellular Na+ (the Na+ pump), and membrane depolarization (Kv7 channels). Individual neurons do not simply contain equal amounts of each system. Rather, neurons under strong control of the Na+ pump are weakly regulated by Kv7 enhancement and vice versa along a continuum. Thus, each motoneuron maintains its characteristic firing rate through a unique combination of the Na+ pump and Kv7 channels, which are dynamically regulated by distinct feedback signals. These results reveal a new organizing strategy for stable circuit output involving multiple fast activity sensors scaled inversely across a neuronal population.

Chapter 16 - Respiratory neuroplasticity: Mechanisms and translational implications of phrenic motor plasticity

Widespread appreciation that neuroplasticity is an essential feature of the neural system controlling breathing has emerged only in recent years. In this chapter, we focus on respiratory motor plasticity, with emphasis on the phrenic motor system. First, we define related but distinct concepts: neuromodulation and neuroplasticity. We then focus on mechanisms underlying two well-studied models of phrenic motor plasticity: (1) phrenic long-term facilitation following brief exposure to acute intermittent hypoxia; and (2) phrenic motor facilitation after prolonged or recurrent bouts of diminished respiratory neural activity. Advances in our understanding of these novel and important forms of plasticity have been rapid and have already inspired translation in multiple respects: (1) development of novel therapeutic strategies to preserve/restore breathing function in humans with severe neurological disorders, such as spinal cord injury and amyotrophic lateral sclerosis; and (2) the discovery that similar plasticity also occurs in nonrespiratory motor systems. Indeed, the realization that similar plasticity occurs in respiratory and nonrespiratory motor neurons inspired clinical trials to restore leg/walking and hand/arm function in people living with chronic, incomplete spinal cord injury. Similar application may be possible to other clinical disorders that compromise respiratory and non-respiratory movements.

Chapter 2 - Electrophysiological assessment of respiratory function

While the traditional lung function tests are used to assess lung capacity and pulmonary function, they cannot evaluate respiratory driving function and the integrity of the conduction pathway from the central nervous system to the respiratory motor neuron in the spinal cord and to the diaphragm. The inspiratory trigger is sent from the central nervous system through the phrenic nerve and drives the diaphragm to generate inspiratory movement. Therefore, phrenic nerve stimulation and diaphragmatic electromyography are two fundamental methods to assess respiratory function. There are several useful tools to assess respiratory motor system including electrical or magnetic phrenic nerve stimulation, diaphragmatic needle electromyography, and diaphragmatic ultrasound. By these means, physicians can assess current respiratory status in different neurological diseases that affect respiratory muscles, follow-up of the severity of respiratory impairment, help to predict the chance of successfully weaning from ventilatory support, and confirm clinical diagnoses such as diaphragmatic myoclonus. Although some of these tests require special training, applying these neurophysiological assessments in clinical practice is highly recommended.

Lactate ions induce synaptic plasticity to enhance output from the central respiratory network.

Lactate ion sensing has emerged as a process that regulates ventilation during metabolic challenges. Most work has focused on peripheral sensing of lactate for the control of breathing. However, lactate also rises in the central nervous system (CNS) during disturbances to blood gas homeostasis and exercise. Using an amphibian model, we recently showed that lactate ions, independently of pH and pyruvate metabolism, act directly in the brainstem to increase respiratory-related motor outflow. This response had a long washout time and corresponded with potentiated excitatory synaptic strength of respiratory motoneurons. Thus, we tested the hypothesis that lactate ions enhance respiratory output using cellular mechanisms associated with long-term synaptic plasticity within motoneurons. In this study, we confirm that 2mM sodium lactate, but not sodium pyruvate, increases respiratory motor output in brainstem-spinal cord preparations, persisting for 2h upon the removal of lactate. Lactate also led to prolonged increases in the amplitude of AMPA-glutamate receptor (AMPAR) currents in individual motoneurons from brainstem slices. Both motor facilitation and AMPAR potentiation by lactate required classic effectors of synaptic plasticity, L-type Ca2+ channels and NMDA receptors, as part of the transduction process but did not correspond with increased expression of immediate-early genes often associated with activity-dependent neuronal plasticity. Altogether these results show that lactate ions enhance respiratory motor output by inducing conserved mechanisms of synaptic plasticity and suggest a new mechanism that may contribute to coupling ventilation to metabolic demands in vertebrates. KEY POINTS: Lactate ions, independently of pH and metabolism, induce long-term increases in respiratory-related motor outflow in American bullfrogs. Lactate triggers a persistent increase in strength of AMPA-glutamatergic synapses onto respiratory motor neurons. Long-term plasticity of motor output and synaptic strength by lactate involves L-type Ca2+ channels and NMDA-receptors as part of the transduction process. Enhanced AMPA receptor function in response to lactate in the intact network is causal for motor plasticity. In sum, well-conserved synaptic plasticity mechanisms couple the brainstem lactate ion concentration to respiratory motor drive in vertebrates.

