Figure 15. Protocol for inducing CPP with sucrose.

The protocol for conditioning and preference testing consisted of four phases over the course of 11 days (sucrose habituation, a baseline place preference (BPP) test, sucrose conditioning, and a conditioned place preference (CPP) test). Food-motivated behavior was assessed during the dark cycle using an unbiased procedure. On the day before BPP, Day −1, sucrose habituation occurred where mice received sucrose pellets overnight (O/N) in their home cage to prevent neophobia. The initial BPP (black vs. white chamber) was assessed on Day 1 in a three-chamber place preference apparatus and the chamber pairing was assigned in an unbiased manner. During sucrose conditioning, mice were given access to sucrose-filled (CS+, Days 2, 4, 6, 8) or empty (CS-, Days 3, 5, 7, 9) lids on alternating days. Mice were given access to sucrose-filled lids in one chamber (e.g. white), then on alternating days they were presented with empty lids in the other chamber (e.g. black). Mice were tested for acquisition of a CPP to sucrose on Day 10, which was indicated by increased time spent in the sucrose-conditioned chamber. Animals were fed ad lib standard low-fat chow in their home cage throughout the study. For cyclical estradiol treatment, animals were given a priming (0.25 μg) and a surge (1.5 μg) dose of 17β-estradiol Benzoate (E2) at 9 AM prior to the BPP and prior to the CPP as indicated. During the sucrose-conditioning (phase 3), the animals were treated twice with a 1 μg maintenance dose of E2.