Skip to main content
. 2018 May 30;7:e30693. doi: 10.7554/eLife.30693

Figure 5. Switch from glutamate to acetylcholine receptors in hindlimb muscles.

(A) Examples of hindlimb innervation patterns and distribution of ACh nicotinic receptors in a whole-mount hindlimb bud, revealed by fluorescence immunolabeling of neurofilament associated protein (Neurofil., red), glutamate receptor (NR2b, blue) and α-bungarotoxin labeling (α-bungaroTx, yellow) respectively, at stages 52 (upper panels) and 57 (lower panels). Inset drawings at bottom left of each panel show bud morphology at the two representative larval stages. Scale bars = 200 µm for stage 52, 100 µm for stage 57. (B) Examples of fluorescence immunolabeling against neurofilament associated protein (red), synaptophysin (Synaptoph., green) and NMDA glutamate receptor subunit 1 (NR1, blue) in whole-mount limb bud at stage 52. White arrowheads indicate sites of apposition of all three markers. Scale bar = 20 µm. (C) Examples of fluorescence immunolabeling against neurofilament associated protein (red), the glutamate vesicular transporter 1 (VGluT1, yellow) and the glutamate receptor subunit NR2b (blue) in whole-mount limb at stage 52. Scale bar = 50 µm.

Figure 5.

Figure 5—figure supplement 1. VGluT1 expression appendicular MNs.

Figure 5—figure supplement 1.

(A) Fluorescence immunolabeling against ChAT (red) and VGluT1 (yellow) in limb MNs retrogradely labeled with Alexa Fluor dextran 647 (AD 647, cyan) at stage 52. Whereas ChAT is absent from retrogradely labeled MNs, VGluT1 is expressed throughout almost the entire ventral horn, including in limb MN somata. Bottom images are magnifications of the area (a) delineated by the dotted line square in the merged image at top right. Punctiform VGluT1 expression occurred around MN somata (indicating labeling of glutamatergic presynaptic terminals) and a more diffuse VGluT1 signal also appeared in MN cytoplasm surrounding cell nuclei. Scale bar = 20 µm. (B) Fluorescence immunolabeling against synaptophysin (Synaptoph., green), NMDA glutamate receptor subunit 1 (NR1, blue), VGluT1 (red) and neurofilament (Neurofil., grey) in hindlimb muscle tissue at stage 52 (B1) and stage 55 (B2). VGluT1 expression colocalized with synaptophysin and postsynaptic glutamate receptors were detected at stage 52, but not at stage 54. The bottom images in B1 are magnifications of the area (a) indicated in the merged image at top right. Scale bars = 200 µm for stage 52 (B1) and 400 µm for stage 55 (B2).