Fig. 3: The ARC^AgRP-dBNST^MC4R circuit mediates metabolism-associated anxiety and depression and feeding of HFD.

a Schematic image of retrograde labeling of AgRP^ARC→dBNST neurons by injecting AAV9-DIO-GTB and RV-EnvA-∆G-mCherry viruses into the dBNST of Mc4r^Cre mice. b, c Overlay of rabies-mCherry and anti-AgRP within the rostral (b) and caudal (c) ARC in mice as described in (a). Scale bar in (b), 50 µm; scale bar in (c), 50 µm. d Diagram showing ablation of the BNST-projecting AgRP neurons via bilateral injection of DT or vehicle into the dBNST of Npy^GFP/+::Agrp^DTR/+ mice. e, f Number (e) and ratio (f) of NPY neurons in mice as described in (e). g Representative images showing AgRP neurons (green) in mice as described in (e). Data represent the mean ± SEM, n = 6–8 per group; *p < 0.05; one-way ANOVA and followed by Tukey post hoc test. h Photostimulation of AgRP axonal terminals within the dBNST of Agrp^Cre::Ai32 mice. i, j Immunostaining of anti-GFP (green) within the ARC (i) and dBNST (J) of Agrp^Cre::Ai32 mice. Scale bar in (i), 400 μm; scale bar in (j), 150 μm. k 1-h food intake of Agrp^Cre::Ai32 mice upon photostimulation in the dBNST for 10 min at 20 Hz. l–q EPT (l–n) and OFT (o–q) test of Agrp^Cre::Ai32 mice upon photostimulation in the dBNST. Error bars represent mean ± SEM. n = 7 per group; *p < 0.05; two-way ANOVA and followed by Bonferroni comparisons test. r Viral transduction pattern of Mc4r^Cre mice with an injection of AAV9-FLEX-hM3Dq-mCherry into the dBNST. Scale bar in (r), 150 μm. s–y EPM (s–u), OFT (v–x), and MBT (y) by the mice as described in (r) upon CNO treatment. Error bars represent mean ± SEM. n = 8–10 per group; *p < 0.05; two-way ANOVA and followed by Bonferroni comparisons test.