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Figure 4 supplement 1. Appendicular MNs are activated centrally by cholinergic inputs.
A. Immuno-labeling of putative cholinergic synapses on limb MNs retrogradely labeled with
Alexa Fluor dextran 647 (AD 647), using antibodies against VAChT and the synaptic marker
synapsin [mouse anti-synapsin (1:200; Synaptic Systems, Germany) revealed by secondary
donkey anti-mouse antibodies coupled to Alexa Fluor 488 (1:500; Thermo Fischer)]. Both
VAChT and synapsin fluorescent signals were found in close apposition surrounding MN cell
bodies, indicating the presence of cholinergic input synapses. Inset: enlargement of synapsin
and VAChT signals in the vicinity of an appendicular MN cell body. Scale bar = 20μm B.
Calcium imaging optical recordings (20 fps) from stage 52 limb MNs retrogradely filled with
Calcium Green Dextran Amine 3kD (CGDA, Invitrogen; see schematic at left) during fictive
swimming (n=3). Right panel: individual calcium signals (δF/F) recorded simultaneously
from 10 MN somata (encircled in x40 image at left) during a swim episode recorded from an
axial VR (lower trace). C. Left panel: average fluorescence variation (black trace; SEM,
shaded area; top) for the 10 MNs illustrated in B during fictive swimming (bottom) in control
(black) and in the presence of 50μM d-tubocurarine (d-tubo.; red). Right panel: mean calcium
transient amplitudes in control (black) and under d-tubocurarine (red) expressed as a
percentage of the maximal control amplitude (i.e.,δF/F peak soon after swim episode onset)
for three stage 52 preparations. Unfilled circles represent single acquisitions; filled circles
denote grand means SEM. Long term calcium recordings were performed in control
experiments without drug application to avoid any fluorescence bleaching effect. |
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Figure 2. Developmental emergence of the molecular ACh phenotype in appendicular
MNs. A, B. Examples of fluorescence immunolabeling against ChAT (magenta) and VAChT
(red) in appendicular MNs (Ap. MN) labeled with retrograde Alexa Fluor dextran 647 (AD
647, cyan) in stages 53 (A) and 57 (B) tadpoles. Insets (b) in B show magnification of stage
57 appendicular MNs. C. Variation of fluorescence (δF/F) of axial (left plot) and
appendicular (right plot) MNs for ChAT and VAChT immuno-signals at stages 49-51 (n=4),
stages 52-54 (n=7) and stage 55-57 (n=8). Grey dots represent the averaged δF/F values for
ChAT (squares) and VAChT (circles) in each preparation, and the black dots are δF/F grand
means ± SEM for all preparations in a given developmental group. ns non significant, ***
p<0.001, Kruskall-Wallis test. D. Examples of in situ hybridization (ISH) labeling for ChAT
mRNA in the appendicular spinal column at stages 51 and 55. E. Example of fluorescence
immunolabeling against ChAT in stage 62 appendicular MNs that were previously labeled
with AD 647 injected into the hindlimb at stage 51 (see schematic at left ). All scale bars =
50μm; D, dorsal; L, lateral. |
