Figure 3. USV emissions during spontaneous morphine withdrawal on P7 in FVB/N pups.

Data are plotted as the mean ± SEM. Closed circles = females. Open circles = males. Data were analyzed using linear mixed models with Saline Treatment, Female Sex, and NCrl Substrain as the reference variables and Pup as a random effect (for repeated measures). The following data are collapsed across Substrain and Sex for simplicity. A. P7 USVs across time: The effect of Morphine Treatment was dependent on Time (β = −5.38, SE = 0.62, t(1573) = −8.63, ****p < 0.001, where Morphine Treatment was associated with an increase in USVs during the first 4 min of the recording session (all **p < 0.0055), and a decrease in USVs during the 6 – 10 min time intervals (all *p < 0.040). B. P7 Total USVs: Overall, there was no effect of Morphine Treatment on the total number of USVs (β = −0.99, SE = 40.64, t(181) = −0.024, p = 0.98). C. P7 Syllable Profile: Morphine Treatment was associated with a decrease in Complex 3 (β = −0.12, SE = 0.025, t(181) = −5.04, ****p < 0.001) and Upward (β = −0.014, SE = 0.006, t(181) = −2.28, *p = 0.024), and an increase in Downward (β = 0.036, SE = 0.0089, t(181) = 4.04, ****p < 0.0001) and Short (β = 0.015, SE = 0.0057, t(181) = 2.71, **p = 0.0074). D. P14 USVs across time: There was a Morphine Treatment x Sex x Time interaction (β = −2.32, SE = 0.53, t(2170) = −4.37, ****p < 0.0001). Both morphine-withdrawn females and males vocalized more over time compared to saline-control pups; however, morphine-withdrawn females vocalized more than morphine-withdrawn males during the 3, 4, and 6 – 15 min intervals (all *p < 0.015). E. P14 Total USVs: There was a Morphine Treatment x Sex interaction (β = −452.5, SE = 153.5, t(151) = −2.95, **p = 0.0037). Both morphine-withdrawn females (β = 883.63, SE = 93.49, t(80) 9.45, ****p < 0.0001) and morphine-withdrawn males (β = 431.1, SE = 124.2, t(71) = 3.47, ***p = 0.00089) vocalized more than saline-control pups. Furthermore, morphine-withdrawn females emitted more USVs than morphine-withdrawn males (β = 471.6, SE = 109, t(151) = 4.32, ****p < 0.0001). F. P14 Syllable Profile: Morphine Treatment was associated with an increase in the proportion of Complex 3 syllables emitted during spontaneous opioid withdrawal (β = 0.14, SE = 0.20, t(155) = 6.89, ****p < 0.0001), and a decrease in Downward (β = −0.015, SE = 0.0077, t(155) = −2.02, *p = 0.045), Flat (β = −0.034, SE = 0.0071, t(155) = −4.73, ****p < 0.0001), Short (β = −0.058, SE = 0.013, t(155) = −4.61, ****p < 0.0001), and Upward (β = −0.056, SE = 0.016, t(155) = −3.48, ***p = 0.00065) syllables. G. Zipf Slope: The Zipf slope of morphine-withdrawn pups decreased from P7 to P14, demonstrating that the syllable repertoire became less random and more repetitious. The Zipf slope of saline-treated pups increased from P7 to P14, depicting increased syllable usage diversity. P7: SAL, n = 88 (50F, 38M); MOR, n = 87 (45F, 42M). P14: SAL, n = 79 (44F, 25M); MOR, n = 76 (38F, 38M).