Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Martin, Melanie; Li, Ké; Wright, Megan C.; Lepore, Angelo C.

doi:10.3791/52605

Cited by 8 publications

(5 citation statements)

References 18 publications

(24 reference statements)

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“…Gastrocnemius, tibialis anterior, and soleus muscles were dissected from SBMA mice and littermate controls and immediately placed into 4% paraformaldehyde (PFA) for 30 min. Muscles were then teased into small bundles under a dissecting microscope in PBS and stained according to Martin et al, 2015 [ 41 ]. Briefly, teased muscle was incubated in 0.1 M glycine solution, incubated in AlexaFluor-647-conjugated α-bungarotoxin, then incubated in pre-cooled methanol.…”

Section: Methodsmentioning

confidence: 99%

Neuromuscular junction pathology is correlated with differential motor unit vulnerability in spinal and bulbar muscular atrophy

Molotsky

Liu

Lieberman

et al. 2022

acta neuropathol commun

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Spinal and bulbar muscular atrophy (SBMA) is an X-linked, neuromuscular neurodegenerative disease for which there is no cure. The disease is characterized by a selective decrease in fast-muscle power (e.g., tongue pressure, grip strength) accompanied by a selective loss of fast-twitch muscle fibers. However, the relationship between neuromuscular junction (NMJ) pathology and fast-twitch motor unit vulnerability has yet to be explored. In this study, we used a cross-model comparison of two mouse models of SBMA to evaluate neuromuscular junction pathology, glycolytic-to-oxidative fiber-type switching, and cytoskeletal alterations in pre- and postsynaptic termini of tibialis anterior (TA), gastrocnemius, and soleus hindlimb muscles. We observed significantly increased NMJ and myofiber pathology in fast-twitch, glycolytic motor units of the TA and gastrocnemius compared to slow-twitch, oxidative motor units of the soleus, as seen by decreased pre- and post-synaptic membrane area, decreased pre- and post-synaptic membrane colocalization, increased acetylcholine receptor compactness, a decrease in endplate area and complexity, and deficits in neurofilament heavy chain. Our data also show evidence for metabolic dysregulation and myofiber atrophy that correlate with severity of NMJ pathology. We propose a model in which the dynamic communicative relationship between the motor neuron and muscle, along with the developmental subtype of the muscle, promotes motor unit subtype specific vulnerability, metabolic alterations, and NMJ pathology.

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Section: Methodsmentioning

confidence: 99%

Neuromuscular junction pathology is correlated with differential motor unit vulnerability in spinal and bulbar muscular atrophy

Molotsky

Liu

Lieberman

et al. 2022

acta neuropathol commun

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“…We did not use the in vivo preparation, described by others ( Lepore et al, 2011 ), because we were concerned that in vivo stimulation of the phrenic nerve, at moderate to high frequencies, would lead to variable and unreliable CMAP recordings, likely due to changes in the electrode position caused by muscle contraction. Moreover, a related method, reported to record from the mouse diaphragm muscle, uses a surface recording electrode, and likely monitors the activity of multiple thoracic muscles ( Martin et al, 2015 ). Thus, following anesthesia with 5% isoflurane, mice were decapitated, and the diaphragm muscle, together with the phrenic nerve, was quickly isolated and transferred to a customized recording chamber.…”

Section: Methodsmentioning

confidence: 99%

Preserving neuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonist antibody

Cantor¹,

Zhang²,

Delestrée

et al. 2018

eLife

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SummaryIn amyotrophic lateral sclerosis (ALS) and animal models of ALS, including SOD1-G93A mice, disassembly of the neuromuscular synapse precedes motor neuron loss and is sufficient to cause a decline in motor function that culminates in lethal respiratory paralysis. We treated SOD1-G93A mice with an agonist antibody to MuSK, a receptor tyrosine kinase essential for maintaining neuromuscular synapses, to determine whether increasing muscle retrograde signaling would slow nerve terminal detachment from muscle. The agonist antibody, delivered after disease onset, slowed muscle denervation, promoting motor neuron survival, improving motor system output, and extending the lifespan of SOD1-G93A mice. These findings suggest a novel therapeutic strategy for ALS, using an antibody format with clinical precedence, which targets a pathway essential for maintaining attachment of nerve terminals to muscle.

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“…Right hemidiaphragms were collected after the terminal EMG experiment, stained and imaged for neuromuscular junctions (NMJs), as previously described (27). Briefly, animals were euthanized with KXA overdose and the right hemidiaphragm was removed and then pinned flat on a silicon dish, fixed in 4% paraformaldehyde (PFA) and washed.…”

Section: Methodsmentioning

confidence: 99%

“…In addition to CMAP recordings, we assessed NMJ morphologies to determine the effects of AAV2-BDNF on PhMN innervation of the diaphragm (23)(24)(25)(26)(27). As with EMGs, we examined NMJs at all 3 subregions of the hemidiaphragm (ventral, medial, dorsal) 8 wk after injury.…”

Section: Aav2-bdnf Did Not Alter Diaphragm Nmj Innervation Phmn Survival or Phmn Somal Morphologymentioning

confidence: 99%

AAV2‐BDNF promotes respiratory axon plasticity and recovery of diaphragm function following spinal cord injury

et al. 2019

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More than half of spinal cord injury (SCI) cases occur in the cervical region, leading to respiratory dysfunction due to damaged neural circuitry that controls critically important muscles such as the diaphragm. The C3-C5 spinal cord is the location of phrenic motor neurons (PhMNs) that are responsible for diaphragm activation; PhMNs receive bulbospinal excitatory drive predominately from supraspinal neurons of the rostral ventral respiratory group (rVRG). Cervical SCI results in rVRG axon damage, PhMN denervation, and consequent partial-to-complete paralysis of hemidiaphragm. In a rat model of C2 hemisection SCI, we expressed the axon guidance molecule, brain-derived neurotrophic factor (BDNF), selectively at the location of PhMNs (ipsilateral to lesion) to promote directed growth of rVRG axons toward PhMN targets by performing intraspinal injections of adeno-associated virus serotype 2 (AAV2)-BDNF vector. AAV2-BDNF promoted significant functional diaphragm recovery, as assessed by in vivo electromyography. Within the PhMN pool ipsilateral to injury, AAV2-BDNF robustly increased sprouting of both spared contralateral-originating rVRG axons and serotonergic fibers. Furthermore, AAV2-BDNF significantly increased numbers of putative monosynaptic connections between PhMNs and these sprouting rVRG and serotonergic axons. These findings show that targeting circuit plasticity mechanisms involving the enhancement of synaptic inputs from spared axon populations is a powerful strategy for restoring respiratory function post-SCI.-Charsar, B. A.,

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Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Cited by 8 publications

References 18 publications

Neuromuscular junction pathology is correlated with differential motor unit vulnerability in spinal and bulbar muscular atrophy

Neuromuscular junction pathology is correlated with differential motor unit vulnerability in spinal and bulbar muscular atrophy

Preserving neuromuscular synapses in ALS by stimulating MuSK with a therapeutic agonist antibody

AAV2‐BDNF promotes respiratory axon plasticity and recovery of diaphragm function following spinal cord injury

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