Fig 2: Photomicrographs of whole-mount preparations of myenteric plexus (MP) of the ileum (a–f) and the colon (g–l) of normothermic (CTRL) (a–c; g–i) and hypothermic (synthetic torpor, ST) rats (d–f; j–l) showing AT8 (a, d, g, j) and neuronal nitric oxide synthase (nNOS) (b, e, h, k) immunolabeling. Open arrows indicate MP neurons expressing AT8 (a, d, g, j) immunoreactivity. Stars indicate nitrergic neurons, which were AT8 negative. White arrows (d–l) indicate some MP neurons co-expressing AT8 and nNOS immunoreactivity. Long white arrows (a–c) indicate nitrergic nerve fibers, which were AT8 negative. In (c, f, i) and ( l ) are the merge images. Bar: (a–l ) = 50 µm

Fig 3: The majority of nitrergic and serotonergic neurons are GFP+. ret:GFP is expressed in the majority of nitrergic (A–A”) and serotonergic (B–B”) neurons (white). Insets in (A”,B”) show close-up of the boxed area, GFP, nNOS or 5-HT, and overlay from left to right. Schematic of boxed area of larval schematic (C) indicates the four gut sub-regions analyzed (D). (E) Quantification of GFP and nNOS colocalization in four gut regions as shown in (D). Bar graph shows % GFP+/nNOS+ out of total nNOS+ cells (yellow), and % GFP-/nNOS+ out of total nNOS+ cells (magenta). Error bars show ±standard error of the mean (2 experiments, 23 larvae). (F) Quantification of GFP and 5-HT colocalization in four gut regions shown in D. Bar graph shows % GFP+/5-HT+ out of total 5-HT+ cells (yellow), and % GFP-/5-HT+ out of total 5-HT+ cells (magenta). Using 2-way ANOVA, we did not find significant differences between the cell population of the different gut regions. Error bars show ±standard error of the mean (2 experiments, 22 larvae). (A–A”,B–B”): maximum projections of dissected guts. Scale bar = 100 µm.