The primary dependent variable was reinforcement rate in stimulations per minute during each frequency trial. To normalize these data, raw reinforcement rates from each trial in each rat were converted to percent maximum control rate (%MCR), with MCR defined as the mean of the maximal rates observed during the second and third baseline components for that rat on that day. Thus, %MCR values for each trial were calculated as (reinforcement rate during a frequency trial) / (MCR) x 100. For each test session, data from the second and third baseline components were averaged to yield a baseline frequency-rate curve, and data from each pair of test components were averaged to generate test frequency-rate curves. Baseline and test curves were then averaged across rats to yield mean baseline and test curves for each manipulation. For statistical analyses, results were compared by repeated measures two-way ANOVA with ICSS frequency as one factor and either dose or time as the second factor. A significant ANOVA was followed by the Holm-Sidak post hoc test, and the criterion for significance was set at P < 0.05. Frequency-rate curves for vehicle (saline), the negative control, were compared to baseline frequency-rate curves, with the expectation of no significant difference. All drug treatments were compared to vehicle. Cocaine was expected to significantly facilitate ICSS responding compared to vehicle[23,24].

To provide a secondary summary measure of drug effects, the total number of stimulations delivered across all 10 frequency-trials of each component was determined for each drug dose at each time point. Mean number of stimulations per component for baseline and vehicle test components at all time points were compared to evaluate the negative control using a repeated measures ANOVA, with significance criterion set at P < 0.05. Summary test data from each day were then normalized to individual baseline data from that day using the equation % baseline total stimulations per component = (mean total stimulations per test component) / (mean total stimulations per baseline component) x 100. Data were then averaged across rats and plotted as a function of time for each drug dose. Results were compared using a repeated measures two-way ANOVA using time as one factor and drug dose as the second factor. A significant ANOVA was followed by the Holm-Sidak post hoc test, with the criterion for significance set at P < 0.05. Statistical analyses were performed using Prism 8.1.2 (GraphPad Software, San Diego, CA, USA).

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