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. 2019 Nov 15;9:16928. doi: 10.1038/s41598-019-53294-z

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© The Author(s) 2019

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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Treatment effects on mouse pup ultrasonic vocalizations (USVs) and call microstructure. Maternal separation was used to evoke USVs in male (A) and female (B) mouse pups on postnatal days (PND) 10, 12, 14, and 16. Total number of USVs per 3 min were quantified; data are expressed as Mean + SEM PND 10 (Male n = VV: 14, PV: 22, VL: 15, PL: 23) (Female n = VV: 18, PV:18, VL: 8, PL: 12), PND12 (Male n = VV: 13, PV: 21, VL: 15, PL: 23) (Male n = VV: 14, PV: 22, VL: 15, PL: 23) (Female n = VV: 16, PV: 16, VL: 8, PL: 12), PND14 (Male n = VV: 8, PV: 8, VL: 14, PL: 25) (Female n = VV: 11, PV: 6, VL: 7, PL: 17) PND16 (Male n = VV: 15 PV: 22, VL: 10, PL: 7) (Female n = VV: 18, PV: 18, VL: 6, PL: 4). Representative tracings on Day 12 from mice that received the control (C) and “two-hit” (D) treatments revealed dramatic differences in USVs. Detailed analyses revealed major differences in call numbers, patterns, and durations in males (E–G) and females (H–J). *P < 0.05, **P < 0.01 compared to VV group, Bonferroni’s tests.