Conditioned taste aversion training

Rats were placed on water restriction with free access to food for a total of 8 days. Rats were habituated to a behavioral chamber where they had 15 min access to a drinking spout with H₂O, followed by 1 hr access to H₂O in their home cage four hours later. The volume consumed was recorded daily throughout training (total volume (ml): juveniles, 12.75 ± 0.70; adults, 16.96 ± 1.17), and rats’ weight was monitored to ensure that it remained within 85% of initial weight. Four days of habituation training was sufficient to stabilize fluid intake levels (Habituation, Figure 1B,C). This was followed by two conditioning trials, with a recovery day in between identical to the habituation days. The recovery day enabled us to confirm that the conditioning procedures did not affect thirst levels. For immunohistochemistry and slice electrophysiology, conditioning consisted of 15 min access to a drinking spout with 0.1M sucrose, followed by an ip injection of 0.15M LiCl (7.5 mL/kg) to induce gastric malaise. Rats in the control group received an ip LiCl injection (0.15M) in the evening on day 4 and 6 of training. The injection was delivered approximately 16 hr before sucrose presentation to ensure lack of association between gastric malaise and sucrose consumption (Figure 1B, pseudo CTA). For experiments in vivo, conditioning for rats in the GFP control group (Figure 6C) and paired 20 Hz opto group (Figure 6D) consisted of 15 min access to a drinking spout with 0.1M sucrose, followed by optogenetic 20 Hz stimulation of BLA terminal fields in GC. Rats in the non-paired 20 Hz opto group (Figure 6E) received 20 Hz stimulation of BLA terminal fields in GC the evening prior to sucrose exposure. Rats in the ramp opto group (Figure 6F) had sucrose exposure followed by optogenetic ramping stimulation of BLA terminal fields in GC. On the 8^th training day, all groups engaged in a 2-bottle test to assess a preference for H₂O or sucrose. In a subset of experiments, the 2-bottle test was repeated on day 9, to assess whether the first test had initiated an extinction process (Figure 1—figure supplement 2).