Figure 1. Overall course of the behavioral experiments.
(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).