Fig. 4

LA neurons activated by a new tone presentation are necessary for fear discrimination processes. a Experimental design for the Daun02 inactivation technique, see Methods for details. Daun02 (n = 9),Vehicle (n = 9), “Daun02-delayed” group (n = 4). b New tone presentation induced Fos (red nuclei) and β-galactosidase (green nuclei) expression in LA neurons. The merged panel shows nuclei that were double-labeled for β-galactosidase and Fos (yellow). Scale bar, 100 µm. c Photomicrographs of Fos-stained LA neurons in the Vehicle- (left) and Daun02-injected (right) rats at 90 min after 7 kHz sound presentation. Scale bar, 50 µm. d There were more Fos-positive cells in the Vehicle- than in the Daun02-injected animals. e Freezing response of the three groups (“Vehicle”, “Daun02-delayed”, and “Daun02”) during new tone (7 kHz) presentation was similar among groups. After Daun02 injection, freezing to the 7 kHz tone was higher than in the two control groups (3 × 2 mixed ANOVA, main effect of group: F_{(2, 19)} = 7.25, P < 0.01, main effect of trial: F_{(1, 19)} = 9.23, P < 0.01, group × trial interaction: F_{(2, 19)} = 20.07, P < 0.001, simple main effect of group in post-injection trial: F_{(2, 19)} = 24.13, P < 0.001). Freezing to the CS was similar between groups (one-way ANOVA: P > 0.05). f, g In open field (OF) and elevated plus maze (EPM) tests, there were no differences between Vehicle and Daun02 groups. h No differences were detected during extinction sessions (n = 6 rats in each group). i In another experimental group, Daun02 (n = 5 rats), Daun02-delayed (n = 5), or Vehicle (n = 6 rats) was injected after CS presentation. Three days later, both a new tone and the CS were presented. Freezing responses to CS in the Daun02-injected animals were decreased (3 × 2 mixed ANOVA, main effect of group: F_{(2, 13)} = 15.36, P < 0.001, main effect of trial: F_{(1, 13)} = 0.80, P > 0.05, group × trial interaction: F_{(2, 13)} = 14.11, P = 0.001, simple main effect of group in post-injection trial: F_{(2, 13)} = 20.57, P < 0.001), but there were no differences between groups during new tone presentation (one-way ANOVA: P > 0.05). j Daun02 administration following the presentation of a new 15 kHz tone did not modify freezing to either this tone or to CS (n = 9 rats in each group). **P < 0.01, ***P < 0.001. All data are mean and SEM. Unpaired t test (d, j); one-way ANOVA (e, i); 3 × 2 mixed ANOVA (e, i); 2 × 2 mixed ANOVA (f, g, j); 2 × 7 mixed ANOVA (h). See Supplementary Note 1 for a more detailed description of statistical results of this figure