Experimental model and participant details
Mice
This study is based on both adult male and female mice (aged > P60). We used five mouse lines: C57BL/6 J (JAX# 000664), VGAT-ChR2-eYFP62 (JAX #14548), Drd1–cre FK150 (ref. 63), Adora2–cre KG126 (ref. 63) and R26-LNL-GtACR1-Fred-Kv2.1 (ref. 47) (JAX #33089). See Supplementary Table 1 for mice used in each experiment.
All procedures were in accordance with protocols approved by the MPFI IACUC committee. We followed the published water restriction protocol64. Mice were housed in a 12–12 reverse light–dark cycle and behaviourally tested during the dark phase. Ambient temperature was 74 °F and humidity ranged between 35% and 60%. A typical behavioural session lasts between 1 h and 2 h. Mice obtained all of their water in the behaviour apparatus (approximately 0.6 ml per day). Mice were implanted with a titanium headpost for head fixation64 and single housed. For cortical photoinhibition, mice were implanted with a clear skull cap37. For bilateral D1/D2-SPN silencing, tapered fibre optics65 (1.0-mm taper, NA 0.37 and core diameter of 200 µm, Doric lenses) were bilaterally implanted during the headpost surgery around the following target coordinates (Bregma): anteroposterior −0.3 mm, mediolateral ±3 mm and dorsoventral 3.5 mm for the VLS; and anteroposterior 0.6 mm, mediolateral ±1.5 mm and dorsoventral 3 mm for the dorsal medial striatum. Craniotomies for recording were made after behavioural training.
Viral injection
To virally express stGtACR1 (ref. 66) in the striatum, we followed published protocols67 for virus injection. AAV2/5 CamKII-stGtACR1-FusionRed (titre: 9.5 × 1012) was injected into anteroposterior −0.3 mm, mediolateral 3 mm, dorsoventral 2.75 and 3.5 mm, 100 nl each depth. The same tapered fibre optics described above were bilaterally implanted at dorsoventral 3.5 mm.
Behaviour
At the beginning of each trial, an auditory cue was presented, which consisted of three repeats of pure tones (3 kHz, 150-ms duration with 100-ms inter-tone intervals, 74 dB). A delay epoch started from the onset of the cue presentation. Licking during the delay epoch aborted the trial without a water reward, followed by a 1.5-s timeout epoch. Licking during the 10-s answer epoch following the delay was considered a ‘correct lick’, and a water reward (approximately 2 µl per drop) was delivered immediately, followed by a 1.5-s consumption epoch. If mice did not lick during the 10-s answer period, the trial would end without a reward. Trials were separated by an ITI randomly sampled from an exponential distribution with a mean of 3 s, with 1-s offset (with a maximum ITI of 7 s). This prevented mice from predicting the trial onset without a cue. Animals had to withhold licking during the full ITI epoch for the next trial to begin (otherwise, the ITI epoch repeated). In approximately 10% of randomly interleaved trials, the auditory cue was omitted to assess spontaneous lick rate (‘no-cue’ trials). No water reward was delivered in no-cue trials.
We followed the protocol described in Majumder et al.68 for training. In brief, the delay duration increased from 0.1 s to 1.8 s gradually based on the performance of the animal68. Once mice reached 1.8-s delay, we started either the switching delay, the random delay or the constant delay conditions (see Supplementary Fig. 3 for example sessions). In the switching delay condition, we switched the delay between 1 s versus 3 s or 1 versus 1.8 s every 30–70 trials (the number of trials was randomly selected from 30 to 70 and not contingent upon behaviour). Similarly, in the random delay condition, we randomly switched the delay among 0.5, 1.0, 1.5, 2.0, 3.0 or 5.0 s every 30–70 trials. For the constant delay condition, mice were trained with a constant delay of 1.5 s across sessions for at least 2 weeks. For the cue-intensity experiments (Extended Data Fig. 5), we changed the cue intensity (3-kHz auditory cue, ±15 dB, lasting 0.6 s) in randomly interleaved test trials (approximately 20%). Except for this modification, the task structure was identical. Cue intensity stayed constant (74 dB) before the cue-intensity experiments. Otherwise, the task design and reward contingency remained the same. ALM and striatal perturbation experiments (Figs. 4 and 5) were performed under the switching delay condition. To avoid human bias, the behaviour was automatically controlled by Bpod (Sanworks) and custom MATLAB codes.
Optogenetics
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