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. 2010 Jan;40(1-2):21-6.
doi: 10.1007/s12031-009-9248-x. Epub 2009 Aug 12.

Postsynaptic development of the neuromuscular junction in mice lacking the gamma-subunit of muscle nicotinic acetylcholine receptor

Yun Liu  1 , Yoshie Sugiura, Daniel Padgett, Weichun Lin
Affiliations

Postsynaptic development of the neuromuscular junction in mice lacking the gamma-subunit of muscle nicotinic acetylcholine receptor

Yun Liu et al. J Mol Neurosci. 2010 Jan.

Abstract

The mammalian muscle nicotinic acetylcholine receptor (AChR) is composed of five membrane-spanning subunits and its composition differs between embryonic and adult muscles. In embryonic muscles, it is composed of two alpha-, one beta-, one delta-, and one gamma-subunit; the gamma-subunit is later replaced by the epsilon-subunit during postnatal development. This unique temporal expression pattern of the gamma-subunit suggests it may play specific roles in embryonic muscles. To address this issue, we examined the formation and function of the neuromuscular junction in mouse embryos deficient in the gamma-subunit. At embryonic day 15.5, AChR clusters were absent and the spontaneous miniature endplate potentials were undetectable in the mutant muscles. However, electrical stimulation of the nerves triggered muscle contraction and elicited postsynaptic endplate potential (EPP) in the mutant muscles, although the magnitude of the muscle contraction and the amplitudes of the EPPs were smaller in the mutant compared to the wild-type muscles. Reintroducing a wild-type gamma-subunit into the mutant myotubes restored the formation of AChR clusters in vitro. Together, these results have demonstrated that functional AChRs were present in the mutant muscle membrane, but at reduced levels. Thus, in the absence of the gamma-subunit, a combination of alpha, beta, and delta subunits may assemble into functional receptors in vivo. These results also suggest that the gamma-subunit maybe involved in interacting with rapsyn, a cytoplasmic protein required for AChR clustering.

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Figures

Figure 1
Figure 1
The pattern of developing neuromuscular junction. Wholemount embryonic diaphragm muscles (wild type; a–b, E15.5) were immunostained with anti-neurofilament antibody (a) and Texas-red conjugated α-bungarotoxin (b). The nerve terminals and AChR clusters were confined to the central region of the muscle. c The pattern of AChR subunit (α-subunit) gene transcription revealed by wholemount in situ hybridization in the diaphragm muscle (E17.5). d The pattern of AChE distribution in wholemount diaphragm muscle (E16.5). Scale bars a, b 400 µm, c 500 µm, d 500 µm
Figure 2
Figure 2
Differentiation of the NMJ in the absence of the γ-subunit. High magnification views of a single endplate from wholemount diaphragm muscle at E18.5, double-labeled with synaptotagmin-2 (Syt-2) antibody (green) and Texas-red conjugated α-bungarotoxin (red). Individual endplate (AChR cluster) in the mutant (e, arrow) appeared less intensely labeled by α-bungarotoxin, but their sizes are bigger, compared to the wild type (b, arrow). In both WT and mutant muscles, Syt-2 antibody staining was highly concentrated at the nerve terminals (arrowheads in a, d). Scale bar 10 µm
Figure 3
Figure 3
Neuromuscular synaptic activity in γ−/− embryos. a mEPPs were detected in the wild type (+/+), but not in the mutant (−/−) at E15.5. b Electrical stimulation of the phrenic nerve elicited EPPs in both +/+ and −/− muscles (E15.5), but with smaller amplitude and slower response in the mutant. Visual inspection of the muscles during stimulation revealed a slow-wavy contraction in the mutant, vs. a fast contraction in the wild type. c MEPPs were recorded from the wild type and the mutant at E18.5. d Quantification of the mEPPs: the amplitudes and half-width of the mEPPs were significantly (*p<0.001) reduced in the mutant, compared to the wild type
Figure 4
Figure 4
Full-length γ-subunit restores AChR clustering in vitro. a, b Live myotube cultures visualized under Nomarski (7 days after plating). Spontaneous myotube fusion was observed in both wild type and γ−/− culture. c, d Fixed myotubes 5 days after serum deprivation. AChR clusters were present in wild-type myotube (e), but not in the γ−/− myotube (f). g, h Rescue of AChR clustering in γ−/− myotubes (h) after transfection with γ/GFP (g). Scales a, b 50 µm, c–h 20 µm
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