![]() With local ethical committee approval and under Home Office Licences, some mice were anaesthetised with isoflurane and volumes of saline (up to 100 µL) containing physiologically-inert, Emerald Fluorescent Protein (emGFP)-tagged tetanus toxin C fragments (emGFP-TetC see below) were injected subcutaneously in one hind limb. However, the intensity and contrast of the fluorescence of these molecules were insufficient for routine visualisation of NMJs using fibre-optic CEM, whose sensitivity is less than that of conventional LCSM systems or wide-field fluorescence microscopes. We confirmed previously that 4-di-2-Asp, a fluorescent styryl dye that passively stains motor nerve terminals, and a green fluorescent protein (GFP)-tagged construct of a botulinum toxin heavy chain that also binds to NMJs both readily enabled visualisation of motor nerve terminals using conventional wide-field or laser scanning confocal microscopy (LSCM). However, wider application of CEM for research, or veterinary or clinical diagnostic applications, would require non-toxic, high-contrast fluorescent ligands for axons and NMJs that can be administered exogenously, for example by local subcutaneous or intramuscular injection. Innervated or denervated NMJs could also be distinguished by combining CEM with conventional electromyography in these mice. We showed previously that CEM visualises motor nerve terminals in mice expressing Yellow Fluorescent Protein (YFP) selectively in motor neurons. ![]() This technology has enabled real time (up to 12 video frames per second), direct imaging of cells either selectively expressing fluorescent proteins or stained with exogenous ligands conjugated to specific fluorochromes. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.Ĭonfocal endomicroscopy (CEM) utilises laser scanning and fluorescence capture via hundreds of optical filaments contained in narrow (0.5–1.5 mm) probes. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized Wld S mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). ![]() Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals.
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