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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 5. — Changes in ultrasonic vocalizations following repeated neonatal morphine exposure. A, There was an increase in normalized syllable duration in morphine-exposed females and males on P14 relative to saline controls. There were main effects of treatment (F_(1,40) = 42.97, ****p < 0.0001), postnatal day (F_(1,40) = 240.0, ****p < 0.0001), and a significant treatment × postnatal day interaction (F_(1,28) = 6.15, *p = 0.02). There was no main effect of sex (F_(1,72) = 0.75, p = 0.39) or any interactions with sex (treatment × sex: F_(1,72) = 0.004, p = 0.95; age × treatment × sex: F_(1,72) = 1.09, p = 0.30). Tukey’s post hoc comparisons revealed the following: P7: saline versus morphine, *p = 0.0249; P14: saline versus morphine, ****p < 0.0001. B, Repeated morphine exposure led to a decrease in normalized intersyllable interval in female and male P14 mice relative to saline controls. There were main effects of treatment (F_(1,40) = 60.06, ****p < 0.0001) and postnatal day (F_(1,40) = 138.3, ****p < 0.0001) as well as a significant treatment × postnatal day interaction (F_(1,28) = 54.82, ****p < 0.0001). Again, there were no main effects of sex (F_(1,144) = 0.15, p = 0.69) or any interactions with sex (treatment × sex: F_(1,144) < 0.001, p = 0.9854; treatment × postnatal day × sex: F_(1,144) = 0.08, p = 0.77). Tukey’s post hoc comparisons revealed the following: P7: saline versus morphine, p = 0.99; P14: saline versus morphine, ****p < 0.0001. C, Chronic opioid exposure led to an increase in normalized minimum frequency in female P14 mice relative to saline controls. There was a main effect of postnatal day (F_(1,40) = 192.10, ****p < 0.0001) and a significant treatment × postnatal day interaction (F_(1,28) = 25.55, ****p < 0.0001). There was no main effect of treatment (F_(1,40) = 3.489, p = 0.0691), no main effect of sex (F_(1,72) < 0.001, p = 0.98), and no interactions with sex (treatment × sex: F_(1,72) = 0.22, p = 0.64; treatment × postnatal day × sex: F_(1,72) = 0.46, p = 0.50). Tukey’s post hoc comparisons revealed the following: P7: saline versus morphine, p = 0.28; P14: saline versus morphine, ****p < 0.0001. D, Repeated morphine exposure led to an overall increase in normalized maximum frequency relative to saline controls. There were main effects of treatment (F_(1,40) = 71.76, ****p < 0.0001) and postnatal day (F_(1,40) = 71.08, ****p < 0.0001) and a significant treatment × postnatal day interaction (F_(1,28) = 29.02, ****p < 0.0001). There was no main effect of sex (F_(1,72) = 0.96, p = 0.33), and there were no interactions with sex (treatment × sex: F_(1,72) = 1.11, p = 0.30; treatment × postnatal day × sex: F_(1,72) = 0.10, p = 0.76). Tukey’s post hoc comparisons revealed the following: P7: saline versus morphine, p = 0.13; P14: saline versus morphine, ****p < 0.0001. E, Chronic opioid exposure led to an increase in normalized mean frequency in female and male P14 mice relative to saline controls. There were main effects of treatment (F_(1,40) = 42.90, ****p < 0.0001) and postnatal day (F_(1,40) = 107.1, ****p < 0.0001), and a significant treatment × postnatal day interaction (F_(1,28) = 33.73, ****p < 0.0001). There was no main effect of sex (F_(1,72) = 0.06, p = 0.82), and there were no interactions with sex (treatment × sex: F_(1,72) = 0.78, p = 0.38; treatment × postnatal day × sex: F_(1,72) = 0.16, p = 0.69). Tukey’s post hoc comparisons revealed the following: P7: saline versus morphine, p = 0.95; P14: saline versus morphine, ****p < 0.0001. F, Chronic opioid exposure led to an overall increase in frequency bandwidth across groups relative to saline controls. There were main effects of both treatment (F_(1,40) = 28.50, ****p < 0.0001) and postnatal day (F_(1,40) = 13.98, ***p = 0.0006), with no significant treatment × postnatal day interaction (F_(1,28) = 1.676, p = 0.2068). There was no main effect of sex (F_(1,72) = 0.4988, p = 0.4823), and there were no interactions with sex (treatment × sex: F_(1,72) = 0.1259, p = 0.7238; treatment × postnatal day × sex: F_(1,72) = 0.3515, p = 0.5551). For all parameters, we performed a three-way ANOVA mixed-effects model followed by a Tukey’s post hoc test.