2.5. Discrete-trials timing task

Training and testing in the discrete-trials task occurred in 2-lever operant chambers (21.6×17.8×12.7 cm, Med Associates Inc., St. Albans, VT). One wall in each chamber contains a food-delivery magazine (Lafayette Instruments, Lafayette, IN) and 2 motor-driven retractable levers 2.2 cm above the grid floor and spaced 10.4 cm apart, with an incandescent house-light located near the ceiling. Two white light-emitting diodes (LEDs), mounted horizontally near the top of the magazine, were used to signal reward availability, and a green LED was mounted horizontally above each lever. Liquid reinforcement (Strawberry Nesquik® plus non-fat milk, 20 μL) was delivered by a peristaltic pump (Lafayette Instruments, Lafayette, IN) to a well located in the floor of the magazine. Magazine entries were monitored using an infrared beam mounted horizontally, 5 mm above the floor and recessed 6 mm into the magazine. The chamber was located in a sound-attenuating box, ventilated by a fan that also provided a low level of background noise. The control of stimuli and recording of responses were managed by a SmartCtrl Package 8-In/16-Out with additional interfacing by MED-PC for Windows (Med Associates Inc., St. Albans, VT) using custom programming.

Mice were maintained at 85% of their free-feeding weight and trained 5 days a week, at the same time each day, during the dark (awake) phase of their light-dark cycle. Mice were first trained using a session in which reinforcement was dispensed every 15 s into the magazine, which was simultaneously illuminated. Magazine entries resulted in the light being extinguished until the next reinforcement is delivered. This training was repeated daily (Monday–Friday) until there were ≥30 magazine entries in 10 min for 2 consecutive days. In the second session, mice were trained to respond on either lever in order to obtain reinforcement under an FR1 schedule. This session was repeated daily (Monday–Friday) until all mice made >70 lever presses within a 30 min session on 2 consecutive days.

The mice were subsequently trained daily (Monday–Friday) in the discrete-trials task, with each training session lasting 30 min or 120 trials (whichever was completed first). At the beginning of each session, the house light was extinguished and the magazine light and the green LEDs above the levers were illuminated; trials were initiated when the mouse entered and then exited the magazine. At the beginning of each trial, the magazine light was extinguished, and then, following a variable duration (2.5, 5.0, 8.0, or 10.5 s, in pseudorandom order), the levers were presented for 10 s. Responding on lever A was reinforced if the interval was <6.5 s, and responding on lever B was reinforced if the interval was >6.5 s. The position of the two levers (left vs. right) was counterbalanced across subjects. An incorrect response or omission (failure to respond within 10 s) resulted in a time-out “punishment” where the levers were retracted, the house light was illuminated, and no aperture was responsive for 4 s. The next trial was initiated when the mouse entered and then exited the magazine. One-fifth of the trials were forced choice trials where only one lever was extended and was not retracted until a response was made. Training occurred until all mice attained a %correct [correct/(correct+incorrect)*100] score of >85% for the 2.5 and 10.5 s trials. Correct, incorrect, omissions, and reward latencies were also recorded. Test sessions were identical to the training sessions except that more durations (2.5, 3.4, 4.3, 5.2, 6.1, 6.9, 7.8, 8.7, 9.6, and 10.5 s) were used, there were no forced choice trials, and the sessions lasted 45 min or 200 trials.

There was a minimum of 7 days between experimental test sessions; group assignments were randomized. DOI and 25CN-NBOH were administered 10 min and 5 min prior to the start of the test session, respectively. For dose-response experiments, M100907 and SB-242,084 were administered 10 min prior to the start of the test session. For blockade experiments, M100907 and SB-242,084 were administered 30 min prior to the start of the test session.

The proportion of responses made on lever B (%B responding) was analyzed by two- or three-way ANOVA with pretreatment and/or treatment as between-subject factors and stimulus duration as a repeated measure. Post-hoc analyses were carried out using Dunnett’s test or Tukey’s test. The alpha level was set at 0.05.

A 2-parameter logistic function was used to fit the data: %B = 1/(1 + exp^{−ε(t−T₅₀)}), where ε is the slope of the function and t is the stimulus duration. The difference limen was calculated as (T₇₅ − T₂₅)/2, and the Weber Fraction was calculated as: (T₇₅ − T₂₅)/(2 × T₅₀). The Weber fraction, difference limen, slope (ε), T₅₀, goodness of fit (r²), and the number of trials completed were analyzed by one- or two-way ANOVA with pretreatment and/or treatment as between-subject factors. Post-hoc analyses were carried out using Dunnett’s test or Tukey’s test. Subjects were excluded from analysis if they failed to complete at least 100 trials during an experiment or if the logistic function failed to fit their %B responding data.