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. 2018 Mar 23;9:1214. doi: 10.1038/s41467-018-03682-2

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — LA activity following the presentation of novel and threatening cues. a Experimental design of behavioral and catFISH experiments, see Methods. Naive n = 9, 15 kHz, n = 5; 7 kHz, n = 9; 3 kHz, n = 5; and 1 kHz, n = 5. CS conditioned stimulus. b The percentage of freezing following the new tone presentation progressively increased in similarity to the CS (F_{(4, 28)} = 36.32, P < 0.001). The freezing of naive and 15 kHz animals was similar (P > 0.05) and, in the meantime, it was lower than freezing of other groups (P < 0.01). Freezing of 7 kHz animals was different also from 3 kHz and 1 kHz (CS) group (P < 0.001). c Freezing in “discriminator” animals (D, n = 5) was lower than that observed in “generalizer” (G, n = 4) animals during the 7 kHz tone delivery (P < 0.001). d Time course of catFISH experiments. e Representative images showing neurons expressing single nuclear H1a (green arrows) and Arc (red) mRNA expression and double-labeled cells (yellow) in the naive, 15 kHz, 7 kHz (discriminators and generalizers), 3 kHz, and 1 kHz groups. Scale bar, 20 µm. f Dot plots showing the percentage of cells activated following new tone presentation (expressing only H1a), CS presentation (only Arc), and during both events (expressing both Arc and H1a). These results revealed an increase in H1a (new tone)- or Arc (CS)-expressing neurons and a decrease in double-labeled cells in the “discriminator” group (F_{(5, 27)} = 13.68 (left), P < 0.001; F_{(5, 27)} = 12.68, P < 0.001 (middle); F_{(5, 27)} = 30.28, P < 0.001 (right)). Raw data were expressed as a number of neurons labeled for Arc, H1a or both mRNA divided for the all counted neuronal nuclei analyzed. For each animal, we then calculated the mean of these raw data. g The total rate of H1a was lower in both the naive and the 15 kHz groups than in the other groups (F_{(5, 27)} = 24.67, P < 0.001). h The total rate of Arc was lower in the naive rats (F_{(5, 27)} = 28.39, P < 0.001). i Venn diagrams showing the percentage of H1a- (green), Arc- (red), and double- (yellow) labeled neurons in LA in the different experimental conditions. Diagrams’ size was scaled on the basis of H1a or Arc total ratios, and percentages were calculated by dividing the number of H1a-, Arc-, and double-labeled neurons for the total number of cells activated in at least one of the two events. *P < 0.05, **P < 0.01, ***P < 0.001. All data are mean and SEM. One-way ANOVA with Newman–Keuls test (b, f, i, j)