Fig. 6: NI NMB neurons project to multiple brain areas.

a The presence of VGAT (slc32a1) mRNA (green) in mCherry-expressing NI NMB neurons (n = 937 VAGT⁺/1234 mCherry⁺ neurons; 937 mCherry⁺ /2089 VGAT⁺ neurons; 20 sections from 4 mice). Arrows, dual labeled cells; Arrowheads, mCherry + cells that clearly lacked VGAT mRNA expression. b Schematic shows that expressing synaptophysin-EGFP in NI NMB neurons labels terminals in several subcortical structures. c The normalized density of innervations (total EGFP⁺ pixels divided by the area of each nucleus; n = 4 mice). d Schematic diagram showing the method of optogenetic stimulation and recordings from the MS, the LH or the IPN in brain slices. e, f Terminal expression of synaptophysin-EGFP in the MS (e) and the effect of activating ChR2-expressing terminals on evoking IPSCs from MS neurons (f). The left panel in (f) shows representative traces of light-evoked IPSCs from an MS neuron before (red; latency, 4.8 ± 1.1 ms), during (black), and after (gray) Gabazine application. The right panel shows the group data on the effect of Gabazine on blocking the light-evoked IPSCs (n = 9 cells from 4 mice). Postsynaptic current was measured holding at −10 mV. g, h Terminal projection pattern of NI NMB neurons in the LH (g) and the effects of activating NI NMB neurons on evoking IPSCs from LH neurons (h; latency, 3.9 ± 1.8 ms; n = 5 cells in 5 mice). i, j Distribution of NI NMB axonal terminals in the IPN (i) and the effects of activating the terminals on the Gabazine-sensitive IPSCs of IPN neurons (j; latency, 6.1 ± 0.5 ms; n = 8 cells from 5 mice). Error bars (c, f, h, j) indicate SEM. Scale bars = 50 µm (a), 200 µm (e, g, i). Source data are provided as a Source Data file.