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. 2021 Sep 20;8(5):ENEURO.0143-21.2021. doi: 10.1523/ENEURO.0143-21.2021

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Copyright © 2021 Borrelli et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Figure 3. — Pronounced increases in USVs during maternal separation following repeated neonatal morphine exposure. A, In examining normalized total USVs emitted over 10 min, RM ANOVA (treatment and sex as factors, postnatal day as the RM) revealed a main effect of treatment (F_(1,69) = 8.77, p = 0.004), postnatal day (F_(1,69) = 70.89, p < 0.001), and treatment × postnatal day interaction (F_(1,69) = 5.10, p = 0.03). There was no effect of sex (F_(1,69) = 0.05, p = 0.82) and no interactions with sex (all p > 0.37). Tukey’s post hoc comparisons revealed no difference between treatment groups on P7 [95% CI for difference (saline – morphine): [−236.51, 118.56]; p = 0.82]. On P14, however, morphine-treated mice emitted more USVs than saline-treated mice [95% CI for difference (saline – morphine): [−531.81, −173.80]; *p < 0.001]. Both the saline- and morphine-treated groups showed a significant reduction in USVs emitted from P7 to P14 (saline group mean difference = −555.40, #p < 0.001; morphine group mean difference = −261.56, %p = 0.002). B, Greenhouse–Geisser corrections were applied when examining normalized USVs per minute on both P7 and P14 (Mauchly’s test; P7: χ²₍₄₄₎ = 117.39, p < 0.001, ε = 0.69; P14: χ²₍₄₄₎ = 155.41, p < 0.001, ε = 0.60). On P7, RM ANOVA (treatment and sex as factors, time as the RM) identified a treatment × sex × time interaction (F_{(6.22,435.22)} = 2.34; p < 0.03). Subsequent RM ANOVA of females only revealed a significant treatment × time interaction (F_{(6.07,230.58)} = 6.91; p < 0.001) that was driven by elevated USVs per minute during the first 3 min in morphine-exposed females compared with saline-control females (*Bonferroni adjusted: p = 0.004, 0.009, and 0.02, respectively). In contrast, RM ANOVA of males only indicated no significant treatment × time interaction (F_{(5.29,169.19)} = 0.83; p = 0.54) but only a main effect of time (F_{(5.29,169.19)} = 3.54; p = 0.004). C, In examining normalized USVs per minute on P14, RM ANOVA (treatment and sex as factors, time as the RM) indicated main effects of treatment (F_(1,70) = 14.09, p < 0.001) and time (F_{(5.41,378.88)} = 34.01, p < 0.001), and a treatment × time interaction (F_{(5.41,378.88)} = 8.19, p < 0.001). There was no main effect or interaction with sex (all p values > 0.47). We performed unpaired t tests between treatment groups (collapsed across sexes) to determine group differences across the 10 min testing period. For minutes 4 and 6–10, morphine-exposed pups emitted significantly more USVs per minute than saline-control pups (*all Bonferroni-adjusted p values < 0.03). At minute 5, the same trend was present, but was not significant (adjusted p = 0.07). Data are presented as the mean ± SEM of raw (non-normalized) data.