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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 4. — Concomitant locomotor activity during USV recordings following repeated neonatal morphine exposure. A, RM ANOVA of normalized total distance during USV recordings (treatment and sex as factors, postnatal day as the RM) indicated no effect of treatment (F_(1,70) = 0.78; p = 0.38), but there was a significant effect of postnatal day (F_(1,70) = 199.52, *p < 0.001), as pups traveled an average of 14.36 ± 1.02 m further on P14 versus P7. There was no effect of sex (F_(1,70) = 0.26, p = 0.61), and there were no significant interactions (all p values ≥ 0.59). B, To detect any time-dependent effects of morphine on locomotor activity during USV recordings, we also assessed distance traveled per min. Again, data were normalized before parametric analysis and Greenhouse–Geisser corrections were applied (Mauchly’s test; P7: χ²₍₄₄₎ = 135.38, p < 0.001, ε = 0.67; P14: χ²₍₄₄₎ = 233.39, p < 0.001, ε = 0.52). On P7, RM ANOVA (treatment and sex as factors, time as the RM) indicated the main effects of treatment (F_(1,71) = 0.67, p = 0.04) and time (F_{(6.03,428.15)} = 77.96, p < 0.001), and significant treatment × time (F_{(6.03,428.15)} = 15.81, p < 0.001) and treatment × sex (F_(1,71) = 4.29, p = 0.04) interactions. The time × treatment interaction was driven by increased distance traveled by morphine-exposed pups during minute 1 through minute 4 of USV recordings (*all adjusted p values ≤ 0.02; unpaired t tests with Bonferroni correction). The treatment × sex interaction was driven by an increase in distance traveled in morphine-exposed males relative to saline males (0.12 ± 0.04 m; Bonferroni-adjusted p = 0.006). C, On P14, RM ANOVA (treatment and sex as factors, time as the RM) indicated a main effect of time (F_(4.7,333.70) = 4.86, p < 0.001) and a near-significant time × treatment interaction (F_(4.7,333.70) = 2.21, p = 0.057). There was no main effect of sex or interactions with sex (p values > 0.23). In contrast to P7, there was no significant difference between saline control and morphine-exposed mice at any time point (all Bonferroni-adjusted p values ≥ 0.53). Data are presented as the mean ± SEM of raw (non-normalized) data.