Supplementary Figure 3: CNO administration has no effect on responses to an appetitive odor in naive and P42-Gi mice trained with the odor.

(a) Flowchart illustrating strategy to test the effects of CNO administration on responses to a novel appetitive odor (Treat A, peanut butter cookie) in P70 naïve mice (not injected with AAV2-Gi at P42). (b) Proportions of successful and failed trials in P70 Nestin-cre mice administered vehicle or CNO over seven days (Fisher’s Exact Test; Number of successful trials, vehicle, 35/42; CNO, 37/42; P=0.76; n=6 animals/group). (c) Temporal plot of responses by all vehicle and CNO treated naïve mice within the 10 minute test period (left y-axis) over the seven days (x-axis). FT, failed trial. Dotted lines represent trendlines calculated from proportions of failed trials (right y-axis) over seven days (shaded regions represent 95% confidence intervals, CI). Significance was tested by Hypothesis test of Linear Regression [Slope (vehicle): -7.14% ± 2.72%, CI: -12.64, -1.64; slope (CNO): -6.55% ± 2.34%/day, CI: -11.28, -1.82; H₀: slope (vehicle) = slope (CNO); F(1,80)=0.027, P=0.87]. Fisher’s Exact Test was used to compare proportions of successful trials each day (days 1 to 7 in order: P=1.0, 0.55, 1.0, 1.0, 1.0, 1.0, 1.0). (d) Flowchart illustrating approach to test the effects of training P42-Gi mice with Treats A and B over seven days prior to CNO testing over a second seven day period. Control mice received vehicle injections in place of CNO. (e) Proportions of successful and failed trials in vehicle and CNO-administered P42-Gi mice during the second seven day test period following seven training days with Treats A and B during which CNO was withheld. There were no failed trials recorded in trained animals; n=6 animals/group.