Discrimination Task

During our experiment, mice went through a series of four two-odor pair discriminations. These were presented as an initial acquisition that was directly followed by a stimulus reversal, where the S+ odor became the S- odor, and vice versa (Table 1). Odor discrimination trials proceeded as the previous training phase four, now including odor stimulus delivery. Individual sessions lasted 30 min, re-entry after a session was blocked for 1 h, and the ITI (inter-trial-interval) remained at the minimum of 1 s. Mice had to respond to the positive stimulus with a “go” response (eight licks within 2 s), a “hit.” Accordingly, they had to suppress their response to the negative odor stimulus with a no-go response (fewer than eight licks within 2 s), considered a “correct rejection.” This no-go response toward the S- odor stimulus was a novel behavioral requirement of the discrimination task that had not been taught previously. A go response to an S- odor (“false alarm”) was negatively reinforced by a 30-s timeout. Not responding led to the ITI, both for correctly rejecting an S- odor and for missing an S+ odor. All hits and correct rejections were counted as correct responses (Figure 3).

Experimental sequence in eight stages with odor pairs in an exemplary order.

The sequence of odor pairs and the initial S+ odor werepseudo-randomly assigned to the mice for counterbalancing. We firstcreated a 4 × 4 Latin square for all odor pairs across thenumber of discriminations and then replicated this Latin square withcontingencies reversed between S+ and S-. Therefore, if an animal had S+ anisole during the third initial acquisition, another animal had S+ eugenol during the third initial acquisition (from the anisole/eugenol pair). As we had 12 subjects, we needed two additional random sequences and their counter-balanced sequences. As one mouse that did not learn was excluded from the analysis, the data shown are for 11 mice. Both the initial acquisition and reversal stages ended when performance reached the criterion of 85% correct responses in 20 consecutive trials. The experimental switch to the next stage (reversal or next odor pair) occurred within ongoing sessions. We implemented this performance-based stage switching in the experimental control software so that it occurred automatically. Otherwise, as commonly done, a mouse could have advanced to the next experimental stage only on the next experimental day. This would have significantly extended the duration of the whole experiment. Also, maintaining training after the criterion is reached could lead to overtraining which may impact later training stages. The experiment ended for a mouse when it had completed all eight stages of odor discrimination learning and reversal. After finishing the experiment, the mouse stayed in the system and was re-started on its discrimination series until all the other mice had completed the experiment.