Supplemental Materials
Subjects
Mice expressing the human D2 receptor under control of the tet-operator (tet-O) were generated on a C57BL/6J: CBA F2 background. tet-O mice were backcrossed for eight generations to the C57BL/6J background and then crossed for behavioral studies with mice expressing the tetracycline transactivator (tTA) transgene under the calcium/calmodulin-dependent kinase IIα (CaMKIIα) promoter (Mayford et al., 1996). CaMKII–tTA mice were on 129SveV(Tac)N17 background. The F1 offspring from an intercross between these 2 strains were used for all experiments. To be consistent with our previous behavioral experiments, female mice were used in all experiments. Littermate mice carrying only one of the transgenes, or neither transgene, were combined and used as controls. Mice were genotyped by triplex polymerase chain reaction (PCR) using primers specific for tTA, tet-O and a fragment of the endogenous D1 receptor gene (to provide a positive control for the PCR). To regulate tet-O-driven gene expression, mice were fed doxycycline-supplemented home-cage chow (40 mg/kg; Bioserv, Frenchtown, NJ) beginning at 12 weeks of age. Behavioral experiments were commenced after a minimum of 2 weeks of feeding with doxycycline chow. To motivate mice to earn rewards in the operant tasks, food was restricted to 1 h daily access in the home cage after testing (regular maintenance mouse chow or the same chow supplemented with doxycycline) except where noted. Water was available ad libitum except where noted. Mice were weighed daily. This food restriction protocol maintained mice at approximately 80-85% of their free-feeding weights during the experiments. We have previously shown that D2R-OE mice do not differ from littermate controls in any aspect of a physical and neurological test battery, and there is also no difference in ad libitum weight or home-cage food consumption between control and D2R-OE mice (Simpson et al., 2011). Mice were housed, bred and tested according to the local IACUC guidelines. They were maintained on a 12 hour light/dark cycle and tested during the light phase.
Apparatus
Gustometer
We used a commercially available multi-channel gustometer (Davis MS160-Mouse; DiLog Instruments, Tallahassee, FL) to record the licking responses of the mice. The gustometer consisted of a testing chamber (14.5 cm wide, 30 cm deep, 15 cm tall), a taste stimulus delivery system and a dedicated computer (with software that controlled the presentation of taste stimuli and recorded the precise timing of each lick; for details, see Glendinning et al 2002). All licks to a sipper tube were recorded by a high-frequency AC contact circuit designed to eliminate ‘electric taste,’ a phenomenon that can occur in DC contact lick detection systems if the current is not sufficiently low. The short response latency of the detection circuit (<1 ms) provided a precise measure of the onset and offset of licks.
Operant chambers
The operant chambers (Med-Associates, St. Albans, VT; model ENV-307w) had internal dimensions 22½ x 18½ x 12½ and were located in a light- and sound- attenuating cabinet equipped with an exhaust fan, which provided 72 dB background white noise. Each chamber was equipped with a feeder trough that was centered on one wall of the chamber. A reward of one drop of evaporated milk could be provided by raising a dipper. An infrared photocell detector was used to record head entries into the trough. A retractable lever was mounted on the same wall as the feeder trough. The chambers were illuminated throughout all sessions with a houselight (Med Associates #1820) located at the top of the chamber. An audio speaker was positioned 8.5 cm from the floor on the wall opposite the feeder trough. The speaker delivered a brief tone (90 db, 2500 Hz, 200 ms) to signal when the liquid dipper was raised.
Gustometer training.
Each mouse was subjected to three days of training. This served to familiarize the mouse with the gustometer and train it to lick from the sipper tube to obtain fluid. The mouse was water-deprived for 22.5 hr prior to each 30-min training session to motivate licking. Each training session began when the shutter opened and the mouse took its first lick. On training day 1, the shutter remained open throughout the test session, permitting the mouse to drink freely from a single stationary spout that dispensed water. Immediately after this training session, the mouse was given 1 hr of ad libitum access to water. Then, it was water-deprived for another 22.5 hr. On training day 2, the mouse was provided with more limited access to two sipper tubes, each of which dispensed water. In this case, once the shutter opened, the mouse initiated each 5-s trial by taking a lick from the sipper tube. At the end of the trial, the shutter closed for 7.5 s (during which time the second sipper tube was positioned in the center of the slot) and then reopened, enabling the mouse to initiate another trial of the same duration. In this manner, the mouse could initiate up to 144 trials across the 30-min test session. On training day 3, the same procedure was repeated. All mice adapted readily to the gustometer across the three training days, as indicated by the fact that they made more than 250 licks per training session. Once training was complete, each mouse was given food and water ad libitum for at least 24 hr.
Operant procedures
Dipper training. All mice were first trained to consume the liquid reward from the dipper located inside the feeder trough. Mice were placed inside the chambers with the dipper in the raised position, providing access to a drop of evaporated milk. The dipper was retracted 10 s after the first head entry into the feeder trough. A variable intertrial-interval (ITI) ensued, followed by a new trial identical to the first. The session ended after 30 min or 20 dipper presentations. On the following day, mice received another session similar to the first, except that the dipper retraction was response-independent. On each trial the dipper was raised for 8 s and then lowered whether or not mice had made a head entry. Sessions like this continued until a mouse made head entries during at least 20 of 30 dipper presentations in one session. There was no significant difference between groups in the number of sessions needed to reach this criterion (overall mean = 2.93). In this and all other segments of the experiment, sessions occurred once per day, 5 days per week.
Lever press training. Mice were required to press a lever to earn the liquid reward. The first phase consisted of three sessions, each containing 60 trials. In each trial the lever was presented after a variable ITI averaging 30 s. A lever press during the first 6 s of lever presentation resulted in immediate reward. After the lever had been extended for 6 s without a press, the dipper was raised for 8 s. In both cases, the lever was retracted at the moment the dipper was raised. Next, mice received 3 sessions in which lever presses were reinforced on a continuous reinforcement (CRF) schedule. The session began with the lever extended. The lever was retracted after every two reinforcements and then re-extended after a variable ITI (average 30 seconds). The session ended when the mouse earned 60 reinforcements, or one hour elapsed. Mice continued receiving sessions like this until they earned 40 rewards in one session. All mice reached this criterion in 2-3 sessions. Mice then moved to one, 120-minute session of variable ratio (VR) training, during which the lever was extended the whole time. In this session, after running on a VR-2 schedule for 20 reinforcements, the training program switched to a VR-5 schedule (range 1-20) for 30 reinforcements. Prior to the beginning of the current experiment, mice participated in a different experiment involving exposure to progressive-ratio schedules (Simpson et al., 2011). Past experimental history of all subjects was equivalent prior to the beginning of the current experiment.
Table S1.
Mean (standard error in parentheses) number of licks per trial as a function of increasing sucrose concentration for control and D2R-OE mice during the gustometer test.
Table S2.
Mean (standard error in parentheses) response and reward data obtained from each pair of concurrent schedules for control and D2R-OE mice during the concurrent schedules experiment.