To exclude the possibility that the lack of representation separation shown in
Fig. 4l–o was due to the presence of the experimenter’s hand during access-restricted trials, we repeated this experiment with the intruder mouse inside a wire mesh container. a, Diagram of experimental setup. b, Percentage of time the imaged mouse spent interacting with the intruder on each of the three days ( n = 2 mice). Aside from day 1, the barrier to free interactions presented by the container did not reduce the time the resident spent investigating the intruder. c, PCC between male and female representations on the third day of the assay (blue bars) showed that the separation of representations did not occur in these two imaged mice. Gray bars show the average PCC between pairs of male trials or pairs of female trials, for comparison. d, Following the three days of interactions with the intruder behind the barrier, mice were given two additional days of free social interaction, before a final day (day 6) in which intruders were again presented inside the mesh container. A third, experienced animal (mouse 18) was also tested with the mesh container. e, PCC between representations of males and females presented within the mesh container on day 6; grey bars show average PCC between pairs of male trials or pairs of female trials. Two out of three tested mice showed clear separation of male and female representations; the third (mouse 17) did not, but failed to fight or mate with conspecifics during the free social interactions on the two days preceding the day 6 test. f, Performance of an SVM decoder trained to predict intruder sex from the data on day 6, showing high accuracy in the two mice that had previously fought and mounted. These data provide additional evidence that olfactory cues from conspecifics are not sufficient to produce ensemble separation, and that behaviors occurring during free social interactions are required.