2.4. Two Bottle Choice Procedure

At the start of the two-bottle choice (TBC) procedure, mice were single-housed and provided two bottles, one “test” and one “control”. Each bottle contained 15–20 ml of drinking liquid that was replenished daily. At least 50 g of standard rodent chow was provided daily, and consumption of liquid from each bottle and food intake was assessed each day between 10:00 am and 12:00 pm. The initial left/right positions of the test and control bottles were distributed evenly among subjects for each experiment, and the position of the test and control bottles was alternated daily to control for any potential positional preference. Initially, both bottles were filled with deionized water, and the mice were acclimated to this set-up for 4–7 days. After acclimation, a vehicle drinking solution containing 1% DMSO (v/v), 0.2% saccharin (w/v), and 0.5% Captisol (w/v) was provided in both test and control bottles for 4 days. The exact dosing paradigms for the different experimental protocols can be found in each figure. Pilot experiments demonstrated 1% DMSO did not significantly change the total liquid intake, and the 0.2% saccharin concentration was selected because previous studies have demonstrated this is a maximally preferred concentration of saccharin for C57BL6 mice. Whenever the contents of the test or control bottles were changed, bottles were flushed with dH₂O at least 3 times to eliminate the previous contents.