Ampakines Stimulate Diaphragm Activity after Spinal Cord Injury.

Respiratory compromise after cervical spinal cord injury (SCI) is a leading cause of mortality and morbidity. Most SCIs are incomplete, and spinal respiratory motoneurons as well as proprio- and bulbospinal synaptic pathways provide a neurological substrate to enhance respiratory output. Ampakines are allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are prevalent on respiratory neurons. We hypothesized that low dose ampakine treatment could safely and effectively increase diaphragm electromyography (EMG) activity that has been impaired as a result of acute- or sub-acute cervical SCI. Diaphragm EMG was recorded using chronic indwelling electrodes in unanesthetized, freely moving rats. A spinal hemi-lesion was induced at C2 (C2Hx), and rats were studied at 4 and 14 days post-injury during room air breathing and acute respiratory challenge accomplished by inspiring a 10% O2, 7% CO2 gas mixture. Once a stable baseline recording was established, one of two different ampakines (CX717 or CX1739, 5 mg/kg, intravenous) or a vehicle (2-hydroxypropyl-beta-cyclodextrin [HPCD]) was delivered. At 4 days post-injury, both ampakines increased diaphragm EMG output ipsilateral to C2Hx during both baseline breathing and acute respiratory challenge. Only CX1739 treatment also led to a sustained (15 min) increase in ipsilateral EMG output. At 14 days post-injury, both ampakines produced sustained increases in ipsilateral diaphragm EMG output and enabled increased output during the respiratory challenge. We conclude that low dose ampakine treatment can increase diaphragm EMG activity after cervical SCI, and therefore may provide a pharmacological strategy that could be useful in the context of respiratory rehabilitation.

Sustained A1 Adenosine Receptor Antagonist Drug Release from Nanoparticles Functionalized by a Neural Tracing Protein.

Respiratory dysfunction is a major cause of death in people with spinal cord injury (SCI). A remaining unsolved problem in treating SCI is the intolerable side effects of the drugs to patients. In a significant departure from conventional targeted nanotherapeutics to overcome the blood-brain barrier (BBB), this work pursues a drug-delivery approach that uses neural tracing retrograde transport proteins to bypass the BBB and deliver an adenosine A1 receptor antagonist drug, 1,3-dipropyl-8-cyclopentyl xanthine, exclusively to the respiratory motoneurons in the spinal cord and the brainstem. A single intradiaphragmatic injection at one thousandth of the native drug dosage induces prolonged respiratory recovery in a hemisection animal model. To translate the discovery into new treatments for respiratory dysfunction, we carry out this study to characterize the purity and quality of synthesis, stability, and drug-release properties of the neural tracing protein (wheat germ agglutinin chemically conjugated to horseradish peroxidase)-coupled nanoconjugate. We show that the batch-to-batch particle size and drug dosage variations are less than 10%. We evaluate the nanoconjugate size against the spatial constraints imposed by transsynaptic transport from pre to postsynaptic neurons. We determine that the nanoconjugate formulation is capable of sustained drug release lasting for days at physiologic pH, a prerequisite for long-distance transport of the drug from the diaphragm muscle to the brainstem. We model the drug-release profiles using a first-order reaction model and the Noyes-Whitney diffusion model. We confirm via biological electron microscopy that the nanoconjugate particles do not accumulate in the tissues at the injection site. We define the nanoconjugate storage conditions after monitoring the solution dispersion stability under various conditions for 4 months. This study supports further development of neural tracing protein-enabled nanotherapeutics for treating respiratory problems associated with SCI.

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.

How Are Adenosine and Adenosine A2A Receptors Involved in the Pathophysiology of Amyotrophic Lateral Sclerosis?

