Figure 1
CS neurons made monosynaptic connections with forearm MNs in P14 animals. (a) Experimental design for retrograde monosynaptic tracing from forearm MNs. RV-ΔG-GFP and AAV6-RFP-f2A-G were co-injected into forearm muscles. Both viruses infect the MNs from the terminals and are retrogradely transported to the somata. RV-ΔG-GFP spreads into presynaptic cells (green) through complementation of G protein encoded by AAV. (b) Infection by RV-ΔG-GFP and AAV6-RFP-f2A-G of forearm MNs revealed by expression of GFP from RV-ΔG-GFP and RFP from AAV6-RFP-f2A-G. Cells in which both fluorescent proteins were co-expressed are starter neurons for monosynaptic tracing (arrows). GFP-labeled neurons in lamina V are shown inside the dotted line. b1, GFP; b2 RFP; b3, merged image (GFP, RFP) and b4, merged image (GFP, RFP, anti-choline acetyl transferase (ChAT) immunostaining). Animals were received intramuscular injection on P7 and sacrificed on P14. The images are representative of three independent experiments. (c) GFP-positive cells in layer V of the cerebral cortex. Superimposed view from 10 sections. Animals were received intramuscular injection of RV-ΔG-GFP and AAV6-RFP-f2A-G on P5 and sacrificed on P14. The image is representative of five independent experiments. (d1) Forearm MNs identified on P10 as cells retrogradely labeled with CTB-Alexa 488 injected intramuscularly on P7. (d2) A targeted CTB-Alexa488-positive cell into which rhodamine-dextran was perfused through a whole-cell pipette. (d3) Merged image of d1 and d2 certifying that whole cell recordings were made from a forearm MN. The images are representative of thirteen independent experiments. (e) EPSCs recorded from a forearm MN in the presence of high concentrations of divalent cations (7 mM Ca2+, 3 mM Mg2+) and showing a fixed latency in response to optogenetic stimulation (blue bar) of CS axons. Scale bars indicate 500 μm in (b), 1 mm in (c) and 20 μm in (d). The traces are representative of seven independent experiments.
