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slc18a3xenopus neuron 

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Experiment details for slc18a3

Functional limb muscle innervation prior to cholinergic transmitter specification during early metamorphosis in Xenopus.

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Gene Clone Species Stages Anatomy
slc18a3.L laevis NF stage 51 to NF stage 65 neuron , motor neuron

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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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Gene Clone Species Stages Anatomy
slc18a3.L laevis NF stage 54 to NF stage 65 neuron , motor neuron

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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.
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