Adenosine is extensively distributed in the central and peripheral nervous systems, where it plays a key role as a neuromodulator. It has long been implicated in the pathogenesis of progressive neurogenerative disorders such as Parkinson’s disease, and there is now growing interest in its role in amyotrophic lateral sclerosis (ALS). The motor neurons affected in ALS are responsive to adenosine receptor function, and there is accumulating evidence for beneficial effects of adenosine A2A receptor antagonism. In this article, we focus on recent evidence from ALS clinical pathology and animal models that support dynamism of the adenosinergic system (including changes in adenosine levels and receptor changes) in ALS. We review the possible mechanisms of chronic neurodegeneration via the adenosinergic system, potential biomarkers and the acute symptomatic pharmacology, including respiratory motor neuron control, of A2A receptor antagonism to explore the potential of the A2A receptor as target for ALS therapy.

Phrenic motor neuron survival below cervical spinal cord hemisection

Cervical spinal cord injury (cSCI) severs bulbospinal projections to respiratory motor neurons, paralyzing respiratory muscles below the injury. C2 spinal hemisection (C2Hx) is a model of cSCI often used to study spontaneous and induced plasticity and breathing recovery post-injury. One key assumption is that C2Hx dennervates motor neurons below the injury, but does not affect their survival. However, a recent study reported substantial bilateral motor neuron death caudal to C2Hx. Since phrenic motor neuron (PMN) death following C2Hx would have profound implications for therapeutic strategies designed to target spared neural circuits, we tested the hypothesis that C2Hx minimally impacts PMN survival. Using improved retrograde tracing methods, we observed no loss of PMNs at 2- or 8-weeks post-C2Hx. We also observed no injury-related differences in ChAT or NeuN immunolabeling within labelled PMNs. Although we found no evidence of PMN loss following C2Hx, we cannot rule out neuronal loss in other motor pools. These findings address an essential prerequisite for studies that utilize C2Hx as a model to explore strategies for inducing plasticity and/or regeneration within the phrenic motor system, as they provide important insights into the viability of phrenic motor neurons as therapeutic targets after high cervical injury.

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

Intrapleural injection of cholera toxin B conjugated to saporin (CTB-SAP) mimics respiratory motor neuron death and respiratory deficits observed in rat models of neuromuscular diseases. Seven-day CTB-SAP rats elicit enhanced phrenic long-term facilitation (pLTF) primarily through TrkB and PI3K/Akt-dependent mechanisms [i.e., Gs-pathway, which can be initiated by adenosine 2A (A2A) receptors in naïve rats], whereas 28-day CTB-SAP rats elicit moderate pLTF though BDNF- and MEK-/ERK-dependent mechanisms [i.e., Gq-pathway, which is typically initiated by serotonin (5-HT) receptors in naïve rats]. Here, we tested the hypothesis that pLTF following CTB-SAP is 1) A2A receptor-dependent at 7 days and 2) 5-HT receptor-dependent at 28 days. Adult Sprague-Dawley male rats were anesthetized, paralyzed, ventilated, and exposed to acute intermittent hypoxia (AIH; 3-, 5-min bouts of 10.5% O2) following bilateral, intrapleural injections at 7 days and 28 days of 1) CTB-SAP (25 µg) or 2) unconjugated CTB and SAP (control). Intrathecal C4 delivery included either the 1) A2A receptor antagonist (MSX-3; 10 µM; 12 µL) or 2) 5-HT receptor antagonist (methysergide; 20 mM; 15 µL). pLTF was abolished with A2A receptor inhibition in 7-day, not 28-day, CTB-SAP rats versus controls (P < 0.05), whereas pLTF was abolished following 5-HT receptor inhibition in 28-day, not 7-day, CTB-SAP rats versus controls (P < 0.05). In addition, 5-HT2A receptor expression was unchanged in CTB-SAP rats versus controls, whereas 5-HT2B receptor expression was decreased in CTB-SAP rats versus controls (P < 0.05). This study furthers our understanding of the contribution of differential receptor activation to pLTF and its implications for breathing following respiratory motor neuron death.NEW & NOTEWORTHY The current study investigates underlying receptor-dependent mechanisms contributing to phrenic long-term facilitation (pLTF) following CTB-SAP-induced respiratory motor neuron death at 7 days and 28 days. We found that A2A receptors are required for enhanced pLTF in 7-day CTB-SAP rats, whereas 5-HT receptors are required for moderate pLTF in 28-day CTB-SAP rats. Targeting these time-dependent mechanisms have implications for breathing maintenance over the course of many neuromuscular diseases.

