Extended Data Fig. 3: Characterization of OPRM1⁺ RVM^SC neurons.

a, Schematic showing spinal injection of AAV8-retro-FLEX(LoxP)-Flp at P1.5 in Oprm1^Cre mice, then four weeks later, RVM injection of AAV8-FLEX(FRT)-Clover3. b, Representative images from three independently performed experiments show the terminals of descending OPRM1⁺ RVM^SC neurons in the spinal cord. Notably, axons of OPRM1⁺ RVM^SC neurons are clustered in lateral funiculus (dash line) before gets into the spinal cord, and its terminals are enriched in the dorsal horn but not in the motor area in the spinal cord. Scale bar: 200 μm. c, Representative TESOS images and single-cell tracing of OPRM1⁺ RVM^SC neurons from three independently performed experiments. Axons of OPRM1⁺ RVM^SC neurons of both red and pink neurons are travelled along the left lateral funiculus, then enter the spinal cord to innervate both sides of the dorsal horn. Scale bar: 500 μm. d, Quantification of molecular markers expressed in the OPRM1⁺ RVM^SC neurons. Related to Fig. 1e. e, Representative images from three independently performed experiments of retrogradely labelled spinal cord projecting Tph2⁺ neurons in the RVM (left) and median raphe (MR, right) after spinal injection of AAV8-retro-mCherry. More Tph2⁺ neurons were labelled in MR than in RVM. mCherry labelled neurons are in red, Tph2⁺ neurons are visualized by immunostaining in green. Scale bar: 500 μm. f, Representative image from three independently performed experiments shows OPRM1⁺ LC^SC neurons labelled by intraspinal injection of AAV8-retro-FLEX(LoxP)-Rpl22-3XHA. RNAscope probes were used to visualize Oprm1 (red), HA tag (green) was visualized by immunostaining. Scale bar: 100 μm.

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