muscle, and that each sMN are posiconed in the holes of the endplate-pretzel or ensheathing the tubular juncconal folds. In vivo Confocal Imaging and 3D ...
In vivo Confocal Imaging and 3D rendering of the Neuromuscular Endplate and its Synap)c Myonuclei Kenth-Arne Hansson, Kris)an Gundersen and Jo C. Bruusgaard
Center for Integrative Neuroplasticity
Department of Biosciences, University of Oslo, Norway
Introduc)on
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The neuromuscular junc/on (NMJ) has been a focus of interest throughout the history of neuroscience, and is probably the most studied of all synapses. Post-synap/cally, numerous acetylcholine receptors (AChRs) occupies the motor endplate on the muscle fiber. Preserving its func/on, motor endplates is supported by a set of synap/c myonuclei (sMN) that have a unique transcrip/onal profile compared to non-sMN. The present study was undertaken with the inten/on of quan/fying sMN and combining in vivo injec/on of fluorescent oligonucleo/des into single muscle fibers, confocal imaging and 3D rendering to study the NMJ and sMN with high resolu/on.
C In vivo microinjec)ons
(A), Mouse prepared for in vivo injec/ons. (B), a micropipeXe is inserted into the muscle fiber and fluorescent oligonucleo/des (magenta) are injected into the muscle fiber of pre-labelled endplates (green), with an subsequent myonuclear uptake of the dye (C).
Methods To stain sMN, motor endplates were pre-labelled with α-bungarotoxin (α-BTX) and mul/ple muscle fibers were injected, in proximity of labelled endplates, in vivo with TRITC-labelled oligonucleo/des. sMN and endplates in single muscle fibers were studied in vivo, and 3D surface visualiza/on of z-stacks were performed using Bitplane Imaris so\ware 8.1. In vivo Confocal images and 3D surface rendering of A
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NMJs Neuromuscular endplate (green) stained with fluorescent α-BTX, and synap/c myonuclei (magenta) stained a\er in vivo injec/ons with TRITC-labelled oligonucleo/des (A). (B), 3D rendering of the neuromuscular endplate with a subsequent rendering of the synap/c myonuclei (C). Removing background fluorescence (A) from (C), the final 3D rendered image appears as shown in (D). In (E) the surface of NMJ is rotated 45° clockwise around its ver/cal axis.
Results & Discussion Our technique exclusively iden/fies sMN and MN. Dual labelling with α-bungarotoxin also highlights the distribu/on of AChRs. Using high-resolu/on image acquisi/on makes it possible to generate detailed 3D rendering of NMJs and sMN. Preliminary results indicate that each motor endplate is supported by 3.9 ± 0.9 (mean ± s.d., N=20) sMN in the EDL muscle, and that each sMN are posi/oned in the holes of the endplate-pretzel or ensheathing the tubular junc/onal folds. A
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SUMMARY:
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Hoechst staining of the motor end-plate En face image of non-specific nuclei in blue and neuromuscular endplate in green. (A) Confocal z-stacks of the NMJ. (B) 3D surface rendering of nuclei, followed by a 3D surface construc/on of the neuromuscular endplate (C). (D) Final image a\er removing background fluorescence from (A). Non-synap/c myonuclei are marked by asterisks.
This labelling technique exclusively highlights sMN and MN, in contrast to e.g. Hoechst and DAPI staining, allowing counterstaining of other cells or cellular structures. Linking in vivo high-resoluJon imaging combined with 3D rendering could be of great aid when studying diseases or illnesses ramifying the neuromuscular juncJon. In addiJon, this technique would be a interesJng tool to apply when studying the growing role of synapJc myonuclei contribuJng to plasJcity of the neuromuscular juncJon.
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Motor endplate and synap)c myonuclei Neuromuscular endplate (green) stained with fluorescent α-BTX, and synap/c myonuclei (magenta) stained a\er in vivo injec/ons with TRITC-labelled oligonucleo/des (A). (B), 3D rendering of the synap/c myonuclei with a subsequent surface rendering of the neuromuscular endplate (C).