Utilization of pectoralis minor accessory inspiratory muscles in a rodent model of respiratory motor neuron loss

Ventilatory failure, and ultimately death, occurs in patients with neuromuscular diseases due to the loss of respiratory motor neurons (e.g., phrenic and intercostal). Prior to ventilatory failure, patients are able to maintain breathing potentially via recruitment of accessory inspiratory muscles (e.g., pectoralis minor, which are responsible for lifting the ribs upward and outward to move the chest wall following increased ventilatory demand). Since genetic rodent models of motor neuron loss develop global symptoms (e.g., dysphagia, etc.), we have developed an inducible model of only respiratory motor neuron death in order to study how breathing is impacted, and to ultimately develop targeted therapeutic interventions. Briefly, adult male rats are intrapleurally injected with cholera toxin B conjugated to saporin (CTB-SAP) that is retrogradely transported to the spinal cord, resulting in selective elimination of phrenic and intercostal motor neurons. Despite deficits in maximal ventilatory capacity and diaphragmatic output following intrapleural CTB-SAP, eupnea is maintained at 7 days (d) and 28d post-injection. Thus, we hypothesize that pectoralis minor accessory inspiratory muscle activity is increased to maintain eupnea over the course of respiratory motor neuron loss in CTB-SAP rats. To test our hypothesis, we intrapleurally injected adult male rats with bilateral CTB-SAP or control (CTB unconjugated to SAP), and 7d and 28d later studied: 1) pectoralis minor motor neuron survival using immunohistochemistry; and 2) pectoralis minor output at baseline and in response to a maximal ventilatory challenge (hypercapnia + hypoxia; max) in anesthetized, spontaneously breathing rats (n=15/group) via electromyography. Thus far, our data suggests that although the pectoralis minor motor nucleus is in close proximity to the phrenic motor nucleus, these pools are exclusive from each other and pectoralis minor motor neuron survival is unaffected following intrapleural CTB-SAP (p>0.05). Additionally, pectoralis minor muscle amplitude is significantly increased in 7d and 28d intrapleurally injected CTB-SAP rats vs. controls (p<0.05) and in 28d vs. 7d CTB-SAP rats at baseline and max (p<0.05). To test whether pectoralis minor muscles are necessary for maintaining eupnea, we evaluated breathing via whole-body plethysmography in unanesthetized adult male rats following bilateral intrapleural and intramuscular pectoralis minor muscle CTB-SAP injections vs. controls (n=3/group). Our preliminary data suggests that eupneic frequency is increased, but tidal volume is decreased in 7d CTB-SAP rats vs. controls, and our 28d experiments are currently being conducted. In conclusion, our data suggests that the pectoralis minor muscles have an independent motor pool that can become recruited to assist in maintaining eupnea following intrapleural CTB-SAP-induced respiratory motor neuron loss. This study furthers our understanding of the contribution of the pectoralis minor muscles to maintaining eupnea following respiratory motor neuron loss, and suggests that these muscles could potentially be further stimulated and harnessed to significantly prolong and/or improve breathing in patients with neuromuscular disease.

Ampakine Pretreatment Enables One Episode of Hypoxia to Produce Phrenic Motor Facilitation with No Added Benefit of Additional Hypoxic Episodes

