Fig. 4. Selective genetic ablation of Insula^Ins neurons disrupts social preference only after 24 h of social isolation.

a, b Experimental design for the viral injection (a) and behavioral assay (b). c Example of a histological control of caspase lesion in the insula identified by NeuN immunofluorescence labeling (green labeling) taken at epifluorescence microscope at low (left, scale bar: 500 µm) and high magnification (right, scale bar: 100 µm). d, e Quantification of NeuN fluorescence density in the control side (contralateral side to the injected lesion side, N = 9 mice) compared to the caspase injection side (N = 9 mice) in the Insula cortex (d, two-tailed Paired-t-test, t(8) = 2.788, p = 0.0236) and in the Somatosensory cortex (e). f, l Quantification of the time spent around the object and social enclosures in the three-chamber test in group-housed condition (f) or after 24 h of social isolation (l, two-way repeated measure Anova, Zone x virus interaction effect, F(1,24) = 8.911, p = 0.0064; zone main effect F(1,24) = 12.94, p = 0.0014; no virus main effect F(1,24) = 1.685, p > 0.05; Bonferroni post hoc test ctrl social vs caspase social p = 0.0059; ctrl social vs control object: p = 0.0001) in Insula control and Insula caspase groups. g, m Social preference ratio in the control group and caspase group in group-housed mice (g, one sample Wilcoxon test for ctrl: W = 105, p = 0.0001, and caspase: W = 76, p = 0.001) or isolated mice (m, two-tailed unpaired t-test, t(24) = 2.806, p = 0.0098; one sample t-test for ctrl: t(13) = 5.211, p = 0.0002; for caspase: t(11) = 0.7564, p > 0.05). h, n Time spent in the social zone between control and caspase mice in group-housed (h) or 24 hours-isolated conditions (n, two-tailed Unpaired t-test, t(24) = 2.792, p = 0.0101), i, o Mean social bout duration in control and caspase group in group-housed (i) and 24 hours-isolated housing condition (o). j, k, p, q Time spent in grooming and rearing in group-housed (j, k) or after 24 h of social isolation (p, q). ctrl control, S time in the social zone, O time in the object zone, contra contralateral, ipsi ipsilateral. Data are presented as mean values ± SEM. ### is used to mention a difference with a social preference ratio equal to 0.5. *p < 0.05, **p < 0.01,***p < 0.001.