Subjects
Male and female mice of C57BL/6J background (Jackson Laboratories, strain 000664) were used in approximately equal numbers for all experiments. Wild-type mice were crossed with DAT:IRES–Cre mice (Jackson Laboratory, strain 006660) or ChAT:IRES–Cre mice (Jackson Laboratory, strain 006410). For slice electrophysiology experiments, ChAT:IRES–Cre mice were crossed with DAT–Flp mice (Jackson Laboratories, strain 035436). All transgenic animals used in experiments were genotyped and found to be heterozygous for Cre-recombinase or Flp-recombinase. Mice were separated by sex and group housed after weaning before surgical procedures or behavioural assays. All behavioural experiments were conducted during the dark cycle (12 h light–dark) when mice were 10–24 weeks old. Mice were housed at approximately 21 °C in 30–70% humidity, and they were food restricted to 85% of ad libitum body weight for all behavioural experiments to facilitate motivated behaviour. All procedures complied with the animal care standards set forth by the National Institutes of Health (NIH) and were approved by the Stanford University Administrative Panel on Laboratory Animal Care.
Behavioural training
Food restriction and behavioural training began at a minimum of 2 weeks after virus injection and fibre implantation. Before starting behavioural experiments, mice were habituated for 2 days, including handling, tethering to the patch cords and allowing exploration of the operant boxes (Med Associates) for 30 min. After habituation, mice were exposed to 1 day of a Pavlovian task in which rewards were delivered at random intervals spanning 25–35 s for 30 min. Operant chambers were equipped with a speaker for white noise and three identical nose-poke ports, each with an associated cue light and an infrared emitter sensor to measure port entry times. Rewards were delivered in conjunction with a 2-s cue comprising white noise and central port light so that mice would associate reward delivery with the light–sound cue. For sucrose rewards, 10 µl of 32% (w/v) sucrose was used, apart from one experiment in which 5% sucrose was used (Extended Data Fig. 9n–w). After 1 day of Pavlovian training, mice progressed to the operant task. Mice trained on FR1 for a minimum of five sessions. The active nose-poke port (left or right) was counterbalanced between mice; each mouse continued with the same active port for all experiments. On days 1–3, the active food port was baited with a crushed portion of fruit loop to encourage exploration of that port. After earning at least 10 rewards at FR1, with an accuracy (% active nose pokes) of more than 80%, mice progressed to FR5. Mice continued FR5 for at least five sessions and until the number of rewards earned per session remained within 20% for 3 consecutive days. Once this criterion was met, mice progressed to the sucrose effort task. We did not attempt to equalize reward consumption between mice, but rather aimed to ensure that all mice had accurate and internally consistent performance. Training for optogenetic self-stimulation was achieved in the same manner as for sucrose, except the sucrose reward was replaced with 5-s optogenetic stimulation paired with the 2-s light and white-noise cue. All behavioural tasks were coded in Med-PC V (Med Associates).
Sucrose effort task
The sucrose effort task comprised five 10-min blocks of FRs including FR46, FR21, FR10, FR5 and FR1. After mice poked in the active port for the required number of times, sucrose reward (10 µl 32% (w/v)) was delivered in a central magazine, accompanied by a 2-s light and white-noise cue. Once the cue stopped and mice entered the magazine to consume the reward, they were free to start the next trial at their own pace by poking in the active port. As in our previous work17, the FRs were presented in descending order to prevent mice from achieving early satiety under low-cost conditions, except for one control experiment with ascending presentation of blocks (Extended Data Fig. 9j–m). Block transitions were not signalled to the mice.
Modified sucrose effort task
For microinjection experiments with PBS or DHβE, due to concern that the behavioural effect of the drug was a result of the drug wearing off over the course of the session, we used a modified sucrose effort task. In the modified task, after PBS or DHβE injection, mice underwent a 30-min session in which the poking requirement was kept constant for the entire session at either FR1 or FR21. The order of the 4 experimental days (PBS versus DHβE; FR1 versus FR21) was counterbalanced between mice.
ChRmine effort task
The ChRmine effort task was structured identically to the sucrose effort task, except that mice worked for optogenetic DA stimulation. ChRmine stimulation was paired with the same 2-s light and white-noise cue in the magazine port, and upon cue cessation, the mice were free to start the next trial at their own pace.
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