Extended Data Figure 2. Comparative analysis of the Gpr83⁺, Tacr1⁺, and Tac1⁺ SPB populations.

a, Distribution of EYFP-expressing Tacr1⁺ (top) or Gpr83⁺ (bottom) spinal neurons and mCherry-expressing Gad2⁺ neurons in the superficial lamina of the spinal cord dorsal horn. b, Quantification of % of Gad2-negative neurons in EYFP⁺ neurons. 97.5 ± 1.4 % of Tacr1⁺ neurons and 99.5 ± 0.5 % of Gpr83⁺ neurons were Gad2-negative. c, Distribution of tdTomato-expressing Tacr1⁺ neurons and GFP-expressing Gpr83⁺ neurons in the spinal cord dorsal horn. d, Quantification of co-expression of tdTomato and GFP. 80.2 ± 1.5% and 87.0 ± 2.5% of tdTomato-expressing Tacr1⁺ neurons are not positive for GFP expression in lamina I+IIo and lamina IIid, respectively. Conversely, 78.0 ± 1.8% and 92.0 ± 1.4% of GFP-expressing Gpr83⁺ neurons are not positive for tdTomato expression in lamina I+IIo and lamina IIid, respectively. e, Distribution of EYFP-expressing Tacr1⁺ neurons, Gpr83⁺ neurons, or both in the superficial lamina of the spinal cord dorsal horn. The SPB neurons were retrogradely labeled with CTB injected into the PBN_L. Arrowheads, CTB and EYFP double-positive neurons. f, Quantification for % of Tacr1⁺ SPB neurons, Gpr83⁺ SPB neurons, and either Tacr1⁺ or Gpr83⁺ SPB neurons. g, % of Tacr1⁺, Gpr83⁺, Tacr1⁺ Gpr83⁺, and Tacr1⁻ Gpr83⁻ SPB neurons calculated from experiments in e, f. h, i, Coronal sections of the ventral brain stem of Tacr1^CreERT2 (f) or Gpr83^CreERT2 (h) mice whose lumbar spinal cords were injected with AAV1-FLEX-Synaptophysin-GFP viruses. MAO, medial accessory olivary nucleus; DAOdf, dorsal accessory olivary nucleus dorsal fold; DAOvf, dorsal accessory olivary nucleus ventral fold; PO, primary olivary nucleus. j, Distribution of tdTomato-expressing Tac1⁺ neurons in the superficial lamina of the spinal cord dorsal horn. The SPB neurons were retrogradely labeled with CTB injected into the PBN_L. Arrowhead, CTB and tdTomato double-positive neuron. k, Quantification of % of Tac1⁺ SPB neurons. l, Schematic of injections of AAV2-retro-FlpO viruses into the PBN_L. m, Distribution of tdTomato-expressing Tacr1⁺ (left) or Gpr83⁺ (right) SPB neurons and Tac1-expressing neurons in the spinal cord dorsal horn. tdTomato (red) and Tac1 (green) mRNA molecules were detected with gene-specific RNAscope probes. Filled arrowheads, double-positive neurons; empty arrowheads, tdTomato⁺ SPB neurons that do not express Tac1. n, Quantification of co-expression of tdTomato and Tac1 in lamina I+IIo. o, Schematic of lumbar injections of an AAV1-FLEX-Synaptophysin-tdTomato virus. p, Distribution of tdTomato-positive synaptic terminals of Tac1⁺ SPB neurons in the PBN_L. q, Quantification of distribution of tdTomato-positive synaptic terminals of Tac1⁺ SPB neurons in the PBN_L. n = number of mice (indicated in the graph). Error bars, s.e.m.