Morphological and functional changes in innervation of a fast forelimb muscle in SOD1-G85R mice

Abstract

Muscle endplates become denervated in mice that express mutations of human superoxide dismutase 1 (hSOD1), models of familial amyotrophic lateral sclerosis. This denervation is especially marked in fast limb muscles, and precedes death of motor neuron somata. This study used mice that expressed yellow fluorescent protein (YFP) in neurons to investigate changes in the morphology and function of axons and motor terminals inervating a fast forelimb muscle (epitrochleoanconeus, ETA) in presymptomatic and symptomatic hSOD1-G85R mice, compared to those in mice that express wild-type (wt) hSOD1. The percentage of endplates (identified using fluorescently-labeled α-bungarotoxin) innervated by motor terminals remained high in presymptomatic SOD1-G85R mice, but fell to ~50% in symptomatic mice. The number of large diameter (≥ 4 µm) axons in the ETA nerve also decreased as mice became symptomatic, and endplate innervation correlated best with the number of large diameter axons. Motor terminal function was assessed using changes in terminal YFP fluorescence evoked by trains of action potentials; different components of the pH-dependent YFP signals reflect stimulation-induced Ca2+ entry and vesicular exo/endocytosis. Most visible motor terminals (>90%) remained capable of responding to nerve stimulation in both pre- and symptomatic hSOD1-G85R mice, but with functional alterations. Responses in presymptomatic terminals suggested reduced acidification and increased vesicular release, whereas symptomatic terminals exhibited increased acidification and reduced vesicular release. The fact that most remaining terminals were able to respond to nerve stimulation suggests that motor terminal-protective therapies might contribute to preserving neuromuscular function in fALS mice.

A Cultured Sensorimotor Organoid Model Forms Human Neuromuscular Junctions

“…One caveat was that the hSYN ::EGFP virus did not report specifically motor neuron outgrowth, although the high percentage of motor neurons in the culture should nonetheless have preserved sensitivity to large effects in motor neurons. While increased numbers of lines will be needed for confirmation, the distinct phenotypes observed – namely a reduced percentage of innervated NMJs in the SOD1 G85R and PFN1 G118V mutations and a decreased area of innervated NMJs in the TARDBP G298S mutation – are similar to prior reports demonstrating that different ALS genetic variants can affect the NMJ in distinct ways 76–78 . Future studies will be necessary to identify the mechanistic connections between these mutations and the NMJ physiological and structural phenotypes.…”

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

“…Accordingly, NMJs can be categorised as fully innervated, partially denervated or fully denervated/vacant (Sleigh et al, 2014a). In addition to studies of synaptic degeneration (Nguyen et al, 2012), the ETA has been used to monitor NMJ development, including the processes of synapse elimination and plaque‐to‐pretzel endplate transition (Figure 5c) (Rodríguez Cruz et al, 2020), as well as terminal sprouting in response to denervation (Nguyen et al, 2012; Rogozhin et al, 2008). While most of these processes can be scored by eye, ImageJ‐based tools, such as NMJ‐morph and NMJ‐Analyser (Jones et al, 2016; Mejia Maza et al, 2021; Minty et al, 2020), provide a more objective and systematic method for analysing NMJs in a quantitative manner, which can facilitate the identification of more subtle morphological phenotypes; for example, NMJ‐Analyser was used to identify structural defects in motor nerve terminals of two different ALS mouse models at a time when no clear denervation was detectable by eye (Mejia Maza et al, 2021).…”

“…The epitrochleoanconeus (ETA), also termed the anconeus epitrochlearis, is a thin, roughly rectangular muscle, located in proximity to the triceps brachii on the medial surface of the upper forelimbs that contributes to forearm supination in rodents (i.e., outward paw rotation). Innervated by a branch of the radial nerve containing ~12 axons (Nguyen et al, 2012 ), the mouse ETA is reported to consist of ~90% fast twitch muscle fibres (Bradley et al, 1989 ). Blood is supplied to the ETA from the brachial artery via a descending branch of the arteria profunda brachii (Greene, 1935 ).…”

“…In rodents, the ETA can be quickly and easily dissected and wholemount processed without the need for sectioning. Consequently, it has been used for electrophysiological recordings (Nanou et al, 2016 ; Rogozhin et al, 2008 ; Wang et al, 2018 ), as well as assessment of pathological changes in NMJ morphology and function in several mouse models of neuromuscular disease (Lyons & Slater, 1991 ; Nguyen et al, 2012 ; Sleigh et al, 2020a ; Tarr et al, 2013 ). Given its superficial, accessible position and relatively large, en face NMJs (Mech et al, 2020 ; van der Pijl et al, 2016 ), the ETA is also an ideal candidate for intravital imaging.…”

Activity-dependent degeneration of axotomized neuromuscular synapses in WldS mice

“…The energetics of maintaining a functional synapse evidently place considerable demands on the metabolism of motor nerve terminals ( Zhu et al, 2012; Schwarz, 2013 ). Synaptic mitochondria are particularly sensitive to transient hypoxia, ischemia and Ca 2+ overload ( Brown et al, 2006; David et al, 2007; Misgeld et al, 2007; Nguyen et al, 2009; Nguyen et al, 2011; Nguyen et al, 2012; Talbot et al, 2012 ). This transient ischemic/hypoxic response is also sensitive to Ca 2+ ( Barrett et al, 2014 ).…”