Behaviour

Behavioural experiments started seven days after surgery. The water restriction regime and the behavioural apparatus were previously described⁵¹. In short, mice were restricted to 85% of their initial weight on the starting date by individually adjusting the daily water ration. First, mice were accustomed to the experimenter and head fixation in the setup by daily handling sessions lasting 10 min. During these sessions, the water ration was offered in a handheld syringe. The remainder was supplemented in an individual drinking cage after a delay of approximately 30 min. After four to seven days of handling, mice were pre-trained to lick for reward, while being head-fixed on the spherical treadmill^52–54 in absence of visual stimulation. Whenever a mouse ceased to run (velocity below 1 cm s⁻¹) and made a lick on the spout, a water reward (drop size 8 μl) was delivered via the spout. A baseline imaging time point (T1) was acquired once the mice consumed more than 50 drops per session (35 to 45 min) on two consecutive days (requiring about three days of pre-training).

Subsequently, daily sessions of visual discrimination training for two initial stimuli started. Each mouse was randomly assigned to one of two groups. One group was first trained on the orientation rule, then on the spatial frequency rule. For the other group, the sequence of the rules was reversed (Extended Data Fig. ​Fig.1).1). Each rule defined a Go category and a NoGo category, separated by a boundary at either 45° (orientation rule) or at 0.043 cyc/° (spatial frequency rule). Trials started with an inter-trial interval of 5 s. After that, the mouse could initiate stimulus presentation by halting and refraining from licking for a minimum of 0.5 s. A single stimulus was subsequently shown for 1.3 ± 0.2 s. At any time during stimulus presentation, the mouse could make a lick to indicate a Go choice. Trials with a Go choice in response to a Go category stimulus triggered a water reward and were classified as hits; trials in which the mice failed to lick during Go category stimulus presentation were considered misses. Correct withholding of a lick to a NoGo category stimulus was classified as a correct rejection, and did not result in a water reward. A lick during a NoGo category stimulus counted as a false alarm. Initially, false alarms only led to the termination of the current trial; later during training, false alarms were followed by a time-out of 5–7 s showing a time-out stimulus (a narrow, horizontal, black bar). Time-outs were included to reduce a Go bias that mice typically showed. The second imaging session (T2) was carried out after a mouse performed at more than 66% correct Go choices in a given session (requiring 11 to 40 sessions).

For the next training stage (leading up to imaging session T3) further stimuli were added (Extended Data Fig. ​Fig.1a),1a), such that both the Go category and the NoGo category consisted of three stimuli differing in the feature either irrelevant to the category rule (T3a, n = 6 mice), or relevant to the category rule (T3b, n = 5 mice). Whenever a mouse’s performance exceeded 66% correct Go choices in one session, we proceeded to the next training (and imaging) stage; 6 stimuli per category, 9 stimuli per category (imaging session T4), and finally 18 stimuli per category (imaging session T5), the latter serving as a crucial test for generalization behaviour.

Rule-switch: After successful learning of rule 1, mice (n = 11) were retrained using the previously irrelevant dimension. This stage, known as rule-switch training, started with two exemplar stimuli for the new rule, and then proceeded with the same steps as for rule 1 and ended with another generalization test of rule 2 (18 stimuli per category, imaging session T8).

Task change: After successful learning of rule 1 (T5), the categorization performance of mice (n = 9) was tested with a different operant response, in a left/right choice task. For this session, the behavioural setup was slightly modified to create a left/right choice task. Instead of one lick spout centred in front of the mouse, the mouse was now presented with two lick-spouts, one offset to the left and one offset to the right. Stimuli of the previous Go category were assigned a new GoRight response (rewarded after a lick on the right lick spout). Stimuli of the previous NoGo category were assigned a new GoLeft response (rewarded after a lick on the left lick spout). The original stimulus to category assignment—that is, the categorization rule—remained the same throughout the task change. Before the first stimulus presentation, ten drops were manually given on each lick spout to motivate the mice to lick on both sides.

Throughout training, stimuli from the Go category and the NoGo category were presented in a pseudorandomized fashion, showing not more than three stimuli of the same category in a row. The behavioural training program was a custom written MATLAB routine (Mathworks).