levator-auris-longus-preparation-for-examination-mammalian

<em>Levator Auris Longus</em> Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Wright State University

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder that is typically fatal within 3-5 years of diagnosis. While motoneuron death is the defining characteristic of ALS, the events that underlie its pathology are not restricted to the nervous system. In this regard, ALS muscle atrophies and weakens significantly before presentation of neurological symptoms. Since the skeletal muscle L-type Ca(2+) channel (CaV1.1) is a key regulator of both mass and force, we investigated whether CaV1.1 function is impaired in the muscle of two distinct mouse models carrying an ALS-linked mutation.

Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis.

Huntington's disease (HD) patients suffer from progressive and debilitating motor dysfunction. Previously, we discovered reduced skeletal muscle chloride channel (ClC-1) currents, inwardly rectifying potassium (Kir) channel currents, and membrane capacitance in R6/2 transgenic HD mice. The ClC-1 loss-of-function correlated with increased aberrant mRNA processing and decreased levels of full-length ClC-1 mRNA (Clcn1 gene). Physiologically, the resulting muscle hyperexcitability may help explain involuntary contractions of HD. In this study, the onset and progression of these defects are investigated in R6/2 mice, ranging from 3 wk old (presymptomatic) to 9-13 wk old (late-stage disease), and compared with age-matched wild-type (WT) siblings. The R6/2 ClC-1 current density and level of aberrantly spliced Clcn1 mRNA remain constant with age. In contrast, the ClC-1 current density increases, and the level of aberrantly spliced Clcn1 mRNA decreases with age in WT mice. The R6/2 ClC-1 properties diverge from WT before the onset of motor symptoms, which occurs at 5 wk of age. The relative decrease in R6/2 muscle capacitance also begins in 5-wk-old mice and is independent of fiber atrophy. Kir current density is consistently lower in R6/2 compared with WT muscle. The invariable R6/2 ClC-1 properties suggest a disruption in muscle maturation, which we confirm by measuring elevated levels of neonatal myosin heavy chain (MyHC) in late-stage R6/2 skeletal muscle. Similar changes in ClC-1 and MyHC isoforms in the more slowly developing Q175 HD mice suggest an altered maturational state is relevant to adult-onset HD. Finally, we find nuclear aggregates of muscleblind-like protein 1 without predominant CAG repeat colocalization in R6/2 muscle. This is unlike myotonic dystrophy, another trinucleotide repeat disorder with similar ClC-1 defects, and suggests a novel mechanism of aberrant mRNA splicing in HD. These early and progressive skeletal muscle defects reveal much needed peripheral biomarkers of disease progression and better elucidate the mechanism underlying HD myopathy.

Progressive Cl- channel defects reveal disrupted skeletal muscle maturation in R6/2 Huntington's mice.

Huntington's disease (HD) is a progressive and fatal degenerative disorder that results in debilitating cognitive and motor dysfunction. Most HD studies have focused on degeneration of the CNS. We previously discovered that skeletal muscle from transgenic R6/2 HD mice is hyperexcitable due to decreased chloride and potassium conductances. The progressive and early onset of these defects suggest a primary myopathy in HD. In this study, we examined the relationship between neuromuscular transmission and skeletal muscle hyperexcitability. We used an  preparation of the levator auris longus muscle from male and female late-stage R6/2 mice and age-matched wild-type controls. Immunostaining of the synapses and molecular analyses revealed no evidence of denervation. Physiologically, we recorded spontaneous miniature endplate currents (mEPCs) and nerve-evoked EPCs (eEPCs) under voltage-clamp, which, unlike current-clamp records, were independent of the changes in muscle membrane properties. We found a reduction in the number of vesicles released per action potential (quantal content) in R6/2 muscle, which analysis of eEPC variance and morphology indicate is caused by a reduction in the number of vesicle release sites () rather than a change in the probability of release (). Furthermore, analysis of high-frequency stimulation trains suggests an impairment in vesicle mobilization. The depressed neuromuscular transmission in R6/2 muscle may help compensate for the muscle hyperexcitability and contribute to motor impersistence. Recent evidence indicates that Huntington's disease (HD) is a multisystem disorder. Our examination of neuromuscular transmission in this study reveals defects in the motor nerve terminal that may compensate for the muscle hyperexcitability in HD. The technique we used eliminates the effects of the altered muscle membrane properties on synaptic currents and thus provides hitherto the most detailed analysis of synaptic transmission in HD. Clinically, the striking depression of neurotransmission we found may help explain the motor impersistence in HD patients. Therapies that target the highly accessible peripheral nerve and muscle system provide a promising new avenue to lessen the debilitating motor symptoms of HD.

Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions.

Patients with myotonia congenita have muscle hyperexcitability due to loss-of-function mutations in the ClC-1 chloride channel in skeletal muscle, which causes involuntary firing of muscle action potentials (myotonia), producing muscle stiffness. The excitatory events that trigger myotonic action potentials in the absence of stabilizing ClC-1 current are not fully understood. Our goal was to identify currents that trigger spontaneous firing of muscle in the setting of reduced ClC-1 current.

Article	Year
Progressive impairment of CaV1.1 function in the skeletal muscle of mice expressing a mutant type 1 Cu/Zn superoxide dismutase (G93A) linked to amyotrophic lateral sclerosis. Skeletal muscle\| PubMed ID: 27340545	2016
Progressive Cl- channel defects reveal disrupted skeletal muscle maturation in R6/2 Huntington's mice. The Journal of general physiology\| PubMed ID: 27899419	2017
Depressed Synaptic Transmission and Reduced Vesicle Release Sites in Huntington's Disease Neuromuscular Junctions. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 28724748	2017
Inhibiting persistent inward sodium currents prevents myotonia. Annals of neurology\| PubMed ID: 28833464	2017

Andrew A. Voss

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