PMC

(A) Olfactory detection experiment. Animals were tested daily in the two-tube experimental device over 16 days (D1–D16). Habituation: On the first 3 days (D1–D3), rats were trained to drink pure water from both tubes (W/W) of the experimental cage. Aversion acquisition: On the following 3 days (D4–D6), rats had access to water odorized with isoamyl-acetate (ISO) diluted at 10⁻⁵ in both tubes (10⁻⁵/10⁻⁵). ISO 10⁻⁵ consumption >0.5 mL was paired with an intraperitoneal injection of LiCl (LiCl IP) to induce a conditioned olfactory aversion (COA) to ISO. Aversion test: On D7, the COA efficiency was tested by giving the animals a choice between water odorized with ISO 10⁻⁵ and pure water (10⁻⁵/W). On Habituation, Aversion acquisition and Aversion test days, animals were trained to receive a daily NaCl ICV injection (NaCl ICV). Olfactory detection test: During the Olfactory detection test period (D8–D11), rats were offered a choice between ISO 10⁻⁹ or ISO 10⁻⁸ and pure water (10⁻⁹/W, 10⁻⁸/W). For a given odorant dilution, the animals were tested on two consecutive days: once 1 h after NaCl ICV injection (D8 and D10) and once 1 h after a 14 mU insulin ICV injection (D9 and D11). Aversion re-test: On D12, the COA stability was assessed by giving the rats the choice between ISO 10⁻⁵ and pure water (10⁻⁵/W). Aversion retention: During the Aversion retention period (D15–D16), three days after the Aversion re-test, rats were offered again the choice between ISO 10⁻⁵ and pure water (10⁻⁵/W) and the animals were tested on two consecutive days: once 1 h after a 14 mU insulin ICV injection (D15) and once after a NaCl ICV injection (D16). (B) Sniffing experiment. Animals were tested daily in a whole-body plethysmograph over 7 days. The rats were first allowed to familiarized with the recording chamber for 4 days (Habituation), D1–D4 without (D1, D2) or with (D3, D4) food odor stimulation. During the sniffing test period (Food odor detection test, D5–D7), the animals were tested either in the fasted state (at 10:00 a.m.), 1 h after 14 mU insulin ICV injection (D5); in the fasted state, 1 h after NaCl ICV injection (D6); in the satiated state, 1 h after NaCl ICV injection (at 4:00 p.m.) (D7).

A) Video image of the place-preference setup. Mice move freely between two compartments divided by a plastic curtain, one containing an odor source (white dish). B) Aversion index reflects the difference in time spent in the odorized chamber in the presence of water (pre-trial) vs odor. Negative values indicate aversion. Control odors are water and the aversive odor 4-methylthiazole (4MT). C) Aversion index values for eA wt and ΔeA mice in response to 0.01% 5-methylhexanoic acid (MHX) and 0.01% 2-methylbutyric acid (MBA), both of which elicit significantly higher aversion in wt than in ΔeA. 1% MHX elicits aversion in both wt and ΔeA. Plots show mean ± SEM.

Conditioned taste aversion. Saccharin-flavored water was paired with an intraperitoneal injection (2% body weight) of PBS (Rin1^−/−, n= 15; WT, n = 17) or 0.3 m LiCl (Rin1^−/−, n = 14; WT, n = 15). Twenty-four hours later, mice were tested for saccharin aversion. Aversion indices were calculated as follows: (saccharin water consumed/(saccharin water consumed + unflavored water consumed)). Rin1^−/−mutants (F_(1,27) = 144.3;p < 0.05) and wild types (F_(1,30) = 35.6; p< 0.05) both acquired a significant aversion to saccharin when paired with 0.3 m LiCl.Rin1^−/− mutants, however, had a significantly higher aversion index than wild types (Rin1^−/−, 0.24 ± 0.03; WT, 0.39 ± 0.04; F_(1,27) = 8.9;p < 0.05). PBS-injected mutants and wild types exhibited equivalent preference for saccharin-flavored water (F_(1,30) = 0.36; p> 0.05). *p < 0.05, indicates significant difference between wild type and mutant under the same conditions.

The GHS-R1A antagonist (6 mg/kg) did not affect the preference for saccharin over water compared to vehicle treatment in the taste aversion experiment. LiCl, on the other hand, decreased the preference for saccharin over water compared to both JMV2959 and vehicle treatment (Square  =  vehicle; triangle  =  JMV2959; Circle  =  LiCl).