Fig. 3: Faithful activation of GFRAL neurons produces sickness-like behaviour.

a Schematic illustrating the Gfral^Cre cross with the loxSTOPlox-hM3Dq-mCherry reporter mouse. b Chemogenetic stimulation of Gfral^{Cre:hM3Dq-mCherry} offspring with CNO (3 mg kg^–1) induces FOS (green) in GFRAL^AP/NTS neurons (magenta) and also in unidentified neurons that lie downstream in the mNTS (asterisk). The percentage of activated GFRAL neurons in the dorsal vagal complex (DVC) is presented on the right (saline n = 3; CNO n = 4; ***p = 0.0002; two-tailed Student’s unpaired t-test). c CNO treatment of Gfral^{Cre:hM3Dq-mCherry} mice decreases cumulative night-time food intake and body weight (saline n = 6; CNO n = 4; **p = 0.0043, **p = 0.0018, ****p < 0.0001; two-way ANOVA followed by Sidak’s multiple comparisons test or two-tailed Student’s unpaired t-test). d Gastric emptying, as measured by the appearance of acetaminophen in the blood, is also reduced. Area under the curve (AUC) for plasma acetaminophen from 0 to 60 min after oral gavage (saline n = 4, CNO n = 6; *p = 0.0408; two-tailed Student’s unpaired t-test). e Activation of Gfral^{Cre:hM3Dq-mCherry} neurons causes aversion, as measured by a conditioned taste avoidance test (saline n = 9, CNO n = 10; ****p < 0.0001; two-way ANOVA followed by Sidak’s multiple comparisons test). f CNO treatment of Gfral^{Cre:hM3Dq-mCherry} mice induced a transient decrease in energy expenditure that lasted for about 2 h (n = 6 per group; *p = 0.0410), and (g) a decrease in RER that lasted for about 10 h (*p = 0.0257, **p = 0.0018, ***p = 0.0008; two-way ANOVA followed by Sidak’s multiple comparisons test). Indirect calorimetry was used to measure metabolic gases. h Top, body temperature measurement by thermal imaging in Gfral^{Cre:hM3Dq-mCherry} mice before and 2 h after saline or CNO (n = 9 per group ***p = 0.0002; two-tailed Student’s unpaired t-test). Thermal images of two Gfral^{Cre:hM3Dq-mCherry} mice before and after saline or CNO injection. arb.units (arbitrary units), AP (area postrema), CC (central canal), DMX (dorsal motor nucleus of the tenth cranial nerve, vagus), NTS (nucleus of the tractus solitarius). All data presented as mean ± SEM. Saline black squares and CNO as red squares throughout. Scale bars indicate 100 μm. Source data are provided as a Source Data file.