Fig. 6: Viral tracing of the CN-RtTg-MNs circuit.

a Schematic for the AAV2/1-Cre-based anterograde transsynaptic tracing from the RtTg to multiple spinal cord segments. b Representative images showing the tdT⁺ neuron labelling in transverse sections of spinal cervical and lumbar segments, and the co-labelling of tdT with ChAT (white arrowheads). Scale bar, 100 μm. c Schematic for rabies virus-based retrograde monosynaptic tracing from hindlimb muscles to the RtTg. d Representative images showing EGFP⁺mCherry⁺ spinal starter MNs (left) and mCherry⁺ neurons in the RtTg (right). Scale bar, 200 μm. e Left: schematic for the multilevel anterograde tracing from the CN to the spinal cord by injecting AAV2/1-Cre (mixed with CTB to visualize injection site) into the CN, followed by the injection of AAV-DIO-EGFP-T2A-Synaptophysin:mCherry into the contralateral RtTg. Right: a representative image showing CN injection site. Scale bar, 200 μm. f A representative image showing Cre-dependent expression of EGFP in RtTg neuron cytosol. Scale bar, 300 μm. g Representative images showing co-existence (white arrowheads) of the mCherry⁺ presynaptic puncta of RtTg neurons and the spinal cervical segment ChAT⁺ MNs. Scale bar, 200 μm (Cervical, Lumbar) and 5  μm (Merged).