Statistics, Data Collection, and Analysis

Rats were randomly assigned to conditioning groups (paired, unpaired) following surgery. Behavioral data from optogenetic conditioning experiments was automatically recorded with Med-PC software (Med Associates) and analyzed using Prism 6.0. Video of conditioning sessions was recorded using Noldus Media Recorder 4.0, and automated behavior tracking data was generated using Noldus Ethovision XT and analyzed in MATLAB. For manual video scoring, experimenters were blind to the anatomical and conditioning group identify. Experimenters were otherwise not blinded. Non-parametric tests were used when data distributions were non-normal. Two-way repeated measures ANOVA was used to analyze changes in behavior among the groups across training. Bonferroni-corrected post hoc comparisons were made to compare groups on individual sessions. No statistical tests were used to predetermine sample sizes, but our sample sizes were similar to previously published studies. Rats were included in optogenetic behavioral analyses if optic fiber tips were no more than ~500 microns dorsal to the target region (VTA or SNc). Photometry data were collected with TDT OpenEx and Synapse software and analyzed using MATLAB. To assess the change in fluorescence across training days we fit a linear mixed-effect model for ΔF/F during each period of interest (0–1 s post-cue and laser omission period), with fixed effects for day and random effects for subject. To assess the relationship between the magnitude of cue-evoked fluorescence and CR latency, we fit a linear mixed-effect model for latency with fixed effects for cue-evoked fluorescence magnitude and random effects for subject. All comparisons were two tailed. Data in figures are expressed as the mean ± SEM. Statistical significance was set at p<0.05. Please see the Life Sciences Reporting Summary for additional information.