Organotypic Culture Assay for Neuromuscular Synaptic Degeneration and Function

Dissanayake, Kosala N.; Chou, Robert Chang-Chih; Brown, Rosalind; Ribchester, Richard R.

doi:10.1007/978-1-0716-0585-1_11

Cited by 2 publications

(4 citation statements)

References 21 publications

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“…Thus, in preparations from Wld S mice, more than 80% of motor endplates are still occupied by motor nerve terminals 24–48 h after incubation in oxygenated MPS at 32°C. By contrast, fewer that 20% of motor nerve terminals remain intact after 24 h when preparations from control mice are similarly organ-cultured ( Brown et al, 2015 ; Di Stefano et al, 2015 ; Dissanayake et al, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…Since muscle aftercontractions and associated increases in endplate Ca 2+ were inhibited by Mg 2+ we therefore asked whether degeneration of NMJs induced by OP compounds would also be inhibited by a similar increase in extracellular [Mg 2+ ]. We tested for this using an “ ex-vivo ” assay of neuromuscular synaptic degeneration ( Brown et al, 2015 ; Di Stefano et al, 2015 ; Dissanayake et al, 2020 ). In the present experiments we utilized double homozygous thy1YFP16 : Wld S mice whose axons and terminals were endogenously fluorescent due to expression of the YFP transgene in motor neurons ( Feng et al, 2000 ; Wong et al, 2009 ; Brown et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

“…Synaptic degeneration was measured in cultures of isolated FDB and DL muscle preparations from 2 to 3 month old thy1YFP16 : Wld S mice as described previously ( Brown et al, 2015 ; Dissanayake et al, 2020 ). Briefly, muscles were pinned to dental wax slabs and placed in plastic vials containing bicarbonate-buffered MPS bubbled continuously with 95%O 2 /5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

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“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Dissanayake

Redman

Mackenzie

et al. 2022

Front. Mol. Neurosci.

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Excitotoxicity is thought to be an important factor in the onset and progression of amyotrophic lateral sclerosis (ALS). Evidence from human and animal studies also indicates that early signs of ALS include degeneration of motor nerve terminals at neuromuscular junctions (NMJs), before degeneration of motor neuron cell bodies. Here we used a model of excitotoxicity at NMJs in isolated mouse muscle, utilizing the organophosphorus (OP) compound omethoate, which inhibits acetylcholinesterase activity. Acute exposure to omethoate (100 μM) induced prolonged motor endplate contractures in response to brief tetanic nerve stimulation at 20–50 Hz. In some muscle fibers, Fluo-4 fluorescence showed association of these contractures with explosive increases in Ca2+ (“calcium bombs”) localized to motor endplates. Calcium bombs were strongly and selectively mitigated by increasing Mg2+ concentration in the bathing medium from 1 to 5 mM. Overnight culture of nerve-muscle preparations from WldS mice in omethoate or other OP insecticide components and their metabolites (dimethoate, cyclohexanone, and cyclohexanol) induced degeneration of NMJs. This degeneration was also strongly mitigated by increasing [Mg2+] from 1 to 5 mM. Thus, equivalent increases in extracellular [Mg2+] mitigated both post-synaptic calcium bombs and degeneration of NMJs. The data support a link between Ca2+ and excitotoxicity at NMJs and suggest that elevating extracellular [Mg2+] could be an effective intervention in treatment of synaptic pathology induced by excitotoxic triggers.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Dissanayake

Redman

Mackenzie

et al. 2022

Front. Mol. Neurosci.

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“…Aided by images and videos (Figures 1 and 2 , Videos [Link] , [Link] , [Link] ), we provide a detailed protocol of how to rapidly dissect the mouse ETA muscle, which can then be used for a variety of neuromuscular analyses, including studies of muscle fibre type composition (Figure 4 ), as well as assessment of motor neuron development and degeneration/regeneration (Figure 5 ). These evaluations can be combined with RNA and protein analyses, electrophysiological recordings and possibly ex vivo synaptic degeneration assays (Dissanayake et al, 2020 ), to name but a few relevant applications, in order to interrogate rodent models of neuromuscular disorders. Furthermore, through comparison with other muscles, a detailed understanding of neuropathology and pertinent features of disease can emerge, especially when correlated with baseline properties of healthy muscles.…”

Section: Discussionmentioning

confidence: 99%

Dissection, in vivo imaging and analysis of the mouse epitrochleoanconeus muscle

2021

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Analysis of rodent muscles affords an opportunity to glean key insights into neuromuscular development and the detrimental impact of disease‐causing genetic mutations. Muscles of the distal leg, for instance the gastrocnemius and tibialis anterior, are commonly used in such studies with mice and rats. However, thin and flat muscles, which can be dissected, processed and imaged without major disruption to muscle fibres and nerve‐muscle contacts, are more suitable for accurate and detailed analyses of the peripheral motor nervous system. One such wholemount muscle is the predominantly fast twitch epitrochleoanconeus (ETA), which is located in the upper forelimb, innervated by the radial nerve, and contains relatively large and uniformly flat neuromuscular junctions (NMJs). To facilitate incorporation of the ETA into the experimental toolkit of the neuromuscular disease field, here, we describe a simple method for its rapid isolation (<5 min), supported by high‐resolution videos and step‐by‐step images. Furthermore, we outline how the ETA can be imaged in live, anaesthetised mice, to enable examination of dynamic cellular processes occurring at the NMJ and within intramuscular axons, including transport of organelles, such as mitochondria and signalling endosomes. Finally, we present reference data on wild‐type ETA fibre‐type composition in young adult, male C57BL6/J mice. Comparative neuroanatomical studies of different muscles in rodent models of disease can generate critical insights into pathogenesis and pathology; dissection of the wholemount ETA provides the possibility to diversify the repertoire of muscles analysed for this endeavour.

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Organotypic Culture Assay for Neuromuscular Synaptic Degeneration and Function

Cited by 2 publications

References 21 publications

“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

“Calcium bombs” as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions

Dissection, in vivo imaging and analysis of the mouse epitrochleoanconeus muscle

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