Excitatory glutamatergic synaptic transmission acting via the α-amino-3-hydro-5-methyl-4-isoxazolepropionate acid receptors (AMPARs) is critical for respiratory rhythmogenesis as well as activation of respiratory motoneurons. A synthetic class of drugs called ampakines can stimulate breathing via positive allosteric modulation of AMPARs which enhances glutamatergic currents. Our laboratory has reported that anesthetized mice pretreated with ampakine CX717 demonstrate enhanced long-term facilitation (LTF) of hypoglossal motor output following repeated bouts of hypoxia. More recently, we reported that ampakine treatment prior to a single bout of hypoxia can produce sustained facilitation of phrenic motor output in anesthetized rats. In the current study, we tested the hypothesis that ampakine pretreatment would enhance the magnitude of phrenic LTF following multiple bouts of hypoxia. Phrenic nerve output, arterial blood pressure and end-tidal CO2 were recorded from urethane-anesthetized, mechanically ventilated and vagotomized adult male Sprague-Dawley rats. Phrenic burst amplitude recorded during the experiments was expressed relative to baseline (pre-treatment) activity (%BL). First, we verified the impact of a single injection of intravenous (iv) dose of ampakine (CX717, 15mg/kg) compared to vehicle solution (10% HPCD). CX717 caused a small and transient increase in phrenic burst amplitude and frequency, confirming previous reports by our group. Phrenic burst amplitude showed a transient increase (70±48%BL after 2 min) that gradually declined and was not different than baseline after 60 min (2±32%BL, n=4). Injection of the HPCD vehicle caused no discernable change in phrenic activity (2 min: 0±15%BL, 60 min: 3±5%Bl, n=4). Second, we investigated if ampakine treatment prior to acute intermittent hypoxia (CX717 + 3 five-minute bouts of hypoxia) would produce a more robust phrenic LTF when compared to ampakine followed by single bout of hypoxia (CX717 + 1 five-minute bout of hypoxia). Similar to our prior report, CX717 + 1 five-minute bout of hypoxia causes a sustained increase in phrenic burst amplitude (60 min: 96±61% Bl, n=8). However, contrary to our hypothesis, little phrenic LTF was observed in the CX717 + 3X hypoxia group (60 min: 24±29%Bl, n=8). Lastly, in a separate series of experiments, we verified the impact of 1 vs. 3 episodes of hypoxia on phrenic motor output, but in the absence of ampakine. Phrenic LTF was present after 3 five-minute bouts of hypoxia (60 min: 88±26%BL, n=3) but not 1 five-minute bout of hypoxia (60 min: 4±1%BL, n=3). These studies of anesthetized and spinal-intact rats indicate that phrenic LTF is not enhanced when ampakine pretreatment is followed by multiple bouts of hypoxia. Rather, the combination of ampakine and a single brief hypoxic episode appears to be ideal for producing sustained increase in phrenic motor output. These findings could have implications for hypoxia-based neurorehabilitation strategies, a method which is gaining traction in spinal cord injury rehabilitation. A single dose of ampakine may serve to “prime” the nervous system such that fewer exposures to hypoxia are required to produce sustained neuroplastic changes.

Serotonergic innervation of respiratory motor nuclei after cervical spinal injury: Impact of intermittent hypoxia

Although cervical spinal cord injury (cSCI) disrupts bulbo-spinal serotonergic projections, partial recovery of spinal serotonergic innervation below the injury site is observed after incomplete cSCI. Since serotonin contributes to functional recovery post-injury, treatments to restore or accelerate serotonergic reinnervation are of considerable interest. Intermittent hypoxia (IH) was reported to increase serotonin innervation near respiratory motor neurons in spinal intact rats, and to improve function after cSCI. Here, we tested the hypotheses that spontaneous serotonergic reinnervation of key respiratory (phrenic and intercostal) motor nuclei: 1) is partially restored 12 weeks post C2 hemisection (C2Hx); 2) is enhanced by IH; and 3) results from sprouting of spared crossed-spinal serotonergic projections below the site of injury. Serotonin was assessed via immunofluorescence in male Sprague Dawley rats with and without C2Hx (12 wks post-injury); individual groups were exposed to 28 days of: 1) normoxia; 2) daily acute IH (dAIH28: 10, 5 min 10.5% O2 episodes per day; 5 min normoxic intervals); 3) mild chronic IH (IH28-5/5: 5 min 10.5% O2 episodes; 5 min intervals; 8 h/day); or 4) moderate chronic IH (IH28-2/2: 2 min 10.5% O2 episodes; 2 min intervals; 8 h/day), simulating IH experienced during moderate sleep apnea. After C2Hx, the number of ipsilateral serotonergic structures was decreased in both motor nuclei, regardless of IH protocol. However, serotonergic structures were larger after C2Hx in both motor nuclei, and total serotonin immunolabeling area was increased in the phrenic motor nucleus but reduced in the intercostal motor nucleus. Both chronic IH protocols increased serotonin structure size and total area in the phrenic motor nuclei of uninjured rats, but had no detectable effects after C2Hx. Although the functional implications of fewer but larger serotonergic structures are unclear, we confirm that serotonergic reinnervation is substantial following injury, but IH does not affect the extent of reinnervation.